The formation of phytochelatins, small metal-binding glutathione-derived peptides, is one of the well-studied responses of plants to toxic metal exposure. Phytochelatins have also been detected in some fungi and some marine diatoms. Genes encoding phytochelatin synthases (PCS) have recently been cloned from Arabidopsis, wheat and Schizosaccharomyces pombe. Surprisingly, database searches revealed the presence of a homologous gene in the Caenorhabditis elegans genome, DDBJ/EMBL/GenBank accession no. 266513. Here we show that C. elegans indeed expresses a gene coding for a functional phytochelatin synthase. CePCS complements the Cd2+ sensitivity of a Schizosaccharomyces pombe PCS knock-out strain and confers phytochelatin synthase activity to these cells. Thus, phytochelatins may play a role for metal homeostasis also in certain animals.

Infection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C (NKG2C(-/-)). Assessment of NK cell repertoires in 60 NKG2C(-/-) donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-γ promoter. We found that both NKG2C(-) and NKG2C(+) adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides "signal 2" in antibody-driven adaptive NK cell responses.

BACKGROUND

HuOKT3gamma1(Ala-Ala) is a genetically-engineered derivative of the parental murine OKT3 monoclonal antibody, in which the six complementarity-determining regions have been grafted within a human IgG1 mAb, and whose C(H)2 region has been altered by site-directed mutagenesis to alter FcR-binding activity, thereby eliminating T cell activation properties. This report describes the results of a phase I trial of huOKT3gamma1(Ala-Ala) treatment of acute renal allograft rejection.

METHODS

Acute renal allograft rejection in kidney and kidney-pancreas transplant recipients was treated with huOKT3gamma1(Ala-Ala). huOKT3gamma1(Ala-Ala) dosing consisted of daily 5- or 10-mg doses adjusted initially to achieve target levels of 1000 ng/ml.

RESULTS

A total of seven patients, five kidney transplant and two kidney-pancreas transplant recipients, were treated with the monoclonal antibody for first rejection episodes. Corticosteroids (500 mg i.v. Solumedrol) were given 2 hr before the first huOKT3gamma1(Ala-Ala) dose only. Banff classification of treated rejections were the following: grade I, 1 patient, grade IIA, 1 patient, grade IIB, 4 patients, and grade III, 1 patient. Median time from transplant to rejection was 15 days, and median follow up 12 months (range 10-17 months). HuOKT3gamma1(Ala-Ala) therapy was given for 10.1+/-2.5 days, and mean total dose was 76+/-27 mg. Rejection was reversed in five of seven patients, and recurrent rejection was observed in one patient. Serum creatinine values peaked on day 1 of huOKT3gamma1(Ala-Ala) therapy, and thereafter demonstrated a progressive decline. Rejection reversal (return of creatinine to baseline) occurred at a median of 4 days and a mean of 4.1+/-2 days. Renal allograft biopsies obtained during huOKT3gamma1(Ala-Ala) therapy provided evidence of rapid rejection reversal. Patient and graft survival were both 100%. First dose reactions were minimal, and anti-OKT3 antibodies were not detected. Elevations in serum IL-10, but not IL-2 levels were observed after the first huOKT3gamma1(Ala-Ala) dose. Marked reductions in circulating CD2+, CD4+, and CD8+ T cells were observed after the first huOKT3gamma1(Ala-Ala) dose, followed by a slow progressive return of cell counts toward pretreatment values. Pharmacokinetic analysis revealed a half-life of 142+/-32 hr.

CONCLUSIONS

HuOKT3gamma1(Ala-Ala) possesses the ability to reverse vigorous rejection episodes in kidney and kidney-pancreas transplant recipients, and in comparison to murine OKT3, possesses minimal first dose reactions and does not seem to induce antibodies that bind the OKT3 idiotype. These results support the conduct of additional clinical trials with the huOKT3gamma1(Ala-Ala) antibody.

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P(5B) ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.

Alefacept, an immunomodulatory recombinant fusion protein composed of the first extracellular domain of LFA-3 fused to the human IgG1 hinge, C(H)2, and C(H)3 domains, has recently been shown in phase II and III clinical trials to safely reduce disease expression in patients with chronic plaque psoriasis. Alefacept modulates the function of and selectively induces apoptosis of CD2(+) human memory-effector T cells in vivo. We have sought to gain further understanding of the mechanisms of action that influence the biological activity of alefacept and may contribute to its efficacy and patient responsiveness. Specifically evaluated is the ability of alefacept to activate intracellular signals mediated via CD2 and/or Fc gamma RIII (CD16). Experimentation using isoforms of alefacept engineered to have amino acid substitutions in the IgG1 C(H)2 domain that impact Fc gamma R binding indicate that alefacept mediates cognate interactions between cells expressing human CD2 and CD16 to activate cells, e.g., increase extracellular signal-regulated kinase phosphorylation, up-regulate cell surface expression of the activation marker CD2CD2 and CD16 and be mediated through CD16, but not CD2. Experimentation using human CD2-transgenic mice and isoforms of alefacept confirmed the requirement for Fc gamma R binding for detection of the pharmacological effects of alefacept in vivo. Thus alefacept acts as an effector molecule, mediating cognate interactions to activate Fc gamma R(+) cells (e.g., NK cells) to induce apoptosis of sensitive CD2(+) target cells.

The numbers, phenotype, and tissue distribution of gamma delta T cells in cattle were studied using two monoclonal antibodies (mAbs) which react with the bovine gamma delta T cell receptor (TCR). Both mAbs stained 20-40% of T cells in peripheral blood, and immunoprecipitated molecules of 44 and 36 kd (reduced) and 70-80 kd (non-reduced). In cattle the majority of circulating gamma delta T cells showed a distinct surface phenotype; they expressed T19, a 215 kd molecule described in sheep and cattle which marks only gamma delta T cells. Bovine gamma delta T cells were also CD2-, CD4-, and mostly CD8-, and failed to express CD6, a molecule possibly involved in T cell activation. The distribution of gamma delta T cells in cattle lymphoid tissues differed markedly from that in humans, in that bovine gamma delta T cells were concentrated around lymph node trabeculae and were usually sparse or absent from the B cell and T cell domains of lymph nodes. Like most other species studied, gamma delta T cells in cattle were localized to epithelial surfaces, particularly within the skin and intestine, indicating that it was at these sites where gamma delta T cells functioned. Our results provide further evidence for the unusual localization, recirculation pattern, and phenotype of gamma delta T cells, and also show that some features of gamma delta T cells can differ quite markedly from species to species.

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton.

Monoclonal antibodies specific for the murine CD2 antigen were identified by an efficient screening method utilizing murine CD2 cDNA transfectants. An unexpected expression of CD2 on murine B cells was revealed by immunofluorescence and immunoprecipitation studies with these monoclonal antibodies and by RNA blot analysis for the murine CD2 transcript.

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.

Magnocellular neurosecretory cells (MNCs) were impaled in perfused explants of rat hypothalamus. Application of a voltage clamp after 1-5 current-evoked spikes revealed a tetrodotoxin-resistant, but Cd2+-sensitive inward current. From a threshold near -85 mV, the amplitude of this current increased as post-spike commands were made to more positive potentials. Following its activation, the current-voltage relation of the cell displayed a region of negative resistance which crossed the spike threshold. This Ca2+-dependent spike after-current can therefore induce and sustain burst firing in MNCs.

It is believed that the pathogenesis of dengue is generated by a deregulation of the immunological response. Dengue virus-infected monocytes/macrophages are likely to secrete monokines, which play a role in clinical features observed in patients with dengue haemorrhagic fever or dengue shock syndrome. This is a report on a study on 45 individuals presenting clinical and laboratory characteristics of dengue virus infection. During the acute phase of infection, immunophenotyping of peripheral mononuclear leukocytes was carried out in 19 patients and demonstrated a reduced frequency of CD2+ lymphocytes and their CD4+ and CD8+ subsets. Normal ratios were recovered during convalescence. Also, during the acute phase, mononuclear cells proliferated poorly in response to mitogens and dengue antigens as detected by incorporation of radiolabeled thymidine. During convalescence the lymphoproliferative response was re-established. In addition, the presence of circulating cytokines was investigated in the plasma of the same 45 patients. Concentrations of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and soluble tumor necrosis factor receptor (sTNF-Rp75) were found to be significantly elevated in patients when compared to normal controls. The increase in TNF-alpha was correlated with haemorrhagic manifestations and the increase in IL-10 with platelet decay. The data demonstrate that during the acute phase of dengue infection subsets of T lymphocytes are depressed in terms of both rate and function and provide evidence that circulating pro-inflammatory cytokines, such as TNF-alpha, are important in the pathogenesis and severity of dengue. IL-10 may be downregulating lymphocyte and platelet function.

The Fc receptor on NK cells, FcγRIIIA (CD16), has been extensively studied for its role in mediating antibody-dependent cellular cytotoxicity (ADCC). A homozygous missense mutation in CD16 (encoding a L66H substitution) is associated with severe herpesvirus infections in rare patients. Here, we identified a new patient with this CD16 mutation and compared the patient's NK cells to those of the originally reported patient. Patients with the L66H mutation had intact ADCC, but deficient spontaneous NK cell cytotoxicity and decreased surface expression of CD2, a coactivation receptor. Mechanistic studies in a human NK cell line, NK-92, demonstrated that CD16 expression correlated with CD2 surface levels and enabled killing of a melanoma cell line typically resistant to CD16-deficient NK-92 cells. An association between CD16 and CD2 was identified biochemically and at the immunological synapse, which elicited CD16 signaling after CD2 engagement. Stable expression of CD16 L66H in NK-92 cells recapitulated the patient phenotype, abrogating association of CD16 with CD2 as well as CD16 signaling after CD2 ligation. Thus, CD16 serves a role in NK cell-mediated spontaneous cytotoxicity through a specific association with CD2 and represents a potential mechanism underlying a human congenital immunodeficiency.

BACKGROUND

CD56 is a lineage-specific marker of human natural killer (NK) cells. There are conflicts in the literature regarding the role of CD56 as a marker of NK cells in non-human primates. In the present study, we examined the role of CD56 in identifying rhesus NK cells.

METHODS

The immunophenotype of normal macaque and human NK cells was analyzed by two- and three-color flow cytometry. Flow cytometric cell sorting was subsequently used to deplete or purify NK cells; the resulting cell populations were then used in standard chromium release assays of NK lytic function.

RESULTS

In peripheral blood mononuclear cells of the rhesus macaque, CD56 was expressed primarily on cells with the light scatter and immunophenotypic profile of monocytes. Flow cytometric depletion of rhesus CD56(+) monocytic cells did not diminish functional activity against K562 cells, whereas depletion of CD8(+) or CD16(+) lymphocytes completely abrogated functional activity. Three-color flow cytometric analysis of CD8(+), CD16(+) lymphocytes showed that they expressed other markers (CD2, CD7, TIA-1) associated with NK cells, but notably, not CD56.

CONCLUSIONS

These studies demonstrate that CD56 is not suitable as a marker of NK cells in the rhesus macaque.

BACKGROUND

The T-lymphocyte cell-surface molecule, CD2, was the first heterophilic cell-adhesion molecule to be discovered and has become an important paradigm for understanding the structural basis of cell adhesion. Interaction of CD2 with its ligands. CD58 (in humans) and CD48 (in mice and rats), contributes to antigen recognition by T cells. CD2, CD48 and CD58 are closely related members of the immunoglobulin superfamily and their extracellular regions are predicted to have very similar structures. The three-dimensional crystal structure of this region of CD2 has been determined, revealing two immunoglobulin domains with the ligand-binding site situated on an exposed beta sheet in the membrane-distal domain. This GFCC'C" beta sheet is also involved in a homophilic 'head-to-head' interaction in the CD2 crystal lattice, which has been proposed to be a model for the interactions of CD2 with its ligands.

RESULTS

We show that the CD2-binding site on rat CD48 lies on the equivalent beta-sheet of its membrane-distal immunoglobulin domain. By making complementary mutations, we have shown that two charged residues in the CD48 ligand-binding site interact directly with two oppositely charged residues in CD2's ligand-binding site. These results indicate that the amino-terminal immunoglobulin domains of CD2 and CD48 bind each other in the same orientation as the CD2-CD2 crystal lattice interaction, strongly supporting the suggestion that CD2 interacts head-to-head with its ligand. Modelling CD48 onto the CD2 structure reveals that the CD2-CD48 complex spans approximately the same distance (134 A) as predicted for the complex between the T-cell receptor and the peptide-bound major histocompatibility complex (MHC) molecule.

CONCLUSIONS

Our results, together with recent structural studies of CD2, provide the first indication of the specific topology of a cell-adhesion molecule complex. The similar dimensions predicted for the CD2-CD48 complex and the complex between the T-cell receptor and the peptide-bound MHC molecule suggest that one of the functions of CD2 may be to position the plasma membranes of the T cell and the antigen-presenting (or target) cell at the optimal distance for the low-affinity interaction between the T-cell receptor and the peptide-bound MHC molecule.

Accelerated arteriosclerosis has emerged as a major life-threatening complication in long-term survivors of heart transplantation. It has been proposed that accelerated arteriosclerosis is an immune-mediated complication of rejection. We observed a striking endothelialitis in the coronary arteries of two explanted hearts obtained from patients with severe transplant-related accelerated arteriosclerosis. This finding prompted us to review the pathologic changes in the coronary arteries of 23 autopsied patients who had received heart transplants. The infiltrate in these vessels was characterized using immunohistochemical stains for lymphocytes (CD45), macrophages (MAC-387), T lymphocytes (CD45RO), B lymphocytes (L-26), and smooth muscle cells (actin). In addition, a full panel of monoclonal antibodies was used on the fresh-frozen tissue available from one of the two explanted hearts. Ten of the eleven recipients with accelerated arteriosclerosis had a moderate to marked lymphocytic endothelialitis compared to 3 of 14 without transplant-related arteriosclerosis (P less than 0.005). Immunohistochemical staining of the paraffin-embedded material demonstrated that most of the lymphocytes in the subendothelial space of these vessels were T lymphocytes and that this infiltrate was associated with an accumulation of macrophages and a proliferation of smooth muscle cells in the intima. In the explanted heart from which fresh-frozen tissue was available for more detailed cell typing, the T cells marked predominantly as cytotoxic T lymphocytes (CD8+, CD2+). These results suggest that accelerated arteriosclerosis may be mediated, in part, by a cytotoxic T-lymphocyte-directed endothelialitis.

The CD2-like receptor-activating cytotoxic cell (CRACC) is a cell surface receptor of the CD2 family that triggers NK cell-mediated cytotoxicity through an undefined signaling pathway. CRACC contains cytoplasmic tyrosine-based motifs, immunoreceptor tyrosine-based switch motifs, which resemble those found in the NK cell receptor 2B4. In 2B4, these motifs recruit the adaptor signaling lymphocytic activation molecule-associated protein (SAP), which initiates a signaling cascade mediating cytotoxicity. However, CRACC does not recruit SAP. In this study, we demonstrate that, upon activation, CRACC associates with a homolog of SAP, Ewing's sarcoma's/FLI1-activated transcript 2 (EAT-2), in human NK cells. We show that association of EAT-2 induces the phosphorylation of CRACC and that this process is partially reduced by a pharmacological inhibitor of Src kinases. We identify PLCgamma1, PLCgamma2, and PI3K as the major signaling mediators downstream of CRACC/EAT-2 implicated in NK cell-mediated cytotoxicity. Moreover, EAT-2 also associates with 2B4 predominantly in resting NK cells, whereas SAP preferentially binds 2B4 upon activation. These results outline a new signaling pathway that triggers CRACC-mediated cytotoxicity and modulates 2B4-mediated activation.

Glomerulosclerosis in a heterogeneous pattern, ie, focal and segmental glomerulosclerosis (FSGS), is a common endpoint in a variety of settings, including idiopathic FSGS, and scarring secondary to other renal or systemic diseases. These different causes contribute to the diverse clinical outcomes of histological focal sclerosis, and the varying histologic manifestations of sclerosis. Numerous models have been established in the rat that aim to mirror the various elements of human glomerulosclerosis. With the availability of knockout gene technology, many, but not all of these models have been translated to mouse species. This review will focus on the remnant kidney model, the podocyte injury models of puromycin aminonucleoside or adriamycin injection, and examples of newly developed genetic models, such as knockout of CD2 associated protein (CD2AP).

Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn2+ and Cd2+. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn2+ and Cd2+, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn2+ or Cd2+, but this mutation markedly reduces sensitivity to block by Ca2+ and Co2+. The double mutant channel (F385C.N388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.

1. Membrane currents were recorded from voltage-clamped, microelectrode-impaled cells of the NG108-15 mouse neuroblastoma x rat glioma clonal cell line, differentiated with prostaglandin E1. 2. A slow outward tail current reversing at post-pulse potentials between -80 and -90 mV was evoked by depolarizing pre-pulses to near 0 mV. The tail current was inhibited by Cd2+ ions (0.2-1 mM) and hence attributed to activation of a Ca2+-dependent K+ current by a priming voltage-activated Ca2+ current. 3. Two components to this tail current could be distinguished pharmacologically: an early (less than or equal to 50 ms) component inhibited by 1-5 mM-tetraethylammonium (TEA), and a late component lasting several hundred milliseconds inhibited by apamin (0.1-0.4 microM) or d-tubocurarine (0.1-0.5 mM). 4. Ionophoretic injection of Ca2+ ions evoked a transient outward current with an apparent reversal potential (from ramped current-voltage curves) of -70 mV. This current was succeeded or sometimes replaced by an inward current with an apparent reversal potential between -20 and -10 mV. 5. The outward current induced by Ca2+ injections was unaffected or partly inhibited by TEA (1-5 mM), but was strongly inhibited by apamin or d-tubocurarine. 6. Hyperpolarizing voltage steps from between -30 and -40 mV induced inward current relaxations reversing at between -80 and -90 mV. These were considered to result from deactivation of the voltage-dependent sustained K+ current, IM. 7. Application of methacholine, muscarine or Ba2+ ions produced an inward current, reduced input conductance and reduced IM deactivation relaxations. 8. It is concluded that differentiated NG108-15 cells possess several of the K+ currents present in sympathetic neurones, including a delayed rectifier current, two species of Ca2+-activated K+ current and the M-current.

1. Calcium currents and intramembrane charge movements were measured in cut twitch muscle fibres of the frog and the time course of activation of the current was studied using various conditioning pulse protocols. 2. When a conditioning activation was produced by a depolarizing pulse which ended before inactivation occurred, a subsequent depolarization led to a faster onset of activation, indicating that the system had not completely returned to the initial state during the interval between the two pulses. 3. The interval between conditioning and test pulse was varied at different subthreshold potentials to study the time course of restoring the steady-state conditions. Complete restoration required a waiting period of about 1 min at the holding potential of -80 mV due to a very slow process but partial recovery was reached within 100 ms. This initial recovery process was strongly voltage dependent and became considerably slower when the interval potential approached the threshold for current activation. 4. Stepping to a roughly 10 mV subthreshold potential without applying a conditioning activation caused no change in the time course of the current produced by a subsequent test depolarization. Depolarizing just to the current threshold caused a slowly progressing acceleration of test current activation. 5. The peak current-voltage relation in the fast gating regime caused by a conditioning activation coincided with the current-voltage relation measured under steady-state conditions, indicating not that a new channel population had become activated but that the same channels showed a different gating behaviour. 6. Intramembrane charge movements measured in 2 mM-Cd2+ and tested at potentials between -40 and +40 mV showed negligible changes when preceded by a strong depolarization. 7. We discuss several possible models which can explain the fact that the current is speeded up by a conditioning activation while the charge movements remain unchanged. It is possible that the fast voltage-dependent transition which becomes visible after conditioning pulses reflects a rapid conformational change of the Ca2+ channel molecule which also occurs during its normal gating mode but remains undetectable in terms of conductance. In view of the hypothesis that the Ca2+ channel molecule forms a voltage sensor for excitation-contraction coupling this fast transition could be coupled to the control of Ca2+ release from the sarcoplasmic reticulum.

TRPM7 is a member of the melastatin-related subfamily of TRP channels and represents a protein that contains both an ion channel and a kinase domain. The protein is ubiquitously expressed and represents the only ion channel known that is essential for cellular viability. TRPM7 is a divalent cation-selective ion channel that is permeable to Ca2+ and Mg2+, but also conducts essential metals such as Zn2+, Mn2+, and Co2+, as well as nonphysiologic or toxic metals such as Ni2+, Cd2+, Ba2+, and Sr2+. The channel is constitutively open but strongly downregulated by intracellular levels of Mg2+ and MgATP and other Mg-nucleotides. Reducing the cellular levels of these regulators leads to activation of TRPM7-mediated currents that exhibit a characteristic nonlinear current-voltage relationship with pronounced outward rectification due to divalent influx at physiologically negative voltages and monovalent outward fluxes at positive voltages. TRPM7 channel activity is also actively regulated following receptor-mediated changes in cyclic AMP (cAMP) and protein kinase A activity. This regulation as well as that by Mg-nucleotides requires a functional endogenous kinase domain. The function of the kinase domain is not completely understood, but may involve autophosphorylation of TRPM7 as well as phosphorylation of other target proteins such as annexin and myosin IIA heavy chain. Based on these properties, TRPM7 is currently believed to represent a ubiquitous homeostatic mechanism that regulates Ca2+ and Mg2+ fluxes based on the metabolic state of the cell. Physiologically, the channel may serve as a regulated transport mechanism for these ions that could affect cell adhesion, cell growth and proliferation, and even cell death under pathological stress such as anoxia.

The epidermis of clinically normal-appearing human skin harbors a phenotypically heterogeneous population of T lymphocytes (TCs), the majority of which are CD2+/CD3+/CD5+ "memory" cells, but in an unactivated state, and express the TCR-alpha/beta. In contrast to murine skin, only a very minor subpopulation of CD3+ cells in the human epidermis bears the TCR-gamma/delta. Epidermal TCs primarily are distributed along the rete ridges in the basal keratinocyte layer and are often in close apposition to Langerhans cells (LCs). These TCs were propagated from epidermal cell suspensions after stimulation with TC activating agents (Con A, rIL-1, rIL-2), then evaluated for phenotypic features and TCR diversity. Similar to the in situ situation, most were CD4-/CD8+/TCR-alpha/beta+. In addition, two cultures contained TCR-gamma/delta+ cells; one of these determined to be an adherent CD4-/CD8+ population. Epidermal TCs were significantly (p less than 0.0001) more abundant in the sole than in the other body regions examined (i.e., 40 vs. 7 CD3+ cells/linear centimeter of epidermis) and seemed to have a particular affinity for the acrosyringial epithelium of eccrine sweat ducts. Moreover, the sole usually contained a greater number of CD8+ relative to CD4+ TCs, whereas the epidermal CD4/CD8 ratio in the trunk and extremities was quite variable, although the trend also was towards a slightly larger percentage of CD8+ cells. Collectively, our data suggest that the volar epidermis has a unique microenvironment which is responsible for both the higher density of TCs, preferentially CD8+, and lower number of LCs. This study has not only provided evidence for significant regional variability in the human epidermal TC population of normal skin, but also strengthens the concept for skin-associated lymphoid tissues (SALT), whereby memory TCs recirculate back to the epidermis and interact with resident antigen-presenting cells (i.e., LC).

Human natural killer (NK) cells are defined as being membrane CD3-, CD16+, and/or CD56+ lymphocytes; however, little is known about the ontogenic development and maturational pathways of human NK cells. The functional, phenotypic, and maturational characteristics of human umbilical cord blood (CB) NK cell subsets were studied to gain insight into the ontogenic and maturational pathways of human NK cells. We have previously shown that there is a novel subset of CD16+ CD56- NK cells present in CB. Here we further demonstrate differences in the expression of the NK-associated molecules <em>CD2</em>, CD7, CD8, and <em>CD2</em>5 between CB and peripheral blood (PB) NK cells and between CB NK cell subsets. Although CB NK cell subsets were deficient in or had less lytic activity against K562 cells compared to PB NK cells, CB NK cells did possess the lytic molecules perforin and granzyme B and when artificially stimulated to secrete their granules during lytic assays, were capable of lytic activity equivalent to that of PB NK cells. Regardless of differences in phenotype and function of CB NK cell subsets, short-term and long-term incubation with cytokines induced functional (adult-like NK activity) and phenotypic (adult-like CD16+56+ or CD16-56+ surface antigen phenotype) maturation, respectively. Interleukin-2 (IL-2), IL-12, and IL-15, but not IL-7, interferon-gamma (IFN-gamma) nor tumor necrosis factor-alpha (TNF-alpha) induced functional and phenotypic maturation of CB NK cell subsets. Interestingly, culture of CB NK cell subsets with IL-2 or IL-15 led to acquisition of predominantly a CD16+56+ phenotype, while culture with IL-12 led to acquisition of both CD16+56+ and CD16-56+ phenotypes. Both functional and phenotypic maturation were not dependent upon proliferation. Studies using neutralizing anti-IFN-gamma and anti-TNF-alpha antibodies showed that survival and phenotypic maturation upon cytokine stimulation is influenced by endogenous production of TNF-alpha but not IFN-gamma. These results demonstrate that CB NK cell subsets are functionally and phenotypically immature but are capable of maturation. Additionally, CD16+56- NK cells are implicated as possible precursors of mature CD16+56+ and CD16-56+ NK cells.

We have previously suggested that sulfated polysaccharides could be used in a vaginal formulation to inhibit infection by human immunodeficiency virus (HIV-1). This supposition was based on studies in which we developed and employed an in vitro model to simulate the mechanism of HIV-1 transmission during coitus. We found that adhesion of mononuclear cells to epithelia was the initial step in infection and speculated that blocking adhesion would prevent HIV-1 transmission. We observed that certain sulfated polysaccharides prevented adhesion of lymphoma cell lines to epithelial cell lines, which were derived from the genital tract, in concentrations of a few milligrams per milliliter; and we theorized that sulfated polysaccharides could thus be used as active ingredients in a topical "microbicide." In the present in vitro study, evidence is presented that a number of sulfated polysaccharides, including carrageenan, dextran sulfate, heparin, fucoidan, and pentosan polysulfate, are capable of blocking infection by mechanisms other than adhesion at concentrations of a thousand times lower than the dosages that are needed to block cell adhesion. One of these compounds, iota carrageenan, is capable not only of blocking infection of epithelia at concentrations of 1-2 micrograms, but of blocking adhesion to a far greater extent than the other sulfated polysaccharides tested. For this reason, as well as for considerations of safety, stability, and gelling properties, we suggest that iota carrageenan may be the best choice of the sulfated polysaccharides tested for use as a vaginal microbicide. The same in vitro model was employed to decipher the cell surface molecules involved in lymphocyte-to-epithelial adhesion. To accomplish this, we screened for the presence of cell adhesion molecules (CAMs), carbohydrates, proteoglycans, and carbohydrate-binding sites. HIV-1-infected lymphocytic cells expressed a CAM profile typical of activated, infected cells (e.g., HLA-DR+, CD4-, LFA-1+, ICAM-1+, LFA-3+, <em>CD2</em>+) whereas epithelia expressed few CAMs (LFA-3, ICAM-1, VLA-5, CD44, <em>CD2</em>6, sLEX). Both cell types expressed heparan sulfate and chondroitin sulfate proteoglycans. A variety of sugars (mannose, fucose, galactose, Nac-galactosamine, Nac-glucosamine) were also present, but these cells expressed few carbohydrate-binding sites; lymphocytes bound beta-galactose. We were unable to block the adhesion with anti-CAM antibodies or with exogenous sugars. When enzymes were used against sulfated cell surface molecules, chondroitinase was found to block the adhesion. Our evidence suggests that this CAM-independent adhesion may be a lectin-glycosaminoglycan interaction.