Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine. Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes. Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody. Conclusions: These results suggest that the murine and human PIF receptors are identical. © 2014 Cancer Research UK.

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxygen free radicals denature human IgG and increase its reactivity with rheumatoid factor antibody

Rheumatoid inflammation is characterised by the production of rheumatoid factor antibodies directed against denatured IgG. Oxygen free radicals have the potential to denature all manner of proteins and can be generated by activated phagocytic cells in the inflamed joint. By modifying routine ELISA and nephelometric procedures for measuring rheumatoid factor, (i.e. substituting free radical altered IgG for rabbit and heat aggregated IgG as antigens) we have observed that oxygen radicals, generated by (1) UV light and (2) PMA-activated neutrophils, give rise to monomeric and polymeric forms of IgG which have increased reactivity towards IgM and IgA polyclonal rheumatoid factor antibodies. We conclude that free radical alteration of IgG may be a stimulus to the formation of immune complexes with rheumatoid factor antibody, thereby promoting and amplifying tissue damage during rheumatoid inflammation.

Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries

Background: Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. The existence of single or multiple VEGF isoforms and receptors suggests that these proteins may have overlapping but distinct functions, which may be reflected in their cell expression and distribution. Methods: The localisation of VEGFs A–C and their receptors (VEGFRs 1–3, respectively) in 30 fresh human atherosclerotic arteries, 15 normal uterine arteries, and 15 saphenous veins using immunohistochemistry and western blotting. Results: Saphenous veins showed no staining for VEGF-B or VEGFR-2. Smooth muscle cells (SMCs) showed the strongest staining for VEGF-A, VEGF-B, VEGFR-1, and VEGFR-2 in all specimens. Conversely, VEGFR-3 and VEGF-C were predominately localised to the endothelial vasa vasorum in normal arteries, whereas medial SMCs showed the strongest staining in atherosclerotic arteries. Western blotting showed variations in VEGF protein localisation, with lower amounts of VEGF-B and VEGF-C in saphenous veins, compared with arterial tissue. Amounts of VEGF-C were lower than those of VEGF-A and VEGF-B in all specimens. Conclusion: This study provides direct evidence of the presence of VEGF proteins and receptors in human physiology and pathology, with variations in both the amounts of VEGF proteins expressed and their cellular distribution in normal arteries compared with atherosclerotic arteries. The presence of VEGFs A–C and their receptors in normal arterial tissue implies that VEGF functions may extend beyond endothelial cell proliferation. Reduced VEGFR-2 staining in atherosclerotic arteries may have implications for the atherosclerosis process and the development of vascular disease and its complications.

Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts:a possible defense against transforming growth factor-β mediated fibrosis

BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.

METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.

CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Autonomous growth of BALB/MK heratinocytes transfected with a retroviral vector carrying the human epidermal growth factor gene

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+ T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.