Addition Of Interferon-gamma Research Articles

Induction of APOBEC3-mediated genomic damage in urothelium implicates BK polyomavirus (BKPyV) as a hit-and-run driver for bladder cancer

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. Mutational signatures show the anti-viral apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes are responsible for the preponderance of mutations in bladder tumour genomes, but no causative viral agent has been identified. BK polyomavirus (BKPyV) is a common childhood infection that remains latent in the adult kidney, where reactivation leads to viruria. This study provides missing mechanistic evidence linking reactivated BKPyV-infection to bladder cancer risk. We used a mitotically-quiescent, functionally-differentiated model of normal human urothelium to examine BKPyV-infection. BKPyV-infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry from G0 into the cell cycle through inhibition of retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, with cells arresting in G2. The single-stranded DNA displacement loops formed in urothelial cells during BKPyV-infection interacted with LT-Ag to provide a substrate for APOBEC3-activity. Addition of interferon gamma (IFNγ) to infected urothelium suppressed expression of the viral genome. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations.

Increase in Efficacy of Checkpoint Inhibition by Cytokine-Induced-Killer Cells as a Combination Immunotherapy for Renal Cancer.

Cytokine-induced killer (CIK) cells are heterogeneous, major histocompatibility complex (MHC)-unrestricted T lymphocytes that have acquired the expression of several natural killer (NK) cell surface markers following the addition of interferon gamma (IFN-γ), OKT3 and interleukin-2 (IL-2). Treatment with CIK cells demonstrates a practical approach in cancer immunotherapy with limited, if any, graft versus host disease (GvHD) toxicity. CIK cells have been proposed and tested in many clinical trials in cancer patients by autologous, allogeneic or haploidentical administration. The possibility of combining them with specific monoclonal antibodies nivolumab and ipilimumab will further expand the possibility of their clinical utilization. Initially, phenotypic analysis was performed to explore CD3, CD4, CD56, PD-1 and CTLA-4 expression on CIK cells and PD-L1/PD-L2 expression on tumor cells. We further treated CIK cells with nivolumab and ipilimumab and measured the cytotoxicity of CIK cells cocultured to renal carcinoma cell lines, A-498 and Caki-2. We observed a significant decrease in viability of renal cell lines after treating with CIK cells (p < 0.0001) in comparison to untreated renal cell lines and anti-PD-1 or anti-CTLA-4 treatment had no remarkable effect on the viability of tumor cells. Using CCK-8, Precision Count Beads™ and Cell Trace™ violet proliferation assays, we proved significant increased proliferation of CIK cells in the presence of a combination of anti-PD-1 and anti-CTLA-4 antibodies compared to untreated CIK cells. The IFN-γ secretion increased significantly in the presence of A-498 and combinatorial blockade of PD-1 and CTLA-4 compared to nivolumab or ipilimumab monotreatment (p < 0.001). In conclusion, a combination of immune checkpoint inhibition with CIK cells augments cytotoxicity of CIK cells against renal cancer cells.

Efficacy of isolated limb perfusion (ILP) in patients with Merkel cell carcinoma (MCC): A multicenter experience

BackgroundMerkel cell carcinoma (MCC) is a rare and potentially aggressive neuroendocrine tumor of the skin, with a propensity for locoregional metastases. In two expert referral centers, isolated limb perfusion (ILP) is used to obtain locoregional control in selected locoregionally advanced MCC patients. This study describes our experience. MethodPatients who underwent ILP for MCC were analyzed. ILP was performed with melphalan and tumor necrosis factor (TNF) combination therapy. Depending on the institution, either a normothermic or a hyperthermic temperature regimen was used. Baseline characteristics, toxicity data, locoregional progression-free survival (LPFS) and overall survival (OS) were assessed. ResultsFour males and 6 females with a median age of 78 years (IQR 61–84 years) were included. Four patients underwent ILP for upper extremity disease and 6 for lower extremity disease. All patients received combination therapy with Melphalan and TNF, one patient with the addition of interferon-gamma. No signs of systemic toxicity were present post-ILP. Severe locoregional toxicity (compartment syndrome) occurred in 1 patient and 1 elderly patient with extensive atherosclerosis had to undergo transfemoral amputation due to critical ischemia. Eight patients could be included for response evaluation. The overall response rate (ORR) was 87.5% with a complete response (CR) rate of 62.5%. Two long-term responses of 53 months and 71 months were observed. Median LPFS was 5 months and median OS was 54 months. ConclusionILP shows a high CR rate that can be durable. Therefore, ILP should be considered an effective treatment modality for locally advanced MCC.

50 Role of reaginic IgE phenomena in Pseudomonas aeruginosa infection in patients with cystic fibrosis

PET using F-FDG is widely used in the diagnosis, monitoring and staging of malignancy. Tissue FDG uptake is determined by metabolic activity and can identify neutrophilic inflammation and in stable patients, enhanced uptake is often absent. We report our experience of PET in three patients with CF and how results helped support clinical decision making. A 38 year old post lung transplant patient presented with recurrent right sided chest pain, fever and elevated CRP. Despite extensive investigation by the transplant unit, no focal abnormality was identified and symptoms failed to resolve with iv antibiotics. PET revealed FDG avidity in the right middle lobe and she required a right middle and upper lobectomy. A 30 year old patient chronically colonised with P. aeruginosa and M. abscessus, developed aggressive lung disease and despite regular intravenous, nebulised and oral antibiotics, continued to decline with a fall in lung function, pyrexia and malaise. PET revealed marked FDP avidity within multiple lung nodules. Treatment was continued with the addition of interferon gamma. Despite a worsening CXR, PET showed significant improvement. These results have helped guide therapy. A 36 year old presented with persistent respiratory symptoms. Sputum was repeatedly culture positive for M. abscessus. There were no clinical or radiological evidence of deterioration. PET was undertaken to exclude active focal inflammation. The scan was unremarkable and a less intense regimen was instituted. Sputum has since been persistently negative for M. abscessus. PET is a useful adjunct to conventional investigations and in selected individuals can be helpful in guiding clinical management.

49 The use of positron emission tomography (PET) in adult patients with cystic fibrosis (CF)

PET using 18F-FDG is widely used in the diagnosis, monitoring and staging of malignancy. Tissue FDG uptake is determined by metabolic activity and can identify neutrophilic inflammation and in stable patients, enhanced uptake is often absent. We report our experience of PET in three patients with CF and how results helped support clinical decision making. A 38 year old post lung transplant patient presented with recurrent right sided chest pain, fever and elevated CRP. Despite extensive investigation by the transplant unit, no focal abnormality was identified and symptoms failed to resolve with iv antibiotics. PET revealed FDG avidity in the right middle lobe and she required a right middle and upper lobectomy. A 30 year old patient chronically colonised with P. aeruginosa and M. abscessus, developed aggressive lung disease and despite regular intravenous, nebulised and oral antibiotics, continued to decline with a fall in lung function, pyrexia and malaise. PET revealed marked FDP avidity within multiple lung nodules. Treatment was continued with the addition of interferon gamma. Despite a worsening CXR, PET showed significant improvement. These results have helped guide therapy. A 36 year old presented with persistent respiratory symptoms. Sputum was repeatedly culture positive for M. abscessus. There were no clinical or radiological evidence of deterioration. PET was undertaken to exclude active focal inflammation. The scan was unremarkable and a less intense regimen was instituted. Sputum has since been persistently negative for M. abscessus. PET is a useful adjunct to conventional investigations and in selected individuals can be helpful in guiding clinical management.

&lt;em&gt;Toxoplasma gondii&lt;/em&gt; Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFNγ). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNγ and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNγ and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

&lt;em&gt;Toxoplasma gondii&lt;/em&gt; Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFNgamma). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNgamma and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNgamma and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

CD4+CD25+TREGS ACTIVATED IN VITRO WITH IL-2 AND IL-12P70 SUPPRESS REJECTION OF FULLY ALLOGENEIC GRAFTS IN ANIMALS WITH NO OTHER IMMUNOSUPPRESSION.

Hall, B. M.1; Verma, N. D.1; Boyd, R.1; Robinson, C. M.1; Tran, G. T.1; Wang, C.2; Bishop, G. A.3; Hodgkinson, S. J.1 Author Information

Toll‐like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar Typhimurium infection, but not for the initiation of bacterial clearance

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF(-/-) mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice in vitro. Pro-inflammatory responses from Mal(-/-) macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF(-/-) macrophages were partially restored by the addition of interferon-gamma (IFN-gamma), and TRIF(-/-) mice produced markedly enhanced IFN-gamma levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4(-/-), MyD88(-/-), TRIF(-/-) and Mal(-/-) mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

Effect of interferon gamma and CD40 ligation on intracellular monocyte survival of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a mucosal pathogen that is a major cause of respiratory infection, including sinusitis, otitis media and bronchitis. This bacterium has evolved a number of mechanisms to facilitate its survival in the human host. Recently it has been recognized that it is capable of intracellular survival in monocytes/macrophages and epithelial cells. Previous work by the authors has demonstrated that the protective response to NTHi is Th1 predominant. This information led to the hypothesis that the intracellular survival of NTHi in human monocytes may be reduced by two key effector mechanisms of Th1-mediated immunity: interferon gamma and ligation of CD40. This study assessed the effect of interferon gamma and ligation of CD40 on the intracellular survival of NTHi in human monocytes. Responses were studied in monocytes from subjects with bronchiectasis and persistent airway infection with NTHi and compared with control subjects. The results demonstrated that different isolates of NTHi were able to survive inside monocytes. Killing of one strain of NTHi could be enhanced by the addition of interferon gamma and CD40 ligation in both control and bronchiectasis subjects. Other strains were more resistant.

Dendritic Cell Function during Chronic Hepatitis C Virus and Human Immunodeficiency Virus Type 1 Infection

Hepatitis C virus (HCV) infection can persist despite HCV-specific T-cell immunity and can have a more aggressive course in persons coinfected with human immunodeficiency virus type 1 (HIV-1). Defects in antigen-presenting, myeloid dendritic cells (DCs) could underlie this T-cell dysfunction. Here we show that monocyte-derived DCs from persons with chronic HCV infection, with or without HIV-1 coinfection, being treated with combination antiretroviral therapy produced lower levels of interleukin 12 (IL-12) p70 in response to CD40 ligand (CD40L), whereas the expression of DC surface activation and costimulatory molecules was unimpaired. The deficiency in IL-12 production could be overcome by addition of gamma interferon (IFN-gamma) with CD40L, resulting in very high, comparable levels of IL-12 production by DCs from HCV- and HIV-1-infected subjects. Smaller amounts of IL-12 p70 were produced by DCs treated with the immune modulators tumor necrosis factor alpha and IL-1beta, with or without IFN-gamma, and the amounts did not differ among the uninfected and infected subjects. Blocking of IL-10 with an anti-IL-10 monoclonal antibody in the CD40L-stimulated DC cultures from HCV-infected persons increased the level of IL-12 p70 production. The ability of DCs from HCV-infected persons to stimulate allogeneic CD4+ T cells or induce IL-2, IL-5, or IL-10 in a mixed lymphocyte reaction was not impaired. Thus, myeloid DCs derived from persons with chronic HCV infection or with both HCV and HIV-1 infections have defects in IL-12 p70 production related to IL-10 activity that can be overcome by treatment of the DCs with CD40L and IFN-gamma. DCs from these infected subjects have a normal capacity to stimulate CD4+ T cells. The functional effectiveness of DCs derived from HCV-infected individuals provides a rationale for the DC-based immunotherapy of chronic HCV infection.

Human adipose derived stem cells suppress lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli

To study immunological properties of adipose derived stem cells (ADSC) and their in vitro immunomodulatory effects on lymphocytes. ADSC was isolated from fat tissue by liposuction and culture expanded. Cells at passage 2 were observed for the expression of HLAI, HLAII by FACs analysis. Peripheral blood lymphocytes (PBL) were cultured with allogeneic ADSCs at dose of 1 x 10(5). A third-party ADSCs at dose of 1 x 10(2), 1 x 10(3), 1 x 10(4), 1 x 10(5) were added to the ongoing two-way mixed lymphocyte reactions (MLR) for 6 days. Value of CPM (count per minute) was calculated with luminescence counter. Flow cytometry showed that undifferentiated ADSCs express HLA class I but not class II. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic ADSCs as stimulators. Results were similar using ADSCs pretreated with IFN-gamma. ADSCs elicited inhibit function of mixed lymphocyte cultures in a dose dependent way. Even if ADSCs were pretreated with IFN-gamma, the suppression ability was maintained. Undifferentiated ADSCs do not elicit alloreactive lymphocyte proliferative responses and could modulate immune responses in vitro.

Gene expression and antimicrobial activity of bovine macrophages in response to Mycobacterium avium subsp. paratuberculosis.

We evaluated gene expression and antimicrobial responses of bovine monocyte-derived macrophages incubated with Mycobacterium avium subsp. paratuberculosis (M. a. ptb), the causative agent of Johne's disease. Gene expression was evaluated by the use of human noncompetitive high-density oligonucleotide microarrays. Bovine messenger RNA hybridized with 14.2-18.2% of the 12,600 oligonucleotide probe sets. When macrophages incubated with M. a. ptb were compared with nonactivated control macrophages, macrophages activated by addition of interferon-gamma and lipopolysaccharide, and macrophages incubated with Mycobacterium avium subspecies avium (M. a. a), 47, 79, and 27 genes, respectively, were differentially expressed. Differential expression of six of these genes was confirmed using reverse transcriptase polymerase chain reaction. Several functional assays were performed to evaluate the potential relevance of differentially expressed genes to host defense. Macrophages phagocytizing M. a. a had a greater capacity to kill the organisms and to acidify phagosomes and a greater degree of apoptosis than did macrophages incubated with M. a. ptb. The results of these studies indicate that multiple genes and metabolic pathways are differentially expressed by macrophages ingesting mycobacterial organisms. Although the intracellular fate of mycobacterial organisms appears to be dependent on a complex interaction between macrophage and organism, phagosome acidification and apoptosis may play central roles in organism survival.

Characterization of M. Tuberculosis-derived IL-12-inducing material by alveolar macrophages

We have investigated the substance derived from Mycobacterium tuberculosis (Mtb) that induces interleukin (IL)-12 production by alveolar macrophages (AMs) in vitro. The cytosol fraction of live Mtb H37Rv induced IL-12 production by AMs in a dose-dependent manner. The addition of interferon-gamma (IFN-γ) augmented IL-12 production. IL-12-inducing activity by AMs (termed as surely active keeping rescue antigen, SAKRA) was purified by gel filtration and ion exchange column chromatography, and the molecular weight of SAKRA was estimated by gel filtration to be more than 700kDa. SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting of SAKRA using rabbit anti-SAKRA antibody suggested that SAKRA is composed with several low molecular weight proteins. Amino acids sequence analysis of several bands after SDS-PAGE suggested that SAKRA is a part of ribosomes. RT-PCR showed that SAKRA induced not only expression of IL-12 p40 mRNA, but expression of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) mRNA at least 6h after stimulation, suggesting that SAKRA activates the bactericidal activity of macrophages. To investigate the potential use of SAKRA as a vaccine against tuberculosis, SAKRA was administered to BALB/c mouse that had been immunized with BCG for 18 months, and mouse were infected with Mtb H37Rv via a respiratory route. Replication of Mtb in lungs and spleens was examined 6 weeks after infection. Administration of SAKRA to BCG-vaccinated mice significantly reduced the numbers of Mtb in lungs and spleens as compared with BCG-vaccinated control mice. Taken together, these results suggest that SAKRA is one of the Mtb-derived immunomodulatory substances which induce IL-12 production during infection and also increases mycobactericidal activities of macrophages, and that SAKRA may be a promising new vaccine candidate against tuberculosis.

THE INFLUENCE OF DIETARY FISH-OIL SUPPLEMENTATION ON CUTANEOUS INFECTION IN MICE

Dietary fish-oil (FO) supplementation has been shown to inhibit inflammation in various clinical disease states and to be beneficial in experimental models of inflammation and bacterial and plasmodial infection. In mice, FO increases macrophage production of tumour necrosis factor alpha (TNF). Production of TNF has been reported to be important in the resistance of mice against various Leishmania spp. We investigated whether dietary supplementation with FO protects susceptible Balb/c mice against infection with Leishmania amazonensis. No influence of the FO diet on the course of infection was observed, as evaluated by the increase in thickness of infected footpads over forty days. Lipopolysaccharide (LPS)-induced TNF production of peritoneal cells was however significantly increased in FO fed mice (P<0.01). When L. amazonensis was used as a stimulus, the in-vitro production of TNF by isolated peritoneal cells was minimal and did not differ between the various treatment groups. Addition of interferon gamma did not restore the effect of FO on TNF production capacity. We conclude that dietary supplementation with FO is of no benefit in Leishmaniasis in susceptible Balb/c mice, and that L. amazonensis is an insufficient trigger for TNF production in this model.

THE INFLUENCE OF DIETARY FISH-OIL SUPPLEMENTATION ON CUTANEOUS LEISHMANIA AMAZONENSIS INFECTION IN MICE

Dietary fish-oil (FO) supplementation has been shown to inhibit inflammation in various clinical disease states and to be beneficial in experimental models of inflammation and bacterial and plasmodial infection. In mice, FO increases macrophage production of tumour necrosis factor alpha (TNF). Production of TNF has been reported to be important in the resistance of mice against various Leishmania spp. We investigated whether dietary supplementation with FO protects susceptible Balb/c mice against infection with Leishmania amazonensis. No influence of the FO diet on the course of infection was observed, as evaluated by the increase in thickness of infected footpads over forty days. Lipopolysaccharide (LPS)-induced TNF production of peritoneal cells was however significantly increased in FO fed mice (P<0.01). When L. amazonensis was used as a stimulus, the in-vitro production of TNF by isolated peritoneal cells was minimal and did not differ between the various treatment groups. Addition of interferon gamma did not restore the effect of FO on TNF production capacity. We conclude that dietary supplementation with FO is of no benefit in Leishmaniasis in susceptible Balb/c mice, and that L. amazonensis is an insufficient trigger for TNF production in this model.

Identification of a clinical-grade maturation factor for dendritic cells.

Dendritic cells (DC) are essential for the generation of primary adaptive immune responses, but their full immunostimulatory capacities are only reached upon maturation. The authors compared several clinical-grade adjuvants of bacterial origin to determine their ability to induce phenotypic and functional maturation of monocyte-derived DC (Dendritophages, Dphi; IDM, Paris, France) differentiated with granulocyte-macrophage colony-stimulating factor and interleukin-13 in single-use cell processors (VacCell; IDM, Paris, France). Monophosphoryl lipid A, Mycobacterium bovis bacillus Calmette-Guerin, and Ribomunyl (Pierre Fabre Medicament, Boulogne, France) all appeared able to provide the signal necessary to initiate Dphi maturation. However, only Ribomunyl (Pierre Fabre Medicament) (containing membrane and ribosomal fractions from four bacterial strains) allowed the authors to obtain a significant enhancement of allostimulatory abilities and cytokine production by Dphi in the absence of active cellular infection. Addition of interferon-gamma (IFN-gamma) to Ribomunyl resulted in more pronounced upregulation of CD83, major histocompatibility complex class I, and B7 molecules by Dphi. Moreover, the IFN-gamma addition modulated their cytokine secretion, allowing higher levels of bioactive interleukin-12 concomitant with lower levels of interleukin-10. In kinetic studies, Dphi contact with Ribomunyl and IFN-gamma for 6 hours was sufficient to trigger a maturation process that completed spontaneously. Thus, Ribomunyl in association with IFN-gamma represents a suitable agent for the ex vivo production of mature monocyte-derived DC that can be used as cellular vaccines to promote a potent type I immune response.

Effects of superantigen and lipopolysaccharide on induction of CD80 through apoptosis of human monocytes.

To investigate the mechanisms underlying superantigen (SAg) stimulation, we analyzed the effect of SAg on monocyte responses with or without lipopolysaccharide (LPS). Addition of gamma interferon (IFN-gamma) to unstimulated cultures induced a marked increase in the number of CD80(+) monocytes, which was inhibited by LPS through the action of interleukin-10. However, CD80(+) monocytes began to increase before IFN-gamma production, observed after 9 h of stimulation with staphylococcal enterotoxin B (SEB). SEB selectively increased the number of apoptotic CD80(-) monocytes, whereas LPS-treated monocytes were resistant to the apoptotic action of SEB. This SEB-induced killing was abrogated by anti-CD95 monoclonal antibody (MAb) ZB4 and anti-CD95 ligand (CD95L) MAb NOK2, suggesting a CD95-based pathway of apoptosis. Furthermore, the numbers of SEB-induced CD80(+) monocytes were partially decreased by anti-CD119 (IFN-gamma receptor) MAb and by anti-CD95L (NOK2) MAb. The CD30 expression of CD27(high) T cells induced by SEB was increased by agonistic anti-CD95 (CH11) MAb. Together, our findings showed that SEB-induced monocyte apoptosis is closely associated with the enrichment of CD80(+) monocytes generated before IFN-gamma production, followed by up-regulation of CD80 by IFN-gamma, and that LPS has negative effects in both cases. These results also suggested that induction of monocyte apoptosis is an important mechanism by which SAg exerts its anti-inflammatory effects.

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells.

Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon gamma (IFNgamma), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNalpha but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis(Y) antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors.

Defective induction of interleukin-12 in human monocytes by germ-tube forms of Candida albicans.

Yeast (Y) to germ-tube (GT) transition of Candida albicans is considered a putative virulence trait. On the other hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen. We studied IL-12 production by human monocytes cocultured in vitro with Y or GT forms of C. albicans. Following stimulation by Y cells, monocytes produced appreciable levels of IL-12, which, upon addition of gamma interferon (IFN-gamma), compared to those achievable by lipopolysaccharide (100 ng/ml) stimulation (140 +/- 65 and 185 +/- 80 pg/ml, respectively [mean +/- standard deviation in four independent experiments]). In contrast, IL-12 production by GT cell-stimulated monocytes was much lower or absent (<5 pg/ml) and could not be brought to the level induced by Y cells by the addition of IFN-gamma (30 +/- 10 pg/ml in the four independent experiments above). Besides being observed as actual cytokine production, this lower response was also observed as specific IL-12 p40 mRNA transcript and was not associated with hyperproduction of the IL-12-competing cytokine IL-10. Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells of C. albicans were not phagocytized by the human monocytes. Importantly, however, Y and GT cells were equally killed by the monocytes. Thus, the virulence trait attributed to the Y-GT transition of C. albicans might also be related to the lack of induction by GT cells of a protective anticandidal immunity through defective IL-12 production.