(-)-Epigallocatechin-3-gallate (EGCG) modulates neurological function when intravenously infused in acute and, chronically injured spinal cord of adult rats.

Spinal cord injury (SCI) causes severe and long lasting motor and sensory deficits, chronic pain, and autonomic dysreflexia. (-)-epigallocatechin-3-gallate (EGCG) has shown to produce neuroprotective effect in a broad range of neurodegenerative disease animal models. This study designed to test the efficacy of intravenous infusion of EGCG for 36 h, in acutely injured rats' spinal cord: within first 4 h post-injury and, in chronically SC injured rats: after one year of injury. Functional outcomes measured using standard BBB scale, The Louisville Swim Scale (LSS) and, pain behavior assessment tests. 72 Female adult rats subjected to moderate thoracic SCI using MASCIS Impactor, blindly randomized as the following: (I) Acute SCI + EGCG (II) Acute SCI + saline. (III) Chronic SCI + EGCG. (IV) Chronic SCI + saline and, sham SCI animals. EGCG i.v. treatment of acute and, chronic SCI animals resulted in significantly better recovery of motor and sensory functions, BBB and LSS (P < 0.005) and (P < 0.05) respectively. Tactile allodynia, mechanical nociception (P < 0.05) significantly improved. Paw withdrawal and, tail flick latencies increase significantly (P < 0.05). Moreover, in the EGCG treated acute SCI animals the percentage of lesion size area significantly reduced (P < 0.0001) and, the number of neurons in the spinal cord increased (P < 0.001). Percent areas of GAP-43 and GFAP immunohistochemistry showed significant (P < 0.05) increase. We conclude that the therapeutic window of opportunity for EGCG to depict neurological recovery in SCI animals, is viable up to one year post SCI when intravenously infused for 36 h.

Incretin response to a standard test meal in a rat model of sleeve gastrectomy with diet-induced obesity.

BACKGROUND: Currently, the most effective treatment for obesity is bariatric surgery. Gastroduodenal bypass surgery produces sustained weight loss and improves glycemic control and insulin sensitivity. Previous studies have shown that sleeve gastrectomy (SG) produces similar results and implicate changes in incretin hormone release in these effects. METHODS: Male Sprague-Dawley rats were divided into four groups; lean control (lean), diet-induced obesity (DIO), DIO animals that had undergone SG (SG), and DIO animals that had undergone a sham operation (sham). RESULTS: After a 2-week recovery period, the incretin response to a standard test meal was measured. Blood sampling was performed in free-moving rats at various time points using chronic vascular access to the right jugular vein. There was a significant increase in the bodyweight of DIO animals fed a high-fat/high-sugar diet compared with the lean animals, which was reversed by SG. DIO caused an impairment of the GLP-1 response to a standard test meal, but not the GIP response. SG resulted in a dramatic increase in the GLP-1 response to a standard test meal but had no effect on the GIP response. CONCLUSIONS: A rapid rise in blood sugar was observed in the SG group following a standard test meal that was followed by reactive hypoglycemia. SG dramatically increases the GLP-1 response to a standard test meal but has no effect on GIP in a rat model of DIO.

Trace elements and cell-mediated immunity in gestational and pre-gestational diabetes mellitus at third trimester of pregnancy.

THE AIM OF THE STUDY: To evaluate the correlations between Zn2+, Cu2+, Mg2+, Se2+ and Cr3+ and alteration in T cell subsets during diabetic and normal pregnancy. METHODS: The study involved 63 women with gestational diabetes mellitus (GD) and 16 pregnant women with Type 2 diabetes and 48 healthy, non-pregnant women were included as controls. Ten ml of whole venous blood from each participant was analyzed for electrolytes by atomic absorption; total antioxidant activity, individual enzymatic antioxidants by spectrophotometry; and lymphocyte sub-populations by flow cytometry. RESULTS: There were significant changes in lymphocyte sub-populations: Naïve T cells were decreased and memory T-cells and activated T cells (CD4+HLA-DR+, CD4+CD29+) were increased in diabetes in pregnancy. Zn2+ and Cr3+ deficiency were observed in Type 2 diabetics with an increase in Cu2+ in all pregnant cohorts. In healthy pregnant subjects, CD4+-HLA-DR+ was increased in direct proportion to serum Mg2+ (p<0.05) and Se2+ (p<0.01). In insulin-treated GD patients, CD4+CD29+ cells were increased proportionally to serum Zn2+ (p<0.05) while in diet controlled GD cohort CD45RO+/ CD45RA+ T cells correlated directly with serum Mg (p<0.05) and Zn2+ (p<0.01) while it correlated inversely with serum Cu2+ (p<0.01). CONCLUSIONS: The results of the present study show a correlation between trace element deficiency and increased lipid peroxidation in diabetes in pregnancy and lymphocyte activation. Dietary manipulation may, therefore, point to improvement in existing approaches to management of diabetes mellitus in pregnancy.

Inhibition of Ras-GTPase farnesylation and the ubiquitin-proteasome system or treatment with angiotensin-(1-7) attenuates spinal cord injury-induced cardiac dysfunction.

Cardiovascular diseases are one of the principal causes of death and disability in people with spinal cord injury (SCI). The present study was designed to investigate if acute treatment with FPTIII (an inhibitor of Ras-GTPase farnesylation) or MG132 (an inhibitor of ubiquitin-proteasome pathway [UPS]) or administration of angiotensin-(1-7), also known as Ang-(1-7), (a known inhibitor of cardiac NF-kB) would be cardioprotective. The weight drop technique produced a consistent contusive injury of the spinal cord at the T13 segment. Hearts were isolated from rats either 6 months (SCI-6) or 12 months (SCI-12) after SCI. Hearts were perfused for 30 min and then subjected to 30 min ischemia followed by 30 min reperfusion (I/R). Recovery of cardiac function after I/R was measured as left ventricular developed pressure (P(max)) and coronary flow (CF). Drugs were given during perfusion before hearts were exposed to ischemia and reperfusion. Percent recovery (%R) in P(max) and CF in hearts from control animals were 48±6 and 50±5, respectively, whereas none of the hearts from animals with SCI recovered after 30 min of ischemia. Treatment with FPTIII, MG 132, or Ang-(1-7) before ischemia for 30 min led to significant recovery of heart function following ischemia in SCI-6 but not in SCI-12 animals. Thus we have shown that acute treatments with FPTIII, MG132, or Ang-(1-7) improve cardiac recovery following ischemic insult in animals with SCI and may represent novel therapeutic agents for preventing ischemia-induced cardiac dysfunction in patients with SCI.

Butyrylcholinesterase activity in gestational diabetes: correlation with lymphocyte subpopulations in peripheral blood.

PROBLEM: Inefficient clearance of pregnancy-threatening toxins may contribute to gestational diabetes (GD) and Type II diabetes mellitus (DM) through mechanisms involving immune dysregulation. METHOD OF STUDY: Peripheral venous blood from pregnant Kuwaiti women in third trimester, including 15 GD and 17 DM patients, 14 healthy pregnant (HP) and eight non-pregnant subjects, was analyzed by two-color flow cytometery for number and percentage representation of T lymphocytes. Buterylcholinesterase (BuChE) activity was measured using buterylthiocholine iodide and spectrophotometry. RESULTS: Relative to HP, GD patients exhibited higher ratios of activated and memory phenotypes, including CD4+ CD25+ (P < 0.01), CD4+ HLA-DR (P < 0.05) and CD4+ CD45RO+ (P < 0.05) cells. Serum BuChE activity exhibited positive correlation within the HP cohort with CD4+ CD25+ (P < 0.05), but not in GD and DM cohorts. CONCLUSIONS: Positive correlation between BuChE and a (presumptive) 'regulatory' T-cell phenotype in HP, but not GD or DM may indicate existence of protective detoxification mechanisms against oxidative stress in normal pregnancies.

Lymphocyte sub-populations in gestational diabetes.

PROBLEM: We hypothesize that the normal immunologic responses by the maternal immune system during pregnancy are not as well-regulated in gestational diabetes (GD) patients as in healthy pregnant women. METHOD OF STUDY: Using two-color flow cytometry we evaluated frequencies of peripheral blood lymphocytes in 20 GD patients being treated with insulin; 43 GD patients treated with dietary therapy but no insulin; 44 women experiencing normal pregnancies; and 48 non-pregnant women. RESULTS: When compared with healthy pregnant women, both GD cohorts showed higher percentages CD4(+)CD25(+) (P < 0.05), CD4(+)CD45RO(+) (P < 0.05) and CD4(+)CD29(+) (P < 0.01) but lower percentages of CD4(+)CD45RA(+) (P < 0.05). Higher percentages of the activated phenotypes CD8(+)CD25(+) and CD8(+)HLA-DR(+) cells in the diet-treated cohort and CD4(+)HLA-DR(+) cells in insulin-treated GB cohort, were observed compared with healthy pregnant subjects (P < 0.05). CONCLUSIONS: Expanded populations of activated peripheral blood T cells are associated with GD, suggesting that normal maternal immunosuppression is less effective in GD-afflicted women.

Major lymphocyte populations and T-cell expression of ICAM-1 and L-selectin adhesion molecules in Kuwaitis with asthma and rhinitis.

The authors evaluate major immunologic features of asthma and allergies in a Kuwaiti population. They analyzed peripheral venous blood from 17 asthmatic and 17 healthy long-term residents of Kuwait by using two-color flow cytometry for major lymphocyte subpopulations; they also evaluated 10 healthy individuals who had recently arrived in Kuwait. Relative to healthy subjects, asthmatics exhibited increased percentages of T+ NK cells (p < .01), T-helper cells (p < .05), T-cytotoxic and NK cells for both total numbers (p < .01-.001) and percentages (p < .05-.01), and increased percentages of T cells expressing CD54 (ICAM-1; p < .001) and CD62 (L-selectin; p < .01). However, B cells were present at significantly lower levels in asthmatics, both in total numbers (p < .05) and percentages (p < .01). In comparison with healthy individuals who had recently arrived in Kuwait, healthy long-term residents exhibited elevated numbers of pan-T cells (p < .01) and T-helper cells (p < .05). These results help establish immunological parameters for asthma and allergies in Kuwaiti populations.

In vitro effects of astaxanthin combined with ginkgolide B on T lymphocyte activation in peripheral blood mononuclear cells from asthmatic subjects.

This study was undertaken to identify novel approaches to pharmacological treatment of asthma. Here we hypothesize that the platelet-activating factor receptor antagonist ginkgolide B (GB) in combination with the antioxidant carotenoid astaxanthin (ASX) suppresses T cell activation comparably to two commonly-used antihistamines: cetirizine dihydrochloride (CTZ) and azelastine (AZE). Peripheral blood mononuclear cells from asthmatics, cultured 24 h with either 50 microg/ml phytohemaglutinin (PHA) or PHA plus selected dosages of each drug are analyzed by flow cytometry for CD25+ or HLA-DR+ on CD3+ (T cells). Results are reported as stimulation indices (SI) of %CD3+CD25+ cells or %CD3+HLA-DR+ cells in cultures treated with PHA alone versus these subpopulations in cultures treated with both PHA and drugs. Combinations of ASX and GB exhibited optimal suppression at 10(-7) M GB + 10(-8) M ASX for CD3+CD25+ (SI = 0.79 +/- 0.04, P = 0.001) and 10(-7) M GB + 10(-7) M ASX for CD3+HLA-DR+ (SI = 0.82 +/- 0.05, P = 0.004). In conclusion, suppression of T cell activation below fully stimulated values by GB, ASX, and their combinations was comparable and for some combinations better than that mediated by CTZ and AZE. These results suggest that ASX and GB may have application as novel antiasthmatic formulations.

Butyrylcholinesterase activity and pregnancy-associated differences in immunologically relevant peripheral blood leukocyte populations.

PROBLEM: Toxic anticholinesterases (AC) are known contributors to negative pregnancy outcome. Impairment of detoxification mechanisms may correlate with occurrence of pregnancy disorders in Kuwait. METHOD OF STUDY: Butyrylcholinesterase (BuChE), an enzyme which detoxifies AC was evaluated in 18 Kuwaiti women with pregnancy-induced hypertension (PIH), compared with 15 healthy pregnant and eight healthy non-pregnant women. T-lymphocyte subpopulations were measured by flow cytometry, and BuChE activity was measured by spectrophotometry. RESULTS: Unlike the PIH group, the normal pregnancy group exhibited a significant increase in BuChE activity compared with non-pregnant control subjects (P = 0.04). Within the PIH cohort, inverse correlations were observed between BuChE activity and percentage of CD4+ CD25+ cells (P = 0.001), and CD8+ CD25+ cells (P = 0.007). CONCLUSIONS: Elevated BuChE activity in normal pregnancy may correlate with better ability to clear pregnancy-threatening toxins, while lesser ability to do this in PIH women may be a contributor to disease. The fact that PIH subjects with large subpopulations of activated T cells also exhibited low BuChE activity further suggests a correlation between susceptibility to pregnancy loss and decreased activity of the enzyme.

Vascular stroma-derived endothelial colony forming progenitor cells adapt to tumour growth.

Under appropriate nutrient agar culture conditions, primary or xenografted human and animal tumour biopsy-derived cell suspensions will form two types of colony. The first type, consisting of tight colonies of round cells which form tumours when introduced into nude mice, is of neoplastic origin. The second type of colony, the cells of which fail to form tumours on injection into nude mice, consists of loose colonies of larger, inter-connecting elongated bi- or tripolar cells and is thought to originate from vascular stroma-derived endothelial colony forming progenitor cells (V-ECPC). The likely importance of V-ECPC to tumour growth is emphasised by a positive correlation between the VECPC-derived endothelial cell colonies and both tumour vascularity and growth rate. A high cloning efficiency obtained from tumours of particularly intense vascular nature indicates that this cell is of importance in vascular adaptation and therefore tumour growth. In contrast, avascular, fibrotic tumour tissue yielded very low numbers of stromal vascular endothelial cell colonies. The results suggest that stromal vascular endothelial cell colonies do not arise from the mature fibroblastic elements of the tumour stroma, but rather from cells within actively growing regions. Tritiated thymidine uptake studies show that the vascular stroma-derived endothelial colony forming progenitor cells cell are cycling. Cell separation studies have characterized the as yet morphologically unidentified V-ECPC as having a sedimentation rate of 4.7 mm./hr and a mean density of 1.068 g/cm3 and hence a calculated volume of 450 microns 3.

Profiles of activated T lymphocytes in peripheral blood of Kuwaiti psoriasis vulgaris patients.

We have previously reported unexpected immunological features of psoriasis among Kuwaitis, suggesting novel patterns of immune reactivity contributing to the disease. To better define this phenomenon, we herein describe profiles of major populations and immunologically activated subsets of peripheral blood lymphocytes in a cohort of Kuwaiti psoriasis vulgaris patients. Whole venous blood from fifteen psoriatic and twenty eight normal, healthy subjects was analyzed by 2-color flow cytometry for levels of major lymphocyte species and their immunologically activated subsets. When compared to normal subjects, psoriatic blood contained lower cell densities of CD2+, CD8+ (p=0.002 respectively) and B lymphocytes (CD19+) (p=0.003), with a trend toward a lower CD4+ density (p=0.072). Within each major lymphocyte population, activated lymphocytes were present at higher percentages in psoriatic than in healthy blood. These included CD4+ HLA-DR+ (p=0.002), CD4+CD25+ (p=0.043), CD4+CD54+ (p=0.005), CD8+CD25+ (p=0.015), CD8+ HLA-DR+ (p=0.046) and CD3+CD16+CD56+ (p=0.023) Additionally, psoriatic patients were found to have an expanded ratio of memory to naive T cells (CD45RO+CD45RA+) relative to control subjects; this was expected on the basis of increased immune activation. Our findings are consistent with a picture of psoriasis as a disease mediated by activated lymphocytes.

Decreased total numbers of peripheral blood lymphocytes with elevated percentages of CD4+CD45RO+ and CD4+CD25+ of T-helper cells in non-segmental vitiligo.

Vitiligo is a disorder involving progressive skin depigmentation caused by host mediated destruction of melanocytes. Its pathogenesis is known to correlate with elevated levels of activated skin-infiltrating T lymphocytes and is presumed to be autoimmune in nature. In the present study, we characterize the immunophenotype of peripheral blood T cells from vitiligo patients, with the objective of developing an investigative and diagnostic tool for the disease, using analysis of peripheral blood. Subjects for this investigation included 32 patients diagnosed with non-segmental vitiligo and 28 age-and gender-matched, normal, healthy control participants. Whole venous blood taken from each subject was analyzed using 2-color flow cytometry for immunologically-relevant lymphocyte subsets. When compared with healthy control subjects, peripheral blood from individuals with vitiligo was found to have lower total numbers of lymphocytes (p<0.039). Vitiligo patients also had elevated percentages of memory (CD4+CD45RO+) T cells; (p<0.05), but NK-T cells (CD3+CD16+CD56+) and naive T cells (CD4+CD45RA+) were present at lower total numbers and percentages than in healthy controls (p<0.01 and 0.05 respectively). Blood from severely afflicted subjects exhibited elevated CD3+HLADR+ and CD4+CD45RO+ as well as lower percentages of NK-T cells (p<0.05) when compared with mild cases. In conclusion, disease-associated, peripheral blood lymphocyte immunophenotypic profiles of vitiligo patients are consistent with the hypothesis of T cell activation as a major feature of the disorder. These include elevated memory and reduced naive T cell percentages and increased expression of the activation-associated surface antigen CD25. These changes presumably reflect increased antigen-mediated activation. Moreover, because a corollary effect is increased activation-induced cell death (AICD), lower overall lymphocyte counts observed in vitiligo-afflicted subjects is also expected.

Antioxidant enzyme level in the testes of cirrhotic rats.

OBJECTIVES: An understanding of the tissue and organ level of antioxidant enzymes that scavenge reactive oxygen species may provide an indication of their susceptibility to free radical-related cytotoxic damage. A direct association between testicular production of excessive reactive oxygen species and male infertility has been noted. We measured the activities of superoxide dismutase and glutathione peroxidase in the testes of thioacetamide-induced cirrhotic rats. METHODS: Antioxidant enzyme activities and trace element levels (copper, zinc, manganese, and selenium) in the testes of thioacetamide-induced cirrhotic and control rats were measured. The statistical difference between the experimental and control groups with regard to the activities of superoxide dismutase and glutathione peroxidase and levels of trace elements was analyzed with Student's t test. RESULTS: Our results showed a significant decrease in the activity of these enzymes in the testes of cirrhotic rats. The testicular levels of copper, zinc, and manganese, which are associated with these antioxidant enzymes, increased, whereas selenium decreased slightly in cirrhotic rats; that decrease was not statistically significant. CONCLUSIONS: Our studies showed a drastic decrease in the level of antioxidant enzymes in the testes of cirrhotic rats that could have deleterious effects on sperm function in these animals. Further studies are necessary to understand the exact pathways of trace element metabolism in the testes of cirrhotic rats.