Grażyna Korczak-Kowalska

Phage as a modulator of immune responses: practical implications for phage therapy.

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Lovastatin potentiates antitumor activity of doxorubicin in murine melanoma via an apoptosis-dependent mechanism

Lovastatin, a drug successfully used in the clinic to prevent and to treat coronary heart disease, has recently been reported to decrease the incidence of melanoma in lovastatin‐treated patients. Lovastatin has also been proved to potentiate antitumor effects of both cisplatin and TNF‐α in murine melanoma models. Recently, an augmented therapeutic effect of lovastatin and doxorubicin has been reported in 3 tumor models in mice. In our preliminary study lovastatin caused retardation of melanoma growth in mice treated with doxorubicin (Feleszko et al. J Natl Cancer Inst 1998;90:247–8). In the present report, we supplement our preliminary observations and demonstrate in 2 murine and 2 human melanoma cell lines that lovastatin effectively potentiates the cytostatic/cytotoxic activity of doxorubicin in vitro via an augmentation of apoptosis (estimated with PARP‐cleavage assay, annexin V assay and TUNEL). The combined antiproliferative activity of lovastatin and doxorubicin was evaluated using the combination index (CI) method of Chou and Talalay, revealing synergistic interactions in melanoma cells exposed to lovastatin and doxorubicin. In B16F10 murine melanoma model in vivo, we have demonstrated significantly increased sensitivity to the combined treatment with both lovastatin (5 mg/kg for 14 days) and doxorubicin (4 × 1 mg/kg) as compared with either agent acting alone. Lovastatin treatment resulted also in significant reduction of the number of experimental metastasis in doxorubicin‐treated mice. The results of our studies suggest that lovastatin may enhance the effectiveness of chemotherapeutic agents in the treatment of malignant melanomas.

Phage as a Modulator of Immune Responses

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs

BACKGROUND A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro. METHODS CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Tregs suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1. CONCLUSIONS Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.

CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood and peritoneal fluid of patients with endometriosis

STUDY QUESTION Is endometriosis associated with changes in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg cells)? SUMMARY ANSWER Endometriosis is associated with disturbed compartmentalization of CD25(high) FOXP3⁺ Treg cells. WHAT IS KNOWN ALREADY Endometriosis is associated with an abrogated immune response and displays some features of an autoimmune disorder. Treg cells play a part in the development of autoimmune reactions; however, their role in pathogenesis of endometriosis is still poorly recognized. STUDY DESIGN, SIZE AND DURATION Case-control study comparing 17 women with laparoscopically and histopathologically confirmed ovarian endometriosis with 15 control women without visible endometriosis foci, pelvic inflammation or related pathology who were subjected to laparoscopic surgery between 2010 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS Peripheral blood and peritoneal fluid were collected during laparoscopy and T cell subpopulations were analysed by flow cytometry using specific monoclonal antibodies recognizing CD4⁺, CD25⁺ and FOXP3⁺ markers. MAIN RESULTS The percentage of CD25(high) FOXP3⁺ Treg cells was significantly decreased in the peripheral blood of women with ovarian endometriosis compared with control women. On the other hand, the proportion of these cells was significantly increased in the peritoneal fluid of women with endometriosis. LIMITATIONS, REASONS FOR CAUTION The present study is limited to patients with ovarian endometrioma and further investigations are needed, including patients with lower grade of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS The present results suggest that Treg cells may play a part in immunopathogenesis of endometriosis being responsible for abrogated local cellular immune responses and facilitation and development of autoimmune reactions. Treg cells may be thus a potential target in the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by 1M15/N/2011 and NK1W grants from the I Faculty of Medicine, Warsaw Medical University. None of the authors has any competing interests to declare.

Association of leptin with inflammatory cytokines and lymphocyte subpopulations in peritoneal fluid of patients with endometriosis

INTRODUCTION Endometriosis is a common, complex and chronic disease related to ectopic implantation and growth of endometrial tissue that may manifest by pelvic inflammatory reactions, chronic pelvic pain and subfertility. Endometriosis may be associated with increased peritoneal fluid leptin levels. Leptin is known to exert immunomodulatory effects; however, an association between leptin and inflammatory reactions in endometriosis has not been documented. Therefore, the aim of this study was to investigate a relationship between leptin concentrations in peritoneal fluid and the levels of peritoneal fluid inflammatory cytokines and mononuclear leukocyte subpopulations. MATERIALS AND METHODS Peritoneal fluid was aspirated by laparoscopy from 46 women in whom endometriosis had been confirmed by clinical and histopathological examinations and from 10 control women qualified for ART in whom pelvic pathology has been excluded. Concentrations of leptin and inflammatory cytokines (IL-1beta, IL-6, IFN-gamma and TNF) in peritoneal fluid were evaluated by specific ELISAs. Percentage of peritoneal leukocyte subpopulations (CD3+, CD4+, CD8+ and CD14+) was analyzed by FACS using specific monoclonal antibodies. RESULTS Leptin concentrations in peritoneal fluid correlated negatively with concentrations of IL-1beta and IFN-gamma (r(s)=-0.38, p=0.01 and r(s)=-0.31, p=0.03, respectively) and correlated positively with the percentage of CD3+ pan-T cells (r(s)=0.69, p=0.009) and CD4+ T helper cells (r(s)=0.74, p=0.036). CONCLUSIONS Increased leptin levels in peritoneal fluid from endometriosis patients may affect local inflammatory/immune reactions, especially infiltration of CD4+ T helper cells. Thus, leptin may play an important role in the immunopathogenesis of endometriosis.

Increased levels of human neutrophil peptides 1, 2, and 3 in peritoneal fluid of patients with endometriosis: association with neutrophils, T cells and IL-8

Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside the uterine cavity. This disease is associated with pelvic inflammation and displays some features of autoimmune disorder. Human neutrophil peptides 1, 2, and 3 (HNP 1-3) belonging to α-defensin family play a crucial role in innate immunity against infections and may exert immunoregulatory effects. They may play a role in various inflammatory reactions; however, their role in endometriosis has not been studied. Therefore, the aim of the present study was to evaluate HNP 1-3 in the peritoneal fluid of 67 patients with endometriosis and 16 healthy control women in relation to peritoneal leukocyte subpopulations (neutrophils, T cells, and macrophages) and inflammatory cytokines (IL-6 and IL-8). HNP 1-3, IL-6 and IL-8 were evaluated in the peritoneal fluid by specific enzyme-linked immunosorbent assays (ELISA), and peritoneal leukocyte subpopulations were evaluated by flow cytometry. We found that the levels of HNP 1-3 were significantly increased in the peritoneal fluid of endometriosis patients, compared with control women, and correlated with severity of the disease. Endometriosis was also associated with increased concentrations of peritoneal neutrophils. In endometriosis the levels of HNP 1-3 strongly correlated with concentrations of neutrophils, T cells and IL-8. HNP 1-3 levels were not associated with peritoneal IL-6 or macrophages. These data suggest that HNP 1-3 and neutrophils might play a role in immunopathogenesis of endometriosis and may be worth evaluating as targets for anti-endometriosis therapy.

Antitumor effects of the combination therapy with TNF-α gene–modified tumor cells and interleukin 12 in a melanoma model in mice

In the present study, TNF-α gene–transduced B78 melanoma cells (B78/TNF) were used as a vaccine and combined with interleukin (IL)-12 in the treatment of B78-melanoma-bearing mice. The combined administration of genetically modified melanoma cells and IL-12 induced specific protective antitumor immunity resulting in a decreased rate of the tumor take following a rechallenge with parental B78 cells. When used therapeutically, intratumoral injections of irradiated B78/TNF melanoma cells and IL-12 exerted strong antitumor effects and led to complete regression of established tumors in 50% of mice. Injections of irradiated B78/TNF cells alone did not influence tumor development and IL-12 itself significantly delayed tumor growth but without curative effect. FACS analysis of parental B78 melanoma cells and its B78/TNF genetically modified variant showed that a proportion of cells of both cell lines expressed B7-1 (CD80) costimulatory molecule and that the expression of this molecule was increased during incubation with IFN-γ. Moreover, IFN-γ markedly augmented expression of major histocompatibility class (MHC) class I and II molecules on B78/TNF cells that were primarily MHC class I and II negative with no substantial effect on MHC-negative parental B78 melanoma. IFN-γ also synergized in cytostatic/cytotoxic effects with TNF-α against B78 melanoma in vitro. Lymphocyte depletion studies in vivo showed reduction of the antitumor response in mice treated with anti-NK monoclonal antibodies (mAbs) as well as in mice treated with anti-CD4+anti-CD8 mAbs. The results suggest that, when used therapeutically, IL-12 and a vaccine containing TNF-α gene–transduced tumor cells may reciprocally augment their overall antitumor effectiveness by facilitating development of systemic antitumor immunity and by stimulating local effector mechanisms of the tumor destruction. Cancer Gene Therapy (2000) 7, 1581–1590.

Immunomodulatory action of human recombinant erythropoietin in man

Recent findings suggest that recombinant human erythropoietin (rhEpo) may have an immunomodulating action. We have studied the in vitro and in vivo effects of rhEpo on immune functions in man. Low pharmacological concentrations of the hormone inhibit T-cell activation and proliferation, while higher ones are without that effect. The same Epo concentrations inhibit mitogen- and alloantigen-driven B-cell differentiation and immunoglobulin synthesis and, to a lesser extent, B-cell proliferation. In vivo treatment with rhEpo causes an initial inhibition of T- and B-cell proliferation, but with prolonged administration improved responsiveness is observed. Our data support the notion that rhEpo can regulate immune functions, a fact of potential clinical application.

The Effects of T4 and A3/R Phage Preparations on Whole-Blood Monocyte and Neutrophil Respiratory Burst

Bacteriophages (viruses of bacteria) are currently considered a promising means of treating antibiotic-resistant infections. The main objective of this study was to evaluate the intensity of the whole-blood monocyte and neutrophil respiratory burst induced by purified preparations and lysates of the bacteriophages T4 and A3/R. While A3/R phage preparations did not induce a significant respiratory burst, T4 phage preparations increased the production of reactive oxygen species in a dose-dependent manner. However, the intensity of the phage-induced respiratory burst was much lower than that triggered by heat-inactivated Staphylococcus aureus cells. These results suggest that phage preparations are not likely to induce oxidative stress following their administration to patients.