Radiation and chemotherapy variable response induced by tumor cell hypoxia: impact of radiation dose, anticancer drug, and type of cancer.

Hypoxia is a condition in which proliferating tumor cells are deprived of oxygen due to limited blood supply from abnormal tumor microvasculature. This study aimed to investigate the molecular changes that occur in tumor cell hypoxia with special emphasis placed on the efficacy of chemotherapeutic and radiation-related effects. Four commercially available chemotherapeutic agents: cisplatin, cyclophosphamide, doxorubicin, and 5-fluorouracil, were tested for their cytotoxic activity on the cancer cell lines PC3 (prostate), HepG2 (liver), and MCF-7 (breast). Tumor cell lines under hypoxia were treated with both IC50 concentrations of the different chemotherapeutic agents and irradiated with 5 and 10 Gy using a 137Cs gamma source. Hypoxia-inducible factor-1α (HIF-1α) protein levels were examined using an ELISA assay. Hypoxic cells showed a significant change in cell viability to all chemotherapeutic agents in comparison to normoxic controls. HepG2 cells were more resistant to the cytotoxic drug doxorubicin compared to other cancer cell lines. The flow cytometric analysis showed that hypoxic cells have lower levels of total apoptotic cell populations (early and late apoptosis) compared to normoxic cells suggesting decreased hypoxia-induced apoptosis in cancer cells. The highest reduction in HIF-1α level was observed in the MCF-7 cell line (95.5%) in response to the doxorubicin treatment combined with 10 Gy irradiation of cells. Chemoradiotherapy could result in minimal as well as a high reduction of HIF-1α based on cell type, type of chemotherapy, and amount of ionizing radiation. This study highlights future research work to optimize a combined chemoradiotherapeutic regime in individual cancer cell hypoxia.

Differential effects of Th17 cytokines during the response of neutrophils to Burkholderia cenocepacia outer membrane protein A.

T helper 17 cells are involved in the immunopathology of cystic fibrosis. They play a key role in recruitment of neutrophils, which is the first line of defence against bacteria. Additionally, Burkholderia cenocepacia outer membrane protein A (OmpA) BCAL2958 is considered a potential protective epitope for vaccine development. The present study aimed to investigate the neutrophil response to OmpA in the presence of Th17 cytokines, IL-17 and IL-22 at different times of activation. Neutrophils were isolated from whole blood of healthy volunteers and activated with OmpA in the presence of IL-17, IL-22 or both cytokines together. Supernatant was collected after 1 h, 2 h, 4 h, 8 h, and 12 h. Neutrophil activation was assessed by measuring MPO, TNF-α, elastase, hydrogen peroxide, catalase and NO. The results revealed that the combination of IL-17 and IL-22 cytokines induced the release of NE, catalase, H2O2 and TNF-α from neutrophils activated with Burkholderia OmpA at late stages of activation. However, IL-22 alone or IL-17 alone decreased the myeloperoxidase (MPO), catalase and NE levels at early stages of neutrophil activation. The presence of IL-17 alone led to a significant increase in TNF-α level after 1 h and 12 h. However, the presence of IL-22 alone led to a significant increase in TNF-α level after only 1 h but a significant decrease after 8 h of activation was observed as compared to OmpA stimulated neutrophils. In conclusion, Th17 cytokines IL-17 and IL-22, have differential effects during the neutrophil response to Burkholderia OmpA.

Liposomal IL-22 ameliorates liver fibrosis through miR-let7a/STAT3 signaling in mice.

The therapeutic effect of liposomal IL-22 versus non-liposomal IL-22 on liver fibrosis was investigated. IL-22 (5 µg/ml) was incorporated into negative charged liposomes. Schistosoma mansoni infected mice were treated with liposomal IL-22 for either 7 or 14 days before decapitation. Liver and spleen were removed and splenocytes were isolated for in vitro investigations. TNF-α, IL-17, IL-22 and IgE levels were assessed. Hepatic granulomas were counted, granuloma index and its developmental stages were calculated. Hepatic expressions of STAT3, ß-catenin and let-7a miRNA were evaluated. Liposomal IL-22 size was clustered around 425.9 ± 58.0 nm with negative zeta potential (-18.8 ± 1.3 mV). After 14 days, 65.5% of IL-22 was released from liposomal IL-22 as was gradually observed in vitro. Liposomal IL-22 significantly (p < 0.05) decreased IL-17 level (-33.1%) of healthy splenocytes compared to non-liposomal IL-22. In vivo therapeutic effect of liposomal IL-22 revealed a significant (p < 0.05) decrease in hepatic granuloma index (-22.1%) and levels of TNF-α (-49.2%) and IL-17 (-57.3%), but a marked increase in IL-22 (64.2%) and IgE (196.1%) levels comparing to non-liposomal IL-22. Three developmental stages of hepatic granuloma (NE, EP, and P) were observed in liposomal and non-liposomal IL-22 groups (79.6 ± 1.7 and 81.8 ± 8.7, respectively, P < 0.05), with higher relative frequency of EP stage. Additionally, liposomal IL-22 treatment increased hepatic expression of STAT3 (21.7 fold change) and let-7a (3.6 fold change) and reduced ß-catenin expression (0.6 fold change) compared to healthy mice. Conclusively, liposomal IL-22 seems more effective in the treatment of liver fibrosis resulting from S. mansoni infection than non-liposomal IL-22.

IL-17 Induced The Recruitment and Functional Activity of Granulocytes Isolated from Patients Coinfected with Schistosoma mansoni and Hepatitis C Virus.

The aim of this study was to investigate the role of Th17 cytokines on granulocytes recruitment and functional activity in Schistosoma mansoni/hepatitis C virus (HCV) co-infected patients. Granulocytes were isolated from the co-infected blood and stimulated overnight with Schistosoma soluble egg antigen (SEA) in the presence of IL-17, IL-22, or both cytokines. In parallel, granuloma was induced in vitro using the isolated granulocytes and SEA-coated polyacrylamide beads in the presence of IL-17 and/or IL-22 then the sizes of granulomas were measured after one week. Overnight culture and granuloma supernatants levels of tumor necrosis factor-alpha (TNF-?, hydrogen peroxide (H2O2) and nitric oxide (NO) were measured using ELISA. Results revealed that Schistosoma/HCV derived granulocytes produced significant (P < 0.0001) higher levels of TNF-? and H2O2 after overnight activation with SEA in the presence of IL-17 as compared to that produced by Schistosoma alone infected granulocytes. Presence of IL-17 increased significantly (P=0.0017) the granuloma index of coinfected patients' granulocytes as compared to that of Schistosoma alone infected granulocytes after one week of granuloma induction. Additionally, TNF-? and H2O2 levels in the granuloma supernatant were increased significantly (P = 0.0009 and P=0.0074 respectively) as compared to that of Schistosoma alone infected granulocytes. However, IL-22 alone or when combined with IL-17 decreased significantly the TNF-? levels (P=0.0112 and P=0.0007 respectively), and H2O2 levels (P=0.002 and P=0.053 respectively) but increased NO levels (P < 0.0001 and P=0.002 respectively) in the granuloma supernatant. In conclusion, IL-17 induces recruitment and functional activity of granulocytes in coinfected with schistosomiasis and hepatitis C virus which may contribute to the progress of fibrosis. Neutralization of this cytokine is suggested as a therapeutic strategy against fibrosis for such category of patients.

HCV Infection Amplified Th2 Bias and Th17 Responses In Schistosoma-Infected Patients.

Schistosomiasis and HCV are the most prevalent infections in Egypt Schistosoma infection is associated with bias towards Th2 responses. Plasma from 17 (Schistosoma-infected), 39 (Schistosoma/HCV co-infected) and 23 controls were collected. Cytokine multiplex array was used to measure 6 plasma cytokine levels representing the surrogate markers of three T helper lymphocytes subpopulations; IL-12 (Th1), IL-4 and IL-10 (Th2), IL-17, IL-22 and IL-23 (Th17). There was a significant increase in plasma levels of IL-17 and IL-23 in co-infected patients compared to Schistosoma-infected patients. As well, there was a significant increase in the Th2 regulatory cytokines IL-10 and IL-4 in co-infected group compared to Schistosoma-infected patients. However, no significant changes were observed in the Th1 cytokine (IL-12) among the groups. In conclusion, presence of HCV infection shifted the response towards Th2/Th17 pathway which may play a role in the progressive pathogenesis of HCV in Schistosoma-co-infected patients.

The Burkholderia cenocepacia OmpA-like protein BCAL2958: identification, characterization, and detection of anti-BCAL2958 antibodies in serum from B. cepacia complex-infected Cystic Fibrosis patients.

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among cystic fibrosis patients, highlighting the need for novel therapeutic strategies. In the present work we have studied the B. cenocepacia protein BCAL2958, a member of the OmpA-like family of proteins, demonstrated as highly immunogenic in other pathogens and capable of eliciting strong host immune responses. The encoding gene was cloned and the protein, produced as a 6× His-tagged derivative, was used to produce polyclonal antibodies. Bioinformatics analyses led to the identification of sequences encoding proteins with a similarity higher than 96 % to BCAL2958 in all the publicly available Bcc genomes. Furthermore, using the antibody it was experimentally demonstrated that this protein is produced by all the 12 analyzed strains from 7 Bcc species. In addition, results are also presented showing the presence of anti-BCAL2958 antibodies in sera from cystic fibrosis patients with a clinical record of respiratory infection by Bcc, and the ability of the purified protein to in vitro stimulate neutrophils. The widespread production of the protein by Bcc members, together with its ability to stimulate the immune system and the detection of circulating antibodies in patients with a documented record of Bcc infection strongly suggest that the protein is a potential candidate for usage in preventive therapies of infections by Bcc.

The immunomodulator AS101 suppresses production of inflammatory cytokines and ameliorates the pathogenesis of experimental autoimmune encephalomyelitis.

We reported that AS101 (organotellurium compound, trichloro(dioxoethylene-O,O') tellurate) inhibited the differentiation of Th17 cells and reduced the production of IL-17 and GM-CSF. In addition, AS101 promoted the production of IL-2 in activated T cells. Flow cytometric analysis showed that AS101 inhibited Th17 cell proliferation. AS101 blocked the activation of transcriptional factor NFAT, Stat3, and RORγt, and increased activation of Erk1/2, suggesting a mechanism of action of AS101. We further demonstrated that AS101 was effective in amelioration of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Finally, by real-time PCR analysis we showed that AS101 reduces the IL-17, IFN-γ, GM-CSF, and IL-6 mRNA expression in inflammatory cells of spinal cords. Additionally, flow cytometry analysis also indicated that the CD4+ T cells and IL-17 and GM-CSF-producing cells were reduced in the spinal cords of AS101 treated mice compared to those treated with PBS.

Interleukin-12 is the optimum cytokine to expand human Th17 cells in vitro.

Recently, a new lineage of CD4+ T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4+ T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R+ cells from the CD4+ T cells. Functional studies revealed that CD4+ IL-23R+ cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-gamma). Furthermore, we expanded the CD4+ IL-23R+ cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4+ IL-23R+ cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN-gamma secreted by IL-23R+ CD4+ cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R+ CD4+ cells and the secretion of IFN-gamma, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.

Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt.

BACKGROUND: Hepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available. OBJECTIVES: The objectives were to determine the HEV genotype(s) currently circulating in Egypt. STUDY DESIGN: AVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype. RESULTS: Of 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9-9.5% divergent from other genotype 1 isolates. ORF2 was 5.3-8.7% divergent from previously reported Egyptian isolates. CONCLUSIONS: This study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.