Sepsis, one of the primary causes of mortality in the intensive care units, occurs due to the host’s dysregulated immune responses to an infection. Consequently, persistent systemic inflammation along with suppressed adaptive immunity ensues, resulting in deranged metabolism, recurrent infections, tissue damage and multi-organ failure. The uncontrolled oxidative stress mediated by the imbalance between the generation of reactive oxygen species and their neutralization by the host’s antioxidant system is involved in inflammation-induced damage. The profound deleterious effects in the host range from mitochondrial dysfunction and endothelial damage to reduced cardiac output. Therefore, antioxidant therapy was actively considered to have therapeutic benefits in sepsis patients. Although some success has been obtained with the use of antioxidants in sepsis patients, considerable ambiguity persists that prevents their routine use. Another key molecule that may dictate the outcome and prognosis during sepsis is nitric oxide (NO). This pleiotropic molecule plays a central role in inflammation and in leukocyte recruitment at the site of inflammation. NO is synthesized by three different isoforms of nitric oxide synthases (NOS) and significantly high and sustained levels of NOS2 have been reported in sepsis. Abundant literature supports the protective roles of NO during sepsis; however, there is uncertainty in various reports. The administration of NO donors in clinical trials for sepsis treatment has encountered limited success. NO, during sepsis, acts like a double-edged sword: increased NO levels can result in hypotension, whereas reduced levels contribute to poor organ perfusion and an elevated susceptibility to infection. Therefore, several parameters need to be evaluated, while considering the potential of antioxidant and NO-based therapy during sepsis.

The thymus is essential for T cell development and maturation. It is extremely sensitive to atrophy, wherein loss in cellularity of the thymus and/or disruption of the thymic architecture occur. This may lead to lower naïve T cell output and limited TCR diversity. Thymic atrophy is often associated with ageing. What is less appreciated is that proper functioning of the thymus is critical for reduction in morbidity and mortality associated with various clinical conditions including infections and transplantation. Therefore, therapeutic interventions which possess thymopoietic potential and lower thymic atrophy are required. These treatments enhance thymic output, which is a vital factor in generating favourable outcomes in clinical conditions. In this review, experimental studies on thymic atrophy in rodents and clinical cases where the thymus atrophies are discussed. In addition, mechanisms leading to thymic atrophy during ageing as well as during various stress conditions are reviewed. Therapies such as zinc supplementation, IL7 administration, leptin treatment, keratinocyte growth factor administration and sex steroid ablation during thymic atrophy involving experiments in animals and various clinical scenarios are reviewed. Interventions that have been used across different scenarios to reduce the extent of thymic atrophy and enhance its output are discussed. This review aims to speculate on the roles of combination therapies, which by acting additively or synergistically may further alleviate thymic atrophy and boost its function, thereby strengthening cellular T cell responses.

BACKGROUND:Leptospirosis, a zoonosis, is a re-emerging disease, affecting populations across the globe. However, the current methods of diagnosis are time- consuming, cumbersome, imprecise or expensive.AIM:To develop an assay for differential and early diagnosis of Leptospirosis.METHODS AND MATERIAL:IgG based ELISA for evaluation of three antigens, namely, a gel-purified recombinant protein (rLipL32), secreted proteins and whole organism sonicates of Leptospira spp. The antigens were evaluated using, rabbit polyclonal antiserum and human sera samples.RESULTS:Studies with a rabbit polyclonal antiserum indicated the utility of these antigens in differentiating Leptospira from other common pathogenic organisms. Evaluation of these antigens with fifteen representative human serum samples indicated gel-purified rLipL32 to be a potentially useful antigen for detection of leptospirosis. The results obtained with IgG ELISA were correlated with the results of microscopic agglutination test (MAT).CONCLUSION:Gel-purified rLipL32 is a valuable antigen for early and accurate diagnosis of leptospirosis. Further evaluation of this assay in field conditions and larger sera samples will indicate its suitability in case of an epidemic.

Major histocompatibility complex encoded class I (MHC-I) molecules display peptides derived from endogenous proteins for perusal by CD8+ T lymphocytes. H6, a mouse hepatoma cell line, expresses low levels of surface H-2Dd but not H-2Kk. Surface H-2Dd molecules are unstable and their levels, but not H-2Kk, are induced at 22°C. Immunoprecipitation experiments revealed that H-2Kk, H-2Dd and β2-microglobulin (β2m) are expressed intracellularly; however no conformed MHC-I are present. Transcriptional profiling of factors required for MHC-I assembly demonstrated greatly reduced levels of the Transporter associated with antigen processing (Tap)2 subunit. The role of key assembly molecules in the MHC-I pathway was investigated by ectopic expression studies. Overexpression of β2m enhanced surface H-2Dd, but not H-2Kk, levels whereas overexpression of TAP2 rescued surface H-2Kk, but not H-2Dd, levels. Interestingly, Tapasin plays a dual role: first, in quality control by reducing the induced surface expression of TAP2-mediated H-2Kk and β2m-mediated H-2Dd levels. Secondly, Tapasin overexpression increases Tap2 transcripts and cooperates with TAPl or human β2m to enhance surface H-2Kk expression; this synergy is TAP-dependent as demonstrated by infected cell protein 47 (ICP47) inhibition studies. Unlike the well studied H-2 MHC-I alleles, H-2Kb, H-2Db, H-2Kd and H-2Dd, a functional TAP is “essential” for H-2Kk cell surface expression.

Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), a fluorogenic endopeptidase substrate, is used to detect 20 S proteasomal activity from Archaea to mammals. An o-phenanthroline-sensitive Suc-LLVY-AMC hydrolyzing activity was detected in Escherichia coli although it lacks 20 S proteasomes. We identified PepN, previously characterized as the sole alanine aminopeptidase in E. coli, to be responsible for the hydrolysis of Suc-LLVY-AMC. PepN is an aminoendopeptidase. First, extracts from an ethyl methanesulfonate-derived PepN mutant, 9218, did not cleave Suc-LLVY-AMC and L-Ala-para-nitroanilide (pNA). Second, biochemically purified PepN cleaves a wide variety of both aminopeptidase and endopeptidase substrates, and L-Ala-pNA is cleaved more efficiently than other substrates. Studies with bestatin, an aminopeptidase-specific inhibitor, suggest differences in the mechanisms of cleavage of aminopeptidase and endopeptidase substrates. Third, PepN hydrolyzes whole proteins, casein and albumin. Finally, an E. coli strain with a targeted deletion in PepN also lacks the ability to cleave Suc-LLVY-AMC and L-Ala-pNA, and expression of wild type PepN in this mutant rescues both activities. In addition, we identified a low molecular weight Suc-LLVY-AMC-cleaving peptidase in Mycobacterium smegmatis, a eubacteria harboring 20 S proteasomes, to be an aminopeptidase homologous to E. coli PepN, by mass spectrometry analysis. "Sequence-based homologues" of PepN include well characterized aminopeptidases, e.g. Tricorn interacting factors F2 and F3 in Archaea and puromycin-sensitive aminopeptidase in mammals. However, our results suggest that eubacterial PepN and its homologues displaying aminoendopeptidase activities may be "functionally similar" to enzymes important in downstream processing of proteins in the cytosol: Tricorn-F1-F2-F3 complex in Archaea and TPPII/Multicorn in eukaryotes.

The proteasome is a large multicatalytic proteinase that plays a role in the generation of peptides for presentation by major histocompatibility complex class I molecules. The 20S proteolytic core of mammalian proteasomes is assembled from a group of 17 protein subunits that generate a distinctive pattern of spots upon two-dimensional gel electrophoresis. The genes for most of these subunits have been cloned from humans and rats. We isolated cDNA clones for the mouse orthologues of ten of the subunits [PSMA1 (C2), PSMA2 (C3), PSMA3 (C8), PSMA4 (C9), PSMA5 (ZETA), PSMA6 (IOTA), PSMA7 (C6-I), PSMB2 (C7-I), PSMB3 (C10-II), and PSMB5 (X)] to complete the cloning of all of the mouse subunits. Using antisera raised against these subunits or their orthologues, we verified the identity of these proteins by two-dimensional NEPHGE-PAGE.

LMP2, LMP7, and MECL are interferon γ–inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous “immunoproteasomes” containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I–binding peptides.

CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain α3 domain. Substitution of a 15 amino acid portion of the H-2Db α3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) β2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26°C in the presence of excess peptide and β2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the α3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.

T lymphocytes, via specific T cell receptors (TCR), recognize antigenic peptides bound to major histocompatibility complex (MHC)-encoded molecules. The recent crystallization of TCR-MHC complexes has enriched our understanding of this recognition process (Garciaet al. 1996a; Garbocziet al. 1996). Broadly speaking, there are two types of MHC molecules, i.e., classical and nonclassical molecules, and the majority of TCR recognize classical MHC molecules. Classical MHC molecules can be further divided into two types, i.e., class I and class II. CD8+ T cells, or cytotoxic T lymphocytes (CTL), recognize MHC class I molecules bound to peptides derived from endogenous proteins (i.e., proteins that are synthesized within the cell or artificially introduced directly into the cytoplasm or nucleus) that are primarily degraded in the cytoplasm. The CD8 accessory molecule present on most CTL appears to augment the formation of TCR-MHC/peptide complexes (Garcia et al. 1996b; Gao et al. 1997). In addition to presenting peptides to T cells, expression of MHC class I molecules protects cells from lysis by natural killer (NK) cells (Lanier 1997). On the other hand, CD4+ T lymphocytes or helper T lymphocytes recognize MHC class II molecules that bind peptides derived from exogenous or membrane proteins which enter the cell via endocytosis or phagocytosis and are primarily degraded by lysosomal proteases.

We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.

LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit‐specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of α, precursor β and processed β subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of α and precursor β but no processed β subunits. These complexes possess short half‐lives, are enzymatically inactive and their molecular weight is ∼300 kDa. Radioactivity chases from these complexes into mature, long‐lived ∼700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing α subunits and the other, precursor β subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor β subunits PSMB2 (C7‐I), PSMB3 (C10‐II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the β subunits and juxtaposition of the two β rings) is accompanied by conformational changes in the (outer) α rings, and may be inefficient. Finally, interferon‐γ had no significant effect on the half‐lives or total amounts of precursor or mature proteasomes.

From the * William S. Rowe Division of Rheumatology, Children’s Hospital Medical Center, Cincinnati, Ohio 45229; ‡ Department of Molecular Genetics and the § Howard Hughes Medical Institute, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267; i Basel Institute for Immunology, CH-4005 Basel, Switzerland; and the ¶ Howard Hughes Medical Institute and Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

LMP-2 and LMP-7, gamma-interferon-inducible subunits of the 20S proteasome, play an important role in antigen processing. To define the molecular basis of their polymorphism, we sequenced Lmp-2 and Lmp-7 cDNA from nine different strains of mice. Three allelic variants of both LMP-2 and LMP-7 were found, but all of the polymorphism in LMP-7 is clustered near the carboxyl terminus of the molecule. We confirmed the nucleotide sequence changes at the protein level in both the unprocessed and processed forms of the molecules by analysis of specific anti-LMP-2, anti-LMP-7 and anti-proteasome immunoprecipitates on two-dimensional PAGE gels. Interestingly, a single amino acid change at position 272 between LMP-7b,d,q and LMP-7k,s,f,x,g7, cas4 from glycine to arginine dramatically affects its migration on SDS-PAGE gels, suggesting the possibility of allele-specific posttranslational modification.

We show that six proteasome-associated proteins are induced by IFN-gamma, corresponding to three proteasome beta-type subunits and their precursors: the MHC-linked subunits (LMP-2 and LMP-7) and LMP-10. Concurrently, incorporation of LMP-9, LMP-17, and LMP-19 into proteasomes is reduced. LMP-10 appears to be the product of a previously cloned proteasome subunit gene, MECL-1. MECL-1 transcription is increased in the presence of IFN-gamma, whereas the transcription of two other proteasome genes, Lmp-15 and Lmp-3, is not affected. The three IFN-gamma-inducible subunits and their constitutively expressed counterparts contain most or all of the catalytic sites of the proteasome. Independent assortment of LMP-2, LMP-7, and LMP-10 into different proteasome complexes may thus generate up to 36 unique proteasome subsets. This may increase the repertoire of potentially antigenic peptides for presentation by MHC class I.