Changes in cerebral endothelial barrier antigen, without alteration of permeability for intravenously injected leptin in diet-induced obesity in rats

Leptin, a potent anorectic, 16-kDa, adipose tissue-derived protein, predominantly acts in hypothalamic nuclei, signaling obesity and modulating ingestive behavior. To reach this brain area, leptin, probably has to cross the blood-brain barrier (BBB). In some cases of obesity, enhanced leptin levels in the blood do not result in anorectic effects, probably due to an altered leptin transport across the BBB. Therefore, we investigated the BBB in lean and diet-induced obese Lewis rats. To obtain information about the presence of microvessels with barrier dysfunction we examined three brain areas (hypothalamus, cortex, hippocampus) using a monoclonal antibody which detects intact microvessels of the BBB (anti-endothelial barrier antigen, anti-EBA). The results showed a significantly reduced EBA staining in the brain sections of the obese animals, except the hippocampus, compared to the control group. In a second step we injected I125-labeled leptin intravenously (i.v.) in permanent i.v.-cannulated, unrestrained Lewis rats (lean and obese). We measured the radioactivity in the cerebrospinal fluid after puncture of the cisterna magna, in the blood and brain tissue 90 min after injection. The leptin content in the cerebrospinal fluid and brain was not reduced in obese compared to lean rats, thus showing a similar transport capacity of the BBB in both experimental groups. Therefore, the results of the in vivo investigations do not indicate an impairment of the BBB in diet-induced obesity, despite the immunohistological findings. Further functional and morphological studies are necessary to evaluate the specific role of other organs and distinct forms of leptin (free and protein-bound) in the pathogenesis of diet-induced obesity.     

Failure of BCL-2 up-regulation in proximal tubular epithelial cells of donor kidney biopsy specimens is associated with apoptosis and delayed graft function

SUMMARY: In renal transplantation, postischemic acute renal failure (ARF) develops in more than 20% of patients. We investigated whether tubular epithelial cells obtained from donor kidneys without subsequent ARF express a different pattern of survival genes, compared with cells from kidneys exhibiting ARF. Donor kidney biopsy specimens were obtained before transplantation from eight recipients of cadaveric kidneys with primary graft function (CAD-PF), eight patients with biopsy-proven ARF without rejection (CAD-ARF), and eight recipients of living donor kidneys with primary graft function (LIV). One thousand proximal tubular epithelial cells per biopsy specimen were isolated by laser capture microdissection. Quantitative analysis of apoptosis and the apoptosis regulatory genes Bcl-2, Bcl-xL, and Bax were performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling staining and real-time PCR, respectively. Primary cultures of human proximal tubular epithelial cells served as calibrator. The number of apoptotic cells was significantly higher in CAD-ARF compared with LIV and CAD-PF (1.5 +/- 1.1% [p < 0.05] vs. 0.3 +/- 0.2% vs. 0.4 +/- 0.2%; mean +/- SD). The apoptosis inhibitors Bcl-2 and Bcl-xL were significantly up-regulated in renal tubular cells of recipients without ARF compared with CAD-ARF. The ratios of Bcl-2/GAPDH normalized to calibrator were as follows: LIV 48 +/- 30, CAD-PF 38 +/- 55, and CAD-ARF 5 +/- 7 (p < 0.05). The corresponding ratios for Bcl-xL were as follows: LIV 6 +/- 6, CAD-PF 5 +/- 3, and CAD-ARF 1 +/- 1 (p < 0.05). No difference in the expression of the proapoptotic Bax could be observed. These data suggest that failure of proximal tubular cells to respond to injury by up-regulation of survival factors from the Bcl-2 family contributes to postischemic ARF in patients after cadaveric renal transplantation.     

House dust mite-specific T cells in the skin of subjects with atopic dermatitis: frequency and lymphokine profile in the allergen patch test.

The mechanism underlying positive patch tests with house dust mite-allergen, Dermatophagoides pteronyssinus (Der p), in patients with atopic dermatitis was investigated by isolating T cells from the test sites of two patients. Eighty-five T cell clones (TCC) were established from the epidermis and dermis of lesional skin by the limiting-dilution method with Der p and interleukin (IL)-2. With restimulation assays, 29 of 60 TCCs tested demonstrated specific proliferation; 85% were of the CD3+, CD2+, and CD4+ phenotype. Der p-specific T cells constituted 0.4% to 2.7% of lesional T cells, and they were more frequent in the skin than in the blood of the patients by one order of magnitude. The mitogen-stimulated lymphokine profile of 55 TCCs was assessed; 42% (11/26) of the allergen-specific TCCs secreted IL-4 but almost no interferon-gamma, as described for the Th2 subset of the mouse. Also, six selected TCCs supported IgE secretion by autologous lymphocytes. Only three of 26 allergen-specific, skin-derived TCCs demonstrated a Th1-like lymphokine profile. These results support the specific nature of Der p-induced patch test lesions in patients with atopic dermatitis, and the results demonstrate also that a considerable proportion of lesional T cells are allergen-specific, IL-4-producing T cells that are capable of enhancing IgE production.     

Immune response against the T-independent antigen alpha (1 leads to 3) dextran. I. Demonstration of an unexpected IgG response of athymic and germ-free-raised euthymic BALB/c mice

The primary antibody response in BALB/c mice to the T-independent bacterial antigen dextran B1355S [alpha(1 leads to 3)dextran] (Dex) was studied by means of isoelectric focusing, hemagglutination and immunodiffusion techniques. In response to a single immunization with 10 micrograms Dex all mice produce specific IgM antibodies. In addition, about 30% of conventionally raised BALB/c and BALB/c nu/ + mice, but 95% of germ-free (GF)-raised normal BALB/c and 100% of athymic BALB/c nu/nu mice produce specific IgG class anti-Dex antibodies. These antibodies include all IgG subclasses, carry predominantly the lambda light chain and the cross-reactive J558 idiotype and are specific for the alpha(1 leads to 3)glucosidic linkage. As compared to athymic and GF-raised mice, conventionally raised mice exhibit only a weak IgG response. The pronounced IgG production of GF-raised mice was not altered when adult mice were removed from their GF environment and housed under conventional conditions for several weeks prior to immunization with Dex. Reconstitution with isolated splenic T cells from conventionally raised, unprimed BALB/c mice reduces the remarkable capacity of BALB/c nu/nu mice to produce IgG anti-Dex antibodies. These findings suggest that the reduced capacity of conventionally raised BALB/c mice to mount an IgG response to the T-independent antigen Dex is due to a T cell-mediated suppressive mechanism which is neonatally induced by contact with environmental, i.e. bacterial, antigens.     

Contribution of coronary endothelial cells to cardiac adenosine production

Experiments were performed in isolated non-working guinea pig hearts perfused according to the Langendorff technique (95% O₂, 5% CO₂), to evaluate the relative contribution of the coronary endothelium to the formation of cardiac adenosine during hypoxia, hypercapnia, and acetylcholine infusion. For this purpose the adenine-nucleotides of the coronary endothelium were prelabeled by perfusion of isolated hearts with³H-adenosine (10^–8 M) for 35 min. Changes in the relative specific radio-activity (RSA) of adenosine released into the coronary effluent perfusate were used to assess changes in the relative contribution of the coronary endothelium and cardiomyocytes to total cardiac adenosine release. Hypoxic perfusion (15% O₂) doubled coronary flow and increased total adenosine release by about two orders of magnitude and in addition, substantially increased the release of³H-adenosine. The RSA of adenosine, however, was consistently depressed. During hypercapnic acidosis (9% CO₂) the increase in coronary flow was associated with only a small and transient rise in cardiac adenosine release, and did not influence the formation of³H-adenosine. In the unpaced heart, acetylcholine (10^–7 and 2×10^–6 M) dose-dependently increased coronary flow and the release of both adenosine and³H-adenosine. Within the first minute, the RSA of adenosine was increased, but thereafter was decreased relative to control. In the paced heart, the effects of acetylcholine (2×10^–6 M) were greatly attenuated. Increasing coronary flow by bradykinin and isosorbide dinitrate or decreasing heart rate by (–)N₆-phenylisopropyl-adenosine did not significantly affect effluent perfusate concentration of adenosine or its RSA. Our findings suggest that coronary endothelium in vivo can contribute to increased cardiac adenosine release in response to hypoxia and acetylcholine but not following hypercapnic acidosis. In addition, the consistent decrease in RSA of adenosine suggests a proportionally greater increase in adenosine release from cardiomyocytes.A preliminary report of part of this work appeared in Pflügers Arch (1984) 402:R19 [Suppl]. This work was supported by the Deutsche Forschungsgemeinschaft SFB 30, Kardiologie Düsseldorf     

The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

Enhancement of death-receptor induced caspase-8-activation in the death-inducing signalling complex by uncoupling of oxidative phosphorylation

Signalling through the death receptor CD95 induces apoptosis by formation of a signalling complex at the cell membrane and subsequent caspase-8 and caspase-3-activation. Treatment of Jurkat T cells with protonophores across the mitochondrial membrane such as 2,4-dinitrophenol (DNP) enhances the death-inducing capacity of CD95. In this study, we show that this enhancement is due to the specific acceleration of caspase-8-processing and activation at the CD95-receptor. DNP-treatment did not affect NF-kappaB-induction by CD95. Immunoprecipitation experiments showed that the amounts of the adapter FADD/MORT1 and pro-caspase-8 at the CD95-receptor were not altered by DNP. Subcellular fractionation studies revealed that the amount of mature caspase-8 but not pro-caspase at the membrane was increased following CD95-stimulation in the presence of DNP. As a consequence of caspase-activation, c-FLIP-levels in the cytosol decreased. In Jurkat cells overexpressing c-FLIPS, DNP was still able to enhance caspase-activation. The enhancing capacity of DNP was seen in some cell lines (Jurkat, CEM and HeLa) but not in SKW6 cells and was also found in mitogen-stimulated human T cells. Furthermore, the enhancement extended to TRAIL-induced caspase-activation. Thus, a mechanism exists by which caspase-8-activation can be accelerated at death receptors and this mechanism can be triggered by targeting mitochondrial oxidative phosphorylation.     

Interferon Induction in Mice by Lipopolysaccharide from Brucella abortus

Endotoxin or lipopolysaccharide isolated from Brucella abortus (strain 456) produced a typical endotoxin-type interferon response in mice with peak levels of the inhibitor occurring 2 h after intravenous injection of the stimuli. Brucella lipopolysaccharide was a much less effective interferon stimulus than the lipopolysaccharide isolated from Salmonella typhimurium (strain LT-2).     

Evaluation of the Determine Rapid Syphilis TP assay using sera

The Abbott Determine Rapid Syphilis TP assay is a treponemal test that can be used in resource-poor settings that lack laboratory facilities. However, this test has not been extensively evaluated. We measured its sensitivity and specificity by using stored serum specimens (n = 567) from all persons who tested Treponema pallidum hemagglutination assay (TPHA) positive (n = 250) or TPHA indeterminate (n = 17) in the year 2001 and the first 300 patients in 2001 who tested TPHA negative at the Evandro Chagas Research Institute in Rio de Janeiro, Brazil. This rapid assay was independently interpreted by three different observers. With TPHA results as the reference, sensitivity ranged between readers from 95.6 to 98.4% and specificity ranged from 97.3 to 95.7%. There was little interreader variability in the interpretation of results, with approximately 98% agreement for all reader combinations. Of samples from persons with human immunodeficiency virus (HIV) infection (n = 198), sensitivity was 96.9 to 99.2% and it was 94.4 to 96.3% among HIV-negative persons (n = 127). Specificity was 92.4 to 95.5% among HIV-positive persons and 97.2 to 100% among HIV-negative persons. We found this test to have high sensitivity and specificity and little interreader variability, indicating that it may be easily used in resource-poor settings without laboratory facilities. Further studies are needed using this test on whole blood and under the clinical conditions for which it is intended.