Lysyl oxidase-a possible role in systemic sclerosis-associated pulmonary hypertension: a multicentre study.

OBJECTIVE: Lysyl oxidase (LOX) is an extracellular enzyme that cross-links collagen fibrils. LOX was found to be increased in serum of SSc patients and was suggested to be related to skin fibrosis, yet a vascular source of LOX has been demonstrated in idiopathic pulmonary arterial hypertension (iPAH). We aimed to validate elevated LOX serum levels in SSc and to study its correlation with clinical characteristics and investigate its main source at the tissue level. METHODS: A total of 86 established SSc patients were compared with 86 patients with very early diagnosis of systemic sclerosis (VEDOSS), 110 patients with primary RP (PRP) and 80 healthy controls. LOX serum levels were determined by ELISA. Five lung and 12 skin biopsies from SSc patients were stained for LOX and compared with controls. RESULTS: Serum levels of LOX in SSc were significantly higher than in VEDOSS, PRP and healthy controls (P < 0.001). LOX inversely correlated with the diffusing capacity of the lung for carbon monoxide diffusing capacity (DLCO) in diffuse SSc (r = -0.376, P = 0.02). Patients with moderate to severe estimated systolic PAH had higher LOX levels (P < 0.01). Lung biopsies demonstrated intense LOX staining in SSc patients with PAH that was predominantly located in the endothelium of the remodelled pulmonary vessels. CONCLUSION: Serum LOX levels are increased in established SSc and inversely correlate with the DLCO. LOX is elevated in patients with moderate to severe PAH and is located in the proliferating endothelium in lung arterioles, suggesting a possible role for LOX in SSc-associated PAH.

Wilms' tumor gene 1: a possible new proangiogenic factor in Hodgkin lymphoma.

INTRODUCTION: Wilms' tumor gene 1 (WT1) was recently found to play a role in solid and hematologic malignancies and serves as a marker of prognosis and minimal residual disease in acute leukemia. WT1 was also found to be involved in tumor angiogenesis. There are no data concerning the involvement of WT1 in angiogenesis in lymphoproliferative tumors. The aim of this study was to explore the involvement of WT1 in Hodgkin lymphoma. METHODS: The expression of WT1, neuropilin 1, and VEGF was tested by immunohistochemistry in lymph nodes biopsies of 20 Hodgkin patients and 7 reactive lymph nodes. RESULTS: WT1 was expressed in endothelial cells, in 95% of the malignant lymph nodes. The average of WT1 expression scale was higher in the malignant lymph nodes than in reactive lymph nodes. We found a positive correlation between WT1 expression scale and the angiogenesis scale (0.53) that was statistically significant (P<0.05). As the number of vessels increases, the expression of WT1 is more intense. CONCLUSIONS: We found, for the first time, that WT1 is expressed in endothelial cells in Hodgkin lymphoma. The clinical implications of these findings should be tested in a future study.

Involvement of the bax and bcl-2 system in the induction of germ cell apoptosis is correlated with the time of reperfusion after testicular ischemia in a rat model.

The objective of this study was to examine the relationship between time of reperfusion and bax/bcl-2-dependent germ cell apoptosis after testicular ischemia-reperfusion injury in the rat. In ischemic testis, bax/bcl-2 ratio did not change significantly, and the elevation of germ cell apoptosis was not marked; in the contralateral testis, germ cell apoptosis increased after 6 hours of reperfusion, achieved statistical significance after 24 hours, and decreased after 72 hours of reperfusion and was initiated by decreased bcl-2 messenger RNA levels and elevated bax/bcl-2 ratio within the first 6 hours of reperfusion.

Leptin accelerates enterocyte turnover during methotrexate-induced intestinal mucositis in a rat.

Gastrointestinal mucositis occurs as a consequence of cytotoxic treatment. In the present study, we tested whether leptin can protect gut epithelial cells from methotrexate (MTX)-induced intestinal damage. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of leptin for 24 h. Cell proliferation and apoptosis were assessed using FACS analysis. Adult rats were divided into three experimental groups: Control rats; MTX-rats were treated with a single dose of MTX, and MTX-LEP rats were also treated with leptin for 3 d. Intestinal mucosal damage (Park score), mucosal structural changes (bowel and mucosal weight, mucosal DNA and protein content, villus height and crypt depth), enterocyte proliferation, and enterocyte apoptosis were measured at sacrifice. RT-PCR was used to determine the level of bax and bcl-2 mRNA expression. In the vitro experiment, treatment with leptin of Caco-2 cells pre-treated with MTX resulted in a significant stimulation of cell proliferation and inhibition of cell apoptosis in a dose-dependent manner. In the vivo experiment, MTX-LEP rats demonstrated a greater jejunal and ileal bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, as well as a greater enterocyte proliferation index compared to MTX-animals. MTX-LEP rats also showed a trend toward an increase in enterocyte apoptosis that was accompanied by an increase in bax mRNA and decrease in bcl-2 mRNA expression. In conclusion, leptin enhances proliferation and decreases apoptosis in Caco-2 cells pretreated with MTX. In a rat model of MTX-induced mucositis, treatment with leptin improves intestinal recovery and enhances enterocyte turnover.

The effect of 100% oxygen on intestinal preservation and recovery following ischemia-reperfusion injury in rats.

OBJECTIVE: Inhalation of oxygen improves the hemodynamic status and attenuates the inflammatory response after intestinal ischemia-reperfusion (IR). Yet, the use of hyperoxia is hindered by concerns that it could exacerbate reperfusion injury by increasing free radical formation. We examined the effect of hyperoxia on enterocyte turnover and intestinal preservation and rehabilitation following IR injury in rats. DESIGN: Animal study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Animals were assigned to four experimental groups: 1) Sham rats underwent laparotomy without vascular occlusion and breathed air, 2) Sham-oxygen rats underwent laparotomy without vascular occlusion and breathed 100% oxygen, 3) IR rats underwent occlusion of the superior mesenteric artery and portal vein for 30 minutes and breathed air, and 4) IR group treated with oxygen (IR-O2)rats underwent IR and breathed 100% oxygen starting 10 minutes before and continued for the first 6 hours after reperfusion. Intestinal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 24 hours after IR. MEASUREMENTS AND MAIN RESULTS: In IR rats, breathing 100% oxygen resulted in a significant decrease in Park's injury score in the ileum (p < 0.05 from untreated IR rats). Rats treated with oxygen also demonstrated a significant increase in mucosal weight (p < 0.05) and mucosal DNA (p < 0.05) in the jejunum and ileum, and an increase in villus height (p < 0.01), and crypt depth (p < 0.05) in the ileum. Enterocyte proliferation (assessed by bromodeoxyuridine uptake) was significantly decreased in the jejunum and ileum in untreated IR rats. Oxygen therapy increased enterocyte proliferation in the ileum, and diminished both the apoptosis index and Bax gene expression in the jejunum and ileum (p < 0.05). Plasma thermochemiluminescence oxidizability assay revealed significantly higher thermochemiluminescence ratios in IR group treated with oxygen than in untreated IR rats (p < 0.05) at 6 hours postreperfusion suggesting a significantly lower prior in vivo molecular oxidation. CONCLUSIONS: Hyperoxia reduces small bowel injury, accelerates enterocyte turnover, and improves intestinal rehabilitation after IR.

Effect of oral glutamine on enterocyte turnover during methotrexate-induced mucositis in rats.

BACKGROUND/AIMS: The objective of this study was to evaluate the effects of oral glutamine in preventing intestinal mucosal damage caused by methotrexate (MTX) in rats. METHODS: Male Sprague-Dawley rats were divided into 3 experimental groups: control rats, rats treated intraperitoneally with MTX (MTX rats) and rats treated with oral glutamine in the drinking water (2%) 72 h following intraperitoneal injection of a single dose of MTX (MTX-glutamine rats). Intestinal mucosal damage (Park's injury score), mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 h following MTX injection. RT-PCR was used to determine Bax and Bcl-2 mRNA expression. RESULTS: MTX-glutamine rats demonstrated greater jejunal and ileal mucosal weight and mucosal DNA, greater ileal villus height and crypt depth, and a greater index of proliferation in the jejunum and ileum compared to MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-glutamine rats (vs. MTX) was accompanied by decreased Bax and increased Bcl-2 mRNA expression. CONCLUSIONS: Treatment with oral glutamine prevents mucosal injury and improves intestinal recovery following MTX injury in the rat.

Effect of lactulose on bacterial translocation and intestinal adaptation in a rat model of short bowel syndrome.

BACKGROUND: It is frequently assumed that dietary nondigestible carbohydrates improve host resistance to intestinal infections by stimulating proliferation of the protective gut microflora. Therefore, the aim of this study was to define the effect of lactulose, a nondigestible carbohydrate, on bacterial translocation and intestinal adaptation in a rat model of short bowel syndrome (SBS). MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into 3 experimental groups. Sham rats underwent bowel transection, SBS rats underwent 75% small bowel resection, and SBS-LAC rats underwent bowel resection and were treated with oral lactulose given in drinking water at a dose of 1.5 g x kg(-1) x day(-1) from the 3rd to the 15th postoperative day. Bacterial translocation (to mesenteric lymph nodes, liver, and portal and peripheral blood) and parameters of intestinal adaptation were determined on day 15. RESULTS: Treatment with lactulose did not change bacterial translocation, but decreased ileal mucosal weight (20%; P < 0.05), ileal mucosal DNA (2-fold decrease; P < 0.05) and protein (26%; P < 0.05), villus height in jejunum (21%; P < 0.05) and crypt depth in ileum (17%; P < 0.05), proliferation rates in jejunum (18%; P < 0.05) and ileum (15%; P < 0.05), and apoptotic index in jejunum (40%; P < 0.05) and ileum (36%; P < 0.05) compared with SBS animals. Bax expression was upregulated in SBS rats compared with control animals, but decreased following treatment with lactulose. CONCLUSIONS: In a rat model of SBS, oral lactulose does not improve bacterial translocation from the intestine, but inhibits intestinal adaptation.

Relationship between time of reperfusion and E-selectin expression, neutrophil recruitment, and germ cell apoptosis after testicular ischemia in a rat model.

OBJECTIVE: To examine the relationship between the time of reperfusion and neutrophil recruitment, E-selectin expression, and germ cell apoptosis in the ischemic and contralateral testis after testicular ischemia-reperfusion (IR) injury in a rat. DESIGN: Laboratory study. SETTING: Research laboratory in a faculty of medicine at Technion-institute of technology in Israel. ANIMAL(S): Sixty adult Sprague-Dawley rats weighing 250-280 g. INTERVENTION(S): Testicular IR. MAIN OUTCOME MEASURE(S): Testicular germ cell apoptosis was assessed by terminal deoxynucleotide transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling immunohistochemical assay, using an in situ cell death detection kit. The recruitment of polymorphonuclear (PMN) cells was calculated per 100 venules. Expression of E-selectin was determined by using immunohistochemical analysis. RESULT(S): E-selectin expression and polymorphonuclear-cell recruitment in the contralateral testis increased significantly after 1 hour of reperfusion and then remained unchanged during the first 24-48 hours, followed by a gradual decrease. Germ cell apoptosis in the contralateral testis increased after 6 hours of reperfusion, achieved statistical significance after 24 hours, and decreased after 72 hours of reperfusion. CONCLUSION(S): Germ cell apoptosis in the contralateral testis increases most significantly within the first 24-48 hours, followed by a gradual decrease, after IR injury. E-selectin expression and neutrophil recruitment increases within the first 6 hours and apparently may initiate the increase in germ cell apoptosis.

Oral insulin up-regulates Toll-like receptor 4 expression and enhances intestinal recovery following lipopolysaccharide-induced gut injury in a rat.

In the present study, we evaluated the protective effect of oral insulin (OI) on intestinal mucosa following lipopolysaccharide-induced intestinal damage in a rat. Male Sprague-Dawley rats were divided into three experimental groups: Sham rats, LPS-rats that were treated with lipopolysaccharide (LPS), and LPS-INS rats that were treated with OI given in drinking water 72 h before and following injection of LPS. Intestinal structural changes, enterocyte proliferation, enterocyte apoptosis, and mucosal expression of Toll-like receptor 4 (TLR4) were determined 24 h after the last LPS injection. LPS-INS animals showed a significantly greater bowel and mucosal weight in jejunum and ileum, mucosal DNA and protein in jejunum and ileum, villus height in ileum, crypt depth in jejunum and ileum, cell proliferation rates in jejunum, and significantly lower apoptotic index in ileum compared to LPS- animals. LPS rats demonstrated 50% increase in TLR4 expression in jejunum compared to sham animals. Treatment with OI resulted in a three-fold increase in TLR4 expression in jejunum, compared to LPS animals. In conclusion, OI improves intestinal recovery after LPS endotoxemia in a rat.

Biopsy-negative giant cell arteritis: Does anti-CD83 immunohistochemistry advance the diagnosis?

BACKGROUND: In situ maturation of adventitial dendritic cells (DC) with expression of CD83 has been proposed as an early event in the pathogenesis of giant cell arteritis (GCA), preceding the appearance of an inflammatory infiltrate. The aim of this study was to evaluate the added value of anti-CD83 staining of temporal artery biopsy (TAB) specimens in patients with biopsy-negative temporal arteritis. METHODS: Fourteen patients with TAB performed in our medical center since 2001 and considered negative for GCA due to the absence of any inflammatory infiltrate were identified by a computerized search of patient records. Their paraffin-embedded TAB specimens were retrieved, reprocessed, and stained with anti-CD83 monoclonal antibody (Serotec, 1:40). Three TAB specimens of patients with biopsy-proven GCA served as positive controls and three specimens of popliteal and/or tibial arteries of patients with atherosclerotic peripheral vascular disease were used as negative controls. Follow-up of the patients was confirmed by personal contact with their rheumatologists and analysis of their hospital charts. RESULTS: Follow-up was available for 12 of 14 patients. Five of these patients were considered to have biopsy-negative GCA: they satisfied the ACR classification criteria, were successfully treated with glucocorticosteroids, and had a follow-up of at least 10 months with no alternative diagnosis established. Anti-CD83 staining was negative in all but one patient who demonstrated a single CD83-positive cell adjacent to the internal elastic membrane. Positive anti-CD83 staining of the inflammatory infiltrate throughout the arterial wall was observed in all patients with biopsy-proven GCA (positive controls). Negative controls did not show any CD83-positive cells. CONCLUSIONS: In this pilot study, anti-CD83 immunohistochemical staining of paraffin-embedded specimens did not improve the yield of TAB in patients with suspected GCA.

Effect of probiotics on intestinal regrowth and bacterial translocation after massive small bowel resection in a rat.

BACKGROUND/PURPOSE: Because of their ability to inhibit intestinal bacterial overgrowth, probiotics (PROs) have been advocated for the treatment of patients with short bowel syndrome (SBS). This study was conducted to determine the effect of PROs on bacterial translocation and intestinal regrowth after massive small bowel resection in a rat. METHODS: Male Sprague-Dawley rats were divided into 3 experimental groups: sham rats underwent bowel transection and reanastomosis, SBS rats underwent 75% small bowel resection, and SBS-PRO rats underwent bowel resection and were treated with a PRO given in drinking water from day 4 through 14. Intestinal structural changes (bowel circumference, overall bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, enterocyte proliferation and enterocyte apoptosis) and bacterial translocation (BT) to mesenteric lymph nodes, liver, portal blood, and peripheral blood were determined on day 15 after operation. RESULTS: Sham rats exhibited a 20% BT to the mesenteric lymph nodes (level I), liver (level II), and blood (level III). Short bowel syndrome rats demonstrated a 100% BT to lymph nodes (level I) and liver (level II) and 40% translocation to peripheral blood (level III). Treatment with PROs resulted in a significant decrease in BT to all 3 target organs and decreased enterocyte apoptosis compared with SBS-untreated animals. Short bowel syndrome rats showed a significant increase (vs sham) in jejunal and ileal bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth. Short bowel syndrome rats also had a greater proliferation index and apoptotic index in both jejunum and ileum compared with sham animals. SBS-PRO rats showed a significant increase (vs SBS rats) in crypt depth in ileum and a mild decrease in apoptotic index in jejunum and ileum, compared with SBS-untreated animals. CONCLUSIONS: In a rat model of SBS, PROs decrease BT through mechanisms which maybe dependent on intestinal mucosal integrity.

Oral glutamine prevents gut mucosal injury and improves mucosal recovery following lipopolysaccharide endotoxemia in a rat.

OBJECTIVE: To evaluate the effects of oral glutamine in preventing mucosal damage caused by lipopolysaccharide (LPS) endotoxemia in a rat. METHODS: Male Sprague Dawley rats, weighing 250 to 280 g, were divided into three experimental groups: CONTR rats (Group A), LPS rats (Group B) were treated with lipopolysaccharide given I.P. at dose 10 mg/kg every 24 h (two injections), and LPS-GLN rats (Group C) were treated with oral glutamine given in drinking water (2%) 72 h before and following injection of LPS. Intestinal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 24 h after last LPS injection. RESULTS: LPS rats demonstrated a significant decrease in bowel and mucosal weight in jejunum and ileum, mucosal DNA and protein in jejunum and ileum, and villus height and crypt depth in jejunum and ileum compared with sham animals. LPS rats also had a significantly greater Park injury score in jejunum and ileum, a lower cell proliferation index in jejunum and ileum, and higher apoptotic index in jejunum and ileum compared with control rats. LPS-GLN animals showed a significant increase in bowel weight in jejunum, mucosal weight in jejunum and ileum, mucosal DNA in jejunum and ileum, villus height in jejunum and ileum, and cell proliferation index compared with LPS animals. The Park injury score was significantly lower in LPS-GLN rats compared with LPS animals. CONCLUSIONS: Oral glutamine supplementation prevents mucosal injury and improves intestinal recovery after LPS endotoxemia in a rat.

Bombesin stimulates enterocyte turnover following massive small bowel resection in a rat.

Recent evidence suggests that bombesin (BBS) is involved in modulation of growth and differentiation of normal small intestine. The purpose of the present study was to evaluate the effects of BBS on enterocyte turnover after massive small bowel resection in a rat. Male Sprague-Dawley rats were divided into three experimental groups: Sham rats underwent bowel transection and re-anastomosis, short bowel syndrome (SBS) rats underwent a 75% small bowel resection, and SBS-BBS rats underwent bowel resection and were treated with BBS given subcutaneously at a dose of 20 mug/kg, once daily, from postoperative day 3 through 14. Parameters of intestinal adaptation (bowel and mucosal weights, mucosal DNA and protein, villus height and crypt depth), enterocyte proliferation and enterocyte apoptosis were determined in jejunum and ileum on day 15 following operation. RT-PCR technique was used to determine Bax and Bcl-2 gene expression in ileal mucosa. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA test, with P less than 0.05 considered statistically significant. Treatment with BBS resulted in a significant increase in ileal bowel and mucosal weight, ileal mucosal DNA and protein, jejunal and ileal villus height, jejunal crypt depth, and jejunal and ileal proliferation index compared to SBS-animals. SBS rats showed a significant increase in Bax and Bcl-2 expression in ileum that was accompanied by a significant increase in cell apoptosis compared to sham animals. SBS-BBS rats demonstrated a significant decrease in Bax and Bcl-2 expression in ileum and a decrease in apoptotic index compared to SBS-animals. In conclusion, in a rat model of SBS, BBS enhances enterocyte turnover and stimulates structural intestinal adaptation. Decreased Bax expression may be responsible for the inhibitory effect of BBS on enterocyte apoptosis.

Dietary glutamine supplementation prevents mucosal injury and modulates intestinal epithelial restitution following ischemia-reperfusion injury in the rat.

The aim of the present study was to evaluate the preventive effect of a 2-day oral glutamine supplementation against intestinal ischemia-reperfusion (IR) injury in a rat. Male Sprague-Dawley rats were divided into four experimental groups: sham rats underwent laparotomy, sham-GLU rats underwent laparotomy and were treaded with enteral glutamine (GLU) given in drinking water (2%) 48 hr before and following operation, IR rats underwent occlusion of both the superior mesenteric artery and the portal vein for 30 min followed by 24 hr of reperfusion, and IR-GLU rats were treated with enteral glutamine 48 hr before and following IR. Intestinal mucosal damage (Park's injury score), mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 24 hr following IR. Sham-GLU rats demonstrated a lower rate of cell apoptosis in jejunum and ileum compared to sham animals. IR-GLU animals demonstrated a greater jejunal and ileal bowel and mucosal weight, mucosal DNA, villous height and crypt depth, and enterocyte proliferation index in ileum and a lower injury score grade in jejunum compared to IR-nontreated rats. In conclusion, pretreatment with oral glutamine prevents mucosal injury and improves intestinal recovery following IR injury in the rat.