Identification of a new group-specific determinant on hepatitis B surface antigen with a synthetic peptide.

In a recent study we demonstrated that a synthetic peptide representing residues 124-147 of the major protein of hepatitis B surface Ag (HBsAg) undergoes spontaneous oligomerization to reconstruct one or more conformational group-specific determinants on HBsAg. The present study was undertaken to identify and characterize the HBsAg-related antigenic determinants on this oligomeric peptide (peptide OS[124-147]). A panel of nine analogs of this peptide was generated by either deleting, substituting, or chemical side chain modification of specific amino acid residues. With HBsAg subtype-specific antisera a single "a" epitope was identified as one that includes Met133 and Lys141. In addition a "d" epitope toward the amino-terminal end of the sequence was also observed. Perturbation of certain amino acid residues was found to enhance a antigenicity and subsequent experiments indicated that maximal expression of this a antigenicity is dependent in part on accessibility of the Lys141 side chain and in part on the primary sequence. With a total of 50 human anti-HBsAg serum samples obtained from individuals vaccinated against hepatitis B, it was demonstrated that these sera recognize the Met133-Lys141-dependent a epitope as the dominant, and in many cases the only, determinant on peptide OS[124-147]. Finally, on immunization, peptide OS[124-147] elicits an anti-HBsAg response that is predominantly anti-a though a lesser contribution from an anti-d response was also obtained.     

Primary multilobated T-cell lymphoma of the breast diagnosed by fine needle aspiration cytology and immunocytochemistry

The fine needle aspiration (FNA) cytologic, immunocytochemical and ultrastructural findings of a primary multilobated T-cell lymphoma arising in the breast of a 61-year-old woman are described. Large pleomorphic multilobated malignant cells were primarily identified as lymphomatous in origin and phenotypically as T-cells by a selected panel of monoclonal antibodies applied to the original smears obtained by FNA biopsy. This appears to be the second report of a multilobated lymphoma arising in the breast and the first with a T-cell phenotype in this anatomic site.     

The primary antibody repertoire represents a linked network of degenerate antigen specificities

In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Importantly, it also ensures that the primary response is composed of Abs with a high degree of "evolvability."     

Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in RA patients with anti-collagen antibodies

IntroductionRheumatoid arthritis (RA) patients with autoantibodies against collagen type II (CII) are characterized by acute RA onset with elevated inflammatory measures and early joint erosions as well as increased production of tumor necrosis factor-α (ΤΝF-α) by peripheral blood mononuclear cells (PBMC) stimulated by anti-CII immune complexes (IC) in vitro. Polymorphonuclear granulocytes (PMN) are abundant in RA synovial fluids, where they might interact directly with anti-CII IC in the articular cartilage, but no studies have investigated PMN responses towards anti-CII IC. The aim was to investigate whether PMN react towards anti-CII IC, and to what extent such reactivity might relate to the clinical acute onset RA phenotype associated with elevated levels of anti-CII.MethodsPMN and PBMC isolated from healthy donors were stimulated with IC made with a set of 72 baseline patient sera (24 anti-CII positive, 48 anti-CII negative) chosen from a clinically well-characterized RA cohort with two-year radiological follow-up with Larsen scoring. PMN expression of cluster of differentiation (CD)11b, CD66b, CD16 and CD32 was measured by flow cytometry, whereas PMN production of myeloperoxidase (MPO) and interleukin (IL)-17, and PBMC production of ΤΝF-α was measured with enzyme linked immunosorbent assay.ResultsPMN expression of CD11b, CD66b and MPO, and PBMC production of ΤΝF-α were upregulated whereas PMN expression of CD16 and CD32 were downregulated by anti-CII IC. CD16, CD66b, and MPO production correlated to serum anti-CII levels (Spearman’s ρ = 0.315, 0.675 and 0.253, respectively). CD16 was associated with early joint erosions (P = 0.024, 0.034, 0.046 at baseline, one and two years) and CD66b was associated with changes in joint erosions (P = 0.017 and 0.016, at one and two years compared to baseline, respectively). CD66b was associated with baseline C-reactive protein and PBMC production of ΤΝF-α was associated with baseline erythrocyte sedimentation rate, in accordance with our earlier findings. No clinical associations were observed for MPO or IL-17.ConclusionPMN responses against anti-CII IC are more closely associated with early joint erosions than are PBMC cytokine responses. PMN reactivity against anti-CII IC may contribute to joint destruction in newly diagnosed RA patients with high levels of anti-CII.     

Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells

The studies on protein–dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA–TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH–HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH–HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH–HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.     

Insulin Receptor Substrates Are Essential for the Bioenergetic and Hypertrophic Response of the Heart to Exercise Training

Insulin and insulin-like growth factor 1 (IGF-1) receptor signaling pathways differentially modulate cardiac growth under resting conditions and following exercise training. These effects are mediated by insulin receptor substrate 1 (IRS1) and IRS2, which also differentially regulate resting cardiac mass. To determine the role of IRS isoforms in mediating the hypertrophic and metabolic adaptations of the heart to exercise training, we subjected mice with cardiomyocyte-specific deletion of either IRS1 (CIRS1 knockout [CIRS1KO] mice) or IRS2 (CIRS2KO mice) to swim training. CIRS1KO hearts were reduced in size under basal conditions, whereas CIRS2KO hearts exhibited hypertrophy. Following exercise swim training in CIRS1KO and CIRS2KO hearts, the hypertrophic response was equivalently attenuated, phosphoinositol 3-kinase (PI3K) activation was blunted, and prohypertrophic signaling intermediates, such as Akt and glycogen synthase kinase 3β (GSK3β), were dephosphorylated potentially on the basis of reduced Janus kinase-mediated inhibition of protein phosphatase 2a (PP2A). Exercise training increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein content, mitochondrial capacity, fatty acid oxidation, and glycogen synthesis in wild-type (WT) controls but not in IRS1- and IRS2-deficient hearts. PGC-1α protein content remained unchanged in CIRS1KO but decreased in CIRS2KO hearts. These results indicate that although IRS isoforms play divergent roles in the developmental regulation of cardiac size, these isoforms exhibit nonredundant roles in mediating the hypertrophic and metabolic response of the heart to exercise.     

Pathogenicity of Mycobacterium tuberculosis Is Expressed by Regulating Metabolic Thresholds of the Host Macrophage

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.     

Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase

Anomalous action of human acetylcholinesterase (hAChE) in Alzheimer’s disease (AD) was restrained by various AChE inhibitors, of which the specific and potent lead candidate Donepezil is used for treating the disease AD. Besides the specificity, the observed undesirable side effects caused by Donepezil invoked the quest for new lead molecules with the increased potency and specificity for AChE. The present study elucidates the potency of six 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives to form a specific interaction with the peripheral anionic site and catalytic anionic subsite residues of hAChE. The NMSMs were prepared in good yield from 1,1-di(methylsulfanyl)-2-nitroethylene and primary amine (or) amino acid esters. In silico interaction analysis reveals specific and potent interactions between hAChE and selected ligand molecules. The site-specific interactions formed between these molecules also results in a conformational change in the orientation of active site residues of hAChE, which prevents them from being accessed by beta-amyloid protein (Aβ), which is a causative agent for amyloid plaque formation and acetylcholine (ACh). In silico interaction analysis between the ligand-bounded hAChE with Aß and ACh confirms this observation. The variation in the conformation of hAChE associated with the decreased ability of Aβ and ACh to access the respective functional residues of hAChE induced by the novel NMSMs favors their selection for in vivo analysis to present themselves as new members of hAChE inhibitors.     

Fine needle aspiration cytology and immunocytochemical characterization of the histiocytes in sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman syndrome)

A fine needle aspiration (FNA) biopsy of an enlarged lymph node in a three-year-old boy who had asymptomatic cervical and submandibular lymphadenopathy showed large histiocytes with abundant pale, eosinophilic cytoplasm containing well-preserved lymphocytes and occasional plasma cells and granulocytes. These features were consistent with a diagnosis of sinus histiocytosis with massive lymphadenopathy (SHML). Immunophenotypic study of the histiocytes, performed on the FNA smears and on paraffin-embedded sections, showed reactivity for S-100 protein and alpha-1-antichymotrypsin and negativity for lysozyme. These features, which are characteristic of SHML, demonstrate the reliability of FNA cytology in making the diagnosis of this disorder.