Safa Taha

TNF-α and IL-8 in Acute Stroke and the Modulation of these Cytokines by Antiplatelet Agents

Stroke is associated with elevation of several proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-8 that are correlated with central nervous system (CNS) injury. Anti-platelet therapies are important agents in stroke management. The role of antiplatelets as anti-inflammatory agents is not known in acute stroke patients. Furthermore, their effect on induction of potential cytokines as TNF-α and IL-8 in those patients is still not clear. Thus, we herein examined the induction of TNF-α and IL-8 in acute stroke patients and examined the effects of the antiplatelets drugs aspirin, clopidogrel and dipyridamole, and piracetam in their induction. Cytokines were detected intracellularly in leukocytes from patients who had first acute ischemic stroke and from matched controls by immunocytochemistry. The results showed significant increase of spontaneously produced TNF-α and IL-8 in patients compared to control. This induction was significantly inhibited differently by each drug and dual drug agents. The data of this work suggest that antiplatelets agents may have a role in inhibition of stroke mediated proinflammatory cytokine effects, which may initiate a new aspect of the role of antiplatelets in the treatment of acute ischemic stroke.

A novel nervous system-induced factor inducing immune responses in the spleen

This study relates to a novel mediator signaling between the nervous system and the spleen following an immune challenge. Using enzyme‐linked immunospot and cell proliferation assays, we found that supernatants of cultured splenocytes prepared from subcutaneously trypanosome‐inoculated rats and mice spleens obtained immediately after inoculation and added to naive cells significantly stimulate interferon‐γ production and cell proliferation compared to phosphate‐buffered saline‐inoculated animals. This action was abrogated by surgical denervation of the spleen. Using the fluorescent differential display technology, the gene involved in this process was identified and further cloned and its sequence was mapped to chromosome 14 (GenBank accession number: EU552928). Protein expression revealed ∼15 kDa molecule with biological activities similar to the cultured supernatants of splenocytes obtained directly from parasite‐inoculated animals. Antibodies raised against the protein blocked the activities of both the protein and the supernatant and also recognized a band in the active supernatant with the same molecular mass as the protein. Furthermore, the protein was able to reactivate experimentally immunosuppressed cells by regaining their ability to proliferate, suggesting that such a nervous system‐induced immune system‐released activating agent (ISRAA) may have a potential therapeutic benefit in immunocompromised situations and in further understanding the mechanism for innate immunity commencement and action.

Chlamydia pneumoniae Induces Chemokine Expression by Platelets in Patients with Atherosclerosis

Objective: In this study, the role of Chlamydia pneumoniae in triggering platelets to induce the inflammatory potential chemokines CCL3, CCL5, CCL7 and CXCL8 in atherosclerotic patients was investigated. Subjects and Methods: Venous blood from control subjects (n = 35) and atherosclerotic patients (n = 35) was collected in tubes with and without EDTA. Platelets from controls and patients were separated from whole blood and then stimulated with lipopolysaccharide (LPS), live C. pneumoniae and heat-treated C. pneumoniae. The ability of C. pneumoniae and its LPS to stimulate platelets and expression of CCL3, CCL5, CCL7 and CXCL8 was assessed with immunofluorescence. Immunosorbent assays were used to detect anti-C. pneumoniae antibodies in sera from patients and healthy subjects. Results: Nonstimulated platelets from patients showed significant expression of CCL3, CCL5, CCL7 and CXCL8 compared to controls (p < 0.0001). Stimulation of platelets from patients with live and heat-treated C. pneumoniae and its LPS demonstrated significant induction of chemokines compared to similarly stimulated platelets from controls (p < 0.01). After stimulation with heat-treated C. pneumoniae chemokine expression in platelets from controls was significantly lower than after stimulation with live C. pneumoniae (p < 0.01), which was not the case when platelets from patients were stimulated. Increased levels of anti-C. pneumoniae antibodies were detected in sera from patients compared to healthy subjects, suggesting prior C. pneumoniae exposure. Conclusion: Our data demonstrated an interactive link between C. pneumoniae and platelets in atherosclerotic patients, leading to induction of potential chemokines and possibly disease development.

Induction of interleukin-18 in atherosclerotic patients: a role for Chlamydia pneumoniae.

Objectives: The present work explored gene expression and spontaneous induction of the inflammatory cytokine interleukin-18 (IL-18) in atherosclerotic patients. In addition, the effect of the chlamydial antigen heat shock protein 60 (HSP60) and lipopolysaccharide (LPS) on the induction of this mediator was examined. Subjects and Methods: Detection of IL-18 mRNA and protein level were assessed by in situ hybridization and immunohistochemistry, respectively, in 15 patients with coronary artery disease undergoing angiograms and 15 matching controls. Results: These experiments showed significantly high levels of spontaneously expressed IL-18 mRNA and high protein levels in patients compared to healthy controls (p < 0.0005). Cells stimulated with chlamydial HSP60 (CHSP60) and LPS showed a significantly high expression of IL-18 at the mRNA level (p < 0.0005 for CHSP60 and p < 0.005 for LPS) and an increased production of IL-18 at protein level (p < 0.0005 for CHSP60 and p < 0.005 for LPS). Conclusion: This study demonstrated de novo synthesis of the inflammatory cytokine IL-18 in atherosclerosis and, furthermore, that chlamydial antigens might play a role in the immunopathological events in this disease by generating more inflammatory mediators such as IL-18.

Modulation of the immune response of BALB/C mice against Leishmania Major by imuvert

This study investigated the effect of Imuvert, a biological response modifier derived from Serratia marcescens, on the progression of Leishmania major infection in Balb/c mice. A single 100 micrograms Imuvert injection was significantly protective in Balb/c mice when challenged 28 days later in the footpad with 5 x 10(5) stationary phase L. major promastigotes. Intraperitoneal (i.p.) and intravenous (i.v.) immunization of mice with heat-killed stationary phase L. major promastigotes significantly reduced lesion development following challenge with L. major promastigotes. Subcutaneous (s.c.) immunization had no protective effect. A single 100 micrograms Imuvert injection significantly reduced lesion development in s.c. immunized mice, but had a lesser effect in mice immunized by i.p. and i.v. routes. Balb/c mice receiving four Imuvert injections 14, 7, 2 and 1 day prior to footpad challenge with L. major promastigotes were not protected, but rather displayed significant exacerbation of infection. Our results suggest the possibility that Imuvert could be useful in stimulating a protective response against L. major when given along with s.c. vaccine, a realistic route for vaccinating humans in contrast to either i.v. or i.p. routes. Since the protective response in Balb/c mice against L. major is dependent on the stimulation of Th1 cells, it is suggested that the observed adjuvant effect of Imuvert given with s.c. vaccine perhaps is due to changes in immunological responses in such a direction.

An interspecies conserved motif of the mouse immune system-released activating agent (ISRAA) induces proliferative effects on human cells

We have recently described an immune system-released activating agent (ISRAA) as a nervous system-induced factor that stimulates immune responses in the mouse spleen. However, the human ISRAA has not yet been identified. In this study, we examined the effects of the mouse ISRAA protein on human peripheral blood mononuclear cells (PBMCs), to observe if the biological activity of this molecule is consistent between the two different species. Mouse ISRAA demonstrated dose-dependent dualistic effects on human cells, as 5 μg exhibited positive apoptosis and 50 pg exhibited significant proliferation (P<0.05). Furthermore, immunosuppressed cells from patients undergoing immunosuppressive therapy demonstrated significant proliferation to 50 pg ISRAA (P<0.05). Studies to compare sequences in different species revealed a preserved motif, exhibiting 72% similarity with the interspecies conserved signal peptide motif of tumor necrosis factor receptor 1 (TNFR1). A mutant ISRAA lacking this motif was produced and tested for its biological effects. The mutant ISRAA demonstrated neither apoptotic nor proliferative effects compared with wild type. Therefore, an interspecies conserved domain of ISRAA constitutes the active site of the molecule, and its effects on immunocompromised cells should be investigated for future therapies in the treatment of immunosuppressive disorders.

NOS 3 894G > T Gene Polymorphism: A Potential Risk Factor of Stroke in Bahraini Patients

The endothelial nitric oxide synthase (eNOS) encoded by the NOS3 gene is responsible for the synthesis of a vasoactive endothelium-derived nitric oxide (NO). The genetic polymorphism of this gene explains, in part, why some people are prone to develop stroke than others. In this study we conducted a case control study in Bahrainis to investigate “for the first time” the relationship between NOS3 894G > T (rs1799983) and 786T > C (rs2070744) polymorphisms with the stroke predisposition in Bahraini population. Detection of NOS3 polymorphism was performed by PCR RFLP genotyping method. The level of NO among cases and controls was measured using ELISA. A total of 93 unrelated stroke patients and 86 controls were included in the study. The three types of stroke; Ischemic, hemorrhagic and transient ischemic attack were reported (91.4%, 7.5% and 1.1% respectively). No significant gender difference was observed (P = 0.74). Having previous stroke was a highly significant risk factor of the disease (P = 0.001, OR = 1.4), where as a family history of stroke was not (OR = 0.11). The analysis provides evidence that the mutant 894GT + TT genotypes of NOS3 894G > T polymorphism were positively associated with stroke predisposition and it increased the risk of stroke nearly two folds (P = 0.037, OR = 1.936). Although we found an association between the mutant genotype786 TC + CC of the NOS3 786T > C polymorphism with the susceptibility to stroke (P = 0.023) suggesting that the mutant C allele might have a protective effect against stroke in this population, the strength of this association was rather low (OR = 0.484). The level of NO in stroke patients was significantly low compared to healthy controls (P 0.005). Diabetes, hypertension, heart diseases were reported in stroke patients (67%, 71.4% and 52.1% respectively). More over 50% of the cases with previous stroke are both diabetic and hypertensive. This indicates that these diseases could be considered as a significant factor in the development of stroke in this population. We concluded that the NOS3-894 G > T polymorphism is a potential risk factor of stroke in Bahraini population, whereas as the NOS3 786T > C polymorphism might have a possible protective role against the disease in this population.

Synthetic cannabinoids nano-micelles for the management of triple negative breast cancer

ABSTRACT Triple‐negative breast cancer (TNBC) is a highly heterogeneous disease with poor prognosis and inadequate therapeutic outcome. This contribution reports the use of a cannabinoid derivative, WIN55,212–2 (WIN) on the growth of TNBC in a 4T1 syngeneic mouse model. To reduce the well‐known psychoactive side effects of cannabinoids, we prepared a nanomicellar formulation of WIN (SMA‐WIN). In vivo biodistribution, in silico ADME predictions, anticancer activity, and psychoactive effect of WIN and SMA‐WIN studies suggest that SMA‐WIN formulation can reduce to greater extent tumor growth with milder psychoactive side effects when compared to free drug. Finally, the effects of WIN and SMA‐WIN in combination with doxorubicin (Doxo), an established chemotherapeutic agent for the treatment of TNBC, were investigated in vitro and in vivo. SMA‐WIN in combination with Doxo showed therapeutic efficacy and was able to reduce the tumor volume of TNBC murine model drastically. Moreover, SMA‐WIN, while favoring drug tumor accumulation, minimized the adverse psychoactive effects that have impeded the use of this agent in the clinic. To our knowledge, this is the first report for the assessment of cannabinoid nanoparticles in vivo for the treatment of TNBC and its enhanced anticancer effect at low doses with Doxo. These findings suggest a new therapeutic strategy in the management of TNBC. HIGHLIGHTSSMA‐WIN has been tested for the first time in vivo for its anticancer activity in a murine model of triple‐negative breast cancers (TNBC)SMA‐WIN micelle showed reduced psychoactive side‐effect in vivo compared to the free drugCombination of doxorubicin (Doxo) and low dosage of SMA‐WIN potentiates the anticancer effect in TNBC in vivo model with negligible psychoactive effectOur findings open up the way to new therapeutic opportunities for TNBC

Interleukin 1 receptor antagonist and 2′-5′-oligoadenylate synthetase-like molecules as novel biomarkers for multiple sclerosis patients in Bahrain

BACKGROUND Multiple sclerosis (MS) is a multi-factorial disease of the Central Nervous System (CNS) affecting young adults leading to significant disabilities over time. MS is now believed to be prevalent in Arabian Gulf area with high incidence due to environmental factors and unknown genetic variations. The objectives of this study was to detect up-regulated potential genes that might be involved in neuroinflammatory process in MS patients in Bahrain and to measure the protein levels of the expressed genes. METHODS A microarray was used to investigate mRNA expression from 12 MS patients and 12 control subjects in Bahrain where the mRNA came from peripheral blood leukocytes. Also, 80 MS patients and 80 control subjects were analyzed to measure serum protein levels of the expressed genes by ELISA. RESULTS The data showed 15,480 genes expressed from over 47,000 transcripts and variants. Only 5 genes were significantly up-regulated in MS patients vs control subjects; namely TNF-AIP6, IL-1RA, OASL, CLC and DOCK4 (p < 0.05). Conversely, KIAA0125 gene was significantly down-regulated (p < 0.0003). Analysis of the effector molecules of the up-regulated genes revealed that 83 MS patients had positive serum level of OASL, 87 MS patients had positive serum levels of IL-1RA, and none of the 88 MS patients showed detectable serum levels of TNF-AIP6, CLC or DOCK4. CONCLUSIONS OASL and IL-1RA genes were strongly expressed in MS patients and that their effector molecules may be considered as biomarkers associated with the inflammatory process of the disease and possibly treatment response.

A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein

The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system.