Addition Of Monoclonal Antibody Research Articles

Localization of Factor VIII Interactive Site within Plasmin/Plasminogen Which Is Responsible for Plasmin-Catalyzed Activation/Inactivation of Factor VIII.

Plasmin (Plm), an active form of plasminogen (Plg), not only functions as a key enzyme in the fibrinolytic system, but also directly inactivates several coagulation factors. Especially, factor VIII is inactivated by Plm immediately after the activation by limited proteolytic cleavage at Lys36, Arg336, Arg372, and Arg740 in the heavy chain, and at Arg1689 and Arg1721 in the light chain (Nogami et al. J. Biol. Chem. 2007, 282, 5287). We recently have identified the plasmin-interactive sites on the A2 domain responsible for cleavages at Arg336 and Arg372, and on the light chain responsible for cleavage at Lys36 (Abst #1991/1709, BLOOD 102/108, 2005/2006). In the present study, we attempted to localize a factor VIII-interactive site on Plm (and Plg). Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of lysine-binding site (LBS) of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the light chain binding to active-site modified Plm (anhydro-Plm), whilst inhibited weakly the A2 binding (by ∼30%). These results suggested that the light chain interaction with Plm was mainly dependent upon LBS, but the A2 interaction was independent. The addition of monoclonal antibody (mAb) against Plg kringle 5-catalytic domain (K5-CD) significantly inhibited Plm-catalyzed activation/inactivation of factor VIII or VIIIa with an ∼4-fold lower rate constant. On the other hand, anti-K1-3 and anti-K4 mAbs any little affected. SDS-PAGE analysis revealed that only anti-K5-CD mAb blocked Plm-catalyzed cleavages at Arg336 and Arg372 by ∼90% in dose-dependent manners (IC50: ∼20 nM). Surface plasmon resonance-based assays showed that the isolated K5-CD bound to factor VIII with an ∼50-fold higher affinity (Kd: 3 nM) compared to the K1-3 and K4, similar to the affinity obtained with anhydro-Plm (Kd: 4 nM). In particular, the K5-CD bound to the A2 domain with an ∼5-fold higher affinity (Kd: 42 nM) than those obtained with the K1-3 and K4. In contrast, both the K1-3 and K4 bound to the light chain predominantly (Kd: 43 and 87 nM, respectively), whilst the K5-CD failed to bind. Furthermore, the addition of a goat antibody against the CD (C-14; Santa Cruz Biotechnology) completely blocked the A2 and K5-CD interaction (by ∼95%). These findings suggest that the CD of Plm (and Plg) interacts with the factor VIII A2 domain through the LBS-independent mechanisms, whilst the K1-3 (and/or K4) interacts with the light chain through the LBS-dependent mechanisms. Furthermore, the CD and A2 interaction would regulate the activation/inactivation of factor VIII by proteolytic cleavages of Arg336 and Arg372.

Immunological Actions of Sophy β‐Glucan (β‐1,3‐1,6 Glucan), Currently Available Commercially as a Health Food Supplement

We examined the immunological actions of Sophy beta-glucan(Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of beta-1,3-1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy beta-glucan significantly (P<0.01) stimulated the (3)H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy beta-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1beta, IL-2, IL-6, IL-12 (p70+40), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy beta-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy beta-glucan-induced production of IL-8 in the U937 cells. Furthermore, a mAb to the beta-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy beta-glucan induced DNA synthesis in the PBMCs, and Sophy beta-glucan-induced production of IL-8 in the U937 cells. The Sophy beta-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy beta-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy beta-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-gamma production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy beta-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy beta-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

P2-046: To overcome T790M associated EGFR TKI resistance by addition of mono-clonal antibodies against HER-1 and/or HER-2 receptors

P2-046: To overcome T790M associated EGFR TKI resistance by addition of mono-clonal antibodies against HER-1 and/or HER-2 receptors

Recent advances in the diagnosis and therapy of richter’s syndrome

Richter's syndrome (RS) denotes the development of aggressive lymphoma that arises in patients with chronic lymphocytic leukemia (CLL). Presenting features typically include a rapid clinical deterioration with fever in the absence of infection, progressive lymph node enlargement, and an elevation in serum LDH. Diagnostic biopsy of affected sites usually reveals large cell lymphomas; however, Hodgkin variant cases have been described. Richter's transformation occurs in approx 5% of CLL patients and may be associated with infection with Epstein-Barr virus (EBV). Chromosome 11 and 14 abnormalities have also been described as well as tumor suppressor gene defects involving p53, p21, and p27. Treatment options for these patients are limited and include combination chemotherapy with or without the addition of monoclonal antibodies and stem cell transplantation. Response to therapy is variable and generally short-lived. Median survival is usually in the order of 5-8 mo. More effective management for RS is needed as well as prognostic models that will identify CLL patients at risk of transformation. This review will address the current status of RS and deal with the pathophysiology, diagnostic approach, and treatment of this challenging disease.

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

Species-Specific Fibroblasts Required for Triggering Invasiveness of Partially Transformed Oral Keratinocytes

This study tests the hypothesis that invasion of partially transformed keratinocytes is initiated by diffusible, proinvasive signals provided by species-specific fibroblasts. In vitro organotypic cultures of neoplastic human oral mucosa were constructed by growing a partially transformed, nontumorigenic keratinocytic cell line isolated from a dysplastic human oral lesion (DOK-ECACC94122104) on top of various types of connective tissue equivalents. Cultured tissues were analyzed by histomorphometry (depth and area of invasion: Dinv, Ainv) and immunohistochemistry. Presence of human fibroblasts in the matrix induced a local invasion of DOK (Dinv = 95.6 +/- 7.1 microm, Ainv = 45.8 +/- 3.5%). Minimal invasion (P < 0.05) was observed when DOK grew on simple collagen matrix (Dinv = 14.1 +/- 2.1 microm, Ainv = 3.7 +/- 0.8%) or matrices containing fibroblasts from mouse (Dinv = 11.5 +/- 4.0 microm, Ainv = 4.3 +/- 1.0%) or rat (Dinv = 15.6 +/- 1.2 microm, Ainv = 6.1 +/- 0.5%). In these cultures, local invasion could be induced by the presence of human fibroblasts in a bottom layer of the collagen matrix (P < 0.05) or by conditioned medium from organotypic cultures of DOK on human fibroblast-containing matrix (P < 0.05) but not by conditioned medium from human fibroblast monocultures (P > 0.05). Deposition of human collagen IV was observed at epithelial-matrix interface only when DOK behaved invasively. In conclusion, invasion of partially transformed oral keratinocytes was triggered by keratinocyte-induced fibroblast-derived diffusible factor(s) in a species-specific manner and associated with de novo synthesis of collagen IV.

Long remissions in hairy cell leukemia with purine analogs

Both pentostatin and cladribine have efficacy in hairy cell leukemia (HCL), but it is not known which agent achieves better results. We reviewed a series of 219 patients with HCL, with median follow-up from diagnosis of 12.5 years (range 1.0 -34.6 yrs), treated with either pentostatin (n = 185) or cladribine (n = 34), to compare these agents and assess the potential for cure. Overall response to pentostatin was 96% with a complete response (CR) in 81% and a median disease-free survival (DFS) of 15 years. Response to first-line cladribine was 100% with a CR in 82% and DFS of 11+ years. The relapse rates at 5 years and 10 years were 24% and 42%, respectively, with pentostatin, and 33% and 48% with cladribine. Survival at 10 years was respectively 96% and 100%. CR rates decreased with each sequential relapse through 69% to 45% (P < or = 0.001). Patients achieving CR after first-line treatment had a significantly longer DFS (P = 0.00007) than those achieving a partial response; a similar result was seen after second-line therapy (P = 0.00001). DFS also declined with sequential treatment (P = 0.00005). We have shown equivalent efficacies for both agents in the treatment of HCL, with DFS showing no plateau. True cure in HCL remains elusive, but the addition of monoclonal antibodies may be beneficial. Our results suggest that achieving CR should remain the main goal of treatment.

Blockade of the Poliovirus-Induced Cytopathic Effect in Neural Cells by Monoclonal Antibody against Poliovirus or the Human Poliovirus Receptor

The poliovirus (PV)-induced cytopathic effect (CPE) was blocked in neural cells but not in HeLa cells by the addition of monoclonal antibody (MAb) against PV or the human PV receptor (CD155) 2 h postinfection (hpi). Since each MAb has the ability to block viral infection, no CPE in PV-infected neural cells appeared to result from the blockade of multiple rounds of viral replication. Pulse-labeling experiments revealed that virus-specific protein synthesis proceeded 5 hpi with or without MAbs. However, in contrast to the results obtained without MAbs, virus-specific protein synthesis with MAbs was not detected 7 hpi. Shutoff of host translation was also not observed in the presence of MAbs. Western blot analysis showed that 2Apro, the viral protein which mediates the cleavage of eukaryotic translation initiation factor eIF4G, was still present 11 hpi. However, intact eIF4G appeared 11 hpi. An immunocytochemical study indicated that 2Apro was detected only in the nucleus 11 hpi. These results suggest that neural cells possess protective response mechanisms against PV infection as follows: (i) upon PV infection, neural cells produce a factor(s) to suppress PV internal ribosome entry site activity by 7 hpi, (ii) a factor which supports cap-dependent translation for eIF4G may exist in infected cells when no intact eIF4G is detected, and (iii) the remaining 2Apro is not effective in cleaving eIF4G because it is imported into the nucleus by 11 hpi.

Addition of Rituximab to Fludarabine Improves Progression Free Survival in Untreated ZAP-70 Negative Chronic Lymphocytic Leukemia (CLL).

Clinical trials of monoclonal antibodies in combination with chemotherapy have reported previously unattained response rates in CLL because this approach reduces disease burden to levels detectable only by flow cytometry or molecular methods. The latest studies from CALGB have demonstrated that rituximab added concurrently or sequentially to fludarabine (Flu) for symptomatic, untreated CLL allows to achieve higher remission rates, longer progression free and overall survival. Nevertheless, the analysis of different biologic parameters could better explain the discordant outcome independent of treatment observed in these studies. Recent literature data indicate that unmutated VH genes, CD38 and/or ZAP-70 protein tyrosine kinase overexpression may predict both a lower response and a shorter survival. We performed at our Institution a phase II study that added rituximab sequentially to Flu as initial therapy for symptomatic, untreated CLL in order to evaluate either the toxicity or the clinical response or outcome. Complete remission (CR) was also assessed by a multiparametric flow cytometric method. ZAP-70 protein and CD38 antigen were determined before chemotherapy on mononuclear cells by flow cytometry using an anti-ZAP-70 and an anti-CD38 antibody, respectively. Forty-nine B-CLL patients, median age 59 years (range 37–74) received six monthly courses of Flu (25 mg/m2 for 5 days) and four weekly doses of rituximab (375 mg/m2) starting on an average of thirty days (range 5–180) after completion of the Flu therapy. According to modified Rai stages, 4 pts had a low stage, 42 an intermediate stage and 3 a high stage. Three out of 49 pts experienced fever, chills and rigors, during the first infusion of rituximab and only 1 patient presented grade 3 infective lung toxicity according to WHO. Hematologic toxicity included neutropenia (grade 1 and/or 2 in 12 pts, grade 3 and/or 4 in 22 pts), thrombocytopenia (grade 1 and/or 2 in 4, grade 3 and/or 4 in 3 pts) and anemia (grade 2 in 3 pts). Based on the NCI criteria, 45/49 (91.8%) pts achieved a CR, 3/49 (6.1%) a partial remission (PR) and 1/49 (2%) no response (NR). The median follow-up duration was 29 months. Median duration of CR and PR has not been reached. Noteworthy, our B-CLL pts treated sequentially with Flu and rituximab experienced a very long progression-free survival (PFS) from treatment (72% at 3 years). ZAP-70 and CD38 were positive (>20%) in 19/48 (39.5%) and in 13/48 (27%) pts, respectively. Minimal residual disease (MRD) performed on bone marrow by flow cytometry was positive (>5% CD19+CD5+CD79b- CLL cells) in 7/37 (19%) analysed pts. A significant shorter PFS was observed in ZAP-70+ pts (38% vs 100% at 3 years; P=0.003), in CD38+ pts (39% vs 92% at 3 years; P=0.007) and also in pts with higher MRD after treatment (50% vs 81% at 2 years; P=0.04). Therefore, the addition of monoclonal antibodies, such as rituximab, to chemotherapy allowed a better outcome in CLL, exerting a key role to eradicate MRD. Moreover, the stratification of pts within different risk classes using novel biologic predictive factors, such as ZAP-70 and CD38, might allow us to offer more tailored treatment strategies, reserving experimental approaches and/or transplantation procedures only to CLL subsets with proved adverse biologic and clinical features.

Advances in chemotherapy for chronic lymphocytic leukemia

While chemotherapy based on alkylating agents has been the standard treatment of chronic lymphocytic leukemia (CLL) for decades, purine analogues and their combinations have emerged as effective new therapies for previously untreated and pretreated patients. As single agents, fludarabine and cladribine are the most promising, showing higher remission rates compared to chlorambucil. For younger and physically fit patients, the combination of fludarabine and cyclophosphamide has shown benefit. Fludarabine plus epirubicin appears equally potent. The addition of monoclonal antibodies, such as rituximab and alemtuzumab, to purine analogues alone or in combination seems to be even more effective for chemotherapy-naive and pretreated CLL patients. Another promising agent in the armamentarium of therapies for CLL is bendamustine, which has properties of both an alkylating agent and a purine analogue. Clinical trials are ongoing with novel drugs that interfere with cell cycle regulation and signaling molecules in CLL, including flavopiridol, UCN-01, bryostatin 1, depsipeptide, and oblimersen. It remains to be seen whether these chemotherapeutic approaches offer real benefit for patients by prolonging survival with an improved quality of life.

Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor.

Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (K(D) = 0.5 microM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.

Role of interaction between Ly49 inhibitory receptors and cognate MHC I molecules in IL2-induced development of NK cells in murine bone marrow cell cultures

Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2). Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures. Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2 b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development. Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2 b haplotype. Blocking Ly49–MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells. Addition of anti-Ly49A (no specificity for H-2 b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2 b) mabs, however, had no effect on IL2-induced NK cell development in BM cells. Mabs specific to Ly49C molecule and MHC I molecules of H-2 b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population. These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development. We propose a model in which Ly49–MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.

Diffuse Aggressive Lymphoma

The aggressive non-Hodgkin's lymphomas can be cured in more than half of the cases. However, there has been great variation in the results reported from individual clinical Phase II trials. This variation in result can be attributed to unrecognized heterogeneity in this group of diseases. Recent clinical and molecular studies have enabled us to define more homogenous population in which new therapies can be studied. For patients with advanced stages of diffuse large B cell lymphoma, a new standard of therapy exists. For patients with localized aggressive non-Hodgkin's lymphomas, heterogeneity in patient selection prevents us from defining a new standard of care. Finally, in mantle cell lymphoma, new opportunities in drug discovery may permit advances in the treatment of this uniformly fatal malignancy. In Section I, Dr. Richard Fisher reviews the development of combination chemotherapy for patients with advanced stage diffuse large B cell lymphoma. Because of great heterogeneity in patients enrolled in Phase II studies, large randomized Phase III studies were required in the 1980s to define CHOP has the standard of care. This heterogeneity has now been defined carefully in the international prognostic factor index and more recently by gene array studies. It will now need to be incorporated prospectively into studies or retrospectively analyzed to understand clinical trial results. The addition of rituximab to CHOP has now been demonstrated to improve survival in two large Phase III studies in elderly patients. A recently presented study in younger patients suggests a similar benefit. Thus CHOP/rituximab has become the established standard of care for all patients with advanced stage diffuse large B cell lymphoma. Other concepts being evaluated to further improve on these results include: dose intensification; initial treatment with chemotherapy plus allogeneic stem cell transplantation; and infusional chemotherapy. Finally, the status of the treatment for relapsed patients will be defined. In Section II, Dr. Thomas Miller defines the treatment for limited stage aggressive non-Hodgkin's lymphoma. Randomized trials have demonstrated the critical importance of initial chemotherapy for treatment of these patients. The amount of chemotherapy given needs to be increased for patients with bulky tumors. In most circumstances radiotherapy after the completion of chemotherapy has been shown to be advantageous. A modification of the international prognostic factor index for patients with early stage disease is presented to permit comparisons among different populations. Recently reported early-stage studies need to be analyzed in terms of the heterogeneity of the patients involved to understand the reported results. The addition of monoclonal antibodies, as well as radioimmunotherapy, are being tested in an effort to improve on the results for the poor prognosis patients. In Section III, Dr. Owen O'Connor describes the pathology immunophenotype and natural history of mantle cell lymphoma. Conventional treatment strategies with combination chemotherapy achieved objective responses in approximately half of the patients but no significant impact on survival. The addition to rituximab to CHOP chemotherapy or other treatment strategies appears to improve the remission rate; however, no major changes in survival have also been reported. Excellent single institution results have been reported with HyperCVAD plus rituximab regimen, which is currently being tested in a national cooperative group trial. The most excitement in this field currently relates to the variety of new agents which appear to have significant activity in relapsed patients with mantle cell lymphoma. This includes the proteosome inhibitor, bortezomib, which is shown to have approximately a 50% response rate with some CRs and reasonable durability in early single institution Phase II studies. Larger national multi-center trials are ongoing. In addition, agents such as thalidomide, flavopiridol, and piroxantrone will be reviewed.

Evaluation of cellular immune response in patients with recurrent candidiasis

Recurrent cutaneous or mucosal candidiasis is characterized by the occurrence of at least four candidiasis episodes within a one-year period. The factors involved in recurrence of infection are still unknown. In the present study the lymphoproliferative response and the IFN-gamma production by candidiasis patients were evaluated. The stimulation index of mononuclear cell cultures of candidiasis patients stimulated with Candida albicans antigen, PPD and TT were 6 +/- 8, 17 +/- 20 and 65 +/- 30, respectively. The addition of monoclonal antibody anti-IL-10 to Candida albicans antigen stimulated cultures raised the lymphoproliferative response from 735 +/- 415 to 4143 +/- 1746 cpm. The IFN-gamma production by cells of candidiasis patients stimulated with Candida albicans antigen was 162 +/- 345 pg/ml. Candidiasis patients have an impairment in the lymphoproliferative response specific to C. albicans antigen and on IFN-gamma production and the lymphoproliferative response can be partially restored, in vitro, by IL-10 neutralization.

Adherence of Mycoplasma bovis to bovine bronchial epithelial cells

Mycoplasma bovis is responsible for considerable economic losses in cattle due to pneumonia, arthritis and mastitis. As the agent was shown to be capable of adhering to neutrophils and embryonic bovine lung (EBL) cells and invading the respiratory epithelium it is highly desirable to improve our understanding of cytadherence processes. Although several surface proteins likely to be directly involved in this initial stage of interaction between pathogen and host cells have been identified, these findings mainly referred to type strain PG45 adhering to the continuous EBL cell line. The present study provides new and complementary data about cytadherence of M. bovis based on adherence of various radiolabeled strains to a primary culture of bovine bronchial epithelial (BBE) cells using a standardized adherence assay. M. bovis was shown to adhere specifically to the primary culture of BBE cells. Inhibition of adherence was observed upon addition of monoclonal antibodies (MAbs), trypsin treatment of mycoplasmas, and competition with non-radiolabeled mycoplasma cells. Interestingly, three MAbs against proteins involved in adherence to EBL cells failed to inhibit significantly the adherence to BBE cells. On the other hand, significant reduction of adherence rates by MAbs 2A8 and 9F1 directed against epitopes of variable surface lipoproteins VspC and VspF, respectively, demonstrated the involvement of these proteins in adherence of M. bovis to primary culture of BBE cells.

Human gingival fibroblasts rescue butyric acid-induced T-cell apoptosis.

We previously demonstrated that butyric acid, an extracellular metabolite from periodontopathic bacteria, induces cytotoxicity and apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we used a cell-to-cell interaction system to examine the contribution of gingival fibroblasts to the regulation of T-cell death induced by butyric acid. Butyric acid slightly suppressed fibroblast viability in a concentration-dependent fashion. However, DNA fragmentation assays indicated that butyric acid did not induce apoptosis for up to 21 h in human gingival fibroblasts (Gin 1, F41-G, and H. pulp cells). The culture supernatants were assayed for interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor alpha, and transforming growth factor beta, but only the IL-6, IL-8, and IL-11 levels were significantly increased by addition of butyric acid. Butyric acid- or Fas-induced Jurkat-cell apoptosis was attenuated when Jurkat cells were cocultured with either F41-G or Gin 1 cells that had been preincubated for 6 h with butyric acid. IL-8 slightly stimulated butyric acid- or Fas-induced Jurkat-cell apoptosis in a dose-dependent manner, although a low dose of IL-8 had a mildly inhibitory effect on apoptosis. In contrast, IL-6 and IL-11 significantly suppressed butyric acid- or Fas-induced apoptosis in a dose-dependent fashion. Furthermore, the addition of monoclonal antibodies against human IL-6 and IL-11 to cocultures of gingival fibroblasts and Jurkat cells partially eliminated T-cell recovery. These results suggest that the proinflammatory cytokines such as IL-6 and IL-11, produced in fibroblasts stimulated with butyric acid, are involved in the attenuation of T-cell apoptosis by gingival fibroblasts.

Costimulatory molecule requirement for bovine WC1+gammadelta T cells' proliferative response to bacterial superantigens.

We have previously shown that the proliferation of freshly isolated bovine WC1+gammadelta T cells to superantigens (SAgs) including staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) or toxic shock syndrome type-1 (TSST-1) required the presence of antigen-presenting cells (APC) and the addition of exogenous interleukin (IL)-2. The costimulatory activity provided by molecules expressed on professional APC for the proliferation of gammadelta T cells has not been addressed hitherto. In the present study, we investigated the ability of two selected APC populations, the dendritic cells (DCs) highly expressing CD80 and CD86 molecules (CD80highCD86high) and the monocytes expressing the same molecules at a rather low level (CD80lowCD86low), to stimulate the proliferation of purified bovine WC1+gammadelta T cells to SAgs. DCs were more efficient than monocytes in inducing gammadelta T-cell proliferation, and this response was dependent on exogenous IL-2 in both presentation modes. Stimulating gammadelta T cells with gradual doses of SAgs or concanavalin A (ConA) resulted in similar dose-dependent reaction profiles suggesting a minimal role of the major histocompatibility complex (MHC). However, significant proliferation was already obtained with the starting doses in the presence of DC compared with monocytes, and higher proliferation was reached with DC at optimal doses. Finally, the addition of monoclonal antibody (MoAb) anti-CD86 markedly inhibited SAgs- and ConA-mediated proliferation, whereas MoAb anti-CD80 had no effect. The combination of both anti-CD80 and anti-CD86, however, suppressed this response. These results suggest that bovine gammadelta T-cell proliferation response requires indubitably CD86 costimulation. The role of CD80 molecule seems less clear.

High dose chemotherapy and autologous transplantation for non-Hodgkin’s lymphoma

Although 40% of patients with aggressive non-Hodgkin’s lymphoma (NHL) can have long term disease-free survival with initial anthracycline-containing chemotherapy, the remainder will not go into a complete remission or relapse at a later time with the need for further therapy. Conventional salvage chemotherapies in these patients will obtain a 30% complete remission rate. However, there are very few long-term disease-free survivors with conventional salvage chemotherapy alone. The use of high-dose care for patients with chemotherapy sensitive relapsed NHL. In this patient group, 45% of patients can expect to be alive and disease-free years after the transplant. Methods to improve the outcomes with transplantation and to extend its usefulness to other high-risk patients are ongoing. Current strategies include the use of transplantation in first complete remission for high-risk patients, the addition of monoclonal antibodies, radioimmunoconjugates, or vaccines to the transplant regimen to enhance the outcome, or the use of multiple cycles of stem cell transplantation. In preliminary trials, some of these modifications appear to improve the outcome of transplantation. Large randomized trials will be needed to document that these modifications will improve the overall outcome for patients with aggressive NHL. *** DIRECT SUPPORT *** A00RC003 00010

A secondary RNA polymerase sigma factor from Streptococcus pyogenes.

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) causes diseases ranging from mild, self-limiting pharyngitis to severe invasive infections. Regulation of the expression of GAS genes in response to specific environmental differences within the host is probably key in determining the course of the infectious process, however, little is known of global regulators of gene expression in GAS. Although secondary RNA polymerase sigma factors act as global regulators of gene expression in many other bacteria, none has yet been isolated from the GAS. The newly available GAS genome sequence indicates that the only candidate secondary sigma factor is encoded by two identical open reading frames (ORFS). These ORFS encode a protein that is 40% identical to the transcription factor ComX, believed to act as an RNA polymerase sigma factor in Streptococcus pneumoniae. To test whether the GAS ComX homologue functions as a sigma factor, we cloned and purified it from Escherichia coli. We found that in vitro, this GAS protein, which we call sigmaX, directed core RNA polymerase from Bacillus subtilis to transcribe from two GAS promoters that contain the cin-box region, required for transcription by S. pneumoniae ComX in vivo. On the other hand, GAS sigmaX did not promote transcription of a GAS promoter (hasA) expected to be dependent on sigmaA, the housekeeping or primary RNA polymerase sigma factor. Addition of monoclonal antibody that inhibited sigmaA-directed transcription had no effect on sigmaX-directed transcription, showing that the latter was not the result of contaminating sigmaA. Transcription of both cin-box-containing promoters initiated downstream of the cin-box and two different single basepair substitutions in the cin-box of the cinA promoter each caused a severe reduction of sigmaX-directed transcription in vitro. Thus, the cin-box is required for sigmaX-directed transcription.

Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes.

The human MAGE-3 gene was originally discovered in melanoma cells that encode tumor antigens, and has been reported to be expressed in various types of tumors, including lung cancer, but not in normal tissues other than testis or placenta. Our aim in this study was to clarify whether HLA-A2 restricted MAGE-3 peptide (FLWGPRALV) could be a lung cancer antigen recognized by cytotoxic T lymphocytes (CTL). MAGE-3-derived peptide-specific CTL were induced from the peripheral blood mononuclear cells (PBMC) of HLA-A0201-positive healthy donors and the regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with lung cancer by multiple stimulations with peptide-pulsed HLA-A0201-positive antigen-presenting cells. Lymphocytes stimulated with MAGE-3 peptide exhibited specific lysis of Epstein-Barr virus-transformed B cells (EBV-B) pulsed with MAGE-3 peptide, but not with control peptide derived from influenza matrix protein, erbB-2, or wild type p53. Specific activity for MAGE-3-presenting targets was found after the second stimulation, and increased depending on the number of stimulations. The peptide-specific activity was inhibited by the addition of monoclonal antibodies against MHC class I and HLA-A2. Such CTL also recognized tumor cell lines expressing both HLA-A2 and MAGE-3 in an MHC class I-restricted manner, but did not recognize tumor cell lines that did not express HLA-A2 or MAGE-3. These results suggested the MAGE-3 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with lung cancer.