The Heat Shock Protein Gp96 - The Immune System's Swiss Army Knife

Das Hitzeschockprotein Glycoprotein-96 (Gp96, Grp94) und andere Hitzeschockproteine (HSP) sind als wirkungsvolle Tumor-Vakzine im Tiermodell beschrieben worden und werden zur Zeit in klinischen Studien untersucht. Der durch HSP vermittelten Immunantwort liegen Peptide zugrunde, die von den HSP intrazellulär während des Prozesses der Antigenprozessierung gebunden werden. In dieser Dissertation werden mehrere neue Eigenschaften von Gp96 beschrieben, die die eindrucksvolle Fähigkeit dieses Moleküls aufzeigen, angeborene und adaptive Immunantworten zusammenzuführen. Erstens wird gezeigt, daß Gp96 an Rezeptoren der Zelloberfläche von antigenpräsentierenden Zellen (APC) bindet. Rezeptorvermittelte Endocytose führt zur Kreuzpräsentation (cross-presentation) von Gp96-gebundenen Peptiden auf Major Histocompatibility Complex (MHC) Klasse-I Moleküle für die Aktivierung von cytotoxischen T-Lymphocyten. Zweitens wird gezeigt, daß Gp96 dendritische Zellen (DC), die nach gängiger Auffassung wichtigsten APC, aktivieren kann, was zu einer Steigerung der immunstimulatorischen Fähigkeiten der DCs führt. An dieser Aktivierung sind die Toll-like Rezeptoren 2 und 4 beteiligt, die zuvor ausschließlich als Rezeptoren zur Verteidigung gegen Mikroorganismen bekannt waren. Basierend auf diesen Beobachtungen schlagen die Autoren dieser Arbeit folgendes Modell vor: Gp96 wird von nekrotischen (z.B. virusinfizierten) Zellen freigesetzt und von umgebenden APC durch rezeptor-vermittelte Endocytose effektiv aufgenommen und kreuzpräsentiert. Gleichzeitig werden die APC über Toll-like Rezeptoren aktiviert. Beides zusammen führt zu einer effizienten Aktivierung von naiven T-Zellen und zum Auslösen einer effektiven Immunantwort.The heat shock protein Glycoprotein-96 (also known as Gp96, Grp94) and other heat shock proteins (HSPs) have been described as potent tumor vaccines in animal models and are currently studied in clinical trials. The underlying immune response relies on immunogenic peptides that the HSPs have acquired intracellularly by interfering with the classical antigen processing pathways. In this dissertation several novel functions of Gp96 are described revealing the powerful nature of Gp96 to bring together innate and adaptive immune responses. First, Gp96 binds to cell surface receptors on antigen-presenting cells (APCs). Receptor-mediated endocytosis leads to the cross-presentation of Gp96-bound peptides on Major Histocompatiblitiy Complex (MHC) class I molecules for activation of cytotoxic T cells. Second, Gp96 can activate dendritic cells (DCs), considered as most potent APCs, to enhance their immunstimulatory capacities. This activation of APCs by Gp96 is facilitated via Toll-like receptors 2 and 4, so far known for their role in defense against microorganisms. Based on these observations the authors of this work propose that HSPs may act as ideal antigen carriers: Gp96 is released from necrotic cells (e.g. that have been infected by virus) leading to cross-priming of the Gp96-attached peptides by neighbouring APCs. Simultaneously, these APCs are activated by Gp96 via Toll-like receptors. Both, cross-presentation and activation of APCs leads to efficient priming of naïve T cells and initiates an effective immune response

Variations in Line Quality of Handwritten Strokes Due to the Photocopying Process- A Preliminary Study

An examination of the line quality of handwritten strokes plays an important role in the detection of forgery. This paper deals with the study of morphological variations in line quality of multi-generation photocopied handwriting (up-to the fifth generation of reproduction) produced by thirty-four different writing instruments. The purpose of the study is to find out the extent to which such line quality features are dependent on the nature of the writing instrument used to prepare the original, as well as the possibility of their survival (or distortion) and, consequently, their detection in the multi-generation photocopies. The overall effect of writing instrument on morphology is seen and felt in varying degrees in photocopier reproductions of all the five generations. Pen characteristics, such as striations, ink gooping, pen skips, ink bleeding/ feathering, nib marks and lead particle deposition have caused a significant difference in the morphology of stroke’s line in photocopy. The effect is much more pronounced in some of the features in photocopies beyond the second generation. There are other features, such as ink feathering and pen skips which could not be reproduced with sufficient clarity in any photocopy generation

Bacteriological profile of acute bacterial meningitis at a tertiary care hospital of North India

Background: Acute bacterial meningitis (ABM) is one of the most severe and potentially life-threatening infectious diseases. It is defined as an inflammation of the meanings, globally distributed as either sporadic or epidemic forms. ABM remains a major cause of mortality and long-term neurological sequel worldwide. Objective of the present study was undertaken to evaluate the bacteriological analysis in term of pathogens frequency and their sensitivity pattern in the cerebrospinal fluid of acute meningitis patients at a tertiary care hospital in eastern Uttar Pradesh, India.Methods: The study was carried out at a tertiary care hospital from June 2014 to November 2015 irrespective of age group. A total of 3803 samples of cerebrospinal fluid (CSF) from clinically suspected cases of meningitis were subjected for bacteriological analysis.Results: During the study period, a total of 3803 CSF samples were studied. Out of these, 343 were confirmed as bacterial meningitis based on Gram staining and or culture showing 9.01% incidence. ABM was more common in paediatric patients than adults. The most common organisms were Gram positive (66.18%) bacteria.Conclusions: Acute bacterial meningitis is a medical emergency and making an early diagnosis and providing early and accurate treatment, are lifesaving and to reduce morbidity. This study may play an important role in the diagnosis and more accurate treatment for the ABM patients

Targeted therapy in renal cell carcinoma: moving from molecular agents to specific immunotherapy

Non-specific immunotherapy has been for a long time a standard treatment option for patients with metastatic renal cell carcinoma but was redeemed by specific targeted molecular therapies, namely the VEGF and mTOR inhibitors. After moving treatment for mRCC to specific molecular agents with a well-defined mode of action, immunotherapy still needs this further development to increase its accuracy. Nowadays, an evolution from a rather non-specific cytokine treatment to sophisticated targeted approaches in specific immunotherapy led to a re-launch of immunotherapy in clinical studies. Recent steps in the development of immunotherapy strategies are discussed in this review with a special focus on peptide vaccination which aims at a tumor targeting by specific T lymphocytes. In addition, different combinatory strategies with immunomodulating agents like cyclophosphamide or sunitinib are outlined, and the effects of immune checkpoint modulators as anti-CTLA-4 or PD-1 antibodies are discussed

Identification of the N-terminal Peptide Binding Site of Glucose-regulated Protein 94

Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, we now locate this binding site by testing predictions of a molecular docking model. The best predicted site was on the opposite face of the β sheet from the pan-HSP90 radicicol-binding pocket, in close proximity to a deep hydrophobic pocket. The peptide and radicicol-binding sites are distinct, as shown by the ability of a radicicol-refractive mutant to bind peptide. When the fluorophore acrylodan is attached to Cys(117)within the hydrophobic pocket, its fluorescence is reduced upon peptide binding, consistent with proximity of the two ligands. Substitution of His(125), which contacts the bound peptide, compromises peptide-binding activity. We conclude that peptide binds to the concave face of the β sheet of the N-terminal domain, where binding is regulated during the action cycle of the chaperone

Perforin Is Required for Innate and Adaptive Immunity Induced by Heat Shock Protein Gp96

Tumor-secreted gp96-Ig is highly immunogenic and triggers CD8 T cell-mediated tumor rejection. In vivo secreted gp96-Ig and gp96-myc cause NK activation and clonal expansion of specific CD8+ CTL in wild-type and in Fas-ligand-deficient (gld) mice but not in perforin- (PKO) or IFN-γ-deficient (GKO) mice. Transfer of perforin-competent NK cells restores the ability of PKO mice to clonally expand CD8 CTL in response to gp96-Ig. The data demonstrate an essential role for perforin-mediated functions in the activation of innate and adaptive immunity by heat shock protein gp96-peptide complexes. Crosspresentation of antigens by heat shock proteins seems to require a perforin-dependent positive feedback loop between NK and DC for both sustained NK activation and clonal CTL expansion. The studies also explain how depressed NK activity in patients with tumors or after viral infections could diminish CTL responses

The mechanisms of androgen effects on body composition: mesenchymal pluripotent cell as the target of androgen action

Testosterone supplementation increases muscle mass primarily by inducing muscle fiber hypertrophy; however, the mechanisms by which testosterone exerts its anabolic effects on the muscle are poorly understood. The prevalent view is that testosterone improves net muscle protein balance by stimulating muscle protein synthesis, decreasing muscle protein degradation, and improving the reutilization of amino acids. However, the muscle protein synthesis hypothesis does not adequately explain testosterone-induced changes in fat mass, myonuclear number, and satellite cell number. We postulate that testosterone promotes the commitment of pluripotent stem cells into the myogenic lineage and inhibits their differentiation into the adipogenic lineage. The hypothesis that the primary site of androgen action is the pluripotent stem cell provides a unifying explanation for the observed reciprocal effects of testosterone on muscle and fat mass

IL-36α induces maturation of Th1-inducing human MDDC and synergises with IFN-γ to induce high surface expression of CD14 and CD11c

We show that IL-36α induced maturation of human MDDCs and stimulated differentiation of IFN-γ producing (Type 1) CD3+ lymphocytes but was not as effective as IL-36β in doing so. For the first time, we also show that IL-36α induced expression of CD14 by MDDCs and this was highly potentiated by co-cultured with IFN-γ. In contrast, lipopolysaccharide (LPS) did not increase CD14 expression by MDDCs, suggesting that if MDDCs represent a physiologically relevant population in vivo, they need to be stimulated by relevant inflammatory cytokines prior to CD14 expression and detection of LPS, expressed by Gram negative bacteria. IFN-γ synergised with IL-36α to restore the high levels of CD11c expression by MDDCs, which was reduced by culture with these cytokines in isolation. IL-36α/IFN-γ synergy also correlated with increased binding of the opsonic complement protein (iC3b) to MDDCs. However although IL-36α increased the phagocytic capacity of MDDCs for Salmonella Typhimurium 4/74 this was not synergistically increased by IFN-γ (P > 0.05). In conclusion we report the hitherto unknown effects of IL-36α on the innate cell function of human MDDCs