Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination.

The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, "tangled" sperm agglutination was used to isolate cDNAs encoding its cognate antigen. Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in lambdagt11. Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3-kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen-36 (SA-36), composed of 558 deduced amino acids. SA-36 cDNA contained a 5' untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3' untranslated region of 1138 nt. SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA. This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from baker's yeast. Moreover, polyclonal, monospecific antibodies produced against beta-galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb. Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination.

Tetrahelical forms of the fragile X syndrome expanded sequence d(CGG)(n) are destabilized by two heterogeneous nuclear ribonucleoprotein-related telomeric DNA-binding proteins.

Formations of hairpin and tetrahelical structures by the trinucleotide repeat sequence d(CGG)(n) might contribute to its expansion in fragile X syndrome. Here we show that tetraplex structures of d(CGG)(n) are destabilized by two mammalian heterogeneous nuclear ribonucleoprotein-related tetraplex telomeric DNA-binding and -stabilizing proteins, quadruplex telomeric DNA-binding protein 42 (qTBP42) (Sarig, G., Weisman-Shomer, P., Erlitzki, R., and Fry, M. (1997) J. Biol. Chem. 272, 4474-4482) and unimolecular quadruplex telomeric DNA-binding protein 25 (uqTBP25) (Erlitzki, R., and Fry, M. (1997) J. Biol. Chem. 272, 15881-15890). Blunt-ended and 3'-tailed or 3'- and 5'-tailed bimolecular tetraplex structures of d(CGG)(n) and guanine-sparse 20-/46-mer partial DNA duplex were progressively destabilized by increasing amounts of qTBP42 or uqTBP25 in time-dependent and ATP- or Mg(2+)-independent reactions. By contrast, tetraplex structures of telomeric and IgG sequences or guanine-rich double-stranded DNA resisted destabilization by qTBP42 or uqTBP25. Increased stability of tetraplex d(CGG)(n) in the presence of K(+) or Na(+) ions or at lowered reaction temperature diminished the destabilizing activity of uqTBP25. The contrasting stabilization of tetraplex telomeric DNA and destabilization of tetraplex d(CGG)(n) by qTBP42 and uqTBP25 suggested that sequence or structural differences between these tetraplexes might serve as cues for the differential stabilizing/destabilizing activities.

Characterization of a potent sperm-agglutinating monoclonal antibody and its cognate antigens.

OBJECTIVE: To identify sperm antigens that are capable of eliciting infertility-related sperm-agglutinating antibodies. DESIGN: In vitro laboratory experiments. SETTING: University research laboratory. PATIENT(S): Fertile semen donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm agglutination, immunofluorescence localization, and flow cytometric analysis of surface expression of A36 antigens. Antigen analysis by Western immunoblotting. RESULT(S): Monoclonal antibody A36 induced intensive head-to-head, tail-to-tail, and head-to-tail agglutination of motile human spermatozoa. Antigens recognized by A36 were localized on the acrosomal cap and in the principal tail regions of motile, noncapacitated human sperm. Changes in subcellular levels and localization of the A36-recognized epitope occurred after capacitation and acrosomal loss. A36 reacted with a polymorphic series of proteins in Western blots of sperm extracts from humans and various other animal species, including mouse testis extracts. A common 53-kd antigen was recognized by the antibody in the different antigenic preparations. CONCLUSION(S): A mouse antibody to human sperm, monoclonal antibody A36, caused intensive agglutination of noncapacitated human spermatozoa and reacted with antigens on the acrosomal cap and in the principal tail regions. Of the multiple polypeptides that were reactive with the monoclonal antibody in sperm extracts from humans and other animal species, a common 53-kd antigen was recognized.

Hematopoietic progenitor cell abnormalities in Hoxc-8 null mutant mice.

The mammalian Hox genes encode a family of conserved transcription factors that control the establishment of the body plan during embryogenesis. Many Hox genes are also known to be expressed in hematopoietic cells. We found that Hoxc-8, a member of the Hox C cluster, is expressed in the mouse hematopoietic organs, fetal liver and adult bone marrow. To determine the role of Hoxc-8 gene in hematopoiesis, we compared progenitor cell numbers in the fetal liver and adult bone marrow cells. We observed a significant reduction in the number of erythroid burst-forming unit (BFU-E) and in granulocyte/macrophage colony-forming unit (CFU-GM) in the Hoxc-8 null mice, although the peripheral blood cell counts were normal. The hematopoietic cells from the homozygote animals exhibited normal expansion capability in a liquid culture system, suggesting that the decreased number of progenitor cells may be due to a defect extrinsic to the hematopoietic cells, such as in the interaction with the microenvironment.

Molecular biology and pathogenicity of mycoplasmas.

The recent sequencing of the entire genomes of Mycoplasma genitalium and M. pneumoniae has attracted considerable attention to the molecular biology of mycoplasmas, the smallest self-replicating organisms. It appears that we are now much closer to the goal of defining, in molecular terms, the entire machinery of a self-replicating cell. Comparative genomics based on comparison of the genomic makeup of mycoplasmal genomes with those of other bacteria, has opened new ways of looking at the evolutionary history of the mycoplasmas. There is now solid genetic support for the hypothesis that mycoplasmas have evolved as a branch of gram-positive bacteria by a process of reductive evolution. During this process, the mycoplasmas lost considerable portions of their ancestors' chromosomes but retained the genes essential for life. Thus, the mycoplasmal genomes carry a high percentage of conserved genes, greatly facilitating gene annotation. The significant genome compaction that occurred in mycoplasmas was made possible by adopting a parasitic mode of life. The supply of nutrients from their hosts apparently enabled mycoplasmas to lose, during evolution, the genes for many assimilative processes. During their evolution and adaptation to a parasitic mode of life, the mycoplasmas have developed various genetic systems providing a highly plastic set of variable surface proteins to evade the host immune system. The uniqueness of the mycoplasmal systems is manifested by the presence of highly mutable modules combined with an ability to expand the antigenic repertoire by generating structural alternatives, all compressed into limited genomic sequences. In the absence of a cell wall and a periplasmic space, the majority of surface variable antigens in mycoplasmas are lipoproteins. Apart from providing specific antimycoplasmal defense, the host immune system is also involved in the development of pathogenic lesions and exacerbation of mycoplasma induced diseases. Mycoplasmas are able to stimulate as well as suppress lymphocytes in a nonspecific, polyclonal manner, both in vitro and in vivo. As well as to affecting various subsets of lymphocytes, mycoplasmas and mycoplasma-derived cell components modulate the activities of monocytes/macrophages and NK cells and trigger the production of a wide variety of up-regulating and down-regulating cytokines and chemokines. Mycoplasma-mediated secretion of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6, by macrophages and of up-regulating cytokines by mitogenically stimulated lymphocytes plays a major role in mycoplasma-induced immune system modulation and inflammatory responses.

Subversion and exploitation of host cells by mycoplasmas.

Mycoplasmas are minute wall-less bacterial parasites that exhibit strict host and tissue specificities. They enter, multiply and survive within the host for extended periods by circumventing host defenses. Their intimate interaction with eukaryotic cells, and in some cases the subsequent invasion into or fusion with these cells, mediates cell damage. Mycoplasmas also modulate the activity of host cells by a variety of direct mechanisms and/or indirectly by cytokine-mediated effects.

Immune complex-like moieties in immunoglobulin for intravenous use (i.v.Ig) bind complement and enhance phagocytosis of human erythrocytes.

Treatment with i.v.Ig can, on rare occasions, lead to detrimental effects such as enhanced erythrocyte sequestration and an increase in serum immune complexes with inflammatory sequellae such as exacerbation of glomerular nephritis. In this study, i.v.Ig (Sandoglobin) was examined for complement binding moieties which resemble immune complexes and can mediate the binding of IgG and C'3b to human erythrocytes via CR1 and enhance erythrocyte susceptibility to sequestration. Sephacryl S-200 HR separated i.v.Ig into two fractions: monomeric IgG (74%) and larger complexes of the molecular weight of an IgG dimer or greater (> or = 300 kD) (26%). In the presence of complement, the 'dimers' bound to human erythrocytes, rendering them susceptible to phagocytosis in vitro. Removal of erythrocyte-specific isoantibodies from the i.v.Ig had no effect on 'dimer' binding to the erythrocytes. Monomeric IgG contained virtually no complement-activating, erythrocyte-binding activity. Erythrocyte binding of complement-bearing IgG 'dimers' and subsequent phagocytosis resembles the binding of complement-bearing immune complexes to erythrocyte CR1. Exposure to Factor I leads to the release of complement-bearing IgG 'dimers' from erythrocyte CR1 and to the abrogation of erythrophagocytosis. Binding of complement-bearing IgG 'dimers' to the erythrocyte is blocked by To5, a CR1-specific monoclonal antibody.

Monoclonal antibodies that inhibit mitogenic activity of Mycoplasma pulmonis.

Previous studies have suggested a correlation between mitogenic, polyclonal activation of host lymphocytes and the respiratory tract inflammatory diseases induced by Mycoplasma pulmonis. This study describes the generation of monoclonal antibodies (MAbs) to M. pulmonis membrane antigens with different capacities to inhibit stimulation of cultured rat lymphocytes by mycoplasmal membranes and with variable effects on M. pulmonis growth. We show that the inhibitory effects exerted on mitogenesis by purified MAbs are inversely related to the effects of MAbs on M. pulmonis growth. Immunoblotting of electrophoretically separated membrane proteins, with both growth- and mitogenesis-inhibiting antibodies, revealed significant changes in the reactions obtained with both types of MAb following short exposure of membranes to heat. Growth-inhibiting MAbs strongly react with heat-labile antigenic complexes with molecular weights of 65,000 to 75,000. Inhibition of mitogenesis is mainly associated with recognition of membrane complexes of 84 to 113 kDa that exhibit disperse smears and variable heat sensitivities. Following brief heating of membranes, more distinct bands of 103, 90, and 84 kDa are obtained with MAbs that inhibit mitogenesis. Experiments with other mitogenic mycoplasma species and MAb 3.3.10.2, a potent inhibitor of mitogenesis reveal that whereas the antigenic epitope recognized by this antibody is present on unheated membranes from different mycoplasmas, with heated membranes the MAb yields reactions only with M. pulmonis and M. arthritidis. Our studies suggest that M. pulmonis mitogens are unique membrane complexes of variable molecular weights, highly susceptible to heat and less sensitive to reducing agents.

Membrane lipids of Mycoplasma fermentans.

Membranes of Mycoplasma fermentans, incognitus strain, were isolated by a combination of osmotic lysis and sonication. Analysis of membrane lipids revealed, in addition to free and esterified cholesterol, six major polar lipids dominated by a de novo synthesized compound (compound X), which accounts for 64% of the total lipid phosphorus. Compound X was labeled by palmitate, but not by oleate. Mass spectrometry and gas liquid chromatography analyses of compound X revealed two molecular species with molecular masses of 1048 and 1076 representing, a dipalmitoyl- and a stearoyl-palmitoyl-glycerodiphosphatidylcholine. Compound X has the ability to stimulate human monocytes to secret TNF alpha and to enhance the fusion of small unilamellar vesicles with MOLT-3 lymphocytes.

Identification of human sperm antigens reacting with antisperm antibodies from sera and genital tract secretions.

OBJECTIVE: To identify sperm antigens reacting with antisperm antibodies relevant in human infertility. DESIGN: The reactions of separated sperm antigens with antibodies present in sera and genital tract secretions from infertile and fertile females and males were examined by immunoblotting techniques. SETTING: The patients were followed in an outpatient setting of a hospital clinic. PATIENTS: One hundred consecutive infertile males and females, referred for determinations of antisperm antibodies, comprised the study group. Fifty hospital and faculty employees with proven fertility served as a control group. RESULTS: A high proportion of sera from fertile and infertile humans contained antibodies reacting with at least one sperm antigen. However, two discrete bands of antigenic proteins with molecular weights of 44 and 72 kd reacted significantly more frequently with serum antibodies from infertile females than from fertile females. No apparent correlation could be demonstrated between any particular antigen and serum antibodies from infertile males. Nevertheless, antigenic proteins of 62 kd were identified as the major sperm antigens reacting with antibodies present in seminal plasmas from infertile males. CONCLUSIONS: The major sperm antigens reacting with systemic antibodies differ from the antigens recognized by local antisperm antibodies. Sperm antigens exhibiting relative molecular weights of 62 kd are major antigens reactive with local antisperm antibodies from infertile humans.

Acute oliguric renal failure complicating type A nonfulminant viral hepatitis. A case presentation and review of the literature.

Nonfulminant acute renal failure very rarely complicates type A viral hepatitis. So far only eight patients have been described in the English language, Japanese, or Israeli literature who had type A viral hepatitis with renal failure without any association with "hepatorenal syndrome," pregnancy, or other types of viral hepatitis. We describe a 52-year-old man with type A viral hepatitis, proven by IgM anti-A virus antibodies complicated by oliguric renal failure, which required hemodialysis. The patient recovered completely after a few weeks. Kidney biopsy demonstrated interstitial nephritis with no glomerular involvement. Review of the literature in relation to the causes and the rarity of the renal failure is included.

A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of antisperm antibodies in sera and genital tract secretions.

A reverse (antibody capture) enzyme-linked immunosorbent assay (ELISA) for detection of antisperm antibodies has been developed. The assay enables detection of immunoglobulin (Ig) M, IgG, IgA, or IgM, IgG, and IgA--antisperm antibodies in serum, cervical mucus, and seminal plasma samples. The reverse ELISA is more specific and sensitive than conventional ELISA in detecting human antisperm antibodies of different isotypes. Using this assay, statistically significant differences in levels of antibodies between infertile and fertile individuals were demonstrated in sera and in genital tract secretions. Studies with 143 infertile couples revealed that the presence of antibodies in sera was not necessarily reflected in individual's genital tract secretion and vice versa. These data emphasize the importance of detecting antisperm antibodies in sera as well as in genital tract secretions for correct evaluation of sperm immunity.

An outbreak of Mycoplasma pneumoniae pneumonia in two kibbutzim: a clinical and epidemiologic study.

During a period a 9 months, 125 individuals with pneumonia due to infection with Mycoplasma pneumoniae were identified among 1,242 individuals in two Israeli kibbutzim. The monthly incidence of M. pneumoniae pneumonia (MPP) was 13.3/1,000 population. Of those infected, 93 (74.4%) were under the age of 18 years. The clinical course of MPP was mostly benign. The prominent signs and symptoms of disease were cough (100%), fine respiratory crepitations (77%), fever (37%), and diminished breathing sounds (25%) above affected lung areas. Leukocytosis was rare (9.6%); however, eosinophilia was observed in 23% of 53 tests performed. Exacerbations of bronchial asthma was observed among 36% of 11 patients with a previous history of asthma. The average duration of disease was 13.5 days, under treatment. A recurrence rate of 11.2% was noted among all MPP patients, with a very high (42.3%) rate among patients treated with cotrimoxazole. All patients with recurrent pneumonia were children under the age of 10 years.

Mycoplasma-induced in vitro production of interleukin-2 and colony-stimulating activity.

Mitogenic doses of membranes purified from Mycoplasma pneumoniae as well as concanavalin A (ConA) were tested for their ability to induce production of interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating activity (GM-CSA) by human peripheral blood mononuclear cells. Unlike ConA, M. pneumoniae exhibited a significantly lower mitogenic effect and lacked the ability to induce production of IL-2. On the other hand, peak levels of GM-CSA produced in the presence of M. pneumoniae and detected in colony assays of human target marrow cells were approximately 80% of those produced in the presence of ConA. Whereas ConA induced a similar time-related effect upon [3H]thymidine uptake and GM-CSA production, maximal GM-CSA production preceded the peak proliferative response to M. pneumoniae, thereby indicating that M. pneumoniae-induced production of GM-CSA is not quantitatively correlated with DNA synthesis. These findings may contribute to the understanding of the inflammatory manifestations induced by this organism.

Direct invasion of the central nervous system by Mycoplasma pneumoniae: a report of two cases.

We studied two patients with involvement of the central nervous system (CNS) associated with Mycoplasma pneumoniae. One patient had encephalitis and acute cerebellar ataxia, whereas the second had a mixed picture of encephalitic reaction superimposed on a disseminated malignancy of unknown origin. Specific IgM antibodies to M. pneumoniae were detected in the patients' sera but not in their cerebrospinal fluid. M. pneumoniae was repeatedly isolated by cultures from throat swabs and cerebrospinal fluid samples from both patients. Our patients add to previous reports suggesting that CNS involvement may result from direct invasion of the CNS by the pathogen.

Comparison of enzyme-linked immunosorbent assay and radioimmunoprecipitation test for detection of immunoglobulin A antibodies to Mycoplasma pneumoniae in nasal secretions.

An immunoglobulin A (IgA) enzyme-linked immunosorbent assay was developed and compared with the radioimmunoprecipitation test in determinations of IgA antibodies to Mycoplasma pneumoniae. Of 45 nasal secretions obtained from infected volunteers, 42 (93.4%) were positive and 2 (4.4%) were negative in both tests. The IgA enzyme-linked immunosorbent assay is at least as sensitive as the IgA radioimmunoprecipitation test but is simpler and safer and should therefore be preferred.

Mycoplasma pneumoniae is a frequent cause of exacerbation of bronchial asthma in adults.

The possible role of infection with Mycoplasma pneumoniae (MP) in exacerbation of bronchial asthma in adults was studied in 95 patients hospitalized due to acute asthma. Twenty (21%) of these patients had evidence of a recent MP infection as determined by the presence of high levels of MP-specific IgM antibodies. In addition, high levels of both IgM and IgG but not IgA serum immunoglobulins were observed in the MP-infected group as compared to a control group of 20 non-MP-infected asthmatics. Five out of 20 MP-infected asthmatics exhibited rheumatoid factor (RF) in their sera while patients in the control group were all negative for RF. It is concluded that MP infection may be significant in exacerbation of asthma in adults.

Induction of interleukin-2 and colony-stimulating factors in lymphoid cell cultures activated by mitogenic mycoplasmas.

Mycoplasmal infections often result in chronic inflammatory diseases that involve both local cell proliferation and chemotactic phenomena. To clarify the mechanisms responsible for these inflammatory reactions, we have examined whether nonspecific activation of host lymphocytes by mitogenic mycoplasmas triggers the production of lymphokines, which influence differentiation and proliferation of lymphocytes, macrophages and granulocytes. Mycoplasma pulmonis and M. neurolyticum were tested for their ability to induce production of interleukin-2 (IL-2) and colony-stimulating factors (CSF) by activated lymphoid cells. Mitogenic doses of membranes purified from M. pulmonis, as well as concanavalin A (ConA), induce rat lymphocytes to produce IL-2. Stimulation with the T cell mitogen ConA induces twice the level of IL-2 than does stimulation with M. pulmonis, which activates both rat B- and T-lymphocytes. Unlike these T cell mitogens, M. neurolyticum, which stimulates rat and mouse B-lymphocytes, lacks the ability to induce production of IL-2. Nevertheless, M. neurolyticum stimulates mouse spleen cells to produce in vitro factors capable of stimulating differentiation of bone marrow cells into colonies of macrophages and granulocytes.