Distinct Proteomic Profile of Spermatozoa from Men with Seminomatous and Non-Seminomatous Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are predominant in young males (15-44 years). Seminomatous and non-seminomatous TGCTs account for about 98% of all TGCTs cases. In this study, we aimed to compare the sperm proteome of patients with seminomatous and non-seminomatous TGCTs to identify possible protein biomarkers that could help distinguish between them in a non-invasive manner. We analyzed semen samples from patients with seminomatous or non-seminomatous TGCTs (n = 15/group) that were cryopreserved before the start of cancer treatment. Quantitative proteomic analysis was conducted on pooled samples (n = 3/group) and a total of 258 differentially expressed proteins (DEPs) were identified. The overexpression of acrosin precursor (ACR) and chaperonin containing TCP1 subunit 6B (CCT6B) as well as the underexpression of S100 calcium-binding protein A9 (S100A9) in the spermatozoa of patients with non-seminomatous TGCTs were validated by western blotting conducted on individual samples (n = 6 for seminomatous group and n = 6 for non-seminomatous group). Our overall results suggest an association between the higher and faster invasiveness of non-seminomatous TGCTs and the altered protein expressions, providing important information for future studies.

Rapid immunoprofiling of cytokines, chemokines and growth factors in patients with active rheumatoid arthritis using Luminex Multiple Analyte Profiling technology for precision medicine.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease of unknown etiology, characterised by symmetric erosive synovitis, leading inevitably to the destruction of cartilage and bone as well as bursa and tendon sheaths of joints. Our aim of this study was to decipher the differential expression of cytokines, chemokines and growth factors in the plasma of RA patients with active disease, using magnetic bead-based Luminex Multiple Analyte Profiling (xMAP) technology, for precision medicine. METHODS: We obtained plasma samples from RA patients (n=25) from the Rheumatology Clinic at the King Abdulaziz University Hospital (KAUH), Jeddah, Kingdom of Saudi Arabia (KSA) after written informed consent for their inclusion in this study. Besides, we have used the plasma samples from inflammatory osteoarthritis (OA) patients (n=10) and healthy volunteers (n=10) for comparison analyses. Plasma samples were examined using the Human Cytokine Magnetic 30-plex panel (Novex®), Invitrogen, USA) and analysed by MAGPIX® instrument (Luminex Corporation, USA). RESULTS: Though several pro-inflammatory cytokines, chemokines and growth factors present in the 30plex magnetic bead panel were not significantly (p>0.05) increased in the plasma of RA patients, the levels of plasma Th1 associated proinflammatory cytokines TNFα, and IL-6 and Th2 associated cytokines such as IL-4, IL-5 and IL-13 were significantly (p<0.05) upregulated compared to OA and normal controls. The proinflammatory IL-12 as well as anti-inflammatory IL-10 and IL-1RA were significantly (p<0.05) upregulated in the plasma of RA patients compared to normal controls. Also, the chemokines such as IP-10, RANTES and IL-8 as well as growth factors such as EGF, and VEGF were significantly (p<0.05) increased in RA. CONCLUSIONS: The MAGPIX data showed that the cytokines, chemokines and growth factors were differentially regulated systemically in patients with active RA compared to OA and normal controls. Hence, the Luminex xMAP technology-based multiplex immunoassays offer clues to formulate effective therapeutic strategies for RA patients with active disease irrespective of their treatment regimen and duration of treatment and, thus, an indispensable tool in precision medicine.

Altered Molecular Pathways in the Proteome of Cryopreserved Sperm in Testicular Cancer Patients before Treatment.

Testicular cancer (TC) represents the most common cancer affecting men within the reproductive age and is often accompanied by major disturbances in semen parameters. Cryopreservation is recommended in these patients before initiating cancer treatment. Currently, there are no studies reporting the molecular mechanisms associated with altered semen quality in these men. The main objective of this study was to compare the sperm proteome of normozoospermic (motility >40%) and asthenozoospermic (motility <40%) TC patients with normozoospermic infertile men without cancer (control group). Pooled sperm samples from normozoospermic (n = 20), asthenozoospermic (n = 11) TC, and a control group (n = 9) were used for quantitative global proteomic profiling using liquid chromatography-tandem mass spectrometry. A total of 1085, 846, and 982 proteins were identified in normozoospermic TC, asthenozoospermic TC, and control groups, respectively. Functional analysis revealed mitochondrial dysfunction and altered cellular pathways in both normozoospermic and asthenozoospermic TC patients. Comparison of pathway analysis showed no significant difference in fertility-associated proteins/mechanism between the normozoospermic TC patients and infertile men. Western blot analysis revealed under-expression of NDUFS1 associated with mitochondrial dysfunction and overexpression of CD63 involved in sperm maturation in both normozoospermic and asthenozoospermic TC patients. Our proteomic results confirm that defective cellular pathways are associated with reproductive functions in both normozoospermic and asthenozoospermic TC patients before the start of cancer treatment.

Proteomic Signatures Reveal Differences in Stress Response, Antioxidant Defense and Proteasomal Activity in Fertile Men with High Seminal ROS Levels.

Elevated levels of reactive oxygen species (ROS) are a major cause of male infertility. However, some men with high seminal ROS levels are still fertile. The main objective of this study was to understand the molecular mechanism(s) responsible for the preservation of fertility in those men. Semen samples from fertile men were divided into two groups: control (n = 10, ROS < 102.2 RLU/s/106 sperm) and ROS+ (n = 10, ROS > 102.2 RLU/s/106 sperm). Proteomic analysis of seminal plasma and spermatozoa was used to identify the differentially expressed proteins (DEPs) between the experimental groups, from which some proteins were validated by Western blot (WB). A total of 44 and 371 DEPs were identified between the study groups in the seminal plasma and spermatozoa, respectively. The identified DEPs were primarily involved in oxidoreductase, endopeptidase inhibitor, and antioxidant activities. We validated by WB the underexpression of NADH:ubiquinone oxidoreductase core subunit S1 (p = 0.01), as well as the overexpression of superoxide dismutase 1 (p = 0.03) and peroxiredoxin 4 (p = 0.04) in spermatozoa of ROS+ group. Our data suggest that fertile men with high ROS levels possess an effective antioxidant defense system that protects sperm proteins, as well as an active proteasomal system for degradation of defective proteins.

Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration.

Musculoskeletal disorders represent a major cause of disability and morbidity globally and result in enormous costs for health and social care systems. Development of cell-based therapies is rapidly proliferating in a number of disease areas, including musculoskeletal disorders. Novel biological therapies that can effectively treat joint and spine degeneration are high priorities in regenerative medicine. Mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs), adipose tissue (AD-MSCs) and umbilical cord (UC-MSCs) show considerable promise for use in cartilage and intervertebral disc (IVD) repair. This review article focuses on stem cell-based therapeutics for cartilage and IVD repair in the context of the rising global burden of musculoskeletal disorders. We discuss the biology MSCs and chondroprogenitor cells and specifically focus on umbilical cord/Wharton's jelly derived MSCs and examine their potential for regenerative applications. We also summarize key components of the molecular machinery and signaling pathways responsible for the control of chondrogenesis and explore biomimetic scaffolds and biomaterials for articular cartilage and IVD regeneration. This review explores the exciting opportunities afforded by MSCs and discusses the challenges associated with cartilage and IVD repair and regeneration. There are still many technical challenges associated with isolating, expanding, differentiating, and pre-conditioning MSCs for subsequent implantation into degenerate joints and the spine. However, the prospect of combining biomaterials and cell-based therapies that incorporate chondrocytes, chondroprogenitors and MSCs leads to the optimistic view that interdisciplinary approaches will lead to significant breakthroughs in regenerating musculoskeletal tissues, such as the joint and the spine in the near future.

Inhibition of Fc epsilon RI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes.

Atopic allergy is characterized by an increase in IgE antibodies that signal through the high-affinity Fcepsilon receptor (FcepsilonRI) to induce the release of inflammatory mediators from mast cells. For unknown reasons, the prevalence of allergic diseases has recently increased steeply in the developed world. However, this increase has not been mirrored in developing countries, even though IgE concentrations are often greatly elevated in individuals from these countries, owing to nonspecific IgE induction by universally present parasitic worms. Here we offer one explanation for this paradox based on the properties of ES-62, a molecule secreted by filarial nematodes. We found that highly purified, endotoxin-free ES-62 directly inhibits the FcepsilonRI-induced release of allergy mediators from human mast cells by selectively blocking key signal transduction events, including phospholipase D-coupled, sphingosine kinase-mediated calcium mobilization and nuclear factor-kappaB activation. ES-62 mediates these effects by forming a complex with Toll-like receptor 4, which results in the sequestration of protein kinase C-alpha (PKC-alpha). This causes caveolae/lipid raft-mediated, proteasome-independent degradation of PKC-alpha, a molecule important for the coupling of FcepsilonRI to phospholipase D and mast cell activation. We also show that ES-62 is able to protect mice from mast cell-dependent hypersensitivity in the skin and lungs, indicating that it has potential as a novel therapeutic for allergy.

Interleukin-33 exacerbates acute colitis via interleukin-4 in mice.

Interleukin-33 (IL-33) and its receptor ST2 are over-expressed in clinical colitis tissue. However, the significance of these observations is at present unknown. Significantly, we demonstrate here that IL33 and ST2 are the primary early genes induced in the inflamed colon of BALB/c mice following dextran sulphate sodium (DSS)-induced experimental ulcerative colitis. Accordingly diarrhoea and DSS-induced colon inflammation were impaired in ST2(-/-) BALB/c mice and exacerbated in wild-type mice by treatment with exogenous recombinant IL-33, associated respectively with reduced and enhanced expression of chemokines (CXCL9 and CXCL10), and inflammatory (IL-4, IL-13, IL-1, IL-6, IL-17) and angiogenic (vascular endothelial growth factor) cytokines in vivo. The exacerbation effect of treatment with recombinant IL-33 on DSS-induced acute colitis was abolished in IL-4(-/-) BALB/c mice. Hence, IL-33 signalling via ST2, by inducing an IL-4-dependent immune response, may be a major pathogenic factor in the exacerbation of ulcerative colitis.

5­Fluorouracil and capecitabine therapies for the treatment of colorectal cancer (Review).

Although 5­fluorouracil (5­FU)­based chemotherapy is the major treatment for colorectal cancer, it has disadvantages such as systemic toxicity, lack of effectiveness and selectivity, and development of resistance. Capecitabine, a prodrug form of 5­FU, was designed to overcome these drawbacks, to fulfill the need for more convenient therapy, and to improve safety, tolerability and intratumor drug concentration levels through a tumor­specific conversion to the active 5­FU drug. The purpose of the present review is to provide a comprehensive comparison between 5­FU therapy and capecitabine. In the current review, anticancer drug classification was discussed and the development of capecitabine from the original fluorinated analogue (5­FU) to overcome its drawbacks was explained. Specifically, 5­FU is compared with capecitabine therapy regarding various properties, including drug metabolism, cellular mechanism, effect on the apoptosis pathway and cell cycle phases, safety and tolerability. Moreover, three metabolizing enzymes required for the activation of capecitabine to 5­FU were discussed. Capecitabine, as monotherapy or in combination with other chemotherapies, exhibited improved drug efficacy and survival. However, the changes that mediate the chemoresistance of capecitabine treatment were classified as intracellular, extracellular or cell surface factors, or cell­phenotype state. Future studies should examine the efficacy of capecitabine combined with novel and safe drugs other than chemotherapeutic agents that play a role in the inhibition of tumor initiation, progression and metastasis.

IL-33 exacerbates autoantibody-induced arthritis.

Rheumatoid arthritis pathogenesis comprises dysregulation in both innate and adaptive immunity. There is therefore intense interest in the factors that integrate these immunologic pathways in rheumatoid arthritis. In this paper, we report that IL-33, a novel member of the IL-1 family, can exacerbate anti-glucose-6-phosphate isomerase autoantibody-induced arthritis (AIA). Mice lacking ST2 (ST2(-/-)), the IL-33 receptor alpha-chain, developed attenuated AIA and reduced expression of articular proinflammatory cytokines. Conversely, treatment of wild-type mice with rIL-33 significantly exacerbated AIA and markedly enhanced proinflammatory cytokine production. However, IL-33 failed to increase the severity of the disease in mast cell-deficient or ST2(-/-) mice. Furthermore, mast cells from wild-type, but not ST2(-/-), mice restored the ability of ST2(-/-) recipients to mount an IL-33-mediated exacerbation of AIA. IL-33 also enhanced autoantibody-mediated mast cell degranulation in vitro and in synovial tissue in vivo. Together these results demonstrate that IL-33 can enhance autoantibody-mediated articular inflammation via promoting mast cell degranulation and proinflammatory cytokine production. Because IL-33 is derived predominantly from synovial fibroblasts, this finding provides a novel mechanism whereby a host tissue-derived cytokine can regulate effector adaptive immune response via enhancing innate cellular activation in inflammatory arthritis.

The cytokine interleukin-33 mediates anaphylactic shock.

Anaphylactic shock is characterized by elevated immunoglobulin-E (IgE) antibodies that signal via the high affinity Fc epsilon receptor (Fc epsilonRI) to release inflammatory mediators. Here we report that the novel cytokine interleukin-33 (IL-33) potently induces anaphylactic shock in mice and is associated with the symptom in humans. IL-33 is a new member of the IL-1 family and the ligand for the orphan receptor ST2. In humans, the levels of IL-33 are substantially elevated in the blood of atopic patients during anaphylactic shock, and in inflamed skin tissue of atopic dermatitis patients. In murine experimental atopic models, IL-33 induced antigen-independent passive cutaneous and systemic anaphylaxis, in a T cell-independent, mast cell-dependent manner. In vitro, IL-33 directly induced degranulation, strong eicosanoid and cytokine production in IgE-sensitized mast cells. The molecular mechanisms triggering these responses include the activation of phospholipase D1 and sphingosine kinase1 to mediate calcium mobilization, Nuclear factor-kappaB activation, cytokine and eicosanoid secretion, and degranulation. This report therefore reveals a hitherto unrecognized pathophysiological role of IL-33 and suggests that IL-33 may be a potential therapeutic target for anaphylaxis, a disease of considerable unmet medical need.

Implications of SARS-CoV-2 infection on the clinical, hematological, and inflammatory parameters in COVID-19 patients: A retrospective cross-sectional study.

BACKGROUND: The current coronavirus pandemic (COVID-19) was caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2). COVID-19 is characterized by atypical pneumonia, mild colds, and more severe illnesses, such as severe acute respiratory distress, thrombosis, organ failure, and various secondary bacterial and fungal infections. Notably, the severity of COVID-19 in different age groups is not well known, and the validity of clinical laboratory data remains unclear. METHODS: In this retrospective cross-sectional study, we examined differential regulation of clinical, hematologic, and inflammatory biomarkers in COVID-19 patients. We divided 104 COVID-19 patients into five different groups according to age (0-17, 18-45, 46-65, 66-79, and >80 years). Baseline data (sex, comorbidities, intensive care admission, and medications), hematologic markers, liver, and renal function tests, coagulation, and inflammatory markers were examined in these groups. Receiver operator characteristic (ROC) analysis was used to determine the optimal threshold for predicting COVID-19 biological markers. RESULTS: We found that the highest percentage (45%) of COVID-19 patients was in the age group of 46-65 years. The hematologic parameters (WBC, HB, and PLT) were normal between the patient groups. The area under the curve in ROC analysis showed significant differences in the levels of creatine, GGT, BUN, CRP, D-dimer, ferritin, AST, and procalcitonin between the patients of age groups 46-65 and 66-79 years. Renal biomarkers were significantly high in most patients, regardless of age. In contrast, the liver biomarkers, did not differ significantly between patient groups. CONCLUSION: The main finding of our study is that laboratory parameters such as GGT, creatinine, BUN, CRP, procalcitonin, ferritin and D-dimer were differentially regulated in COVID -19 patients of different age groups. Importantly, these laboratory parameters may help as clinical predictors to assess the severity of the disease in the population. We conclude here that age is an important factor influencing COVID-19 severity.

Molecular determinants of etoposide resistance in HL60 cells.

Chemotherapy resistance is the main reason for treatment failure in acute myeloid leukemia (AML) and the major cause of its mortality. Etoposide is a DNA topoisomerase-II inhibitor that is used either as a single agent or in combination with cytarabine, azacytidine, vinca alkaloids, and anthracyclines for the treatment of relapsed /refractory AML. In this study, we sought to determine and understand the mechanism of etoposide resistance in AML using the HL60 cell line.HL60 cells were treated with incremental doses of etoposide and resistant colonies were isolated by culturing the resistant cells in semi-solid culture media. Three clones were selected for etoposide resistance namely, HL60-EtopR H1A, HL60-EtopR H1B, and HL60-EtopR H1C which demonstrated 4.78, 2.39, and 4.42-fold higher resistance to etoposide compared with the parental cells. To determine molecular differences between the etoposide-resistant HL60-EtopR cells and the parental cells, microarray-based gene expression profiling was performed. We found up regulation of members of the src tyrosine kinase family genes in the etoposide resistant cells. Further studies are required to evaluate the role of Src inhibitors in targeting etoposide resistant cells.

The Prognostic Value of the Developmental Gene FZD6 in Young Saudi Breast Cancer Patients: A Biomarkers Discovery and Cancer Inducers OncoScreen Approach.

Wnt signalling receptors, Frizzleds (FZDs), play a pivotal role in many cellular events during embryonic development and cancer. Female breast cancer (BC) is currently the worldwide leading incident cancer type that cause 1 in 6 cancer-related death. FZD receptors expression in cancer was shown to be associated with tumour development and patient outcomes including recurrence and survival. FZD6 received little attention for its role in BC and hence we analysed its expression pattern in a Saudi BC cohort to assess its prognostic potential and unravel the impacted signalling pathway. Paraffin blocks from approximately 405 randomly selected BC patients aged between 25 and 70 years old were processed for tissue microarray using an automated tissue arrayer and then subjected to FZD6 immunohistochemistry staining using the Ventana platform. Besides, Ingenuity Pathway Analysis (IPA) knowledgebase was used to decipher the upstream and downstream regulators of FZD6 in BC. TargetScan and miRabel target-prediction databases were used to identify the potential microRNA to regulate FZD6 expression in BC. Results showed that 60% of the BC samples had a low expression pattern while 40% showed a higher expression level. FZD6 expression analysis showed a significant correlation with tumour invasion (p < 0.05), and borderline significance with tumour grade (p = 0.07). FZD6 expression showed a highly significant association with the BC patients' survival outcomes. This was mainly due to the overall patients' cohort where tumours with FZD6 elevated expression showed higher recurrence rates (DFS, p < 0.0001, log-rank) and shorter survival times (DSS, p < 0.02, log-rank). Interestingly, the FZD6 prognostic value was more potent in younger BC patients as compared to those with late onset of the disease. TargetScan microRNA target-prediction analysis and validated by miRabel showed that FZD6 is a potential target for a considerable number of microRNAs expressed in BC. The current study demonstrates a potential prognostic role of FZD6 expression in young BC female patients and provides a better understanding of the involved molecular silencing machinery of the Wnt/FZD6 signalling. Our results should provide a better understanding of FZD6 role in BC by adding more knowledge that should help in BC prevention and theranostics.

VAMP8 is essential in anaphylatoxin-induced degranulation, TNF-alpha secretion, peritonitis, and systemic inflammation.

VAMP8, a member of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) family of fusion proteins, initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. VAMP8 physiological function in inflammation has not been elucidated. In this paper, we show that deficiency of VAMP8 protects mice from anaphylatoxin (C5a)-induced neutropenia, peritonitis, and systemic inflammation. We show that, in vivo, VAMP8 deletion inhibits neutropenia and phagocyte recruitment. We also show that in macrophages, VAMP8 localizes on secretory granules and degranulation is inhibited in VAMP8-deficient macrophages. Moreover, VAMP8(-/-) mice show reduced systemic inflammation with inhibition of serum TNF-alpha levels, whereas IL-1beta, IL-6, and MIP1alpha release are not affected. In wild-type macrophages, TNF-alpha colocalizes with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, the TNF-alpha release is inhibited. Furthermore, VAMP8 regulates the release of TNF-alpha and beta-hexosaminidase triggered by fMLP, and VAMP8(-/-) mice are protected from fMLP-induced peritonitis. These data demonstrate that the VAMP8 vesicle-associated-SNARE is required for the proper trafficking of secretory lysosomal granules for exocytosis in macrophages and for the release of the potent proinflammatory cytokine, TNF-alpha.

Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo.

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

Human Wharton's Jelly Stem Cell Secretions Inhibit Human Leukemic Cell Line K562 in vitro by Inducing Cell Cycle Arrest and Apoptosis.

Emerging resistance to the tyrosine kinase inhibitors that target the BCR-ABL1 oncoprotein has prompted research for novel therapeutics against chronic myeloid leukemia (CML). Herein, we evaluated the tumor inhibitory properties of the human Wharton's jelly stem cells (hWJSCs) co-culture (hWJSC-CC) and their extracts, namely, the hWJSC-conditioned medium (hWJSC-CM; 100%) and hWJSC-lysate (hWJSC-L; 15 µg/ml), on a CML cell line K562 in vitro. The hWJSCs expressed mesenchymal stem cell (MSC)-related cluster of differentiation (CD) markers and demonstrated mesodermal tissue differentiation potential. The cell metabolic activity showed a mean maximal decrease in the K562 cells by 49.12, 41.98, and 68.80% following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L, respectively, at 72 h. The sub-G1 population in the cell cycle was decreased by 3.2, 4.5, and 3.8% following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L, whereas the G2/M cell population was increased by 13.7 and 12.5% with the hWJSC-CM and hWJSC-L, respectively, at 48 h. Annexin V-allophycocyanin (APC) assay showed an increase in the apoptotic cells by 4.0, 3.9, and 4.5% at 48 h. The expression of pro-apoptotic BAX and CASP3 genes were increased, whereas BIRC5 (Survivin) was decreased compared with the control. The pro-inflammation-related genes, namely, IFN-γ, TNF-α, IL-1ß, IL-6, IL-8, and IL-12A, were decreased, whereas the anti-inflammatory genes, namely, IL-4 and IL-10, were increased following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L at 48 h. Multiplex bead-based cytokine assay also demonstrated decreases in the pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, IL-6, and IL-12) and an increase in the anti-inflammatory cytokine (IL-10) compared with the control. The pro-inflammatory cytokine IL-8 showed an increase with the hWJSC-CC and decreases with both the hWJSC-CM and the hWJSC-L. The hWJSCs and their extracts inhibited the K562 cells by causing cell cycle arrest and inducing apoptosis via the soluble cellular factors. However, an in vivo evaluation is necessary to unravel the true potential of the hWJSCs and their extracts before its use in CML inhibition.

Resveratrol attenuates C5a-induced inflammatory responses in vitro and in vivo by inhibiting phospholipase D and sphingosine kinase activities.

The anti-inflammatory activity of the phytoalexin resveratrol (RSV) was evaluated in C5 anaphylatoxin (C5a)-stimulated primary neutrophils and in a mouse model of acute peritonitis. Pretreatment of human and mouse neutrophils with RSV significantly blocked oxidative burst, leukocyte migration, degranulation, and inflammatory cytokine production. The anti-inflammatory activity of RSV was a function of inhibition of sphingosine kinase (SphK) activity (IC(50) approximately 20 microM) within 5 min of exposure, its membrane localization, and SphK1-mediated Ca(2+) release. As an experimental control, the SphK1 pharmacological inhibitor N,N-dimethyl sphingosine (DMS) was used to compare the inhibitory effect of RSV. We also provide evidence that the SphK inhibitory effect of RSV was mediated via its ability to block phospholipase D (PLD) activity and membrane recruitment. Furthermore, RSV blocked ERK1/2 phosphorylation, which functioned independently of SphK1 in this study. To provide in vivo relevance to these data, C5a-induced model of acute peritonitis was established, and the effects of prior injection of RSV were investigated. Indeed, prior injection of RSV virtually completely attenuated the effects of C5a on vascular permeability, neutrophil migration, release of interleukin 1beta, tumor necrosis factor alpha, interleukin 6, and the chemokine MIP-1alpha. Taken together, these data demonstrate strong anti-inflammatory activity of RSV in vitro and in vivo and highlight SphK1 as a potential target of this remarkable phytoalexin. These data could have tremendous implications for the clinical use of RSV in inflammatory pathologies.

New Insights on the Mechanisms Affecting Fertility in Men with Non-Seminoma Testicular Cancer before Cancer Therapy.

PURPOSE: Patients with non-seminoma testicular cancer (NSTC) cancer can be subfertile or infertile, and present reduced sperm quality, but the underlying mechanisms are unknown. The aim of this study was to compare the sperm proteome of patients with NSTC, who cryopreserved their sperm before starting cancer treatment, with that from healthy fertile men. MATERIALS AND METHODS: Semen volume, sperm motility and sperm concentration were evaluated before the cryopreservation of samples from patients with NSTC (n=15) and the control group (n=15). Sperm proteomic analysis was performed by liquid chromatography-tandem mass spectrometry and the differentially expressed proteins (DEPs) between the two groups were identified using bioinformatic tools. RESULTS: A total of 189 DEPs was identified in the dataset, from which five DEPs related to sperm function and fertilization were selected for validation by Western blot. We were able to validate the underexpression of the mitochondrial complex subunits NADH:Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1) and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2), as well as the underexpression of the testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4) in the NSTC group. CONCLUSIONS: Our results indicate that sperm mitochondrial dysfunction may explain the observed decrease in sperm concentration, total sperm count and total motile count in NSTC patients. The identified DEPs may serve as potential biomarkers for the pathophysiology of subfertility/infertility in patients with NSTC. Our study also associates the reduced fertilizing ability of NSTC patients with the dysregulation of important sperm molecular mechanisms.