Jacob Bejar

Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat

BackgroundArginine (ARG) and nitric oxide maintain the mucosal integrity of the intestine in various intestinal disorders. In the present study, we evaluated the effects of oral ARG supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat.MethodsMale rats were divided into four experimental groups: Control rats, CONTR-ARG rats, were treated with oral ARG given in drinking water 72 hours before and 72 hours following vehicle injection, MTX rats were treated with a single dose of methotrexate, and MTX-ARG rats were treated with oral ARG following injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. RT-PCR was used to determine bax and bcl-2 mRNA expression.ResultsMTX-ARG rats demonstrated greater jejunal and ileal bowel weight, greater ileal mucosal weight, greater ileal mucosal DNA and protein levels, greater villus height in jejunum and ileum and crypt depth in ileum, compared to MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-ARG rats (vs MTX) was accompanied by decreased bax mRNA and protein expression and increased bcl-2 protein levels.ConclusionsTreatment with oral ARG prevents mucosal injury and improves intestinal recovery following MTX- injury in the rat.

Dietary transforming growth factor-beta 2 (TGF-β2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat.

Background/Aims Dietary supplementation with transforming growth factor-beta (TGF-β) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-β2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model. Methods Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-β2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-β rats were treated with diet enriched with TGF-β2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-β rats were treated with diet enriched with TGF-β2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, β-catenin, IL-1B and bax protein expression. Results Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-β2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-β2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-β2 also led to increased pERK, IL-1B and β-catenin protein levels in intestinal mucosa. Conclusions Treatment with TGF-β2 prevents mucosal-injury, enhances p-ERK and β-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.

The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs).

Background and Aims Immune semaphorins are a large family of proteins involved in the pathogenesis of inflammatory diseases through the regulation of immune homeostasis and tissue inflammation. We aim to assess the possible involvement of semaphorin3A (sema3A) and 4A (sema4A) in peripheral immune responses and bowel tissue inflammation of patients suffering from Crohn’s disease (CD) and ulcerative colitis (UC). Patients and Methods Twenty-seven CD patients and 10 UC patients were studied and compared to 10 patients followed for acute diverticulitis (disease control) and 12 healthy individuals. All were evaluated for sema3A expression on T regulatory cells (Tregs), serum levels of sema3A and sema4A, and tissue expression of sema3A and sema4A in bowel biopsies. Results The percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals. Conclusions Altered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.

Glutamine attenuates the inhibitory effect of methotrexate on TLR signaling during intestinal chemotherapy-induced mucositis in a rat

Toll-like receptor 4 (TLR-4) is crucial in maintaining intestinal epithelial homeostasis, participates in a vigorous signaling process and heightens inflammatory cytokine output. The objective of this study was to determine the effects of glutamine (GLN) on TLR-4 signaling in intestinal mucosa during methotrexate (MTX)-induced mucositis in a rat. Male Sprague–Dawley rats were randomly assigned to one of four experimental groups of 8 rats each: 1) control rats; 2) CONTR-GLN animals were treated with oral glutamine given in drinking water (2%) 48 hours before and 72 hours following vehicle injection; 3) MTX-rats were treated with a single IP injection of MTX (20 mg/kg); and 4) MTX-GLN rats were pre-treated with oral glutamine similar to group B, 48 hours before and 72 hours after MTX injection. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. The expression of TLR-4, MyD88 and TRAF6 in the intestinal mucosa was determined using real time PCR, Western blot and immunohistochemistry. MTX-GLN rats demonstrated a greater jejunal and ileal mucosal weight and mucosal DNA, greater villus height in ileum and crypt depth and index of proliferation in jejunum and ileum, compared to MTX animals. The expression of TLR-4 and MyD88 mRNA and protein in the mucosa was significantly lower in MTX rats versus controls animals. The administration of GLN increased significantly the expression of TLR-4 and MyD88 (vs the MTX group). In conclusion, treatment with glutamine was associated with up-regulation of TLR-4 and MyD88 expression and a concomitant decrease in intestinal mucosal injury caused by MTX-induced mucositis in a rat.

Significance of bone marrow reticulin fibrosis in chronic lymphocytic leukemia at diagnosis: a study of 176 patients with prognostic implications.

Bone marrow (BM) biopsies from patients with chronic lymphocytic leukemia (CLL) may show reticulin fibrosis at diagnosis, but its significance remains unclear. This study sought to assess the prognostic impact of BM reticulin fibrosis in patients with previously untreated CLL.

Alendronate preserves femoral head shape and height/length ratios in an experimental rat model: A computer-assisted analysis

Background: Surgical osteonecrosis of the rat femoral head was induced by detaching the ligamentum teres and stripping the femoral neck periosteum. Bone and marrow necrosis were found from the fifth postoperative day and replaced by creeping substitution. Osteonecrosis of the femoral head results in the flattening to various degrees of roundness and osteoarthritic changes of the hip joint. Alendronate, an osteoclast inhibitor, slows down bone resorption and remodeling. The purpose of this study was to evaluate objectively the influence of alendronate treatment on the rat femoral head shape after six weeks of daily treatment, when compared with controls. Materials and Methods: The blood circulation of right femoral head of 20 female Sprague-Dawley rats was interrupted. Twelve were treated by alendronate injections of 200 µg/kg/day and eight controls were treated with saline, both for a total of 42 days. Both femoral head specimens were obtained for computed-assisted morphometry. For each rat, the right operated head was compared with the left, and the alendronate treated group was compared with the control group. Results: No differences were found in shape factor and femoral head height/length ratios in the alendronate treated femoral heads. Among the nontreated control group, shape-factor differences were found between the operated and the nonoperated femoral heads. Conclusion: Alendronate treatment prevented the distortion and destruction of the femoral head. Osteoclast inhibition might prolong the bone creeping substitution process and could enable secondary bone maturity and mineralization that increases bone strength. Alendronate preserved the femoral head architecture, which might reduce morbidity and disability due to femoral head collapse.

Prevention of distortion of vascular deprivation-induced osteonecrosis of the rat femoral head by treatment with alendronate

IntroductionIn animals with a disrupted blood supply and drainage of the femoral head, the dead epiphyseal bone undergoes osteoclastic osteolysis and is replaced by newly synthesized, immature, and weak bone, which cannot withstand the daily loads and, therefore, the articular surface caves in.MethodsFemale Sprague–Dawley rats with interrupted blood circulation of the femoral head were treated with alendronate and compared to controls.ResultsThere was no distortion of the femoral heads in the alendronate-treated animals.InterpretationAlendronate medication interferes with osteoclastic activities, slowing down bone turnover. These observations verify our hypothesis that osteoclastic activity is detrimental to the conservation of a hemispherical femoral head because of the rapidly occurring replacement of the dead by living tissues. Hence, halting the activities of the osteoclasts by alendronate stops the hasty new bone formation which is responsible for early femoral capital disfigurement.

Primary duodenal mucosa-associated lymphoid tissue (MALT) lymphoma - A rare presentation of gastric outlet obstruction

Malignant lymphoma of mucosa-associated lymphoid tissue (MALT) can arise in a variety of anatomical sites. The majority of these tumors arise in the stomach, with fewer than 30% arising in the small intestine. Primary duodenal MALT lymphoma is a very rare neoplasm. There are very few cases of duodenal MALT lymphoma reported in the literature. This is the third published case presenting clinically as a gastric outlet obstruction. The patient was successfully treated with a combination of chemotherapy and rituximab.

PDGF-α stimulates intestinal epithelial cell turnover after massive small bowel resection in a rat

Numerous cytokines have been shown to affect epithelial cell differentiation and proliferation through epithelial-mesenchymal interaction. Growing evidence suggests that platelet-derived growth factor (PDGF) signaling is an important mediator of these interactions. The purpose of this study was to evaluate the effect of PDGF-α on enterocyte turnover in a rat model of short bowel syndrome (SBS). Male rats were divided into four groups: Sham rats underwent bowel transection, Sham-PDGF-α rats underwent bowel transection and were treated with PDGF-α, SBS rats underwent a 75% bowel resection, and SBS-PDGF-α rats underwent bowel resection and were treated with PDGF-α. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at euthanasia. Illuminas Digital Gene Expression analysis was used to determine PDGF-related gene expression profiling. PDGF-α and PDGF-α receptor (PDGFR-α) expression was determined by real-time PCR. Western blotting was used to determine p-ERK, Akt1/2/3, bax, and bcl-2 protein levels. SBS rats demonstrated a significant increase in PDGF-α and PDGFR-α expression in jejunum and ileum compared with sham animals. SBS-PDGF-α rats demonstrated a significant increase in bowel and mucosal weight, villus height, and crypt depth in jejunum and ileum compared with SBS animals. PDGF-α receptor expression in crypts increased in SBS rats (vs. sham) and was accompanied by an increased cell proliferation following PDGF-α administration. A significant decrease in cell apoptosis in this group was correlated with lower bax protein levels. In conclusion, in a rat model of SBS, PDGF-α stimulates enterocyte turnover, which is correlated with upregulated PDGF-α receptor expression in the remaining small intestine.

Severe pneumonia associated with ibrutinib monotherapy for CLL and lymphoma

In recent years, there have been major advances in the treatment of chronic lymphocytic leukemia (CLL) particularly since the development of novel therapeutic agents, mostly “biological drugs.” One of the obvious advantages of these agents is the decreased rate of infectious complications occurring during the course of therapy, compared to the use of standard immuno‐chemotherapy regimens. Here, we describe 3 patients with CLL and 1 with mantle cell lymphoma who developed severe life‐threatening pneumonias, during monotherapy with ibrutinib. The first case was a 70‐year‐old woman with relapsed CLL who developed bilateral pneumonia with hypoxia 1 week after starting ibrutinib. She did not respond to broad‐spectrum antibiotics and was treated empirically with trimethoprim‐sulphamethoxazole and improved. In the second case, we describe a 76‐year‐old woman with relapsed CLL who developed recurrent pneumonia after 3 years of treatment with ibrutinib. Presuming that ibrutinib was the cause of pneumonitis with secondary infection, it was stopped with subsequent improvement. The third patient a 67 year‐old man died because of severe bilateral necrotizing pneumonia due to invasive aspergillosis and mucormycosis with pulmonary hemorrhage. The fourth patient with relapsed mantle cell lymphoma died because of severe bilateral pneumonia, caused by pseudomonas and candida, despite receiving appropriate antibiotics. From this experience, we hypothesize that the etiology of severe pneumonia associated with ibrutinib treatment is probably multifactorial, involving factors like preexisting immune‐suppression, drug induced pneumonitis and infections. We suggest that patients with CLL or other lymphoproliferative disorders with suspected pneumonia during monotherapy with ibrutinib should be very carefully evaluated and need to undergo complete diagnostic workup to establish an exact diagnosis. Understanding which patients with CLL or lymphoma treated with kinase inhibitors are at a higher risk for developing pulmonary complications could be one of the important future challenges, when selecting the best available therapy for these patients.