Distinct myeloid suppressor cell subsets correlate with plasma IL-6 and IL-10 and reduced interferon-alpha signaling in CD4<Superscript>+</Superscript> T cells from patients with GI malignancy

Interferon-alpha (IFN-α) promotes anti-tumor immunity through its actions on immune cells. We hypothesized that elevated percentages of myeloid-derived suppressor cells (MDSC) and increased pro-inflammatory cytokines in peripheral blood would be associated with impaired response to IFN-α in patients with gastrointestinal (GI) malignancies. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets, and IFN-α-induced signal transduction in 40 patients with GI malignancies. Plasma IL-6 and IL-10 were significantly higher in patients versus normal donors. CD33⁺HLADR⁻CD11b⁺CD15⁺ and CD33⁺HLADR^−/lowCD14⁺ MDSC subsets were also elevated in patients versus normal donors (P < 0.0001). Plasma IL-6 was correlated with CD33⁺HLADR⁻CD15⁺ MDSC (P = 0.008) and IL-10 with CD33⁺HLADR⁻CD15⁻ MDSC (P = 0.002). The percentage of CD15⁺ and CD15⁻ but not CD14⁺ MDSC subsets were inversely correlated with IFN-α-induced STAT1 phosphorylation in CD4⁺ T cells, while co-culture with in vitro generated MDSC led to reduced IFN-α responsiveness in both PBMC and the CD4⁺ subset of T cells from normal donors. Exploratory multivariable Cox proportional hazards models revealed that an increased percentage of the CD33⁺HLADR⁻CD15⁻ MDSC subset was associated with reduced overall survival (P = 0.049), while an increased percentage of the CD33⁺HLADR^−/lowCD14⁺ subset was associated with greater overall survival (P = 0.033). These data provide evidence for a unique relationship between specific cytokines, MDSC subsets, and IFN-α responsiveness in patients with GI malignancies.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

TNK cells (NKG2D<Superscript>+</Superscript> CD8<Superscript>+</Superscript> or CD4<Superscript>+</Superscript> T lymphocytes) in the control of human tumors

Innate and adaptive immune responses have many interactions that are regulated by the balance of signals initiated by a variety of activatory and inhibitory receptors. Among these, the NKG2D molecule was identified as expressed by T lymphocytes, including most CD8⁺ cells and a minority of CD4⁺ cells, designated TNK cells in this paper. Tumor cells may overexpress the stress-inducible NKG2D ligands (NKG2DLs: MICA/B, ULBPs) and the NKG2D signaling has been shown to be involved in lymphocyte-mediated anti-tumor activity. Aberrant expression of NKG2DLs by cancer cells, such as the release of soluble form of NKG2DLs, can lead to the impairment of these immune responses. Here, we discuss the significance of NKG2D in TNK-mediated anti-tumor activity. Our studies demonstrate that NKG2D⁺ T cells (TNK) are commonly recruited at the tumor site in melanoma patients where they may exert anti-tumor activity by engaging both TCR and NKG2D. Moreover, NKG2D and TCR triggering was also observed by peripheral blood derived T lymphocyte- or T cell clone-mediated tumor recognition, both in melanoma and colorectal cancer (CRC) patients. Notably, heterogeneous expression of NKG2DLs was found in melanoma and CRC cells, with a decrease of these molecules along with tumor progression. Therefore, through the mechanisms that govern NKG2D engagement in anti-tumor activity and the expression of NKG2DLs by tumor cells that still need to be dissected, we showed that NKG2D expressing TNK cells are a relevant T cell subtype for immunosurveillance of tumors and we propose that new immunotherapeutic interventions for cancer patients should be aimed also at enhancing NKG2DLs expression by tumor cells. This paper is a focused research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany, 15–16 May 2008.     

CD4<Superscript>+</Superscript> T cell response against a non-tumor antigen is unaffected in melanoma-bearing mice

The tumor microenvironment is complex and creates an immunosuppressive network to tolerize tumor-specific immune responses; however, little information is available regarding the response against non-tumor antigens in tumor-bearing individuals. The goal of the present study was to evaluate if tumor burden could influence a CD4⁺ T cell response against a soluble protein, not expressed by the tumor, in the absence of in vitro stimulation. Using an experimental system in which we can compare CD4⁺ T cell responses to the Ea antigen when it is either expressed by B16F10 melanoma cells (B16EaRFP cells) or is an exogenous, non-tumor antigen (soluble EaRFP protein), in immunizations of B16F10 tumor-bearing mice, we observed that the tumor can modulate the CD4⁺ T cell-specific response to the antigen when it is expressed by the tumor cells. TEa cells proliferated poorly and produced less IFN-γ in mice bearing B16F10 melanoma expressing Ea peptide, and tumor growth was impervious to this response. However, in mice bearing 7 days B16F10 tumors, not expressing the Ea antigen, priming of TEa cells was similar to that observed in tumor-free mice, based on the total number of cells recovered and proliferation assessed by CFSE dilution after EaRFP immunization. We also investigated if tumor burden could influence recall responses of already differentiated effector cells. We immunized mice with EaRFP antigen and after a few days injected B16F10 cells. After 10 days of tumor growth, we challenged the mice with the non-tumor antigen. We found that the number of TEa cells producing IFN-γ in tumor-bearing mice was not different compared to tumor-free mice. No differences in antigen presentation, assessed by YAe antibody staining, were verified in the draining lymph node of these two groups. Collectively, our data indicate that tumor burden does not affect immune responses to non-tumor antigens. These results have important implications in the design of anti-cancer therapy.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Human CD80/IL2 lentivirus transduced acute myeloid leukaemia cells enhance cytolytic activity in vitro in spite of an increase in regulatory CD4<Superscript>+</Superscript> T cells in a subset of cultures

Immunotherapeutic strategies are increasingly being explored as a method of enhancing anti-tumour immune responses in patients with acute myeloid leukaemia (AML). Regulatory CD4⁺ T cells (Tregs) suppress effector T and natural killer (NK) cells and therefore pose a potential challenge to the efficacy of immunotherapy. AML cells transduced with a lentivirus expressing CD80 (B7.1) and IL2 (LV-CD80/IL2) are capable of stimulating T and NK cell cytotoxicity in vitro. This study examines the effect of CD80/IL2 modified AML cells on Treg number and function. We report a significant increase in the number of CD8⁺ T cells (P = 0.046) CD3⁻CD56⁺ NK cells (P = 0.028) and CD3⁺CD4⁺CD25^highFoxp3⁺ Tregs (P = 0.043) following stimulation for 7 days with allogeneic LV-CD80/IL2 AMLs. In contrast, autologous LV-CD80/IL2 AML cell cultures provide a weaker stimulation with a lower number of CD8⁺ T cells (P = 0.011) and no change in NK cell or Treg numbers. However, an increase in cytotoxic CD8⁺ T cells and NK cells are detected following both allogeneic and autologous LV-CD80/IL2 stimulation as demonstrated by an increase in IFN-γ and CD107a expression. Despite the presence of increased numbers of Tregs with suppressive activity in a subset of cultures, increased lysis of unmodified AMLs was still achieved following allogeneic (day 0, 2.2%; day 7, 20.4%) and more importantly, autologous LV-CD80/IL2 culture in which AML patients had recently received intensive chemotherapy (day 0, 0%; day 7, 16%). Vaccination with LV-CD80/IL2 therefore provides a potential strategy to enhance anti-leukaemia immune responses without a concomitant stimulation of Treg-mediated inhibition of cytotoxic immunological responses.     

Cancer/testis antigens can be immunological targets in clonogenic CD133<Superscript>+</Superscript> melanoma cells

“Cancer stem cells” that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133⁺ melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133⁺ clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8⁺ T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

Metagenome Cloning and Functional Analysis of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Genes from Keke Salt Lake in China

Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA ⁻ , nhaB ⁻ , chaA ⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA ⁻ , nhaB ⁻ , chaA⁻) grow in the LBK medium containing 0.2–0.6 M Na⁺ or with 0.05–0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.     

Dynamic Changes of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells in NOD.H-2<Superscript>h4</Superscript> Mice with Iodine-Induced Autoimmune Thyroiditis

Iodine is an essential trace element for thyroid hormone synthesis and metabolism, either low or high intake may lead to thyroid disease, but the pathogenetic mechanisms by which iodine interacts with the thyroid autoimmune are poorly understood. We investigated the dynamic changes of CD4⁺CD25⁺ regulatory T cells in NOD.H-2^h4 mice with iodine-induced autoimmune thyroiditis (AIT), and explore potential immune mechanism of AIT induced by iodine. NOD.H-2^h4 mice were randomly divided into two groups, and received plain water or water containing 0.005% sodium iodide. Eight weeks after iodine provision, the incidences of thyroiditis, relative weights of thyroids, and serum thyroglobulin antibody titers in the iodine-supplied groups were significantly increased compared to the control groups (p < 0.05). The AIT mice had fewer CD4⁺CD25⁺Foxp3⁺ T cells and reduced Foxp3 mRNA expression in splenocytes compared with the controls (p < 0.01), and maintained relatively low levels during the development of thyroiditis. The changes described above aggravated gradually with the extension of iodine treatment. These data suggest that CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis and development of AIT induced by iodine.     

Oxygen consumption rate and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in early developmental stages of the sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Lam.

Changes in oxygen consumption rate and Na⁺/K⁺-ATPase activity during early development were studied in the sea urchin Paracentrotus lividus Lam. The oxygen consumption rate increased from 0.12 μmol O₂ mg protein⁻¹ h⁻¹ in unfertilized eggs to 0.38 μmol O₂ mg protein⁻¹ h⁻¹ 25 min after fertilization. Specific activity of the Na⁺/K⁺-ATPase was significantly stimulated after fertilization, ranging up to 1.07 μmol P_i h⁻¹ mg protein⁻¹ in the late blastula stage and slightly lower values in the early and late pluteus stages.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

Polyfunctionality of a DKK1 self-antigen-specific CD8<Superscript>+</Superscript> T lymphocyte clone in lung cancer

Polyfunctionality is the capacity of a T-cell to execute a variety of effector functions mainly mediated by production of cytokines, chemokines, and cytolytic enzymes. Studies in anti-viral immunity have acknowledged the importance of polyfunctionality in the clearance of infections and maintenance of protection. Although accepted in the field, this concept has not been as well characterized in cancer immunology. Here, we report the polyfunctionality profile analysis of a CD8⁺ T-cell clone isolated from a lung cancer patient and directed against Dickkopf-1, a potentially new tumor-associated antigen (TAA). The clone showed Tc1/Th1 effector tendencies confirmed by secretion of cytokines such as IFN-γ, IP-10, MIP-1β, MIP-1α, IL-2, GM-CSF, and expression of cytolytic enzyme granzyme B. This secretion profile is of particular interest in the context of an anti-tumor response. Although secretion of IL-5 and IL-13 was also detected, absence of IL-4 and IL-10 opposes the idea of cytokine-dependent Th1 inhibition. Establishing a comprehensive cytokine secretion profile may help predict T cells’ specific response against a novel TAA in a peptide vaccination context. It may further help in selecting clones with an optimal functional profile from the peripheral blood of cancer patients for expansion and adoptive cell transfer therapy.     

State-Dependent Block of Na<Superscript>+</Superscript> Channels by Articaine Via the Local Anesthetic Receptor

Articaine is widely used as a local anesthetic (LA) in dentistry, but little is known regarding its blocking actions on Na⁺ channels. We therefore examined the state-dependent block of articaine first in rat skeletal muscle rNav1.4 Na⁺ channels expressed in Hek293t cells. Articaine exhibited a weak block of resting rNav1.4 Na⁺ channels at −140 mV with a 50% inhibitory concentration (IC₅₀) of 378 ± 26 μM (n = 5). The affinity was higher for inactivated Na⁺ channels measured at −70 mV with an IC₅₀ value of 40.6 ± 2.7 μM (n = 5). The open-channel block by articaine was measured using inactivation-deficient rNav1.4 Na⁺ channels with an IC₅₀ value of 15.8 ± 1.5 μM (n = 5). Receptor mapping demonstrated that articaine interacted strongly with a D4S6 phenylalanine residue, which is known to form a part of the LA receptor. Thus the block of rNav1.4 Na⁺ channels by articaine is via the conserved LA receptor in a highly state-dependent manner, with a ranking order of open (23.9×) > inactivated (9.3×) > resting (1×) state. Finally, the open-channel block by articaine was likewise measured in inactivation-deficient hNav1.7 and rNav1.8 Na⁺ channels, with IC₅₀ values of 8.8 ± 0.1 and 22.0 ± 0.5 μM, respectively (n = 5), indicating that the high-affinity open-channel block by articaine is indeed preserved in neuronal Na⁺ channel isoforms.     

CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> immunoregulatory T cells may not be involved in controlling autoimmune arthritis

Accumulating evidence suggests that regulatory T cells play a crucial role in preventing autoimmunity. Recently, a naturally occurring CD4⁺CD25⁺ T-cell subset that is anergic and also suppressive has been shown to suppress autoimmunity in several animal models. We used proteoglycan-induced arthritis (PGIA) as a study model to investigate the role of the CD4⁺CD25⁺ regulatory T cells in autoimmune arthritis. There was no significant change in the percentage of CD4⁺CD25⁺ T cells during the immunization period when proteoglycan- or ovalbumin-immunized BALB/c and C57BL/6 mice were compared. An adoptive transfer study showed that the CD4⁺CD25⁺ T cells did not protect severe combined immunodeficient mice from arthritis when they were cotransferred with splenocytes from arthritic animals. Similarly, depletion of the CD4⁺CD25⁺ T cells did not enhance the onset of the disease or disease severity in severe combined immunodeficient mice. Moreover, CD28-deficient mice, which have very few CD4⁺CD25⁺ T cells, were highly resistant to PGIA. These findings indicate that the CD4⁺CD25⁺ regulatory T cells may not play a critical role in controlling PGIA.     

Association between HLA-DRB1, HLA-DRQB1 alleles,and CD4<Superscript>+</Superscript>CD28<Superscript>null</Superscript> T cells in a Chinese population with coronary heart disease

CD4⁺CD28^null T cells are present in increased numbers in the peripheral blood of patients with acute coronary syndrome. However, the triggers of expansion of these cells are unclear. Susceptibility to coronary heart disease (CHD) is strongly associated with alleles of human leukocyte antigen (HLA), but it is not equally strong in different human populations. The objective of the study was to investigate association between CD4⁺CD28^null T cells and HLA-DRB1 alleles. The HLA alleles were determined by polymerase chain reaction with sequence-specific primers (PCR-SSP) method, in a group of CHD patients and control subjects from the same area. The number of CD4⁺CD28^null T cells was measured using the flow cytometry technique. The HLA-DRB1*01 (RR = 4.705, P < 0.005) and DRB1*04 (RR = 3.554, P < 0.005) alleles showed the strongest association with CHD in the Chinese population, and increased numbers of CD4⁺CD28^null T cells were found in association with HLA-DRB1*04 (17.1%) and DRB*01 (12.9%), while decreased numbers of CD4⁺CD28^null T cells were present in subjects with DRB1*15 (0.8%). CHD in Chinese population is strongly associated with HLA-DRB1*01 and DRB1*04 haplotypes, and formation of CD4⁺CD28^null T cells was related to HLA-DRB1*01, DRB1*04, and DRB1*15 alleles.     

Clozapine Administration Modifies Neurotensin Effect on Synaptosomal Membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript> -ATPase Activity

Na⁺, K⁺-ATPase is inhibited by neurotensin, an effect which involves the peptide high affinity receptor (NTS1). Neurotensin effect on cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase activity of rats injected i.p. with antipsychotic clozapine was studied. Whereas 3.5 × 10⁻⁶ M neurotensin decreased 44% Na⁺, K⁺-ATPase activity in the controls, the peptide failed to modify enzyme activity 30 min after a single 3.0, 10.0 and 30.0 mg/kg clozapine dose. Neurotensin decreased Na⁺, K⁺-ATPase activity 40 or 20% 18 h after 3.0 or 5.6 mg/kg clozapine administration, respectively, and lacked inhibitory effect 18 h after 17.8 and 30.0 mg/kg clozapine doses. Results indicated that the clozapine treatment differentially modifies the further effect of neurotensin on synaptosomal membrane Na⁺, K⁺-ATPase activity according to time and dose conditions employed. Taken into account that clozapine blocks the dopaminergic D2 receptor, findings obtained favor the view of an interplay among neurotensinergic receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase at synaptic membranes.     

Associations between common variants in <Emphasis Type="Italic">GC</Emphasis> and <Emphasis Type="Italic">DHCR7</Emphasis>/<Emphasis Type="Italic">NADSYN1</Emphasis> and vitamin D concentration in Chinese Hans

Recent studies have identified common variants in or near GC, CYP2R1 and NADSYN1/DHCR7 to be associated with 25-hydroxyvitamin D [25(OH)D] levels in European populations. We aimed to examine whether these variants also influence 25(OH)D levels in Chinese. Seven common variants were successfully genotyped and tested for associations with plasma 25(OH)D levels in a population-based cohort of 3,210 Chinese Hans from Beijing and Shanghai. Six common variants at GC (rs4588, rs7041, rs2282679 and rs1155563) and NADSYN1/DHCR7 (rs3829251 and rs1790349) loci were all significantly associated with lower plasma 25(OH)D levels (−0.036 ≤ β ≤ −0.076 per risk-allele, P ≤ 5.7 × 10⁻⁵), while CYP2R1-rs2060793 showed a trend toward association with 25(OH)D levels in the Shanghai subpopulation (P = 0.08), but not in the Beijing subpopulation (P = 0.82). Haplotype-based association analyses of the four GC variants showed that only the haplotype that contained all risk-alleles (TACC) was significantly associated with lower plasma 25(OH)D levels (β = −0.085, P = 2.3 × 10⁻⁹), while the haplotype containing the risk-alleles of rs4588 and rs2282679 (TATC) was marginally associated with lower 25(OH)D levels (β = −0.054, P = 0.0562) when compared with GCTA haplotype carrying the four protective alleles. Most notably, conditional analyses showed that only GC-rs4588 and GC-rs2282679 (r ² = 0.97) remained significantly associated with 25(OH)D concentrations (P ≤ 1.9 × 10⁻⁵) after adjusting for the other two SNPs in GC. In conclusion, GC and NADSYN1/DHCR7 loci individually and collectively contribute to variation in plasma vitamin D levels in Chinese Hans.     

Prevalence,clinical relevance and characterization of circulating cytotoxic CD4<Superscript>+</Superscript>CD28<Superscript>-</Superscript> T cells in ankylosing spondylitis

Circulating CD3⁺CD4⁺CD28^- cells exhibit reduced apoptosis and were found to be more enriched in patients with ankylosing spondylitis than in age-matched healthy control individuals (7.40 ± 6.6% versus 1.03 ± 1.0%; P < 0.001). Levels of CD4⁺CD28^- T cells correlate with disease status as measured using a modified metrology score, but they are independent of age and duration of ankylosing spondylitis. CD4⁺CD28^- T cells produce IFN-γ and perforin, and thus they must be considered proinflammatory and cytotoxic. These T cells share phenotypic and functional properties of natural killer cells, strongly expressing CD57 but lacking the lymphocyte marker CD7. MHC class I recognizing and activating natural killer cell receptors on the surface of CD4⁺CD28^- T cells may be involved in a HLA-B27 mediated co-stimulation of these proinflammatory and cytotoxic cells.