Serological distinction of integral plasma membrane proteins as a class of mycobacterial antigens and their relevance for human T cell activation

This study pertains to classification and antigenic analysis of mycobacterial plasma membrane proteins in relation to human T cell proliferative responses, using a ‘fast grower’ Mycobacterium fortuitum as model. Membrane vesicles, prepared by sonication and differential centrifugation, were subjected to biphasic Triton X-1 14 extraction for isolation of integral (detergent phase) and peripheral (aqueous phase) proteins. Neither protein pool showed any appreciable overlap serologically. SDS-PAGE showed five prominent bands in peripheral and three in the integral protein pool, whereas immunoblotting with rabbit antisera identified only two major antigens (60 and 67kD) in the former and five (24, 34, 42, 51 and 54kD) in the latter, ELISA with a panel of anti-mycobacterial MoAbs revealed that nine out of 12 previously known antigens were present in the peripheral protein pool. Only two of them (33 and 40 kD) were additionally detected amongst integral proteins. The membrane-associated immunosuppressive moiety lipoarabinomannan was semiquantitatively located in aqueous phase. In bulk T cell proliferation assays, seven out of 10 subjects belonging to a ‘responder’ background (BT-BB leprosy patients and healthy contacts) showed high responses for Myco. fortuitum antigens. Proliferative response with integral proteins was comparable to that with whole membrane, hut it was significantly higher (P < 0.0005) than t he response with peripheral proteins. The distinction and relevance of integral membrane proteins as a class of mycobacterial antigens make them worthy of consideration in a subunit vaccine design.     

Inhibitory effect of plasma obtained from hypophysectomized and control women on the assay of bioactive luteinizing hormone

The purpose of this study was to determine the effect of components of female plasma on the value of bioactive luteinizing hormone (LH), especially in the presence of low immunological LH value. Using both an immunoradiometric assay (IRMA) and rat Leydig cell bioassay, immunoreactive (I) and bioactive (B) LH were assessed in plasma collected from women during a gonadotrophin releasing hormone (GnRH) test performed on day 7 of a spontaneous cycle. Two modes of response to an acute administration of GnRH were defined: normal production of gonadotrophins (group I) and excessive secretion (group II) associated with a significant difference in the B/I-LH ratio between the two groups. The B/I-LH ratio did not vary with sampling time during the test in either group. The addition of LH-free plasma collected from hypophysectomized women caused a 30% decrease in testosterone production compared to control values (in the presence or absence of hLH standard). A partial restoration of testosterone production was observed if plasma was first treated with PEG 12%. The inhibitory factor(s) was also present in plasma from ovulatory women, even after treatment by an antibody against the entire LH molecule. The effect of normal (A) or low I-LH plasma (B) on testosterone production varied strongly according to the plasma volume added to the bioassay, as well as to plasma treatments. Diethylether treatment caused a 50% decrease in testosterone secretion for plasma B (but not for A) whereas a diminution of the steroidogenesis is observed after a PEG treatment of plasma A (but not for B), suggesting that different inhibitory factors are present in plasmas A and B. Therefore the LH bioactivity measured in the rat Leydig cell assay, in terms of testosterone output, seems to represent a balance between the LH molecule and the presence of inhibitory factors in the plasma.     

Increased seminal plasma lead levels adversely affect the fertility potential of sperm in IVF

BACKGROUND: Lead remains in high levels in the environment and is known to reduce fertility in animal models, but a direct link between lead exposures and human infertility has not yet been established. METHODS: In a prospective, double-blind study of the metal ion levels and sperm function, semen was obtained from partners of 140 consecutive women undergoing their first IVF cycle. Lead in seminal plasma was determined by atomic absorption spectroscopy. Motile sperm populations were assessed for surface receptors for mannose binding, and the ability to undergo premature ('spontaneous'), and free mannose-induced acrosome reactions. Fertile donor (n = 9) sperm were exposed to exogenous lead during capacitating incubations and then assessed for mannose receptor expression and acrosome loss. RESULTS: Lead levels were negatively correlated with IVF rates. Lead levels were negatively correlated to two of the three sperm function biomarkers (mannose receptors, mannose-induced acrosome reactions). Lead levels positively correlated with the spontaneous acrosome reaction. These findings were mimicked by in-vitro exposure of fertile donor sperm to lead. CONCLUSIONS: Multiple sperm parameters are affected as lead levels rise. Increased lead levels may contribute to the production of unexplained male infertility.     

Antibody response in laying hens with small amounts of antigen

Chicken antibodies offer many advantages over the traditional mammalian ones. A laying hen produces large amounts of yolk antibodies and the use of yolk antibodies eliminates the painful procedure of collecting blood from the animal. Thus, the use of chicken antibodies will reduce both the number of animals required to produce antibodies and also animal distress. Chicken antibodies also have several biochemical advantages compared to mammalian antibodies: they often increase the signal and reduce interference in many assays. However, the species chosen for antibody production have usually been mammals. This is probably due to tradition, but also to limited knowledge about the production of chicken antibodies. We studied the immune response in the chicken using small amounts of mammalian antigen, and show that a good immune response can be obtained with 0.1–1.0 μg of bovine serum albumin.     

Alterations of plasma antioxidants and mitochondrial DNA mutation in hair follicles of smokers

The effects of long-term smoking on mitochondrial DNA (mtDNA) deletions in hair follicles were investigated in subjects with different antioxidant capacity. Twenty-two male smokers with a smoking index of greater than 5 pack-years and without any known systemic diseases were recruited for this study. Forty healthy nonsmoking males were included as controls. We found that the concentrations of ascorbate and alpha-tocopherol and the activities of glutathione S-transferase (GST) and glutathione peroxidase in blood plasma were significantly decreased in smokers. The levels of glutathione and protein thiols in whole blood and the incidence of a 4,977 bp deletion of mtDNA (dmtDNA) in hair follicles were significantly increased in smokers. A significantly higher incidence of the 4,977 bp dmtDNA was found in smokers with plasma GST activity less than 5.66 U/l (OR = 7.2, P = 0.020). Using multiple covariate ANOVA and logistic regression, we found that age and low plasma GST activity were the only two risk factors for the 4,977 bp dmtDNA. These results suggest that smoking depletes antioxidants and causes mtDNA deletions and that plasma GST may play an important role in the preservation of the mitochondrial genome in tissue cells of smokers.     

Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.     

Assessment of HBV persistent infection in an adult population in Taiwan

In order to study the prevalence of hepatitis B virus (HBV) in the adult population of Taiwan, we screened for the presence of HBV DNA in 205 blood samples from adult (20-59-year-old) volunteers. According to the serological markers of HBV, samples were divided into three groups: group I (173 subjects) was negative for both HBsAg and HBeAg; group II (14 subjects) was positive for both HBsAg and HBeAg; and group III consisted of 18 subjects who were HBsAg-positive but HBeAg-negative. Plasma HBV DNA was not detected in group I, but it was found in 85.7% and 11.8% of samples in group II and group III, respectively. A free-form HBV DNA was found in 14.3% of the leukocyte samples in group II. Furthermore, an integrated form of HBV DNA was detected in the leukocytes of two cases of group I who remained healthy based on clinical data. HBV DNA was also detected in the spermatozoa and liver cells of one of the cases.     

Phosphate transport in brush border membrane vesicles isolated from renal cortex of young growing and adult rats

Recent clearance studies have demonstrated that the maximal tubular reabsorption of inorganic phosphate (Pi) per ml of glomerular filtrate (max. TRPi/ml GF) of the whole kidney is markedly lower in adult than in young growing rats fed either normal (0.8 g%) or low (0.2 g%) phosphorus diet. In addition, in adult rats clearance studies indicate that enhancement of max. TRPi/ml GF is observed 21 days but not 8 days after starting the low (0.2%) phosphorus diet. In the present work we have studied in the same experimental condition the Na⁺-dependent Pi uptake in brush border membrane vesicles (BBMV) isolated from renal cortex of either young growing or adult rats. The results of this study indicate that under the low (0.2%) but not under the normal (0.8%) phosphorus diet the Na⁺-dependent Pi uptake by BBMV was significantly depressed in adult as compared to young growing rats. In adult rats the Pi transport response to Pi restriction monitored at the brush border membrane level was different from that observed by clearance studies in the whole kidney. Indeed, the Pi uptake by BBMV was already enhanced after 8 days of Pi restriction and it did not increase further when studied 21 days after starting the low (0.2%) phosphorus diet. These results suggest that the regulation of the overall transfer of Pi across the renal epithelium may involve other additional modulating factors than the Na⁺-dependent Pi transport system present in the luminal membrane of the proximal tubule.     

Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection

Human immunodeficiency virus (HIV) has been isolated from plasma in 6 of 7 patients showing clinical symptoms of a primary HIV infection. Parallel cultures from peripheral blood mononuclear cells (PBMC) yielded virus in 5 patients. In one case, virus could only be isolated from the cerebrospinal fluid but not from peripheral blood. Detectable viremia was transient and preceded the appearance of HIV specific antibodies. After cessation of acute symptoms, the frequency of HIV isolations was similar to that of asymptomatic carriers (23 and 26%, respectively). The role of the immune response in terminating detectable viremia remains to be established.