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M Kotb

Publications and source records attributed to M Kotb.

At least 91 records · Page 5Linked to original sources
PubMed1993

Role of antigen-presenting cells in activation of human T cells by the streptococcal M protein superantigen: requirement for secreted and membrane-associated costimulatory factors.

The requirements for T-cell activation by the streptococcal superantigen (SAg), pepsin-extracted M protein from type 5 streptococci (pep M5), were studied by monitoring Ca2+ influx and cell proliferation. Cells from a pep M5-specific T-cell line showed no change in intracellular Ca2+ levels in response to pep M5 when added alone or with freshly isolated autologous antigen-presenting cells (APC). However, after being incubated with pep M5 overnight, the APC secreted soluble factors that together with pep M5 induced a marked increase in intracellular Ca2+ levels in pep M5-specific T cells or freshly isolated, purified T cells. Removal of the SAg from the overnight APC-derived supernatants resulted in loss of the Ca(2+)-mobilizing activity, which was restored within seconds of addition of SAg, suggesting that both the SAg and the soluble factors synergize to induce the Ca2+ influx. Induction of cell proliferation required additional signals inasmuch as the activated APC-derived supernatant failed to synergize with pep M5 to induce the proliferation of purified T cells and required the presence of phorbol myristate acetate for this activity. Metabolically inactive, fixed APC were impaired in their ability to present pep M5 to T cells. Presentation of pep M5 by fixed APC was, however, restored when the APC-derived soluble costimulatory factors were added to the culture. Our data suggest that pep M5-induced activation of T cells is dependent on APC-derived soluble factors and an APC membrane-associated costimulatory molecule(s). These interactions may be important in regulating the in vivo responses to M proteins, could contribute to the severity or progression of infections with Streptococcus pyogenes, and may influence the susceptibility of individuals to its associated nonsuppurative autoimmune sequelae.

Antigen-Presenting Cells↗
PubMed1993

Temporal relationship of cytokine release by peripheral blood mononuclear cells stimulated by the streptococcal superantigen pep M5.

We undertook this study to determine the quality, quantity, and temporal relationship of pep M5-induced cytokine release. The ability of pep M5 to stimulate interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) production by a T-cell-depleted, monocyte- and B-cell-enriched cell population was dependent on the presence of T cells. The requirement for T cells could be met by addition of exogenous gamma interferon (IFN-gamma). In the presence of IFN-gamma, pep M5 induced the release of TNF-alpha, IL-1, and IL-6, TNF-alpha levels peaked at 24 h, while IL-1 and IL-6 levels peaked at 48 h. pep M5 induced T cells to produce IFN-gamma, which may have accounted for the ability of the super antigen to induce the production of IL-1, IL-6, TNF-alpha, and TNF-beta by peripheral blood mononuclear cells (PBMC). The addition of excess IFN-gamma to cultures of pep M5 and PBMC did not further increase the release of these cytokines at 24 and 48 h but resulted in sustained higher levels at 72 h. Interestingly, TNF-beta production occurred only in the presence of pep M5 and exogenous IFN-gamma. The ability of pep M5 to induce cytokine production was compared with that of a potent super antigen, staphylococcal enterotoxin B (SEB). SEB was a 2- to 14-fold-more-potent inducer of IFN-gamma production. Furthermore, the profile of cytokine released by PBMC in response to this super antigen mimicked that seen with pep M5 in the presence of exogenous IFN-gamma. In conclusion, pep M5 induces the production of cytokines that are involved in immune regulation and inflammation. These cytokines also play a major role in human T-cell responses to this super antigen.

Adult↗
PubMed1993

A prospective study of postoperative fever--a clinical epidemiologic study.

During a 12 months period 693 patients undergoing surgical procedures were prospectively studied to investigate the etiology of postoperative fever. The overall rate of fever was similar for the three categories of surgical procedures studied. No cause of fever was found in 5%, 2.7% and 1.7% of patients who underwent clean, clean contaminated and contaminated surgical procedures.

Cross Infection↗
PubMed1992

Induction of interleukin 2 production but not methionine adenosyltransferase activity or S-adenosylmethionine turnover in Jurkat T-cells.

We have recently reported that methionine adenosyltransferase (MAT) in resting human peripheral blood T-cells is primarily present in the form of a precursor which we named lambda. This protein decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits of the enzyme increase. When resting cells are activated by phytohemagglutinin, the decrease in lambda and increase in alpha/alpha' occurs after interleukin 2 (IL-2) production and before DNA synthesis. The human T-leukemia cell line, Jurkat, is unique in its ability to produce IL-2 in response to exogenous stimuli such as T-cell mitogens and therefore provides a convenient model for studying biochemical reactions involved in T-cell activation. In this study the regulation of MAT activity and S-adenosylmethionine (AdoMet) in resting and activated Jurkat cells was investigated. Here we report that MAT activity in unstimulated Jurkat cells is about 10- and 3-fold higher than the activity in resting and activated peripheral blood mononuclear cells, respectively. Activation of Jurkat cells with phytohemagglutinin resulted in increased IL-2-production, but not an increase in MAT activity. Identical results were obtained using freshly isolated cells from acute lymphoblastic leukemia patients. AdoMet utilization and pool size were approximately 3- and 10-fold higher, respectively, in Jurkat cells compared to peripheral blood mononuclear cells, and both parameters were unaffected by phytohemagglutinin stimulation. Jurkat MAT was determined to be structurally indistinguishable from enzyme from T- or B-leukemia cells but was different from resting, normal T-cells in that it lacked the lambda form. Furthermore, unlike MAT in resting T-cells, the relative amounts of the alpha, alpha', and beta subunits of the enzyme did not change throughout the course of IL-2 induction. We conclude that AdoMet metabolism and MAT activity in Jurkat cells are constitutively high and that induction of IL-2 synthesis in these cells is independent of changes in AdoMet synthesis or turnover. The lack of the lambda form and the difference in MAT regulation between leukemic T-cells and peripheral blood mononuclear cells may be exploited in the design of specific chemotherapeutic agents.

Enzyme Precursors↗
PubMed1992

Changes in the relative amount of subunits of methionine adenosyltransferase in human lymphocytes upon stimulation with a polyclonal T cell mitogen.

Activation of resting human peripheral blood T lymphocytes by the lectin phytohemagglutinin results in an increase in methionine adenosyltransferase (MAT) activity, accompanied by an increase in the amount of the alpha/alpha' catalytic subunits of the enzyme. In contrast, the amount of the noncatalytic beta subunit remains constant throughout the course of the response. Using both polyclonal antibodies to the holoenzyme and monoclonal antibodies to the alpha/alpha' subunits, we detected a cross-reactive 68-kDa protein, which we refer to as lambda. This protein is present in high abundance in resting T cells but decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits increase. The decrease in lambda and increase in alpha/alpha' occurs after interleukin-2 production and before DNA synthesis. lambda virtually disappears when the cells are actively dividing. Several continuous T cell lines (HPB-ALL, MOLT-4, and Jurkat) as well as a freshly isolated T cell leukemia (ALL-2) had no detectable lambda. The Km for L-methionine for enzyme from resting peripheral blood mononuclear cells was 19-23 microM, which is 3-8-fold higher than purified MAT from fresh leukemic cells or enzyme from Jurkat cells, both of which have a Km of 3.5-3.8 microM. Kinetic analysis of enzyme activity from activated peripheral blood mononuclear cells suggested the presence of two forms of enzyme catalyzing the synthesis of AdoMet. After separation of lambda from the alpha and beta subunits by hydrophobic chromatography, it was determined that lambda has MAT activity but that it is significantly less active than the form containing the alpha subunit. It therefore appears that in resting T cells MAT is sequestered as a less active form. We hypothesize that lambda is a precursor to the catalytic subunits of human lymphocyte MAT and propose that the transition from lambda to alpha/alpha' may be important in the response of T cells to mitogenic signals.

Antibodies, Monoclonal↗
PubMed1992

Metabolically active antigen presenting cells are required for human T cell proliferation in response to the superantigen streptococcal M protein.

M protein from type 5 group A streptococci has been identified as a member of the family of polyclonal T cell activators termed superantigens because it preferentially stimulates T cells bearing specific V beta elements of the T cell receptor (TCR). In this study the molecular and cellular requirements for presentation of this protein to T cells were investigated. Only accessory cells (AC) expressing class II major histocompatibility complex (MHC) molecules were capable of supporting T cell activation in response to a 22 kDa fragment of M protein (pep M). Despite the need for class II elements, processing of pep M5 by the antigen-presenting cells (APC) was not required for T cell proliferation induced by pep M5. Fixation of APC by paraformaldehyde (PF) treatment impaired their ability to induce optimal T cell proliferation in response to pep M5 without significantly affecting interleukin (IL-2) production. In contrast, PF-fixation of cells from the B cell lymphoma line, Raji, did not affect their ability to present pep M5 to human T cells. Addition of rIL-1 and IL-6 to PF-treated APC restored pep M5-induced blastogenesis. Our data suggest that pep M5 directly associates with HLA class II molecules forming a complex that can induce IL-2 production but not optimal proliferation by T cells. Additional signals provided by the AC are required to trigger optimal T cell proliferation in response to this superantigen.

Antigen-Presenting Cells↗
PubMed1992

Distinct T-cell receptor V beta gene usage by human T lymphocytes stimulated with the streptococcal pyrogenic exotoxins and pep M5 protein.

A number of streptococcal products, including the streptococcal pyrogenic exotoxin (SPE) types A, B, and C as well as a 22-kDa fragment of M type 5 protein (pep M5), are potent stimulants of human T-lymphocyte blastogenesis and belong to the newly designated family of superantigens. The V beta usage of human T cells stimulated with these toxins was investigated by using the polymerase chain reaction. We demonstrate that SPE A, B, and C as well as pep M5 stimulate the proliferation of T cells in a dose-dependent manner. pep M5 stimulates cells bearing V beta 2, 4, and 8 elements of the T-cell receptor (TCR), whereas SPE A stimulates TCR V beta 2-, 12-, 14-, and 15-bearing cells. SPE B stimulated only cells expressing TCR V beta 8 elements, while SPE C stimulated cells expressing V beta 1, 2, 5.1, and 10. These studies reveal that the preferential usage of particular V beta elements is distinct for these different superantigens, which may be important in the pathogenesis of various streptococcal diseases.

Antigens, Bacterial↗
PubMed1991

T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein.

M proteins, the major virulence factor of group A streptococci, have been implicated in the pathogenesis of acute rheumatic fever (ARF) and other streptococcal related autoimmune diseases. A 22-kD fragment of M type 5 protein is a potent stimulant of human T cells and has recently been shown by our laboratory to belong to the newly designated family of superantigens. Using flow cytometry and the polymerase chain reaction, we demonstrate that this molecule reacts with subsets of human T cells expressing specific T cell receptor (TCR) V beta elements, namely V beta 2, 4, and 8. We employed similar techniques to analyze the TCR V alpha usage of pep M5-stimulated T cells. These studies revealed that the preferential usage of particular V alpha elements is not specific for the superantigen; rather, it may reflect the repertoire of the individual being tested. The expansion of a large number of T cells bearing specific TCR V beta sequences by M protein may account for its role in mediating the pathogenesis of post-streptococcal diseases. Furthermore, the preferential usage of TCR V alpha elements in certain individuals may be an important factor that predisposes them to development of self-reactivity.

Antigens, Bacterial↗
PubMed1991

Regulation of S-adenosylmethionine synthetase activity in cultured human lymphocytes.

S-Adenosylmethionine (AdoMet), inorganic pyrophosphate (PPi) and inorganic phosphate (Pi) are potent product inhibitors of AdoMet synthetase and have been postulated to play a role in increasing AdoMet levels and turnover in peripheral blood mononuclear cells (PBM) after stimulation with phytohemagglutinin (PHA). Measurements of these metabolites in PHA-stimulated PBM showed the expected 2- to 3-fold increases in AdoMet after 8 h, and smaller increases in PPi and Pi. Since the kinetic model requires substantial decreases in PPi and Pi in response to PHA, product inhibition cannot explain the observed changes in AdoMet metabolism in this system. A 2.5-fold increase in AdoMet synthetase catalytic activity was found in crude extracts of PBM within 8 h of PHA-stimulation and probably accounts for increased cellular levels and utilization of AdoMet. Immunochemical analyses with a monoclonal antibody specific for the alpha/alpha' subunits of human lymphocyte AdoMet synthetase showed that these increases in catalytic activity were not associated with increases in immunoreactive protein. The ratio of catalytic activity to immunoreactivity in stimulated cells was 4-fold higher than in unstimulated controls and almost identical to that found in extracts from the human B-lymphocyte line WI-L2. Unstimulated PBM appear to contain substantial amounts of AdoMet synthetase alpha/alpha' subunit with reduced or absent catalytic activity, which can be activated by PHA-stimulation.

Adenosine Triphosphate↗
PubMed1991

Production of a factor, or factors, suppressing IL-2 production and T cell proliferation by Sertoli cell-enriched preparations. A potential role for islet transplantation in an immunologically privileged site.

Isolated islet allografts survive indefinitely in the abdominal testis of nonimmunosuppressed diabetic rats. The predominant feature of these testes is that the presence of Sertoli cells, but not Leydig cells, is required for extended survival of the islet allografts. Sertoli cells cultures were therefore established in vitro and we examined the effects of the conditioned media on Con A--stimulated spleen lymphocyte proliferation. These studies revealed that a product(s) secreted by Sertoli cells inhibits Con A-stimulated lymphocyte proliferation in a dose-dependent manner. The synthesis of this product is both temperature-dependent, occurring predominantly at 37 degrees C, and hormone-dependent, requiring the presence of follicle stimulating hormone, in the culture medium. We further examined the mechanism of inhibition of lymphocyte proliferation and showed that Sertoli cell-enriched media inhibit the production of IL-2 in a dose-dependent manner. Furthermore, the finding that the addition of exogenous IL-2 is not able to reverse this inhibition indicates that the Sertoli cell-enriched media inhibit both IL-2 production and IL-2 responsiveness of T lymphocytes.

Animals↗
PubMed1991

Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.

A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.

Amino Acid Sequence↗
PubMed1990

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Amino Acid Sequence↗
PubMed1990

Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen.

Stimulation of T cells by superantigens has been reported to be dependent on the presence of APC where binding to class II molecules is a prerequisite to recognition by the TCR. We examined the response of human T cells and a leukemic T cell line, Jurkat to the superantigen, streptococcal M protein. We show that immobilized or cross-linked streptococcal M protein stimulates Jurkat cells (V beta 8), but not normal purified human T cells, to produce IL-2. Activation of purified T cells by this superantigen required costimulatory signals provided by PMA, IL-1, and IL-6. These cytokines and growth factors alone can induce IL-2 production by T cells; however, proliferation occurred only in the presence of superantigen, which together with PMA, IL-1, and IL-6 induced the expression of IL-2R alpha on T cells. Similar results were obtained when the response of purified T cells to another known superantigen, staphylococcal enterotoxin B were examined, indicating that this phenomenon is not unique to M protein. Superantigens interact with a large number of T cells with particular V beta, and thus provide excellent models for studies of the role of biochemical events and signal transduction in T cell activation. Understanding these events may also explain the pathogenesis of autoimmune diseases associated with certain superantigens, such as streptococcal M protein that is thought to be involved in rheumatic fever and rheumatic heart disease.

Antigen-Presenting Cells↗
PubMed1990

Superantigenicity of streptococcal M protein.

M proteins that define the serotypes of group A streptococci are powerful blastogens for human T lymphocytes. The mechanism by which they activate T cells was investigated and compared with the conventional T cell mitogen phytohemagglutinin, and the known superantigen staphylococcal enterotoxin B. Although major histocompatibility complex (MHC) class II molecules are required for presentation, there is no MHC restriction, since allogeneic class II molecules presented the bacterial protein to human T cells. Type 5 M protein appears to bind class II molecules on the antigen-presenting cells and stimulate T cells bearing V beta 8 sequences. Our results indicate that this streptococcal M protein is a superantigen and suggest a possible mechanism of its role in the pathogenesis of the postinfectious autoimmune sequelae.

Antibodies, Monoclonal↗
PubMed1990

Regulation of human lymphocyte S-adenosylmethionine synthetase by product inhibition.

A steady-state kinetic analysis of human S-adenosylmethionine synthetase indicates that the reaction is Bi Ter with ordered addition of ATP and L-methionine and release of S-adenosylmethionine as the first product. Pyrophosphate and phosphate are then released randomly. I-Parabolic inhibition by phosphate with respect to ATP indicates that this product must bind to more than one site. A model in which phosphate binds to the pyrophosphate site gives a rate equation that is consistent with the kinetic data. Values have been determined for those constants in the equation that are large enough to evaluate, and the in vitro kinetic behavior of S-adenosylmethionine synthetase can be predicted at substrate and product concentrations that are expected intracellularly. Inhibition by combinations of products, especially pyrophosphate and phosphate, is synergistic. Of particular interest is the ability of pyrophosphate and phosphate to increase the sensitivity of the enzyme to inhibition by S-adenosylmethionine. This phenomenon may play a role in regulating steady-state cellular concentrations of S-adenosylmethionine.

Diphosphates↗
PubMed1990

Differential signal requirements in T-cell activation by mitogen and superantigen.

The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca2+ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca2+ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.

Antigen-Presenting Cells↗