Lymphocyte Signal Transduction Research Articles

PERICARDIAL TUBERCULOSIS IN A PATIENT WITH REFRACTORY PERICARDIAL EFFUSION AND USE OF DASATINIB: CASE REPORT

AbstractCardiovascular events have been reported in patients with chronic myeloid leukemia (CML) using tyrosine kinase inhibitors (TKIs). This study reports the occurrence of pericardial effusion (PE) refractory to usual therapy, with a final diagnosis of extrapulmonary tuberculosis (TB). IntroductionBy suppressing cellular immunity and compromising T lymphocyte signal transduction, TKIs are associated with tuberculosis. In vitro, dasatinib's action on Src-family kinases can impair cellular function and increase lysosomal acidification in macrophages, reducing Mycobacterium tuberculosis (Mtb) intracellular proliferation. In parallel, pleural effusion is a significant adverse effect of dasatinib. Its pathophysiology may involve increased vascular permeability via endothelial ABL kinases, Src kinases, and off-target inhibition of PDGFR-β. IgE-mediated histamine release by basophils has also been observed in vitro and may participate in dasatinib-related PE. ReportA 45-year-old male patient was diagnosed with low-risk CML in 2019 (EUTOS, SOKAL, HASFORD). After loss of major molecular response to imatinib and grade 4 toxicity to nilotinib, he was started on dasatinib 100 mg/day. Two years and eight months later, he presented with moderate pleural and marked pericardial effusion with hemodynamic repercussions. The medication was discontinued, with pleural effusion improvement after ten days of diuretics and prednisone. Conversely, the pericardial effusion persisted. Pericardiocentesis was performed, draining 460 ml of citrine fluid. Analysis revealed glucose 126 mg/dL (serum 165 mg/dL); RBC < 10, WBC 196 cells/μL, MN 95%, PMN 3.9%; pH 7.49; Xpert MTB/RIF assay: negative. After clinical improvement and a brief reintroduction of reduced-dose dasatinib, a new severe PE occurred. Partial pleurectomy and pleuropericardial window were performed, draining 500 ml of serohematic fluid. Cytology disclosed macrophages and Xpert MTB/RIF assay trace result. The diagnosis of pericardial tuberculosis prompted the initiation of an alternative treatment with levofloxacin, isoniazid, pyrazinamide, and ethambutol due to the rifampicin-TKI interaction. Treatment resumed with bosutinib 100 mg/day, increasing to 300 mg/day, with satisfactory tolerance. A new BCR::ABL1 evaluation is in progress. DiscussionDasatinib affects Src kinases expressed by lymphocytes and macrophages, which are crucial in the response to Mycobacterium tuberculosis. The incidence of TB during TKI use varies geographically, and its incidence with dasatinib is not yet known. Nonetheless, extrapulmonary TB has been reported. The median time to development of TB after initiation of TKI treatment is not established. Dasatinib is associated with PE in 1-4% of cases. The complication usually occurs 8-12 months after treatment initiation. In severe cases, management includes dose interruption or reduction, diuretics, corticosteroids, and pericardiocentesis. ConclusionDasatinib is effective in CML, but its immunological effect requires further study. The incidence of TB varies geographically and possibly among TKIs since the dual action of dasatinib appears to interfere with cellular immunity and reduce Mtb growth in vitro. Cavitary effusions are reported at all doses of dasatinib and are generally mild and manageable, even if grade ≥3. Refractory effusions should raise suspicion of infection, especially in areas with a high prevalence of TB.

Covalent docking-driven virtual screening of extensive small-molecule libraries against Bruton tyrosine kinase for the identification of highly selective and potent novel therapeutic candidates

Bruton tyrosine kinases (BTKs) play critical roles in various diseases, including chronic lymphatic leukemia (CLL), Waldenström Macroglobulinemia, Marginal Zone Lymphoma, Mantle Cell Lymphoma (MCL), and Graft Versus Host diseases. BTKs are a family of tyrosine kinases involved in B lymphocyte signal transduction, development, and maturation. Their overexpression can lead to cancer as they are essential for the activation of the B Cell Receptor (BCR) signaling pathway. Blocking the activation of BTKs presents a promising approach for treating CLL. This study was centered around the identification of small-molecule therapeutics that have an impact on human BTK. The covalently bound Ibrutinib molecule, recognized for its ability to inhibit BTK, was used as the query molecule. IUPAC text files containing molecular fragments of Ibrutinib were employed to virtually screen five different libraries comprising small-molecules, resulting in the screening of over 2.4 million synthesized compounds. Covalent docking simulations were applied to the selected small-molecules obtained through text mining from databases. Potent hit molecules capable of inhibiting BTKs through virtual screening algorithms were identified, paving the way for novel therapeutic strategies in the treatment of CLL.

The prohibitin complex regulates macrophage fatty acid composition, plasma membrane packing, and lipid raft-mediated inflammatory signaling

Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure. SummaryProhibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.

Association of Tuberculosis in Patients with Chronic Myeloid Leukemia, a Treatment Proposal Based on Literature Review

Introduction: Chronic Myeloid leukemia (CML) is a chronic myeloproliferative neoplasm with an increased undifferentiated proliferation of granulocytic lineage of the white blood cells. It is an acquired disorder of the hematopoietic stem cells due to a cytogenetic aberration known as the Philadelphia chromosome. It occurs due to a phenomenon of a reciprocal translocation between chromosomes t(9,22) and results in BCR/ABL fusion gene. The product of this gene has tyrosine kinase property, which predominantly causes the disease. Tyrosine kinase inhibitors (TKI) are the mainstay of the treatment to achieve remission, while bone marrow transplant is the only proven cure. Tuberculosis (TB), both active and reactivation of latent TB, remains the most common infectious disease worldwide and leads to high mortality. Currently, one-fourth of the global population has been infected by Mycobacterium tuberculosis. The incidence of TB has been reportedly increasing in patients with cancer. It has been mostly found in association with non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Recently, the incidence increased in patients with CML during treatment with Imatinib (IM) has also been reported in a retrospective cohort study in Taiwan by Lui C-J et. al. and few other published cases in literature. This might be due to impairment of the T lymphocytes signal transduction leading to increased host susceptibility to TB, hepatitis B, and varicella-zoster infection. TB treatment is mainly based on the susceptibility of the Mycobacterium Tuberculosis to the anti-TB medications. First-line therapy includes of four drugs, Isoniazid (INH), Rifampicin (RIF), Pyrazinamide (PZA), and Ethambutol (EMB). Anti TB drugs administration also needs close monitoring because of potential side effects. These include, but not limited to hepatoxicity, optic neuritis, impaired color vision, peripheral neuropathy, and gastrointestinal disturbances. Their occurrence necessitate discontinuation of the culprit drug from the regimen. Certain anti TB drugs are also known to disrupt bioavailability of other drugs metabolized in the liver when concomitantly administered by influencing the hepatic cytochrome P450 enzyme. This happens while using anti-TB medication to treat concomitant TB infection in CML patients on tyrosine kinase inhibitors therapy. Methods and critical analysis of the reviewed study articles: The authors reviewed the current literature focusing on all articles addressing TB infections and their diagnosis and management in patients with CML (PubMed and Google Scholar between 2000 and 2020). Our review aimed to provide the necessary data to improve clinical diagnosis and clarify the management strategy of this condition. Conclusion and treatment proposal: Throughout our review of multiple research articles, it has been realized that patients with CML and other hematological malignancies and solid tumors (e.g., non-Hodgkin lymphoma, chronic lymphocytic leukemia, metastatic gastric tumors), have a higher risk of having TB infection. TB infection (pulmonary and extra-pulmonary) occurs 2-9 times more than the general population in patients with hematological malignancies. In CML patients, treatment therapy with Imatinib posed a higher risk of development of TB due to defective cellular immunity. Co-administration of 1st line anti Tb medications mainly rifampicin with standard TKIs therapy Imatinib and Dasatinib has resulted in sub-therapeutic levels of these drugs due to induction of CYP4503A enzyme. Either by changing the TB treatment regimen to a non-rifampicin based therapy line with a combination of a fluoroquinolone, increasing the dose, or switching to a different TKI class may result in clinical improvement. To the best of our knowledge, there are no randomized control trials, or FDA approved guidelines to treat concomitant TB infection in CML patients. Therefore, a clear strategy to deal with such a clinical condition is lacking. The immediate treatment for the concomitant disease has been suggested based on clinical experience and clinical response of some patients published in few case reports, case series, and retrospective cohort studies. After careful literature review, we put forth our management proposals, However it need further research studies and clinical trials to bring recommendations and guidelines. Disclosures No relevant conflicts of interest to declare.

Lymphocyte mass cytometry identifies a CD3-CD4+ cell subset with a potential role in psoriasis.

Psoriasis (PS) is a systemic, immune-mediated inflammatory disorder. However, the whole lymphocyte compartment and the potential pathologies of PS have not been fully characterized. In the present study, we examined whole lymphocyte subsets and signal transduction proteins using high-dimensional single-cell mass cytometry and a bioinformatics pipeline for an in-depth characterization of the immune cell subsets and protein profiles involved in pathways in the peripheral blood of patients with PS. We identified 15 major immune cell populations in T cell lineages and characterized various CD3+CD4+ Th and CD3+CD8+ T cytotoxic cell populations simultaneously across 24 leukocyte markers and 7 proteins related to the signal transduction pathways. High-dimensional analysis identified 3 new subsets that are abundant in PS peripheral blood, resembling CD3-CD4+ lymphoid tissue inducer cells, Tc17 cells, and CD8+CXCR3+ Tregs. We confirmed the CD3-CD4+ cells, and their features and functions, in an independent PS cohort. The use of single-cell mass cytometry allows systemic-level characterization of lymphocyte subpopulations and dysregulated signaling pathways in the blood of patients with PS, identifying abnormalities of different immune cell subsets. We validated that the CD3-CD4+ cells had elevated OX40 and decreased FRA2 expression, which were positively associated with the PS area and severity index.

Pharmacological inhibition of MALT1 protease activity suppresses endothelial activation via enhancing MCPIP1 expression

Mucosa associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is not only an intracellular signaling scaffold protein but also a paracaspase that plays a key role in the signal transduction and cellular activation of lymphocytes and macrophages. However, its role in endothelial cells remains unknown. Here we report that pharmacological inhibition of MALT1 protease activity strongly suppresses endothelial activation via enhancing MCPIP1 expression. Treatment with MALT1 protease inhibitors selectively inhibited TNFα-induced VCAM-1 expression in HUVECs and LPS-induced VCAM-1 expression in mice. In addition, Inhibition of MALT1 protease activity also significantly inhibited TNFα-induced adhesion of THP-1 monocytic cells to HUVECs. To explore the mechanisms, MALT1 inhibitors does not affect the activation of NF-κB signaling pathway in HUVEC. However, they can stabilize MCPIP1 protein and significantly enhance MCPIP1 protein level in endothelial cells. These results suggest that MALT1 paracaspase also targets MCPIP1 and degrade MCPIP1 protein in endothelial cells similar as it does in immune cells. Taken together, the study suggest inhibition of MALT1 protease activity may represent a new strategy for prevention/therapy of vascular inflammatory diseases such as atherosclerosis.

Immune Tolerance Effect in Mesenteric Lymph Node Lymphocytes of Geniposide on Adjuvant Arthritis Rats.

Rheumatoid arthritis (RA) is a systemic, Th1 cytokine-predominant autoimmune disease result in a chronic and inflammatory disorder. Geniposide (GE), an iridoid glycoside compound that is purified from Gardenia jasminoides Ellis, has antiinflammatory and other immunoregulatory effects, but its exact mechanism of actions on RA is unknown. The aim of this study was to elucidate antiinflammation effects of GE on adjuvant arthritis (AA) rats and its possible immune tolerance mechanisms. Male Sprague-Dawley rats were administered with GE (30, 60, and 120mg/kg) orally from day 17 to 24 after immunization. Lymphocyte proliferation was assessed by MTT. Levels of interleukin-2 (IL-2), IL-4, and transforming growth factor-β1 were tested by ELISA. The expression of β2-AR, GRK2, and β-arrestin-1 and β-arrestin-2 was detected by western blot. Geniposide was found to relieve the secondary hind paw swelling and arthritis scores, along with attenuating histopathologic changes and decreasing IL-2 and increasing IL-4, transforming growth factor-β1 in mesenteric lymph node (MLN) lymphocytes of AA rats. In addition, GE in vivo increased the expression of β2-AR and decreased the expression of GRK2, β-arrestin-1 and β-arrestin-2, and level of cyclic adenosine monophosphate of MLN lymphocytes in AA rats. From these results, we can infer that GE on immune tolerance effects, β2-AR desensitization, and β2-AR-AC-cyclic adenosine monophosphate transmembrane signal transduction of MLN lymphocytes plays crucial roles in antiinflammatory and immunoregulatory pathogeneses of RA. Copyright © 2017 John Wiley & Sons, Ltd.

Protein Kinase C-Theta (PKCθ): A Rheostat in T cell Signaling and Cancer

Protein kinase C-theta (PKCθ) is a key enzyme in T lymphocytes signal transduction pathway that works downstream of the activated T cell receptor (TCR) and the CD28 receptor. This protein translocates to the center of the immunological synapse (IS) as T cells encounter an antigen. Depending on the quality and quantity of extracellular antigenic stimuli, PKCθ differentially phosphorylates and activates different effector molecules that mediate signal transduction into distinct subcellular compartments and activate the major T cell responsive transcription factors, NF-κB, NFAT and AP-1. Besides having a major biological role in T cells, PKCθ is also expressed at high levels in gastrointestinal stromal tumors, although the functional importance is not fully clear. The present manuscript shades light on the current understanding on PKCθ in T cell signaling and cancer.

Abnormal lipid rafts related ganglioside expression and signaling in T lymphocytes in immune thrombocytopenia patients

Aberrant T lymphocytes signaling is considered to play a crucial role in the abnormal immune state of primary immune thrombocytopenia (ITP). Lipid raft has been verified to engage in the T cell receptor (TCR)-mediated T lymphocytes signal transduction. Whether lipid raft-associated T cells signal transduction has impact on the pathogenesis of ITP is still unconfirmed. In this study, we aimed to reveal the abnormality in structure and function of lipid rafts (LRs) in CD4+ and CD8+ T lymphocytes of patients with ITP. Our results showed that there was an increased lipid raft aggregation in ITP patients, while this kind of increase would not be influenced by platelet counts or therapeutic regimes. Stimulation by anti-CD3/CD28 monoclonal antibodies promoted enhanced lipid raft clustering in T lymphocytes of ITP patients compared with negative controls. Methyl-β-cyclodextrin (MβCD) could block the abnormal lipid raft aggregation and disrupt the TCR-mediated T cells proliferation and cytokines secretion, including both proinflammatory cytokines and anti-inflammatory cytokines. The spontaneous activation of T lymphocytes from ITP patients might be due to the elevated co-localization of protein tyrosine phosphatase (PTP) CD45 and lipid rafts in patients’ CD4+ and CD8+ T lymphocytes. These findings suggest that the autoactivation of T lymphocytes from ITP patients may lead to the abnormality in lipid raft structure and raft-anchored proteins, and the changes conversely promote the TCR-mediated T cells activation of ITP patients.

Wiskott-Aldrich syndrome complicated with demyelinating disease of the central nervous system:report of one case and literature review

Objective To discuss the correlation of Wiskott-Aldrich syndrome (WAS) with autoimmune disease of the central nervous system and its possible pathogenesis by reporting one case of the disease and reviewing related literature. Methods One case of WAS complicated with demyelinating disease of the central nervous system was reported. The patient's clinical symptoms, laboratory examinations (such as blood tests, immune function tests, etc) and imaging features were analyzed. The patient's blood DNA was extracted and performed gene testing. And related literature was reviewed. Results The patient showed typical clinical symptoms of WAS, including eczema, thrombocytopenia and immune deficiency, complicated with demyelinating disease of the central nervous system. The DNA testing showed C400G>C p. (ALa134Pro) mutation, which is a missense mutation. The 134th amino acid in protein was changed from alanine to proline. The patient also showed the symptoms of demyelinating disease of the central nervous system, which drew our attention. This was the first report on WAS complicated with demyelinating disease of the central nervous system, which was perhaps caused by a gene mutation. Conclusions WAS complicated with demyelinating disease of the central nervous system is possibly resulted from the gene mutation, which leads to the expression disorder of WAS protein. And then non-red hematopoietic cells lead to signal transduction and cytoskeleton recombination disorders in response to environment stimulus, which produces lymphocytes immigration, signal transduction and immune synaps formation disorders. Key words: Wiskott Aldrich syndrome; Demyelinating diseases; Central nervous system diseases; Autoimmune diseases

JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4(+)IL-17A-F(+) and T CD4(+)IL-23R(+) Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4(+)IL-17A-F(+) cells, as well as of T CD4(+) cells expressing IL-23Rp19 (T CD4(+) IL-23R(+)), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4(+)IL-17A-F(+) and T CD4(+)IL-23R(+) Th17 Teff cells in SF of clinically active joints of PsA patients.

Successful Treatment with Tacrolimus in a Case of the Glucocorticoid-Dependent Recurrent Cutaneous Eosinophilic Vasculitis

Successful Treatment with Tacrolimus in a Case of the Glucocorticoid-Dependent Recurrent Cutaneous Eosinophilic Vasculitis

Protein Kinase C-θ Clustering at Immunological Synapses Amplifies Effector Responses in NK Cells

In lymphocytes, stimulation of cell surface activating receptors induces the formation of protein microclusters at the plasma membrane that contain the receptor itself, along with other signaling molecules. Although these microclusters are generally thought to be crucial for promoting downstream cellular responses, evidence that specifically links clustering potential to signaling output is lacking. We found that protein kinase C-θ (PKCθ), a key signaling molecule in multiple lymphocyte subsets, formed microclusters in activated NK cells. These microclusters coalesced within the immunological synapse between the NK cell and its target cell. Clustering was mediated by the regulatory region of PKCθ and specifically required a putative phosphotyrosine-binding site within its N-terminal C2 domain. Whereas expression of wild-type PKCθ rescued the cytokine production defect displayed by PKCθ-deficient NK cells, expression of a PKCθ point-mutant incapable of forming microclusters had little to no effect. Hence, PKCθ clustering was necessary for optimal effector function. Notably, only receptors containing ITAMs induced PKCθ microclusters on their own, explaining previous observations that ITAM-coupled receptors promote stronger activating signals and effector responses than do receptors lacking these motifs. Taken together, our results provide a cell biological basis for the role of PKCθ clustering during NK cell activation, and highlight the importance of subcellular compartmentalization for lymphocyte signal transduction.

Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling

BackgroundT lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.ResultsCalcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells.ConclusionsThis study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.

Normalization of deranged signal transduction in lymphocytes of COPD patients by the novel calcium channel blocker H-DHPM

Investigations on the role of intracellular Ca 2+ ion concentration in the mechanism of development of COPD in smokers and non-smokers were carried out. The intracellular Ca 2+ levels were found to be increased in human lymphocytes in patients with COPD as compared to non-smokers and smokers without COPD. The investigations reveal an association in altered intracellular Ca 2+ regulation in lymphocytes and severity of COPD, by means of significant activation of Protein kinase C and inducible nitric oxide synthase (iNOS). The effect of a novel calcium channel blocker ethyl 4-(4′-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM) as a potential candidate for the treatment of COPD was also investigated. H-DHPM treated cells showed a decrease in intracellular Ca 2+ level as compared to the control cells. Molecular studies were carried out to evaluate the expression profile of NOS isoforms in human lymphocytes and it was shown that H-DHPM decreases the increased iNOS in COPD along with reestablishing the normal levels of endothelial nitric oxide synthase (eNOS). The results of H-DHPM were comparable with those of Amlodipine, a known calcium channel blocker. Calcium channel blocker H-DHPM proves to be a potential candidate for the treatment of COPD and further clinical studies are required to prove its role in the treatment of pulmonary hypertension (PH).

Replicated associations of TNFAIP3, TNIP1 and ETS1 with systemic lupus erythematosus in a southwestern Chinese population

IntroductionRecent genome-wide and candidate gene association studies in large numbers of systemic lupus erythematosus (SLE) patients have suggested approximately 30 susceptibility genes. These genes are involved in three types of biological processes, including immune complex processing, toll-like receptor function and type I interferon production, and immune signal transduction in lymphocytes, and they may contribute to the pathogenesis of SLE. To better understand the genetic risk factors of SLE, we investigated the associations of seven SLE susceptibility genes in a Chinese population, including FCGR3A, FCGR2A, TNFAIP3, TLR9, TREX1, ETS1 and TNIP1.MethodsA total of 20 SNPs spanning the seven SLE susceptibility genes were genotyped in a sample of 564 unrelated SLE patients and 504 unrelated healthy controls recruited from Yunnan, southwestern China. The associations of SNPs with SLE were assessed by statistical analysis.ResultsFive SNPs in two genes (TNFAIP3 and ETS1) were significantly associated with SLE (corrected P values ranging from 0.03 to 5.5 × 10-7). Through stratified analysis, TNFAIP3 and ETS1 showed significant associations with multiple SLE subphenotypes (such as malar rash, arthritis, hematologic disorder and antinuclear antibody) while TNIP1 just showed relatively weak association with onset age. The associations of the SNPs in the other four genes were not replicated.ConclusionsThe replication analysis indicates that TNFAIP3, ETS1 and TNIP1 are probably common susceptibility genes for SLE in Chinese populations, and they may contribute to the pathogenesis of multiple SLE subphenotypes.

Impact of the cellular protein Dlg 1 on HIV infectivity and cell to cell transfer

HIV-1 spread in an infected organism is highly dependent on proper virus assembly and budding for the efficient formation of infectious particles. To achieve this goal, the virus hijacks numerous cellular proteins such as members of the ESCRT pathway, Tip47 or AP complexes. These are generally partners for Gag, Env or both, mediating their intracellular traffic to the site of release, or even favouring their interaction with one another. We have previously identified Dlg1, the human homologue of Drosophila Discs Large protein as a new partner for HIV-1 Gag [1]. Dlg1 underlies the plasma membrane of different cell types (epithelial, neural cells, T lymphocytes) and acts as a scaffold for the formation of multiprotein complexes. We reported that depleting endogenous Dlg1 in T cells does not affect Gag production, maturation or release. Nevertheless, Dlg1 knockdown enhances the infectivity of released virions, which correlates with an increase of the amount of Env in producer cells as well as in viral particles. Depletion of Dlg1 also induces a modification of Gag and Env cellular localisation from the plasma membrane to an apparent internal punctate structure where they colocalize. Thus Dlg1 can be considered as a new negative regulator of HIV-1 virions infectivity. Dlg1 regulates the immunological synapse, playing a role in lymphocyte polarisation, signal transduction and effector function. Besides, Dlg1 is a partner of Zap70, a kinase involved in both the immunological and the virological synapse formation, also known to facilitate HIV-1 cell to cell transmission. Overall these data led us to question the impact of Dlg1 in HIV-1 cell to cell transmission. To this end we used a cell culture system in which virus release from donor cells is unaffected while virus transfer from donor to recipient cells is severely impaired. The impact of Dlg 1 under these culture conditions will be presented.

Lipoic acid attenuates high fat diet-induced chronic oxidative stress and immunosuppression in mice jejunum: A microarray analysis

A high fat diet (HFD) has long been linked to immune dysfunction, including diminished numbers or reactivity of lymphocytes, increased susceptibility to infection, inhibited lymphocytes function during antigen-specific responses and developed oxidative stress. Whereas the molecular mechanistic events associated with immune deficiency remain to be fully determined. Using the DNA microarray system, we analyzed the gene expression patterns of lymphocyte related signal transduction proteins in jejunum of C57BL/6 mice in order to gain insight on the possible molecular mechanism by which HFD induced oxidative stress effects on signal transduction of lymphocytes. Results of present study showed that HFD induced oxidative stress and immunosuppression in jejunum. Antioxidant lipoic acid (LA) supplement ameliorated that HFD induced oxidative stress and immunosuppression by recovering transcriptional levels of the gene involved in B cell receptor, T cell differentiation signaling pathway, and free radical scavengers. The present study indicates that a HFD can induce chronic oxidative stress, suppress signal transduction of gut-associated lymphocytes, and lead to an inhibition of mucosal immunity.

An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activation of src-family protein tyrosine kinases (PTK) such as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10(-9) M) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c-src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4-associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes.

A FLIPR-Based Assay to Assess Potency and Selectivity of Inhibitors of the TEC Family Kinases Btk and Itk

Bruton's tyrosine kinase (Btk) and interleukin-2-inducible T cell kinase (Itk) are members of the TEC family of nonreceptor tyrosine kinases and are expressed primarily in B and T cells, respectively. Both kinases are critically involved in lymphocyte development and signal transduction. In particular, Btk and Itk regulate calcium mobilization subsequent to antigen receptor stimulation. Small molecule antagonists that specifically inhibit either Btk or Itk may allow for selective modulation of B cell or T cell activity and may be useful in treating inflammatory and autoimmune conditions. We have developed a medium-throughput fluorescent imaging plate reader (FLIPR®)- based calcium flux assay that can be used to assay potential Btk and Itk inhibitors. This assay takes advantage of Btk-deficient DT40 (DT40-Btk−/−) chicken B cells, which are unable to mobilize calcium in response to cross-linking of their B cell receptor (BCR). Ectopic expression of TEC family kinases can restore antigen receptor signaling in these cells. We have generated stable DT40-Btk−/− lines expressing either wild-type human Btk (huBtk) or a chimeric Btk-Itk kinase (huBtk-Itk) molecule—a Btk protein whose kinase domain has been replaced by the kinase domain of Itk. Expression of either huBtk or huBtk-Itk in DT40-Btk−/− cells restores calcium flux in response to BCR engagement. Using Btk- and Itk-selective inhibitors, we show that inhibition of calcium responses in huBtk-Itk-DT40-Btk−/− cells and huBtk-DT40-Btk−/− cells is dependent on the Itk or Btk kinase domain, respectively. Thus, the FLIPR assay described here can be used to assess, compare, and rank the potency and selectivity of inhibitors of Itk and Btk kinases.