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Vitro Production (vitro + production)
Selected AbstractsIn Vitro Production of Equine Embryos: State of the ArtREPRODUCTION IN DOMESTIC ANIMALS, Issue 2010K Hinrichs Contents In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development similar to those for cattle (25% to 35%) may be obtained. Pregnancy rates after transfer of in vitro -produced blastocysts are similar to those for embryos recovered ex vivo. [source] Male and Female Effects on the In Vitro Production of Bovine EmbryosANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 5 2004G. A. Palma Summary A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3,93.4%) and blastocysts on day 7 (6.9,51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F -value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme. [source] Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodiesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2005Bernd Franke Abstract The prodromal phase of type 1 diabetes is characterised by the appearance of multiple islet-cell related autoantibodies (Aab). The major target antigens are islet-cell antigen, glutamic acid decarboxylase (GAD), protein-tyrosine phosphatase-2 (IA-2) and insulin. Insulin autoantibodies (IAA), in contrast to the other autoimmune markers, are the only ,-cell specific antibodies. There is general consensus that the presence of multiple Aab (, 3) is associated with a high risk of developing diabetes, where the presence of a single islet-cell-related Aab has usually a low predictive value. The most commonly used assay format for the detection of Aab to GAD, IA-2 and insulin is the fluid-phase radiobinding assay. The RBA does not identify or measure Aab, but merely detects its presence. However, on the basis of molecular studies, disease-specific constructs of GAD and IA-2 have been employed leading to somewhat improved sensitivity and specificity of the RBA. Serological studies have shown epitope restriction of IAA that can differentiate diabetes-related from unrelated IAA, but current assays do not distinguish between disease-predictive and non-predictive IAA or between IAA and insulin antibodies (IA). More recently, phage display technology has been successful in identifying disease-specific anti-idiotopes of insulin. In addition, phage display has facilitated the in vitro production of antibodies with high affinity. Identification of disease-specific anti-idiotopes of insulin should enable the production of a high affinity reagent against the same anti-idiotope. Such a development would form the basis of a disease-specific radioimmunoassay able to identify and measure particular idiotypes, rather than merely detect and titrate IAA. Copyright © 2005 John Wiley & Sons, Ltd. [source] Endocrine responses of Fundulus heteroclitus to effluent from a bleached-kraft pulp mill before and after installation of reverse osmosis treatment of a waste streamENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2000Monique G. Dubé Abstract Implementation of process changes on the nonbleaching side of bleached kraft pulp mill (BKPM) operations has increased in recent years to maximize resource use and to minimize residual environmental effects of discharged effluents. The objective of this study was to determine if reverse osmosis (RO) treatment of evaporator and digester clean condensates reduced or removed the effects of a BKPM effluent on reproductive endocrine function of the estuarine killifish, Fundulus heteroclitus (mummichog). Comparison of data collected before (1997) and after (1998), the years of the process change, showed that the potential of the combined mill effluent to depress plasma testosterone levels after 30 and 57 d of exposure to an environmentally relevant effluent concentration (1%) was reduced after RO treatment of condensates. However, in vitro production of some sex steroids was depressed with a 1% effluent exposure after the process change. In addition, in 1998, depression of plasma testosterone levels in effluent-exposed fish was present at higher effluent concentrations (5%). These results are significant because they suggest that condensates may be a source of endocrine-disrupting compounds in BKPM effluents and RO may reduce their discharge. [source] Pregnancy, but not the allergic status, influences spontaneous and induced interleukin-1, (IL-1,), IL-6, IL-10 and IL-12 responsesIMMUNOLOGY, Issue 1 2006Petra Amoudruz Summary In this study, we investigated how pregnancy influences cytokine production in response to stimulation of the innate and the adaptive immune system, respectively. Peripheral blood mononuclear cells (PBMCs) from allergic (n = 44) and non-allergic (n = 36) women were collected at three time-points: during the third trimester, at delivery and at a non-pregnant state 2 years after delivery. The production of interleukin-1, (IL-1,), IL-6, IL-10 and IL-12 was measured by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay (ELISPOT). The spontaneous cytokine production, and the response following stimulation with agents that primarily activate the adaptive part of the immune system [phytohaemagglutinin (PHA), allergen extracts from cat and birch], or lipopolysaccharide (LPS) that activate innate immunity was measured in vitro. There was a significantly higher spontaneous in vitro production of IL-1,, IL-6 and IL-10 by PBMCs during pregnancy than 2 years after pregnancy, and this was not affected by the allergic status of the women. Conversely, in PHA-stimulated cell cultures there was a lower production of IL-10 and IL-12 during pregnancy than 2 years after pregnancy. LPS-induced IL-6 levels were significantly lower in PBMCs obtained during pregnancy than at 2 years after pregnancy. In addition, we made the interesting observation that in allergic women total immunoglobulin E (IgE) levels were significantly lower 2 years after pregnancy compared to the levels during pregnancy. Taken together, our results indicate that while atopic allergy in women does not have a substantial effect on cytokine production, pregnancy has an obvious effect on the immune system in terms of cytokine production as well as on the total IgE levels. [source] Genetically engineered normal flora for oral polypeptide delivery: Dose,absorption responseJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2009Gagan Kaushal Abstract Genetically modified Lactococcus lactis (L. lactis), a probiotic bacterium, able to secrete ,-lactamase (29 kDa), was used as a vector for the oral delivery of ,-lactamase to the rats. Three different doses of L. lactis were administered to the rats, and the resulted ,-lactamase oral bioavailability was studied, and compared to the solution form. The oral administration of 1.2,×,107, 3,×,107, and 8,×,107 colony-forming units of L. lactis led to 145, 209, and 364 mU of ,-lactamase absorbed, and the corresponding bioavailability was 8.7%, 15.5%, and 20.8% based on the in vitro production of ,-lactamase by L. lactis. The oral administration of 504 mU and 1008 mU ,-lactamase free solution resulted in 30 and 47 mU absorbed, a bioavailability of 5.9% and 4.7%, respectively. L. lactis significantly (p,<,0.01) increased the oral bioavailability compared to the free solution form. A significant (p,<,0.01) increase in the MAT value as compared to the solution, demonstrated that L. lactis can be used as a sustained delivery system. In conclusion, there is a linear relationship between L. lactis dose and these absorption PK parameters within L. lactis dose range of the current study. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2573,2580, 2009 [source] Pretransplantation tumor necrosis factor-, production predicts acute rejection after liver transplantationLIVER TRANSPLANTATION, Issue 6 2000Andrew J. Bathgate Immunosuppressive therapy has many adverse effects in both the short and longer term. Tailoring immunosuppression might be possible if pretransplantation parameters predicted rejection. We investigated production of the proinflammatory cytokine, tumor necrosis factor-, (TNF-,), and the anti-inflammatory cytokine, interleukin-10 (IL-10), pretransplantation to determine whether there is a relation with acute rejection. Peripheral-blood mononuclear cells were obtained from patients with chronic liver disease on the waiting list for orthotopic liver transplantation and healthy controls. Cells (0.5 × 106) were stimulated with 200 ng of lipopolysaccharide. Preincubation for 30 minutes with tacrolimus, cyclosporine, and dexamethasone at concentrations of 10 and 100 ng was also performed. TNF-, and IL-10 levels were measured by enzyme-linked immunosorbent assay. Acute rejection was defined on clinical and histological grounds. Pretransplantation in vitro production of TNF-, significantly (P < .05) increased in the group of patients with acute rejection (n = 9) compared with those who did not develop rejection (n = 12). Preincubation with dexamethasone significantly (P < .001) reduced TNF-, and IL-10 production in both patients and controls (n = 8). IL-10 production pretransplantation was not different in those who developed acute rejection (n = 9) compared with those who did not (n = 9). Preincubation with tacrolimus augmented (P < .05) the production of IL-10 in patients (n = 18), but not controls (n = 6). Pretransplantation TNF-, production is increased in patients who go on to develop acute rejection posttransplantion. [source] The effects of selected probiotic strains on the development of eczema (the PandA study)ALLERGY, Issue 9 2009L. Niers Background:, Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to prevent allergic disease. We aimed to study primary prevention of allergic disease in high-risk children by pre- and postnatal supplementation of selected probiotic bacteria. Methods:, In a double-blind, randomized, placebo-controlled trial, a mixture of probiotic bacteria selected by in-vitro experiments (Bifidobacterium bifidum, Bifidobacterium lactis, and Lactococcus lactis; Ecologic® Panda) was prenatally administered to mothers of high-risk children (i.e. positive family history of allergic disease) and to their offspring for the first 12 months of life. Results:, Parental-reported eczema during the first 3 months of life was significantly lower in the intervention group compared with placebo, 6/50 vs 15/52 (P = 0.035). After 3 months, the incidence of eczema was similar in both groups. Cumulative incidence of parental-reported eczema at 1 and 2 years was 23/50 (intervention) vs 31/48 (placebo) and 27 (intervention) vs 34 (placebo), respectively. The number needed to treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis. Furthermore, at age 3 months, in vitro production of IL-5 (146 pg/ml vs 72 pg/ml; P = 0.04) was decreased in the probiotic-group compared with the placebo-group. Conclusions:, This particular combination of probiotic bacteria shows a preventive effect on the incidence of eczema in high-risk children, which seems to be sustained during the first 2 years of life. In addition to previous studies, the preventive effect appears to be established within the first 3 months of life. [source] Several signaling pathways are involved in the control of cattle oocyte maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Céline Vigneron Abstract The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment. Mol. Reprod. Dev. 69: 466,474, 2004. © 2004 Wiley-Liss, Inc. [source] Expression of peroxiredoxins in bovine oocytes and embryos produced in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2004Gregory Leyens Abstract Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine-tuning peroxide levels in embryos during in vitro production. In this study, RT-PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up-regulated after maturation. Single embryos were analyzed from the two-cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9- to 16-cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9- to 16-cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 243,251, 2004. © 2004 Wiley-Liss, Inc. [source] Protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relativesPEDIATRIC DIABETES, Issue 5 2007Zuzana Vrabelova Background:, Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis. Objectives:, The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. Subjects:, Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. Methods:, PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247,279, 509,528, and 524,543; proinsulin a.a. 9,23; and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853,872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-,, tumor necrosis factor ,, transforming growth factor ,1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. Results:, Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-, (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. Conclusion:, By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy. [source] Thymoquinone supplementation attenuates hypertension and renal damage in nitric oxide deficient hypertensive ratsPHYTOTHERAPY RESEARCH, Issue 5 2007Mahmoud M. Khattab Abstract The present study was undertaken to evaluate the protective effect of thymoquinone (TQ), the main constituent of the volatile oil from Nigellasativa seeds, in rats after chronic inhibition of nitric oxide synthesis with N, -nitro- l -arginine methyl esters (l -NAME). Rats were divided randomly into different treatment groups: control, l -NAME, TQ and l -NAME + TQ. Hypertension was induced by 4 weeks administration of l -NAME (50 mg/kg/day p.o.). TQ was administered alone or in combination with l -NAME and continued for 4 weeks. The animals were killed, and the serum and kidney tissues were isolated for the determination of creatinine and glutathione (GSH), respectively. Rats receiving l -NAME showed a progressive increase in systolic blood pressure compared with control rats. Concomitant treatment with TQ (0.5 and 1 mg/kg/day p.o.) reduced the increase in systolic blood pressure induced by l -NAME in a dose dependent manner. Kidney injury was demonstrated by a significant increase in serum creatinine and a decrease in GSH in kidney tissue from l -NAME treated rats. Treatment of rats with TQ decreased the elevated creatinine and increased GSH to normal levels. TQ inhibited the in vitro production of superoxide radical in enzymatic and non-enzymatic systems. In conclusion, TQ is effective in protecting rats against l -NAME-induced hypertension and renal damage possibly via antioxidant activity. Copyright © 2007 John Wiley & Sons, Ltd. [source] Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the importance of xylanase and polygalacturonasePLANT PATHOLOGY, Issue 1 2007M.-N. Douaiher Eight Mycosphaerella graminicola isolates were investigated for correlations between pathogenicity and the in vitro production of cell wall-degrading enzymes. Isolate pathogenicity was evaluated in terms of lesion and production of pycnidia in wheat leaves. Additionally, the isolates were compared over time for their ability to produce in vitro significant levels of xylanase (EC 3·2·1·8), ,-xylosidase (EC 3·2·1·37), ,-1,3-glucanase (EC 3·2·1·6), cellulose (EC 3·2·1·4) and polygalacturonase (EC 3·2·1·15) activities when grown in a liquid medium. Correlation tests and principal component analysis revealed a significant correlation between the in vitro production of xylanase and pectinase and pathogenicity components. Xylanase was correlated to necrosis frequency (r = 0·795), ,-xylosidase was correlated to the mean of the lesion length (r = ,0·787), whereas polygalacturonase was correlated to the time when 50% of the leaves contained a lesion (r = 0·776), the lesion frequency (r = 0·646) and the time when 50% of the leaves showed pycnidia (r = ,0·711). The results suggest that these two groups of cell wall-degrading enzymes are therefore likely to be key determinants of pathogenicity in M. graminicola. [source] Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA MicroarrayREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010OM Kandil Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source] Pregnancy-Specific Glycoproteins Function as Immunomodulators by Inducing Secretion of IL-10, IL-6 and TGF-,1 by Human MonocytesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2001SARA K. SNYDER PROBLEM: Low levels of pregnancy-specific glycoproteins (PSGs) in maternal serum have been correlated with complications of pregnancy. We investigated the ability of human PSGs to regulate in vitro production of cytokines. METHOD OF STUDY: Human monocytes and murine RAW 264.7 cells were treated with recombinant PSG1, PSG6, PSG11, or a truncated PSG6 consisting of only the N-terminal domain (PSG6N). Cytokine production in response to PSG-treatment was measured by ELISA and/or reverse transcriptase-PCR. RESULTS: All PSGs tested induced secretion of interleukin (IL)-10, IL-6 and transforming growth factor (TGF)-,1 by both human and murine cells, but not IL-1,, tumor necrosis factor (TNF)-, or IL-12. The N-terminal domain of PSG6 was sufficient for induction of monocyte cytokine secretion. Induction of IL-10 and IL-6 was preceded by an increase in the specific mRNAs. CONCLUSIONS: PSG1, PSG6, PSG6N, and PSG11 induce dose-dependent secretion of anti-inflammatory cytokines by human monocytes. Human and murine PSGs exhibit cross-species activity. Our results are consistent with a role for PSGs in modulation of the innate immune system. [source] Male and Female Effects on the In Vitro Production of Bovine EmbryosANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 5 2004G. A. Palma Summary A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3,93.4%) and blastocysts on day 7 (6.9,51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F -value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme. [source] Outside-to-inside signaling through transmembrane tumor necrosis factor reverses pathologic interleukin-1, production and deficient apoptosis of rheumatoid arthritis monocytesARTHRITIS & RHEUMATISM, Issue 9 2009Undine Meusch Objective Monocytes are a major source of proinflammatory cytokines in rheumatoid arthritis (RA), and inhibitors of monocytic cytokines are highly efficient agents for treatment of the disease. The aim of this study was to analyze the effects of a therapeutic anti,tumor necrosis factor , (anti-TNF,) antibody on monocytes from patients with RA and healthy control subjects. Methods Peripheral blood monocytes from patients with RA and healthy control subjects were incubated in the presence of anti-TNF, antibody or IgG. Annexin V staining, caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA staining with propidium iodide were used to analyze apoptosis. The signaling events elicited in monocytes by infliximab were analyzed by Western blotting and electromobility shift assay. Results Peripheral blood monocytes from patients with RA were characterized by increased expression of transmembrane TNF,, spontaneous in vitro production of interleukin-1, (IL-1,), and a decreased rate of spontaneous ex vivo apoptosis. Incubation with infliximab induced significantly increased apoptosis in monocytes from patients with RA but not in monocytes from healthy control subjects. This apoptosis was triggered by reverse signaling of transmembrane TNF after ligation by infliximab and was independent of caspase activation. Instead, transmembrane TNF reverse signaling inhibited the constitutive NF-,B activation in RA monocytes, suppressed IL-1, secretion, and normalized spontaneous in vitro apoptosis. This normalization was reversible by the addition of exogenous IL-1,. Conclusion This study demonstrates that outside-to-inside signaling through transmembrane TNF after ligation by infliximab inhibits constitutive NF-,B activation and suppresses spontaneous IL-1, production by monocytes from patients with RA. Besides the induction of monocyte apoptosis, this inhibition could also contribute to the therapeutic effects observed during treatment with TNF, inhibitors. [source] | ||||||||||||||||||||||