The electrophysiological properties of glial cells were examined in primary culture in the presence of tetraethylammonium and Ba2+, a treatment that reduces K+ permeability of the membrane and enhances currents through voltage-dependent Ca2+ channels. Under these conditions, glial cells showed both spontaneous action potentials and action potentials evoked by the injections of current. These responses appear to represent entry of Ba2+ through Ca2+ channels because they were resistant to tetrodotoxin but were blocked by Mn2+ or Cd2+.

The immune system has a variety of regulatory/suppressive processes, which are decisive for the development of a healthy or an allergic immune response to allergens. NK1 and NK2 subsets have been demonstrated to display counterregulatory and provocative roles in immune responses, similar to Th1 and Th2 cells. T regulatory cells suppressing both Th1 and Th2 responses have been the focus of intensive research during the last decade. In this study, we aimed to investigate regulatory NK cells in humans, by characterization of NK cell subsets according to their IL-10 secretion property. Freshly purified IL-10-secreting NK cells expressed up to 40-fold increase in IL-10, but not in the FoxP3 and TGF-beta mRNAs. PHA and IL-2 stimulation as well as vitamin D3/dexamethasone and anti-CD2/CD16 mAbs are demonstrated to induce IL-10 expression in NK cells. The effect of IL-10+ NK cells on Ag-specific T cell proliferation has been examined in bee venom major allergen, phospholipase A2- and purified protein derivative of Mycobecterium bovis-induced T cell proliferation. IL-10+ NK cells significantly suppressed both allergen/Ag-induced T cell proliferation and secretion of IL-13 and IFN-gamma, particularly due to secreted IL-10 as demonstrated by blocking of the IL-10 receptor. These results demonstrate that a distinct small fraction of NK cells display regulatory functions in humans.

Efficient expression of genes transferred by retroviral vectors is a prerequisite for gene therapy, especially when the biological effect depends on the amount of transgene product. High-level gene expression is desirable for several gene therapy approaches involving T lymphocytes. We evaluated standard retroviral vectors with cis-regulatory control elements of the Moloney murine leukemia virus (Mo-MLV) with or without the human T cell-specific CD2 enhancer. For comparison, vectors containing the long terminal repeat (LTR) of myeloproliferative sarcoma virus (MPSV) and an improved 5' untranslated region were used (MP71 vectors), with or without the woodchuck hepatitis virus posttranscriptional regulatory element (PRE). All vectors expressed the enhanced green fluorescent protein (GFP) to measure transgene expression. In mouse T cells MP71 vectors with and without the PRE yielded an up to 10-fold higher expression level compared with the Mo-MLV-based vectors currently used for gene transfer into T lymphocytes. A high multiplicity of infection (MOI) of standard Mo-MLV vectors could not reach expression levels obtained with a low MOI of MP71 vector. Ex vivo-transduced mouse T lymphocytes maintained the vector-dependent differences in level of transgene expression in Rag-1-deficient mice when adoptively transferred. In four human T cell lines and human primary T lymphocytes MP71 vectors yielded an up to 75-fold higher GFP expression level in comparison with the standard Mo-MLV vector. In contrast to mouse T cells, the integration of the PRE into MP71 vectors induced in human T cells a further significant increase in transgene expression level. Southern blot analysis of CEM T cells revealed that the superior performance of MP71 vectors was not due to a higher rate of viral integration. In summary, MP71 vectors are useful tools for stable, high-level gene expression in T lymphocytes, for example, in the expression of T cell receptor genes.

Tetracycline uptake into inverted membrane vesicles from Tn10-bearing Escherichia coli cells required divalent cations. The degree of the stimulation of tetracycline uptake by various divalent cations showed the following decreasing order: Co2+ greater than Mn2+ greater than Mg2+ greater than Cd2+ greater than Ca2+. This order is consistent with the increasing order of the dissociation constants for metal chelate complexes of tetracycline. The Hill constants for the tetracycline uptake rate with various divalent cation concentrations were one. These observations strongly suggested that a 1:1 complex of tetracycline and a divalent cation was transported by a tetracycline resistance protein. This notion was confirmed by our observations that 60Co2+ was actively taken up with tetracycline by the membrane vesicles prepared from resistant cells. In the absence of tetracycline, no uptake of 60Co2+ was observed. It is clear that the 60Co2+ uptake was mediated by the tetracycline resistance protein, because the membrane vesicles from tetracycline-sensitive cells did not show the uptake of 60Co2+ and tetracycline. The 60Co2+ uptake was inhibited in the presence of other divalent cations, without any significant effect on tetracycline uptake, indicating that these cations are also transported with tetracycline by the tetracycline resistance protein.

1. The bursting pacemaker neurone R-15 of Aplysia was injected with the Ca2+ sensitive dye arsenzo III. Changes in absorbance were measured with a differential spectrophotometer to monitor changes in free intracellular Ca2+ during membrane depolarization under voltage clamp conditions. 2. Dye absorbance increased linearly for depolarizing pulse durations up to 100 msec and approximately linearly between 100 and 300 msec, but for longer durations the absorbance change decreased. 3. The absorbance change vs. voltage relation increased steeply between -20 and 0 mV (e-fold per 8.5 mV), peaked at +36 mV and declined non-linearly to an estimated null or suppression potential of about +139 mV. 4. TTX (5 x 10(-5 M) had no effect on the change in dye absorbance produced by brief or long duration stimuli whereas Ca2+ free ASW abolished all changes in dye absorbance. 5. The absorbance change saturated with increasing external Ca2+ concentrations. The relation between dye absorbance and external Ca2+ concentration was hyperbolic and for a small range of external Ca2+ concentration and membrane potentials could be fitted by a Michaelis--Menten expression where the dissociation constant and the maximum absorbance change are voltage dependent. 6. The absorbance change was reduced by external divalent ions which block the Ca2+ channel (e.g. Cd2+ and Ni2+). The suppression of dye absorbance was increased by membrane depolarization and suggests that there is a voltage dependent site within the Ca2+ channel which binds divalent ions. 7. The decline of the absorbance--voltage relation from its peak to the suppression potential showed a greater nonlinearity when longer duration voltage clamp pulses were used. The non-linearity can be explained if the accumulation of Ca2+ ions next to the inner surface of the membrane during depolarization reduces the driving force on Ca2+ ions and thus decreases Ca2+ ion influx. 8. The suppression potential estimated from the absorbance--voltage relation increased 29 mV per tenfold change in the external Ca2+ concentration and thus can be used to estimate the Ca2+ equilibrium potential. 9. The change in dye absorbance produced by brief depolarizing voltage clamp steps was inactivated at positive holding potentials (50% inactivation at about -14 mV). Our results suggest that the slow decrease in dye absorbance during prolonged depolarization is caused by inactivation of the Ca2+ channel.

Sciatic nerve section (axotomy) increases the excitability of rat dorsal root ganglion (DRG) neurons. The changes in Ca2+ currents, K+ currents, Ca2+ sensitive K+ current, and hyperpolarization-activated cation current (I(H)) that may be associated with this effect were examined by whole cell recording. Axotomy affected the same conductances in all types of DRG neuron. In general, the largest changes were seen in "small" cells and the smallest changes were seen in "large" cells. High-voltage-activated Ca2+ channel current (HVA-I(Ba)) was reduced by axotomy. Although currents recorded in axotomized neurons exhibited increased inactivation, this did not account for all of the reduction in HVA-I(Ba). Activation kinetics were unchanged, and experiments with nifedipine and/or omega-conotoxin GVIA showed that there was no change in the percentage contribution of L-type, N-type, or "other" HVA-I(Ba) to the total current after axotomy. T-type (low-voltage-activated) I(Ba) was not affected by axotomy. Ca2+ sensitive K+ conductance (g(K,Ca)) appeared to be reduced, but when voltage protocols were adjusted to elicit similar amounts of Ca2+ influx into control and axotomized cells, I(K,Ca)(s) were unchanged. After axotomy, Cd2+ insensitive, steady-state K+ channel current, which primarily comprised delayed rectifier K+ current (I(K)), was reduced by about 60% in small, medium, and large cells. These data suggest that axotomy-induced increases in excitability are associated with decreases in I(K) and/or decreases in g(K,Ca) that are secondary to decreased Ca2+ influx. Because I(H) was reduced by axotomy, changes in this current do not contribute to increased excitability. The amplitude and inactivation of I(Ba) in all cell types was changed more profoundly in animals that exhibited self-mutilatory behavior (autotomy). The onset of this behavior corresponded with significant reduction in I(Ba) of large neurons. This finding supports the hypothesis that autotomy, that may be related to human neuropathic pain, is associated with changes in the properties of large myelinated sensory neurons.

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.

BACKGROUND

The use of molecular biological techniques has demonstrated the importance of enteroviral infection of the myocardium in the pathogenesis of myocarditis and dilated cardiomyopathy in adults and adenovirus and enterovirus infection in children. The aim of this study was to determine the frequency of adenoviral infection of the myocardium of adults with impaired left ventricular function of unknown origin.

RESULTS

Nested polymerase chain reaction (nPCR) was used to determine the frequency of detection of adenoviral DNA and enteroviral RNA in myocardial tissue samples from 94 adult patients with idiopathic left ventricular dysfunction and 14 control patients. Histological and immunohistological analyses were performed to detect myocardial inflammation. Adenoviral genomic DNA was detected by nPCR in 12 of the 94 patients with left ventricular dysfunction (in each case, adenovirus type 2), whereas enteroviral RNA was detected in another 12 patients. All control samples were negative for both viruses. In all patients, active myocarditis was excluded according to the Dallas criteria. However, there was significantly decreased CD2, CD3, and CD45RO T lymphocyte counts in the adenovirus-positive group compared with the adenovirus-negative group (P<0.05), whereas no differences were associated with enterovirus infection.

CONCLUSIONS

Although enteroviruses are an important causative agent in the pathogenesis of myocarditis and dilated cardiomyopathy, this study shows that adenovirus infection is also important in the pathogenesis of left ventricular failure in adults. However, the pathogenetic basis of disease associated with adenovirus infection may be different than that after infection with other agents, particularly with respect to activation of the host immune response.

We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan's disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and non-Hodgkin's lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families.

Antibody-dependent cellular cytotoxicity (ADCC) is a potentially effective adaptive immune response to human immunodeficiency virus (HIV) infection. The study of ADCC responses has been hampered by the lack of simple methods to quantify these responses and map effective epitopes. We serendipitously observed that standard intracellular cytokine assays on fresh whole blood from a cohort of 26 HIV-infected subjects identified non-T lymphocytes expressing gamma interferon (IFN-gamma) in response to overlapping linear peptides spanning HIV-1 proteins. The effector cells were CD3(-) CD4(-) CD8(-) CD14(-) CD2(+) CD56(+/-) NK lymphocytes and degranulated granzyme B and perforin in response to antigen stimulation. Serum transfer assays demonstrated that the specific response was mediated by immunoglobulin G. Fresh blood samples from half of the HIV-infected cohort demonstrated robust HIV peptide-specific IFN-gamma expression by NK cells, predominately to Env, Pol, and Vpu HIV-1 proteins. Responses were readily mapped to define minimal epitopes utilizing this assay. Antibody-dependent, HIV-specific NK cell recognition, involving components of both innate and adaptive immune systems, represents a potentially effective immune response to induce by vaccination.

Voltage-clamp experiments were carried out with the objective of identifying and characterizing the time- and voltage-dependent properties of a transient outward current recorded in single myocytes from the crista terminalis region of the rabbit heart. A collagenase enzymic dispersion procedure similar to that described by Desilets & Horackova (1982) was used to obtain these viable individual myocytes. Transmembrane ionic currents were recorded using a single micro-electrode voltage-clamp technique. In experiments aimed at studying a tetrodotoxin-resistant transient inward current, (ICa); a transient outward current was consistently recorded following blockade of ICa with Cd2+ (5 X 10(-4) M). The time and voltage dependence of the activation and inactivation of this current were measured. Its steady-state inactivation curve spans the voltage range -70 to -10 mV, and it is activated between -20 and +10 mV. The reversal potential of this transient outward current is approximately -75 mV in [K+]O 5 mM, suggesting that it is carried mainly by K+. This transient outward current can be inhibited completely by external application of 4-aminopyridine (4-AP, 3 mM). The time- and voltage-dependent properties, the reversal potential, and the sensitivity to 4-AP of this transient outward current are all very similar to those of a transient outward current first identified in molluscan neurones. Hence, we have labelled it, IA. Selective inhibition of IA and knowledge of its voltage- and time-dependent properties yield specific predictions concerning its role in the action potential of isolated crista terminalis cells. Consistent with these predictions, a decrease in stimulus rate is found to decrease the duration of the action potential and vice versa; and application of effective doses of 4-AP results in a substantial lengthening of the action potential. These results are discussed in terms of the possible physiological role of IA in subsidiary or follower pace-maker tissue, and the anatomical and physiological heterogeneity of the sino-atrial node region of the rabbit heart.

The capacity of the monoclonal antibodies (Mab) 64.1 and OKT3 directed at CD3 molecules to induce T4 cell proliferation and interleukin 2 (IL 2) production was examined. Each was tested in soluble form or was immobilized by adhering it to the wells of plastic microtiter wells. Soluble anti-CD3 did not induce proliferation of accessory cell (AC)-depleted T4 cells. In contrast, immobilized anti-CD3 induced T4 cell IL 2 production and proliferation in the complete absence of AC. When T4 cells were stimulated with high density immobilized anti-CD3, responses did not require AC, IL 2, or Mab directed at the Tp44 molecule (9.3). In contrast, responses stimulated by lower densities of immobilized anti-CD3 were enhanced by IL 2, AC, and 9.3, and with even lower densities of immobilized anti-CD3 proliferation, required these additional signals. A variety of other immobilized Mab directed at T cell surface proteins including class I major histocompatibility complex encoded gene products, CD2, CD5, 4F2, and Tp44, did not induce proliferation even in the presence of IL 2. Anti-CD4 Mab (66.1) inhibited immobilized anti-CD3-stimulated T4 cell responses, with a greater degree of inhibition noted when lower densities of immobilized anti-CD3 were used to stimulate T4 cells. The data demonstrate that stimulation of T4 cells by anti-CD3 is completely AC independent when the antibody is immobilized onto a surface. Furthermore, the results indicate that maximal stimulation requires multiple interactions with anti-CD3 without internalization of the CD3 molecule. The observation that additional signals are required to support T4 cell proliferation when the density of immobilized anti-CD3 is diminished suggests that these are necessary only when insufficient interactions with the CD3 molecule have occurred to transmit a maximal activation signal to the cell. Finally, the results indicate that anti-CD4 provides a direct inhibitory signal to the T4 cell, the effect of which is inversely proportional to the intensity of the activation signal.

BACKGROUND

The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule.

RESULTS

The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM.

CONCLUSIONS

The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.

The VLA-4 (CD49d/<em>CD2</em>9) integrin is the only member of the VLA family expressed by resting lymphoid cells that has been involved in cell-cell adhesive interactions. We here describe the triggering of homotypic cell aggregation of peripheral blood T lymphocytes and myelomonocytic cells by mAbs specific for certain epitopes of the human VLA alpha 4 subunit. This anti-VLA-4-induced cell adhesion is isotype and Fc independent. Similar to phorbol ester-induced homotypic adhesion, cell aggregation triggered through VLA-4 requires the presence of divalent cations, integrity of cytoskeleton and active metabolism. However, both adhesion phenomena differed at their kinetics and temperature requirements. Moreover, cell adhesion triggered through VLA-4 cannot be inhibited by cell preincubation with anti-LFA-1 alpha (CD11a), LFA-1 beta (CD18), or ICAM-1 (CD54) mAb as opposed to that mediated by phorbol esters, indicating that it is a LFA-1/ICAM-1 independent process. Antibodies specific for <em>CD2</em> or LFA-3 (CD58) did not affect the VLA-4-mediated cell adhesion. The ability to inhibit this aggregation by other anti-VLA-4-specific antibodies recognizing epitopes on either the VLA alpha 4 (CD49d) or beta (<em>CD2</em>9) chains suggests that VLA-4 is directly involved in the adhesion process. Furthermore, the simultaneous binding of a pair of aggregation-inducing mAbs specific for distinct antigenic sites on the alpha 4 chain resulted in the abrogation of cell aggregation. These results indicate that VLA-4-mediated aggregation may constitute a novel leukocyte adhesion pathway.

The electrical properties of nodose ganglion cells acutely isolated from adult rats were studied using the whole-cell patch-clamp recording method. Current-clamp recordings revealed a mean resting membrane potential of -54.3 mV and an input resistance of 527 M omega. Depolarizing current steps evoked action potentials with the following properties (mean): amplitude 111 mV, threshold -36 mV, and rate of rise 117 V/s. Two types of action potentials were observed, short and long duration. These properties, with the exception of input resistance (527 M omega cf. 50 M omega), are similar to those reported previously using intracellular recording methods in intact nodose ganglia (11, 20, 28). Brief application of 10 microM 5-hydroxytryptamine resulted in a rapid depolarization and burst of action potentials in the majority of cells. With voltage-clamp recording, step depolarizations to potentials positive to -10 mV elicited a transient inward current that was followed by a sustained outward current. Inward Na+ current was isolated by ion substitution and pharmacological agents. Two types of Na+ current were observed. One current was completely abolished by 3-15 microM tetrodotoxin (TTX), had a rapid time course, activated over the potential range -60 to -10 mV, and attained half-maximal conductance at -30 mV. The other current persisted in the presence of 15 microM TTX, had a slower time course, activated over the potential range -30 to 0 mV, and attained half-maximal conductance at -15 mV. In addition, 500 microM Cd2+ and 5.0 mM Co2+ reduced the TTX-insensitive current to 53 and 42% of control, respectively. Inward Ca2+ current was isolated by ion substitution and pharmacological agents and was identified by a dependence on external Ca2+. Cd2+ (500 microM) and Co2+ (5 mM) reduced the maximal inward current to 5 and 20% of control, respectively. When Ba2+ was substituted for Ca2+ as the charge carrier, the maximal inward current increased to 175% of control. Some cells had two Ca2+ current components, an inactivating component that activated near -60 mV and a large sustained current that activated near -40 mV. The initial inactivating current appeared as a "hump" on the current-voltage (I-V) curve over the potential range of -60 to -30 mV. The results indicate that, following isolation of these adult mammalian neurons, the membrane surfaces are sufficiently clean to allow patch-clamp recording.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Layer II of the medial entorhinal cortex is composed of two electrophysiologically and morphologically distinct types of projection neurons: stellate cells (SCs), which are distinguished by rhythmic subthreshold oscillatory activity, and non-SCs. The ionic mechanisms underlying their differential electroresponsiveness, particularly in the subthreshold range of membrane potentials, were investigated in an "in vitro" slice preparation. 2. In both SCs and non-SCs, the apparent membrane input resistance was markedly voltage dependent, respectively decreasing or increasing at hyperpolarized or subthreshold depolarized potential levels. Thus the neurons displayed inward rectification in the hyperpolarizing and depolarizing range. 3. In the depolarizing range, inward rectification was blocked by tetrodotoxin (TTX, 1 microM) in both types of neurons and thus shown to depend on the presence of a persistent low-threshold Na+ conductance (gNap). However, in the presence of TTX, pronounced outward rectification became manifest in the subthreshold depolarizing range of membrane potentials (positive to -60 mV) in the SCs but not in the non-SCs. 4. The rhythmic subthreshold membrane potential oscillations that were present only in the SCs were abolished by TTX and not by Ca2+ conductance block with Cd2+ or Co2+. Subthreshold oscillations thus rely on the activation of voltage-gated Na+, and not Ca2+, conductances. The Ca2+ conductance block also had no effect on the subthreshold outward rectification. 5. Prominent time-dependent inward rectification in the hyperpolarizing range in the SCs persisted after Na(+)- and Ca2+ conductance block. This rectification was not affected by Ba2+ (1 mM), but was blocked by Cs+ (1-4 mM). Therefore, it is most probably generated by a hyperpolarization-activated cationic current (Q-like current). However, the Q-like current appears to play no major role in the generation of subthreshold rhythmic membrane potential oscillations, because these persisted in the presence of Cs+. 6. On the other hand, in the SCs, the fast, sustained, outward rectification that strongly developed (after Na+ conductance block) at the oscillatory voltage level was not affected by Cs+ but was blocked by Ba2+ (1 mM). Barium was also effective in blocking the subthreshold membrane potential oscillations. 7. In the non-SCs, which do not generate subthreshold rhythmic membrane potential oscillations or manifest subthreshold outward rectification in TTX, Ca2+ conductance block abolished spike repolarization and caused the development of long-lasting Na(+)-dependent plateau potentials at a high suprathreshold voltage level. At this level, where prominent delayed rectification is present, the Na+ plateaus sustained rhythmic membrane potential oscillations.(ABSTRACT TRUNCATED AT 400 WORDS)

1. In dissociated and cultured 2-5-day-old chick dorsal root ganglion cells, a large transient inward current could be activated in response to a 'step' increase in [H+]o. 2. Using the single-electrode patch clamp technique in its whole-cell configuration, the proton-induced current was graded with [H+]o and relaxed in 1-2 s. 3. The pH dependence of the current was sigmoid with activation occurring at around pH 7.0 (at[Ca2+]o = 1 mM) and a maximum at pH 6.0-5.5. 4. Small increases of [H+]o, which by themselves failed to activate a significant amount of current, inactivated the proton-induced current. The half-maximum of inactivation occurred at pH 7.11 at [Ca2+]o = 5 mM, but this changed to pH 7.32 at [Ca2+]o = 1 mM. 5. The proton-induced inward current reversed direction at the Na+ equilibrium potential and was suppressed in the absence of [Na+]o. Measurement of the reversal potential at different [Na+]o and/or [Na+]i showed a linear relation with a slope of 58 mV/decade as predicted from the Nernst equation. Thus, proton-induced current was carried by Na+ and was abbreviated as INa(H). 6. The membrane conductance associated with INa(H) showed no voltage dependence, but did change in parallel with the activation of the current. The membrane conductance increased by a factor of 10-20-fold at the peak of the inward current. 7. INa(H) was blocked by organic and inorganic Ca2+ channel blockers (diltiazem, Cd2+ and Ni2+), but was unaffected by high concentrations of tetrodotoxin (TTX) or steady-state increases of the [Ca2+]i to 10(-4) M or the [H+]i to 10(-6) M. 8. In outside-out membrane patches, the single channel associated with the proton-induced current opened in bursts, with long pauses. The mean open time during the bursts was 1.26 ms and the channel had a conductance of 20-25 pS at -80 mV (120 mM [Na2+]o, 20 mM [Na2+]i). 9. Measurement of the voltage-gated Ca2+ current using short (30-50 ms) depolarizing pulses to zero showed that the Ca2+ current (ICa) but not the fast Na+ current (INa) was completely suppressed during the time course of activation of INa(H). 10. INa(H) was completely blocked by high (35-40 mM) [Ca2+]o. 11. Simultaneous elevation of [H+]o and [Ca2+]o failed to activate INa(H) but enhanced the voltage-gated Ca2+ channel. 12. Our data show that the proton-induced current is carried by Na+ flowing through a transformed Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Wegener's granulomatosis (WG) is a granulomatous vasculitis that affects the upper respiratory tract, lung, and kidney. Since T cells make up a significant proportion of cells infiltrating granulomatous lesions in WG, we investigated the proliferative response and cytokine profile of T cells from these patients. PBMCs were isolated from 12 patients with active WG, 7 patients with inactive disease, and 12 healthy normal donors. PBMCs from clinically active WG patients exhibited increased proliferation following stimulation with either PMA/ionomycin or anti-<em>CD2</em> and anti-<em>CD2</em>8, when compared with normal donors. In addition, these PBMCs exhibited increased secretion of IFN-gamma, but not of IL-4, IL-5, or IL-10. Furthermore, TNF-alpha production from PBMCs and CD4+ T cells isolated from patients with WG was elevated, when compared with healthy donors. In further studies, we investigated the ability of WG patients' monocytes to produce IL-12 and showed that both inactive and active patients produced increased amounts of IL-12. Finally, the in vitro IFN-gamma production by WG PBMC is inhibited in a dose-dependent manner by exogenous IL-10. These data suggest that T cells from WG patients overproduce IFN-gamma and TNF-alpha, probably due to dysregulated IL-12 secretion, and that IL-10 may therefore have therapeutic implications for this disease.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.

Focal segmental glomerulosclerosis (FSGS) is a histologic diagnosis in several kidney diseases characterized by proteinuria and a severe decrease in kidney function. Mutations in several genes were found in patients with primary FSGS, one of which is a CD2-associated protein CD2AP (originally referred to as CMS). This gene encodes an adaptor protein that plays a role in endocytosis, cell motility, and cell survival. Mice deficient in Cd2ap (the mouse homolog) die due to kidney failure, while heterozygous mice develop lesions similar to those of FSGS patients. In the kidney, CD2AP regulates the actin cytoskeleton. The only previously described patient with CD2AP mutation had a severely truncated protein. In this study, we describe a patient with a novel mutation resulting in a premature stop codon yielding a protein truncated by only 4%. This shortened CD2AP protein displays a significantly decreased F-actin binding efficiency in vitro with no expression of the mutated allele in the patient's lymphocytes. Heterozygous expression of the CD2AP mutation in both parents did not lead to any kidney pathology, as both have normal glomerular filtration rates and no proteinuria.

We have investigated the changes of cytosolic [Ca2+] and the secretory activity in single glomus cells dispersed from rabbit carotid bodies during exposure to solutions with variable O2 tension (Po2). In normoxic conditions (Po2 = 145 mmHg; 1 mmHg = 133 Pa), intracellular [Ca2+] was 58 +/- 29 nM, and switching to low Po2 (between 10 and 60 mmHg) led to a reversible increase of [Ca2+] up to 800 nM. The response to hypoxia completely disappeared after removal of external Ca2+ or with the addition of 0.2 mM Cd2+ to the external solution. These same solutions also abolished both the Ca2+ current of the cells and the increase of internal [Ca2+] elicited by high external K+. Elevations of cytosolic [Ca2+] in response to hypoxia or to direct membrane depolarization elicited the release of dopamine, which was detected by amperometric techniques. Dopamine secretion occurred in episodes of spike-like activity that appear to represent the release from single secretory vesicles. From the mean charge of well-resolved secretory events, we estimated the average number of dopamine molecules per vesicle to be approximately 140,000, a value about 15 times smaller than a previous estimate in chromaffin granules of adrenomedullary cells. These results directly demonstrate in a single-cell preparation the secretory response of glomus cells to hypoxia. The data indicate that the enhancement of cellular excitability upon exposure to low Po2 results in Ca2+ entry through voltage-gated channels, which leads to an increase in intracellular [Ca2+] and exocytotic transmitter release.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the <em>CD2</em>8 and <em>CD2</em> proteins, respectively. Supporting a role for Lck in <em>CD2</em>8 signaling, we demonstrate that <em>CD2</em>8 signaling in both transformed and primary T cells requires Lck as well as proline residues in <em>CD2</em>8. These data suggest that Lck plays an essential role in <em>CD2</em>8 costimulation.

We describe the clinical and laboratory findings of 78 adult patients with T-prolymphocytic leukemia (T-PLL) studied over the last 12 years. The main disease features were splenomegaly (73%), lymphadenopathy (53%), hepatomegaly (40%), skin lesions (27%), and a high leukocyte count (greater than 100 x 10(9)/L in 75%) with nucleolated prolymphocytes. A variant form with small, less typical cells was recognized in 19%. Membrane markers defined a postthymic phenotype TdT-, CD2+, CD3+, CD5+, CD7+; in 65%, the cells were CD4+ CD8-, in 21%, they coexpressed CD4 and CD8, and, in 13%, they were CD4- CD8+. Serology for human T-cell leukemia/lymphoma virus Type-I (HTLV-I) was negative in the 27 cases investigated. Cytogenetic analysis in 30 cases showed a consistent abnormality of chromosome 14, usually inv (14), with breakpoints at q11 and q32 in 76% of cases. Trisomy 8, including iso8q, was shown in 53%; t (11;14)(q13;q32) was documented in one case; and one had a normal karyotype. The clinical course was progressive with a median survival of 7.5 months. Thirty-one patients were treated with 2' deoxycoformycin and 15 responded (3 complete remissions and 12 partial remissions); the response rate (48%) increased to 58% in patients with a CD4+ CD8- phenotype. The median survival of responders was 16 months and of nonresponders 10 months; other treatments were less effective. T-PLL is a distinct clinico-pathologic entity with aggressive course and characteristic chromosome abnormalities. A subgroup of patients may benefit from deoxycoformycin.