ISOLATION, CHARACTERIZATION AND IMMUNOCHEMICAL STUDIES ON FIBROUS PROTEINS FROM COWRY SHELL (CYPRAEA MONETA, LINNAEUS).

BACKGROUND: Biomaterials are non-drug substances used to treat, enhance or replace functions of body tissues or organs. Natural sources of biomaterials have recently become the focus of several research activities. Cowry shell constitutes one of the most promising natural sources of biomaterials because of its chemical stability, biodegradability and biocompatibility in the body. However, its applications may be limited due to immunogenic and toxic responses that may occur following implantation, hence this study. MATERIALS AND METHODS: Crude fibrous protein extracted with citrate buffer from pulverised cowry shells (Cypraea moneta (L)), was resolved into two components (CSP1 and CSP2) by gel filtration. Immunological studies were performed with antisera obtained from rabbits by double immunodiffusion and immunoelectrophoresis techniques. Mice treated with the proteins were observed for signs of toxicity and their liver, kidney, lungs and spleen were processed histologically. RESULTS: The native molecular weight of CSP1 and CSP2 determined by gel filtration were 91kDa and 33kDa respectively. CSP1 and CSP2 displayed single bands on SDS-PAGE with subunit molecular weight values of 19kDa and 19.5kDa respectively. Antisera obtained from rabbits immunised with the crude citrate buffer extracts precipitated the antigen in double immunodiffusion tests. Histopathological examinations revealed a dose-dependent damaging effect of the shell proteins on liver, kidney, lung and spleen tissues of the treated mice. CONCLUSION: This study showed that cowry shells contain fibrous proteins which are immunogenic and toxic in mice at relatively high concentrations, causing visible organ damage without concurrent physical manifestations.

Preliminary report of HLA (DNA) typing of Nigerians using sequence-specific primer technique.

AIMS AND OBJECTIVES: This communication is an attempt to present the experience and a preliminary report of results over a one-year period. PATIENTS AND METHODS: From December 2011 to December 2012, a prospective determination of the HLA types of 20 individuals referred to the Tissue Typing Laboratory of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife was done. These consisted of prospective transplant recipients, their donors, and a migrant pair for kinship determination. DNA was extracted from the client's peripheral blood sample, using the QIAmp Blood DNA Mini kit, (Qiagen). PCR was done using OlerupR low-resolution PCR-SSP typing kit. The PCR product was resolved in 2% agarose gel, and the bands visualised under UV light. The HLA types were determined using provided tables and/or Helmberg software. Data were presented using descriptive statistics whileHLA antigen frequency (AF) was expressed in percentage and gene frequency (GF) was determined using square root method (1-(1-AF)1/2). RESULTS: A total of 20 individuals (13males and 7females) consisting of seven renal transplant recipients and seven prospective donors; a stem cell recipient and three donors and a migrant pair for kinship determination were typed. Age ranged from 4-65 years. 44 HLA alleles were detected, while HLA-A, B, C, DRB1 and DQB1 were 7, 10, 11, 8, 8 alleles respectively. The alleles were heterogeneous in distribution while 6 antigens (HLA-A*02, B*30, C*15, DRB1*03, DRB1*08 and DQB1*06) were having frequencies e"25%. CONCLUSION: This report confirms that DNA-based HLA typing is feasible locally, andit was observed that renal transplantation procedure is the most frequent indication. The HLA antigens observed to have very high frequencies (e"25% frequency) in this population were HLA-A*02, B*30, C*15, DRB1*03, DRB1*08 and DQB1*06. There is a strong need to develop a broad-based HLA data bank for Nigeria to further strengthening her transplantation programmes.

On the possible function of Telfairia occidentalis agglutinin in the plant.

The fate of Telfairia occidentalis seed Agglutinin, TOA, has been monitored during germination. While the level of the Agglutinin in the cotyledons decreased sharply in the first three days to about half of the initial level, it stabilises at this level for the following twelve days. In this interval, Agglutinin activity becomes manifest in the radicle on the fourth day, peaking on the fifth and decreasing rapidly thereafter. In the plumule, the lectin activity becomes manifest on the sixth day, peaks on the seventh and decreases rapidly thereafter. No lectin activity is detectable in any plant tissue including the rump of the cotyledons twenty-seven days after germination. The implications of these observations on the possible role of lectins in plants are discussed.

Activation of CD8+ T lymphocytes in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease directed against the insulin-secreting beta cells of the islets of Langerhans of the pancreas. We have previously shown that in organ-specific autoimmune diseases, Graves' disease (GD), and IDDM, the antigen that is specific for each of these disorders (i.e., TSH receptor for GD, glutamic acid decarboxylase-65 (GAD65) for IDDM) does not activate the disease-specific CD8+ cells as fully as CD8+ cells from normal persons. In order to identify the specific antigen responsible for triggering or maintaining autoimmunity in patients afflicted with the disease, we have studied the effects of islet (beta) cell-specific antigens GAD65, insulin, pancreatic antigen (P69), T cell epitope 69 (Tep69), and a milk-derived bovine serum albumin (BSA)-peptide-ABBOS (pre-BSA positions 157-169) on the activation of CD8+ T lymphocytes in IDDM patients. We compared the patterns of T cells activation with those mediated by an irrelevant peptide antigen, P348 (amino-terminal region of human cardiac myosin light chain-1), and also tetanus toxoid. We also studied the responses of CD8+ T lymphocytes to these IDDM-relevant and -irrelevant antigens in Hashimoto's thyroiditis patients (HT), rheumatoid arthritis patients (RA), and normal control subjects (N) to compare the pattern of responses in the other autoimmune diseases. Activation of lymphocytes was monitored by measuring the expression of the activation molecule-major histocompatibility complex class II antigen (HLA-DR) on the surfaces of CD8+ T lymphocytes by flow cytometry. Peripheral blood mononuclear cells (PBMC) obtained from 14 patients with IDDM, 14 N, 14 with HT, and 13 with RA were cultured for 7 days in the presense or absence of antigens. The stimulation index (SI) of activation of the lymphocytes was determined. When the response of CD8+ T lymphocytes of IDDM patients to each of the IDDM-relevant antigens was compared to that of the irrelevant antigen, only GAD65 and ABBOS showed a significantly reduced activation compared to P348 and tetanus toxoid. Other relevant antigens, insulin, P69, and Tep69, did not show any significant differences in their SI compared to those of the irrelevant antigens. In the N, HT, and RA groups, there was no significant difference in the SI of the responses of CD8+ cells to any of the relevant antigens compared to that of the irrelevant antigens. Moreover, CD8+ T lymphocytes of IDDM patients showed a significantly lower activation by GAD65 than those from N, HT, and RA. In conclusion, our data suggest that CD8+ T lymphocytes of IDDM patients but not those from N, HT, and RA groups have specifically reduced potential for activation in response to GAD65 but not to insulin, P69, and Tep69, whereas ABBOS exerts a less well-defined reductive effect on the activation of CD8+ lymphocytes of IDDM patients. Since CD8+ cells have been shown to contain suppressor activity, our data support the notion that a disease-specific defect in GAD65 autoantigenic induction of suppressor T lymphocytes may be important in the pathogenesis of IDDM.

Characterization of isolectins in Tetracarpidium conophorum seeds (Nigerian walnut).

A lectin preparation obtained from Tetracarpidium conophorum (Nigerian walnut) by affinity chromatography of seed extracts on lactose-agarose has been shown to contain two components by gel filtration on Sephadex G150. The larger component Tetracarpidium conophorum agglutinin I (TCAI) is a disulphide-bonded 70 kDa homodimer whereas the second component TCAII is a 34 kDa monomeric protein. Amino terminal aminoacid sequencing shows identity in TCAI and TCAII for the first fifteen residues after which the sequences diverge. The N-terminal sequences of TCAI and TCAII show identity with sequences in the B-chains of ricin and Ricinus communis agglutinin I (RCAI) in eleven of the initial fifteen residues. Thereafter TCAI appears to be homologous to the ricin B chain whereas TCAII is more homologous with the B chain of RCAI. A limited screening of the carbohydrate-binding specificity of TCAII by affinity chromatography of defined oligosaccharides on TCAII Sepharose columns shows that the binding specificity reported earlier for affinity purified Tetracarpidium conophorum isolectins (Sato S, Animashaun T, Hughes RC (1991) J Biol Chem 266:11485-94) reflects the binding properties of TCAII which is the major isolectin in unfractionated lectin preparations.

Some factors affecting the quantitation of rheumatoid factors by enzyme immunoassay.

Some factors affecting the use of enzyme-labelled antibody to human IgM or enzyme-labelled human IgG for the detection and assay of IgM RFs by enzyme immunoassay have been investigated. IgM RF levels, obtained by standard radioimmunoassay techniques, showed a better agreement with those obtained with enzyme-labelled antibody than with enzyme-labelled human IgG. Immuno-adsorption studies using a sheep cell agglutination test (SCAT) confirmed a partial loss of antigenicity of the IgG molecules due to glutaraldehyde treatment, which increased with the length of time of exposure to glutaraldehyde.