Joanne M. Manns

Amelioration of inflammation, angiogenesis and CTGF expression in an arthritis model by a TSP1-derived peptide treatment.

Objective: To evaluate the effect of a thrombospondin 1 (TSP1)‐derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan‐polysaccharide (PG‐PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1‐derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT‐PCR. Results: Histological data indicated that the TSP1‐derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1‐derived peptide treatment. Also, immunofluorescence and RT‐PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1‐derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1‐derived peptide treated animals. Moreover, macrophage/monocyte‐specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1‐derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1‐derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. J. Cell. Physiol. 211: 504–512, 2007.

An antifungal compound from Solanum nigrescens

The antifungal activity of Solanum nigrescens extracts has been traced to the presence of a spirostanol glycoside, cantalasaponin-3.

Thrombospondin-1 and transforming growth factor beta are pro-inflammatory molecules in rheumatoid arthritis

Thrombospondin-1 (TSP1/THBS1) plays a major role in the pathophysiology of rheumatoid arthritis (RA); however, its interface with the cytokine network involved in RA has not been delineated. Correlations were performed between plasma levels of TSP1 and selected cytokines from blood samples collected from 20 patients affected by RA and 13 healthy donors (control). Plasma levels of TSP1 and tissue growth factor beta (TGFbeta) were determined by standard enzyme-linked immunosorbent assay, and cytokines were measured by protein profiling rolling-circle amplification (RCA). TSP1 circulating levels in plasma were found significantly increased in the RA patients when compared with control individuals (P = 0.039). The plasma levels of TGFbeta were also increased in the RA patients, which indicates a statistical trend. Cytokine levels of interleukin (IL)-4, IL-5, IL-12, chemokine CXC 10 (CXCL10/IP10), and chemokine CC 4 (CCL4)/MIP1beta were significantly increased in the RA patients when compared with the control group. In summary, this study demonstrates increased plasma levels of TSP1, which correlated with increased levels of proinflammatory cytokines in plasma of RA patients. More detailed research is required to explore the cytokine imprint yielded by this study and its interface with TSP1 and TGFbeta.

SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE) of Proteins

Electrophoresis is a method by which a complex mixture of proteins can be separated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) is a technique used to move charged molecules through a gel matrix by means of an electric current. This procedure is used to determine protein subunit composition, verify homogeneity of the protein sample, and purify proteins for use in other applications. The migration rate of the proteins during SDS‐PAGE is determined by the pore size of the gel matrix and charge, size, and shape of the protein. In this unit, the protocol covers the casting of gels, preparation of the protein samples, staining and drying of the gels, and calculation of molecular mass of the proteins based on electrophoretic mobility. Curr. Protoc. Microbiol. 22:A.3M.1‐A.3M.13.

Assembly of the prothrombinase complex on the surface of human foreskin fibroblasts: Implications for connective tissue growth factor

Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its auto-activation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7±0.8nM) and thrombin (7.9±0.04 U/mL×10-3) generation. Addition of TSP1 significantly enhanced thrombin (23.3±0.7 U/mL×10-3) but not FXa (8.5±0.6nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as shown by phosphorylation of the SMAD pathway, an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa- and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI. In summary, assembly of the prothrombinase complex occurs on fibroblasts surface leading to serine proteases generation, an event enhanced by TSP1 and associated with CTGF/CCN2 upregulation. These mechanisms may play an important role in human diseases associated with fibrosis.

Immunotherapy in a hypercholesterolemic rabbit model of Alzheimer's disease

acid sequence of beta amyloid (Abeta), which is close to identical to the human sequence. Also, rabbits have an extensively characterized profile on a measure of learning and memory, classical eye blink conditioning, that closely parallels human performance and that is impaired in AD. In two separate studies, we examined the effect of various durations of the cholesterol/ trace copper diet on the development of intracellular Abeta, extracellular Abeta plaques, eye blink conditioning, and the integrity of the blood brain barrier (BBB).Methods: For intracellular brain Abeta and Abeta plaque determination, the research design is shown in Table 1. Rabbits received the cholesterol copper diet in periods varying from 2 to 12 weeks. Immunohistochemistry was used to evaluate brain pathology. EBCC was tested. For BBB studies, rabbits were tested 4 or 10 weeks after cholesterol/trace copper diet onset. Changes in BBB permeability were assessed using the fluorescent tracer, sodium-fluorescein. Results: Intracellular brain Abeta increased with disease progression as shown in Figure 1. In spite of the developing neuropathology, EBCC was not different in rabbits exposed longer to the diet. There was a progression of impairment in the BBB over the 10week course of diet administration, with a 1.6 fold increase in permeability at 4 weeks and over a 4-fold increase in permeability at 10 weeks. Conclusions: Brain pathology increases with longer time on the cholesterol/trace copper diet, and the BBB is impaired in AD model rabbits as it is impaired in human AD. Cognitive changes reflecting the increases of Abeta pathology and BBB impairment were not reflected in EBCC performance.