M1 protein triggers a phosphoinositide cascade for group A Streptococcus invasion of epithelial cells

Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics. M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A Streptococcus (GAS) and also renders the bacterium resistant to phagocytosis. In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry. LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by Salmonella enterica serovar Typhimurium. Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS. To separate the influence of other streptococcal virulence factors from M protein, Lactococcus lactis was engineered to express M1 protein on its surface. L. lactis(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion. Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells. LY294002 and wortmannin inhibited these cellular changes. A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS. Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.     

First Case of Subcutaneous Phaeohyphomycosis Caused by Scytalidium lignicola in a Human

A case of subcutaneous phaeohyphomycosis in a human, involving the ankle and caused by Scytalidium lignicola, is described. The isolate was found to be sensitive to amphotericin B, 5-fluorocytosine, miconazole, and ketoconazole in vitro.     

The group B streptococcal C5a peptidase is both a specific protease and an invasin

The group B streptococcus (GBS) is a major cause of pneumonia, sepsis, and meningitis in neonates and a serious cause of mortality or morbidity in immunocompromised adults. Although these streptococci adhere efficiently and invade a variety of tissue-specific epithelial and endothelial cells, adhesins and invasins are still unknown. All serotypes of GBS studied to date express C5a peptidase (SCPB) on their surface. This investigation addresses the possibility that this relatively large surface protein has additional activities. Rabbit anti-SCPB serum inhibited invasion of lung epithelial A549 cells by the serotype Ia strain O90R, suggesting that SCPB is an invasin. This was confirmed by inserting an in-frame 25-amino-acid deletion into the scpB gene. Invasion of HEp2 and A549 human cell lines was significantly reduced by the mutation. Enzyme-linked immunosorbent assays were used to demonstrate that purified SCPB protein binds directly to HEp2 and A549 cells and also binds the extracellular matrix protein fibronectin. Binding was dose dependent and saturable. These results suggested that SCPB is one of several potential invasins essential for GBS colonization of damaged epithelium.     

Expression in non-Hodgkin's lymphoma of the bcl-2 protein associated with the t(14;18) chromosomal translocation

For many non-Hodgkin's lymphomas, the bcl-2 gene has been implicated as a likely proto-oncogene, since it is consistently located at or near the breakpoint sites of t(14;18) chromosomal translocations. To define the role of the protein product of the bcl-2 gene in lymphoid cancers, we used anti-bcl-2 antibodies to perform immunohistochemical studies of frozen sections of 136 tissue specimens affected by lymphoma or non-neoplastic lymphoid disorders. Immunoreactive bcl-2 protein was observed in the neoplastic cells in almost all the follicular lymphomas, whereas no bcl-2 protein was detected in follicles affected by non-neoplastic processes or in normal lymphoid tissue. Every tumor with molecular-genetic evidence of t(14;18) translocation expressed detectable levels of bcl-2 protein, regardless of whether the breakpoint was located in or at a distance from the bcl-2 gene. These data show consistent expression of a proto-oncogenic protein in a large proportion of non-Hodgkin's lymphomas and provide further support of a role for bcl-2 in the pathogenesis of all lymphomas with the t(14;18) karyotypic abnormality. Increased expression of bcl-2 after t(14;18) translocations may be a specific marker for B-cell cancers, and demonstration of the protein with use of anti-bcl-2 antibodies could be useful in the diagnosis of many non-Hodgkin's lymphomas.     

Expression of M type 12 protein by a group A streptococcus exhibits phaselike variation: evidence for coregulation of colony opacity determinants and M protein.

Three major categories of colony opacity were observed for natural variants of the M type 12 (M12) group A streptococcus strain CS24. Colony opacity variants that switched between two alternative categories at significantly high frequencies were identified and are referred to as switching between more opaque (Op+) and less opaque (Op-) phenotypes. Twenty lineages of such variants were derived for analysis and were assessed for resistance to phagocytosis, acid-extractable M12 antigen, and M12 mRNA, criteria which define the M protein-positive phenotype (M+). Transition from the M+ to the M protein-negative phenotype (M-) correlated with a change from Op+ to Op-. Reversion to the Op+ phenotype was accompanied by reversion to the M+ state in all variants except one and occurred at a higher frequency than the forward M+ to M- switch. These data demonstrate the existence of M12 protein phaselike switching in the group A streptococcus strain CS24. The discovery of an Op+ M- revertant confirmed that colony opacity and M protein can be expressed independently and are distinct gene products. We suggest that coregulation of colony opacity and M protein expression accounts for their association among descendents of strain CS24. Southern blot hybridization analyses of digested genomic DNA from 27 M- variants and 15 M+ revertants were performed with DNA probes containing M12 protein and adjacent upstream sequences. DNA deletions were identified only in two stable M- variants, approximately 1.3 and 1.4 kilobases upstream from the M12 gene, respectively, whereas all unstable M- variants lacked detectable rearrangements. This suggests that deletions within or adjacent to the structural gene are unlikely to be responsible for the reversible switch in M protein expression. However, the association with the stable M- phenotype and the location of these deletions, as well as two other deletions, approximately 0.5 kilobase upstream from the M12 promoter in two previously described variants of strain CS24 suggests that a second gene product is required for full expression of M12 protein synthesis in this strain.     

Monocytoid B-cell lymphoma: a study of 36 cases

We have studied 36 cases of monocytoid B-cell lymphoma (MBCL). We confirm the predilection for females (30 of 36; ratio, five women to one man). The median age was 65 years (range, 29 to 85 years). Monocytoid B-cell lymphoma characteristically involves peripheral lymph nodes (30 of 36) with a propensity for paraparotid or intraparotid nodes. Salivary glands were affected in five patients. Other extranodal sites of involvement included breast, thyroid, stomach, and soft tissue of chest wall. Eight patients manifested with Sj?gren's syndrome, one had systemic lupus erythematosus, one presented initially with Raynaud's phenomenon, and two had a monoclonal gammopathy. "Composite lymphomas" were encountered in seven patients. In addition, association with or progression to a higher-grade lymphoma, ie, mixed small and large cell (one) and large cell (six), was observed in seven patients and was associated with a more aggressive behavior of the lymphoma. Immunohistochemical studies performed on biopsy sections from 20 patients confirmed the B-cell nature of MBCL. An average reactivity of less than 10% of the monocytoid B cells with the proliferation marker Ki-67 was demonstrated, in keeping with the indolent behavior of MBCL. Despite our observation of follicular lymphomas frequently accompanying MBCL, the t(14;18) chromosomal translocation does not appear to play a pathogenetic role for MBCL, as determined by molecular studies for the t(14;18) chromosomal translocation and immunologic studies for the BCL2 protein. Our observations also provide support for the proposal that there is an overlap between MBCL and "MALT lymphomas" (those arising from mucosa-associated lymphoid tissue).     

Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk

To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 ± 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-¹⁴C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-³H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.     

Presence of the AZF region in a female with an idic(Y)(q11)

In a child with some features of Turner's syndrome, gonosomal mosaicism with an isodicentric nonfluorescent (idic)Y chromosome was detected (mos 45,X/47,X,idic(Y)(q11),idic(Y)(11)/46,X,idic(Y)(q11)). Histopathological examination showed streak gonads with some evidence of ovarian stroma and no sign of gonadoblastoma. Polymerase chain reaction (PCR) analysis in blood lymphocytes and gonadal tissues using primers of seven loci along the Y chromosome, including the sex determined region (SRY), azoospermia factor region (AZF) and the deleted in azoospermia ( DAZ ) gene was positive for all loci tested, confirming the isodicentric character of the Y chromosome and indicating the presence of the AZF region. It is remarkable that the existence of spermatogenesis controlling genes does not play an important role in gonadal development and differentiation in a phenotypic female with some Turner stigmata. The data presented here are briefly discussed with previously-described patients.     

Mapping of translocation breakpoints on the short arm of chromosome 19 in acute leukemias by in situ hybridization

Non-random translocation involving the short arm of chromosome 19 are frequently observed in acute leukemias. Recent studies have shown that the 19p13 genes E2A and LYLl, both of which encode helix-loop-helix proteins, lie at two different translocation breakpoints in acute lymphoblastic leukemias (ALL). The E2A gene is involved by the t(1;19)(q23;p13) in acute pre-B-cell leukemias and the LYL1 gene is structurally altered by a t(7;19)(q34;p13) in T-cell ALL. To assess the role of these genes in other leukemia-associated translocations we mapped their locations with respect to the t(11;19)(q23;p13) and t(4;19)(q21;p13) translocation breakpoints carried by T-ALL cell lines SUP-T13 and SUP-T8a, respectively. In situ hybridization studies indicated that the E2A and LYL1 genes are physically distinct from the t(4;19) and t(11;19) breakpoints. Using these and other 19p13 translocation breakpoints as landmarks, we established a partial physical map of 19p: 19pter-E2A-INSR-LYL1-[t(4;19)]-19cen. These data should help guide molecular studies to further characterize 19p13 breakpoints and mapping of genes in this chromosomal region.