Phase II trial of single-agent panobinostat consolidation improves responses after sub-optimal transplant outcomes in multiple myeloma.

Panobinostat is a pan-deacetylase inhibitor that modulates the expression of oncogenic and immune-mediating genes involved in tumour cell growth and survival. We evaluated panobinostat-induced post-transplant responses and identified correlative biomarkers in patients with multiple myeloma who had failed to achieve a complete response after autologous transplantation. Patients received panobinostat 45 mg administered three-times weekly (TIW) on alternate weeks of 28-day cycles commencing 8-12 weeks post-transplant. Twelve of 25 patients (48%) improved their depth of response after a median (range) of 4·3 (1·9-9·7) months of panobinostat. In responders, T-lymphocyte histone acetylation increased after both three cycles (P < 0·05) and six cycles (P < 0·01) of panobinostat when compared to baseline, with no differences in non-responders. The reduction in the proportion of CD127+ CD8+ T cells and CD4:CD8 ratio was significantly greater, after three and six cycles of panobinostat compared to pre-transplant, in non-responders when compared to responders. Whole marrow RNA-seq revealed widespread transcriptional changes only in responders with baseline differences in genes involved in ribosome biogenesis, oxidative phosphorylation and metabolic pathways. This study confirmed the efficacy of panobinostat as a single agent in multiple myeloma and established acetylation of lymphocyte histones, modulation of immune subsets and transcriptional changes as pharmacodynamic biomarkers of clinical benefit.

Association of synovial tissue polyfunctional T-cells with DAPSA in psoriatic arthritis.

OBJECTIVE: This study examines polyfunctional T-cells in psoriatic arthritis (PsA) synovial tissue and their associations with clinical disease and implications for therapy. METHODS: PsA synovial tissue was enzymatically/mechanically digested to generate synovial tissue single cell suspensions. Frequencies of polyfunctional CD4, CD8, T-helper 1 (Th1), Th17 and exTh17 cells, using CD161 as a marker of Th17 plasticity, were determined by flow cytometry in matched PsA synovial tissue and peripheral blood. Synovial T-cell polyfunctionality was assessed in relation to Disease Activity in PSoriatic Arthritis (DAPSA) and in synovial cell suspensions cultured with a current mode of treatment, phosphodiesterase 4 (PDE4) inhibitor. RESULTS: PsA synovial tissue infiltrating CD4+ T-cells expressed higher levels of interleukin (IL)-17A, interferon gamma (IFN-γ), GM-CSF and CD161, with parallel enrichment of Th1, Th17 and exTh17 T-helper subsets (all p<0.05). Interestingly, a significant proportion of synovial T-cell subsets were triple-positive for GM-CSF, tumour necrosis factor (-TNF), -IL-17 or IFN-γ compared with matched blood (all p<0.05). Importantly, frequencies of polyfunctional T-cells correlated with DAPSA: Th1-GM-CSF+/TNF+/IFN-γ+ (r=0.7, p<0.01), Th17-GM-CSF+/TNF+/IL-17+ (r=0.6, p<0.057) and exTh17-GM-CSF+/TNF+/IFN-γ+ (r=0.7, p=0.0096), with no associations observed for single cytokine-producing T-cells. Following ex vivo culture of PsA synovial tissue cell suspensions, polyfunctional GM-CSF+TNFα+IL-17A+ or/IFN-γ+-producing T-cells (p<0.05), but not single cytokine-producing T-cells, were inhibited with a PDE4 inhibitor. CONCLUSION: These data demonstrate enrichment of polyfunctional T-cells in PsA synovial tissue which were strongly associated with DAPSA and ex vivo therapeutic response.

Soluble SLAMF6 Receptor Induces Strong CD8+ T-cell Effector Function and Improves Anti-Melanoma Activity In Vivo.

SLAMF6, a member of the SLAM (signaling lymphocyte activation molecules) family, is a homotypic-binding immune receptor expressed on NK, T, and B lymphocytes. Phosphorylation variance between T-cell subclones prompted us to explore its role in anti melanoma immunity. Using a 203-amino acid sequence of the human SLAMF6 (seSLAMF6) ectodomain, we found that seSLAMF6 reduced activation-induced cell death and had an antiapoptotic effect on tumor-infiltrating lymphocytes. CD8+ T cells costimulated with seSLAMF6 secreted more IFNγ and displayed augmented cytolytic activity. The systemic administration of seSLAMF6 to mice sustained adoptively transferred transgenic CD8+ T cells in comparable numbers to high doses of IL2. In a therapeutic model, lymphocytes activated by seSLAMF6 delayed tumor growth, and when further supported in vivo with seSLAMF6, induced complete tumor clearance. The ectodomain expedites the loss of phosphorylation on SLAMF6 that occurs in response to T-cell receptor triggering. Our findings suggest that seSLAMF6 is a costimulator that could be used in melanoma immunotherapy. Cancer Immunol Res; 6(2); 127-38. ©2018 AACR.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Effect of intermittent interleukin-2 therapy on CD4+ T-cell counts following antiretroviral cessation in patients with HIV.

BACKGROUND: Interleukin (IL)-2 therapy impacts T-cell homeostasis. Whether IL-2 expanded CD4(+) T cells may persist following viral rebound has not been fully investigated. METHODS: Patients with CD4(+) T cells 500/µl or more and HIV RNA less than 50 copies/ml were randomized to continue antiretroviral therapy (ART) either alone (n = 67) or combined with three IL-2 cycles (n = 81; 6 million units) twice daily for 5 days at weeks 0, 8, and 16 before stopping ART (week 24). Patients were followed up to 168 weeks. RESULTS: At week 24, median CD4(+) T-cell counts were 1198 and 703 cells/µl in the IL-2 and control groups, respectively (P < 0.001). At week 72, 27% (IL-2 group) and 45% (control group; P = 0.03) of patients were in failure (defined as no interruption of ART at week 24, CD4 drop below 350 cells/µl or ART resumption). After week 24, a biphasic decline (before and after week 32) of CD4 was noted -106 and -7 cells/µl per month in controls and -234 and -17 in IL-2 group (all P ≤ 0.0001). At week 96, IL-2-expanded CD4(+)CD25(+) T cells remained higher than in the control group (26 vs. 16%, P = 0.006). CONCLUSION: In IL-2-treated patients, CD4(+)CD25(+) T cells persisting despite viral replication allow a longer period of ART interruption.

Loss of P2X7 receptor plasma membrane expression and function in pathogenic B220+ double-negative T lymphocytes of autoimmune MRL/lpr mice.

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220(+) CD4(-)CD8(-) (DN) T lymphocytes. The precise ontogeny of B220(+) DN T cells is unknown. B220(+) DN T lymphocytes could be derived from effector CD4(+) and CD8(+) T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220(+) DN T-cell numbers in lymphoid organs. Importantly, we found that this B220(+) DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220(+) T cells observed in normal MRL(+/+) and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220(+) T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.

[Effect of enterosorption on immunologic parameters of patients with colorectal cancer in the postoperative period].

Our investigation was carried out on an assumption that end results among patients radically-treated for colorectal cancer might be improved by use of enteroabsorption. The study group included 17, controls--13 patients with diagnostically verified stage I-III tumors. Mixed sorbent (microcellulose + polysorb) (6g) was administered, once a week, on the average of 20 days after operation. Immunological vigor was assayed 3 weeks after surgery: immunoglobulin levels--by turbodimetric method, cellular profile of lymphocytes--monoclonal antibodies to cell markers CD3, CD4, CD8, CD16 and CD22. As a result of adjuvant treatment CD22 (B-lymphocytes) concentration increased significantly--from 17.70 to 21.66 (22%), while CD16 (innate killers) both in absolute numbers (19%) and by percentage points (9%). Circulating immunocomplex levels in the sorbent-treatment group were significantly lower (37.44 ths units) than in control (48 ths units) (average 28%). No relapse or metastases were reported in either group.

Involvement of central immunity in uncomplicated diverticular disease.

OBJECTIVE: The pathogenesis of symptoms of uncomplicated diverticular disease (UDD) is unclear, but changes in gut microflora and physiologic inflammation may be implicated. The objective of the study was to investigate the distribution of gut homing lymphocytes in peripheral blood and intestinal mucosa of UDD patients, and the effects of luminal antibiotic treatment. MATERIAL AND METHODS: Ten UDD patients and 10 age- and gender-matched healthy subjects underwent peripheral blood sampling, and colonoscopy with biopsies taken from the transverse and sigmoid colon. Treatment consisted of a 2-month course of rifaximin 1.2 g/day for 15 days/month. Blood sample and mucosal biopsies were repeated in UDD patients at the end of treatment. Flow cytometry was performed using monoclonal antibodies (CD3, CD4, CD8, CD25, CD19, CD45, CD62L, CD103). RESULTS: In peripheral blood, both CD4+ and CD8+/CD103+ were significantly higher in patients at baseline than in controls (0.95% versus 0.36%, and 0.5% versus 0.09%, respectively). After treatment, peripheral CD4+/CD103+ decreased (0.27%), while CD8+/CD103+ did not change (0.35%); on the contrary, peripheral CD25+ increased, the CD4+ subpopulation showing significantly higher levels than those in controls. No difference was found between lymphocytes in the diverticular sigmoid mucosa of patients at baseline and those in controls, but there was a significant decrease in CD8+/CD62L+ after treatment. In the normal transverse colon, CD4+/CD62L+ of patient at baseline were significantly lower than in controls. After treatment, CD4+/CD103+ levels significantly increased, while CD8+/CD62L+ levels significantly decreased. CONCLUSIONS: Both central and mucosal immunity may be modified in UDD patients, with an increased recruitment of CD103+ lymphocytes. A 2-month course of rifaximin appears to reduce CD103+ levels, suggesting a decrease in mobilization of mucosal homing.

Effect of somatostatin on immune inflammatory response in patients with severe acute pancreatitis.

OBJECTIVE: Somatostatin regulates immune inflammatory response via apoptosis and adhesion of leukocytes in many diseases. This article reported a study that aimed to observe the mechanism and effect of somatostatin on the immune inflammatory response through apoptosis and adhesion of leukocytes in severe acute pancreatitis. METHODS: Thirty-eight patients with severe acute pancreatitis, that fulfilled the guidelines for the treatment of severe acute pancreatitis of China and Balthazar computed tomography severity index (>or=5) were enrolled consecutively. Nineteen of these patients received our routine treatment and 19 received additional somatostatin. In all patients the expressions of CD4, CD8, CD95/CD95 ligand and CD18/CD62 ligand on leukocytes were determined by flow cytometry, both upon admission and on the fourth day. Thirty healthy volunteers constituted the normal healthy group. RESULTS: In the treatment group, CD4, CD4:CD8 ratio and CD62 ligand on leukocytes increased from 11.4+/-8.2, 0.47+/-0.10 and 25.5+/-9.2 to 22.1+/-9.7, 0.68+/-0.11 and 36.2+/-11.7 (P<0.05) respectively, while CD95 ligand on both lymphocyte and polymorphonuclear cells increased from 0.65+/-0.21 and 0.76+/-0.29 to 1.18+/-0.32 and 1.58+/-0.43 after treatment with somatostatin (P<0.05). Furthermore, lactate dehydrogenase, aspartate aminotransferase, amylase, C reactive protein and acute physiology and chronic healthy evaluation (APACHE II) score in the treatment group reduced faster than those in the control group (P<0.05), though there was no difference in mortality (15.7% vs 5.3%) between the two patient groups (P>0.05). CONCLUSION: Somatostatin can modulate the immune inflammatory response and the severity of severe acute pancreatitis through apoptosis and adhesion of leukocytes, but this modulatory effect by itself is not strong enough to improve the final.

Glatiramer acetate (GA) therapy induces a focused, oligoclonal CD8+ T-cell repertoire in multiple sclerosis.

We have demonstrated that GA therapy induces a differential upregulation of GA-specific, cytotoxic/suppressor CD8+ T-cell responses in MS patients. We utilized a novel combination of flow sorting and anchored PCR to analyze the evolving clonal composition of GA-specific CD4+ and CD8+ T-cells. TCRbeta chain analysis revealed the development of an oligoclonal GA-specific CD8+ repertoire with persistence of dominant clones over long periods. Interestingly, some sequences resembled published oligoclonal CD8+ TCR sequences from MS lesions. In contrast, GA-specific CD4+ responses were polyclonal and showed continual evolution of their repertoire. This clonotypic and functional analysis provides mechanistic insights into GA therapy.

The effect of Juzen-taiho-to/TJ-48 on the expression of killer-cell immunoglobulin-like receptors (CD158a/b) on peripheral lymphocytes in vitro experiment.

Juzen-taiho-to (TJ-48), a mixture of extracts from 10 medicinal herbs, has been used traditionally to treat patients with anemia, anorexia or fatigue. It is well known that the treatment of TJ-48 result in the decrease of patient's complaints, as well as the increase of NK cytolytic activity (NK activity) although its augmentation is not clear in the other kampo formula from the clinical viewpoint. To investigate its biological activities, such as the augmentation of NK activity, we analyzed the effects of TJ-48 on the expression of killer-cell immunoglobulin-like receptors (KIRs) in vitro experiment. The peripheral lymphocytes were incubated in medium alone, or medium containing TJ-48 or interleukin-2 (IL-2) plus TJ-48 at several concentrations for 48 h. After each incubation, cells were collected and their KIRs were detected by flow cytometry using monoclonal antibodies CD158a and CD158b. TJ-48 increased the populations of CD16+CD158a+ and CD16+CD158b+ cells in a dose-dependent manner. In contrast, CD16-CD158a/b+ cells did not increase. Additionally, the extract of TJ-48 enhanced the increase of KIRs expression induced by IL-2. These actions contribute to the augmentation of NK cytolytic activity by TJ-48, and might explain, in part, its antitumor effects which has been observed in vivo.

Antitumor and antiangiogenic effects of interleukin 12 gene therapy in murine head and neck carcinoma model.

Interleukin-12 (IL-12) plays a critical role in producing an immune response, as indicated in many ways, e.g., induction of interferon-gamma (IFN-gamma), and augmentation of the cytotoxic activity of resting activated T cells and natural killer (NK) cells. In this study, we examined whether intratumoral injection of a recombinant retrovirus vector expressing IL-12s induce antitumor and antiangiogenic effects in a murine model using a murine head and neck squamous cell carcinoma (NR-S1). In vitro the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression were decreased in IL-12 gene transfected NR-S1 cell. in vivo direct IL-12 gene therapy resulted in significantly remarkable inhibition of tumor growth compared to the control group. The tumor regression by direct IL-12 gene therapy was also associated with decreased vessel density, and apoptosis and increased infiltration of CD8(+) T cells and CD56(+) NK cells in the tumor increased. Also, the number of IFN-gamma expressed cells of spleen cells was increased in the treatment group compared with the control group. These results suggested that direct IL-12 gene therapy appears to be effective in reducing tumor growth by triggering both antiangiogenic effects and an immunological enhancing mechanism through induction of IFN-gamma.

Low-level sarin-induced alteration of immune system reaction in inbred BALB/c mice.

To study the influence of low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to low concentrations of sarin for 60 min in the inhalation chamber. Two concentrations of sarin were chosen-asymptomatic concentration (LEVEL 1) and non-convulsive symptomatic concentration (LEVEL 2). The evaluation of immune functions was carried out using phenotyping of CD3 (T-lymphocytes), CD4 (helper T-lymphocytes), CD8 (cytotoxic T-lymphocytes) and CD19 cells (B-lymphocytes) in the lungs, blood and spleen, lymphoproliferation of spleen cells stimulated in vitro by various mitogens (concanavalin A, lipopolysaccharides), phagocyte activity of peritoneal and alveolar macrophages, production of N-oxides by peritoneal macrophages and the measurement of the natural killer cell activity at 1 week following sarin exposure. The results were compared to the values obtained from control mice exposed to pure air instead of sarin. The results indicate that not only symptomatic but also asymptomatic dose of sarin is able to alter the reaction of immune system at 1 week following exposure to sarin. While the number of CD3 cells in the lungs was slightly decreased, an increase in CD19 cells was observed especially in the lungs and blood. The reduced proportion of T-lymphocytes is caused by decay of CD4 positive T-cells. Lymphoproliferation was significantly decreased regardless of the mitogen and sarin concentration used. The production of N-oxides by peritoneal macrophages was stimulated after exposure to LEVEL 2 of sarin whereas their ability to phagocyte the microbes was increased after exposure to LEVEL 1. The natural killer cell activity was significantly higher in the case of inhalation exposure of mice to LEVEL 2 of sarin. Thus, not only organophosphorus insecticides but also nerve agents such as sarin are able to alter immune functions even at a dose that does not cause clinically manifested intoxication following the inhalation exposure. Nevertheless, the alteration of immune functions following the inhalation exposure to a symptomatic concentration of sarin seems to be more pronounced.

Butyric acid derivative induces allospecific T cell anergy and prevents graft-versus-host disease.

Graft-versus-host-disease (GVHD) is a common and potentially fatal complication following bone marrow transplantation. This study was initiated to test whether MEB [n-butyrate 2-(4-morpholinyl) ethyl butyrate hydrochloride], a derivative of the G1 blocker butyric acid, could specifically inactivate the alloantigen-specific T cells that mediate GVHD. MEB was shown to inhibit proliferation in a one-way mixed lymphocyte reaction (MLR) in which spleen cells from C57BL/6 mice (H-2b) were stimulated with spleen cells from DBA/2 mice (H-2d). The addition of MEB to the MLR prevented the expansion of alloantigen-stimulated CD8+ and CD4+ T cells in association with decreased IL-2 production. In addition, MEB inhibited the CTL activity of CD8+ T cells from the MLR. Most importantly, T cells from the MEB-treated MLR, unlike T cells from an untreated MLR, were unable to induce the splenomegaly and increased serum TNF-alpha levels characteristic of acute GVHD when injected into B6D2F1 mice. The splenomegaly found in the B6D2F1 mice injected with T cells from an untreated MLR encompassed the expansion and activation of CD8+ T cells, CD4+ T cells, B cells and macrophages. In contrast, the spleens of mice injected with T cells from MEB-treated MLR looked essentially identical to those of control B6D2F1 mice in terms of the numbers and activation state of the spleen cell populations. Similarly, the increase in IFN-gamma and TNF-alpha production by CD4+ and CD8+ T cells from the spleens of mice undergoing acute GVHD was not observed if the mice were injected with T cells from an MEB-treated MLR instead of an untreated MLR. The use of MEB to inactivate host-specific T cells ex vivo underlines the potential clinical importance of this compound in the prevention and treatment of unwanted immune responses such as GVHD.

Immunoregulatory and antioxidant performance of alpha-tocopherol and selenium on human lymphocytes.

The role of alpha-tocopherol (alpha-toco) and selenium (Se) on human lymphocyte oxidative stress and T-cells proliferation were studied by flow cytometry. We measured the hydrogen peroxide and glutathione levels in cultured human T-lymphocytes and the proliferation of their subsets: T-helper/inducer, T-suppressor/cytotoxic, and natural killer and interleukin-2 receptors upon stimulation by the mitogens phytohemaglutinin (PHA) and lipopolysaccharide (LPS). The results indicate that early stimulation by mitogens is affected by the glutathione and hydrogen peroxide status of the T-lymphocytes. The addition of 100 microM or 500 microM alpha-toco or 0.5 microM Se alone shows weak antioxidant and immunostimulant properties. When combined, an enhanced antioxidant and immunoregulatory effect was observed. The present findings indicate that alpha-toco and Se have interactive effects as oxygen radical scavengers, thus promoting human lymphocyte response to antigens. This suggests that micronutrient status is an important factor in considering when interpreting the results of in vitro assays of lymphocyte function.

Ribavirin enhances interferon-gamma levels in patients with chronic hepatitis C treated with interferon-alpha.

Some patients with chronic hepatitis C respond to interferon (IFN)-alpha treatment, and the efficiency can be improved by combining it with ribavirin. The mechanism of this improvement is unknown. To investigate the effects of these two regimens on the immune responses in 51 patients with chronic hepatitis C, we examined the hepatitis C core antigen-specific proliferative response and cytokine production profiles, natural killer (NK) cell cytotoxicity and cytotoxic T cell function during treatment. The results are as follows: (1) both viral clearance and biochemical normalization occurred more frequently in patients receiving combination therapy; (2) the function of NK cells increased after treatment in the responders of both groups (p < 0.05); (3) the level of IFN-gamma produced by hepatitis C core antigen-stimulated peripheral blood mononuclear cells was higher in patients receiving combination therapy, especially in responders; (4) the core antigen-specific proliferative response decreased after treatment, and (5) in addition, the core-specific cytotoxic T cell activities of five responder patients also increased significantly after therapy. In conclusion, enhancement of immune responses, especially those related to type-1 T helper cell activity, may contribute to better efficacy in combining ribavirin with IFN-alpha for treatment of chronic hepatitis C.

Synergistic immunopotentiating effects induced by T-cell and B-cell superantigen in mice.

Staphylococcal Enterotoxin-A(SEA), a 27kDa monomeric protein, produced by some strains of Staphylococcus aureus, is a prototype T-cell superantigen which causes proliferation of cytotoxic T-lymphocytes and produces cytokines like TNF-alpha and IFN-gamma. Recently Protein A (PA), a 42 kDa membrane protein of the Staphylococcus aureus Cowan-I strain, has been termed a B-cell super antigen. It has been shown to cause multiple immunological responses. In the present study we examined the effect of these two superantigens used separately as well as combination in a normal mouse system. It has been shown that combination treatment of PA and SEA is more effective than that of each individual one. FACS analyses of cell cycles showed that a finely turned cellular collaboration occurred in various phases of cell growth and proliferative response compared with controls (P<0.01). It has also been shown that the percentage of various cell types bearing different clusters of differentiation markers, e.g., CD8+, CD34+ increases considerably due to the combined effect of PA and SEA. We also observed that co-administration of both the elicits different soluble mediators like cytokines (TNF-alpha, INF-gamma, IL-1beta). No apoptotic phenomenon was observed (from the cell cycle analysis) for the dose of PA and SEA, used for the experiments, suggesting that these doses of PA and SEA should be non-toxic.

Effect of mycophenolate mofetil in heart transplantation.

OBJECTIVE: To study the effect of mycophenolate mofetil (MMF), a new immunosuppressive drug that acts by inhibiting de novo pathways of purine synthesis, and rabbit antithymocyte globulin (RATG) on the lymphocyte subpopulation after heart transplantation. DESIGN: A review of clinical and laboratory records. SETTING: The Montreal Heart Institute. PATIENTS: Thirty-one patients who underwent heart transplantation. In 9 patients, neoral cyclosporine, prednisone and azathioprine were administered (group 1). In 14 patients RATG was added during the first 3 postoperative days (group 2) and in 8 patients RATG and combination immunosuppression was given, but MMF was used instead of azathioprine (group 3). The demographic characteristics of donors and recipients were similar among the 3 groups. MAIN OUTCOME MEASURES: The proportion of CD2, CD4 and CD8 receptor-positive lymphocytes, expressed as a mean (and standard deviation) percentage of the total lymphocyte population, measured at 7, 15 and 30 days and 6 months after transplantation. RESULTS: At 7 days after transplantation, CD2 lymphocytes averaged 55% (18%), 16% (15%) and 14% (11%) in groups 1, 2 and 3 respectively (p < 0.05), CD4 averaged 36% (11%), 9% (12%) and 7% (8%) in groups 1, 2 and 3 (p < 0.05), and CD8 averaged 14% (6%), 4% (3%) and 4% (3%) in groups 1, 2 and 3 (p < 0.05). At 15 days after transplantation CD2 averaged 69% (10%), 42% (16%) and 47% (20%) in groups 1, 2 and 3 respectively (p < 0.05), and CD8 averaged 16% (7%), 16% (6%) and 19% (7%) (p = NS). At 30 days after transplantation the percentages of CD2, CD4 and CD8 lymphocytes were similar among the groups. The freedom rate from acute rejection averaged 22% (14%), 9% (8%) and 50% (18%) (p < 0.05) in groups 1, 2 and 3 at 6 months after transplantation, and the freedom rate from infection averaged 56% (17%), 36% (13%) and 38% (17%) for the 3 groups at this time period (p = NS). CONCLUSIONS: A short course of RATG causes severe, transitory depletion of CD2, CD4 and CD8 lymphocyte subpopulations. MMF decreases the incidence of early acute rejection after heart transplantation without affecting the lymphocyte subpopulation when compared with azathioprine.

Phase I trial of combined immunotherapy with subcutaneous granulocyte macrophage colony-stimulating factor, low-dose interleukin 2, and interferon alpha in progressive metastatic melanoma and renal cell carcinoma.

The purpose of our study was to determine the maximally tolerated dose (MTD) and DLT of combined administration of granulocyte macrophage colony-stimulating factor (GM-CSF), low-dose interleukin 2 (IL-2) and IFN-alpha in patients with progressive metastatic melanoma or renal cell carcinoma (RCC). In addition, the activation and expansion of effector cells were measured. Cohorts of three patients were treated with increasing doses of IL-2 (1, 4, and 8 MIU/m2) and GM-CSF (2.5 and 5 microg/kg) with a constant dose of IFNalpha (5 million units) s.c. for 12 days every 3 weeks. An additional six patients were treated at the MTD. Immune activation was monitored during the first cycle. Response was evaluated after two cycles. The MTD was found to be 2.5 microg/kg GM-CSF, 4 MIU/m2 IL-2, and 5 mega units of IFNalpha. DLT was grade 4 fever, chills with hypotension, grade 3 fatigue/malaise, and fluid retention. Dose reduction of IL-2 to 2 MIU/m2 was necessary in three of nine patients who initially received the MTD. Treatment was initiated in the hospital but could be continued at home after 3-4 days. Significant increases in lymphocytes, (activated) T cells (CD4+ and CD8+), NK cells, monocyte DR expression, neutrophils, and eosinophils were found. CD8+ T-cell activation (sCD8) and NK cell expansion was mainly present in patients receiving 2 or 4 MIU/m2 IL-2. Of eight patients with progressive metastatic RCC after nephrectomy, three achieved a complete remission, and 1 of 7 patients with metastatic melanoma achieved a partial remission. In our study, the MTD of combined immunotherapy with GM-CSF, IL-2, and IFNalpha was established; DLT was: (a) grade 4 fever with hypotension needing i.v. fluid support; and (b) grade 3 fluid retention and/or fatigue/malaise. The scheme resulted in considerable expansion and/or activation of various effector cells. The complete responses in RCC patients are promising but need to be confirmed in Phase II studies.