Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation of Cd79a (mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences of Ebf1 haploinsufficiency together with haploinsufficiency of Runx1 genes in mice. Although numbers of "committed" pro-B cells were maintained in Ebf1(+/-)Runx1(+/-) (ER(het)) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, kappa0 sterile transcripts, <em>CD2</em> and <em>CD2</em>5 were reduced and delayed in ER(het) pro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells in ER(het) mice. Late pre-B and immature and mature B cells were decreased in the bone marrow of Ebf1(+/-) (E(het)) mice and were nearly absent in ER(het) mice. Although we did not observe significant effects of haploinsuficiencies on IgH or Igkappa rearrangements, a relative lack of Iglambda rearrangements was detected in E(het) and ER(het) pre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow of E(het) and ER(het) mice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reduced Ebf1 dosage and the compounding effects of reduced Runx1 dosage. Our data confirm and extend the importance of EBF1 in regulating target genes and Ig gene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.

Action potential durations, measured at one-third peak amplitude, were examined during intracellular recordings in 134 supraoptic nucleus neurones maintained in vitro in perfused hypothalamic explants. Spike durations ranged between 1.2 and 3.9 ms and were dependent on firing frequency. Shortest measurements (1.74 +/- 0.03 ms; mean +/- S.E. of mean) were obtained during relative quiescence, i.e. less than or equal to 0.5 Hz. A gradual increase in firing frequency through continuous injection of depolarizing current prolonged spike duration, with maximum levels (2.68 +/- 0.05 ms) achieved at 20 Hz. When interspike interval variability was eliminated and firing was more precisely regulated by brief 15-20 ms intracellular current pulses given at pre-determined frequencies, a proportional relationship between increasing spike duration and firing frequency was retained but the change in spike duration at frequencies between 2 and 10 Hz was less pronounced. Once action potentials had achieved the long duration configuration, their return to the shorter duration took place gradually during any succeeding silent interval with a time constant of 4.9 s. Action potential broadening occurred progressively and was most pronounced at the onset of spontaneous or current-induced bursts. In thirty-six phasically active neurones, spike broadening at the onset of a burst was concurrent with the presence of 5-10 consecutive short (less than or equal to 100 ms) interspike intervals; thereafter, despite a greater than 50% reduction in firing frequency, action potential durations remained prolonged throughout the burst. In all of nineteen cells tested, frequency-dependent changes in spike duration were reversibly decreased or blocked by Cd2+, Co2+ and Mn2+, or when CaCl2 was exchanged for equimolar amounts of EGTA in the perfusion medium. These observations indicate that a Ca2+ conductance contributes to frequency- and firing-pattern-dependent changes in spike duration in rat supraoptic nucleus neurones.

The enterotoxins produced by Staphylococcus aureus (SE) are the most potent mitogens known. Triggering of proliferation or cytotoxicity by SE requires the presence of MHC class II molecules on accessory or target cells. In this study we have investigated the role of HLA class II molecules in the activation of human T cells by SE and the nature of the target structure on the responding T lymphocyte for SE. This dependence on class II molecules is not due to an immunological "recognition" of SE since there is no restriction by polymorphic determinants of HLA molecules and since even xenogeneic class II molecules can reconstitute the human T cell response to SE. Furthermore, HLA class II-positive but not -negative cells absorb the mitogenic activity from SE solutions and significant binding of 125I-labeled SE can be demonstrated to class II-positive but not to class II-negative cells. Enterotoxin molecules react directly with T cells since they cause an increase in cytosolic Ca2+ concentration similar to anti-CD3 mAb. This increase is abrogated by prior modulation of the TCR/CD3 complex. Antibodies to CD2, CD3 and the TCR that block antigen-specific activation also block T cell activation by SE. Moreover, preincubation of purified resting accessory cell-free T cells with SE leads to modulation of the TCR/CD3 complex. Taken together these data indicate that SE interact selectively with HLA class II molecules on accessory or target cells and with a TCR-associated structure on the T cell.

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7-specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept-based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection-free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor-specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation.

1. Two kinds of transient outward currents were observed upon depolarization of single smooth muscle cells isolated from guinea-pig ureter. The major transient outward current was through Ca2(+)-activated K+ channels (IK(Ca) which had a large conductance (130 pS; 126 mM [K+]i/5.9 mM [K+]o). 2. The smaller transient outward current (ITO) was pharmacologically separated from other membrane currents in the presence of 1 mM-Cd2+ and 2 mM-tetraethylammonium(TEA+) and was selectively blocked by 3 mM-4-aminopyridine. It peaked (approximately 200 pA) within 10 ms upon depolarization from -80 to +20 mV and its half-inactivation time was approximately 50 ms at +20 mV. Half-maximum voltages (V 1/2) for activation and inactivation were about -8 and -50 mV, respectively, in the presence of 1 mM-Cd2+ and 2 mM-TEA+. The time course of recovery from inactivation of ITO was fitted with a single-exponential function (tau = 100 ms at -80 mV). A tenfold change of [K+]o resulted in a 53 mV change in the reversal potential of the tail of ITO. 3. Cadmium reduced peak ITO and shifted the voltage dependence of activation and inactivation in the positive direction in a concentration-dependent manner. The V 1/2 for inactivation in the absence of Cd2+ was estimated to be approximately -64 mV. 4. Single-channel outward currents which appeared only in the initial part of a depolarizing pulse from about -100 mV were recorded using the cell-attached patch clamp. The decay of the ensemble average of the current was similar to the macroscopic ITO under whole-cell clamp. When the holding potential was less negative, the opening probability of the channel greatly decreased. The channel conductance in normal extracellular medium was 14 pS. 5. In ureter cells ITO resembles A-type current. ITO does not contribute significantly to the repolarization of the action potential but it may regulate membrane excitability by opposing Ca2+ current activated around the threshold of the action potential.

To delineate factors involved in NK cell development, we established an in vitro system in which lineage marker (Lin)-, c-kit+, Sca2+ bone marrow cells differentiate into lytic NK1.1+ but Ly49- cells upon culture in IL-7, stem cell factor (SCF), and flt3 ligand (flt3L), followed by IL-15 alone. A comparison of the ability of IL-7, SCF, and flt3L to generate IL-15-responsive precursors suggested that NK progenitors express the receptor for flt3L. In support of this, when Lin-, c-kit+, flt3+ or Lin-, c-kit+, flt3- progenitors were utilized, 3-fold more NK cells arose from the flt3+ than from the flt3- progenitors. Furthermore, NK cells that arose from flt3- progenitors showed an immature NK1.1dim, CD2-, c-kit+ phenotype as compared with the more mature NK1.1bright, CD2+/-, c-kit- phenotype displayed by NK cells derived from flt3+ progenitors. Both progenitors, however, gave rise to NK cells that were Ly49 negative. To test the hypothesis that additional marrow-derived signals are necessary for Ly49 expression on developing NK cells, flt3+ progenitors were grown in IL-7, SCF, and flt3L followed by culture with IL-15 and a marrow-derived stromal cell line. Expression of Ly49 molecules, including those of which the MHC class I ligands were expressed on the stromal or progenitor cells, as well as others of which the known ligands were absent, was induced within 6-13 days. Thus, we have established an in vitro system in which Ly49 expression on developing NK cells can be analyzed and possibly experimentally manipulated.

The effects of low concentrations of 4-aminopyridine (4-AP) on the membrane properties of guinea pig cerebellar Purkinje cells were investigated in slice preparation using intracellular recordings. It was found that 1-10 microM 4-AP did not affect the resting potential or the input resistance of the cells, but reduced markedly the duration of the slowly depolarizing potential (SDP), and thus the latency to the firing of Ca2+ spikes in response to intracellular current pulses. Intradendritic recordings in the presence of tetrodotoxin, Cd2+, and low [Ca2+]o, which blocked all the regenerative responses, exhibited prominent membrane outward rectification in response to depolarizing current pulses. Under these conditions, the SDP was abolished and, in contrast, a slowly developing hyperpolarization was consistently observed. Application of 10 microM 4-AP reduced the outward membrane rectification in a reversible manner, but did not affect the transient hyperpolarization, which is usually attributed to the activation of potassium "A" current. These results demonstrate, for the first time, the presence of a highly 4-AP sensitive delayed rectifier in guinea pig cerebellar Purkinje cells, which prominently affects their excitability. The results also indicate that the slowly depolarizing potential of guinea pig Purkinje cells does not involve inactivation of transient potassium currents, which has been suggested previously as an underlying mechanism for this phenomenon in turtle Purkinje cells.

Southern blot screening of a genomic Helicobacter pylori library was employed to find a P type ATPase using a mixture of 16 DNA oligonucleotides coding for the DKTGT(I/L)T consensus sequence specific for the phosphorylation site of this family of ATPases. A positive clone, pRH439, was isolated and sequenced. The inserted 3.4-kb H. pylori DNA contained an intact open reading frame encoding a protein of 686 amino acids carrying the consensus sites for phosphorylation and ATP binding. The amino acid sequence exhibits a 25-30% identity with bacterial Cd2+ and Cu2+ ATPases. Genomic Southern blot analysis showed that this ATPase was present in all H. pylori strains examined, whereas it was not detectable in Campylobacter jejuni and other bacteria. The membrane topology of this ATPase was investigated using in vitro transcription/translation of fusion vectors to find signal anchor and/or stop transfer sequences. Eight regions of the H. pylori ATPase acted as signal anchor and/or stop transfer sequences and were ordered pairwise along the polypeptide chain placing the N and C-terminal amino acids in the cytoplasm. These transmembrane segments are contained between positions 73 and 92 (H1), 98 and 125 (H2), 128 and 148 (H3), 149 and 176 (H4), 309 and 327 (H5), 337 and 371 (H6), 637 and 658 (H7), and 659 and 685 (H8). The membrane domain of the ATPase, therefore, consists of at least four pairs of transmembrane segments with the phosphorylation site and ATP binding domain located in the large cytoplasmic loop between H6 and H7. The cytoplasmic domain contains several histidines and cysteines, perhaps indicative of divalent cation binding sites. There are several charged amino acids (3 Lys, 2 Glu, 2 Asp), predicted to be in the membrane domain mainly in H2, H3, and H4 and a Cys-Pro-Cys putative metal ion site in H6. The extracytoplasmic domain also has several charged amino acids (5 Glu, 1 Asp, 1 Lys, 1 Arg). It is likely that this novel protein is a heavy metal cation transporting ATPase and belongs to a family of P type ATPases containing eight transmembrane segments.

1. Whole-cell recordings were made from cultured cerebellar granule neurons using perforated patch clamp techniques. The primary cultures were prepared using 6- to 9-day-old Sprague-Dawley rats. 2. Neurons in culture for less than 48 h possessed resting membrane potentials of -29 mV. However, neurons in culture for 7 days had much more hyperpolarized resting membrane potentials (-89 mV). Over the same period, these neurons developed an additional component of outward current. 3. This non-inactivating current was activated by depolarization, exhibited outward rectification and reversed close to the potassium equilibrium potential. The kinetics of activation and deactivation were very rapid. 4. Muscarine ((+)-muscarine chloride) reversibly inhibited the current with an EC50 of 0.17 microM. The inhibition by muscarine was unaffected by pre-incubation for 17-20 h with 120 micrograms ml-1 pertussis toxin. 5. The current and its inhibition by muscarine were unaffected by 100 microM Cd2+. In Ca(2+)-free conditions, the current was significantly larger than in 0.5 mM Ca2+, but inhibition by 10 microM muscarine was significantly reduced. 6. The standing outward current was not obviously affected by 50 microM 5-HT, 50 microM noradrenaline, 50 microM 2-chloroadenosine or 5 mM tetraethylammonium. It was reduced by 10 microM La3+, 10 microM Zn2+ and 1 mM Ba2+. 7. Muscarinic agonists increased the input resistance of neurons and shifted the zero current level in the depolarized direction when voltage clamped. This enhanced excitability was evident under current clamp, where 10 microM muscarine depolarized granule neurons such that action potentials became evident.

Apparently chromosomally located mercury resistance determinants in five methicillin-resistant Staphylococcus aureus strains of different geographical origin were structurally homologous to plasmid-located mercury resistance determinants in S. aureus. These were all located on a 6.3-kilobase (kb) Bg/II fragment, as evident from Southern hybridization experiments with the 6.3-kb Bg/II fragment of plasmid pI258 as the probe. These methicillin-resistant S. aureus strains exhibited similar phage susceptibility patterns and biochemical reactions. They differed, however, in the DNA location of the mercury resistance determinants, as evidenced by neighboring cleavage sites for restriction endonucleases EcoRI, HindIII, and PstI. In an environmental (nonhospital) strain in which mercury resistance was also apparently chromosomally conferred, these determinants were also homologous to pI258 DNA, but they were located on a 6.6-kb Bg/II fragment. Cadmium resistance determinants in the five methicillin-resistant S. aureus strains and the environmental S. aureus strain were not similar to the known plasmid-located determinants cadA and cadB. Cd2+ resistance was based on an efflux mechanism for Cd2+. However, no parallel resistance to zinc was conferred. The 3.2-kb XbaI-Bg/II fragment obtained from plasmid pI258 and used as a cadA-specific probe did not hybridize to total DNA digests of the strains with apparently chromosomally determined cadmium resistance.

Antilymphocyte or antithymocyte globulins were shown to be immunosuppressive when administered to recipients of organ transplants as prophylactic or rescue treatment of acute rejection or in patients with acute graft versus host reactions following bone marrow transplantation. Several monoclonal antibodies specific for activation or adhesion molecules of the T lymphocyte surface can also inhibit experimental or clinical allograft reactions. We have investigated the presence of some antibodies, of defined specificity and documented biological activity, in polyclonal antilymphocyte and antithymocyte globulins in order to get further insight into the mechanism of action of these polyclonal antibodies. Using a quantitative immunofluorescence assay by flow cytometry we could estimate the minimal amounts of antibodies to LFA-1 (CD11a and CD18), CD45, CD3, and CD5. Antibodies to HLA-DR, <em>CD2</em>, CD4, CD8, and <em>CD2</em>5 were also demonstrated but could not be quantified. Antibodies to beta 2-microglobulin were determined by ELISA. These data suggest that interference with functional lymphocyte surface molecules may account at least in part for the immunosuppressive activity of antilymphocyte and antithymocyte globulins.

With 90 mM Ba2+, the main Ca2+ current in frog sympathetic neurons peaks near +30 mV and is blocked by omega-conotoxin GVIA (omega-CgTx). It is modulated by norepinephrine (NE) in a voltage-dependent manner via a membrane-delimited mechanism. Surprisingly, a different current dominates at more negative voltages (-30 to +10 mV). That novel current is not sensitive to selective blockers of L- or N-type channels (respectively, dihydropyridines or omega-CgTx) and is inhibited weakly if at all by NE. It is selectively inactivated at -40 mV and is selectively blocked by Ni2+, whereas Cd2+ is slightly more potent against the main current. The novel current is associated with a 19 pS channel (0.6 pA at 0 mV). This channel may have been misidentified as the single-channel correlate of the whole-cell N-type Ca2+ current in some previous studies.

Integrins are a superfamily of related molecules whose function, where known, is to mediate adhesion. The so-called very-late-activation antigen (VLA) family includes at least five distinct heterodimers, each composed of a unique alpha-subunit non-covalently associated with a common beta-subunit. Several members of the family have been shown to bind extracellular matrix proteins, but the function of VLA-4 is so far unknown. VLA-4 is the only member of the family detected on thymocytes and resting T cells. We show here that an antibody which recognizes the beta-subunit of VLA-4 (<em>CD2</em>9) on T cells can inhibit CD4+ cell proliferation triggered by <em>CD2</em> or CD3, and that binding of this antibody to activated T cells leads to an increase in cyclic AMP levels which is comparable to that elicited by forskolin. These negative signalling effects are unique to this antibody: other <em>CD2</em>9 antibodies do not affect the growth of activated CD4 cells but enhance the proliferation of whole T cell populations and abrogate the suppressive effects of mitomycin-treated CD8 cells on CD4-cell growth. Taken together, our results indicate that VLA-4 functions in cell-cell interactions and that it is the target for the suppressive effects of CD8 cells on CD4 cells.

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+>>) Li+>>) TMA+>> NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage-dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.

The GABA rho 1 subunit, cloned from a human retina library, can form homooligomeric receptors with properties similar to GABAc receptors characterized in retinal cells. The divalent cation Zn2+, abundant in the CNS and retina, was found to inhibit GABA rho 1 receptors in a voltage-independent manner. Varying the extracellular pH from 7.4 to 5.6 significantly reduced this inhibitory effect. This pH profile suggested that one or more histidine residues might play a role in the interaction between Zn2+ and the GABA rho 1 receptor. Site-directed mutagenesis revealed that a single histidine residue (His 156) in the putative extracellular domain of rho 1 was critical for Zn2+ sensitivity. Substitution of this amino acid with tyrosine (H156Y) created a functional GABA receptor with agonist and channel properties indistinguishable from wildtype. However, the H156Y mutant was insensitive to Zn2+, even at concentrations as high as 1 mM. Mutation to aspartic acid, an amino acid that can interact with Zn2+ in other proteins, preserved sensitivity to Zn2+ but abolished the pH-dependent effect. This histidine residue is also involved in Ni2+ and Cd2+ interaction since the H156Y mutation completely suppressed the inhibition effects of these two cations. These data demonstrate that an extracellular histidine residue is critical for transition metal cation sensitivity of GABA rho 1 receptors.

Kidney podocytes and their slit diaphragms (SDs) form the final barrier to urinary protein loss. There is mounting evidence that SD proteins also participate in intracellular signaling pathways. The SD protein nephrin serves as a component of a signaling complex that directly links podocyte junctional integrity to actin cytoskeletal dynamics. Another SD protein, CD2-associated protein (CD2AP), is an adaptor molecule involved in podocyte homeostasis that can repress proapoptotic TGF-beta signaling in podocytes. Here we show that dendrin, a protein originally identified in telencephalic dendrites, is a constituent of the SD complex, where it directly binds to nephrin and CD2AP. In experimental glomerulonephritis, dendrin relocates from the SD to the nucleus of injured podocytes. High-dose, proapoptotic TGF-beta1 directly promotes the nuclear import of dendrin, and nuclear dendrin enhances both staurosporine- and TGF-beta1-mediated apoptosis. In summary, our results identify dendrin as an SD protein with proapoptotic signaling properties that accumulates in the podocyte nucleus in response to glomerular injury and provides a molecular target to tackle proteinuric kidney diseases. Nuclear relocation of dendrin may provide a mechanism whereby changes in SD integrity could translate into alterations of podocyte survival under pathological conditions.

Binding monoclonal antibodies (MAb) both to D66 and 9.6/T11(1) epitopes on the CD2 [T,gp50]-defined molecule produces a high level of T cell mitosis. This was observed with a battery of MAb of different isotypes. In contrast, none of the anti-D66 or anti-9.6/T11(1)Ab could trigger T cell proliferation in combination with anti-T11(3). Moreover, all anti-D66-9.6/T11(1) pairs of MAb tested required monocytes to activate T cells which were recruited through their Fc receptors. Variations among normal individuals were observed in the level of response to anti-D66-9.6/T11(1) pairs of Ab, 75% of a population of French Caucasians giving a high response. The level of response of a given individual was determined by his accessory cells. However, the level of response of an individual appeared to be minimally influenced by the isotype of a peculiar anti-D66 or anti-9.6/T11(1) Ab. The addition of exogeneous IL 2 could overcome the removal of accessory cells or the modulation of CD3 molecules. In contrast, IL 2 receptor appearance was not overcome by removal of monocytes. Thus, T cell activation via CD2 seems to be produced by "touching" several definite regions of this molecule which trigger a cascade of events similar to those produced by mitogenic lectins. One can assume that the appropriate conformational changes of the CD2 molecule induced by anti-D66-9.6/T11(1) pairs of Ab are solely produced when they are presented by accessory cells. This leaves open the question of whether accessory cells would also play a more active role.

Natural killer (NK) cell neoplasms, which include extranodal NK/T-cell lymphoma (nasal and extranasal) and aggressive NK cell leukemia, are generally rare, but they are more common in people of Oriental, Mexican and South American descent. These neoplasms are highly aggressive, and show a strong association with Epstein-Barr virus. Extranodal NK/T-cell lymphoma most commonly affects the nasal cavity and other mucosal sites of the upper aerodigestive tract. Patients present with nasal obstruction or midfacial destruction. Despite the early stage of disease at presentation, overall survival is poor. Patients with the extranasal form of the lymphoma often present with high-stage disease, commonly involving the skin, gastrointestinal tract, testis, and soft tissue, and the prognosis is even worse. Histologically, the lymphoma can show a broad cytologic spectrum, but apoptosis, necrosis, and angioinvasion are common. The most common immunophenotype is CD2(+), surface CD3(-), cytoplasmic CD3(+), CD56(+). Based on currently available data, treatment of nasal NK/T-cell lymphoma should consist of radiotherapy, with or without multiagent chemotherapy. More research is required to ascertain the role of high-dose chemotherapy with stem cell rescue and that of non-multidrug resistance-related chemotherapeutic agents. Aggressive NK cell leukemia affects younger patients, who present with poor general condition, fever, and disseminated disease; they often die within a short time from systemic disease or complications such as multi-organ failure. The peripheral blood and bone marrow show atypical large granular lymphocytes, which exhibit an immunophenotype similar to that of extranodal NK/T-cell lymphoma. Aggressive NK cell leukemia must be distinguished from T-cell large granular lymphocyte leukemia and indolent NK cell lymphoproliferative disorder, both of which are indolent.

Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6 beta-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41,000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 microM and 43 microM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 microM and 90 microM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 microM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.

The chronic fatigue syndrome is a disorder of unknown aetiology which is characterized by debilitating fatigue. Recent evidence has suggested that viruses may persist in the tissues of patients with chronic fatigue syndrome. A concurrent immunological disturbance is likely to be associated with the persistence of viral antigens. Therefore, the humoral and cellular immunity of 100 patients who were suffering from chronic fatigue syndrome and that of 100 healthy, age- and sex-matched control subjects were compared. This study documents the frequent occurrence of abnormalities within the cellular and humoral immune systems of patients with well-defined chronic fatigue syndrome. Disordered immunity may be central to the pathogenesis of chronic fatigue syndrome. In patients with chronic fatigue syndrome, a significant (P less than 0.01) reduction was found in the absolute number of peripheral blood lymphocytes in the total T-cell (CD2), the helper/inducer T-cell (CD4) and the suppressor/cytotoxic T-cell (CD8) subsets. A significant (P less than 0.001) reduction also was found in T-cell function, which was measured: in vivo by delayed-type hypersensitivity skin-testing (reduced responses were recorded in 50 [88%] of 57 patients); and in vitro by phytohaemagglutinin stimulation. Reduced immunoglobulin (Ig) levels were common (56% of patients), with the levels of serum IgG3- and IgG1-subclasses particularly (P less than 0.05) affected.

The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo.

OBJECTIVE

We conducted a retrospective analysis to evaluate the safety and efficacy of Campath-1H, an anti-CD52 humanized monoclonal antibody, in previously treated T-prolymphocytic leukemia (T-PLL) patients in a compassionate-use program.

METHODS

Seventy-six patients with T-PLL (including four chemotherapy-naive patients) received 3, 10, and 30 mg of Campath-1H on sequential days, followed by 30 mg three times weekly, as 2-hour intravenous infusions, for 4 to 12 weeks.

RESULTS

Median patient age was 60 years (range, 35 to 84). Spleen liver, lymph node, and skin involvement were present in 64%, 40%, 54%, and 18% of patients, respectively. All tested patients had CD2, CD7, CD4, and/or CD8 positivity, whereas CD5 and CD3 were positive in 98% and 96% of tested patients, respectively. The objective response rate was 51% (95% confidence interval [CI], 40% to 63%), with a 39.5% complete response (CR) rate (95% CI, 28% to 51%). The median duration of CR was 8.7 months (range, 0.13+ to 44.4), and median time to progression was 4.5 months (range, 0.1 to 45.4) compared with 2.3 months (range, 0.2 to 28.1) after first-line chemotherapy. The median overall survival was 7.5 months (14.8 months for CR patients). The most common Campath-1H-related adverse events were acute reactions during or immediately after infusions. Fifteen infectious episodes occurred during treatment in 10 patients (13%), leading to treatment discontinuation in three. Eight patients experienced possibly related, late-onset infections. Severe thrombocytopenia and/or neutropenia occurred in six patients (8%), leading to treatment discontinuation in four. Two treatment-related deaths occurred.

CONCLUSIONS

Campath-1H is an active drug in T-PLL patients for whom first-line therapy has failed. It has a favorable risk/benefit ratio and should be prospectively investigated in chemotherapy-naive patients.

Influence on the immune system activity by sex hormones has been widely reported. Fertile women are proner to the onset of autoimmune diseases than men, but this increased susceptibility disappears after menopause. The hormonal changes are very likely to be responsible for this event, but precise correlations between sex hormone levels and immune functions have not been defined. For this reason we have analyzed phenotype and natural cytotoxicity of peripheral blood lymphocytes (PBL) from 35 women in menopause, comparing them with the same parameters of 28 fertile and 8 postmenopausal women and correlating them with the hormonal pattern of each group. We have also considered 8 women with premature menopause. Hormonal levels have been detected by radioimmune assays, while PBL phenotype has been studied by immunofluorescence and FACS analysis. The natural killer (NK) cell activity has been calculated on the basis of a chromium release assay. Postmenopausal women showed a reduction of the number of total lymphocytes (1650 +/- 215 cells/mmc) in comparison to fertile women (2081 +/- 200 cells/mmc, P < 0.01). The decrease mainly involved B and CD4+ T lymphocyte subpopulations (P < 0.05 and P < 0.01, respectively). Women with premature menopause had lower percentage of CD4 lymphocytes (34% vs 47%, P < 0.01) and higher percentage of CD8 (30% vs 22%, P < 0.02) and NK cells (32% vs 14%, P < 0.009) than fertile women of the same age. The percentage of circulating lymphocytes expressing HLA class II antigens also resulted as being increased (22% vs 9%, P < 0.01). The number of total, CD2, CD4 T lymphocytes, B and NK cells correlated positively with LH and negatively with FSH serum levels (P < 0.05 and P < 0.002, respectively). PRL positively influenced CD2, CD4 and B lymphocyte numbers (P < 0.001). FSH and 17 beta-estradiol inversely affected CD8 and B lymphocyte numbers (P < 0.005 and P < 0.02, respectively). In conclusion, the increase of FSH and the decrease of PRL levels appear to be involved in the reduction of B and CD4 T lymphocytes thus lowering the risk for the onset of autoimmune diseases during and after menopause. Generalized activation of the immune system (raised expression of HLA class II antigens) with elevated numbers of cytotoxic subpopulations (CD8 and NK lymphocytes) is present in women affected by premature menopause suggesting the involvement of autoimmune dysregulation in the pathogenesis of this syndrome.