1. Potassium conductances were studied in large layer V neurons using an in vitro slice preparation of cat sensorimotor cortex. The kinetics and pharmacological sensitivity of K+ currents were studied directly using single microelectrode voltage clamp and indirectly by evoking single or multiple spikes and recording the spike repolarization and subsequent afterhyperpolarizations (AHPs). 2. A fast-decaying afterhyperpolarization (fAHP) and a subsequent medium-duration afterhyperpolarization (mAHP) followed a single spike. The amplitude and duration of the mAHP increased when multiple spikes were evoked at a fast rate (e.g., 100 Hz), and a slower afterhyperpolarization (sAHP) appeared only after sustained repetitive firing. 3. All AHPs were reduced by membrane potential hyperpolarization and raised extracellular K+ concentration, suggesting they were caused by an increased K+ conductance. Only the mAHP and sAHP reversed at the estimated value of potassium equilibrium potential (-100 mV), whereas the mean reversal potential of the fAHP was nearly identical to the mean value of resting potential (-71 mV). 4. Mechanisms underlying spike repolarization, the fAHP, and the mAHP were investigated. Two rapidly activating outward currents, a fast-inactivating current and a slowly inactivating delayed rectifier, were detected by voltage clamp. Both currents were reduced rapidly by tetraethylammonium (TEA). The fast transient current was reduced slowly after divalent cations were substituted for Ca2+ (through a mechanism unrelated to blockade of Ca2+ channels), whereas the delayed rectifier was unaffected. 5. Spike duration was increased and the fAHP was abolished only by blocking agents that reduced the fast outward currents. Effects of extracellular and intracellular TEA were similar. Effects of TEA and Ca2+-free perfusate were additive and resembled the effects of intracellular Cs+. The addition of apamin, d-tubocurare, or Cd2+ was ineffective. We conclude that the two fast outward currents reflect pharmacologically and kinetically separate K+ conductances that are primarily responsible for spike repolarization and the fAHP. 6. Voltage-clamp studies revealed two additional outward currents, which were persistent and Ca2+-mediated. Each current activated and deactivated slowly, but the kinetics of one component were approximately 10 times slower than the other. The decay of these currents gave rise to AHPs resembling the mAHP and the early sAHP. 7. Neither the mAHP nor the sAHP was reduced by TEA. The mAHP was reduced when divalent cations were substituted for Ca2+ or when Cd2+, apamin, or d-tubocurare were added.(ABSTRACT TRUNCATED AT 400 WORDS)

CD45, a hematopoietic cell-specific surface antigen, has recently been shown to be a protein tyrosine phosphatase. Expression of CD45 is essential for the T-cell antigen receptor to couple with the phosphatidylinositol second messenger pathway and for antigen-mediated proliferation of T lymphocytes. In this report we describe a CD45-deficient mutant of the human T-cell leukemia line Jurkat. CD45 expression is required for the activation of a T-cell receptor-associated tyrosine kinase as well as the phosphatidylinositol pathway. Additionally, stimulation of T lymphocytes by way of the accessory molecule <em>CD2</em> requires the expression of CD45. The mutation in the CD45-deficient cell specifically impairs signal transduction by the T-cell receptor and <em>CD2</em> because activation events by way of another accessory molecule, <em>CD2</em>8, are unimpaired.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide present in the walls of growing plant cells. We now report that RG-II, released by endopolygalacturonase treatment of the walls of suspension-cultured sycamore cells and etiolated pea stems, exists mainly as a dimer that is cross-linked by a borate ester. The borate ester is completely hydrolyzed at room temperature within 30 min at pH 1, partially hydrolyzed between pH 2 and 4, and stable above pH 4. The dimer is formed in vitro between pH 2.4 and 6. 2 by treating monomeric RG-II (0.5 mM) with boric acid (1.2 mM); the dimer formed after 24 h at pH 3.4 and 5.0 accounts for approximately 30 and approximately 5%, respectively, of the RG-II. In contrast, the dimer accounts for approximately 80 and approximately 54% of the RG-II when the monomer is treated for 24 h at pH 3.4 and 5.0, respectively, with boric acid and 0.5 m Sr2+, Pb2+, or Ba2+. The amount of dimer formed at pH 3.4 or 5.0 is not increased by addition of 0.5 mM Ca2+, Cd2+, Cu2+, Mg2+, Ni2+, and Zn2+. Steric considerations appear to regulate dimer formation since those divalent cations that enhance dimer formation have an ionic radius >1.1 A. Our data suggest that the borate ester is located on C-2 and C-3 of two of the four 3'-linked apiosyl residues of dimeric RG-II. Our results, taken together with the results of two previous studies (Kobayashi, M., Matoh, T., and Azuma, J.-I. (1996) Plant Physiol. 110, 1017-1020; Ishii, T., and Matsunaga, T. (1996) Carbohydr. Res. 284, 1-9) provide substantial evidence that this plant cell wall pectic polysaccharide is covalently cross-linked.

The breathing motor pattern in mammals originates in brainstem networks. Whether pacemaker neurons play an obligatory role remains a key unanswered question. We performed whole-cell recordings in the preBotzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (I(NaP))-mediated bursting in approximately 5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. I(NaP)-mediated bursting was voltage dependent and blocked by 20 mum riluzole (RIL). We found Ca2+ current (I(Ca))-dependent bursting in 7.5% of inspiratory neurons in P8-P10 slices, but in P0-P5 slices these cells were exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 microm Cd2+ or flufenamic acid (FFA) (10-200 microm), which suggests that a Ca2+-activated inward cationic current (I(CAN)) underlies burst generation. These data substantiate our observation that P0-P5 slices exposed to RIL contain few (if any) pacemaker neurons, yet maintain respiratory rhythm. We also show that 20 nm TTX or coapplication of 20 microm RIL + FFA (100-200 microm) stops the respiratory rhythm, but that adding 2 mum substance P restarts it. We conclude that I(NaP) and I(CAN) enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When I(NaP) and I(CAN) are removed pharmacologically, the rhythm can be maintained by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the preBotzinger complex.

Bacterial plasmids encode resistance systems for toxic metal ions, including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO3(2-), Tl+ and Zn2+. The function of most resistance systems is based on the energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The Cd(2+)-resistance ATPase of Gram-positive bacteria (CadA) is membrane cation pump homologous with other bacterial, animal and plant P-type ATPases. CadA has been labeled with 32P from [alpha-32P] ATP and drives ATP-dependent Cd2+ (and Zn2+) uptake by inside-out membrane vesicles (equivalent to efflux from whole cells). Recently, isolated genes defective in the human hereditary diseases of copper metabolism, namely Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to bacterial CadA than to other ATPases from eukaryotes. The arsenic resistance efflux system transports arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. The triple-polypeptide Czc (Cd2+, Zn2+ and Co2+) chemiosmotic efflux pump consists of inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell.

Detection of positive Darwinian selection has become ever more important with the rapid growth of genomic data sets. Recent branch-site models of codon substitution account for variation of selective pressure over branches on the tree and across sites in the sequence and provide a means to detect short episodes of molecular adaptation affecting just a few sites. In likelihood ratio tests based on such models, the branches to be tested for positive selection have to be specified a priori. In the absence of a biological hypothesis to designate so-called foreground branches, one may test many branches, but a correction for multiple testing becomes necessary. In this paper, we employ computer simulation to evaluate the performance of 6 multiple test correction procedures when the branch-site models are used to test every branch on the phylogeny for positive selection. Four of the methods control the familywise error rates (FWERs), whereas the other 2 control the false discovery rate (FDR). We found that all correction procedures achieved acceptable FWER except for extremely divergent sequences and serious model violations, when the test may become unreliable. The power of the test to detect positive selection is influenced by the strength of selection and the sequence divergence, with the highest power observed at intermediate divergences. The 4 correction procedures that control the FWER had similar power. We recommend Rom's procedure for its slightly higher power, but the simple Bonferroni correction is useable as well. The 2 correction procedures that control the FDR had slightly more power and also higher FWER. We demonstrate the multiple test procedures by analyzing gene sequences from the extracellular domain of the cluster of differentiation 2 (CD2) gene from 10 mammalian species. Both our simulation and real data analysis suggest that the multiple test procedures are useful when multiple branches have to be tested on the same data set.

Cadmium is a dangerous metal distributed widely in the environment. Members of our laboratory recently identified the ZIP8 transporter protein, encoded by the mouse Slc39a8 gene, to be responsible for genetic differences in response to cadmium damage of the testis. Stable retroviral infection of the ZIP8 cDNA in mouse fetal fibroblast cultures (rvZIP8 cells) leads to as much as a 10-fold increase in the rate of intracellular cadmium influx and accumulation. In the present study, we showed that cadmium uptake operated maximally at pH 7.5 and a temperature of 37 degrees C and was inhibited by cyanide. Of more than a dozen cations tested, manganese(II) was the best competitive cation for cadmium uptake. The Km for Cd2+ uptake was 0.62 microM, and the Km for Mn2+ uptake was 2.2 microM; thus, manganese is probably the physiological substrate for ZIP8. Cadmium uptake was independent of sodium, potassium or chloride ions, but strongly dependent on the presence of bicarbonate. By Western blot analysis of rvZIP8 cells, we showed that ZIP8 protein was glycosylated. Using Z-stack confocal microscopy in Madin-Darby canine kidney polarized epithelial cells, we found that ZIP8 was localized on the apical side-implying an important role for manganese or cadmium uptake and disposition. It is likely that ZIP8 is a Mn2+/HCO3- symporter, that a HCO3- gradient across the plasma membrane acts as the driving force for manganese uptake, and that cadmium is a rogue hitchhiker displacing manganese to cause cadmium-associated disease.

In response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha, cord blood CD34+ hematopoietic progenitor cells differentiate along two unrelated dendritic cell (DC) pathways: (1) the Langerhans cells (LCs), which are characterized by the expression of CD1a, Birbeck granules, the Lag antigen, and E cadherin; and (2) CD14+ cell-derived DCs, characterized by the expression of CD1a, CD9, CD68, CD2, and factor XIIIa (Caux et al, J Exp Med 184:695, 1996). The present study investigates the functions of each population. Although the two populations are equally potent in stimulating naive CD45RA cord blood T cells through apparently identical mechanisms, each also displays specific activities. In particular CD14-derived DCs show a potent and long-lasting (from day 8 to day 13) antigen uptake activity (fluorescein isothiocyanate dextran or peroxidase) that is about 10-fold higher than that of CD1a+ cells, which is restricted to the immature stage (day 6). The antigen capture is exclusively mediated by receptors for mannose polymers. The high efficiency of antigen capture of CD14-derived cells is coregulated with the expression of nonspecific esterase activity, a tracer of lysosomial compartment. In contrast, the CD1a+ population never expresses nonspecific esterase activity. The most striking difference is the unique capacity of CD14-derived DCs to induce naive B cells to differentiate into IgM-secreting cells, in response to CD40 triggering and interleukin-2. Thus, although the two populations can allow T-cell priming, initiation of humoral responses might be preferentially regulated by the CD14-derived DCs. Altogether, those results show that different pathways of DC development might exist in vivo: (1) the LC type, which might be mainly involved in cellular immune responses, and (2) the CD14-derived DC related to dermal DCs or circulating blood DCs, which could be involved in humoral immune responses.

BACKGROUND

Although glucocorticoids are the most effective treatment for chronic inflammatory diseases, such as asthma, some patients show a poor response. IL-2 combined with IL-4 can alter glucocorticoid receptor (GR) ligand-binding affinity and modulate glucocorticoid function.

OBJECTIVE

We sought to confirm the altered ligand-binding affinity in a distinct group of steroid-dependent asthmatic subjects and examine the mechanism by which IL-2 and IL-4 modify the ligand-binding affinity of the GR.

METHODS

We examined PBMCs from healthy subjects, subjects with mild asthma, and steroid-dependent subjects with severe asthma using dexamethasone-binding assays and Western blot analysis of GR and phosphorylated activated transcription factor 2 expression. GR phosphorylation was measured after orthophosphate labeling and immunoprecipitation and cytokine production by means of ELISA.

RESULTS

GR ligand-binding affinity was reduced in the nucleus but not in the cytoplasm of steroid-dependent asthmatic subjects compared with that seen in healthy subjects (dissociation constant, 39.8 +/- 4.6 vs. 6.79 +/- 0.8 nmol/L). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 cotreatment and was blocked by the p38 mitogen-activated kinase (MAPK) inhibitor SB203580. Activation of p38 MAPK by IL-2 and IL-4, as shown by means of phosphorylation of activated transcription factor 2, resulted in GR phosphorylation and reduced dexamethasone repression of LPS-stimulated GM-CSF release. p38 MAPK phosphorylation of CD2(+) T cells occurred on serine residues. The ability of dexamethasone to modulate IL-10 release was also inhibited by IL-2 and IL-4 cotreatment. These effects were also inhibited by SB203580.

CONCLUSIONS

These data show that p38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and reestablishing the beneficial effects of glucocorticoids in patients with severe asthma.

A workshop jointly sponsored by the University of Hong Kong and the Society for Hematopathology explored the definition, differential diagnosis, and epidemiology of angiocentric lymphomas presenting in the nose and other extranodal sites. The participants concluded that nasal T/natural killer (NK) cell lymphoma is a distinct clinicopathologic entity highly associated with Epstein-Barr virus (EBV). In situ hybridization for EBV an be very valuable in early diagnosis, especially if tissue is sparse. The cytologic spectrum is broad, ranging from small or medium-sized cells to large transformed cells. Histologic progression often occurs with time. Necrosis is nearly always present, and angioinvasion by tumor cells is seen in most cases. Nasal T/NK cell lymphoma has a characteristic immunophenotype: CD2-positive, CD56-positive, but usually negative for surface CD3. Cytoplasmic CD3 can be detected in paraffin sections. Clonal T-cell receptor gene rearrangement is not found. Tumors with an identical phenotype and genotype occur in other extranodal sites, most commonly in the skin, subcutis, and gastrointestinal tract, and should be referred to as nasal-type T/NK cell lymphomas. The differential diagnosis includes lymphomatoid granulomatosis, blastic or monomorphic NK cell lymphoma/leukemia, CD56-positive peripheral T-cell lymphoma, and enteropathy-associated T-cell lymphoma.

Stimulation of antigen receptors of lymphocytes triggers a transitory release of Ca2+ from internal stores and the opening of a transmembrane Ca2+ conductive pathway. The latter underlies the sustained increase of intracellular free calcium concentration, and it seems to be a key event in the Ca(2+)-dependent biochemical cascade leading to T cell proliferation. Alternatively, pharmacological depletion of internal stores by itself activates Ca2+ influx. This has led to the hypothesis that antigen-triggered Ca2+ influx is secondary to Ca2+ release from internal stores. However, the precise relationship between antigen and Ca2+ release-activated Ca2+ currents remains unclear, particularly since neither of them has been electrophysiologically recorded in normal lymphocytes. Using the whole-cell and the perforated configurations of the patch clamp technique on peripheral blood lymphocytes, we found that a low amplitude Ca(2+)-selective current was triggered when intracellular stores were depleted by stimuli such as the intracellular perfusion of inositol triphosphate or thapsigargin and the extracellular perfusion of ionomycin. A similar current was elicited by the cross-linking of the T cell receptor-CD3 complex. This current displayed an inward rectification below 0 mV and was completely blocked by the divalent cation Cd2+. It was very selective for Ca2+ over Na+ and insensitive to changes in chloride concentration. The physiological relevance of this conductance was investigated with the analysis of abnormal Ca2+ signaling in lymphocytes from a patient suffering from a primary immunodeficiency associated with a defective T cell proliferation. Using fura-2 video imaging, an absence of Ca2+ influx was established in the patient's lymphocytes, whereas the Ca2+ release from internal stores was normal. This was the case whether cells were stimulated physiologically through their antigen receptors or with store depleting pharmacological agents. Most importantly, no Ca(2+)-selective current was elicited in these cells. Our data strongly suggest that the Ca2+ release-activated current underlies the sustained Ca2+ influx during antigenic stimulation and that it plays a key role in the immune function.

Antiviral cell-mediated immunity is initiated by the dendritic cell (DC) network in lymph nodes (LNs). Plasmacytoid DCs (pDCs) are known to migrate to inflamed LNs and produce interferon (IFN)-alpha, but their other roles in antiviral T cell immunity are unclear. We report that LN-recruited pDCs are activated to create local immune fields that generate antiviral cytotoxic T lymphocytes (CTLs) in association with LNDCs, in a model of cutaneous herpes simplex virus (HSV) infection. Although pDCs alone failed to induce CTLs, in vivo depletion of pDCs impaired CTL-mediated virus eradication. LNDCs from pDC-depleted mice showed impaired cluster formation with T cells and antigen presentation to prime CTLs. Transferring circulating pDC precursors from wild-type, but not CXCR3-deficient, mice to pDC-depleted mice restored CTL induction by impaired LNDCs. In vitro co-culture experiments revealed that pDCs provided help signals that recovered impaired LNDCs in a CD2- and CD40L-dependent manner. pDC-derived IFN-alpha further stimulated the recovered LNDCs to induce CTLs. Therefore, the help provided by pDCs for LNDCs in primary immune responses seems to be pivotal to optimally inducing anti-HSV CTLs.

Voltage-activated K+ channels are integral membrane proteins containing a potassium-selective transmembrane pore gated by changes in the membrane potential. This activation gating (opening) occurs in milliseconds and involves a gate at the cytoplasmic side of the pore. We found that substituting cysteine at a particular position in the last transmembrane region (S6) of the homotetrameric Shaker K+ channel creates metal binding sites at which Cd2+ ions can bind with high affinity. The bound Cd2+ ions form a bridge between the introduced cysteine in one channel subunit and a native histidine in another subunit, and the bridge traps the gate in the open state. These results suggest that gating involves a rearrangement of the intersubunit contacts at the intracellular end of S6. The recently solved structure of a bacterial K+ channel shows that the S6 homologs cross in a bundle, leaving an aperture at the bundle crossing. In the context of this structure, the metal ions form a bridge between a cysteine above the bundle crossing and a histidine below the bundle crossing in a neighboring subunit. Our results suggest that gating occurs at the bundle crossing, possibly through a change in the conformation of the bundle itself.

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. We demonstrate now that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria.

Regulation of proliferation of normal human T lymphocytes (T cells) by glutathione (GSH) was explored with T-cell activation models that do not require accessory cell signals. L-Buthionine-(S,R)-sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and therefore inhibits GSH synthesis, inhibited proliferation elicited by monoclonal antibodies directed at cluster designation 2 (CD2) and CD3 antigens, or by sn-1,2-dioctanoylglycerol and ionomycin. L-Buthionine-(R)-sulfoximine, which does not inactivate gamma-glutamylcysteine synthetase, did not affect proliferation. BSO-induced inhibition of accessory cell-independent T-cell proliferation was not reversed by recombinant human interleukin 2, despite activation-dependent expression of interleukin 2 receptor alpha by T cells treated with BSO. However, BSO-associated inhibition of T-cell proliferation was reversed by GSH or GSH ester. These studies, which show that GSH can directly modulate proliferation of highly purified T cells, suggest that GSH is essential for steps close to or at DNA synthesis. The availability of methods for decreasing and for increasing GSH levels suggest therapies to produce (i) immunosuppression (of value in organ transplantation), and (ii) immunopotentiation (of potential value in treatment of immunodeficiency states such as AIDS).

Memory T cells promote allograft rejection particularly in co-stimulation blockade-based immunosuppressive regimens. Here we show that the CD2-specific fusion protein alefacept (lymphocyte function-associated antigen-3-Ig; LFA -3-Ig) selectively eliminates memory T cells and, when combined with a co-stimulation blockade-based regimen using cytotoxic T lymphocyte antigen-4 (CTLA-4)-Ig, a CD80- and CD86-specific fusion protein, prevents renal allograft rejection and alloantibody formation in nonhuman primates. These results support the immediate translation of a regimen for the prevention of allograft rejection without the use of calcineurin inhibitors, steroids or pan-T cell depletion.

The chronic fatigue syndrome (CFS), formerly known as chronic Epstein-Barr virus syndrome, is a clinical state of some complexity and uncertain etiology. In order to characterize in a comprehensive manner the status of laboratory markers associated with cellular immune function in patients with this syndrome, 30 patients with clinically defined CFS were studied. All of the subjects were found to have multiple abnormalities in these markers. The most consistent immunological abnormality detected among these patients, when compared with normal controls, was low natural killer (NK) cell cytotoxicity. The number of NK cells, as defined by reactivity with monoclonal antibody NKH.1 (CD56), was elevated, but the killing of K562 tumor cells per CD56 cell was significantly diminished. Lymphoproliferative responses after stimulation with phytohemagglutinin and pokeweed mitogen were decreased in most patients when compared with those in normal controls, as was the production of gamma interferon following mitogen stimulation. Lymphocyte phenotypic marker analysis of peripheral blood lymphocytes showed that there were significant differences between patients with CFS and controls. There was an increase in the percentage of suppressor-cytotoxic T lymphocytes, CD8, and a proportionally larger increase in the number of CD8 cells expressing the class II activation marker. Most patients had an elevated number of <em>CD2</em> cells which expressed the activation marker CDw26. The numbers of CD4 cells and the helper subset of CD4+<em>CD2</em>9+ cells in patients with CFS were not different from those in controls. There was, however, a significant decrease in the suppressor inducer subset of CD4+ CD45RA+ cells. The number of B cells, <em>CD2</em>0 and <em>CD2</em>1, were elevated, as were the numbers of a subset of B cells which coexpressed <em>CD2</em>0 and CD5. The patterns of immune marker abnormalities observed was compatible with a chronic viral reactivation syndrome.

Optimal proliferation of T cells although initiated via ligation of the CD3/TCR complex requires additional stimulation resulting from adhesive interactions between costimulatory receptors (R) on T cells and their counter-R on APC. At least four distinct adhesion molecules (counter-R) present on APC, B7, ICAM-1 (CD54), LFA-3 (CD58), and VCAM-1 have been individually shown to costimulate T cell activation. Because some of these molecules may be expressed simultaneously on APC, it has been difficult to examine relative contributions of individual counter-R during the induction of T cell proliferation. We have produced soluble IgC gamma 1 fusion chimeras (receptor globulins or Rg) of B7, ICAM-1, LFA-3, and VCAM-1 and compared their relative abilities to costimulate proliferation of resting or Ag-primed CD4+ T cells. When co-immobilized with mAb directed at TCR alpha beta or CD3 but not <em>CD2</em> or <em>CD2</em>8, each Rg induced proliferation of both resting and Ag-primed CD4+ cells. In contrast, similarly co-immobilized CD7 Rg or ELAM-1 Rg were ineffective. Resting CD4+ T cells produced more IL-2, expressed significantly higher levels of IL-2R alpha, and proliferated more efficiently when costimulated with either ICAM-1 Rg or VCAM-1 Rg than with B7 Rg or LFA-3 Rg. CD4+ CD45RO+ memory T cells proliferated more vigorously in response to the costimulation by each of the four Rg than CD4+ CD45RA+ naive T cells. In contrast with the behavior of resting CD4+ T cells, proliferation of Ag-preactivated CD4+ T cells was most efficient when costimulated by B7 Rg. The costimulatory effect of LFA-3 Rg on Ag-primed CD4+ T cells was weaker than that of B7 Rg but was significantly greater than that of either ICAM-1 Rg or VCAM-1 Rg. These results suggest that resting and Ag-primed CD4+ T cells preferentially respond by proliferation to different costimulatory counter-R. ICAM-1 and VCAM-1 may be involved in the initiation of proliferation of Ag-responsive T cells, and B7 and LFA-3 may facilitate sustained proliferation of Ag-primed T cells. The cumulative costimulation by the above counter-R may facilitate optimal expression of various regulatory and effector functions of T cells.

A recently proposed model for voltage-dependent activation in K+ channels, largely influenced by the KvAP X-ray structure, suggests that S4 is located at the periphery of the channel and moves through the lipid bilayer upon depolarization. To investigate the physical distance between S4 and the pore domain in functional channels in a native membrane environment, we engineered pairs of cysteines, one each in S4 and the pore of Shaker channels, and identified two instances of spontaneous intersubunit disulfide bond formation, between R362C/A419C and R362C/F416C. After reduction, these cysteine pairs bound Cd2+ with high affinity, verifying that the residues are in atomic proximity. Molecular modeling based on the MthK structure revealed a single position for S4 that was consistent with our results and many other experimental constraints. The model predicts that S4 is located in the groove between pore domains from different subunits, rather than at the periphery of the protein.

We have determined the gene structure for the NMDA receptor subunit gene NMDAR1. We found eight splice variants that arise from different combinations of a single 5' terminal exon insertion and three different 3' terminal exon deletions, relative to NMDAR1. We analyzed the modulation by Zn2+ of currents through homomeric receptors assembled from these splice variants and found that, in addition to its well-known inhibitory effect at high concentrations, Zn2+ potentiates agonist-induced currents at submicromolar concentrations (EC50 = 0.50 microM). This potentiation is observed only with a subset of NMDAR1 splice variants that show additional differences in pharmacological properties. Zn2+ potentiation is rapidly reversible, noncompetitive with either glutamate or glycine, and voltage independent. Zn2+ potentiation is mimicked by Cd2+, Cu2+, and Ni2+, but not by Mn2+, Co2+, Fe3+, Sn2+, or Hg2+. Our results suggest a possible role for Zn2+ as a positive modulator of NMDA receptors in certain regions of the brain.

One of the mechanisms to terminate a specific immune response may involve elimination of antigen activated T cells by programmed cell death, apoptosis. Apoptosis in activated T cells may be induced via the TCR-CD3 complex or/and cell surface molecules like the APO-1 (Fas) antigen, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily. To investigate apoptosis in activated T cells we studied expression of APO-1 and sensitivity to APO-1 mediated apoptosis in human peripheral T lymphocytes. APO-1 is not expressed on cord blood and the majority of resting T cells, but on activated T cells. One day activated T cells in culture showed activation induced resistance to apoptosis (ARA). However, after prolonged in vitro culture, 6 day activated T cells acquired sensitivity to activation induced sensitivity to apoptosis (ASA). Restimulation of the ASA+ activated T cells by triggering TCR-CD3 or CD2 induced proliferation and apoptosis in a fraction of the cells. In the surviving fraction of ASA+ activated T cells, however, this treatment reinduced a transient ARA+ phenotype. Thus, activation of resting mature T cells or restimulation of activated T cells may induce a transient resistance to apoptotic signals. Activation signals may interfere with the APO-1 pathway and may prevent elimination of activated T cells in the periphery (peripheral selection).

In previous studies we identified a surface molecule (termed GL183) capable of mediating cell activation and selectively expressed by a subset of human CD3-CD16+ natural killer (NK) cells. In this study we analyzed whether other subset-specific functional molecules were expressed in GL183- NK cells. To this end, mice were immunized with the PE29 (CD3-CD16+GL183-) NK clone. Monoclonal antibodies (mAbs) were selected by screening the hybridoma supernatants for their ability to trigger the cytolytic activity of clone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgG1) triggered the PE29 clone, but not a GL183+ clone used as a control. EB6+ cells ranged between 1 and 13% of peripheral blood lymphocytes and were largely included in the CD3-CD16+CD56+ cell populations (only less than 2% of EB6+ cells were CD3+). Analysis of resting or activated CD3-CD16+ populations, or clones for the expression of EB6 or GL183 mAbs, allowed us to identify four distinct, phenotypically stable, NK subsets (EB6+GL183-; EB6+GL183+; EB6-GL183+; EB6-GL183-). Similar to GL183 mAb, the EB6 mAb selectively triggered the NK subset expressing the corresponding surface antigen to lyse human tumor cell lines including U937, IGROV-I, M14, and A549. In addition, EB6 mAb sharply inhibited the cytolytic activity of EB6+ clones against P815, M12, and P3U1 murine target cells. In EB6+GL183+ ("double-positive") clones both EB6 and GL183 mAb inhibited the redirected killing of P815 cells induced by anti-CD16, anti-CD2 mAbs and phytohemagglutinin (PHA). Similar to GL183 molecules, molecules precipitated by EB6 mAb were represented by either single 58-kD chain or double chains of 55 and 58 kD (with no detectable differences in EB6+GL183- or EB6+GL183+ clones). In sequential immunoprecipitation experiments using the double-positive clones CEG52 and CA25.50, preclearing of cell lysates with EB6 or GL183 mAb removed only EB6 or GL183 molecules, respectively, thus indicating that the two antigenic determinants are carried by two distinct molecules. Peptide map analysis indicated that EB6 (or GL183) molecules precipitated from double-positive clones were identical to the corresponding molecules isolated from single-positive ones. On the other hand, comparison of the EB6 and GL183 maps revealed peptides that were unique to each molecule, although most of the major peptides migrated to identical positions. We further investigated whether correlation existed between the phenotypic assignment of NK clones and their ability to mediate specific lysis of normal allogeneic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorescein isothiocyanate-conjugated dextran was introduced preferentially into hepatic lysosomes by intraperitoneal injection into rats. The pH in isolated lysosomes, measured by fluorescein fluorescence, was approximately 5 and gradually increased in KCl (to 7.0) at 25 degrees C. In the presence of Mg2+, ATP caused acidification of lysosomes that was reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Mn2+, Co2+, and Fe2+ could replace Mg2+ but Ca2+ could not. Cu2+, Zn2+, and Cd2+ were inhibitory. A membrane-permeant anion, in practice chloride, was required for this acidification. ATP analogues, including 5'-adenylyl imidodiphosphate, could not be substituted for ATP. ATP-driven acidification was sensitive to N-ethylmaleimide and quercetin but insensitive to oligomycin, ouabain, and vanadate. There were some differences between "normal" lysosomes and tritosomes; the acidification was resistant to azide and N,N'-dicyclohexylcarbodiimide in normal lysosomes but sensitive to these reagents in tritosomes. These results provide evidence for the presence of an electrogenic proton pump driven by MgATP (H+-ATPase) on lysosomes.