Category: 8. Health

“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.”

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

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.

References

1. Biliński J, Jasiński M, Basak GW. The role of fecal microbiota transplantation in the treatment of acute graft-versus-host disease. Biomedicines. 2022;10(4):837. doi:10.3390/biomedicines10040837

2. Khoury HJ, Wang T, Hemmer MT, et al. Improved survival after acute graft-versus-host disease diagnosis in the modern era. Haematologica. 2017;102(5):958–966. doi:10.3324/haematol.2016.156356

3. Braidotti S, Curci D, Maestro A, Zanon D, Maximova N, Di Paolo A. Effect of early post- hematopoietic stem cell transplant tacrolimus concentration on transplant outcomes in pediatric recipients: one facility’s ten-year experience of immunosuppression with tacrolimus. Int Immunopharmacol. 2024;138:112636. doi:10.1016/j.intimp.2024.112636

4. Braidotti S, Granzotto M, Curci D, Faganel Kotnik B, Maximova N. Advancing allogeneic hematopoietic stem cell transplantation outcomes through immunotherapy: a comprehensive review of optimizing non-CAR donor T-lymphocyte infusion strategies. Biomedicines. 2024;12(8):1853. doi:10.3390/biomedicines12081853

5. Li A, Abraham C, Wang Y, Zhang Y. New insights into the basic biology of acute graft-versus-host-disease. Haematologica. 2020;105(11):2540–2549. doi:10.3324/haematol.2019.240291

6. Cooke KR, Luznik L, Sarantopoulos S, et al. The biology of chronic graft-versus-host disease: a Task Force Report from the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in chronic graft-versus-host disease. Biol Blood Marrow Transplant. 2017;23(2):211–234. doi:10.1016/j.bbmt.2016.09.023

7. Naymagon S, Naymagon L, Wong SY, et al. Acute graft-versus-host disease of the gut: considerations for the gastroenterologist. Nat Rev Gastroenterol Hepatol. 2017;14(12):711–726. doi:10.1038/nrgastro.2017.126

8. Jansen SA, Nieuwenhuis EES, Hanash AM, Lindemans CA. Challenges and opportunities targeting mechanisms of epithelial injury and recovery in acute intestinal graft-versus-host disease. Mucosal Immunol. 2022;15(4):605–619. doi:10.1038/s41385-022-00527-6

9. Anderson JM, Van Itallie CM. Physiology and function of the tight junction. Cold Spring Harb Perspect Biol. 2009;1:a002584. doi:10.1101/cshperspect.a002584

10. Nalle SC, Zuo L, Ong MLDM, et al. Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability. J Clin Invest. 2019;129(2):902–914. doi:10.1172/JCI98554

11. Bhat AA, Uppada S, Achkar IW, et al. Tight junction proteins and signaling pathways in cancer and inflammation: a functional crosstalk. Front Physiol. 2019;9:1942. doi:10.3389/fphys.2018.01942

12. Malard F, Huang XJ, Sim JPY. Treatment and unmet needs in steroid-refractory acute graft-versus-host disease. Leukemia. 2020;34:1229–1240. doi:10.1038/s41375-020-0804-2

13. Pescador R, Capuzzi L, Mantovani M, et al. Defibrotide: properties and clinical use of an old/new drug. Vascul Pharmacol. 2013;59:1–10. doi:10.1016/j.vph.2013.05.001

14. Coutsouvelis J, Avery S, Dooley M, Kirkpatrick C, Spencer A. Defibrotide for the management of sinusoidal obstruction syndrome in patients who undergo haemopoietic stem cell transplantation. Cancer Treat Rev. 2016;50:200–204. doi:10.1016/j.ctrv.2016.09.014

15. Corbacioglu S, Carreras E, Mohty M, et al. Defibrotide for the treatment of hepatic veno-occlusive disease: final results from the international compassionate-use program. Biol Blood Marrow Transplant. 2016;22:1874–1882. doi:10.1016/j.bbmt.2016.07.001

16. Richardson PG, Smith AR, Triplett BM, et al. Defibrotide for patients with hepatic veno-occlusive disease/sinusoidal obstruction syndrome: interim results from a treatment IND study. Biol Blood Marrow Transplant. 2017;23:997–1004. doi:10.1016/j.bbmt.2017.03.008

17. Palomo M, Mir E, Rovira M, et al. What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance. Blood. 2016;127:1719–1727. doi:10.1182/blood-2015-10-676114

18. García-Bernal D, Palomo M, Martínez CM, et al. Defibrotide inhibits donor leucocyte-endothelial interactions and protects against acute graft-versus-host disease. J Cell Mol Med. 2020;24:8031–8044. doi:10.1111/jcmm.15434

19. Bracht F, Schrör K. Isolation and identification of aptamers from defibrotide that act as thrombin antagonists in vitro. Biochem Biophys Res Commun. 1994;200:933–937. doi:10.1006/bbrc.1994.1539

20. Coccheri S, Nazzari M. Defibrotide as a possible anti-ischemic drug. Semin Thromb Hemost. 1996;22(Suppl 1):9–14.

21. Morabito F, Gentile M, Gay F, et al. Insights into defibrotide: an updated review. Expert Opin Biol Ther. 2009;9:763–772. doi:10.1517/14712590903008507

22. Abraham C, Abreu MT, Turner JR. Pattern recognition receptor signaling and cytokine networks in microbial defenses and regulation of intestinal barriers: implications for inflammatory bowel disease. Gastroenterology. 2022;S0016–5085(22):00125.

23. Clayburgh DR, Barrett TA, Tang Y, et al. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J Clin Invest. 2005;115:2702–2715. doi:10.1172/JCI24970

24. Bruewer M, Utech M, Ivanov AI, et al. Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like process. FASEB J. 2005;19:923–933. doi:10.1096/fj.04-3260com

25. Zihni C, Mills C, Matter K, et al. Tight junctions: from simple barriers to multifunctional molecular gates. Nat Rev Mol Cell Biol. 2016;17(9):564–580. doi:10.1038/nrm.2016.80

26. Palaniyandi S, Kumari R, Strattan E, et al. Role of defibrotide in the prevention of murine model graft-versus-host disease after allogeneic hematopoietic cell transplantation. Transplant Cell Ther. 2023;S2666–6367(23):01435.

27. Yamamoto-Furusho JK, Barnich N, Hisamatsu T, Podolsky DK. MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function. Inflamm Bowel Dis. 2010;16(5):736–742. doi:10.1002/ibd.21144

28. Brugnoli F, Grassilli S, Al-Qassab Y, et al. PLC-β2 is modulated by low oxygen availability in breast tumor cells and plays a phenotype dependent role in their hypoxia-related malignant potential. Mol Carcinog. 2016;55:2210–2221. doi:10.1002/mc.22462

29. Turner JR. Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 2009;9:799–809. doi:10.1038/nri2653

30. Brunner J, Ragupathy S, Borchard G. Target specific tight junction modulators. Adv Drug Deliv Rev. 2021;171:266–288.

31. Zuo L, Kuo WT, Turner JR. Tight junctions as targets and effectors of mucosal immune homeostasis. Cell Mol Gastroenterol Hepatol. 2020;10:327–340. doi:10.1016/j.jcmgh.2020.04.001

32. Van Itallie CM, Tietgens AJ, Krystofiak E, Kachar B, Anderson JM. A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction. Mol Biol Cell. 2015;26:2769–2787. doi:10.1091/mbc.E15-04-0232

33. Van Itallie CM, Tietgens AJ, Anderson JM. Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1. Mol Biol Cell. 2017;28:524–534. doi:10.1091/mbc.e16-10-0698

34. Kuo WT, Zuo L, Odenwald MA, et al. The tight junction protein ZO-1 is dispensable for barrier function but critical for effective mucosal repair. Gastroenterology. 2021;161:1924–1939. doi:10.1053/j.gastro.2021.08.047

35. Nighot P, Ma T. Endocytosis of intestinal tight junction proteins: in time and space. Inflamm Bowel Dis. 2021;27:283–290. doi:10.1093/ibd/izaa141

36. Lameris AL, Huybers S, Kaukinen K, et al. Expression profiling of claudins in the human gastrointestinal tract in health and during inflammatory bowel disease. Scand J Gastroenterol. 2013;48:58–69. doi:10.3109/00365521.2012.741616

37. Luettig J, Rosenthal R, Barmeyer C, Schulzke JD. Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation. Tissue Barriers. 2015;3:e977176. doi:10.4161/21688370.2014.977176

38. Zhao X, Zeng H, Lei L, et al. Tight junctions and their regulation by non-coding RNAs. Int J Biol Sci. 2021;17:712–727. doi:10.7150/ijbs.45885

39. Henden AS, Hill GR. Cytokines in graft-versus-host disease. J Immunol. 2015;194(10):4604–4612. doi:10.4049/jimmunol.1500117

40. Zeiser R. Advances in understanding the pathogenesis of graft-versus-host disease. Br J Haematol. 2019;187(5):563–572. doi:10.1111/bjh.16190

41. Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J Immunol. 2005;174:6571–6576. doi:10.4049/jimmunol.174.11.6571

42. Snyder KM, Mackall CL, Fry TJ. IL-7 in allogeneic transplant: clinical promise and potential pitfalls. Leuk Lymphoma. 2006;47:1222–1228. doi:10.1080/10428190600555876

43. Dean RM, Fry T, Mackall C, et al. Association of serum interleukin-7 levels with the development of acute graft-versus-host disease. J Clin Oncol. 2008;26(35):5735–5741. doi:10.1200/JCO.2008.17.1314

44. Kielsen K, Jordan KK, Uhlving HH, et al. T cell reconstitution in allogeneic haematopoietic stem cell transplantation: prognostic significance of plasma interleukin-7. Scand J Immunol. 2015;81:72–80. doi:10.1111/sji.12244

45. Poiret T, Rane L, Remberger M, et al. Reduced plasma levels of soluble interleukin-7 receptor during graft-versus-host disease (GVHD) in children and adults. BMC Immunol. 2014;15:25. doi:10.1186/1471-2172-15-25

46. New JY, Li B, Koh WP, et al. T cell infiltration and chemokine expression: relevance to the disease localization in murine graft-versus-host disease. Bone Marrow Transplant. 2002;29:979986. doi:10.1038/sj.bmt.1703563

47. Lo BK, Yu M, Zloty D, Cowan B, Shapiro J, McElwee KJ. CXCR3/ligands are significantly involved in the tumorigenesis of basal cell carcinomas. Am J Pathol. 2010;176:2435–2446. doi:10.2353/ajpath.2010.081059

48. Chirumbolo G, Dicataldo M, Barone M, et al. A multiparameter prognostic risk score of chronic graft-versus-host disease based on CXCL10 and plasmacytoid dendritic cell levels in the peripheral blood at 3 months after allogeneic hematopoietic stem cell transplantation. Transplant Cell Ther. 2023;29(5):302. doi:10.1016/j.jtct.2023.02.008

49. Piper KP, Horlock C, Curnow SJ, et al. CXCL10-CXCR3 interactions play an important role in the pathogenesis of acute graft-versus-host disease in the skin following allogeneic stem- cell transplantation [published correction appears in Blood. 2008 Aug 15;112(4):1546]. Blood. 2007;110(12):3827–3832. doi:10.1182/blood-2006-12-061408

50. Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in inflammation and cancer. Eur J Cancer. 2005;41:2502–2512. doi:10.1016/j.ejca.2005.08.016

51. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 2014;6:a016295. doi:10.1101/cshperspect.a016295

52. Saadi MI, Ramzi M, Hosseinzadeh M, et al. Expression levels of Il-6 and Il-18 in acute myeloid leukemia and its relation with response to therapy and acute GvHD after bone marrow transplantation. Indian J Surg Oncol. 2021;12:465–471. doi:10.1007/s13193-021-01358-w

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Medicines containing a type of PFAS or ‘forever chemical’ called fluorine are not leading to higher numbers of adverse drug reactions according to new data analysis.

In a new paper published in PLOS ONE today, researchers from the University of Birmingham studied data from the MHRA’s Yellow Card system on 13 drugs containing carbon-fluorine bonds as well as six drugs which were structurally similar but not containing this forever chemical.

Using five years of data from 2019-2024, the research team analyzed the number of adverse drug reactions (ADRs) per 1 million medicines dispensed. They found that most of the ADRs that were listed for drugs containing fluorine were associated with conditions outside of the scope of usual side effects from PFAS, and the highest prescribed drug lansoprazole, a proton pump inhibitor to reduce stomach acid, had a low rate of 14.1 ADRs per 1m items.

“In this study we explored adverse drug reactions reported to the MHRA Yellow Card scheme relative to their prescribing rate. Reassuringly, no statistical correlation between the fluorine content of the medicine and type of side effect emerged,” Dr. Alan Jones, lead author of the study.

Dr. Alan Jones from the School of Pharmacy at the University of Birmingham and corresponding author of the paper said: “Per- and poly-fluoroalkyl substances (PFAS) are often referred to as “forever chemicals” due to their persistence in the environment and effects on human health. PFAS are found in a range of everyday products such as cookware or clothing. Recent changes to the classification of PFAS means certain essential medicines are now deemed to contain forever chemicals.

“In this study we explored adverse drug reactions reported to the MHRA Yellow Card scheme relative to their prescribing rate. Reassuringly, no statistical correlation between the fluorine content of the medicine and type of side effect emerged.”

Among the 13 drugs selected for study, the team observed no relationship between the amount of fluorine atoms in the medicine and the number of ADRs reported. Among the drugs with the highest level of fluorine, sitagliptin and flecainide didn’t see highest levels of reactions.

The team also looked at specific types of ADRs across the 13 fluorinated drugs studied, and found that while reactions have been associated with PFAS-like side effects, the comparison with non-fluorinated drugs suggests that the way the drug acted was more likely to result in that ADR.

The researchers note that limitations of the study include the self-reported nature of the Yellow Card system which could lead to underreporting of adverse drug reactions.

Reference: Balasubramaniam B, Jones AM. Observational suspected adverse drug reaction profiles of fluoro-pharmaceuticals and potential mimicry of per- and polyfluoroalkyl substances (PFAS) in the United Kingdom. PLOS One. 2025. doi: 10.1371/journal.pone.0331286

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Early birds have long basked in the glory of health superiority, sometimes even tinged with a hit of moral righteousness. It’s easier for them to snooze at night and rise with the sun, allowing them to tick through their to-do list, maybe knock out a self-care routine or morning workout, before night owls even drag themselves out of bed. And a new study just granted them even more aura points: Researchers found that older adults who maintained an early breakfast time as they aged were at less risk of dying during a roughly 20-year period than those who pushed back that morning meal over time.

The study followed nearly 3,000 older folks in the United Kingdom who filled out questionnaires at various points during the study period, recording lifestyle details like their typical meal and sleep timing, as well as any symptoms of physical and psychological illness they were experiencing. Some of them also did blood testing, allowing researchers to track who among them had certain genes linked with having an evening chronotype (a.k.a. night owl tendencies). To no surprise, the night-owl people tended to eat all their meals at later times. But more illuminating were the consistent associations the researchers found between mealtimes and health outcomes: Delaying breakfast was linked with depression, higher levels of fatigue, and greater frequency of illness and, yep, mortality risk.

Further stacking the evidence in favor of an early breakfast, the researchers also pinpointed two general clusters of participants: an early-eating group that had breakfast around 7:50 a.m. and a later-eating group that had their morning meal at 8:50 a.m. And it turned out, the earlier-eaters had a higher survival rate than the later-eaters. In fact, when the researchers crunched the numbers, they found that with each hour later that participants ate breakfast, they had an 11% increased risk of dying during the study period.

It’s worth noting, studies like this one can only prove correlation, not causation—so it might be that health issues pushed some participants to eat breakfast later, rather than a delayed breakfast causing them to be worse off, health-wise. That change in meal timing among older adults “could be an easy marker, something that a family member could even pick up on, of an underlying health condition,” lead author Hassan Dashti, PhD, RD, a nutrition scientist and circadian biologist at Massachusetts General Hospital, tells SELF.

But at the same time, Dr. Dashti holds that a consistent, early breakfast may have a positive effect on health and longevity, particularly by sharpening the circadian rhythm. As we age, that rhythm gets blunted, which can have a negative ripple effect on various body systems. A routine morning meal “is a strong environmental cue that tells your body it’s daytime,” Dr. Dashti says, “which signals each of your organs to shift from evening functioning into daytime mode.” That helps keep everything chugging along in optimal form.

This isn’t the first study to suggest the importance of breakfast for living a long life—research has shown that regularly eating a morning meal is linked with lower overall and heart-related mortality (and that bypassing it can up your heart-disease risk).

Aspirin is one of the most widely taken medicines in the world, having been recommended for decades as a way of protecting against heart attacks and strokes in at-risk patients. However, a new study has revealed that clopidogrel, another commonly used blood thinner, or anticoagulant, is more effective in preventing serious heart attacks and strokes and carries no additional risk.

The finding is the result of research conducted by an international team of scientists from the US, UK, Switzerland, Australia, and Japan. Their work is a meta-analysis—a study that collects and analyzes the results of multiple smaller studies, with the aim of reaching a more reliable conclusion by looking at a larger amount of data. In total, this meta-analysis looked at clinical data from nearly 29,000 patients diagnosed with coronary artery disease (CAD), a condition where fat builds up in the arteries, which can lead to secondary effects such as heart attacks and heart failure.

The specialists conducted a systematic search of medical databases such as PubMed, Scopus, Web of Science, and Embase to find randomized trials of treatments for CAD published up to April 12, 2025. The aim was to identify papers comparing the efficacy of aspirin versus clopidogrel in the prevention of cardiovascular deaths, heart attacks, and strokes.

The analysis focused on seven investigations that included clinical information from persons with confirmed cases of CAD treated with aspirin or clopidogrel for an average of 2.3 years. After a follow-up of 5.5 years, the researchers observed that those who received clopidogrel had a 14 percent lower risk of a major cardiovascular event compared with those treated with aspirin.

Ultimately, the team concluded that these findings “add to the evidence” that clopidogrel is superior to aspirin for preventing major adverse cardiac and cerebrovascular events. In the researchers’ view, these findings support using clopidogrel over aspirin in patients with established CAD to try to prevent them going on to have major complications as a result of their condition, such as a heart attack. The findings were published in the journal The Lancet.

In terms of mortality and bleeding risk, the meta-analysis concluded that the rates were similar in both groups, confirming that clopidogrel is as safe as aspirin.

“To the best of our knowledge, clopidogrel monotherapy is the only antiplatelet treatment that has consistently demonstrated greater efficacy than aspirin without compromising safety,” the researchers wrote in the paper.

The discovery could transform medical guidelines internationally. Clopidogrel is a widely available, affordable drug with reliable generic versions, characteristics that would make it easy to incorporate into routine clinical practice. Nevertheless, specialists stress that more extensive research is needed to evaluate the cost-effectiveness of clopidogrel and its performance in diverse populations in order to support its inclusion in treatment standards.

Cardiovascular diseases are the leading cause of death in the world. According to the World Health Organization, an estimated 17.9 million people die each year from these conditions. More than four out of every five of these deaths are due to coronary heart disease or stroke. The new research suggests that clopidogrel could become a key alternative to combat this public health problem, the incidence of which continues to rise around the world.

This story originally appeared on WIRED en Español and has been translated from Spanish.