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Biomedical subjects

M Kotb

Publications and source records attributed to M Kotb.

At least 19 recordsLinked to original sources
PubMed1999

The use of positive B cell flow cytometry crossmatch in predicting rejection among renal transplant recipients.

We performed retrospective flow cytometry crossmatch (FCXM) on 106 renal graft recipients who were transplanted based on current T cell negative serologic crossmatch. T and B cell FCXMs were performed on current and historical peak reactive post-transplant sera using 1024-channel flow cytometer and the shift in median channel fluorescence (SMCF) over the negative control was calculated. Cut-off values for a positive T and B crossmatch, > 40 and > 80 SMCF, respectively, were determined based on previous retrospective analysis of the data in the context of clinical outcome in our center, and were 1.5 times the standard deviation (SD) above the mean median channel fluorescence (MCF) of normal sera controls. The 1-yr graft survival was 95% for the total group of patients studied, and 87% for the recipients who had a positive T cell FCXM. To focus on the influence of a positive B cell FCXM on the incidence of rejection, primary transplant recipients who had a negative T cell FCXM (n = 81) were studied. Fifteen of 30 (50%) recipients with a positive B cell FCXM experienced at least one rejection episode within the first year. By contrast, only 15 of 51 (29.4%) of patients with a negative B cell FCXM experienced rejection (p = 0.05). The mean B cell SMCF in the group of patients who had no rejections was 45 +/- 59, while that of the group of patients who experienced at least one rejection was 97 +/- 97 (p = 0.012). By comparison, the rejection rate among the retransplant patients was 44.4%, and the mean B cell SMCF in the group with rejection was 94 +/- 75 while it was 5 +/- 7 among retransplant patients who did not have rejection (p = 0.031). Eighty-six percent of sensitized (panel reactivity antibodies (PRA) > 10%) patients who had a B positive/T negative FCXM experienced rejection, compared to 33% (n = 6 out of 16) of the B negative/T negative sensitized patients (p = 0.03). Furthermore, 62% (n = 13 out of 21) of donor-recipient mismatched patients with a B positive/T negative FCXM experienced rejection, compared to 38% (n = 13 out of 35) of patients with T negative/B negative FCXM who were similarly mismatched (p = 0.064). These data demonstrate the value of a positive B cell FCXM for predicting post-transplant rejections particularly when evaluated in the context of prior sensitization and/or DR mismatching. Our results suggest that B cell FCXM may have significant clinical implications, justifying its use in post-transplant management of recipients who have other risk factors of rejection.

Adult↗
PubMed1999

Risk factors in the pathogenesis of invasive group A streptococcal infections: role of protective humoral immunity.

An impressive change in the epidemiology and severity of invasive group A streptococcal infections occurred in the 1980s, and the incidence of streptococcal toxic shock syndrome cases continues to rise. The reason for the resurgence of severe invasive cases remains a mystery-has there been a change in the pathogen or in host protective immunity? To address these questions, we have studied 33 patients with invasive infection caused by genotypically indistinguishable M1T1 strains of Streptococcus pyogenes who had different disease outcomes. Patients were classified as having severe (n = 21) and nonsevere (n = 12) invasive infections based on the presence or absence of shock and organ failure. Levels of anti-M1 bactericidal antibodies and of anti-streptococcal superantigen neutralizing antibodies in plasma were significantly lower in both groups than in age- and geographically matched healthy controls (P < 0.01). Importantly, the levels of these protective antibodies in plasma samples from severe and nonsevere invasive cases were not different. Together the data suggest that low levels of protective antibodies may contribute to host susceptibility to invasive streptococcal infection but do not modulate disease outcome. Other immunogenetic factors that regulate superantigen responses may influence the severity of systemic manifestations associated with invasive streptococcal infection.

Animals↗
PubMed1998

Differential effect on polyamine metabolism in mitogen- and superantigen-activated human T-cells.

Polyamines are important for regulation of lymphocyte differentiation and proliferation. Mitogens induce synthesis of ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis. Since mitogens stimulate T-cells by non-physiological routes, the role of polyamine metabolism in T-cell receptor (TCR)-mediated T-cell activation has not been adequately evaluated. The effect of phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) on T-cell ODC and polyamine synthesis was compared. ODC activity was 6-11-fold higher in PHA compared to SEB stimulated T-cells. These differences were not attributed to differences in the magnitude of T-cell proliferation. Kinetics of ODC and polyamine synthesis were also different in PHA- and SEB-stimulated T-cells. In PHA-stimulated cells ODC levels and the induction of putrescine and spermidine synthesis peaked 6 h prior to peak IL-2 production, while in SEB-stimulated cells, ODC levels and polyamine synthesis peaked 6-12 h after IL-2 production. Differences in the temporal relationship between IL-2 production and polyamine induction in mitogen- versus superantigen-stimulated cells may account for the significant inhibition of the proliferative response by alpha-difluoromethylornithine following PHA but not SEB stimulation. Polyamine metabolism is regulated differently in T-cells stimulated via TCR engagement than with polyclonal mitogens.

Eflornithine↗
PubMed1998

Superantigens of gram-positive bacteria: structure-function analyses and their implications for biological activity.

Just as we thought that we know everything about superantigens, new molecular and structural studies indicate that we have only just begun to unravel the secrets of these fascinating molecules. Recent structure-function analysis of superantigens from Gram-positive bacteria, with emphasis on their interaction with major histocompatibility complex molecules, could help us decipher the role of superantigens in disease, identify host factors that potentiate their effects and design drugs that specifically block their activity.

Amino Acid Sequence↗
PubMed1998

Varying titers of neutralizing antibodies to streptococcal superantigens in different preparations of normal polyspecific immunoglobulin G: implications for therapeutic efficacy.

Inasmuch as normal intravenous polyspecific immunoglobulin G (IVIG) neutralizes the activity of a wide spectrum of superantigens, it may be an efficient adjunctive therapy for diseases associated with superantigen-producing organisms, including severe group A streptococcal diseases. The neutralizing activity against purified superantigens, streptococcal pyrogenic exotoxins (Spe), and a mixture of superantigens present in culture supernatant of clinical group A streptococcal isolates was determined for five IVIG preparations. A significant variation among different IVIG preparations (P < .05) and different lots of the same IVIG brand (P < .044) was found. Neutralization of SpeA activity was significantly lower than that of other streptococcal superantigens (P < .05); however, there was no correlation between SpeA binding and SpeA neutralizing activity in different IVIGs. Plasma samples obtained from patients after IVIG infusion varied in their titers of neutralizing antibodies to culture supernatants prepared from their respective isolates, and this variation paralleled differences in the neutralizing titer of the IVIG lot administered to each patient studied. The study suggests that complete neutralizing activity may be achieved by optimizing the type and/or dose of IVIG used in treatment.

Antibodies, Bacterial↗
PubMed1998

Acute pancreatitis induces cytokine production in endotoxin-resistant mice.

OBJECTIVE: The purpose of this study was to determine whether pathologic progression and cytokine responses in acute pancreatitis (AP) are altered in the absence of endotoxemia. SUMMARY BACKGROUND DATA: Previous studies have demonstrated that AP is characterized by rapid production and release of inflammatory cytokines, which play a major role in the local pancreatic and systemic complications of this disease. Infection and endotoxemia have been implicated as a major source of morbidity and death in AP and as possible stimuli for the overwhelming cytokine response seen in this disease. METHODS: AP was induced by a choline-deficient and ethionine-supplemented diet for 4 days in normal C57BL/6J mice (controls, n = 23) and in CD14 knockout mice (CD14KO, n = 23), which cannot produce circulating cytokines in response to endotoxin. Control and endotoxin-resistant mice were killed at time 0, then at 24, 48, 72, and 96 hours after the start of the diet. At each time point serum was collected for amylase, glucose, and cytokine measurements (tumor necrosis factor-alpha [TNFalpha] and interleukin-1beta [IL1beta]), and the pancreas was removed for histologic examination. TNFalpha was measured with a bioassay using WEHI-2F cells and IL1beta with a bioassay using D10.G4.1 cells. RESULTS: CD14KO mice developed biochemical manifestations of AP with alterations in amylase levels, hypoglycemia, weight loss, and histologic changes of pancreatitis similar to the pattern seen in control mice. TNFalpha and IL1beta production had similar kinetics in both groups, with significant peak TNFalpha serum levels at 72 hours and a progressive rise of IL1beta levels throughout the study period. Histologic changes appeared earlier and were more pronounced in the control versus the CD14KO mice. However, the mortality rate was identical (20% at 96 hours) for both groups. CONCLUSIONS: These results demonstrate that the progression of AP, the cytokine response associated with the disease, and early death are independent of endotoxin action. These findings, which suggest that an uncharacterized stimulus is responsible for triggering the cytokine cascade in this disease, may have significant implications for the management of patients with AP.

Acute Disease↗
PubMed1998

Opsonic antibodies to the surface M protein of group A streptococci in pooled normal immunoglobulins (IVIG): potential impact on the clinical efficacy of IVIG therapy for severe invasive group A streptococcal infections.

The surface M protein of group A streptococci (GAS) is one of the major virulence factors for this pathogen. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in human blood. We investigated whether pooled normal immunoglobulin G (IVIG) contains antibodies that can opsonize and enhance the phagocytosis of type M1 strains of GAS and whether the levels of these antibodies vary for different IVIG preparations. We focused on the presence of anti-M1 antibodies because the M1T1 serotype accounts for the majority of recent invasive GAS clinical isolates in our surveillance studies. The level of anti-M1 antibodies in three commercial IVIG preparations was determined by enzyme-linked immunosorbent assay (ELISA), and the opsonic activity of these antibodies was determined by neutrophil-mediated opsonophagocytosis of a representative M1T1 isolate. High levels of opsonic anti-M1 antibodies were found in all IVIG preparations tested, and there was a good correlation between ELISA titers and opsonophagocytic activity. However, there was no significant difference in the levels of opsonic anti-M1 antibodies among the various IVIG preparations or lots tested. Adsorption of IVIG with M1T1 bacteria removed the anti-M1 opsonic activity, while the level of anti-M3 opsonophagocytosis was unchanged. Plasma was obtained from seven patients with streptococcal toxic shock syndrome who received IVIG therapy, and the level of anti-M1 antibodies was assessed before and after IVIG administration. A significant increase in the level of type M1-specific antibodies was found in the plasma of all patients who received IVIG therapy (P < 0.006). The results reveal another potential mechanism by which IVIG can ameliorate severe invasive group A streptococcal infections.

Animals↗
PubMed1998

MHC class II-independent, Vbeta-specific activation of T cells by superantigen mutants fused to anti-tumor Fab fragments: implications for use in treatment of human colon carcinoma.

Genetically engineered fusion proteins of the super-antigen staphylococcal enterotoxin A (SEA) and tumor-reactive monoclonal antibodies, C215Fab-SEA and C242Fab-SEA, have been generated and shown to be effective in mediating superantigen-antibody directed cellular cytotoxicity against human carcinoma cells expressing the CA215 or CA242 antigens in an MHC class II-independent manner. In an attempt to reduce the in vivo toxicity of superantigen administration, alanine substitution mutations in SEA at residues F47 and D227 that affect SEA binding to class II molecules have been created and genetically linked to C215Fab or C242Fab. The purpose of this study was to determine whether these Fab-SEA mutant fusion proteins, that have low MHC class II binding affinities, were still able to stimulate human T cells in a Vbeta-specific manner in the presence or absence of MHC class II molecules. The SEA wt- and SEA-D227A-based fusion proteins shared the ability to activate V beta5. 2-, Vbeta6-, Vbeta7-, Vbeta9- and Vbeta18-bearing T cells, whereas Fab-SEA-F47A protein activated only Vbeta6- and Vbeta7-bearing T cells. The fusion of Fab fragments onto SEA wt, SEA-F47A or SEA-D227A had no effect on the Vbeta specificity of these superantigens. Fab fusion proteins containing either SEA wt or SEA mutants were presented, in the absence of class II molecules, by CHO cells transfected with CA215 and CD80 and all induced the expansion of only Vbeta6-, Vbeta7- and Vbeta 18-bearing T cells. Fab-SEA mutant fusion proteins may provide attenuated therapeutic agents that, while still able to specifically target high affinity T cells for MHC class II-independent local tumor killing, will not induce excessive systemic toxicity.

Animals↗
PubMed1997

Differential regulation of methionine adenosyltransferase in superantigen and mitogen stimulated human T lymphocytes.

Superantigens interact with the T cell receptor for antigen (TCR) and are, therefore, more physiological stimulators of T lymphocytes than nonspecific polyclonal T cell mitogens. The effects of these two classes of T cell stimulators on methionine adenosyltransferase (MAT) and S-adenosylmethionine (AdoMet) levels were investigated. Activation of resting human peripheral blood T lymphocytes by the mitogen phytohemagglutinin (PHA) or the superantigen staphylococcal enterotoxin B (SEB) caused a 3- to 6-fold increase in MAT II specific activity. Although the proliferative response was higher in cultures stimulated with PHA compared with SEB, MAT II activity was comparable in both cultures. Both stimuli caused down-regulation of the MAT 68-kDa lambda subunit expression and induced a comparable increase in the expression of the catalytic alpha2/alpha2' subunit mRNA and protein. However, in superantigen-stimulated cells, the expression of the noncatalytic beta subunit was down-regulated and virtually disappeared by 72 h post-stimulation; whereas, no change in the expression of this subunit was noted in PHA-stimulated cells. Thus, at 72 h following stimulation, PHA-stimulated cells expressed MAT II alpha2/alpha2' and beta subunits while SEB-stimulated cells expressed the alpha2/alpha2' subunits only; the beta subunit was no longer expressed in superantigen-stimulated cells. Kinetic analysis of MAT II in extracts of PHA- and SEB-stimulated cells using reciprocal kinetic plots revealed that in the absence of the beta subunit the Km of the enzyme for L-methionine (L-Met) was 3-fold higher than in the presence of the beta subunit. Furthermore, AdoMet levels were 5-fold higher in cell extracts lacking the beta subunit (SEB-stimulated cell extracts) compared with extracts containing MAT II alpha2/alpha2' and beta subunits. We propose that the increased levels of AdoMet in superantigen-stimulated cells may be attributed to the absence of the beta subunit, which seems to have rendered MAT II less sensitive to product feedback inhibition by (-)AdoMet. The data suggest that the beta subunit of MAT II, which has no catalytic activity, may be a regulatory subunit that imparts a lower Km for L-Met but increases the sensitivity to feedback inhibition by AdoMet. The down-regulation of the beta subunit, which occurred when T cells were stimulated via the TCR, may be an important mechanism to regulate AdoMet levels at different stages of T cell differentiation under physiological conditions.

Cells, Cultured↗
PubMed1997

Differential induction of Th1 versus Th2 cytokines by group A streptococcal toxic shock syndrome isolates.

The majority of group A streptococcal (GAS) isolates from patients with streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) express numerous virulence factors, including several superantigens (SAgs). Purified SAgs are potent inducers of inflammatory (Th1) cytokines that contribute to the pathogenesis of severe infections. However, GAS-infected individuals are likely to be exposed to a mixture of GAS SAgs as well as other virulence factors produced by the bacteria, and therefore, our goal was to characterize the mitogenic and cytokine induction profiles of this mixture. All GAS isolates tested had brisk mitogenic activity and induced potent cytokine responses, with higher frequencies of Th1 than Th2 cytokine-producing cells. The mitogenic activity produced in culture supernatants of three selected clinical GAS isolates was significantly different, but no marked difference was found in their overall cytokine induction profiles. However, significant differences (P < 0.0062) were noted in the induction of Th2 cytokines between GAS supernatants and recombinant streptococcal pyrogenic exotoxin A (rSpeA), suggesting that the presence of other SAgs and/or the production of additional virulence factors may alter the overall cytokine induction profile of SAgs. A significant individual variation in the level of proliferative and cytokine responses to the same GAS culture supernatants or to rSpeA was noted. Individuals with higher frequencies of cells producing Th2 cytokines mounted lower levels of Th1 cytokine responses, and vice versa. Furthermore, quantification of the intensity and cell area of interleukin-1beta (IL-1beta)-producing cells by image analysis revealed that individuals with higher Th2 responses had significantly lower IL-1beta production (P < 0.0001) than the individual with a strong Th1 response. Differences in the ability to induce Th1 versus Th2 cytokines, as well as the individual variations in cytokine responses to streptococcal SAgs, may play a central role in determining the severity of invasive GAS infections.

Cytokines↗
PubMed1997

Preferential recognition of human myocardial antigens by T lymphocytes from rheumatic heart disease patients.

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune sequelae of upper respiratory infections with group A streptococci (GAS). To gain a better understanding of the pathogenesis of these diseases, we examined the in vitro proliferative responses of peripheral blood mononuclear cells (PBMC) from RHD patients to human myocardial proteins in a T-cell Western assay. A number of myocardial proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were recognized by PBMC from both patients and controls. However, PBMC from a significant percentage of RHD patients (40%) responded to a discrete band of myocardial proteins migrating with an apparent molecular mass of 50 to 54 kDa while none of the control subject PBMC responded to this protein band (P < or = 0.0001). To further investigate the link between infections with GAS and autoimmune carditis, we studied the proliferative responses of PBMC from patients and controls to myocardial proteins before and after in vitro stimulation of the cells with opsonized GAS isolated from ARF patients. Priming of PBMC with rheumatogenic GAS caused the percentage of RHD patients responding to the 50- to 54-kDa myocardial proteins to increase from 43 to 90% (P < or = 0.0284). By contrast, PBMC from control subjects failed to recognize the 50- to 54-kDa myocardial proteins even after stimulation with the opsonized streptococci (P < or = 0.0001). The assay sensitivity was increased from 40 to 90% after priming of a patient's cells with opsonized GAS, but the positive predictive value was 100% in both unprimed and primed cultures. Antibodies generated to partially purified 50- to 54-kDa myocardial proteins did not cross-react with either streptococcal homogenates, purified M protein, myosin, laminin, or vimentin, suggesting a lack of cross-reactivity at the humoral level. This study suggests that the 50- to 54-kDa myocardial proteins contain a putative antigen that is preferentially recognized by T cells from RHD patients and demonstrates that exposure to streptococcal antigens enhances the ability of patients to recognize these proteins.

Animals↗
PubMed1997

Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer.

PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS: Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.

Adult↗
PubMed1996

Selective T cell receptor Vbeta gene usage by alloreactive T cells responding to defined HLA-DR alleles.

Previous studies have provided strong evidence for restricted Vbeta gene usage in response to DR synthetic peptides presented in context by self MHC molecules, i.e., via the indirect pathway of allorecognition. Although numerous studies have suggested a role for the T cell receptor (TCR) beta chain in the direct pathway of allorecognition, it is not clear whether a particular HLA allele elicits a consistent pattern of restricted Vbeta gene usage by resting T cells from different individuals. To address this problem, HLA class II homozygous cell lines that do not express class I antigens were used to study the role of the TCR Vbeta elements in direct recognition of specific DR alleles. Analysis of TCR V gene usage revealed that each DR allele tested elicited the same pattern of preferential Vbeta gene usage in all individuals studied. For example, the DRB1*0101 allele was preferentially recognized by T cells expressing Vbeta2, Vbeta13.1, Vbeta18, and Vbeta20, and the DRB1*1301 caused expansion of Vbeta4-, Vbeta6-, Vbeta8-, and Vbeta18-bearing T cells. Similarly, analysis of TCR V gene usage in response to defined DR alleles expressed on homozygous cell lines that express class I was also made possible by using anti-class I antibodies to block class I recognition and focus the response on the DR molecule. The results showed that the DRB1*1501 allele expressed on two distinct homozygous lines elicited the preferential expansion of Vbeta2, Vbeta8, and Vbeta13.2 T cells. Similarly, the DRB1*0301 allele expressed on normal, Epstein-Barr virus-transformed, or transfected fibroblasts was found to elicit the same pattern of Vbeta expansion and to selectively stimulate T cells bearing Vbeta2, Vbeta16, and Vbeta17 elements. In contrast to this highly reproducible pattern of Vbeta gene usage elicited by specific DR alleles, extensive heterogeneity in the CDR3 region was found and no preferential Jbeta or Valpha gene usage was observed in response to any of the DR alleles tested. The data suggest that, similar to the situation with the indirect pathway, TCR Vbeta elements are involved in the direct pathway of allorecognition. Although some overlap may exist, different sets of Vbeta elements may be preferentially used for each of these pathways. Inasmuch as HLA-identical siblings, but not HLA-identical unrelated individuals, express a very similar TCR repertoire, our data suggest that additional factors are involved in shaping the repertoire. Preferential activation of T-cell subsets by specific DR alleles may play an important role in primary alloresponses, e.g., in mixed lymphocyte reactions, and organ transplantation. Elucidation of the Vbeta specificity of each DR allele may have an impact on therapeutic strategies aimed at blocking specific alloresponse and prolonging graft survival in transplant recipients while avoiding the hazardous sequelae of nonspecific immunosuppression.

Alleles↗