Identification of a Promiscuous T-Cell Epitope in Mycobacterium tuberculosis Mce Proteins

The characterization of Mycobacterium tuberculosis antigens inducing CD4(+) T-cell responses could critically contribute to the development of subunit vaccines for M. tuberculosis. Here we performed computational analysis by using T-cell epitope prediction software (known as TEPITOPE) to predict promiscuous HLA-DR ligands in the products of the mce genes of M. tuberculosis. The analysis of the proliferative responses of CD4(+) T cells from patients with pulmonary tuberculosis to selected peptides displaying promiscuous binding to HLA-DR in vitro led us to the identification of a peptide that induced proliferation of CD4(+) cells from 50% of the tested subjects. This study demonstrates that a systematic computational approach can be used to identify T-cell epitopes in proteins expressed by an intracellular pathogen.     

Sequence distribution of poly(ether-sulfone)/poly(ether-ketone) copolymers by mass spectrometry and 13C NMR

The sequence distributions of four poly(ether-sulfone)/poly(ether-ketone) (PES/PEK) copolymer samples were determined by means of fast atom bombardment mass spectrometry (FAB-MS) and by ¹³C NMR spectroscopy. The controlled partial degradation of the copolymer chains to produce oligomers suitable of FAB-MS analysis was performed with sodium methoxide in dimethyl sulfoxide solution at 130°C, and the experimental conditions were optimized. The sequential arrangements of ether-sulfone/ether-ketone units present in these materials were estimated by a best-fit minimization method using the MACO4 computer program which compares the experimental FAB-MS spectral intensities with theoretical intensities. Random PES/PEK copolymer samples showed quite the same sequence arrangements as expected from monomer feed-ratios used in the syntheses. Instead, a PES/PEK copolymer sample expected to be exactly alternating (from the synthesis procedure) showed 44% of random sequences owing to the transetherification reaction which occurred in the synthesis. The results of sequential analysis obtained from ¹³C NMR data compare well with the data obtained by the FAB-MS analysis.     

No association between the Pro197Leu polymorphism in the glutathione peroxidase (GPX1) gene and schizophrenia

OBJECTIVE: Oxidative stress such as free radical-mediated neuronal dysfunction may be involved in the pathophysiology of schizophrenia. The human glutathione peroxidase (GPX1) is a selenium-dependent enzyme, which plays an important role in the detoxification of free radicals. We therefore hypothesized that the GPX1 gene, which is located on chromosome 3p21.3, may be involved in the pathophysiology of schizophrenia. The aim of this study is to examine whether a potentially functional polymorphism, a proline (Pro) to leucine (Leu) substitution at codon 197 (Pro197Leu) of the human GPX1 gene, is associated with susceptibility to schizophrenia. METHODS: We genotyped the Pro197Leu polymorphism in a total of 113 nuclear families that had a proband with schizophrenia. Genetic association was tested using the transmission disequilibrium test (TDT), the sib transmission disequilibrium test (STDT), and the family-based association test (FBAT). RESULTS: The minor allele (Leu) frequency was calculated to be 0.282. We could not find significant transmission disequilibrium of the alleles for the Pro197Leu polymorphism in the GPX1 gene in association with the presence of schizophrenia in our family sample (TDT, chi2=0.03, degrees of freedom=1, P=0.86; combined TDT-STDT, Z'=-0.052, P=0.47; FBAT, Z=0.000, P=1.000). CONCLUSION: The results of this study suggest that the GPX1 polymorphism is unlikely to be associated with susceptibility to schizophrenia.     

The enzyme responsible for the respiratory burst in elicited guinea pig peritoneal macrophages

The enzymatic basis of the respiratory burst induced by phorbol myristate acetate in elicited peritoneal macrophages of the guinea-pig has been studied. The following evidence suggests that a membrane-bound oxidase that preferentially uses NADPH as substrate is the main enzyme responsible for activation of the oxidative metabolism: (1) The supernatant of postnuclear fractions of resting macrophages oxidises NADH and NADPH with formation of O. The activity with both substrates is very low and does not change in the supernatant obtained from activated cells. (2) The cell-free particles of resting macrophages also oxidise both NADH and NADPH with formation of O. The activity of the cell-free particles from activated macrophages does not change when NADH is the substrate. By contrast, the activity of the cell-free particles from activated cells is markedly increased when NADPH is the substrate. (3) In cell-free particles from activated macrophages the K_m for NADPH is about one order of magnitude lower than that for NADH and the V_max with NADPH is double that with NADH. (4) The NADPH oxidase of cell-free particles is insensitive to azide, cyanide, antimycin A and rotenone and is sensitive to the sulphydryl reagent PCMB. All these drugs have the same effect on the respiratory response of intact macrophages. (5) A direct correlation is found between the degree of activation of the respiratory metabolism of intact macrophages and the extent of activation of the NADPH oxidase. A new approach designed to measure the activity of the oxidase soon after the activation of the enzyme has taken place, shows that the NADPH oxidase can account for the respiratory burst of intact macrophages.     

Depolarization-induced retrograde synaptic inhibition in the mouse cerebellar cortex is mediated by 2-arachidonoylglycerol

Endocannabinoids acting on CB₁ cannabinoid receptors are involved in short- and long-term depression of synaptic transmission. The aim of the present study was to determine which endocannabinoid, anandamide or 2-arachidonoylglycerol (2-AG), is involved in depolarization-induced suppression of inhibition (DSI) in the cerebellar cortex, which is the most widely studied form of short-term depression. Depolarization of Purkinje cells in the mouse cerebellum led to an increase in intracellular calcium concentration and to suppression of the inhibitory input to these neurons (i.e. DSI occurred). Orlistat and RHC80267, two blockers of sn -1-diacylglycerol lipase, the enzyme catalysing 2-AG formation, abolished DSI by acting downstream of calcium influx. In contrast, DSI occurred also in the presence of a phospholipase C inhibitor. Intact operation of the calcium-dependent messengers calmodulin and Ca²⁺–calmodulin-dependent protein kinase II were necessary for DSI. DSI was potentiated by an inhibitor of the main 2-AG-degrading enzyme, monoacylglycerol lipase. Interference with the anandamide metabolizing enzyme, fatty acid amide hydrolase, did not modify DSI. Thus, three kinds of observations identified 2-AG as the endocannabinoid involved in DSI in the mouse cerebellum: DSI was abolished by diacylglycerol lipase inhibitors; DSI was potentiated by a monoglyceride lipase inhibitor; and DSI was not changed by an inhibitor of fatty acid amide hydrolase. Further experiments indicated that 2-AG is the endocannabinoid mediating short-term retrograde signalling also at other synapses: orlistat abolished DSI in the rat cerebellum, DSI in the mouse substantia nigra pars reticulata and depolarization-induced suppression of excitation in the mouse cerebellum.     

Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells

Murine plasmacytoid dendritic cells (pDCs) have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) function and mediate immunosuppression in specific settings; yet, the conditions of spontaneous versus induced activity have remained unclear. We have used maneuvers known to up-regulate IDO in different cell types and have examined the relative efficacy and mechanisms of the induced activity in splenic pDCs, namely, after specific receptor engagement by CTLA-4-Ig, CD200-Ig or CD28-Ig, the latter in combination with silenced expression of the suppressor of cytokine signaling 3 (SOCS3) gene. We found that pDCs (CD11c+ mPDCA-1+ 120G8+) do not express IDO and are not tolerogenic under basal conditions. B7-1 engagement by CTLA-4-Ig, CD200R1 engagement by CD200-Ig and B7-1/B7-2 engagement by CD28-Ig in SOCS3-deficient pDCs were each capable of initiating IDO-dependent tolerance via different mechanisms. IFN-gamma was the major cytokine responsible for CTLA-4-Ig effects, and type I IFNs for those of CD200-Ig. Immunosuppression by CD28-Ig in the absence of SOCS3 required IFN-gamma induction and IFN-like actions of IL-6. Therefore, although pDCs do not mediate IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by pDCs in the mouse.     

A B7-1-transfected human melanoma line stimulates proliferation and cytotoxicity of autologous and allogeneic lymphocytes

B7 co-stimulation is necessary to activate resting T cells upon antigen recognition by the T cell receptor. To see whether expression of B7 may render human melanoma cells able to stimulate T cells, a cloned melanoma line (Me1B6), which did not express B7-1, was transfected with the human B7-1 gene. In proliferation assays, B7-1 transfected cells (Me1B6/B7) showed greater stimulatory activity of allogeneic and autologous peripheral blood lymphocytes (PBL) compared to parental, non-transfected tumor cells. This effect was also seen when allogeneic CD8⁺ and CD4⁺ subpopulations were used as effectors. In these studies, activation of lymphocytes was B7-1-dependent and HLA classes I and II mediated. The higher proliferation correlated with an increased lytic activity by PBL stimulated with B7-1⁺ tumor cells against the untransfected Me1B6. Furthermore, PBL from a metastatic melanoma patient stimulated by Me1B6/B7 developed an higher lytic activity not only against Me1B6 but also against their autologous, B7-1^? tumor. Finally, after Me1B6/B7 stimulation, PBL released interleukin (IL)-2 and interferon-γ, but not IL-4, suggesting a Th1-mediated response. These data support the use of B7-1 transfected melanoma cells in the therapeutic vaccination of melanoma patients.     

Early increase precedes a depletion of VIP and PGP-9.5 in the skin of insulin-dependent diabetics—correlation between quantitative immunohistochemistry and clinical assessment of peripheral neuropathy

Diabetic neuropathy affects both sensory and autonomic peripheral nerve fibres. Vasoactive intestinal polypeptide (VIP) is present in autonomic fibres which modulate sweat secretion, while calcitonin gene-related peptide (CGRP) is localized to cutaneous sensory fibres. In this study, immunohistochemistry and image analysis were used to assess changes of VIP and CGRP, and of the pan-neuronal marker protein gene-product (PGP)-9.5, in skin biopsies of 18 patients affected by type 1 diabetes (age range 18–46 years) and from seven aged-matched controls. Patients were divided into three groups: group 1 (n=6), with diabetes for 6 months to 3 years; group 2 (n=5), with the disease for 5–10 years; and group 3 (n=7), with diabetes for more than 10 years. VIP immunoreactivity (IR) and PGP-9.5-IR were significantly reduced around sweat glands (P <0.005) in groups 2 and 3. Epidermal CGRP-IR and PGP-9.5-IR were significantly reduced in group 3 (P <0.05). Twenty-eight per cent (5/18) of all patients showed high VIP-IR around sweat glands (>95 per cent confidence limits of controls) and all of these patients had diabetes for less than 3 years. Conversely, 55 per cent (10/18) of patients had low VIP-IR (<5 per cent confidence limit of controls). The latter, compared with the former, showed a significantly longer duration of diabetes (Fisher exact test P=0·002), presence of clinical autonomic neuropathy (Fisher exact test P=0.04), and a reduced sural nerve conduction velocity (Fisher exact test P=0.04). These results suggest that quantitative immunohistochemical analysis of peptide-containing cutaneous nerves allows an objective evaluation of nerve fibre alterations at early stages of diabetes than is currently possible with neurophysiological functional tests.     

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.