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G M Winslow

Publications and source records attributed to G M Winslow.

18 recordsLinked to original sources
PubMed2001

Outer membrane protein-specific monoclonal antibodies protect SCID mice from fatal infection by the obligate intracellular bacterial pathogen Ehrlichia chaffeensis.

Previous studies of Ehrlichia chaffeensis infection in the mouse have demonstrated that passive transfer of polyclonal Abs from resistant immunocompetent mice to susceptible SCID mice ameliorated infection and disease, even when Abs were administered during established infection. To identify particular Abs that could mediate bacterial clearance in vivo, E. chaffeensis-specific mAbs were generated and administered to infected SCID mice. Bacterial infection in the livers was significantly lowered after administration of either of two Abs of different isotypes (IgG2a and IgG3). Moreover, repeated administration of one Ab (Ec56.5; IgG2a) rescued mice from an otherwise lethal infection for at least 5 wk. Both protective Abs recognized the E. chaffeensis major outer membrane protein (OMP)-1g. Further studies revealed that both Abs recognized closely related epitopes within the amino terminus of the first hypervariable region of OMP-1g. Analyses of human sera showed that E. chaffeensis-infected patients also generated serological responses to OMP-1g hypervariable region 1, indicating that humans and mice recognize identical or closely related epitopes. These studies demonstrate that OMP-specific mAbs can mediate bacterial elimination in SCID mice, and indicate that Abs, in the absence of cell-mediated immunity, can play a significant role in host defense during infection by this obligate intracellular bacterium.

Amino Acid Sequence↗
PubMed2000

Detection of viral superantigen-class II MHC interactions at the cell surface.

Mouse mammary tumor virus superantigens (vSAgs) must bind to class II MHC proteins to activate T cells. Although direct interaction of vSAgs with class II proteins has been demonstrated biochemically, the details of this interaction are largely unresolved. To facilitate the study of class II-vSAg interactions, a sensitive assay has been developed that can detect binding of vSAgs to class II proteins on the cell surface. The assay measures changes in vSAg surface expression upon enzymatic removal of a co-expressed glycan-phosphatidyl inositol-anchored form of the class II molecule IE(k). Because the vSAgs are synthesized as integral membrane proteins that undergo proteolytic processing, an event that is likely required to eliminate membrane tethering, the data provide further evidence that a proteolytic fragment of vSAg is bound to class II proteins on the cell surface. The assay was utilized to identify mutant vSAgs that either did not associate with IE(k) molecules, or did not undergo furin-dependent proteolytic processing. Class II protein binding was detected using vSAg7 mutants that lacked furin endoprotease recognition sites, and after expression of vSAg in furin-deficient cells. The data demonstrate that furin-mediated processing is not necessary for association of vSAg7 with class II proteins, supporting previous studies that have indicated a role for alternative endoproteases in vSAg activation. However, because class II interactions were also noted in the apparent absence of proteases that are known to activate vSAgs, the data suggest that yet other proteases may process vSAgs in a fashion that does not necessarily lead to activation of T cells.

Animals↗
PubMed2000

Antibody-mediated elimination of the obligate intracellular bacterial pathogen Ehrlichia chaffeensis during active infection.

It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection by Ehrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6 scid mice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms.

Animals↗
PubMed2000

Intercellular transfer of a soluble viral superantigen.

Mouse mammary tumor virus (MMTV) superantigens (vSAgs) can undergo intercellular transfer in vivo and in vitro such that a vSAg can be presented to T cells by major histocompatibility complex (MHC) class II proteins on antigen-presenting cells (APCs) that do not express the superantigen. This process may allow T-cell activation to occur prior to viral infection. Consistent with these findings, vSAg produced by Chinese hamster ovary (CHO) cells was readily transferred to class II IE and IA (H-2(k) and H-2(d)) proteins on a B-cell lymphoma or mouse splenocytes. Fixed class II-expressing acceptor cells were used to demonstrate that the vSAg, but not the class II proteins, underwent intercellular transfer, indicating that vSAg binding to class II MHC could occur directly at the cell surface. Intercellular transfer also occurred efficiently to splenocytes from endogenous retrovirus-free mice, indicating that other proviral proteins were not involved. Presentation of vSAg7 produced by a class II-negative, furin protease-deficient CHO variant (FD11) was unsuccessful, indicating that proteolytic processing was a requisite event and that proteolytic activity could not be provided by an endoprotease on the acceptor APC. Furthermore, vSAg presentation was effected using cell-free supernatant from class II-negative, vSAg-positive cells, indicating that a soluble molecule, most likely produced by proteolytic processing, was sufficient to stimulate T cells. Because the membrane-proximal endoproteolytic cleavage site in the vSAg (residues 68 to 71) was not necessary for intercellular transfer, the data support the notion that the carboxy-terminal endoproteolytic cleavage product is an active vSAg moiety.

Animals↗
PubMed1998

Self-restricted dual receptor memory T cells.

Enhanced immune responses during secondary exposure to Ag result from the development of memory cells. In the present report we show that stimulation through one receptor on dual receptor CD4 cells can promote the generation of T cells capable of giving a memory response through the second receptor, even though the cells had not been previously exposed to the Ag recognized by the second receptor. Cloned cells generated from dual receptor memory T cells proliferated and secreted the same lymphokines after stimulation with either Ag. Independent recognition of both Ags by distinct TCRs was shown by production of variants that had lost either Ag specificity along with the corresponding TCR. Recognition of both Ags is MHC restricted, since the cells recognize Ag presented by self, but not non-self, MHC class II molecules. These results raise the possibility that one potential mechanism of maintaining specific memory to a given Ag is through stimulation by an unrelated Ag via the second TCR.

Animals↗
PubMed1998

Redundant proteolytic activation of a viral superantigen.

Proteolytic activation of viral superantigens (vSAgs)4 expressed in Chinese hamster ovary (CHO) cells is required for T cell stimulation, and is mediated primarily by the protein convertase (PC) furin. Three PC recognition sites are highly conserved in vSAgs, but it was not known which sites are required for PC dependent vSAg activation. Moreover, because the PC recognition sites are not conserved in all functional vSAgs it was possible that activation could occur by processing at any of several sites. To identify the location(s) where processing of vSAg7 generates an active superantigen, each of two PC recognition sites, and a third related site were altered by in vitro mutagenesis, and the mutant proteins were tested for their abilities to activate T cells. Mutation of the PC recognition site at position 68-71 in vSAg7 had no effect on its ability to activate T cells. Mutation of the processing site at position 169-172 completely abolished T cell activation, and indicated that cleavage at this position was obligatory for proteolytic activation of vSAg7. However, introduction of a PC recognition site at position 192-195, a position that in many other vSAgs encodes a PC recognition site, restored activity to a vSAg7 protein that lacked a recognition site at position 169-172. The data revealed that processing of vSAgs at either position 169-172 or 192-195 was sufficient for vSAg7 activation, and explain how vSAgs that lack some PC recognition sites can be activated by proteolytic processing.

Animals↗
PubMed1998

Infection of the laboratory mouse with the intracellular pathogen Ehrlichia chaffeensis.

To determine the basis of susceptibility and resistance to human monocytic ehrlichiosis (HME), immunocompetent and immunocompromised mice were infected with Ehrlichia chaffeensis and bacterial loads were measured by PCR and by immunohistochemistry. Immunocompetent (C. B-17 and C57BL/6) mice cleared the bacteria within 10 days, but immunocompromised SCID and SCID/BEIGE mice developed persistent infection in the spleen, liver, peritoneal cavity, brain, lung, and bone marrow and became moribund within 24 days. Both immunocompromised strains lack T and B lymphocytes, but the SCID/BEIGE strain is also deficient in natural killer (NK) cell function. During advanced stages of disease, the infections were associated with wasting, splenomegaly, lymphadenopathy, liver granulomas and necroses, intravascular coagulation, and granulomatous inflammation. Histochemical and immunohistochemical localization studies confirmed the presence of bacteria in tissues, and viable bacteria were cultured from infected animals. The data reveal that T and/or B cells play an essential role during resistance of immunocompetent mice to infection with E. chaffeensis and demonstrate the utility of immunocompromised mice as an experimental model for the study of HME.

Animals↗
PubMed1996

Proteolytic processing activates a viral superantigen.

Mouse mammary tumor virus (MMTV) superantigens (vSAg) undergo proteolytic processing at residues that have been demonstrated in vitro to be recognition sites for the endoprotease furin. To examine the role of furin in the presentation of vSAg7 to T cells, the vSAg7 and class II MHC IEk genes were introduced into Chinese Hamster Ovary (CHO) cells (furin-positive) and into a furin-negative CHO variant (FD11). Both transfected cell lines efficiently presented peptide antigen and bacterial superantigens to T cell hybridomas. However, while the furin-positive cells presented vSAg7 well, the furin-negative cells presented poorly. Transient transfection of the furin-negative cells with an expression plasmid containing the furin gene restored the ability to present vSAg7 efficiently. The marginal presentation of vSAg7 observed using the furin-negative transfectants was eliminated after culture with the protease inhibitor leupeptin, suggesting that one or more endoproteases other than furin have a detectable but limited capacity to proteolytically activate vSAg7. Biochemical analyses revealed that vSAg7 was largely unprocessed in the absence of furin. Thus, viral superantigens, unlike bacterial superantigens, require proteolytic processing to activate T cells.

Animals↗
PubMed1995

Proteolytic processing is required for viral superantigen activity.

The mouse mammary tumor virus-7 superantigen (vSAG7) is proteolytically processed in B cells at as many as three positions. Proteolytic processing appears to be important for superantigen activity because a processed form of vSAG7 was predominant among those forms that were found to bind to major histocompatibility complex class II molecules. To determine the functional significance of proteolytic processing, a mutation was introduced in vSAG7 at one of the sites where proteolytic cleavage is thought to take place in B cells. Elimination of the putative processing site at position 171 abrogated detectable vSAG7 surface expression in B cells, indicating that proteolytic processing is required for vSAG7 function. Coexpression in insect cells of vSAG7 and furin, a proprotein-processing enzyme, also demonstrated that furin could process vSAG7 at position 171.

Amino Acid Sequence↗
PubMed1994

Processing and major histocompatibility complex binding of the MTV7 superantigen.

Mouse mammary tumor viruses produce superantigens (vSAGs) which interact with class II major histocompatibility complex (MHC) proteins and stimulate T cells. vSAGs are synthesized as Type II membrane proteins, but at least one of these proteins (vSAG7) is found on the cell surface in a proteolytically processed form. Monoclonal antibodies (MAbs) were used to characterize vSAG7 and its binding to class II molecules. vSAG7 is synthesized in the endoplasmic reticulum (ER) as a 45 kd glycoprotein containing N-asparagine-linked oligomannosyl carbohydrates. vSAG7 transits the golgi complex, where it is modified by the addition of complex-type glycans and proteolysed at three positions. After proteolysis, the amino and carboxyl termini remain noncovalently associated. The ER, golgi, and surface forms of vSAG7 are stably bound to class II, but one of the proteolysed forms comprises the majority of the class II-bound material.

Amino Acid Sequence↗
PubMed1993

Direct evidence for the role of COOH terminus of mouse mammary tumor virus superantigen in determining T cell receptor V beta specificity.

It has recently been shown that open reading frames in the 3' long terminal repeats of mouse mammary tumor viruses encode superantigens. These viral superantigens (vSAGs) stimulate most T cells expressing appropriate V beta s almost regardless of the rest of the variable components of the T cell receptors (TCR) expressed by those cells. vSAGs produce a type II integral membrane protein with a nonessential short cytoplasmic domain and a large glycosylated extracellular COOH-terminal domain, which is predicted to interact with major histocompatibility complex class II molecules and the TCR. The transmembrane region of vSAG also has an internal positively charged lysine residue of unknown significance. A set of chimeric and mutant vSAG genes has been used in transfection experiments to show that only the extreme COOH-terminal portion of vSAGs determine their TCR V beta specificities, and to show that the lysine residue in the transmembrane domain is not essential for the function of vSAG.

Amino Acid Sequence↗
PubMed1993

The bacterial and mouse mammary tumor virus superantigens; two different families of proteins with the same functions.

In conclusion, the bacterial toxins are completely unlike the MTV superantigens in primary sequence and structure. The former are soluble globular proteins which do not have to be proteolytically cleaved before they act. The latter are synthesized as type II membrane proteins and may be clipped before they reach the cell surface and act to stimulate T cells. Table III summarizes the similarities and differences between the two sets of superantigens. The most notable quality of these molecules is that both sets of families have developed strategies whereby they bind to Class II and engage V beta. As far as the microorganisms which produce them are concerned, these two properties appear to be essential since they are absolutely conserved over proteins of a number of different structures. Several questions can now be addressed as follows. a. Why do all known superantigens bind to Class II? For the microorganism which produces them, the function of superantigens appears to be T-cell and perhaps directly or indirectly B-cell and macrophage stimulation. Activation of virgin T cells requires engagement with antigen plus MHC on professional antigen-presenting cells. Unlike other cell surface proteins, for example Class I, most Class II in animals is expressed on such cells. Therefore it is likely that superantigens have evolved to engage Class II because presentation to T cells by Class II-bearing cells offers the superantigen the best chance of activating its target T cells. b. Why do superantigens engage TCR V beta and not V alpha or CD3? It is possible that superantigens bind to the V beta portion of the TCR rather than V alpha because the latter does not have a consistently well exposed face for engagement. The fact that it is perhaps relatively easier to produce anti-V beta rather than anti-V alpha antibodies supports this idea. We have shown that N-glycosylation of V beta can interfere with recognition by vSAGs (Pullen et al. 1991), perhaps glycosylation of V alpha tends to conceal otherwise available sites. As far as C beta, C alpha or CD3 engagement is concerned, this may be just too dangerous for MTVs. The role of MTVs SAgs in the life history of the virus seems to be to stimulate T cells in the suckling recipient and thereby create a pool of activated lymphocytes in which the virus may survive until the mouse gives birth and transmits the virus to her own progeny (Hainaut et al. 1990, Golovkina et al. 1992).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Detection and biochemical characterization of the mouse mammary tumor virus 7 superantigen (Mls-1a).

Mouse mammary tumor viruses encode superantigens that bind to class II major histocompatibility complex proteins and engage T cells that bear particular V beta s. Among these superantigens is the long known, but previously uncharacterized, Mls-1a product, encoded by Mtv-7. Using a monoclonal antibody, we detect the Mtv-7 superantigen on the surface of activated B cells, but not on T cells or resting B cells. The superantigen is synthesized as a 45 kd transmembrane glycoprotein precursor, but is proteolytically processed to yield an 18.5 kd surface protein that we suggest is the functional form of the superantigen.

Animals↗
PubMed1989

Transcriptional activation by the Antennapedia and fushi tarazu proteins in cultured Drosophila cells.

Drosophila homeodomain proteins bind to specific DNA sequences in vitro and are hypothesized to regulate the transcription of other genes during development. Using a cotransfection assay, we have shown that homeodomain proteins encoded by the homeotic gene Antennapedia (Antp) and the segmentation gene fushi tarazu, as well as a hybrid homeodomain protein, are activators of transcription from specific promoters in cultured Drosophila cells. Sequences downstream of the Antp P1 and Ultrabithorax transcription start sites mediate the observed activation. A TAA-rich DNA sequence to which the Antp protein binds in vitro is sufficient to confer regulation on a heterologous promoter. The results demonstrate that homeodomain proteins are transcriptional regulators in vivo and that in cultured cells, different homeodomain-containing proteins can act upon a common sequence to modulate gene transcription.

Animals↗
PubMed1988

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo.

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.

Abdomen↗
PubMed1987

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo.

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.

Animals↗
PubMedDate not supplied

Maternal control of Drosophila segmentation gene expression.

Several genes have been identified that are involved in establishing the segmented body pattern during development of the fruit-fly Drosophila melanogaster. These fall into several classes on the basis of the kind of alteration to the wild-type segmentation pattern observed in mutant embryos. For example, mutations of the pair-rule class, such as fushi tarazu (ftz), cause the deletion of pattern elements with a two-segment periodicity; those of the gap class, such as knirps, cause the deletion of contiguous groups of segments. The availability of antibodies against the ftz protein has allowed its spatial pattern of expression to be studied during the development of wild-type and mutant embryos. The aim of the latter kind of experiment is to investigate possible interactions between these important genes. We have recently reported that knirps mutations cause a striking alteration to the pattern of transverse stripes of ftz expression usually seen during embryogenesis. Knirps is a zygotically-expressed gene, but recently a class of maternally-active genes has been identified that causes similar defects in pattern formation. We have now investigated the pattern of ftz expression in mutants of this class and have found that while they do have features seen in knirps mutants, they also exhibit significant differences between the different mutations reflecting the distinct but overlapping domains of gene activity. These observations demonstrate that maternally-active segmentation genes regulate zygotic gene expression, and that some of their effects on ftz may be directed through the knirps gene.

Animals↗