Evidence for low androgenicity among Indian (South Asian) men.

There are increasing data indicating profound ethnic differences in the levels of virilization of males. It is well understood that the intensity of testosterone-mediated effects is modulated by sex hormone binding globulin (SHBG) and the CAG repeat lengths in the androgen receptor (AR) gene. We determined the serum testosterone, estradiol and SHBG levels and average CAG repeat lengths among a group of healthy older Indian men living in Connecticut, USA and compared these parameters with those of a reference group of white Caucasian men. We also compared various parameters that represent the end-manifestations of testosterone activity - serum prostate-specific antigen (PSA) levels, lean body mass, skeletal mineralization and visceral fat. Our data suggest that men from the Indian subcontinent are smaller, manifest lower levels of circulating free testosterone, lower mean PSA levels and lean body mass, but are comparable to white Caucasian men in terms of SHBG, estradiol, levels of visceral fat and CAG repeat length. These data suggest that Indian men manifest a lower level of virilization compared to white Caucasian males and that this might be due to lower mean circulating testosterone levels rather than higher AR CAG repeat length or SHBG.

Granuloma formation around filarial larvae triggered by host responses to an excretory/secretory antigen.

In previous studies using a murine model of filarial infection, granuloma formation was found to be a most important host-protective mechanism. We have also shown that in vitro cytoadherence is a surrogate for the formation of antifilarial granulomas in vivo and that it requires "alternatively activated" host cells and a source of antifilarial antibody. We show here that antibodies against L3 excretory/secretory (E/S) products can facilitate in vitro cytoadherence. We generated a set of hybridomas reactive with filarial E/S products and screened them for their ability to mediate in vitro cytoadherence. One clone (no. 1E9) was positive in this assay. We then screened a novel expression library of filarial antigens displayed on the surface of T7 bacteriophage for reactivity with 1E9. Phage expressing two filarial antigens (TCTP and BmALT-2) reacted with 1E9. Immunization of mice showed that the cohort immunized with BmALT-2 cleared a challenge infection with infective Brugia pahangi L3 in an accelerated manner, whereas cohorts immunized with TCTP cleared larvae with the same kinetics as in unimmunized mice. These data confirm that BmALT-2 is the antigenic target of granuloma-mediated killing of B. pahangi L3. Our findings also confirm previous studies that BmALT-2 is a potential vaccine candidate for filarial infection. Our data reinforce the work of others and also provide a possible mechanism by which immune responses to BmALT-2 may provide host protection.

Importance of bone mineral density measurements in evaluating fragility bone fracture risk in Asian Indian men.

UNLABELLED: We evaluated the effect of BMD on fracture risk prediction using FRAX® among Asian Indian men when used in conjunction with clinical risk factors. A majority of our subjects were either osteopenic or osteoporotic, and their fracture risk increased when FRAX® was used in conjunction with femur neck T-scores. INTRODUCTION: Asian Indian men living in the United States may represent a population that is at high and underappreciated risk for fragility bone fractures. PURPOSE: To evaluate the effect of BMD on fracture risk prediction using FRAX® among Asian Indian men when used in conjunction with clinical risk factors. METHODS: Forty four Asian Indian men (mean age 64.9 (±8.4) years) who had lived in the United States for an average of 33.6 (±10.6) years underwent BMD measurement at the proximal femur. Subjects were subjected to a general physical exam and history of fracture, hip fracture in a parent, current smoking and alcohol use, and diagnosis of inflammatory arthritis was obtained. Data from each subject were entered into the FRAX® algorithm and 10-year fracture probabilities were calculated using clinical risk factors (CRFs) alone and in combination with femur neck T-scores. RESULTS: Thirteen subjects (29.5%) had femur neck T-scores ≥ -1.0, 28 (63.6%) T-scores between -1.0 and -2.5, and three (6.8%) T-scores < -2.5. The 10-year probability of a major osteoporotic fracture based on a combination of clinical risk factors and femur neck T-scores was significantly higher than the fracture probability based on clinical risk factors alone (t(43) = 2.58, p = 0.01). CONCLUSIONS: Among Asian Indian men, the 10-year probability of a major osteoporotic fracture increases when femur neck T-scores are added to clinical risk factors in the FRAX® algorithm, and this population have a high fracture probability even in the absence of clinical risk factors.

B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites.

Host defense against multicellular, extracellular pathogens such as nematode parasites is believed to be mediated largely, if not exclusively, by T lymphocytes. During our investigations into the course of Brugia malayi and Brugia pahangi infections in immunodeficient mouse models, we found that mice lacking B lymphocytes were permissive for Brugian infections, whereas immunocompetent mice were uniformly resistant. Mice bearing the Btk(xid) mutation were as permissive as those lacking all B cells, suggesting that the B1 subset may be responsible for host protection. Reconstitution of immunodeficient recombination activating gene (Rag)-1(-/)- mice with B1 B cells conferred resistance, even in the absence of conventional B2 lymphocytes and most T cells. These results suggest that B1 B cells are necessary to mediate host resistance to Brugian infection. Our data are consistent with a model wherein early resistance to B. malayi is mediated by humoral immune response, with a significant attrition of the incoming infectious larval load. Sterile clearance of the remaining parasite burden appears to require cell-mediated immunity. These data raise the possibility that the identification of molecule(s) recognized by humoral immune mechanisms might help generate prophylactic vaccines.

Maintenance of the human malarial parasite, Plasmodium falciparum, in scid mice and transmission of gametocytes to mosquitoes.

The study of human malaria has been hampered by the lack of small animal models for the human-infecting malarial parasites. To approach this problem, the erythrocytic stages of the human malarial parasite Plasmodium falciparum were adapted to in vitro growth in the presence of ascites fluid from mice homozygous for the severe-combined immunodeficiency (scid) mutation. Human red blood cells (hRBCs) infected with these adapted parasites were then injected i.p. into nonobese diabetic scid/scid (NOD/LtSz-scid) mice. With daily supplemental intraperitoneal boosts of uninfected hRBCs, parasites were detected in the peripheral circulation of these mice for an average of 7 d after injection. Splenectomy of NOD/LtSz-scid mice increased both the level and duration of parasitemia in the periphery, and it also promoted the circulation of mature sexual stage parasites (gametocytes). When Anopheline mosquitoes were allowed to feed on the splenectomized mice, the gametocytes were ingested by the mosquitoes and developed into oocysts in the mosquito midguts. To our knowledge, these results are the first demonstration of human malarial parasite propagation in mice and transmission of these parasites to the invertebrate vector.

A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Expression of H-2Db on the cell surface in the absence of detectable beta 2 microglobulin.

In this report we describe a variant of the C57BL/6 T lymphoma EL4 (EL4/Mar) which, in contrast to the parental cell line, expresses neither H-2Kb nor beta2-microglobulin (beta2m) but which does express H-2Db detectable by serology and by alloreactive cytotoxic T lymphocytes (CTL). This observation raises the possibility that H-2Db and perhaps other major histocompatibility complex class I molecules are normally not associated with beta2m on the cell surface. In addition, this report is the first to indicated that alloreactive CTL can interact with a beta2m-free class I antigen.

The immunodeficient scid mouse as a model for human lymphatic filariasis.

The C.B.-17-scid/scid mouse (hereafter referred to as the scid mouse) is homozygous for a recessive mutation at a locus that influences the assembly of intact immunoglobulin and T cell receptor genes. Therefore, scid mice cannot generate functional B or T lymphocytes, are profoundly immunodeficient, and have been reported to be receptive to reconstitution with human immune cells. In the present study, we injected scid mice with infective larvae of the human filarial parasite Brugia malayi. Within 6-10 wk after subcutaneous injection of infective L3 larvae, both male and female worms were observed in various stages of development in 90% of the mice. In animals tested 8 weeks or more after infection, microfilariae were detected in the blood or peritoneal cavity of 52% of the mice examined. Adult worms were observed in the lymphatics of the infected scid mice, where their presence was associated with lymphangitis and lymphangiectasia. These results suggest that the scid mouse model of lymphatic filariasis may be important in investigation of the interaction of the murine, and possibly the human, immune system with the lymphatic filarial parasite.

Epitopes on H-2Dd somatic cell mutants recognized by cytotoxic T cells.

We have generated several cell lines that express an altered H-2Dd molecule. These cell lines, which were selected for by the failure to express the serological specificity reacting with the monoclonal antibody 34-2-12, have also undergone alterations in epitopes recognized by CTL. One of the mutants, 2.12(-4) was not killed by an allogeneic anti-Dd CTL line, CTLL-A2, even though this line was cytotoxic for the parental cell line and two other 34-2-12- mutant lines. Two of the 34-2-12- mutant lines had an identical serological profile using other monoclonal Dd antibodies, however these two mutants differed markedly in their susceptibility to cytotoxicity by CTLL-A2. In addition to the determinants recognized by allogeneic CTL we also examined the effect of the mutation on the determinants involved in restricting the anti-FITC modified-self-cytotoxic response. An anti-FITC-Dd CTL line did not react with two of the mutants and reacted only weakly with the other mutant, demonstrating not only that the Dd epitopes recognized by this cell line and the allogeneic CTL were different, but also that it is possible for a H-2 class I molecule to express epitopes recognized by allogeneic CTL but not epitopes that function as restricting elements to certain antigens. The observation that both T cell- and B cell-defined determinants were altered in these mutant cell lines is in contrast to the findings, with the mutant mouse strains which were selected for by changes in T cell-defined determinants, which show few, if any, alterations to serological specificities. Characterization of T cell-recognized epitopes expressed on serologically selected somatic cell variants may therefore prove to be most useful for the study of structure-function relationships of H-2 class I molecules.

Neonatal tolerance and patent filarial infection.

Lymphatic filariasis occurs in endemic pockets. Patent infections with long-term, high-grade microfilaremia do not develop in nonendemic individuals. It is tempting to speculate that individuals with intact immune responses to filarial antigens are capable of dealing with filarial exposure without developing persistent infection. There are published data that support the idea that only those individuals who are impaired in their immune defense against these parasites owing to neonatal tolerization become productively infected with the filarial parasites. If the model is correct, there are profound implications for global eradication.

Recapitulation of normal and abnormal BB rat immune system development in scid mouse/rat lymphohemopoietic chimeras.

Mice homozygous for the mutation "severe combined immune deficiency" (C.B17-scid/scid) lack functional T and B lymphocytes and readily accept tumor xenografts. Partial lymphohemopoietic scid/human and mouse/rat chimeras have been described, but complete chimerization with thymic engraftment and generation of donor-origin thymocytes has not been achieved. We now report that low-dose irradiation permits the engraftment of BB rat fetal liver stem cells in scid recipients. We observed that BB rat fetal liver cells injected into irradiated scid mice establish a rat hemopoietic system in the scid mouse bone marrow and populate the scid mouse thymus. These stem cells generated rat-origin thymocytes that migrated to the scid mouse spleen, a peripheral lymphoid organ. Finally, we found that xenogeneic chimeras created using fetal liver cells from the abnormal (lymphopenic, diabetes prone) subline of BB rats recapitulated both the quantitative and phenotypic abnormalities of the donor rat. Xenogeneic lymphohemopoietic chimeras established in scid mice may provide a powerful new tool in the study of immune system development and autoimmunity.

Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.

Heterozygous mammalian cell lines normally express both parental alleles at most autosomal loci. However, mutants can be isolated that fail to express one of the alleles. Using a murine pre-B cell line that is heterozygous for several loci on chromosome 12, including one encoding the cell surface antigen Ly-18, we found that one of the two Ly-18 antigenic forms was lost at a rate of 1.5 x 10(-5) per cell per generation. Molecular analysis revealed that a genetic marker distal to Ly-18 became homozygous. Analysis of the genotype of the mutants at the rDNA cluster, located close to the centromere, strongly suggests that the mutants arose by mitotic recombination within this multicopy locus.

Regulatory variants for the expression of H-2 antigens. II. Level of discrimination of cell-mediated cytolysis.

We used alloantigenic variants to analyze whether killer T-cells and antibody in the presence of complement (two effector components of the immune system) recognized the same determinants on target tissues. The variants were selected from mouse myeloma cells by immune selection and differed from the parent cell in that they did not express H-2 antigens. The results suggested that both killer T-cells and humoral effectors recognized the same determinants, though the killer T-cells may have been more sensitive either in recognizing these determinants or mediating lysis.

Regulatory variants for the expression of H-2 antigens. I. Isolation and characterization.

By subjecting "mutagenized" mouse myeloma cells repeatedly to selection with antiserum against H-2Kd, we isolated variants that expressed little or no H-2Kd alloantigens. The phenotype was unstable, with the culture accumulating revertants in the absence of selective pressure. The variants not only failed to express the selected antigen, but also the antigen coded by the closely liked H-2Dd gene. The cells reexpressed this antigen with the same kinetics as for the selected antigen. Several physiologic parameters (karyotype, cell morphology, size, and growth curves) were not altered by the loss of H-2 antigens.However,when injected iv, the H-2-deficient cells did not lodge in the spleen as readily as did the wild-type cells.

An RT6a gene is transcribed and translated in lymphopenic diabetes-prone BB rats.

T-cells expressing the RT6 surface alloantigen appear to perform important immunoregulatory functions in the rat. Diabetes-prone BB rats lack circulating RT6+ T-cells and spontaneously develop autoimmune diabetes mellitus and thyroiditis. The coisogenic diabetes-resistant BB rat does circulate RT6+ T-cells and is free of disease. Transfusions leading to engraftment of RT6+ T-cells prevent both diabetes and thyroiditis in the diabetes-prone rat. To investigate the absence of this subset in the lymphopenic BB rat, we used both molecular and biochemical procedures and made the following observations: 1) an mRNA encoding RT6 protein is present in diabetes-prone spleen cells; 2) nucleotide sequencing of this transcript reveals an intact coding sequence for the RT6.1 alloantigen; 3) sensitive chemiluminescent assay of diabetes-prone lymph node cell detergent extracts shows that diabetes-prone RT6 mRNA is translated in vivo; 4) quantitatively, diabetes-prone lymph node cells express < or = 10% of the RT6.1 protein found on similar numbers of diabetes-resistant BB cells; and 5) finally, we obtained evidence of an intact phosphatidylinositol linkage of the molecule to the cell surface and successfully immunoprecipitated the phosphatidylinositol-linked protein with DS4.23 monoclonal antibody, indicating that the RT6.1 antigen is correctly processed and folded in diabetes-prone lymph node cells. We conclude that the near total absence of RT6+ T-cells in the diabetes-prone BB rat is unlikely to be because of a defect in RT6 gene expression per se. Defects in RT6 gene regulation or other cellular defects leading to premature cell death in the T-cell lineage, alone or in combination, may instead be responsible.

Natural course of lymphatic filariasis: insights from epidemiology, experimental human infections, and clinical observations.

Lymphatic filariasis has been described as a "spectral disease". Analysis of the natural course of infection in nonendemic individuals as well as experimental infections of "volunteers" suggests that the filarial parasites are not inherently aggressive infectious agents. Experimental infections of humans with infective larvae result in transient, low-level microfilaremia, if at all. Nonendemic individuals with limited exposure show no evidence of persistent infection or pathology. Nonendemic individuals exposed to repeated infections show accelerated pathology. It is tempting to speculate that normal, immunocompetent residents in an endemic area show either (a) no pathology (endemic normals) because they are subject to the relatively low levels of infection or (b) chronic pathology if they are repeatedly infected. It would appear that only those individuals rendered immunologically tolerant to filarial parasites become productively infected with the filarial parasites. The intensity of transmission may underlie the differences in clinical presentation seen in diverse global pockets of endemicity.

The L3 to L4 molt of Brugia malayi: real time visualization by video microscopy.

Brugia malayi and other filarial parasites have been studied in great detail, especially in the context of human disease. In common with other nematodes, these organisms molt 4 times in their life cycles, but details of this process have not been described. We have recently developed an in vitro culture system that supports the L3 to L4 molt at high efficiency. This has permitted us to visualize, for the first time, details of this molt using real-time video microscopy. Molting is preceded by a phase of altered motility during which the larva exhibits contractile, coiling movements. The earliest evidence of ecdysis is a clearing at one end, more frequently caudal, caused by the larva retracting from that end. A cleavage develops in the cuticle near the head end, forming a rostral cap, which is continuous with the pharyngeal cuticle. Simultaneously, it retracts out of the cuticle using coiling and writhing movements. This process takes 5 to 10 min. Finally, it retracts out of the cap and extrudes the pharyngeal cuticle. Detachment of the pharyngeal cuticle is the final event in the process and continues up to an hour after the rest of the cuticle has been shed.

Neonatal outcome in the United Arab Emirates: the effect of changes in resources and practices.

Selective improvements in neonatal care resources and practices were instituted between 1992/1994 (period 1) and 1995/1998 (period 2) following a neonatal audit in the United Arab Emirates. We evaluated the effect of these changes on neonatal mortality rate (NNMR), birth-weight-specific mortality rates and causes of mortality. Overall there was a 17% decline in the NNMR from periods 1 to 2. Mortality rates in infants with birth weight < 1000 g and > 2500 g decreased by 36% and 35% respectively from periods 1 to 2. Modest declines in deaths from asphyxia, sepsis and complications of preterm births occurred from periods 1 to 2 but the differences were not statistically significant.

Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency.

Spontaneous mouse mutations that cause severe immunodeficiency or autoimmunity are invaluable tools with which to investigate the mammalian immune system. Mutations at the 'motheaten' locus result in severe immunological dysfunction due to disruption of the structural gene encoding Src-homology 2-domain phosphatase-1 (SHP-1). This natural model for a specific protein-tyrosine-phosphatase deficiency is being widely utilized to determine the role of SHP-1 in the negative regulation of multiple signaling pathways in a number of hematopoietic lineages.

Pristane retards clearance of particulate materials from the peritoneal cavity of laboratory mice.

The effect of intraperitoneal pristane on the movement of India ink particles and water-based radio-opaque dye injected into the peritoneal cavity of mice was examined. There was a marked difference between pristane-treated mice and unmanipulated controls in terms of particle retention; unmanipulated mice cleared India ink particles via lymphatic drainage within 24 h following injection, whereas pristane-treated mice retained particles for as long as 22 days post-injection. In contrast, all mice displayed similar kinetics of removal of radio-opaque dye. We conclude that the movement of particulate matter from the peritoneal cavity is retarded in pristane-treated mice whereas removal of water-based materials is unaffected. The implications for generation of ascites using hybridoma cells in pristane-primed mice is discussed.