Modified Dynabeads method for enumerating CD4+ T-lymphocyte count for widespread use in resource-limited situations

The Dynabeads method showed the potential for enumerating CD4 T lymphocytes (CD4 count) in HIV-1-infected individuals. The large volume of Dynabeads required for 1 sample and complex procedure made the method expensive and not easy for use, however. To decrease the cost and simplify the procedure, we reduced the volume of the Dynabeads, added wash times, and skipped over the staining step so as to count the CD4 cells directly under an optical microscope. The CD4 count of 246 blood samples using our modified Dynabeads method (DynabeadsCD4) showed a significant correlation with that obtained by flow cytometry (FlowcytoCD4) (r = 0.91 [P < 0.0001]; slope = 1.03, intercept = -16). The sensitivity and specificity for a CD4 count less than 200 cells/microL were 79% and 94%, and for a CD4 count less than 350 cells/microL, the sensitivity and specificity were 95% and 88%, respectively. The positive and negative predictive values for a CD4 count less than 350 cells/microL were 97% and 83%, respectively. The systematic error was 8 cells/microL (95% confidence interval [CI]: 0.4-16). The cost of Dynabeads for 1 sample was less than $1.00; thus, the estimated cost per DynabeadsCD4 test is less than $3.00, including the cost of other disposable materials. Our modified method is simple, economic, and accurate enough to monitor antiretroviral therapy in resource-limited situations.     

Low grade malignant peripheral nerve sheath tumour: varied cytological and histological patterns

BACKGROUND: A small number of malignant peripheral nerve sheath tumours (MPNSTs) are low grade, and the nature of these low grade tumours has never been systematically assessed. AIMS: To describe the clinicopathological, immunohistochemical, and ultrastructural features of low grade MPNST and to discuss the main differential diagnoses. METHODS: Four cases of low grade MPNST were studied, including one coexistent with neurofibromatosis type 1. The tumours were analysed with respect to nuclear atypia, cellularity, nuclear enlargement, hyperchromasia, mitotic rate, and necrosis. Immunohistochemistry was performed by standard techniques, and an ultrastructural study was performed on one tumour. RESULTS: The ages of the patients ranged from 32 to 72 years (mean, 58). Two were male and two were female. Three tumours occurred in the deep tissue, including one in the retroperitoneum, and one was located in the dermal and subcutaneous tissue. The maximum diameters of the tumours ranged from 3.5 to 8.0 cm. Microscopically, all tumours showed moderate hypercellularity, an increased nuclear to cytoplasmic ratio, and hyperchromasia, but exhibited varied growth patterns, including those that were atypical neurofibroma-like, low grade fibromyxoid sarcoma-like, low grade epithelioid, and haemangiopericytoma-like. All tumours showed immunoreactivity for S-100 protein and vimentin. CONCLUSIONS: These findings suggest that careful clinical and histological evaluation, along with S-100 protein immunostaining, are essential for the accurate diagnosis of low grade MPNST.     

Bcl11b is required for differentiation and survival of alphabeta T lymphocytes

The gene Bcl11b, which encodes zinc finger proteins, and its paralog, Bcl11a, are associated with immune-system malignancies. We have generated Bcl11b-deficient mice that show a block at the CD4-CD8- double-negative stage of thymocyte development without any impairment in cells of B- or gammadelta T cell lineages. The Bcl11b-/- thymocytes showed unsuccessful recombination of V(beta) to D(beta) and lacked the pre-T cell receptor (TCR) complex on the cell surface, owing to the absence of Tcrb mRNA expression. In addition, we saw profound apoptosis in the thymus of neonatal Bcl11b-/- mice. These results suggest that Bcl11b is a key regulator of both differentiation and survival during thymocyte development.     

Temperature-dependent Cu2+-Cu2+ paired structure in ethylene/methacrylic acid copolymer neutralized with Cu(II). Ionic crystallite disordering and polyethylene crystallite melting

Temperature-dependent ESR spectra of Cu²⁺-Cu²⁺ pairs in ethylene/methacrylic acid copolymer neutralized with Cu(II) were reexamined in detail. The resonance positions and the linewidths of one of the ESR fine-structure lines showed thermal distension of the Cu²⁺-Cu²⁺ distance, and the slopes in the temperature variations changed at the temperature associated with melting of the polymer crystallites. No meaningful anomalies were observed around the temperature at which the preceding endothermic transition takes place. In this transition, the Cu²⁺-Cu²⁺ pairs seems to enter a disordered state, keeping almost the same paired structure. In contrast to this irreversible order-disorder transition, the melting process in the most part of the polyethylene crystallite phases starts to impose stress upon the Cu²⁺-Cu²⁺ pairs, accompanying the slope changes of the ESR parameters. These reversible variations with remarkable thermal hysteresis are compatible with the DSC analyses.     

Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-kappaB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-kappaB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.     

Abnormal IgG galactosylation in MRL-lpr/lpr mice: pathogenic role in the development of arthritis

MRL-lpr/lpr (MRL/lpr) mice spontaneously develop arthritis by an increase in the incidence of agalactosylated oligosaccharides in serum IgG, similar to rheumatoid arthritis patients. However, whether this association has a pathogenic significance is still unknown. In this study, we analyzed the oligosaccharide structure of serum IgG in various MRL mice with or without arthritis, to clarify the relationship between the oligosaccharide abnormality and the development of arthritis. The level of agalactosylation in serum IgG was comparable in both arthritis-free MRL/lpr and MRL-+/+ (MRL/+) mice at 6 weeks of age. In contrast, the incidence of IgG lacking galactose markedly increased in MRL/lpr mice at 6 months of age (the age at which arthritis occurred), compared with that from age-matched MRL/+ mice without arthritis. However, the proportion of agalactosylated IgG increased similarly in anti-CD4 monoclonal antibody-treated MRL/lpr mice at 6 months of age, despite the absence of the development of arthritis, because of depletion of CD4+ T cells. These results suggest that the abnormality in IgG galactosylation of MRL/lpr mice developed in an age-dependent manner, but it did so independently of CD4+ T cell-dependent B-cell activation and is not a consequence of the development of arthritis.     

Specificity of co-promoting effects of caffeine on thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine

The specificity of copromotion effects of caffeine with known goitrogenic factors on thyroid carcinogenesis was examined in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Male F344 rats were divided into 8 groups, each consisting of 10 animals, and received a single sc injection of 2,800 mg/kg DHPN. From one week after the DHPN initiation, they were given basal diet, iodine deficiency (ID) diet, 500 ppm phenobarbital (PB) solution or 1,000 ppm sulfadimethoxine (SDM) solution with or without 1,500 ppm caffeine feeding for 12 weeks. The caffeine, PB, SDM, and ID treatments significantly (p < 0.05 or 0.01) increased the relative thyroid weights, and the increases with PB or ID were further (p < 0.05 or 0.01) enhanced in combination with caffeine. SDM drastically promoted thyroid carcinogenesis in association with increased serum TSH levels regardless of the caffeine treatment. Thyroid follicular carcinomas and adenomas were more frequently observed in the additional caffeine groups than in the ID alone groups. The incidence and multiplicity of focal thyroid follicular hyperplasias in the ID-treated groups were significantly (p < 0.05 and 0.01) elevated in the case of combination with caffeine. Increases in serum TSH levels with PB or ID were also further enhanced in combination with caffeine. Serum thyroid hormone levels were significantly (p < 0.01) decreased by SDM but significantly (p < 0.05 or 0.01) increased by caffeine, PB or ID. Our results clearly indicate that dietary caffeine at a high dose of 1,500 ppm interacts with ID, but neither SDM nor PB, to promote rat thyroid carcinogenesis although the combined caffeine + PB treatment somewhat affected thyroid weights as well as thyroid hormone levels.     

Clostridium sordellii phospholipase C: gene cloning and comparison of enzymatic and biological activities with those of Clostridium perfringens and Clostridium bifermentans phospholipase C

The gene encoding Clostridium sordellii phospholipase C (Csp) was cloned and expressed as a histidine-tagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of Clostridium perfringens phospholipase C (Cpa) and Clostridium bifermentans phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.     

DIISOPROPANOLNITROSAMINE (DIPN) INDUCED RAT THYROID LESIONS

DIPN induced localized lesions of the rat thyroid gland are described histologically and classified into three major types: type 1, foci of cellular alteration; type 2, proliferative nodules; and type 3, overt carcinomas. Follicular, papillary and mixed papillofollicular subtypes are recognized in the type 2 lesions, and follicular, papillary, anaplastic and mixed subtypes in the type 3 lesions. The nature of the individual lesions is discussed.     

Generation of thyroglobulin-specific T-cell clones derived from F1 Fisher rats.

We established T-cell clones from 'poor-responder' Fisher rats specific for thyroglobulin (Tg) with a view to examining Tg presentation by cloned Fisher rat thyroid (FRTL) cells. From the screening of 60 T-cell clones, three high-responding Tg-specific clones (B21.01, B21.04 and B21.05) were isolated from the lymph nodes of F1 generation (Fisher x 'high-responder' Buffalo) female rats immunized with murine Tg in complete Freund's adjuvant (CFA). Three T-cell clones expressed a W3/25+, OX-8- phenotype and responded specifically to murine and rat Tg in T-cell proliferation assays with Fisher rat antigen-presenting cells and secreted IL-2 as measured using a murine cytotoxic T-lymphocyte line (CTLL-2). Both B21.04 and B21.05 T-cell clones were capable of providing helper T-cell function for rat Tg antibody production in syngeneic reconstitution cultures in vitro. In contrast, clone B21.01 inhibited Tg antibody secretion. These data demonstrate that 'poor-responder' Fisher rats are capable of mounting significant T-cell responses to Tg in an F1 generation. Such Tg-specific T-cell clones will allow us to analyse their interaction with cloned Fisher rat thyroid cells and determine the role, if any, of thyroid cell antigen self-presentation to the immune system.