Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24-27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis.     

CTLA-4 up-regulation of lymphocyte function-associated antigen 1 adhesion and clustering as an alternate basis for coreceptor function

Although cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T cell activation, the full range of functions mediated by this coreceptor has yet to be established. In this study, we report the surprising finding that CTLA-4 engagement by soluble antibody or CD80 potently up-regulates lymphocyte function-associated antigen 1 (LFA-1) adhesion to intercellular adhesion molecule-1 (ICAM-1) and receptor clustering concurrent with IL-2 inhibition. This effect was also observed with CTLA-4 ligation and not with other coreceptors. T cell antigen receptor (TcR)-induced lymphocyte function-associated antigen 1 function was also dependent on CTLA-4 expression as observed with reduced adhesion/clustering on CTLA-4(-/-) primary T cells. CTLA-4 up-regulated adhesion was mediated by regulator for cell adhesion and polarization type 1 (Rap-1) as shown by anti-CTLA-4-induced Rap-1 activation as well as Rap-1-N17 blockade and Rap-1-V12 mimicry of adhesion/clustering. Our findings identify a potent role for CTLA-4 in directing integrin adhesion and provide an alternate mechanism to account for aspects of CTLA-4 function in T cell immunity.     

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants

A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.     

Effects of OP2113 on Myocardial Infarct Size and No Reflow in a Rat Myocardial Ischemia/Reperfusion Model

Cardiovascular Drugs and Therapy - The present study was to determine whether OP2113 could limit myocardial infarction size and the no-reflow phenomenon in a rat myocardial ischemia/reperfusion...     

Solution‐Processable Donor‐Acceptor‐Donor Oligomers with Cross‐Linkable Functionality

Electron‐acceptor units, combined with bithiophene substituted with flexible chains end‐functionalized with cross‐linkable moieties, provide soluble donor‐acceptor‐donor (DAD) π‐conjugated oligomer‐type molecules with cross‐linking ability and broad absorption in the visible spectrum. A study on the cross‐linking conditions of the new oligomers to yield insoluble polymer networks is presented, including conditions for obtaining polymer films over poly(3,4‐ethylenedioxythiophene):polystyrene sulfonate‐covered substrates. The combination of the DAD molecular design and cross‐linking functionality opens prospects for applications in solution‐processed small‐molecule solar cells with morphologically‐stable organic layers.

     

Hyperhomocysteinemia and inflammatory bowel disease: prevalence and predictors in a cross-sectional study

OBJECTIVE: Homocysteine is a sulfur-containing amino acid formed during the demethylation of methionine. Vitamin B12 and folate deficiency and therapy with antifolate drugs may predispose patients with inflammatory bowel disease (IBD) to hyperhomocysteinemia. The known associations between hyperhomocysteinemia and smoking, osteoporosis, and thrombosis make it an interesting candidate as a pathogenetic link in IBD. The aim of this study was to identify the prevalence and risk factors of hyperhomocysteinemia in patients with IBD. METHODS: Sixty-five consecutive IBD patients were recruited from a tertiary outpatient gastroenterology practice. Fasting plasma homocysteine levels were measured, along with vitamin B12 and folate. Data regarding medication use, multivitamin use, disease location and severity, and extraintestinal manifestations of IBD were gathered. Homocysteine levels in 138 healthy control subjects were compared with the IBD cohort, and adjustments for age and sex were made using logistic regression. Multivariate analysis was performed to seek predictors of homocysteine levels. RESULTS: The mean age in the IBD cohort was 42+/-13.4 yr (+/-SD), and 43% were male. The mean disease duration was 13.8+/-9.4 yr, and 32% had used steroids within the last 3 months. Immunomodulator therapy had been used in 32%, and 75% had had an intestinal resection. Osteoporosis was present in 33% of patients. Five patients had experienced venous thrombosis or stroke, but only one of these had hyperhomocysteinemia. Of the 10 IBD patients (15.4%) with hyperhomocysteinemia, only two had vitamin B12 deficiency. The homocysteine levels in the IBD cohort cases and controls were 8.7 and 6.6 micromol/L, respectively (p < 0.05). IBD significantly increased the risk of hyperhomocysteinemia (adjusted odds ratio = 5.9 [95% CI: 1.5-24]). Advanced age, male sex, vitamin B12 deficiency or lower vitamin B12 serum levels, and multivitamin therapy were independently associated with higher homocysteine levels in the multivariate analysis (R2 = 0.55; p = 0.001). CONCLUSIONS: Hyperhomocysteinemia is significantly more common in patients with IBD compared with healthy controls, and is associated with lower (but not necessarily deficient) vitamin B12 levels.     

Ectopic expression of rhombotin-2 causes selective expansion of CD4-CD8- lymphocytes in the thymus and T-cell tumors in transgenic mice

Although the proto-oncogene rhombotin-2 (RBTN-2) is widely expressed in most tissues, it is not expressed in T cells. We investigated the potential for overexpression of RBTN-2 to cause tumors in T cells and other tissues by constructing transgenic mice that expressed RBTN-2 under control of the metallothionein-1 promoter. Despite overexpression of RBTN-2 in all tissues, transgenic mice developed T-cell tumors only, thus indicating that tumorigenesis caused by RBTN-2 is T-cell-specific. Thymic tumors were found between 37 and 71 weeks and were invariably associated with metastasis to nonlymphoid organs. Thymuses from apparently healthy transgenic mice were also examined. In some mice there was an 10-fold increase in the CD4-CD8- thymocyte subset, yet the total number of thymocytes was the same as that in wild-type mice. Thymic homeostasis was maintained by a compensatory reduction in the CD4+CD8+ subset. The expansion of CD4-CD8- thymocytes was associated with increased expression of RBTN-2 and with increased cell proliferation. No differences were found in the proportion of thymocytes undergoing apoptosis in transgenic mice. Furthermore, RBTN-2- induced expansion of CD4-CD8- cells did not block differentiation of these cells. Thymuses with 30% CD4-CD8- cells were essentially monoclonal, indicating that all thymic immunophenotypes were derived from a single clone. Overall, our data are consistent with the following scenario: (1) RBTN-2 expression in T cells causes selective and polyclonal proliferation of CD4-CD8- thymocytes accompanied by a compensatory decrease in other thymocyte subsets; (2) a clone with growth advantage and differentiation potential is selected and populates the thymus; and (3) this clone eventually breaches homeostasis of the thymus, accompanied or followed by metastasis to other organs.     

Emerging risk factors and early atherosclerosis indices in subjects with impaired glucose tolerance

AIM: To evaluate the response to an oral lipid overload, inflammatory markers and carotid intima-media thickness in subjects with impaired glucose tolerance. METHODS: 54 subjects, both sexes, 58 y-old average were submitted to 1) Clinical evaluation 2) Glucose tolerance test with 75 g glucose; classified as normal (2 h plasma glucose<140 mg/dl, n=37) or IGT (2 h G 140-200 mg/dl, n=17), 3) 12 h fasting sample (plasma glucose, lipids, C-reactive protein, fibrinogen and HOMA-IR calculation); 4 and 6 h after the oral lipid overload (1000 kcal, lipids 65 g) glycemia, fibrinogen and triglycerides were reevaluated. Intima-media thickness was calculated by the average of 6 measurements (3 highest of each carotid) evaluated by ultrasonography (7 MHZ transducer). RESULTS: The IGT group had higher (P<0.001) fasting plasma glucose (89.4 +/- 13 vs 104.4 +/- 8 mg/dl), HOMA-IR (1.69 +/- 1.2 vs 2.93 +/- 2.2) and waist (91 +/- 14 vs 101 +/- 9 cm), similar fasting lipids, intima-media thickness (P=0.58) and post-oral lipid overload triglycerides (P=0.74), but higher fibrinogen (284.3 +/- 6 and 305 +/- 10 mg/dl, P=0.05) and C-reactive protein (2.11 +/- 0.33 and 4.19 +/- 0.65 mg/l, P=0.003). C-reactive protein was positively correlated with HOMA-IR (r=0.45, P=0.001), fasting plasma glucose (r=0.43, P=0.002) and waist (r=0.45, P=0.0006), but not with postprandial lipids.CONCLUSION: A higher C-reactive protein in IGT, and its positive correlation with insulin resistance indices, but not with postprandial lipaemia, suggests that the clustering of these factors, characteristic of the metabolic syndrome, occurs earlier than postprandial lipid abnormalities.