Category: 8. Health

HaDEA opens 2025 EU4Health proposals focused on CBRN threats and vector-borne diseases and crisis preparedness 

The European Health and Digital Executive Agency (HaDEA) has announced new funding opportunities under the 2025 EU4Health Work Programme. This new funding will improve the European Union’s crisis preparedness and response to future health emergencies.

These calls for proposals are focused on the development of innovative medical countermeasures for chemical, biological, radiological, and nuclear (CBRN) threats, as well as advanced diagnostics for vector-borne diseases.

Applications are open until 4 December 2025 at 17:00 CEST, through the EU Funding and Tenders Portal.

Focusing on CBRN medical countermeasures

The first central funding line, EU4H-2025-HERA-PJ-1, invites proposals to support the development of cutting-edge medical tools to counter CBRN threats. This call, along with the Health Emergency Preparedness and Response Authority (HERA), is divided into three subtopics:

• Medicinal Products (EU4H-2025-HERA-PJ-1-a): 
  • Funding will support the development of innovative medicinal products to prevent or treat illnesses resulting from CBRN exposure.
• Reusable Respiratory PPE and Protection Suits (EU4H-2025-HERA-PJ-1-b):
  • This subtopic targets innovation in high-quality, reusable personal protective equipment designed for use in CBRN environments.
• Detection and Diagnosis (EU4H-2025-HERA-PJ-1-c):
  • This part of the call supports the development of technologies for rapid detection and accurate diagnosis of CBRN-related threats.

These initiatives aim to enhance the EU’s crisis preparedness for health emergencies, enhance civilian protection, and ensure the availability of effective countermeasures during crises.

Diagnostics for vector-borne diseases

The second major funding call, EU4H-2025-HERA-PJ-2, addresses the growing concern of vector-borne diseases (VBDs), which are transmitted by insects such as mosquitoes and ticks. The call focuses on the development of innovative diagnostic tests that can help health systems detect and manage these diseases more effectively.

With climate change and increased global travel contributing to the spread of VBDs like dengue, chikungunya, and Lyme disease, the EU aims to strengthen health surveillance and early detection capabilities. Improved diagnostics are expected to play a key role in reducing the burden of these diseases and enhancing public health resilience.

Information session on 15 September

To support potential applicants, HaDEA and HERA will host an Info Session on Monday, 15 September 2025, from 14:30 to 17:00 CEST. The session will provide an overview of the calls, including their policy background, objectives, expected outcomes, and application procedures. Interested participants are encouraged to register in advance to secure their attendance.

These calls form part of the EU4Health programme, the EU’s largest health funding initiative to date. Launched in response to the COVID-19 pandemic, EU4Health aims to build more resilient healthcare systems across Europe. By supporting innovation in medical technologies, public health infrastructure, and cross-border cooperation, the programme contributes to a healthier and more secure Europe.

From 2021 to 2027, HaDEA is responsible for implementing most of the EU4Health budget, managing grants and tenders to support a wide range of health-related projects.

Scientists are hailing promising breakthroughs in the fight against cancer, with one new therapy appearing to kill tumors without damaging healthy flesh.

Novartis’s radioligand therapy targets mutations in tumors, delivering radiation only where it is needed, unlike ordinary radiotherapy which kills non-cancerous cells as well as cancerous ones. In a trial, the Novartis treatment removed all disease from 21% of patients whose cancers had spread around the body, which an oncologist told The New York Times was “never seen before.”

In other progress in the fight against cancer, The Economist reported that scientists are attempting to prevent the disease by boosting the metabolism of non-cancerous cells so they grow faster, “levelling the arms race between unhealthy and healthy cells.”

Introduction

Community-acquired pneumonia (CAP), a significant health concern around the globe, represents a leading cause of hospitalization in children.^1,2 The overall incidence rate of CAP was 15.97 per 1000 person-years in children below 5 years old in southeastern China from January 1, 2015 to December 31, 2020.³ Pediatric CAP is characterized by heterogeneous clinical presentations, ranging from mild respiratory or systemic symptoms (eg, fever, cough, and wheezing) to severe complications, such as acute renal injury, sepsis, and multiorgan failure.⁴ Intricate molecular mechanisms including inflammatory responses, oxidative reactions, and cellular apoptosis play pivotal roles in the progression of childhood CAP.⁵ The pediatric critical illness score (PCIS) is summed based on 10 indicators from laboratory tests and physical examination.⁶ The clinical pulmonary infection score (CPIS) is calculated at a basis of clinical, analytical, imaging and microbiological data.⁷ Both PICS and CPIS are conventionally used to evaluate CAP severity in children.^8,9 Complicated CAP, a severe form manifested by local or systemic complications, signifies disease progression and necessitates aggressive treatments.^10–12 Accordingly, early identification of complicated CAP may be of utmost significance in the clinical practice of CAP treatment in children. However, complexities of PICS and CPIS calculations may limit their clinical feasibility in clinical work, necessitating continued search for blood biomarkers owning to easy obtainability of blood samples in terms of discrimination of complicated CAP in children.

Inflammasomes have been implicated in a spectrum of pathophysiological processes, including the occurrence and development of pulmonary infections.^13,14 Absent in melanoma 2 (AIM2), a key component of the inflammasome complex, is a critical mediator of the inflammatory responses in various inflammation-related diseases.^15,16 AIM2 expression in the lung tissues was substantially elevated.¹⁷ In addition, lung injury was attenuated, and survival was significantly improved in AIM2-deficient mice with influenza-induced lung injury.¹⁸ Similarly, AIM2-driven alveolar macrophage pyroptosis markedly aggravated experimental lung injury, whereas genetic silencing of AIM2 notably diminished inflammation.¹⁹ Moreover, higher AIM2 levels in the bronchoalveolar lavage fluid were associated with pulmonary fibrosis.²⁰ Intriguingly, increased serum AIM2 levels were independently associated with stroke-associated pneumonia in adults with acute intracerebral hemorrhage.²¹ These data suggest that AIM2 could be specifically derived from lung injury, therefore leading to the conception that serum AIM2 may be a potential biomarker of lung injury. Here, serum AIM2 levels were measured in a group of children with CAP to investigate serum AIM2 as a biomarker for assessing severity and identifying complicated CAP in children.

Materials and Methods

Study Design and Subject Selection

This prospective cohort study was done at the Hangzhou Children’s Hospital between January 2022 and June 2023. All children with CAP were enrolled consecutively. The inclusion criteria were as follows: (1) newly diagnosed CAP, (2) 3 months < age <14 years in consideration of blood-sampling obtainability and physiological traits of children, and (3) admission of children with CAP to the hospital. The exclusion criteria were (1) other respiratory diseases, such as allergic pneumonia, asthma, or tuberculosis; (2) use of immunosuppressive drugs, underlying immune system disorders, congenital illnesses, and severe illness in other organs; and (3) other specific conditions, such as reluctance to participate, loss to follow-up, incomplete information, and unqualified blood samples. Children who underwent routine examinations at Hangzhou Children’s Hospital were recruited as controls. This study was conducted in accordance with the principles of the Declaration of Helsinki, and the research protocol was approved by the Ethics Committee of the Hangzhou Children’s Hospital (Ethics Approval Number: 2021–47) and written informed consent was obtained from the children’s guardians.

Data Collection

Some basic information, including age, sex, weight, height, preterm birth, family smoking status, vaccination, preadmission antibiotic use, preadmission fever and cough durations, were registered. Disease severity was assessed using the PCIS⁶ and the CPIS.⁷ Pathogens were classified into bacteria, virus, mycoplasma pneumoniae and mixed type. Complicated CAP was considered when any local or systemic complication was identified.^10–12 Local complications included parapneumonic effusion, empyema, necrotizing pneumonia, and lung abscess, and systemic complications included bacteremia, metastatic infection, multiorgan failure, acute respiratory distress syndrome, and disseminated intravascular coagulation and so forth.^10–12

Immune Analysis

Peripheral venous blood samples were collected at admission from children with CAP and at the entrance of the study from the control children. The blood samples were centrifuged to separate the serum for storage at −80 °C until subsequent testing. Serum AIM2 levels were measured using enzyme-linked immunosorbent assay (Catalog No. ZY-E6125H; Shanghai Zeye Biotechnology Co. Ltd., Shanghai, China). The detection range of this kit was 0.156–10 ng/mL with a sensitivity of 0.094 ng/mL, and both intra- and inter-assay coefficients of variation were less than 10%. All samples were tested in duplicate by identical proficient technicians, who were inaccessible to clinical details. The two measurements were averaged for subsequent analyses.

Statistical Analysis

Statistical analyses were completed applying SPSS 25.0 (IBM Corporation, Armonk, NY, USA), GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA), R 4.2.2 (https://www.r-project.org), and MedCalc 20.305 (MedCalc Software, Mariakerke, Belgium). The Kolmogorov–Smirnov test was used to determine the distribution normality of the quantitative variables. Normally distributed variables are presented as mean±standard deviation, whereas non-normally distributed variables are presented as median (25th-75th percentiles). Qualitative data are reported as counts (proportions). Based on the different data types, the χ2 test, Fisher’s exact test, Mann–Whitney U-test, or t-test was employed for intergroup comparisons, as applicable. Bivariate correlation analysis was performed using the Spearman correlation test. A multivariate linear regression model was used to identify variables that were independently associated with serum AIM2 levels. Serum AIM2 levels were dichotomized according to their median values as high and low levels. The relevant variables were compared between the two groups to determine substantially different variables. These markedly different factors were included in the binary logistic regression model to reveal the independently associated parameters. In order to ascertain whether linear model was appropriate for statistical analysis, the restricted cubic spline was drawn to discern the possible linear correlation between serum AIM2 levels and risk of complicated CAP; and if P value was above 0.05 for nonlinear assumption, the linear model should be adopted for data analysis. To compare the differences of data between children with and without complicated CAP, a binary logistic regression model was used to investigate independently associated variables. Odds ratios (OR) and corresponding 95% confidence intervals (CI) were calculated to show associations. Subgroup analyses were performed to investigate whether the association was moderated by other variables, such as age, sex, weight, height and so forth. E-value, a component of sensitivity analysis, was computed based on OR value in regression analysis for reflecting the robustness of the association, with higher value signifying more strong result association.²² A variance inflation factor (VIF) was generated to evaluate multicollinearity in the regression model; a VIF value < 10 indicates the absence of multicollinearity.²³ Receiver operating characteristic (ROC) curves were constructed to explore the discrimination efficiency. Z-test was used to compare the area under the curve. The independent predictors of complicated CAP were consolidated to develop the model. The model was pictorially represented by the nomogram, so as to predict CAP risk, in which each independent predictor corresponded to the respective point and all points were aggregated to mirror risk. A calibration curve was plotted to demonstrate the stability of the model and a decision curve was drawn to assess the clinical applicability of the model. Meanwhile, the Hosmer-Lemeshow test was done and brier score was computed in order to unveil whether the model was performed stably. Net reclassification improvement and integrated discrimination improvement indices were calculated to determine the improvement rate of the model. Here, the sample size was estimated at a type 1 error value (alpha) of 0.05, test power (1-beta) of 0.95, and Cohen’s d of at least 0.8 for effect size in comparison of serum AIM2 levels across complicated CAP. A priori power analysis was performed to validate the adequate sample size by employing the G*Power 3.1.9.4 (Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, Düsseldorf, Germany). Differences were considered statistically significant at a two-sided P-value of <0.05.

Results

Subject Selection and Features

An initial assessment was performed on 362 children with CAP who met pre-established inclusion criteria. In accordance with the prespecified exclusion criteria, fifty-seven children were excluded from this study because of other respiratory diseases (17 cases), use of immunosuppressive drugs (6 cases), underlying immune system disorders (7 cases), congenital illnesses (8 cases), severe sickness in other organs (8 cases), reluctance to participate in this study (3 cases), missed visits (2 cases), incomplete information (2 cases), and unqualified blood samples (4 cases). Ultimately, 305 children were included in the epidemiological survey. Baseline patient characteristics are outlined in Table 1. A group of 100 healthy children was used as a control. This group of controls consisted of 54 boys and 46 girls, encompassed 24 children experiencing family smoking, included 10 suffering from preterm birth, were aged at mean value of 43.4 months (standard deviation, 33.4 months), had mean weight of 16.8 kg (standard deviation, 7.8 kg) and showed mean height of 101.8 cm (standard deviation, 25.3 cm). The above six variables did not differ significantly between the diseased children and the controls (all P>0.05).

Serum AIM2 Levels and Disease Severity

Serum-based AIM2 levels were markedly higher in children with CAP than in the controls (P<0.001; Figure 1). Serum AIM2 levels were significantly negatively correlated with the PCIS (P<0.001; Figure 2) and were substantially positively related to CPIS (P< 0.001; Figure 3). In addition to the PCIS and CPIS, body temperature, blood procalcitonin levels, white blood cell counts, and blood C-reactive protein levels were closely related to serum AIM2 levels (all P<0.05; Table 1). By incorporating the six aforementioned factors, that is the PCIS, CPIS, body temperature, blood procalcitonin levels, white blood cell counts and blood C-reactive protein levels, into the multivariable linear regression model, the PCIS (beta, −0.020; 95% CI, −0.025–0.015; VIF, 1.408; P=0.001) and CPIS (beta, 0.092; 95% CI, 0.069–0.115; VIF, 1.553; P=0.002) were independently correlated with serum AIM2 levels. Next, diseased children were divided into two groups according to the median serum AIM2 level, that is the levels ≥ 1.45 ng/mL and < 1.45 ng/mL. As compared to children with serum AIM2 levels < 1.45 ng/mL, those with the levels ≥ 1.45 ng/mL displayed substantially elevated PCIS, CPIS, body temperature, blood procalcitonin levels, white blood cell counts and blood C-reactive protein levels (all P<0.05; Table 2). Subsequently, those significant variables, encompassing PCIS, CPIS, body temperature, blood procalcitonin levels, white blood cell counts and blood C-reactive protein levels, were included in the binary logistic regression model, and then PCIS (OR, 0.864; 95% CI, 0.824–0.907; VIF, 1.982; P=0.002) and CPIS (OR, 1.924; 95% CI, 1.531–2.417; VIF, 2.103; P=0.003) were independently associated with serum AIM2 levels ≥ 1.45 ng/mL.

Table 2 Baseline Features Between Community-Acquired Pneumonia Children with High and Low Serum Absent in Melanoma 2 Levels

Figure 1 Differences in serum levels of absent in melanoma 2 between healthy controls and children with community-acquired pneumonia. Serum absent in melanoma 2 levels are expressed as the median (upper quartile-lower quartile). Using the Mann–Whitney U-test, serum absent in melanoma 2 levels in children with community-acquired pneumonia were significantly higher than those in healthy controls (P<0.001). AIM2 indicates absent in melanoma 2.

Figure 2 Relationship between serum absent in melanoma 2 levels and pediatric critical illness score after community-acquired pneumonia in children. Using Spearman correlation coefficient, serum absent in melanoma 2 levels were strongly inversely correlated with the pediatric critical illness score after childhood community-acquired pneumonia (P<0.001). AIM2 means absent in melanoma 2. Abbreviation: PCIS, pediatric critical illness score.

Figure 3 Relationship between serum absent in melanoma 2 levels and clinical pulmonary infection score after pediatric community-acquired pneumonia. Using Spearman correlation coefficient, serum absent in melanoma 2 levels were intimately positively correlated with the clinical pulmonary infection score of children with community-acquired pneumonia (P<0.001). AIM2 denotes absent in melanoma 2. Abbreviation: CPIS, clinical pulmonary infection score.

Serum AIM2 Levels and Complicated CAP

In contrast to children without complicated CAP, those with the adverse event had notably increased serum AIM2 levels (P<0.001; Figure 4). Alternatively, serum-based AIM2 levels effectively anticipated complicated CAP, and its threshold was selected at 1.58 ng/mL using the Youden approach, generating the maximum Youden index of 0.535 for outcome prediction (Figure 5). In the context of the restricted cubic spline analysis, serum AIM2 levels were linearly related to the probability of complicated CAP (P for nonlinearity > 0.05; Figure 6), signifying suitability of linear model in the next statistical analysis. As shown in Table 3, children presenting with complicated CAP, relative to those without such an event, had obviously decreased age and height, as well as held apparently increased serum AIM2 levels, PCIS, CPIS, blood procalcitonin levels, white blood cell counts, and blood C-reactive protein levels (all P<0.05). When all eight significantly different parameters, encompassing age, height, serum AIM2 levels, PCIS, CPIS, blood procalcitonin levels, white blood cell counts and blood C-reactive protein levels, were integrated into the binary logistic regression module, we found that serum AIM2 levels (OR, 6.162; 95% CI, 1.752–21.670; VIF, 2.312; P=0.005), PCIS (OR, 0.907; 95% CI, 0.867–0.949; VIF, 2.419; P=0.001), and CPIS (OR, 1.391; 95% CI, 1.114–1.738; VIF, 2.375; P=0.004) independently predicted complicated CAP. In the subgroup analysis framework, the association between serum AIM2 levels and complicated CAP was not moderated by certain factors, such as age, sex, weight, height, family smoking, preadmission fever duration, and cough duration (all P interaction > 0.05; Figure 7). As for the sensitivity analysis in Figure 8, the E-value was 11.8 (95% CI, 2.90, 42.83), denoting enough high E-value versus OR value. In the next step, we modelled a prediction system by integrating the three independent predictors of complicated CAP, namely, serum AIM2, PCIS, and CPIS. The model was pictorially exhibited via the nomogram to instruct clinicians to prognosticate complicated CAP, with higher total scores corresponding to higher risk (Figure 9). In the milieu of the calibration curve analysis, the model had satisfactory goodness of fit, as confirmed by a small mean absolute error at 0.025 (Figure 10). Using the Hosmer-Lemeshow test, P value equaled to 0.235. And, brier score was 0.258. Based on the background of the decision curve analysis, the model presented good clinical validity, as opposed to serum AIM2, PCIS, CPIS, and PCIS combined with CPIS (Figure 11). Under the ROC curve (Figure 12 and Table 4), predictive ability of serum AIM2 resembled those of PCIS and CPIS (both P>0.05); combination of CPIS and PCIS significantly outperformed serum AIM2, PCIS and CPIS (all P<0.05); as well as predictive capability of the model, in which three predictors were integrated, substantially surpassed those of serum AIM2, PCIS, CPIS, and PCIS combined with CPIS (all P<0.05). Also, the conventional biomarkers, that is blood procalcitonin levels, white blood cell counts and blood C-reactive protein levels, were not in possession of obvious advantages in identifying childhood complicated CAP (all P<0.001; Table 4). Next, the model improvement rate was estimated. As shown in Figure 13, the net reclassification improvement was 0.126 (95% CI, 0.011–0.242) (P=0.032) and the integrated discrimination improvement was 0.066 (95% CI, 0.018–0.114) (P=0.007).

Table 3 Factors Associated with Complicated Community-Acquired Pneumonia

Table 4 Areas Under Receiver Operating Characteristic Curve for Identifying Complicated Community-Acquired Pneumonia in Children

Figure 4 Differences in serum absent in melanoma 2 levels between children with complicated community-acquired pneumonia and those without such an adverse event. Using the Mann–Whitney U-test, serum absent in melanoma 2 levels were substantially higher in children with complicated community-acquired pneumonia than in those not presenting with such an affair (P<0.001). AIM2 signifies absent in melanoma 2. Abbreviation: CAP, community-acquired pneumonia.

Figure 5 Receiver operating characteristic curve evaluating discrimination efficiency of serum absent in melanoma 2 levels on complicated community-acquired pneumonia in children. Complicated community-acquired pneumonia was effectively anticipated due to the absence of serum absent in melanoma 2 levels in children. The Youden approach was applied to determine the threshold value of serum absent in melanoma 2 levels to make predictions with medium-to-high sensitivity and specificity. Circle refers to the cutoff value of serum absent in melanoma 2 levels. Abbreviation: CAP, indicates community-acquired pneumonia; AUC, area under the curve; 95% CI, 95% confidence interval.

Figure 6 Restricted cubic spline assessing linear relationship between serum absent in melanoma 2 levels and risk of complicated community-acquired pneumonia in children. Serum absent in melanoma 2 levels were linearly correlated with the likelihood of pediatric complicated community-acquired pneumonia (P for nonlinear > 0.05), indicating that result association could be verified in regression model. AIM2 is indicative of absent in melanoma 2. Abbreviation: CAP, community-acquired pneumonia.

Figure 7 Subgroup analyses examining interactional effects of some conventional variables on association of serum absent in melanoma 2 levels with childhood complicated community-acquired pneumonia. Age, sex, weight, height, family smoking, pre-admission fever duration, and pre-admission cough duration did not show a markedly moderate relationship between serum absent in melanoma 2 levels and pediatric complicated community-acquired pneumonia (all P interaction > 0.05). OR stands for odds ratio; 95% CI, 95% confidence interval.

Figure 8 Diagrammatic sketch showing E-value for expressing robustness of association between serum absent in melanoma 2 levels and childhood complicated community-acquired pneumonia. For sensitivity analysis, the E-value was 11.8 (95% confidence interval, 2.90–42.83) for displaying a robust association of serum absent in melanoma 2 levels with pediatric complicated community-acquired pneumonia.

Figure 9 Nomogram exhibiting model of complicated community-acquired pneumonia in children. The three predictors of complicated community-acquired pneumonia, that is, serum absent in melanoma 2, pediatric critical illness score, and clinical pulmonary infection score, were consolidated to develop a combined model for outcome anticipation in children. The model was visualized via the nomogram, with the summed scores reflecting risk. AIM2 denotes absent in melanoma 2. Abbreviations: PCIS, Pediatric Critical Illness Score; CPIS, Clinical Pulmonary Infection Score; CAP, community-acquired pneumonia.

Figure 10 Calibration curve determining stability of the merged model for forecasting complicated community-acquired pneumonia in children. A model containing serum absent in melanoma 2, pediatric critical illness score, and clinical pulmonary infection score was established to predict complicated pediatric community-acquired pneumonia. In accordance with low mean absolute error at 0.025, the model remained stable for outcome prediction. CAP is indicative of community-acquired pneumonia.

Figure 11 Decision curve observing validity of the combined model in prognosticating complicated community-acquired pneumonia in children. The model was composed of serum absent in melanoma 2, pediatric critical illness score, and clinical pulmonary infection score. In contrast to serum absent in melanoma 2, pediatric critical illness score, clinical pulmonary infection score, and combination of pediatric critical illness score with clinical pulmonary infection score, the model was demonstrated to benefit the clinical prediction of pediatric complicated community-acquired pneumonia on account of biggest area occupied by the model. AIM2 denotes absent in melanoma 2. Abbreviations: PCIS, Pediatric Critical Illness Score; CPIS, Clinical Pulmonary Infection Score.

Figure 12 Receiver operating characteristic curve investigating predictive strength of the model on pediatric complicated community-acquired pneumonia. The model was formed by combining the serum absent in melanoma 2, pediatric critical illness score, and clinical pulmonary infection score. In contrast to serum absent in melanoma 2, pediatric critical illness score, clinical pulmonary infection score, and the combination of pediatric critical illness score with clinical pulmonary infection score, the model was confirmed to possess significantly efficacious prediction ability in childhood complicated community-acquired pneumonia. AIM2 signifies absent in melanoma 2. Abbreviations: PCIS, Pediatric Critical Illness Score; CPIS, Clinical Pulmonary Infection Score.

Figure 13 Plot showing calculation of net reclassification improvement and integrated discrimination improvement. The standard model was composed of the pediatric critical illness and clinical pulmonary infection scores. The new model comprised serum absent in melanoma 2, pediatric critical illness score, and clinical pulmonary infection score. The net reclassification improvement was 0.126 (95% confidence interval, 0.011–0.242) and the integrated discrimination improvement was 0.066 (95% confidence interval, 0.018–0.114), meaning that the combined model may be in possession of markedly higher improvement rate.

Discussion

To the best of our knowledge, this may be the first study to explore the relationship between serum AIM2 levels, disease severity, and complicated CAP in children diagnosed of CAP. First, a profound increase in serum AIM2 levels after childhood CAP has been demonstrated in comparison to controls. Second, PCIS and CPIS were independent correlates of serum AIM2 levels, whether serum AIM2 was identified as a continuous variable or transformed into a binary variable. Third, serum AIM2, PCIS, and CPIS levels were independently predictive of complicated CAP in children. Finally, the model combining serum AIM2, PCIS, and CPIS showed a good performance in forecasting complicated CAP in children. Taken together, serum AIM2 levels may represent a promising biomarker for estimating CAP severity and predicting complicated CAP in children.

AIM2 functions as a cytosolic receptor for double-stranded DNA and is extensively involved in inflammasome activation.²⁴ It is widely expressed in epithelial and immune cells, particularly under infection and stress.²⁵ AIM2 is upregulated in lung tissues during infections such as tuberculosis and idiopathic pulmonary fibrosis.^26,27 Furthermore, increased expression of AIM2 has been documented in alveolar macrophages and lung epithelial cells in inflammatory and fibrotic lung diseases.^17–20 In adults with acute intracerebral hemorrhage, markedly enhanced admission serum AIM2 levels were strongly associated with a higher risk of stroke-associated pneumonia.²¹ Based on our finding that serum AIM2 levels are significantly higher following pediatric CAP, AIM2 may be actively involved in the host immune response to pulmonary tissue injury secondary to childhood CAP. Although it is unclear about detailed mechanisms of AIM2’ involvement in CAP or its complications, evidence about inflammasome signaling activation in other diseases implies that AIM2 activation may result in the synthesis of active interleukin-1beta and interleukin-18, thereby inducing pyroptosis, with subsequent participation in pathophysiological processes of pneumonia.^28–30 However, such a hypothesis needs to be demonstrated in future studies.

Compelling data suggest that AIM2 may be a deleterious factor in pulmonary infections,^18,19 and therefore AIM2 may be a potential therapeutic target of CAP and even its complications. On the other hand, it leads to the assumption that serum AIM2 levels may be positively related to CAP severity. CPIS and PCIS are two highly acknowledged severity assessment systems for childhood CAP.^8,9 In this cohort of children with CAP, serum AIM2 levels were strongly associated with CPIS and PCIS in univariate analysis. Using multivariate analysis, serum AIM2 was present in two forms: continuous and binary variables. Finally, it was affirmed that CPIS and PCIS were independently related to serum AIM2 levels in two multivariate modules, namely, the multivariate linear regression model and binary logistic regression model. These data strongly support the notion that serum AIM2 levels are highly correlated with CAP severity in children.

Complicated CAP encompasses one or more of the local or systemic complications of CAP.^10–12 Complicated CAP, which is marked by severe conditions, may massively protract from the disease course, thereby prolonging the length of hospitalization.^10–12 In this study, complicated CAP was identified as the outcome variable of interest. The two CAP severity scaling metrics, CPIS and PCIS, together with serum AIM2, were fully corroborated using multivariate analysis as the three associative factors of pediatric complicated CAP. A restricted cubic spline assessment was initiated in advance to verify the linear relationship between serum AIM2 levels and the possibility of complicated CAP in children. Moreover, the VIF for scaling multicollinearity was less than 10 in the current study, thereby avoiding multicollinearity.²³ Subgroup analysis was performed to investigate the moderating effect, and the association of serum AIM2 levels with complicated CAP was not affected by age, sex, weight, height, or other factors. E-value calculation is a sensitivity analysis modality.²² The E-value, relative to the OR value, was within the rational range in this cohort of subjects with childhood CAP. This series of statistical measurements ensured the validity and reliability of the results. Therefore, serum AIM2 may be an encouraging biomarker for identifying the risk of childhood complicated CAP.

Early and accurate recognition of the likelihood of pediatric complicated CAP is of the utmost importance in clinical practice.^10–12 Serum AIM2, PCIS, and CPIS levels are three determinants of childhood complicated CAP here. Serum AIM2 levels had a predictive ability comparable to that of PCIS and CPIS. Also, serum AIM2 levels transcended the conventional biomarkers, that is blood procalcitonin levels, white blood cell counts and blood C-reactive protein levels, in terms of identification ability of childhood complicated CAP. The prediction model was composed of independent predictors. As demonstrated by the ROC curve, calibration curve and decision curve, the model was clinically efficient, steady, and beneficial for prognosticating complicated CAP in children. Addition of the Hosmer-Lemeshow test and brier score calculation to statistical analysis further supports the steadiness of the model. Moreover, by estimating the net reclassification improvement and integrated discrimination improvement, the model, as opposed to PCIS combined with CPIS, achieved a significantly elevated improvement rate. Overall, accumulating statistical analyses showed that, from the perspective of additive effects possessed by serum AIM2, serum AIM2 may be an effective predictor of complicated CAP in children.

Several strengths and weaknesses should be mentioned. The strengths are shown below. First, the novelty of our study is pointed out here. To the best of our knowledge, this may be a first series of investigating serum AIM2 in children diseased of CAP and therefore finding that serum AIM2 may be a potential biomarker in relation to severity and complicated CAP in childhood. Second, the clinical values of our study should be elucidated here. In accordance with the cutoff value of serum AIM2 levels, a risk stratification could be done for children with CAP. If serum AIM2 levels are greater than the cutoff value, these diseased children may be at high risk of complicated CAP; so, this group of children should be actively monitored and even admitted into intensive care unit, followed by an aggressive treatment. And, based on numerous statistical methods, the integrated model containing serum AIM2 may be effective in clinical practice of pediatric complicated CAP because the model is able to facilitate risk stratification of complicated CAP in children and assists with aggressive intervention of childhood complicated CAP. The weaknesses are displayed in the following. First, because the risk of overfitting may be existent in model construction in a single-center design lacking external validation, and there are different populations or settings in clinical applications, particularly potential ethnic and environmental differences; these unstable factors possibly lead to difficulty in generalization of model in clinical use. And accordingly, a larger cohort study is warranted to validate effectiveness and stability of the model before the model is applied in prediction of pediatric complicated CAP. Second, even if serum AIM2 alone or the combined model integrating serum AIM2 is demonstrated to be a potential tool for discriminating children at risk of complicated CAP and subsequently instructing clinical treatments, its clinical practicability should be validated in future interventional study.

Conclusions

In children with CAP, significantly elevated serum AIM2 levels are independently correlated with PCIS and CPIS. Serum AIM2 levels are independent predictors of complicated pediatric CAP. The integrated model containing serum AIM2, PCIS, and CPIS has high clinical effectiveness in forecasting childhood complicated CAP. In summary, serum AIM2 level may be a potential biochemical indicator for pediatric CAP severity appraisal and anticipation of complicated CAP in children; and the combined model incorporating serum AIM2 may be a good tool for risk stratification of pediatric complicated CAP.

Abbreviations

AIM2, absent in melanoma 2; CAP, community-acquired pneumonia; PCIS, pediatric critical illness score; CPIS, clinical pulmonary infection score; ROC, receiver operating characteristic; AUC, area under the curve; OR, odds ratio; 95% CI, 95% confidence interval.

Data Sharing Statement

The raw data supporting the conclusions of this study will be provided by the authors without undue retention.

Funding

This study was financially supported by Zhejiang Provincial Medical and Health Science and Technology Plan (No. 2023RC248).

Disclosure

The authors declare that they have no competing interests in this work.

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Introduction

Acute ischemic stroke is one of the most prevalent diseases in the world, affecting 77 million people worldwide with a mortality rate of 3.3 million each year, becoming the second leading cause of death after heart disease.¹ The disease affected 8.3% of the population, causing a death toll of up to 192 deaths per 100,000 people and costs up to 2.57 trillion rupiahs.² The burden of stroke extends beyond death tolls because it is a leading cause of long-term disability. Around five million of stroke survivors suffer from permanent disabilities, such as vision loss, speech loss, paralysis, confusion, and thus lowers productivity and quality of life.³ These debilitating effects of stroke can be mitigated by early and effective interventions for acute ischemic stroke.⁴

Alteplase is the standard therapy for acute stroke. As a recombinant tissue plasminogen activator (rt-PA), alteplase must be administered within less than 4.5 hours of onset in stroke patients with no contraindications. Delayed treatment with alteplase will only diminish the effects or even worsen, and it will increase the risk of intracranial hemorrhage.⁵ This poses challenges for developing countries with their problems in recognition, admission, and diagnosis of stroke patients. Knowledge regarding stroke symptoms, awareness of the window period, and decision-making continue to contribute to delays.^6,7 Other than that, inaccessible areas and low-income status have restricted stroke patients from accessing costly thrombolytic agents.⁷ These problems are aggravated by the limited availability of hospitals performing thrombolysis.^2,8 As the gold standard therapy for acute ischemic stroke, alteplase still has its own limitations, especially when applied in developing countries. Therefore, alternative and adjuvant therapies have been recommended.

Lumbrokinase, a fibrinolytic agent, has been deemed a potential adjuvant therapy for ischemic stroke. This fibrinolytic enzyme, specifically Lumbricus sp., has inhibitory effects on platelet aggregation and is currently being rigorously studied for the treatment of various diseases, including cardiovascular and cerebrovascular diseases. It has the ability to hydrolyze both fibrin and fibrinogen which will prevent the formation of blood clots.⁹ Lumbrokinase has qualities superior to other similar fibrinolytic agents, such as urokinase and streptokinase, due to its higher thermal stability, alkali resistance, no conversion of plasminogen into plasmin, and highly specific to fibrin. Due to that, lumbrokinase can be administered orally, can reduce the risk of hemorrhage, and does not induce hyperfibrinolysis.^10,11 Other than that, due to its natural availability and non-invasive nature, lumbrokinase cuts off unnecessary expenses and does not pose various risks.¹² The overall ability and characteristics of lumbrokinase exhibit valuable potential as a therapeutic agent in treating stroke.

Lumbrokinase is typically administered orally in capsule form, which makes it more practical and non-invasive for stroke patients, particularly in low-resource settings. Studies have also explored intravenous and intramuscular routes, but oral administration remains the most common and accessible approach. Compared to alteplase – and even the newer drug, tenecteplase – lumbrokinase is considered less expensive, particularly in developing countries where healthcare costs are a major barrier to standard thrombolytic therapy. However, robust evidence for lumbrokinase as an adjunctive therapy for ischemic stroke is scarce. This increases the urgency for the systemic evaluation of lumbrokinase to treat ischemic stroke patients, including its efficacy and safety, when compared with standard supportive management. This paper unveils the efficacy and safety of lumbrokinase as an adjunctive therapy combined with supportive management for acute stroke when compared with standard management alone.

Methods

Study Design

A systematic review and meta-analysis was conducted using the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) 2020.^13,14

Data Sources and Search Strategy

A literature search was conducted independently and comprehensively up to July 2024 throughout several databases, including PubMed, Science Direct, EMBASE, EBSCO, Clinical key, Scopus, Proquest, MedRxiv, BioRxiv, SSRN, ClinicalTrials, PsycInfo, PsycNet, Web of Science, Google Scholar, and Cochrane. The keywords that were used are “lumbrokinase”, “acute ischemic stroke”, and “randomized controlled trials”. Any discrepancies were further discussed by the authors. Subsequently, the results were scrutinized for duplication and screened using the predetermined eligibility criteria.

Study Selection

All articles were independently reviewed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. A screening process was started through title and abstract and continued with full-text screening of selected studies to exclude studies that met the exclusion criteria. We included RCTs comparing lumbrokinase with supportive management to supportive management alone and excluded any other study designs such as non-RCTs and observational studies. All the selected studies were validated to ensure their eligibility for the next step.

Although older and smaller RCTs were included, they were retained based on predefined eligibility criteria and the limited availability of large, high-quality trials evaluating lumbrokinase in acute ischemic stroke. These studies contribute valuable data to a relatively under-researched area, and their inclusion allows for a more comprehensive understanding of the intervention’s efficacy and safety.

Outcomes Measured

This study measured both the primary and secondary outcomes to evaluate the effectiveness and safety of the intervention. The primary outcomes included the Barthel Index and NIHSS score, which assess functional independence and neurological deficits, respectively. Secondary outcomes included adverse events, such as gastrointestinal discomfort and bleeding, along with laboratory parameters, such as activated partial thromboplastin time (aPTT) and D-dimer levels.

Data Analysis

To ensure the reliability of the findings, the risk of bias in each included study was assessed using the Cochrane Risk of Bias 2.0 (RoB 2.0) tool.^15,16 Statistical analysis was conducted using a random effects model to account for potential variability among studies. The results were reported as odds ratios (OR) for dichotomous outcomes and mean differences (MD) for continuous outcomes, each with corresponding 95% confidence intervals (CI).

To evaluate publication bias, funnel plots were visually inspected for asymmetry, which may have indicated selective reporting. Heterogeneity among the included studies was assessed using the I² statistic, which quantifies the proportion of total variation in the effect estimates owing to heterogeneity rather than chance. An I² value of 0% indicated no observed heterogeneity, values above 50% indicated substantial heterogeneity, and values above 75% indicated considerable heterogeneity. The presence of significant heterogeneity may reflect clinical, methodological, or statistical differences between studies, which informs the interpretation of the pooled estimates in the meta-analysis.

Results

A total of 64 studies^17–80 were included in the analysis as illustrated in the PRISMA flowchart (Figure 1). These studies were selected after a comprehensive screening process that involved the identification of 1836 records through database searches and manual reviews, followed by the exclusion of 1773 duplicates and irrelevant studies. The final dataset comprised randomized controlled trials evaluating the efficacy and safety of the intervention across various clinical and laboratory outcomes, which are summarized in Table 1. The risk of bias is shown in Figure 2.

Figure 1 PRISMA 2020 flowchart of the included studies.

Figure 2 Risk of bias of the included studies.

Adverse events were grouped into five groups; three studies^60,69,79 reported GI discomfort, and the results shown in Figure 3 showed that there was no significant difference between the experimental and control groups, with an OR of 1.00 [95% CI 0.32; 3.16]. Seven studies^{36,38,47,56,58,69,79} reported vomiting, and the results shown in Supplementary Figure 1 showed that the experimental group was favorable with an OR of 2.00 [95% CI 0.74; 5.39], rash with an OR of 1.64 [95% CI 0.38; 7.01],^47,56,69 and GI tract bleeding with an OR of 1.42 [95% CI 0.55; 3.67].^19,65,79 Heterogeneity was absent across groups, and the funnel plot showed no evidence of heterogeneity.

Figure 3 Forest plot of adverse events.

Eight studies^{26,38,57,66,69,73,77,80} reported aPTT, and the results as seen in Supplementary Figure 2 showed that the control group is favorable with MD of 1.93 [95% CI 1.58; 2.28]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Nine studies^{19,27,38,51,63,64,69,79,80} reported the Barthel Index, and the results as seen in Supplementary Figure 3 showed that the control group is favorable with MD of 15.17 [95% CI 14.60; 15.74]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Three studies^20,75,77 reported carotid artery intima media thickness, and the results as seen in Supplementary Figure 4 showed that the experimental group is favorable with MD of −0.27 [95% CI −0.36; −0.17]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity.

Four studies^47,48,53,63 reported cell aggregation rate, and the results as seen in Supplementary Figure 5 showed that the control group is favorable with MD of 0.31 [95% CI 0.26; 0.36]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Two studies^54,55 reported coagulation factor, and the results as seen in Supplementary Figure 6 showed that the experimental group is favorable with MD of −0.62 [95% CI −0.88; −0.35]. Heterogeneity was low, and the funnel plot showed no evidence of true heterogeneity. Five studies^{17,18,21,65,77} reported CRP, and the results as seen in Supplementary Figure 7 showed that the experimental group is favorable with MD of −1.40 [95% CI −1.47; −1.34]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Thirty five^{19,33–42,44–46,48,51,52,54–62,64–66,69–74} studies reported curative effect, and the results as seen in Supplementary Figure 8 showed that experimental group is favorable with odds ratio of 2.77 [95% CI 2.33; 3.29]. Heterogeneity was moderate, and the funnel plot showed few outliers, indicating true heterogeneity.

Six studies^{38,66,69,74,77,78} reported D-dimer, and the results shown in Supplementary Figure 9 showed that the experimental group was favorable, with an MD of −0.04 [95% CI −0.05; −0.03]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Three studies^48,55,62 reported erythrocyte sedimentation rate, and the results as seen in Supplementary Figure 10 showed that the experimental group is favorable with MD of −0.58 [95% CI −2.55; −1.40]. Heterogeneity was low, and the funnel plot showed no evidence of true heterogeneity.

Eight studies^{33,36,43,51,54,55,62,73} reported hematocrit, and the results as seen in Supplementary Figure 11 showed that the control group is favorable with MD of 0.07 [95% CI 0.06; 0.08]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Three studies^19,77,80 reported INR, and the results as seen in Supplementary Figure 12 showed that the control group is favorable with MD of 0.05 [95% CI 0.02; 0.09]. Heterogeneity was low, and the funnel plot showed no evidence of true heterogeneity. Three studies^65,66,79 reported mRS score, and the results as seen in Supplementary Figure 13 showed that the experimental group is favorable with MD of −1.28 [95% CI −1.54; −1.05]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity.

Twenty four studies^{19,25,28,32,37,38,42,47,48,50,55,56,59,60,64–69,71,76–79} reported NIHSS Score, and the results as seen in Supplementary Figure 14 showed that the experimental group is favorable with MD of −2.01 [95% CI −2.06; −1.97]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Two studies^20,77 reported the number of carotid plaques, and the results shown in Supplementary Figure 15 showed that there was no difference between the experimental and control groups (MD −0.00 [95% CI −0.13; 0.12]). Heterogeneity was low, and the funnel plot showed no evidence of true heterogeneity. Thirty three^{18–21,25,36,37,39,40,43,45,47–50,52,53,56,57,59,62,63,66–69,71–74,76,77} studies reported plasma fibrinogen, and the results as seen in Supplementary Figure 16 showed that the experimental group is favorable with MD of −1.00 [95% CI −1.03; −0.96]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Twenty two studies^{29,33,35,36,39,40,45,47,48,51–55,57,59,62,63,66,68,73,77} reported plasma specific viscosity, and the results as seen in Supplementary Figure 17 showed that the experimental group is favorable with MD of −0.16 [95% CI −0.17; −0.14]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity.

Five studies^{27,38,66,77,78} reported plasminogen activator inhibitor, and the results as seen in Supplementary Figure 18 showed that the experimental group is favorable with MD of −0.77 [95% CI −0.85; −0.69]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Three studies^53,54,62 reported platelet aggregation rate (0.5 min), and the results as seen in Supplementary Figure 19 showed that the experimental group is favorable with MD of −205.86 [95% CI −206.77; −204.96]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity. Nine studies^{36,38,42,53,63,66,67,73,74} reported platelet aggregation rate (1 min), and the results as seen in Supplementary Figure 20 showed that the experimental group is favorable with MD of −7.25 [95% CI −8.61; −5.90]. Heterogeneity was high, and the funnel plot showed few outliers, indicating evidence of true heterogeneity.

Two studies^18,72 reported platelet aggregation rate, and the results as seen in Supplementary Figure 21 showed that the control group is favorable with MD of 6.59 [95% CI 3.22; 9.96]. Heterogeneity was high, and the funnel plot indicated evidence of true heterogeneity. Seven studies^{19,26,28,35,37,43,71} reported platelet count, and the results as seen in Supplementary Figure 22 showed that the experimental group is favorable with MD of −8.78 [95% CI −13.49; −4.07]. The heterogeneity was moderate, and the funnel plot indicated evidence of true heterogeneity. Seventeen studies^{21,26,28,31,35,38–40,57,63,66,69,71,77,80} reported PT (second), and the results as seen in Supplementary Figure 23 showed that the experimental group is favorable with MD of −0.17 [95% CI −0.22; 0.12]. Heterogeneity was high, and the funnel plot indicated evidence of true heterogeneity.

Three studies^47,55,62 reported red blood cell deformation coefficient, and the results as seen in Supplementary Figure 24 showed that the experimental group is favorable with MD of −0.33 [95% CI −0.39; −0.27]. Heterogeneity was low, and the funnel plot showed no evidence of true heterogeneity. Two studies^18,21 reported thrombosis precursor protein, and the results as seen in Supplementary Figure 25 showed that the experimental group is favorable with MD of −3.10 [95% CI −3.99; −2.21]. Heterogeneity was absent, and the funnel plot showed no evidence of true heterogeneity. Four studies^26,38,66,77 reported tissue plasminogen activator, and the results shown in Supplementary Figure 26 showed that the control group was favorable (MD 0.22 [95% CI 0.19; 0.24]). The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity.

Two studies^20,54 reported total cholesterol, and the results as seen in Supplementary Figure 27 showed that the experimental group is favorable with MD of −0.92 [95% CI −1.20; −0.65]. The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity. Two studies^20,54 reported triacylglycerol, and the results as seen in Supplementary Figure 28 showed that the experimental group is favorable with MD of −0.46 [95% CI −0.66; −0.25]. Heterogeneity was low, and the funnel plot showed no evidence of true heterogeneity. Two studies^38,66 reported TT (second), and the results shown in Supplementary Figure 29 showed that the experimental group was favorable, with an MD of −0.08 [95% CI −0.48; 0.32]. Heterogeneity was absent, and the funnel plot showed no evidence of true heterogeneity.

Seventeen studies^{23,29,33,35,36,38,48,51–55,57,59,69,73,77} reported whole blood high viscosity shear, and the results as seen in Supplementary Figure 30 showed that the experimental group is favorable with MD of −0.14 [95% CI −0.16; −0.12]. The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity. Fifteen studies^{23,29,33,35,36,48,52–55,57,59,69,73,77} reported whole blood low viscosity shear, and the results as seen in Supplementary Figure 31 showed that the experimental group is favorable with MD of −1.29 [95% CI −1.40; −1.17]. The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity. Six studies^{48,52,55,62,66,68} reported whole blood reduced viscosity, and the results as seen in Supplementary Figure 32 showed that the control group is favorable with MD of 0.03 [95% CI 0.01; 0.05]. The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity. Five studies^{18,21,28,66,68} reported whole blood specific viscosity, and the results as seen in Supplementary Figure 33 showed that the experimental group is favorable with MD of −0.67 [95% CI −0.78; −0.57]. The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity. Four studies^36,38,53,77 reported whole blood viscosity mid-cut, and the results as seen in Supplementary Figure 34 showed that the experimental group is favorable with MD of −0.42 [95% CI −0.53; −0.30]. The heterogeneity was high, and the funnel plot showed evidence of true heterogeneity.

Clinical efficacy was divided into seven groups, as shown in Supplementary Figure 35, and eight groups showed that the results favored the experimental group. Heterogeneity was high across the groups, and the funnel plot showed true evidence of heterogeneity.

Discussion

Lumbrokinase has been known to have anti-ischemic properties through inhibition of platelet aggregation and promotion of platelet disintegration.⁸¹ Based on the result of this meta-analysis, it is shown that lumbrokinase can improve functional outcomes in acute ischemic stroke patients compared to those with standard therapy. These were concluded by significant improvements in the functional outcome components, such as improvements in the Barthel Index and lowered NIHSS and mRS scores. These results suggest a positive efficacy of lumbrokinase in the treatment of acute ischemic stroke.

In addition to its fibrinolytic properties, lumbrokinase can lower stress in the endoplasmic reticulum. This meta-analysis found improvements in the Barthel Index in the lumbrokinase group compared to that in the control group, suggesting higher independence in the lumbrokinase group. The Barthel Index, which is commonly used to observe improvements during or after rehabilitation, can depict a patient’s dependency in daily life. A study in mice showed that stroke-induced endoplasmic reticulum stress was reduced by decreasing the phosphorylation of inositol-requiring enzyme-1 (IRE1) and attenuating autophagy and inflammation. Lower IRE1 levels are associated with lower activity of apoptosis-promoting pathways.⁸² Our study is in agreement with previous studies. Thus, lumbrokinase exhibits neuroprotective functions, prevents neuronal death, and further halts the worsening of Barthel Index score. Therefore, it can be inferred that lumbrokinase can increase independence in daily life in patients with acute ischemic stroke compared to those treated with standard therapy. In addition, the NIHSS and mRS scores, which reflect neurological deficits, were lower in the lumbrokinase group. Despite its clear effect favoring the lumbrokinase group, we also found high heterogeneity, which indicated variability in the study results. Differences in the study design, follow-up duration, and baseline data could potentially affect the results. More studies are needed to explore the neuroprotective and neurorehabilitative effects of lumbrokinase in patients with acute stroke.

Current standard therapies for acute ischemic stroke, such as antiplatelet and thrombolytic agents, have shown significant clinical benefits in daily usage. However, adverse effects such as an increased risk of bleeding complications remain a major clinical concern. Alternatives with similar efficacies have been rigorously investigated to minimize complications. In this meta-analysis, we found that there was no significant increase in adverse events in the lumbrokinase group compared with the control group. This suggests that despite its high fibrinolytic activity, lumbrokinase does not cause a higher adverse effect than the standard therapy. Previous studies have shown that the fibrinolytic properties of lumbrokinase are highly specific for fibrin; plasminogens are not activated into plasmin with lumbrokinase. Therefore, they are only active in the presence of fibrin.⁸³ Meanwhile, other thrombolytics, such as tPA, are not specific for fibrin. Thus, adverse effects, especially bleeding effects, are minimized in the lumbrokinase group by this mechanism.⁸⁴ We also did not find any significant differences in other adverse effects, such as gastrointestinal discomfort, vomiting, and rash between lumbrokinase therapy and standard therapy.

Various laboratory test results were compared between the two groups. One such test is aPTT, which reflects the ability of platelets to form blood clots and stop bleeding. Our study found that aPTT was significantly longer in the lumbrokinase group. Lower platelet aggregation rates, whether at 0.5 second or 1 second, were observed in the lumbrokinase group. Moreover, D-dimer levels were lower in the kinase group. This supports previous studies that stated its antithrombotic potential.⁸¹ Whilst laboratory results might have statistically significant differences between groups, several precautions should be accounted for. A previous study stated that only aPTT is likely to develop moderate to severe bleeding.⁸⁵ Our study shows that the mean time for aPTT lies between 22 and 39.9 seconds in the lumbrokinase group, while in the standard therapy group it ranged from 24 to 37.72 seconds. Another study also found that shortened aPTT, defined as shorter than 28.4 seconds, is an independent factor for ischemic stroke, stroke severity, and neurological decline.⁸⁶ Previous study conducted by Nurindar et al found that there was only a weak positive correlation between lower platelet aggregation rate and the degree of neurological deficit, and no statistically significant result was observed in the study.⁸⁷ A study conducted by Zi et al found that the range of D-dimer in acute ischemic stroke was 0.28 to 2.11 mg/L,⁸⁸ while our study reports a range from 0.007 to 0.91 mg/L in the lumbrokinase group and 0.07 to 0.65 mg/L in the control group. Thus, even when lumbrokinase is found to statistically improve laboratory findings, it should not be interpreted alone, and the clinical context should be considered. Although statistically significant, the three indicators also had high heterogeneity, which might be affected by different baseline characteristics, differences in measurement techniques, and study designs.

As previously described, we can infer that lumbrokinase holds potential as an adjuvant or alternative therapy for acute ischemic stroke compared to the standard therapy currently available, especially when resources are limited and access to alteplase might be minimal. Lumbrokinase was isolated and extracted from the earthworm, Lumbricus rubellus. These earthworms can be found in several places with limited resources.⁸⁹ have favorable outcomes in improving neurological status and laboratory findings, while having no statistical difference in adverse effects when compared to standard therapy demonstrated its potential. However, the clinical findings should be considered when implementing these findings. Our findings should not be overstated because of our moderate quality of evidence, and alteplase remains the gold-standard therapy based on international guidelines. Further robust research for lumbrokinase in treating acute ischemic stroke with less heterogeneity should be conducted, for our findings of high heterogeneity remain our limitations. We also found that there was a lack of direct comparison between lumbrokinase and alteplase and the moderate-to-high risk of bias studies used in our study, which might have affected our study.

Several limitations must be acknowledged. First, although a large number of RCTs were included, many of them presented a moderate to high risk of bias, which may impact the reliability of pooled estimates. Second, the lack of direct comparisons between lumbrokinase and alteplase limits our ability to determine its effectiveness relative to standard thrombolytic therapy. Third, heterogeneity in study designs, follow-up durations, and baseline characteristics was substantial in several outcomes. Finally, as most included studies were conducted in specific regions, external validity and generalizability remain limited. Future high-quality, multicenter studies are needed to address these concerns.

The findings of this study have notable implications for clinical practice, particularly in low- and middle-income countries where access to intravenous thrombolytics is limited. Lumbrokinase, as an orally administered agent with a favorable safety profile, presents a promising adjunctive or alternative therapy. Its affordability, non-invasive administration, and comparable efficacy in improving outcomes could help expand stroke care beyond major urban centers. However, clinicians must still interpret laboratory improvements cautiously and consider patient-level characteristics, comorbidities, and timing of therapy.

Lumbrokinase has the potential to improve outcomes and reduce stroke severity, particularly when used in combination with standard supportive care. Further high-quality RCTs with direct comparisons to alteplase, exploration of long-term outcomes, and broader safety profiles may be beneficial for further validation of lumbrokinase use. Although lumbrokinase shows promise in improving outcomes in acute ischemic stroke when combined with supportive management, the results must be interpreted with caution due to low-to-moderate evidence quality, and it should not be considered as a replacement for established thrombolytic agents, such as alteplase, but rather as a supplementary treatment.

Conclusion

Lumbrokinase, a fibrinolytic enzyme derived from earthworms, appears to improve neurological function and reduce laboratory markers of thrombosis in patients with acute ischemic stroke when used alongside supportive care. It offers a safe and potentially cost-effective alternative in settings with limited access to conventional thrombolytic agents. However, high heterogeneity, risk of bias, and lack of direct comparisons with current standard therapies limit definitive conclusions. Future research should focus on large-scale randomized trials comparing lumbrokinase directly with established treatments and evaluating its long-term safety and clinical benefits.

Data Sharing Statement

All data generated or analyzed during the study are included in this published article.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

There is no funding to report.

Disclosure

The authors declare no conflicts of interest in this work.

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11. Nguyen QTT, Rhee H, Kim M, Lee MY, Lee EJ. Lumbrokinase, a fibrinolytic enzyme, prevents intra-abdominal adhesion by inhibiting the migrative and adhesive activities of fibroblast via attenuation of the AP-1/ICAM-1 signaling pathway. Biomed Res Int. 2023;2023:4050730. doi:10.1155/2023/4050730

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29. Wei Z, Linhong Z, Wuping X. Effects of lumbrokinase on blood lipids and blood rheology in patients with ischemic cerebrovascular disease in the recovery period combined with hyperlipidemia. Cap Med. 2010;2010:51–52.

30. Hongbin L, Liyan J, Guangna Y. Lumbrokinase combined with atorvastatin calcium in the treatment of transient ischemic attack. China Prac Med. 2010;5(20):172.

31. Huiyu Z, Wei T, Weidong Z. Observation on the efficacy of Bio-lumbrokinase combined with ligustrazine in the treatment of 38 cases of acute cerebral infarction. Zhou Huiyu. 2008;141:101–102.

32. Liqin Z, Chunhua Q. Clinical observation of lumbrokinase capsule and compound danshen injection in preventing recurrence of cerebral infarction. J Changchin Univ Tradit Chin Med. 2008;24:290.

33. Xinhong L. Comparative study on the efficacy of aspirin alone and in combination with lumbrokinase enteric-coated capsules in the treatment of cerebral infarction. J Clin Exp Med. 2008;7(2):68–69.

34. Hui X. Lumbrokinase capsule combined with probucol tablets in the treatment of patients with cerebral infarction: observation on the efficacy of unstable atherosclerotic plaques in the carotid artery. Treat Obs. 2016;10(11):82–83.

35. Qiangfeng L, Hong X, Shuizhong J. Observation on the clinical effect of lumbrokinase enteric-coated capsules in preventing recurrence of cerebral infarction in the elderly. Chin J Clin Ration Drug Use. 2013;6(5A):94.

36. Li W, Jianglong T, Yousheng X, Yunhua L. Observation on the efficacy of lumbrokinase combined with Ginkgo biloba in the treatment of 48 cases of cerebral infarction. New Med. 2007;38(8):550.

37. Jing L. Observation on the efficacy of lumbrokinase capsule in the treatment of acute cerebral infarction. Cap Med. 2011;2011:38–39.

38. Yongwei Z, Wen’an W, Zheng P, Wei C, Ge Y, Genfa W. Observation on the efficacy and safety of lumbrokinase capsule combined with aspirin in the treatment of patients with acute cerebral infarction. J Clin Intern Med. 2006;23(6):413–414.

39. Zihan C, Jaihui M. Clinical efficacy of lumbrokinase in the treatment of ischemic cerebrovascular disease. Contemp Med. 2016;22(27):143–144.

40. Jiajiao H, Yongqiu L. Observation on the efficacy of atorvastatin or lumbrokinase combined with aspirin in the treatment of ischemic cerebrovascular disease. Chin J Clin Ration Drug Use. 2015;8(7B):50.

41. Conghua L, Qiongxiao X. Clinical observation of lumbrokinase combined with ginkgo leaf injection in the treatment of acute ischemic cerebral infarction. Prac Chin W Med Clin. 2005;5(1):13–14.

42. Liang J, Yu W, Guofeng L, Yongmei K. Clinical observation of lumbrokinase capsule in the treatment of cerebral infarction. Heli Med J. 2015;37:246–247.

43. Sheng A, Wang X, Xu Z, et al. Study of the effects of lumbrokinase on fibrinogen, D-dimer & platelet aggregation on patients with ischemic cerebral vascular disease. Chin New Drugs J. 2002;11(1):82–84.

44. Chunxia W. Clinical observation of lumbrokinase in the treatment of cerebral infarction. Cap Med. 2008;8(10):49.

45. Zhenlei J, Miaofen L. Clinical analysis of 99 cases of acute cerebral infarction treated with Bio-lumbrokinase. China Pharm. 2012;21(12):17.

46. Qinfeng G. Clinical efficacy analysis of lumbrokinase combined with ozagrel sodium in the treatment of acute cerebral infarction. Strait Pharm. 2012;24(4):78–79.

47. Lijun L, Guohua C, Yulan J, Hanyun Y, Junhua M. Clinical observation of lumbrokinase in the treatment of acute cerebral infarction. Cap Med. 2011;7:28–29.

48. Baixue, Yundong J, Sijin Y. Clinical observation of lumbrokinase combined with xuesaitong in the treatment of acute cerebral infarction. Cap Med. 2010;1:46–47.

49. Zhai W, Song L, He X, et al. The effect of long-term fibrinogen-depleting on the carotid atherosclerosis and cerebral infarction. Chin Med Guid. 2011;9(20):221–222.

50. Xia Z, Xingchen W, Huikui Z. Clinical observation of sequential fibrinolytic enzyme lumbrokinase combined with aspirin in preventing recurrence of cerebral infarction. Cap Med. 2011;2:42.

51. Xu R, Tian M. Clinical observation of lumbrokinase combined with aspirin in secondary prevention of ischemic cerebrovascular disease. Cap Med. 2010;2010:39–40.

52. Min X, Yangbai F. Clinical efficacy and safety of lumbrokinase capsule combined with aspirin in the sequelae of ischemic stroke. Cap Med. 2010;2010:39–40.

53. Zhijie C, Lirong J, XInjian W, Hui J, Wei F, Guoping Z. Study on the effect of lumbrokinase on blood rheology in patients during stroke recovery period. J Chin Microcircul. 2011;5(4):288–289.

54. Huang Z, Li Z, Zhang W. Lumbrokinase in the treatment of cerebral infarction. Chin J New Drugs Clin Rem. 2000;19(6):453–455.

55. Liu J, Li L. Lumbrokinase in the treatment of acute ischemic cerebral infarction. Chin J New Drug Clin Rem. 1998;17(2):79–80.

56. Jianyong L. Clinical observation of lumbrokinase capsule in the treatment of acute cerebral infarction. Capital Med. 2004;2004:38–39.

57. Ma X, Zhan P, Xu J. Clinical observation of lumbrokinase combined with Ginkgo biloba extract in the treatment of acute cerebral infarction. Chin J Tradit Chin Emerg Med. 2007;16(2):269–270.

58. Zhang Z, Peng X. Clinical observation of Bio-lumbrokinase capsule combined with ozagrel sodium in the treatment of acute cerebral infarction. Capital Med. 2007;12:28–29.

59. Zhu X, Tan J, Zhu Y, et al. Clinical study on the treatment of acute cerebral infarction with Bio-lumbrokinase capsules. Capital Med. 2009;2:46–47.

60. Wang T, Yang S, Zhu B, Yuan Y; Chen Health Care. Clinical study on treatment of acute cerebral infarction with hydrochloric fasudil and lumbrokinase. Chin J Pract Nerv Dis. 2009;12(17):15–17.

61. Dongju Z, Zhiqiang Z, Hongyuan M. Observation on the efficacy of lumbrokinase, ticlopidine, and aspirin with alprostadil in preventing recurrence of acute cerebral infarction. Capital Med. 2004;11(8):43–44.

62. Jiping Z. Clinical observation of lumbrokinase in the treatment of cerebral infarction. Capital Med. 2004;11(10):29–30.

63. Ying D. Observation on the efficacy of batroxobin followed by lumbrokinase in the treatment of cerebral infarction. Hainan Med. 2007;18(1):101–103.

64. Mu Z, Kong F, Hou L. Clinical study of Shuxuening injection combined with lumbrokinase in the treatment of cerebral infarction. Drugs Clinic. 2018;33(2):238–241.

65. Tong J, Chen E, Yu Z, Jiang J. Observation on the efficacy of lumbrokinase in the treatment of acute cerebral infarction. Lingnan J Emerg Med. 2004;9(3):174–175.

66. Dong Q, Qiao J, Shi L, et al. Efficacy and safety of lumbrokinase capsules in patients with cerebral infarction. Chin J New Drugs. 2004;13(3):257–260.

67. Ding Y, Yin X. Small-dose aspirin plus lumbrukinase in improving neurological function of patients with acute cerebral infarction. Chin J Clin Rehabil. 2006;10:60.

68. Duan H, Liu H. Efficacy and safety observation of lumbrokinase combined with aspirin in secondary prevention of cerebral infarction. Capital Med. 2011;2011:43–44.

69. Qong L, Xian G. Effects of lumbrokinase enteric-coated capsules on coagulation indexes and neurological function in patients with acute cerebral infarction. Heilonghiang Med J. 2023;36(3):610–612.

70. Huawei F. Study and analyze the clinical value of lumbrokinase capsule in treating patients with cerebral infarction. China Rural Health. 2021;18:44–45.

71. Han Y, Wu X. Observation on the efficacy of ozagrel sodium combined with lumbrokinase in the treatment of progressive cerebral infarction. Chin J Pract Nerv Dis. 2012;15(14):46–47.

72. Feng S, Min T. Observation on the efficacy of lumbrokinase combined with ozagrel in the treatment of acute cerebral infarction. Capital Med. 2012;41:1.

73. Liu X, Zhang L. Evaluation of the efficacy and safety of lumbrokinase combined with clopidogrel in the treatment of diabetic acute cerebral infarction. Capital Med. 2012;2012:36–38.

74. Gao Z, Park Y, Kong Y, Zhu Z. Effects of clopidogrel combined with lumbrokinase on plasma D-dimer, platelet aggregation and fibrinogen in patients with acute cerebral infarction. Jiangsu Medicine. 2014;40(14):1707–1708.

75. Liu C, Chen J. The intervention and preventive effects of lumbrokinase capsule on patients with cerebral infarction. Shandong Med. 2011;51(33):85–86.

76. Sui X. Batroxobin combined with lumbrokinase: observation on the efficacy of treating acute cerebral infarction. Capital Med. 2006;2006:47–48.

77. Cao Y, Zhang X, Wang WH, et al. Oral fibrinogen-depleting agent lumbrokinase for secondary ischemic stroke prevention: results from a multicenter, randomized, parallel-group and controlled clinical trial. Chin Med J. 2013;126(21):4060. doi:10.3760/cma.j.issn.0366-6999.20131332

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79. Pinzon RT, Tjandrawinata RR, Wijaya VO, Veronica V. Effect of DLBS1033 on functional outcomes for patients with acute ischemic stroke: a randomized controlled trial. Stroke Res Treat. 2021;2021:5541616. doi:10.1155/2021/5541616

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Central Africa  has approved 25 priority diseases for targeted prevention, detection, and response in a major step towards stronger epidemic preparedness.

Africa CDC in partnership with the European Centre for Disease Prevention and Control (ECDC) developed the list—covering threats such as viral hemorrhagic fevers, measles, dengue, cholera, yellow fever, mpox, and meningitis  through a rigorous risk-ranking exercise.

Experts from nine African Union Member States in Central Africa, together with regional and international partners, assessed diseases using epidemiological, socio-economic, and operational criteria.

The rating exercise carefully considered factors including frequency of outbreaks and cross-border spread, severity, case fatality rates and inclusion in the International Health Regulations (IHR, 2005) list of notifiable diseases.

 “This prioritization is a crucial step toward building a resilient health system that is ready to respond to emerging threats. It will enable more targeted planning, faster outbreak response, and more strategic resource allocation, while fostering regional coordination in the face of cross-border health risks,” said Dr. Brice Bicaba, Director of the Africa CDC Regional Coordinating Centre for Central Africa.

This risk-ranking exercise was first applied in 2024 by Africa CDC at continental level. Since then, it has shaped strategic public health preparedness and response initiatives. Building on this, Africa CDC is now focusing on regional-level risk assessments and prioritizations to ensure preparedness and response planning that is better adapted to the specific contexts of each region.

Given the evolving epidemiological landscape in Central Africa—characterized by a large population, the effects of climate change, and the persistent threat of emerging and re-emerging diseases—this regional prioritization exercise comes at a critical time in the region.

Beyond validation, representatives from the Ministries of Health strengthened their capacity to use the risk-ranking methodology and tool, developed through wide expert consultations across the continent. This tool has been applied to rank risks at both continental and regional levels.

“This workshop has helped enrich the evidence base on priority risks, capacity gaps, and the next steps needed to mitigate these risks. The ECDC looks forward to continuing its collaboration with Africa CDC in this area, which is a priority pillar of our partnership,” said Jonatan Suk, Head of the Health Security Projects Group on behalf of the European Centre for Disease Prevention and Control.

Added to this prioritization, a regional roadmap was developed to strengthen early detection, rapid response, and multisectoral coordination in the face of epidemics. It includes: enhancing epidemiological surveillance and health monitoring; establishing cross-border coordination mechanisms; improving national diagnostic and case management capacities; and developing multisectoral preparedness plans aligned with national and regional contexts.

The meeting helped consolidate an integrated regional approach by facilitating and strengthening exchanges between Central African countries, regional health institutions, and partners. The classification of epidemic-prone diseases will now serve as a foundation for planning, resource mobilization, capacity allocation, and community engagement in the region.

Through this initiative, Central Africa is reinforcing its collective ability to anticipate, prevent, and contain epidemics, aligning with the objectives of the International Health Regulations (IHR) and the Global Health Security Agenda.

Digestive issues such as bloating, gas, and indigestion are everyday concerns, and while medicines offer relief, natural remedies can often provide lasting support without side effects. Herbs and spices used for centuries in Indian kitchens are now being recognised by modern science for their gut-healing properties. Dr Saurabh Sethi, an AIIMS, Harvard, and Stanford-trained gastroenterologist, strongly believes that “real gut healing starts in your kitchen.” He recently shared eight herbs he personally relies on for better digestion. From turmeric to cumin, these simple additions to daily meals can help soothe discomfort and strengthen gut health naturally.

8 gut-healing herbs recommended by an AIIMSs doctor

On Instagram, AIIMS, Harvard, and Stanford-trained gastroenterologist Dr. Saurabh Sethi shared eight herbs he personally uses to improve gut health, reminding followers that “real gut healing starts in your kitchen.”

Turmeric supports digestion and reduces inflammation

Turmeric, a staple in Indian households, is well known for its anti-inflammatory compound curcumin. Dr Sethi suggests adding turmeric to warm milk or curries to soothe the gut, reduce inflammation, and support bile flow, which helps break down fats. This golden spice not only calms an irritated digestive tract but also promotes overall gut lining health. Regular use may reduce the risk of long-term inflammatory gut conditions. A human pilot study published in study found that supplementation with turmeric or curcumin significantly altered gut microbiota composition, including a notable increase in species diversity; curcumin increased detected species by 69% compared to a placebo, which saw a 15% decrease

Ginger relieves bloating and nausea

Ginger has long been used as a natural digestive aid. It stimulates gastric emptying, reduces bloating, and relieves nausea. Dr Sethi recommends steeping fresh ginger in hot water to make a soothing tea, especially after heavy meals. Its warming properties help settle the stomach, making digestion smoother and more comfortable. For people with sluggish digestion, ginger can act as a gentle stimulant.

Fennel seeds ease gas and bloating

Chewing fennel seeds after meals is a time-tested Indian practice, and science confirms its benefits. These seeds contain compounds that relax gut muscles, helping release trapped gas and easing bloating. Dr Sethi recommends chewing a teaspoon of fennel seeds after meals or making a calming tea. This simple habit can help reduce discomfort and improve digestion naturally.

Cumin improves bile flow and eases cramps

Cumin is another household spice with powerful gut benefits. It stimulates the release of bile, which aids digestion of fats. It is also useful for people with irritable bowel syndrome (IBS), as it helps relieve cramps. Dr Sethi suggests toasting cumin seeds and adding them to dals, curries, or vegetable stir-fries. Apart from improving flavour, this enhances nutrient absorption and digestive function.

Cinnamon regulates digestion and blood sugar

Cinnamon adds warmth and sweetness to foods, but it also has medicinal value. It helps regulate gut motility, making digestion smoother, and plays a role in stabilising blood sugar levels. Dr. Sethi advises sprinkling cinnamon on oats, kefir, or even coffee. Its ability to calm the gut makes it particularly helpful for people who experience erratic digestion.

Peppermint relaxes gut muscles

Peppermint has a cooling effect and works as a natural gut muscle relaxant. It helps reduce spasms and discomfort caused by digestive issues. Dr Sethi recommends drinking peppermint tea or using peppermint oil capsules to ease gut irritation. However, he cautions against using peppermint if you experience reflux, as it may worsen the symptoms by relaxing the lower oesophageal sphincter.

Garlic nourishes gut bacteria

Garlic is a natural prebiotic, meaning it feeds beneficial gut bacteria, helping them thrive. At the same time, it has antibacterial, antifungal, and antiparasitic properties that keep harmful microbes under control. Dr. Sethi advises lightly crushing garlic before cooking to activate its gut-boosting compounds. Regular consumption can improve microbial balance, which is essential for long-term gut and immune health.

Coriander reduces bloating and adds flavour

Coriander, also known as cilantro, is another herb that promotes gut comfort. It helps reduce gas, bloating, and indigestion while adding freshness to meals. Dr Sethi recommends adding coriander to curries, chutneys, and salads. Beyond its digestive benefits, coriander provides antioxidants that protect gut cells and support overall wellbeing.Gut health is deeply influenced by what we eat daily. Instead of relying solely on medicines, incorporating herbs like turmeric, ginger, fennel, cumin, cinnamon, peppermint, garlic, and coriander can make digestion smoother and more comfortable. These natural remedies work best when used consistently as part of a balanced diet.Dr Sethi’s advice is clear: healing starts in the kitchen. By rotating these herbs weekly and using them in everyday cooking, you can take simple, natural steps towards better gut health.Also Read: Don’t follow these 9 cooking habits that harm digestion and trigger Irritable Bowel Syndrome

“An advanced test for AIDS, the deadly acquired immune deficiency syndrome, could be available in Hongkong by May,” reported the South China Morning Post on February 19, 1985. “American researchers have developed a commercial kit to detect the disease and hope to receive an official go-ahead to begin production this month.

“The US Federal Drug Administration (FDA) is expected to license the test within the next two weeks, paving the way for its export abroad. The Australian Government has already placed an order and plans to have units operating before April to screen blood donors.

“The chairman of a task force set up in Australia to combat a threatened AIDS epidemic, said yesterday he had been told by US medical authorities the test kit could be marketed worldwide. ‘I would think that it would be available to Hongkong or any country wanting it,’ Professor David Penington said.”

On August 17, the Post confirmed that “blood tests for AIDS can begin on Monday, the Medical Health Department announced yesterday. Laboratory screening will be run at Queen Mary Hospital and Yan Oi Polyclinic in Tuen Mun – charging $220 or $250 a time depending on what type of test. The facilities will be open for all doctors and hospitals in the territory.

“The Hongkong Red Cross and the Family Planning Association both welcomed the news.”

Introduction

Hematopoietic stem cell transplantation (HSCT) has been demonstrated to be an effective treatment for several hematological malignant and non-malignant diseases. Despite its proven efficacy and the use of immunosuppressive prophylaxis, it is associated with early and late complications, among which there is the graft-versus-host disease (GVHD; OMIM#614395; ORPHA:39812). Acute GVHD (aGVHD) is the second most common cause of death in allogeneic HSCT recipients after the primary disease recurrence.^1,2 Understanding the mechanisms responsible for the initiation and progression of this complication is fundamental to developing effective prevention and treatment strategies.^3,4

The aGVHD involves a cascade of events, including early inflammation and tissue injury, dysregulated immunity, until aberrant tissue repair with fibrosis that leads to irreversible tissue damage.^5,6 The intestine is one of the organs most affected by aGVHD.^7,8 Epithelial barrier loss can occur due to direct epithelial cells damage or through more subtle changes in paracellular tight junction permeability.⁹ When dysregulated, these forms of intestinal barrier loss are thought to contribute to the initiation and propagation of the inflammation and damage progression, and it is considered a driving mechanism in aGVHD.^10,11

Despite the remarkable progress achieved in developing new and effective therapeutic strategies for treating severe aGVHD, no unique and safer treatment options are available nowadays. Steroid therapy is the first-line therapy, as well as the only one universally recommended. About 35–50% of patients with aGVHD develop refractory to systemic steroid therapy, and only 1–2% of patients with grade IV aGVHD survive more than two years.¹²

Defibrotide is a mixture of phosphodiester oligonucleotides (90% single-stranded and 10% double-stranded), obtained from controlled depolymerization of porcine intestinal mucosal DNA,¹³ indicated for treating severe hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) following allogenic-Hematopoietic cell transplantation (allo-HCT).^14–16

It has been demonstrated that defibrotide exerts a protective role on activated endothelial cells^13,17 mainly decreasing leukocyte extravasation and downregulating the expression of endothelial surface proteins involved in leukocyte recruitment.¹⁸

Defibrotide also has anti-inflammatory, anti‐thrombotic and pro‐fibrinolytic activities.^13,19–21

Even though clinical and experimental studies demonstrate that defects in the intestinal tight junction barrier and increased permeability are observed in various intestinal acute and chronic diseases, and systemic disorders, currently, the best therapy for barrier loss should target the disease itself.²² In this context, early reports suggest that restoring tight junction barrier function may have therapeutic benefits.^23,24 Therapies targeted to restore the barrier function precisely may provide a substitute or supplement to immunologic-based treatments. However, the mechanisms of tight junction regulation will have to be defined in greater detail to make them viable as pharmacological targets.²⁵

The primary objective of this study was to evaluate in vitro on cells of the intestinal mucosa the effect of defibrotide, that, in a recent study,²⁶ has shown the ability to reduce the cytokine levels in a murine model of GVHD. In this work we have demonstrated for the first time that defibrotide can act in vitro towards damaged intestinal tight junctions leading to their rapid restoration supporting the repositioning of this drug for complete remission in patients with aGVHD of grade IV after failure of advanced-line therapies, including total lymphoablation with antithymocyte globulins.

Materials and Methods

Drug and Chemicals

Defibrotide (Defitelio^® 80 mg/mL, Gentium Srl, Villa Guardia, Italy) derives from a kind concession of soon-to-expire waste lots by the Jazz Pharmaceuticals, for preclinical research purposes only. It was stored at room temperature according to the manufacturer’s instructions.

MDP (N-Acetylmuramyl-L-alanyl-D-isoglutamine hydrate, Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in saline solution, according to manufacturer instructions. MDP (10 μM) was added to cell culture to mimic the inflammatory condition.²⁷

Cell Culture

HCT116 cells (human colon carcinoma cell line) were obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified eagle medium (DMEM; Corning, New York, NY, USA) supplemented with 10% fetal bovine serum and with 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (all from GIBCO, Grand Island, NY, USA). Cells were left untreated or treated with 100 or 200 μg/mL defibrotide, alone or in combination with 10 μM MDP, for 24 hours. Where specified, the defibrotide was added both at the beginning of the experiments and after 8 hours.

Immunocytochemical Analysis

For immunocytochemical analysis, HCT116 cells were grown on coverslips in complete medium, treated for 24 hours, as described above, then fixed with freshly prepared 4% paraformaldehyde (for 10 minutes at room temperature) and washed in PBS 1X.

The cells on coverslips were then incubated with a Net Gel solution (150 mM NaCl, 5 mM EDTA, 50 mM TRIS-HCl pH 7.4, 0.05% NP40, 0.25% Carrageenan Lambda gelatin, and 0.02% Na azide) for 1 hour at room temperature to block non-specific binding.²⁸ Then, the cells were incubated with anti-zonulin-1 (ZO-1) polyclonal antibody, anti-Occludin monoclonal antibody (OC-3F10), both from ThermoFisher Scientific (Waltham, MA, USA) for 3 hours in Net Gel at room temperature. Samples were subsequently incubated with the specific secondary FITC and TRITC-conjugated antibodies in Net Gel for 45 min at room temperature. After two washes with NET gel and PBS, nuclei were counterstained with DAPI (0.5 μg/mL) and coverslips were dried with ethanol and mounted in glycerol containing 1,4-diazabicyclo [2.2.2] octane (DABCO). The slides were analyzed with a Nikon Eclipse TE2000-E microscope (Carl Zeiss, Oberkochen, Germany).

Western Blot Analysis

HCT116 cells, cultured and treated as reported above, were lysed in ice-cold RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.25% sodium deoxycholate) supplemented with Pierce Protease and Phosphatase Inhibitor mini tablets (Thermo Scientific, Rockford, IL, USA) on ice for 45 minutes. Protein determination was performed by using the BCA Protein Assay (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. Samples were supplemented with the loading buffer (250 mM Tris pH 6.8, 2% SDS, 40% Glycerin, 20% b-mercaptoethanol) and boiled for 2 minutes. Equal amounts of proteins (50 μg) for each sample were migrated in acrylamide gels and blotted onto nitrocellulose filters. Western blot analysis was performed according to standard procedures using the following primary antibodies: anti-ZO-1 polyclonal antibody, anti-Occludin monoclonal antibody (OC-3F10), anti-Claudin 3 polyclonal antibody, anti-Claudin 4 monoclonal antibody (3E2C1), all from ThermoFisher Scientific, and anti-Tubulin monoclonal antibody from Sigma-Aldrich (St. Louis, MO, USA). After incubation with secondary antibodies (anti-mouse or -rabbit IgG HRP-conjugated; Sigma-Aldrich), a specific band detection was performed with the WesternBright Quantum kit (Advansta, Menlo Park, CA, USA). Image acquisitions were performed using the ImageQuant™ LAS 4000 imager and TL software (GE Healthcare, Buckinghamshire, UK). Densitometry of the Western blotting bands were analyzed with the Image J software (NIH). Western blotting was repeated at least three times with similar results and bands of interest were quantified with ImageJ software (NHI, USA), after normalizing with tubulin.

In-vivo Defibrotide Treatment: Therapeutic Approach and Ethical Approval

Two patients who underwent allogeneic HSCT after standard myeloablative conditioning were included in the study. Both the patients selected for the study had grade IV multisystem aGVHD with predominant involvement of the gastrointestinal tract. Defibrotide (Defitelio^® 80 mg/mL, Gentium Srl, Villa Guardia, Italy) was administered as two-hour intravenous infusions of 6.25 mg/kg (25 mg/kg/day) every six-hour. The protocol followed the guidelines approved for VOD treatment, in accordance with the recommendations of the Haemato-oncology subgroup of the British Committee for Standards in Hematology (BCSH) and the British Society for Blood and Marrow Transplantation (BSBMT).

The transplant procedures and defibrotide treatment were performed at the Pediatric Bone Marrow Transplant Center (IRCCS Burlo Garofolo in Trieste), while all experiments with cell cultures were conducted at University of Ferrara. The Ethical Committee of the Institute for Research in Maternal and Child Health Burlo Garofolo of Trieste approved the study (reference no. 1105/2015). All laboratory experiments were carried out of the clinical study DF VOD-2013-03-REG, which investigates the efficacy of defibrotide to prevent conditioning-related organ injury in the course of allogeneic myeloablative HSCT.

The patient’s parents provided informed written consent for the off-label defibrotide use and for the anonymous publication of clinical data and images.

All procedures were performed in accordance with the requirements of the Declaration of Helsinki.

Clinical Recovery of GVHD Patients Refractory to Conventional Treatments

Both patients were prednisone-resistant and had failed to respond to numerous treatments including ruxolitinib, tacrolimus, mycophenolate mofetil, infliximab, as well as rescue treatment with fludarabine and rabbit anti-thymocyte globulin (Thymoglobulin). During the third-line treatment, the first patient developed diffuse intestinal pneumatosis involving massively the ileum and entire large bowel, the sigma, and the rectum up to the rectal ampulla included, with the presence of free air under the diaphragm, in the retroperitoneum, and mesenteric fat, bringing her to discouraging clinical conditions. The second patient continued to deteriorate despite several lines of aggressive immunosuppressive treatment after ten days of continuous severe bleeding, which required exceptional transfusional support. Additionally, worsening of liver function occurred. In the absence of valid therapeutic alternatives, we decided to try off-label treatment with defibrotide to preserve liver function at least. Both patients made full gut and liver recoveries within two weeks of continuous defibrotide administration associated with morphine infusion only.

Collection of Serum Samples

Peripheral blood samples were collected during the acute phase and remission of intestinal aGVHD, as part of diagnostic procedures, and used for research purposes only when clinical procedures had been completed. Patients’ samples (∼3–5 mL) were collected in sterile, serum-separator tubes and allowed to clot at room temperature for 30 minutes. Following clotting, the samples were centrifuged at 1500 × g for 10 minutes at room temperature, to separate the serum from the cellular components. The resulting serum was carefully aspirated and transferred into sterile cryovials. Each vial was labelled with the patient identification number and stored at −80°C for long-term preservation until further analysis.

Cytokine Profile Evaluation

Patients’ sera were tested for the evaluation of the following cytokines/chemokines (expressed in pg/mL): Interleukin (IL)1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12 (p70), IL13, IL17, granulocyte-colony stimulating factor (G-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF), Interferon (IFN)-γ, Monocyte chemotactic and activating factor (MCP1; MCAF), Macrophage Inflammatory Protein (MIP)1β and Tumor Necrosis Factor (TNF)-α, using the human cytokine BioPlex assay (BioRad Laboratories, Milan, Italy), a magnetic bead-based multiplex kit. Samples used for the immunoassay test were frozen and thawed only once. Cytokine evaluation was performed according to the manufacturer’s instructions on a Bio-Plex 200 instrument equipped with the Bio-Plex Manager software, using a five-parameters not-linear regression formula to compute sample concentrations from the standard curves.

For the patients’ sera analysis, the control donors, for ethical reasons, were limited to infants and young children who had to undergo a medically indicated peripheral venous blood sampling before elective surgical interventions or with the scope of diagnostic procedures. Moreover, we excluded subjects affected by an acute or chronic infectious disease.

Statistical Analysis

All results are expressed as the mean±standard deviation (SD). Statistical analysis of bands densitometry was carried out using one-way analysis of variance (ANOVA), followed by Bonferroni multiple comparison test. We used, also, t-test to compare the cytokine levels of the two independent groups (remission and control groups) to determine if there was a statistically significant difference between them. Statistical analyses were performed using GraphPad Prism (version 5.0; GraphPad Software Inc., La Jolla, CA, USA).

Results

Effect of Defibrotide on Tight Junction Proteins in HCT116 Cells

To evaluate the potential efficacy of defibrotide in modulating intestinal permeability, we conducted in vitro experiments on a colorectal carcinoma cell line (HCT116 cells) used as an epithelium model of the large intestine. In a first step of experiments, we analyzed the expression of tight junction proteins in untreated and defibrotide-treated cells.

As shown in Figure 1, immunoblot results revealed that the protein expressions of ZO-1 and Occludin were increased in cells treated with defibrotide compared to untreated cells. ZO-1 levels were significantly increased with 100 μg/mL of defibrotide treatment (p<0.05, Figure 1A), while Occludin expression was significantly induced when defibrotide was added at a concentration of 200 μg/mL (p<0.01, Figure 1B). In line with these results, immunohistochemical analysis also showed an increase in the expression levels of ZO-1 and Occludin in HCT116 cells treated with defibrotide (Figure 2).

Figure 2 Immunofluorescence analysis of ZO-1 and Occludin in HCT116 cells. Representative images of HCT116 cells expressing ZO-1 (green) and Occludin (red). DAPI staining (blue) indicates nuclei. Magnification=40X/0.95. UNT=untreated; D100=defibrotide 100 μg/mL; D200=defibrotide 200 μg/mL, scale bar = 50 μm.

Effect of Defibrotide on Tight Junction Proteins Under Inflammatory Conditions

To assess the activity of defibrotide in counteracting the damage induced by inflammation, HCT116 cells were treated with 10 μM MDP alone to mimic inflammation, or in combination with defibrotide (100–200 μg/mL). After 24 hours of treatment, the cells were harvested and the protein levels of ZO-1, Occludin, Claudin 3 and Claudin 4 were analyzed by Western blot (Figure 3).

Figure 3 Effects of defibrotide on the expression of tight junctions’ proteins in conditions of inflammation. Representative Western blotting images of ZO-1, Occludin, Claudin 3 and Claudin 4 are shown. Immunoblotting was performed using 50 μg of HCT116 cell lysate. Tubulin staining is used as loading control. MDP= 10 μM (inflammatory condition); D100=defibrotide 100 μg/mL; D100 t0/t8=defibrotide 100 μg/mL added at time 0 and after 8 hours; D200=defibrotide 200 μg/mL; D200 t0/t8=defibrotide 200 μg/mL added at time 0 and after 8 hours. The experiments were performed at least in triplicate.

As previously described, defibrotide induced an increase in ZO-1 and Occludin levels, as well as Claudin 3 and Claudin 4, compared to untreated cells (Figure 3). Notably, except for ZO-1, the increase was more evident when defibrotide was added twice, at the beginning of the experiment and after 8 hours. Conversely, treatment with MDP induced a significant decrease in Occludin and Claudin 3 protein levels, while it seemed to minimally affect Claudin 4. As shown in Figure 3, the addition of defibrotide counteracted the effect of MDP, restoring the protein levels when added twice. Importantly, ZO-1 was not decreased by MDP treatment, and defibrotide was able to increase the levels of this protein at both concentrations, confirming the results previously described.

Clinical Efficacy of Defibrotide in GVHD Patients

Sera of two patients were collected during the acute phase and remission to analyse changes in cytokine and chemokine levels.

In Figure 4, cytokines and chemokines that present significantly different levels in the acute phase compared to remission are shown (the first patient is represented in red on the graphs, and the second patient in blue).

Figure 4 Levels of IL-7, MIP-1β, IP-10, G-CSF, Eotaxin and IL-6 measured in acute phase, remission and control groups. Cytokines downregulated in the remission group in comparison to the acute phase group were measured in serum samples by multiplex immunoassays. Statistically significant p-values are shown in all comparisons (*p<0.05, **p<0.01, ***p<0.001).

As represented in Figure 4, IL-7, MIP-1β, IP-10, G-CSF, Eotaxin, and IL-6 had significantly different levels in the acute phase of the disease compared to the remission phase in at least one of the two patients. The results show that IL-7 levels were significantly higher in both patients than in the controls during the acute phase of the disease. Defibrotide treatment was successful, as cytokine levels significantly decreased in both patients, with a lower decrease in the first patient compared to the second, where cytokine levels had fallen below control (acute phase vs remission: Pt#1, p<0.05; Pt#2, p<0.001).

In our analyses, MIP-1β levels were elevated in the acute phase of both patients and then significantly decreased after treatment with defibrotide. In the first patient, there was a more significant decrease of this chemokine, so that after therapy, MIP-1β levels fell below the controls. The second patient, on the other hand, showed a less significant decrease in MIP-1β, but still remained above the level of controls (acute phase vs remission: Pt#1, p<0.001; Pt#2, p<0.05).

A similar trend was observed for IP-10. Indeed, serum levels of this chemokine had increased in all patients during the acute phase and significantly decreased after resolution of the disease. Moreover, defibrotide treatment was able to decrease G-CSF levels, with significance in the first patient (acute phase vs remission: Pt#1, p<0.01), where the glycoprotein levels during remission were like control.

Eotaxin and IL-6 were elevated in the serum of the first patient during the acute phase, but they were significantly reduced after defibrotide treatment (acute phase vs remission: Pt#1, p<0.001). The second patient, on the other hand, showed a reverse trend, with IL-6 and eotaxin levels below the controls both during the acute and remission phases.

Discussion

The integrity of the intestinal barrier is crucial for maintaining overall gut health and preventing the translocation of harmful pathogens and toxins into the bloodstream.²⁹ In our study, we analyzed the effects of defibrotide on colon physical barrier in experimental conditions mimicking the inflammatory state typical of bowel diseases, to evaluate the anti-inflammatory efficacy of this drug.

First, we analyzed the effect of defibrotide on the tight junctions (TJ), which consist of integral transmembrane proteins such as claudins, Occludin, and junctional adhesion molecules (JAMs), as well as zonula occludens (ZOs) cytoplasmic proteins, like ZO-1, ZO-2, and ZO-3, that connect transmembrane proteins to the actin cytoskeleton.^30,31

The results of our study demonstrate that defibrotide significantly enhances the expression of tight junction proteins, such as ZO-1 and Occludin, in HCT116 cells. This effect was observed both under normal conditions and in an inflammatory environment, suggesting that defibrotide may play a crucial role in maintaining and restoring intestinal barrier integrity.

In particular, ZO-1 is a cytoplasmic peripheral membrane isoform that forms a scaffold anchoring the actin cytoskeleton and transmembrane proteins of the tight junctions.^32,33 A recent study demonstrated, in a mouse model with intestinal epithelial-specific ZO-1 knockout, that ZO-1 is not required for epithelial barrier function but is crucial for the repair process of the mucosal epithelium.³⁴ In this context, the ability of defibrotide to increase the basal levels of ZO-1 could be important in hypothesizing a role for this compound in restoring the intestinal barrier.

TJ permeability is determined by the combination of different components, including barrier-forming junctional proteins Occludin, Claudins-1, −3, −4, and −8.³⁵ Claudins are a family of proteins distributed with distinct expression patterns in many organs and segments, such as the gastrointestinal tract. Claudins-3 and −4, predominantly expressed in the distal regions of the intestine,^36,37 are sealing claudins³⁸ that prevent the passage of molecules through the TJ and, like Occludin, are downregulated in diseases affecting the small and large intestine.³⁷ In line with these studies, Occludin, Claudins-3 and −4 were downregulated in our in vitro model mimicking intestinal inflammation, and this effect was counteracted by treatment with defibrotide.

We have also verified the efficacy of the systemic administration of defibrotide in clinical settings, evaluating the cytokine spectrum of two patients, who were successfully treated with defibrotide, as it has been demonstrated that the release of these molecules play an important role in the onset of GVHD.^39,40

In our study we identified different cytokines upregulated during the acute phase, with respect to controls, and downmodulated after the treatment with defibrotide: IL-7, MIP-1β, IP-10, G-CSF, Eotaxin and IL-6.

IL-7 is a cytokine involved in T cell lymphopoiesis and in the homeostatic and extrathymic expansion of T cells in lymphopenic hosts.^41,42 The immune benefits of this cytokine are, however, counterbalanced by the evidence that elevated plasma levels of IL-7, after allogenic-HSCT, are predictive of increased risk of aGVHD and mortality.^43,44 IL-7 may promote the expansion of alloreactive T cells mediating GVHD.⁴⁵ In line with these studies, both patients present high levels of this cytokine during the acute phase, decreasing at the remission after the treatment with defibrotide.

MIP-1β is a pro-inflammatory chemokine that increases the release of cytokines, such as IL-6, from fibroblasts and macrophages, as well as chemotaxis and trans-epithelial migration, mechanisms that contribute to the onset of inflammation.⁴⁶ For this chemokine, the levels before and after treatment of the first patient were significantly reduced, while in the second patient there was always a decrease, although less marked. A similar trend was also identified for IP-10, a CXC chemokine released by antigen presenting cells (APC), epithelial cells as well as endothelial and stromal cells.⁴⁷ It has been suggested that the onset of GVHD is triggered by activation of APCs by damage-associated molecular patterns and pathogen-associated molecular patterns, leading to the production of inflammatory cytokines, such as IFNs, which in turn can induce the production of chemokines, such as IP-10.⁴⁸

An effect similar to that shown for IL-17, MIP-1β, IP-10 and G-CSF can also be observed in the first patient for eotaxin, that plays a role in promoting organ-specific migration of inflammatory cells in GVHD pathophysiology.⁴⁹ Our results support this hypothesis: indeed, the first patient showed significantly higher levels in the acute period of the disease than in the control. After treatment with defibrotide, the levels of chemokine decreased significantly in conjunction with the remission phase, where a reduction in inflammation is observed. In contrast, the second patient shows extremely low levels, comparable to control, already during the acute phase. This may be due to the different onset and severity of GVHD in the two patients.

IL-6 is a cytokine involved in multiple mechanisms such as inflammation, cancer and immunity.^50,51 IL-6 is a pleiotropic cytokine that plays a key role in inflammatory diseases and the onset of GVHD.⁵² In line with the results obtained by Palaniyandi et al,²⁶ the first patient showed a significant reduction in IL-6 levels after treatment with defibrotide, supporting the hypothesis that the drug has an anti-inflammatory activity.

The results obtained in our study are in line with literature, as the levels of pro-inflammatory cytokines and chemokines during the acute phase of GVHD were higher than those found in the controls. Furthermore, from the results obtained, even if preliminary for the limited number of patients analyzed, it is possible to assume that defibrotide may lead to a reduction of the pro-inflammatory cytokine and chemokine profile, leading to an improvement in symptoms of GVHD and allowing its remission.

Conclusion

In conclusion, defibrotide was effective in lowering levels of proinflammatory cytokines to a baseline similar to the controls and also in restoring the expression of structural TJ’s proteins. From the data obtained, it can therefore be assumed that this drug could be used in cases of aGVHD resistant to first- and second-line drugs and could resolve the condition of leaky gut. Consequently, defibrotide could become a repositioned drug, which would have the advantage of reducing development costs and time, since toxicological, pharmacokinetic and safety data had already been collected earlier.

The results presented in the study lay the basis for a more complex clinical investigation involving a broader range of cases, and further research will be necessary to validate our findings and to analyze the molecular mechanisms involved in defibrotide interaction with intestinal TJ.

Data Sharing Statement

The data underlying this study will be shared on request to the corresponding author.

Funding

This research was supported by University of Ferrara local fundings.

Disclosure

Erika Rimondi and Elisabetta Melloni are co-first authors for this study. Natalia Maximova and Annalisa Marcuzzi are co-last authors for this study. The authors report no conflicts of interest in this work.

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