Higher cytotoxic activities of CD8+ T cells and natural killer cells from peripheral blood of early diagnosed lung cancer patients.

INTRODUCTION: Cytotoxic (CD8+) and natural killer (NK) cells play critical roles in anti-tumor immunity. Dysfunction in these cells is considered as one of the extrinsic mechanisms for tumor relapse. AIM: We aimed in this study to assess cytotoxic activities of CD8 + T and NK cells in the peripheral blood from lung cancer patients before and after induction of chemotherapy. SUBJECTS AND METHODS: Healthy (n = 5) volunteers and lung cancer patients (n = 15:5 before, 5 during, and 5 after induction of chemotherapy) were recruited. Flow cytometry was used to analyze the numbers of CD8 + T cells, NK and CD56+T cells and their intracellular expression of granzyme B (GzB) in fresh peripheral blood mononuclear cells (PBMCs) and after 72 h of their culture in vitro and stimulation with 5 µg/ml Concanavalin A (Con A) and 50ng/ml IL-2). In addition, the plasma levels of inflammatory cytokines were measured using luminex. RESULTS: After culture, significant increases in the number of GzB expressing cells gated on CD3+, CD4+, CD8 + and NKCD8 + T cells in the PBMCs from lung cancer patients before induction of chemotherapy as compared to control individuals as well as patients during and after induction of chemotherapy. Serum levels of IL-1 and CXCL8 in patients before induction of chemotherapy showed 37- and 40-fold increases, respectively, as compared to control individuals. Both GzB expression and cytokines levels in patients during and after chemotherapy were similar. CONCLUSION: Polyclonal stimulation of PBMCs can restore the cytolytic activities of cytotoxic CD8 and NK cells from lung cancer patients even after chemotherapy.

Brief in vitro IL-12 conditioning of CD8 + T Cells for anticancer adoptive T cell therapy.

Cancer immunotherapy represents a potential treatment approach through non-specific and specific enhancement of the immune responses. Adoptive cell therapy (ACT) is a potential modality of immunotherapy that depends on harvesting T cells from the tumor-bearing host, activating them in vitro and infusing them back to the same host. Several cytokines, in particular IL-2, IL-7 and IL-15, have been used to enhance survival T cells in vitro. Although effective, conditioning of T cells in vitro with these cytokines requires long-term culture which results in the loss of expression of their trafficking receptors mainly CD62L. It also results in exhaustion of the activated T cells and reduction in their functions upon adoptive transfer in vivo. Our recent studies and those of other groups showed that brief (3 days) conditioning of CD8+ T cells by IL-12 in vitro can result in enhancing function of tumor-reactive CD8+ T cells. Adoptive transfer of these IL-12-conditioned CD8+ T cells into tumor-bearing mice, preconditioned with cyclophosphamide, 1 day before ACT, induced tumor eradication that was associated with generation of tumor-specific memory response. In this review, we summarize studies that indicated to the superiority of IL-12 as a potential cytokine for conditioning T cells for ACT. In addition, we discuss some of the cellular and molecular mechanisms that govern how IL-12 programs CD8+ T cells to enhance their functionality especially in vitro and its implication in combination with other ACT modalities, opening a avenue for the clinical application of this cytokine.

Recent Research on Flavonoids and their Biomedical Applications.

Flavonoids, commonly found in various plants, are a class of polyphenolic compounds having a basic structural unit of 2-phenylchromone. Flavonoid compounds have attracted much attention due to their wide biological applications. In order to facilitate further research on the biomedical application of flavonoids, we surveyed the literature published on the use of flavonoids in medicine during the past decade, documented the commonly found structures in natural flavonoids, and summarized their pharmacological activities as well as associated mechanisms of action against a variety of health disorders including chronic inflammation, cancer, cardiovascular complications and hypoglycemia. In this mini-review, we provide suggestions for further research on the biomedical applications of flavonoids.

WITHDRAWN: The effect of anti IL-2/IL-2 complex versus stand-alone low dose of IL-2 on imiquimod induced psoriasis like skin inflammation model.

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Correction to: MicroRNA signature in hepatocellular carcinoma patients: identification of potential markers.

The correct spelling of the 7th authors' name is Mona Watany.

MicroRNA signature in hepatocellular carcinoma patients: identification of potential markers.

MicroRNAs (miRNAs) play important roles in liver pathologies and they are potential biomarkers for diagnosis of liver diseases progression. Changes in miRNA sera expression can be used as non-invasive biomarkers for hepatocellular carcinoma (HCC). The aim of the study was to identify the miRNome profiling of HCC and its diagnostic value in distinguishing HCC from healthy individuals. Expression profiles of miRNAs in serum samples of 20 HCC patients and 10 healthy controls were detected. Whole miRNome profiling was done using next generation sequencing. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of the deregulated miRNAs for discriminating HCC cases from healthy controls. MiRNA 142 was highly expressed in HCC (P value = 0.023) while miRNAs 191, 22, and 126 were higher in the controls (P value = 0.005, 0.034, 0.010 respectively). We have identified 5 novel miRNAs and they were highly expressed in HCC than controls. Analysis of ROC curve demonstrated that these deregulated miRNAs can be used as a reliable biomarker for detection of HCC with high diagnostic accuracy (AUC = 0.93). We have detected a panel of serum miRNAs that can be used as a reliable noninvasive screening biomarker of HCC. The study recommends further research to shed light on a possible role of the newly discovered novel miRNAs in HCC pathogenesis.

Fibroblast Growth Factor 2 Augments Transforming Growth Factor Beta 1 Induced Epithelial-mesenchymal Transition in Lung Cell Culture Model.

Impaired lung epithelial cell regeneration following injury may contribute to the development of pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) is a critical event in embryonic development, wound healing following injury, and even cancer progression. Previous studies have shown that the combination of transforming growth factor beta-1 (TGFß1) and fibroblast growth factor 2 (FGF2) induces EMT during cancer metastasis. However, this synergy remains to be elucidated in inducing EMT associated with wound healing after injury. We set out this study to determine the effect of fibroblast growth factor 2 (FGF2) on TGFß1-induced EMT in the human lung epithelium. BEAS-2B and A549 cells were treated with TGFß1, FGF2, or both. EMT phenotype was investigated morphologically and by measuring mRNA expression levels; using quantitative real-time PCR. E-cadherin expression was assayed by western blot and immunofluorescence staining. Cell migration was confirmed using a wound-healing assay. TGFß1 induced a morphological change and a significant increase in cell migration of BEAS-2B cells. TGFß1 significantly reduced E-cadherin (CDH1) mRNA expression and markedly induced expression of N-cadherin (CDH2), tenascin C (TNC), fibronectin (FN), actin alpha 2 (ACTA2), and collagen I (COL1A1). While FGF2 alone did not significantly alter EMT gene expression, it enhanced TGFß1-induced suppression of CDH1 and upregulation of ACTA2, but not TNC, FN, and CDH2. FGF2 significantly inhibited TGFß1-induced COL1A1 expression. Furthermore, FGF2 maintained TGFß1-induced morphologic changes and increased the migration of TGFß1-treated cells. This study suggests a synergistic effect between TGFß1 and FGF2 in inducing EMT in lung epithelial cells, which may play an important role in wound healing and tissue repair after injury.

Comparative expression of caspases and annexin V in benign and malignant ovarian tumors.

AIM OF STUDY: Ovarian cancer (OC) leads to high mortality rate if diagnosed at late stage. The aim of the present study was to increase the survival rate by early disease diagnosis. MATERIALS AND METHODS: A total of 21 females were divided into three groups. The control (n = 3), benign (n = 8), and malignant group (n = 10). We used flow cytometry to analyze cell cycle, caspase-3, -8, and annexin V. RESULTS: The results showed that the annexin V expressed in malignant group more than benign and normal groups with (P = 0.000 and P = 0.007), respectively. Caspase-3 and 8 expression decreased in benign and malignant group than in normal group (P = 0.012 and P = 0.007), respectively. Furthermore, sub-G1 apoptosis level decreased statistically significant in benign and malignant group than in normal group (P = 0.012 and P = 0.007), respectively. These data showed that (S phase) level had statistically significant increase in malignant group (P = 0.007) than the control group and marked statistically significant decrease (P = 0.000) in benign group than malignant group. This study explained changes in sub-G1 apoptosis for benign group increase statistically significant (P = 0.003) than malignant group level. CONCLUSION: Caspase-3 and -8 and annexin V may serve as diagnostic markers in OC, also explained that the decrement in control of the S phase in the cell cycle may considered one of the significant factors in the development of ovarian tumors.

Chemotherapy alters the increased numbers of myeloid-derived suppressor and regulatory T cells in children with acute lymphoblastic leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. The precise mechanism behind the relapse in this disease is not clearly known. One possible mechanism could be the accumulation of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) which we and others have reported to mediate suppression of anti-tumor immune responses. AIM: In this study, we aimed to analyze the numbers of these cells in a population of B-ALL pediatric patients. METHODS: Peripheral blood samples withdrawn from B-ALL pediatric patients (n = 45 before, during and after the induction phase of chemotherapy. Using multi parametric flow cytometric analysis. MDSCs were identified as Lin-HLA-DR-CD33+CD11b+; and Treg cells were defined as CD4+CD25+CD127-/low. RESULTS: Early diagnosed B-ALL patients showed significant increases in the numbers of MDSCs and Tregs as compared to healthy volunteers. During induction of chemotherapy, however, the patients showed higher and lower numbers of MDSCs and Treg cells, respectively as compared to early diagnosed patients (i.e., before chemotherapy). After induction of chemotherapy, the numbers of MDSCs and Treg cells showed higher increases and decreases, respectively as compared to the numbers in patients during chemotherapy. CONCLUSION: Our results indicate that B-ALL patients harbor high numbers of both MDSCs and Tregs cells. This pilot study opens a new avenue to investigate the mechanism mediating the emergence of these cells on larger number of B-ALL patients at different treatment stages.

High numbers of myeloid derived suppressor cells in peripheral blood and ascitic fluid of cirrhotic and HCC patients.

BACKGROUND: Hepatocellular carcinoma (HCC) is the 3rd most common cause of cancer-related death worldwide. It has evolved different immune escape mechanisms, which might include emergence of lymphoid and myeloid regulatory cells. Aim of this work: To determine the numbers of Myeloid-derived suppressor cells (MDSCs) in peripheral blood and ascitic fluid in cirrhosis and HCC and their relation to IFN-γ and α-fetoprotein (α-FP). PATIENTS AND METHODS: Sixty individuals were enrolled in this study; forty cirrhotic patients with ascites; twenty without HCC (Group I), and twenty with HCC (group II) as well as twenty healthy individuals as a control group (group III). The phenotype and numbers of MDSCs were analyzed in peripheral blood of all the individuals and ascitic fluid of the patients using flow cytometry. Intracellular IFN-γ and serum alfa-fetoprotein were measured. RESULTS: Significant increases in the relative and the mean number of peripheral blood MDSCs were found in the cirrhosis and HCC groups than in the control group, with the HCC group showing the highest number. MDSC count was negatively correlated with IFN-γ levels, while α-FP was positively correlated with MDSC% in the HCC group. MDSC count was low in ascitic fluid of both HCC and cirrhosis groups with no significant difference between the 2 groups. CONCLUSION: A high frequency of MDSCs was detected in the peripheral blood of cirrhotic and HCC patients, indicating presence of immunosuppressive arms. These cells could be targeted to develop a new effective immunotherapy or an adjuvant to current therapies.

Stem cell therapy as a novel therapeutic intervention for resistant cases of alopecia areata and androgenetic alopecia.

BACKGROUND: Management of alopecia areata (AA) and androgenetic alopecia (AGA) is often challenging as patients may be resistant to currently available modalities of treatment. The use of stem cells may be a novel option for resistant cases. OBJECTIVE: To evaluate the safety and efficacy of the use of autologous bone marrow derived mononuclear cells (including stem cells) as compared to follicular stems cells for the management of resistant cases of AA and AGA. METHODS: This study included 40 patients (20 AA patients and 20 AGA patients), all patients were treated with a single session of intradermal injection of autologous stem cells (SCs) therapy. They were divided into four groups according to the applied modality [either autologous bone marrow derived mononuclear cells (bone marrow mononuclear cells [BMMCs] or autologous follicular stem cells [FSC]). RESULTS: Six months after stem cell therapy (SCT) injection, there was a significant improvement, confirmed by immunostaining and digital dermoscopy. The mean improvement in all groups was "very good". There was no significant difference between both methods in either type of alopecia. No serious adverse events were reported. CONCLUSION: Autologous BMMCs and FSC seem to be a safe tolerable and effective treatment for the management of both resistant AA and AGA.

IFN-α-based treatment of patients with chronic HCV show increased levels of cells with myeloid-derived suppressor cell phenotype and of IDO and NOS.

INTRODUCTION: Hepatitis C virus (HCV) infection causes chronic hepatitis, which is often associated with suppressed anti-HCV immune responses. We have recently reported accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed immunity in cancer patients. AIM: The main aim of this study was to determine whether chronic HCV patients harbor high of MDSCs in general and in nonresponders to IFN-based therapy in particular as well as to analyze the immune suppressive molecules. METHODS: Peripheral blood samples withdrawn from 154 patients with chronic HCV infection and were categorized into responders and nonresponders based on viral titer upon IFN-α treatment. RESULTS: The relative and absolute numbers of MDSCs defined as Lin-/HLA-DR-/CD33+/CD11b+ increased in all HCV patients, where they were higher in nonresponders than in responders. Additionally, the levels of MDSCs after 4-6 months of treatment in responders were lower than during the course of treatment. The responders also showed higher levels of IL-2 coincided with increased numbers of dendritic cells (DCs), CD4+ and CD8+ T cells. The levels of total NOS and IDO were also higher in nonresponders as compared to responders and healthy controls, while the expression levels of CD3ζ was lower in responders as compared to nonresponders and healthy volunteers. CONCLUSION: Chronic HCV patients harbor high numbers of MDSCs, which are higher in nonresponders than in responders. The higher numbers of MDSCs associated with increases in the suppressing factors.

Anti-fibrotic potential of human umbilical cord mononuclear cells and mouse bone marrow cells in CCl4- induced liver fibrosis in mice.

Liver fibrosis is the consequence of hepatocyte injury that leads to the activation of hepatic stellate cells (HSC). The treatment of choice is Liver transplantation; however, it has many problems such as surgery-related complications, immunological rejection and high costs associated with the procedure. Stem cell-based therapy would be a potential alternative, so the aim of this study is to investigate the therapeutic potential of human umbilical cord mononuclear cells (MNC) and mouse bone marrow cells (BMC) against carbon tetrachloride (CCl4) induced liver fibrosis in mice and compare it with that of silymarin. In the present study, male albino mice (N=60) were divided into six groups (10 mice each), the first group served as the normal control group while the remaining five groups were rendered fibrotic by intraperitoneal injections of CCl4 and being left for 6 weeks to develop hepatic fibrosis. Thereafter, the mice were divided into CCl4 group, CCl4 group receiving MNC or BMC or silymarin or MNC and silymarin combination. After the specified treatment period, animals were then euthanized, blood and tissue samples were collected for measurement of alanine aminotransferase(ALT), aspartate aminotransferase(AST), malondialdehyde(MDA), reduced glutathione(GSH), collagen, Laminin, transforming growth factor ß1(TGFß1), tumor necrosis factor alpha(TNFα). MNC, BMC, and the combination therapy showed a significant decrease in ALT, AST, MDA, collagen, Laminin, TGFß1, and TNFα and a significant increase in GSH. The data displayed a similar regression of fibrosis with the histological and immunohistological parameters. In conclusion, MNC, BMC and the combination therapy showed a potential therapeutic effect against liver fibrosis via reducing oxidative stress, inflammatory mediators, and fibrogenic markers.

Higher Activities of Hepatic Versus Splenic CD8+ T Cells in Responses to Adoptive T Cell Therapy and Vaccination of B6 Mice with MHC Class-1 Binding Antigen.

The liver has unique microenvironment which is known to induce tolerance of cytolytic CD8+ T cells to hepatic and extra hepatic antigens, resulting in persistence of infection of the liver by the hepatitis B and C viruses. However, under some conditions, functional immune responses can be elicited in the liver in particular to show preferential retention of activated CD8+ T cells. It is not clear whether this retention depends on the type of the exogenous immunostimulatory or the endogenous innate immune cells. The T cell receptor (TCR) transgenic OT-1 (CD8+) mouse model was used in which OT-1 cells were harvested from the spleen of the donor and transferred into recipient mice followed by immunization with OVA peptide followed by injection of GM-CSF, CCL21 chemokine, or cytokines (IL-2, IL-12, or IL-15), or the toll-like receptor 3 agonist poly(I:C). Co-administration of any of these immunostimulatory agents relatively augmented the retention of CD8+ T cells with different levels of effects. Compared to spleen, the Ag-specific CD8+ T cells in the liver showed higher activities including expansion, proliferation, apoptosis and memory responses as well as cytolytic function. While depletion of natural killer cells significantly decreased the hepatic retention of the antigen-specific T cells, depletion of Kupffer cells showed opposite effect. Taken together, the antigen reactive T cells in the liver have higher activities than their counterparts in the peripheral tissues such as spleen. These data have important clinical implications for designing immunotherapeutic protocols toward the liver diseases.

Effect of administration timing of postchemotherapy granulocyte colony-stimulating factor on host-immune cell recovery and CD8+ T-cell response.

Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor, is a standard supportive therapy given during cancer treatment. It induces acceleration in neutrophil recovery through stimulation of mobilization of hematopoietic progenitors. Given that the latter is also induced by chemotherapy itself, the timing of administration of G-CSF postchemotherapy might impact the resultant overall effects. The present study aimed to determine the optimal timing of G-CSF postchemotherapy to exert its optimal effects on the immune cell recovery and its impact on antigen-specific CD8+ T-cell response. B6 mice were treated once with cyclophosphamide (4 mg/mouse; CTX) and then daily with G-CSF (5 g/mouse) from Days 1-5, 2-5 or 5-9 post-CTX treatment. The total numbers of various immune cell types were analyzed on Days 7, 9 and 12 post-CTX treatment. To evaluate effects on CD8+ T-cell response, a pmel-1 transgenic mouse model was used in combination with prime boost peptide vaccination therapy. The total number of white blood cells (WBC), neutrophils, monocytes, lymphocytes, granulocytes and dendritic cells (DC) were significantly increased after G-CSF treatment in particular when G-CSF was administered from Days 2-5 post-CTX treatment. Application of this timing of G-CSF and CTX treatment after adoptive transfer of T-cells followed by prime-boost vaccination with antigenic peptide did not block the expansion of the donor pmel-1 CD8+ T-cells. In conclusion, adjusting the timing of treatment with G-CSF postchemotherapy can optimize its promoting effects on recovery of myeloid cells without altering the associated antigen-specific immunity.

Correlation of viral load with bone marrow and hematological changes in pale patients with chronic hepatitis C virus.

Liver is considered the target of hepatitis C virus (HCV), which has marked tropism for hepatocytes. In this study, we investigated changes in bone marrow (BM) and blood and their correlation with viremia level in 30 pale patients with chronic HCV who were selected before antiviral therapy. Patients with BM positive for HCV RNA (53.33 %) showed moderate to high viremia, while patients with BM negative for RNA (46.67 %) had low viremia. There was no significant difference in the liver histopathology between patients with HCV-RNA-negative and positive BM. Patients with BM positive for HCV RNA showed significant changes in BM cells, including the degree of immune complex deposition and alterations in peripheral blood counts compared to patients with BM negative for RNA and healthy controls, suggesting that BM changes could be a sequel or a reservoir for HCV viremia.

Triggering of toll-like receptor signaling pathways in T cells contributes to the anti-tumor efficacy of T cell responses.

Traditionally, expression of toll-like receptors (TLRs) has been associated with innate immune cells in particular professional antigen presenting cells and natural killer cells. This led to the concept that the adjuvant effects of ligation of TLR in a host occur mainly in innate immune cells. However, this concept has been challenged by recent studies including ours demonstrating that T cells express appreciated levels of different TLRs, which can serve as costimulatory co-receptors during polyclonal and antigen-specific stimulation of T cells. Because T cells express low levels of TLRs as compared to innate immune cells, increasing the expression levels of TLRs in T cells can significantly maximize their responses to the costimulatory effects of TLR ligation. This review article focuses on the potential role of TLR expression in T cells in their responses to vaccination regimen containing TLR agonists and how it can be modulated to optimize anti-tumor immunity.