Role of Nijmegen Breakage Syndrome Protein in Specific T-Lymphocyte Activation Pathways

Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by immunodeficiency, microcephaly, and “bird-like” facies. NBS shares some clinical features with ataxia telangiectasia (AT), including increased sensitivity to ionizing radiation, increased spontaneous and induced chromosome fragility, and strong predisposition to lymphoid cancers. The mutated gene that results in NBS codes for a novel double-stranded DNA break repair protein, named nibrin. In the present work, a Spanish NBS patient was extensively characterized at the immunological and the molecular DNA levels. He showed low CD3⁺-cell numbers and an abnormal low CD4⁺ naive cell/CD4⁺ memory cell ratio, previously described in AT patients and also described in the present report in the NBS patient. The proliferative response of peripheral blood lymphocytes in vitro to mitogens is deficient in NBS patients, but the possible link among NBS mutations and the abnormal immune response is still unknown.     

Low Ca2+-sensitive maxi-K+ channels in human cultured fibroblasts

The patch clamp technique was used to reveal single channel activity in the membrane of human cultured fibroblasts. The most frequently detected ion channel type was a Ca²⁺-dependent K⁺ channel with a conductance of 287±38 pS in symmetrical 130 mM KCl. The channel showed a peculiar low Ca²⁺-sensitivity compared to that of similar channels in other preparations. In fact micromolar values of internal Ca²⁺ were not effective in the channel activation, except at high depolarizing membrane potentials. The activity was highly increased only when the channel was exposed to relatively high internal Ca²⁺ concentrations (0.2–2.0 mM).     

Treponema denticola Outer Membrane Inhibits Calcium Flux in Gingival Fibroblasts

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca²⁺ concentration ([Ca²⁺]_i) was not affected by treatment with OM extract, but normal spontaneous [Ca²⁺]_i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca²⁺ to the extracellular pool following depletion of intracellular Ca²⁺ by thapsigargin and extracellular Ca²⁺ by EGTA yielded 59% less replenishment of [Ca²⁺]_i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca²⁺]_i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca²⁺]_i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca²⁺]_i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60°C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.     

New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia

Despite recent progress in our understanding of renal magnesium (Mg²⁺) handling, the molecular mechanisms accounting for transepithelial Mg²⁺ transport are still poorly understood. Mutations in the TRPM6 gene, encoding the epithelial Mg²⁺ channel TRPM6 (transient receptor potential melastatin 6), have been proven to be the molecular cause of hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014). HSH manifests in the newborn period being characterized by very low serum Mg²⁺ levels (<0.4 mmol/l) accompanied by low serum calcium (Ca²⁺) concentrations. A proportion of previously described TRPM6 mutations lead to a truncated TRPM6 protein resulting in a complete loss-of-function of the ion channel. In addition, five-point mutations have been previously described. The aim of this study was to complement the current clinical picture by adding the molecular data from five new missense mutations found in five patients with HSH. To this end, patch-clamp analysis and cell surface measurements were performed to assess the effect of the various mutations on TRPM6 channel function. All mutant channels, expressed in HEK293 cells, showed loss-of-function, whereas no severe trafficking impairment to the plasma membrane surface was observed. We conclude that the new TRPM6 missense mutations lead to dysregulated intestinal/renal Mg²⁺ (re)absorption as a consequence of loss of TRPM6 channel function.     

Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein

Dispersed single smooth muscle cells of rabbit portal vein were prepared by treatment with collagenase and trypsin. The muscle cells were 100–300 m in length, 5–10 m in maximum width and cylindrical in shape. In insideout membrane patches, two different amplitudes of ionic currents were recorded, and these single channel conductances were 273 pS (K_l-channel) and 92 pS (K_s-channel), when both sides of the membrane were exposed to 142 mM K⁺ solution. The channel conductances depended on concentrations of K⁺ on both sides of the membrane. When K⁺ were replaced with Na⁺ or Tris⁺, these single-channel currents were abolished. When the concentration of Ca²⁺ inside the membrane was greater than 10^–7 M, the channel activity was enhanced but there was enhancement when Ca²⁺ was applied to the extracellular membrane surface, in concentrations ranging between 10^–9 and 10^–3 M. During application of tetraethylammonium (TEA⁺; 1–10 mM) to the intracellular membrane surface, amplitudes of the single-channel current of both types of the K-channel were not modified. By contrast application of TEA⁺ (0.1–1 mM) to the extracellular membrane surface, reduced the amplitudes of the current and increased noise levels during the open-state of the K_l-channels, but did not have such an effect on the K_s-channel. We conclude that there are at least two different Ca-dependent K-channels distributed on the smooth muscle membrane of the rabbit portal vein. TEA⁺ applied to the extracellular membrane surface blocks activation of the K_l-channel, but not that of the K_s-channel. These two Ca-dependent K-channels do not seem to be important for maintenance of the resting membrane potential, but do play an important role in the repolarizing stage of the Ca spikes, in the rabbit portal vein.     

Effect of Malnutrition on K+ Current in T Lymphocytes

Severe malnutrition in children is frequently associated with infectious diseases. Animal models have been useful for studying the effects of malnutrition. One of the immunosuppressive mechanisms of malnutrition is inhibition of the activation of T lymphocytes. The voltage-dependent K(V) potassium channels are vital for the activation of T lymphocytes. The blockade of K(V) channels inhibits the activation of T lymphocytes. Malnutrition could affect the suitable synthesis of K(V) channels in T lymphocytes, producing changes in the magnitude and/or dependency of the voltage of the K⁺ current. We reported a significant decrease in the K⁺ current and activation to a 20 mV more positive membrane potential in T lymphocytes of rats with severe malnutrition. These results indicate that the diminution in the K⁺ conductance by alteration of K(V) channels in severe malnutrition is one of the mechanisms that inhibit the activation of T lymphocytes.     

Enhanced Na+/H+ exchange activity contributes to the pathogenesis of muscular dystrophy via involvement of P2 receptors

A subset of muscular dystrophy is caused by genetic defects in dystrophin-associated glycoprotein complex. Using two animal models (BIO14.6 hamsters and mdx mice), we found that Na⁺/H⁺ exchanger (NHE) inhibitors prevented muscle degeneration. NHE activity was constitutively enhanced in BIO myotubes, as evidenced by the elevated intracellular pH and enhanced ²²Na⁺ influx, with activation of putative upstream kinases ERK42/44. NHE inhibitor significantly reduced the increases in baseline intracellular Ca²⁺ as well as Na⁺ concentration and stretch-induced damage, suggesting that Na⁺_i-dependent Ca²⁺overload via the Na⁺/Ca²⁺ exchanger may cause muscle damage. Furthermore, ATP was found to be released continuously from BIO myotubes in a manner further stimulated by stretching and that the P2 receptor antagonists reduce the enhanced NHE activity and dystrophic muscle damage. These observations suggest that autocrine ATP release may be primarily involved in genesis of abnormal ionic homeostasis in dystrophic muscles and that Na⁺-dependent ion exchangers play a critical pathological role in muscular dystrophy.     

Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G₁ cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3⁺-T-cell apoptosis followed by similar levels of both CD4⁺- and CD8⁺-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3⁺ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4⁺ and CD8⁺ cells.     

Clinical and immunopathologic alterations in rhesus macaques affected with globoid cell leukodystrophy

Globoid cell leukodystrophy, or Krabbe’s disease, is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical, and immunohistological features observed in rhesus macaques affected with Krabbe’s disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits, and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68⁺, HAM56⁺, LN5⁺, CD163⁺, IBA-1⁺, and Glut-5⁺, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-α, and IL-1β were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP⁺ cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of certain cytokines may contribute to the pathogenesis of Krabbe’s disease.     

Potassium single-channel properties in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

Ion channels in normal and Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts (CEFs) were examined by using the patch-clamp technique. Three different types of ion channels were observed with single-channel conductances in symmetrical 140 mM KCl (with frequencies of occurrence in parentheses) of 186 pS (70%), 110 pS (10%), and 65 pS (20%), which are identical in normal and RSV-transformed CEFs. The total channel density in both cell types is about 0.13 per m⁺. All three types of channels are highly selective for K⁺ ions, they are Ca²⁺- and voltage-dependent, and they can be completely blocked by external tetraethylammonium (10 mM) in both normal and RSV-transformed cells. Some channel properties, however, are different in normal and RSV-transformed CEFs. The K₁₈₆ channel of normal CEFs is almost completely activated in the presence of about 1 nM free internal Ca²⁺ and is insensitive to charybdotoxin (100 nM). In contrast, the K₁₈₆ channel of RSV-transformed CEFs has an EC₅₀ value for activation by internal Ca²⁺ of about 100 nM and is highly sensitive to charybdotoxin (IC₅₀=9 nM). In normal CEFs, the K₁₈₆ channel activity starts at membrane potentials more positive than –50 mV and reaches a high open state probability of 0.94 at +50 mV. In RSV-transformed CEFs, the threshold of K₁₈₆ channel activity is also –50 mV but the maximal open state probability is only 0.70 at +50 mV membrane potential. Averages of current traces of K₁₈₆ channels show the typical features of the macroscopic K⁺ currents described previously for normal and RSV-transformed CEFs. However, one of the properties of the whole-cell current of normal CEFs, i.e. the inactivation during prolonged membrane depolarization, could not be observed at the single-channel level. Nevertheless, we suggest that the K₁₈₆ channel is the main contributor to the macroscopic K⁺ currents both in normal and RSV-transformed CEFs.     

The Potassium Transporter Trk and External Potassium Modulate Salmonella enterica Protein Secretion and Virulence

Potassium (K⁺) is the most abundant intracellular cation and is essential for many physiological functions of all living organisms; however, its role in the pathogenesis of human pathogens is not well understood. In this study, we characterized the functions of the bacterial Trk K⁺ transport system and external K⁺ in the pathogenesis of Salmonella enterica, a major food-borne bacterial pathogen. Here we report that Trk is important for Salmonella to invade and grow inside epithelial cells. It is also necessary for the full virulence of Salmonella in an animal infection model. Analysis of proteins of Salmonella indicated that Trk is involved in the expression and secretion of effector proteins of the type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) that were previously shown to be necessary for Salmonella invasion. In addition to the role of the Trk transporter in the pathogenesis of Salmonella, we discovered that external K⁺ modulates the pathogenic properties of Salmonella by increasing the expression and secretion of effector proteins of the SPI1-encoded TTSS and by enhancing epithelial cell invasion. Our studies demonstrated that K⁺ is actively involved in the pathogenesis of Salmonella and indicated that Salmonella may take advantage of the high K⁺ content inside host cells and in the intestinal fluid during diarrhea to become more virulent.     

Dendritic cell-based immunotherapy for colon cancer using an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope from tumor-associated antigen 90K

Tumor-associated antigen 90K is implicated in cell–cell and cell-extracellular matrix adhesion through its interaction with galectin-3 and integrin-β1 and is highly expressed in malignant tissues, making it a novel target for the development of new immunotherapies. We investigated a potential immunotherapy treatment for colon cancer using 90K-specific cytotoxic T lymphocytes induced by autologous dendritic cells and pulsed with 90K peptides. We selected three peptides (90K₃₅₁, 90K₅ and 90K₅₂₃) that bind to HLA-A*0201 molecules on the basis of their binding affinity, as determined by a peptide-T2 binding assay. Dendritic cells pulsed with 90K peptides resulted in the efficient generation of mature dendritic cells and exhibited enhanced T-cell stimulation and polarization of naive T cells toward Th1. Dendritic cells pulsed with 90K peptides generated potent cytotoxic T-lymphocytes that lysed T2 cells loaded with each 90K peptide, and 90K⁺/HLA-A2⁺ colon cancer cell lines, including HCT116 and SW480, in a dose-dependent and HLA-A*0201-restricted manner. No killing was observed in 90K⁺/HLA-A2⁻ DLD1 or 90K⁻/HLA-A2⁻ K562 cells. Therefore, we believe that cytotoxic T-lymphocytes stimulated by 90K peptide-pulsed dendritic cells naturally recognize the 90K peptide presented by colon cancer cells in the context of HLA-A2, and kill 90K-positive tumor cells. Dendritic cells pulsed with 90K peptides led to the induction of granzyme B and perforin positive CD8⁺ T cells against HCT116 and SW480 cells, but not DLD1 cells. In conclusion, 90K-specific cytotoxic T lymphocytes, generated by stimulating T cells with 90K peptide-pulsed dendritic cells, could be useful effector cells for the immunotherapy treatment of colon cancer.     

Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca²⁺ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca²⁺ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca²⁺ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca²⁺ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca²⁺ in a concentration-dependent fashion by influx of extracellular Ca²⁺ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca²⁺ even in the absence of extracellular Ca²⁺. Leukotoxin-induced intracellular Ca²⁺ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A₂ and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca²⁺ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca²⁺ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A₂ and C and release of arachidonic acid in bovine alveolar macrophages. Ca²⁺ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca²⁺ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.     

Paradox response of frog muscle membrane to changes in external potassium

Applying conventional microelectrode technique the anomalous behaviour of membrane potential in response to changes in [K⁺]_o was demonstrated in normal and cevadine-treated muscles bathed in Cl^–-free medium. Partial repolarization of the cevadine-depolarized membrane and reappearance of the slow membrane potential oscillation (SMPO) were induced by elevating [K⁺]_o from 2.5 mM to 10–20 mM. Both effects were reversed by return to 2.5 mM [K⁺]_o. The K-induced repolarization was markedly reduced by 20 mM Cs⁺, but not by 0.1 mM ouabain, 1 mM 4-aminopyridine, or 1 mM diethyl-pyrocarbonate. The elevation of [K⁺]_o failed to repolarize muscle fibers that had been depolarized only to a small extent. No K-induced repolarization has been observed in Cl^–-containing fluid. In cevadine-free experiments the omission of potassium from the extracellular space in Cl^–-free solution hyperpolarized some of the fibers, while depolarized others. Strong electrical stimuli applied in zero K-zero Cl solution turned all the fibers into depolarized state; on returning to 2.5 mM [K⁺]_o complete repolarization was achieved in most of the fibers. It has been concluded that the paradox response of the muscle membrane to changes in [K⁺]_o can be attributed to the K-dependent conductance changes of the inward rectifier K channel providing an explanation for the plateau-formation of SMPO and for the existence of two stable levels of membrane potential of the skeletal muscle bathed in Cl^–-free medium.     

Bretylium causes a K-Na pump activation that is independent of exchange in depolarized rat, mouse and human lymphocytes

We have studied a bretylium tosylate induced increase of the membrane potentials of partially depolarized rat, mouse and human lymphocytes, using the potential sensitive dye, bis [1,3, dibutylbarbituric acid-(5) trimethine oxonol]. The extent of this repolarization is dose-dependent and decreased in magnitude as the temp was reduced from 37°C to room temp. The repolarizing effect is inhibited by K⁺-Na⁺-pump blockers or lack of extracellular Na⁺.Sodium ion channel blockers are effective in abolishing repolarization only if applied prior to, or simultaneously with, bretylium. Activation of exchange is not involved in the mechanism of the phenomenon as the latter is completely eliminated in the presence of 10μM amiloride (concn of the diuretics having no measurable inhibition on the action of the exchanger). These data suggest that bretylium opens ligand- and voltage-gated Na⁺ channels, and repolarization occurs due to higher activity of the K⁺-Na⁺-pump stimulated by the enhanced intracellular Na⁺ accumulation.     

Large conductance calcium-activated potassium channels in cultured retinal pericytes under normal and high-glucose conditions

Pericytes are considered to contribute to the regulation of retinal microcirculation which is impaired in diabetic retinopathy. Single, large-conductance, Ca²⁺-dependent K⁺ channels (BK) were studied in cultured bovine retinal capillary pericytes using the patch-clamp method. In excised patches with symmetrical 135-mmol/l K⁺ solutions a single channel conductance of 238±9.9 pS was measured. With a K⁺ gradient of 4/ 135 mmol/l (extracellular/intracellular) the slope conductance averaged 148±2.9 pS at 0 mV. The mean permeability was 4.2×10^–13 cm³/s. The channel was highly selective for K⁺ with a permeability ratio for K⁺ over Na⁺ of 1/0.02. The mean open time and the open probability (P_o) of the BK channel increased with depolarization and with increasing internal [Ca²⁺] showing a maximal sensitivity to Ca²⁺ between 10^–4 and 10^–5 mol/l Ca²⁺. Ba²⁺ (5 mmol/l), quinine (5 mmol/l), and verapamil (Michaelis constant 1.5×10^–5 mol/l) blocked from the intracellular side. Tetraethylammonium induced a dose-dependent block from the outside only with a halfmaximal blocking concentration of 2.5×10^–4 mol/l. Charybdotoxin (10^–8 mol/l) blocked completely from the extracellular side. The channel activity was not changed by either internal adenosine triphosphate (ATP, 10^–4 mol/l) or the putative opener of ATP-sensitive K⁺ channels Hoe 234 (10^–6 mol/l). In cell-attached patches channelP _o was less than 3%. After a 3-day incubation in culture medium containing an elevated glucose concentration (22.5 mmol/l) the channel activity in attached patches was markedly increased. These data indicate that cultured retinal pericytes possess a BK channel. The activity of the channel increases after incubation with elevated glucose concentrations, which could indicate altered regulation of the channel under these conditions. The implications of altered function of BK channels are discussed with respect to haemodynamic changes observed in diabetic retinopathy.     

Regeneration and Tolerance Factor: a Correlate of Human Immunodeficiency Virus-Associated T-Cell Activation

Human immunodeficiency virus (HIV) infection causes extensive phenotypic alterations in lymphocytes. Cellular markers that are normally absent or expressed at low levels on quiescent cells are upregulated throughout the disease course. The transmembrane form of regeneration and tolerance factor (RTF) is expressed at negligible levels on resting T cells but is quickly upregulated following in vitro stimulation and activation. Recently, we reported that expression of RTF was significantly higher in cells from HIV-seropositive (HIV⁺) individuals than in cells from HIV-seronegative (HIV⁻) individuals. Because T cells from HIV⁺ individuals express markers reflecting chronic activation, we hypothesized that these in vivo-activated cells would coexpress RTF. Flow cytometry was used to assess RTF expression on activated (CD38⁺ and HLA-DR⁺) CD4⁺ and CD8⁺ T cells. HIV⁺ individuals had higher percentages of RTF⁺ CD38⁺ (P < 0.0001) or RTF⁺ HLA-DR⁺ (P = 0.0001) CD4⁺ T cells than HIV⁻ individuals. In HIV⁺ individuals, increased percentages of CD4⁺ T cells that were RTF⁺, RTF⁺ CD38⁺, and RTF⁺ HLA-DR⁺ correlated inversely with the absolute number and percentage of CD4⁺ T cells and correlated positively with plasma β₂-microglobulin concentrations. HIV⁺ individuals had higher percentages of CD8⁺ T cells that were RTF⁺ CD38⁺ (P = 0.0001) or RTF⁺ HLA-DR⁺ (P = 0.0010). In HIV⁺ individuals, increased percentages of CD8⁺ T cells that were RTF⁺ HLA-DR⁺ correlated inversely with the percentage of CD4⁺ T cells, and high percentages of CD8⁺ T cells that were RTF⁺ CD38⁺ correlated positively with plasma β₂-microglobulin levels. These findings strongly suggest that increased RTF expression is a correlate of HIV-associated immune system activation.     

CD4+VEGFR1HIGH T cell as a novel Treg subset regulates inflammatory bowel disease in lymphopenic mice

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4⁺ Tregs have been identified, including Foxp3⁺, Tr1, Th3, and Foxp3neg iT(R)35 cells. In this study, we identified a new subset of CD4⁺VEGFR1^high Tregs that have immunosuppressive capacity. CD4⁺VEGFR1high T cells, which constitute approximately 1.0% of CD4⁺ T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4⁺VEGFR1^high T cells are distinct from known Tregs. CD4⁺VEGFR1^high T cells suppressed the proliferation of CD4⁺CD25⁻ T cell as efficiently as CD4⁺CD25^high natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4⁺VEGFR1⁺ T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4⁺ VEGFR1^high T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.     

Expression of Regeneration and Tolerance Factor Correlates Directly with Human Immunodeficiency Virus Infection and Inversely with Hepatitis C Virus Infection

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3⁺ T cells of HIV-seropositive (HIV⁺) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV⁺ HCV-seropositive (HCV⁺), HIV- and HCV-seropositive (HIV⁺ HCV⁺), and HIV- and HCV-seronegative (HIV⁻ HCV⁻) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV⁺ individuals is upregulated and that its expression on T cells from HCV⁺ individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3⁺ T cells was seen in both HIV⁺ and HIV⁺ HCV⁺ individuals compared to HIV⁻ HCV⁻ individuals. HCV⁺ individuals had lower levels of RTF expression than HIV⁻ HCV⁻ individuals (P < 0.005 for CD4⁺; P < 0.0005 for CD8⁺). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV⁺ > HIV⁺ HCV⁺ > HIV⁻ HCV⁻ > HCV⁺. The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.     

Immunocytochemical Studies of Aquaporin 4, Kir4.1, and α1-syntrophin in the Astrocyte Endfeet of Mouse Brain Capillaries

One of the most important physiological roles of brain astrocytes is the maintenance of extracellular K⁺ concentration by adjusting the K⁺ influx and K⁺ efflux. The inwardly rectifying K⁺ channel Kir4.1 has been identified as an important member of K⁺ channels and is highly concentrated in glial endfeet membranes. Aquaporin (AQP) 4 is another abundantly expressed molecule in astrocyte endfeet membranes. We examined the ultrastructural localization of Kir4.1, AQP4, α1-syntrophin, and β-spectrin molecules to understand the functional role(s) of Kir4.1 and AQP4. Immunogold electron microscopy of these molecules showed that the signals of these molecules were present along the plasma membranes of astrocyte endfeet. Double immunogold electron microscopy showed frequent co-localization in the combination of molecules of Kir4.1 and AQP4, Kir4.1 and α1-syntrophin, and AQP4 and α1-syntrophin, but not those of AQP4 and β-spectrin. Our results support biochemical evidence that both Kir4.1 and AQP4 are associated with α1-syntrophin by way of postsynaptic density-95, Drosophila disc large protein, and the Zona occludens protein I protein-interaction domain. Co-localization of AQP4 and Kir4.1 may indicate that water flux mediated by AQP4 is associated with K⁺ siphoning.