Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Purpose  The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α could also enhance PBMC cytotoxicity toward bladder cancer cells. Materials and methods  PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-α or control MV261 BCG, and used as effector cells in ⁵¹Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. To determine the role of natural killer (NK) and CD8⁺ T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in ⁵¹Cr-release assays. Results  Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8⁺ T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant. Conclusions  rBCG-IFN-α is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.     

Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b⁺ myeloblasts. Both disease manifestations are more pronounced in interferon-γ receptor knock-out (IFN-γR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b⁺ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-γR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-κB ligand (RANKL) or with tumour necrosis factor-α (TNF-α). Significantly larger numbers of such cells could be generated from splenocytes of IFN-γR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-α-induced osteoclast formation. Splenocyte cultures of IFN-γR KO mice also produced more TNF-α and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-γR KO mice contained comparatively low levels of pro-interleukin-1β (pro-IL-1β) and pro-caspase-1, indicating more extensive conversion of pro-IL-1β into secreted active IL-1β. These observations provide evidence that all conditions are fulfilled for the expanding CD11b⁺ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-γR KO mice to CIA.     

Flowcytometric assessment of intracellular interferon -γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation

Interferon-(IFN-γ) has been considered to be a critical protective immunomodulatory component against Mycobacterium tuberculosis (M. tb.) infection. In this study T-cell proliferation and IFN-γ production upon stimulation with M. tb. were assessed in patients of pulmonary tuberculosis and healthy contacts. The studies were based on lymphocyte transformation test and detection of intracellular IFN-γ production by CD4 + ve T-cells by flowcytometry. Patients showed lower levels of proliferation, the stimulation index being in the range of 2.17 1.1 (mean + SD) compared to the contacts (SI = 4 59±1.6) (P < 0.01). The kinetics of intracellular induction of IFN-γ on M. tb. stimulation showed a proportional increase in the CD4 + ve T-cell population. The increase was maximal between 96–120 h of culture. In healthy contacts the number of IFN-γ expressing CD + ve T-cells increased to 2.5 to 41 × 10⁴ cells/ml in M. tb. stimulated cultures compared to control cultures (0.1 – 15 × 10⁴). In contrast patients showed no/marginal increase in CD4 + ve T-cell population expressing intracellular IFN-γ Thus the lack of induction of IFN in CD4 + ve T-cells in patients could be a critical shortcoming in their ability to combat tubercle bacilli infection.     

Distinct myeloid suppressor cell subsets correlate with plasma IL-6 and IL-10 and reduced interferon-alpha signaling in CD4<Superscript>+</Superscript> T cells from patients with GI malignancy

Interferon-alpha (IFN-α) promotes anti-tumor immunity through its actions on immune cells. We hypothesized that elevated percentages of myeloid-derived suppressor cells (MDSC) and increased pro-inflammatory cytokines in peripheral blood would be associated with impaired response to IFN-α in patients with gastrointestinal (GI) malignancies. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets, and IFN-α-induced signal transduction in 40 patients with GI malignancies. Plasma IL-6 and IL-10 were significantly higher in patients versus normal donors. CD33⁺HLADR⁻CD11b⁺CD15⁺ and CD33⁺HLADR^−/lowCD14⁺ MDSC subsets were also elevated in patients versus normal donors (P < 0.0001). Plasma IL-6 was correlated with CD33⁺HLADR⁻CD15⁺ MDSC (P = 0.008) and IL-10 with CD33⁺HLADR⁻CD15⁻ MDSC (P = 0.002). The percentage of CD15⁺ and CD15⁻ but not CD14⁺ MDSC subsets were inversely correlated with IFN-α-induced STAT1 phosphorylation in CD4⁺ T cells, while co-culture with in vitro generated MDSC led to reduced IFN-α responsiveness in both PBMC and the CD4⁺ subset of T cells from normal donors. Exploratory multivariable Cox proportional hazards models revealed that an increased percentage of the CD33⁺HLADR⁻CD15⁻ MDSC subset was associated with reduced overall survival (P = 0.049), while an increased percentage of the CD33⁺HLADR^−/lowCD14⁺ subset was associated with greater overall survival (P = 0.033). These data provide evidence for a unique relationship between specific cytokines, MDSC subsets, and IFN-α responsiveness in patients with GI malignancies.     

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

 The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3⁺PBL) and TIL (CD3⁺TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3⁺TIL and CD3⁺PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3⁺TIL produced significantly lower levels of IL-2 than CD3⁺PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3⁺TIL as compared to the CD3⁺PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3⁺TIL and CD3⁺PBL derived from patients with renal cell carcinomas. Received: 8 August 1995 / Accepted: 23 January 1996     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4⁺ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-γ or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-γ producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-γ alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-γ- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-γ and IL-4 producers.     

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo

Background We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)⁺ T cells is reflected by changes of selected immune parameters measured in peripheral blood. Methods ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)⁺ T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. Results Prior to infusion, scFv(G250)⁺ T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX⁺ RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-γ were produced, whilst production of TNF-α, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-γ was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-γ increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. Conclusion ScFv(G250)-receptor-mediated functions of the scFv(G250)⁺ T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-γ levels.     

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I–restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8⁺ splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8⁺ tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8⁺ TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8⁺ splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8⁺ TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8⁺ anti-tumor response.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Age related changes in T cell mediated immune response and effector memory to Respiratory Syncytial Virus (RSV) in healthy subjects

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA^-CCR7⁺) and effector (CD45RA^-CCR7^-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4⁺25⁺FoxP3⁺) T cells (T_reg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in T_reg was observed with aging. The finding of T_reg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.     

Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4⁺ memory T cells, whereas CD4⁺ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4⁺ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α. A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.     

The Phosphoenolpyruvate Carboxykinase of Mycobacterium Tuberculosis Induces Strong Cell-Mediated Immune Responses in Mice

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes guanosine or adenosine mononucleotide-dependent reversible conversion of oxaloacetate (OAA) and phosphoenolpyruvate (PEP). Mycobacterium (M) tuberculosis possesses a putative GTP-dependent PEPCK. To analyze the immune responses caused by PEPCK, the effects of PEPCK on the induction of CD4⁺ T cells and cytokines such as IFN-γ, IL-12 and TNF-α were evaluated in mice. It was found that the number of CD4⁺ T cells was increased in the PEPCK immunized mice although the change of the number of CD8⁺ T cells was not significant. The cytokines IFN-γ, IL-12 and TNF-α were increased significantly in the mice immunized with PEPCK than those of incomplete adjuvant. These characteristics were further demonstrated in the mice infected by pckA mutated BCG strain. The results indicate that PEPCK can effectively induce cell-mediated immune response by increasing activity of cytokines and PEPCK may be a promising new subunit vaccine candidate for tuberculosis.     

Characterisation of the immune response to type I collagen in scleroderma

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSE^low). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4⁺, activated (CD25⁺), memory (CD45RO⁺) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.     

Immunomodulatory effects ofBacillus firmus

3-day-old miniature piglets were stimulatedin vivo withBacillus firmus by the intraperitoneal or intragastric route for 1 d. Cells containing IgA and IgG₂ were detected in the ileum in all stimulated but not in control animals. The frequency of blood CD3⁺ cells increased after intraperitoneal administration ofB. firmus, the ratio of polymorphonuclears to lymphocytes increased in all stimulated piglets.B. firmus induced antitumor immunity in rats with transplanted Yoshida sarcoma cells. Granular lymphocytes and dead tumor cells were found in peritoneal exudate of stimulated animals.B. firmus induced IFN-γ synthesis in human blood lymphocytes stimulatedin vitro for 1 d. The amount of TNF-α produced by these stimulated human peripheral blood mononuclears (PBMC) was lower than that of PBMC stimulated with some other bacterial immunomodulators. Cells containing TGF-β or IL-8 were not found in human PBMC stimulated withB. firmus.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Interferon-γ- and lipopolysaccharide-induced tumor necrosis factor-α is required for nitric oxide production: Tumor necrosis factor-α and nitric oxide are independently involved in the killing ofMycobacterium microti in interferon-γ-and lipopolysaccharide-treated J774A.1 cells

A comparative study was done using J774A.1 and J774A. 1-derived transfected cells (J774A.1 C.1) containing antisense tumor necrosis factor α (TNF-α) plasmid to determine the role of endogenous TNF-α on nitric oxide production as well as on the growth ofMycobacterium microti in interferon γ (IFN-γ)- and lipopolysaccharide (LPS)-treated cells. On stimulation with IFN-γ and LPS a higher level of NO was observed in J774A.1 cells compared to J774A.1 C.1 which indicated that endogenous TNF-α is required for the production of NO. Comparing the effect of IFN-γ and LPS on the intracellular growth ofM. microti, the growth-reducing activity was higher in J774A.1 cells than in J774A.1 C.1 cells and was not completely abrogated in the presence of the nitric oxide inhibitorN ^G-methyl-l-arginine (l-NMA). J774A.1 C.1 cells infected withM. microti produced a significant amount of NO when exogenous TNF-α was added along with IFN-γ and LPS and the concentration of intracellular bacteria decreased almost to that in IFN-γ and LPS treated parental J774A.1 cells. Addition of exogenous TNF-α even in the presence ofl-NMA in J774.1 C.1 cells could also partially restore intracellular growth inhibition ofM. microti caused by IFN-γ and LPS. TNF-α is probably required for the production of NO in J774A.1 cells by IFN-γ and LPS but TNF-α and NO are independently involved in the killing of intracellularM. microti with IFN-γ and LPS.     

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 × 10⁷ CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-γ, IL-1α, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-γ production in mice, as well as the CFA, were used to stimulate IFN-γ production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-γ production in the infected group. In humans, only the CFA-induced IFN-γ production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.     

Characteristic Immune Response in Peyer’s Patch Cells Induced by Oral Administration of Bifidobacterium Components

We demonstrate immunomodulatory effects, especially those involving murine intestinal IgA secretion, in Peyer's patch cells following oral administration of Bifidobacterium immunomodulator (BIM) derived from sonicated B. pseudocatenulatum 7041. BALB/c mice were administered BIM orally for 7 consecutive days. The PP cells demonstrated upregulated secretion of total IgA including BIM-specific IgA following BIM administration. In observing the response of PP cells co-cultured with BIM, we found enhanced secretion of interferon-γ (IFN-γ) and interleukin (IL)-6 in the CD4⁺ T cells. In contrast, IL-12 secretion by Thy1.2⁻ PP cells was enhanced, but secretion of IFN-γ, IL-5, and IL-6 was not significantly affected. Furthermore, the population of CD4⁺ CD45RB^high T cells in PP increased following oral administration of BIM. These data suggest that CD4⁺ T cells were affected by BIM administration. Overall, the results show that oral administration of BIM induced CD4⁺ PP cells to change their expression of cell surface antigen and cytokine production.     

Fungal Infections in Phagocytic Defects

Delineating the infection susceptibility of primary immunodeficiencies allows insight into host immunity. Filamentous mold infections are seen most frequently in chronic granulomatous disease, a neutrophil disorder characterized by impaired superoxide production. Mucocutaneous candidiasis occurs in disorders of impaired interleukin (IL)-17 and IL-22 signaling, such as seen in autosomal dominant hyper-IgE (Job’s) syndrome and in disorders with autoantibodies to these cytokines. The endemic dimorphic fungi are in part controlled by disorders of the IL-12/interferon (IFN)-γ pathway, such as IFN-γ receptor and STAT1 defects. Understanding the pathways involved in these primary immunodeficiency disorders will also provide insight into these infections in secondary immunodeficiencies and allow guidance for novel therapies.     

Interferon-α/β upregulate IL-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis C patients during interferon-α/β treatment

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8⁺ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response.