Defective expression of p56lck in an infant with severe combined immunodeficiency.

Severe combined immune deficiency (SCID) is a heterogeneous disorder characterized by profound defects in cellular and humoral immunity. We report here an infant with clinical and laboratory features of SCID and selective CD4 lymphopenia and lack of CD28 expression on CD8(+) T cells. T cells from this patient showed poor blastogenic responses to various mitogens and IL-2. Other T cell antigen receptor- induced responses, including upregulation of CD69, were similarly inhibited. However, more proximal T cell antigen receptor signaling events, such as anti-CD3 induced protein tyrosine phosphorylation, phosphorylation of mitogen-associated protein kinase, and calcium mobilization were intact. Although p59fyn and ZAP-70 protein tyrosine kinases were expressed at normal levels, a marked decrease in the level of p56lck was noted. Furthermore, this decrease was associated with the presence of an alternatively spliced lck transcript lacking the exon 7 kinase encoding domain. These data suggest that a deficiency in p56lck expression can produce a SCID phenotype in humans.

Sulindac-induced aseptic meningitis.

A 22-year-old woman with systemic lupus erythematosus experienced generalized pruritus, shortness of breath, pleuritic chest pain, visual blurring, severe photophobia, a stiff neck, an occipital headache, and a temperature of 39.4 degrees C within one hour after taking sulindac (Clinoril). Findings from a CSF examination disclosed a notable elevation of protein and a polymorphonuclear pleocytosis. All symptoms disappeared within 24 hours. Inhibition of prostaglandin synthesis did not seem to be the mechanism of this adverse reaction, since the patient tolerated aspirin.

Substance P does not alter interleukin-1 expression by splenic or granuloma macrophages in murine schistosomiasis.

Substance P (SP) is an undecapeptide with neurotransmitter and immunoregulatory properties. In murine schistosomiasis, ova naturally induce liver and intestinal granulomas. These granulomas contain macrophages, and eosinophils that produce SP. A report showed that human blood monocytes isolated by adherence release interleukin-1 (IL-1) in response to SP (Lotz et al. (1989) Science 241, 1218). IL-1 is important for initiation of hypersensitivity granulomas. Therefore, it was determined whether SP modulates granuloma macrophage IL-1 production in murine schistosomiasis. Macrophages were obtained from lung and liver granulomas, and from spleens of infected mice. A thymocyte proliferation assay measured IL-1 activity in culture supernatants. Total RNA, extracted from macrophages, was assayed for IL-1 alpha and beta mRNA by Northern blotting using cDNA probes. In response to lipopolysaccharide (LPS), splenic macrophages and macrophages from young lung granulomas released appreciable IL-1. Macrophages from liver granulomas, that were lesions older than the lung granulomas, were unresponsive to LPS with regard to IL-1 secretion. Yet, granuloma macrophages spontaneously expressed IL-1 alpha and beta mRNA. LPS enhanced IL-1 mRNA expression in both splenic and granuloma macrophages. Exposure of macrophages from all sources to SP did not alter IL-1 secretion or gene expression. Similarly, the responsiveness of macrophages to LPS was not affected by concomitant exposure to SP. It is concluded that, in the murine system, SP does not directly influence splenic or granuloma macrophage IL-1 secretion or gene expression. Also, it appears that macrophage secretion of IL-1 is rapidly down-regulated following granuloma elicitation.

Alteration of human accessory cell function by heat treatment: role of IL-1 and class II MHC antigens.

Heat-treated monocytes (1 hr, 45 degrees C) cannot present soluble antigen or mitogen to purified autologous T cells. This is despite normal viability and normal expression of class II MHC antigens. They do not secrete IL-1 nor stimulate secretion of IL-2 by T cells. Addition of exogenous IL-1 or IL-2 does not, however, reconstitute the response to soluble antigen. Furthermore, even after overnight pulsing with antigen prior to heat treatment under circumstances in which the antigen is known to be appropriately processed, stimulation of T-cell proliferation still does not occur. Thus there appear to be at least two discrete lesions produced by heating: failure of IL-1 production, per se, and intrinsic failure to present previously processed antigen. It is also hypothesized that heat treatment may produce alterations in Ia molecules which specifically disallow transduction of the proliferation signal to T cells.

Complete heart block in association with graft-versus-host disease.

An infant who received haploidentical BM for severe combined immunodeficiency (SCID) developed acute, reversible complete heart block in association with an exacerbation of GVHD. Respiratory distress and myocardial dysfunction were also seen with this and previous GVHD exacerbations. The patient had not received chemotherapy or radiation prior to BMT. The complete heart block resolved after 1 week of intensive immunosuppression. The association of complete heart block with GVHD is important because the heart block is potentially reversible with prompt, aggressive control of the GVHD.

Clinical evaluation of ketorolac tromethamine 0.5% ophthalmic solution for the treatment of seasonal allergic conjunctivitis.

We evaluated 148 patients with allergic conjunctivitis in a double-masked, paired comparison clinical trial comparing ketorolac 0.5% ophthalmic solution with vehicle. Patients received one drop of each study medication in preassigned eyes, four times a day, for seven days. Both treatments showed significant changes from baseline in the signs and symptoms associated with allergic conjunctivitis. Evaluations at the final visit (day 7 or 8) showed that ketorolac-treated eyes had a significant treatment response when compared to vehicle-treated eyes for conjunctival inflammation (p = 0.010), ocular itching (p = 0.006), swollen eyes (p = 0.002), discharge/tearing (p = 0.021), foreign body sensation (p = 0.035), and conjunctival injection (p = 0.016). Mean scores evaluating the overall therapeutic effect of the study treatments at the completion of the study were higher for ketorolac-treated eyes than for vehicle-treated eyes as rated by investigators (p = 0.004) and study patients (p < 0.001). Results of this study confirmed the trends of a previous study showing that ketorolac 0.5% ophthalmic solution applied topically is an effective therapy for allergic conjunctivitis.

Effect of a single ethanol exposure on HIV replication in human lymphocytes.

BACKGROUND: Alcoholism is known to cause perturbations in cellular and humoral immunity, and some data suggest that acute alcohol ingestion enhances HIV replication in the lymphocytes of drinkers. METHODS: To study the acute effects of alcohol ingestion on HIV replication, oral ethanol (1 g/kg) was administered to 12 healthy volunteers in a controlled clinical setting. In vitro replication of HIV in the subjects' cultured lymphocytes and changes in lymphocyte phenotypes were evaluated. RESULTS: Statistically significant increases in peripheral lymphocytes and natural killer cell numbers were identified after the initial ethanol trial. HIV replication also increased in the isolated lymphocytes of some subjects after ethanol ingestion, but most subjects in the second trial showed essentially no changes in any of these parameters. CONCLUSIONS: Our results are consistent with either a subtle, study-induced stress-related enhancement in HIV replication or significant individual variation in response to ethanol. The results do not provide evidence for a general increase in HIV replication in the lymphocytes of subjects following a single in vivo ethanol dose of 1 g/kg.

Immune dysfunction in refractory sinusitis in a tertiary care setting.

OBJECTIVE: To examine the contribution of the primary immunodeficiency states, which are uncommon in the general population, to refractory sinusitis. STUDY DESIGN: We retrospectively reviewed the charts of 316 patients with sinusitis who were referred to the Allergy and Immunology Clinic for immunological evaluation from 1991 to 1997. METHODS: Of the 316 patients, 79 were selected for further study. Inclusion criteria included at least one sinus surgery and/or sinusitis diagnosed by endoscopy and/or computed tomography (CT) scan at least three times in the previous year. Patients with human immunodeficiency virus (HIV), allergic fungal sinusitis, cystic fibrosis, and primary ciliary dyskinesia were excluded. The results of their immunological evaluation for atopy, T-lymphocyte function, and immunoglobulin levels were examined. RESULTS: The average age of these 79 patients was 44 years (+/- 14.5 standard deviation [SD]). They had, on average, 2.94 (+/- 2.19 SD) previous operations and had mean sinus CT scores (Lund-McKay) of 11.2 (+/- 5.0 SD). Forty of 79 (50.6%) patients had at least one positive result on skin test to an aeroallergen. Delayed hypersensitivity skin testing revealed that 22 of 55 patients (40%) were anergic. Of the 60 patients with in vitro T-lymphocyte function testing, 54.8% showed abnormal proliferation in response to recall antigens, 11.3% had decreased response to alloantigen, and 26.3% demonstrated decreased response to T-cell mitogens. Determination of quantitative immunoglobulins showed low immunoglobulin G in 14 of 78 patients (17.9%), low immunoglobulin A in 13 of 78 (16.7%), and low immunoglobulin M in 4 of 78 (5.1%). Common variable immunodeficiency (CVID) was diagnosed in 9.9% of patients, and selective IgA deficiency was found in 6.2%. CONCLUSIONS: This retrospective review reveals an unexpectedly high incidence of immune dysfunction. These results suggest that immunological testing should be an integral part of the evaluation of patients with refractory sinusitis.

TH1 cytokine response of CD57+ T-cell subsets in healthy controls and patients with alcoholic liver disease.

Patients with chronic inflammatory diseases, including Crohn's disease and rheumatoid arthritis, as well as those with certain viral infections, and patients who are transplant recipients or who have certain hematologic malignancies have been observed to have CD57+ T cell expansion in both CD4+ and CD8+ subsets. We have reported previously that alcoholic patients also have CD57+ T cell expansion. Because many alcoholics become seriously deficient in cell-mediated immunity, it is of interest to determine whether the expanded CD57+ subsets can respond to stimulation with normal T helper cell subtype 1 (TH1) cytokine production. We report evaluation of the CD57 T-cell subsets of patients with alcoholic liver disease (ALD) with the use of cytoplasmic staining after stimulation through the T-cell receptor (TCR). The CD57+ subsets of the T cells of both healthy individuals and patients with ALD express significantly higher amounts of cytoplasmic tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) after 6 h of stimulation than do the CD57- subsets. This increased production can persist up to 46 h of continuous stimulation. Under these assay conditions, very little cytoplasmic interleukin (IL)-4 is observed in the T cells of either healthy control subjects or patients with ALD. Measurement of cytokine secretion by sort-purified CD57 T-cell subsets with the use of enzyme-linked immunosorbent assay (ELISA) shows that the CD57+ T-cell subset produces 18- to 30-fold more TNF- and IFN-, respectively, than does the CD57- subset in the first 12 h of stimulation. This response requires only stimulation through the TCR for the CD57+ subset, whereas significant secretion by the CD57- subset requires added IL-2 or anti-CD28 antibody. These results are consistent with the concept of the CD57+ T-cell subset as a differentiated effector cell and demonstrate that patients with ALD who are not drinking at the time of evaluation have normal or increased immediate TH1 T-cell responses.

Natural killer cell function in women with vestibulitis.

OBJECTIVE: To assess natural killer (NK) cell activity in patients with vestibulitis. STUDY DESIGN: Twenty-two patients who met the International Society for the Study of Vulvar Disease criteria for vestibulitis and 17 age-, sex- and race-matched controls were recruited. NK cell activity was examined using a standard, four-hour 51Cr-release assay, freshly and after stimulation with interleukin 2 (IL2) or alpha interferon (IFN alpha). RESULTS: The subject samples had significantly decreased fresh NK cell activity (mean lytic units [LU]/10(6) peripheral blood leukocytes [PBLs] of 0.93 vs. 4.19, P < .001). This activity was augmented in response to either IFN or IL2. However, it remained significantly lower than in the control samples (12.07 vs. 20.6 LU/10(6) PBL, P = .007 for IL2 and 5.98 vs. 15.33 LU/10(6) PBL, P < .001 for IFN). This difference was not universal since the major histocompatibility-nonrestricted T killer cell activity of the subject samples was not significantly different from that in the control samples. CONCLUSION: This pilot study suggests that patients with vulvar vestibulitis have markedly decreased NK cell activity. Although this activity is increased in response to IL2 or IFN, it remains significantly impaired in comparison to the control samples.

Lymphokine-activated killer (LAK) cells. I. Differential recovery of LAK, natural killer cells, and cytotoxic T lymphocytes after a sublethal dose of cyclophosphamide.

The effect of a sublethal dose of cyclophosphamide (CTX) administered in vivo on murine natural killer (NK) cells, lymphokine-activated killer (LAK) cells, and cytotoxic T lymphocytes (CTL) was examined. It was found that such a dose of CTX abolished all of the killer cell responses examined. The recovery of each response was then examined as a function of time. Allogeneic CTL responses were the first to recover and could be generated from spleen cells obtained 6 days after CTX administration. Fresh NK cell activity recovered by days 9 to 12. However, when spleen cells obtained 12 days after CTX administration were cultured with interleukin 2 (IL 2) for 5 days, they lost the ability to lyse YAC-1 cells, suggesting a distinction between "augmented" and "fresh" NK cell activity. H-2-restricted, trinitrophenyl-specific CTL activity could be generated by day 14 after CTX, provided the cultures were supplemented with exogenous IL 2. LAK could not be induced until day 21 post-CTX treatment. These data suggest that LAK precursors, CTL precursors, and NK cells are distinct cell populations. Additionally, this report introduces a model that may be useful in examining the differential contribution of NK and LAK to successful adoptive tumor immunotherapy.

Limitations of recognition of modified-self by alloantigen-activated cytotoxic T lymphocytes.

Alloantigen-activated CTL from CBA (H-2k), but not from C57BL/6 (H-2b), mice were able to lyse autologous cells modified by incubation with TNP-HSA. Moreover, allogeneic CTL of the H-2k haplotype lysed syngeneic cells modified with high or low concentrations of TNBS. Allogeneic CTL of the H-2b haplotype, however, did not lyse autologous cells modified with low concentrations of TNBS, even though they lysed syngeneic cells modified with high concentrations of TNBS. These limitations of recognition by the different H-2 haplotypes paralleled the restrictions evident for MHC-restricted, TNBS-specific CTL. It was thus apparent that alloantigen-activated CTL are governed by the same limitations of recognition operant for the MHC-restricted, TNBS-specific CTL.

Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation.

Lymphocyte proliferation and differentiation result from ordered cellular interactions governed by soluble products (lymphokines). Dissecting the individual steps in these processes has been difficult, due to a paucity of pure lymphokines. Recently, it was reported that the derivatized ribonucleoside 8-mercaptoguanosine (8MGuo) has both mitogenic and differentiative effects on murine B cells. In the present studies, we tested 8MGuo for its ability to stimulate both B and T cell responses. In contrast to the murine studies, 8MGuo does not stimulate rat B cells to proliferate and, when tested for B cell growth factor-like activity, no stimulation was observed. The addition of 8MGuo (0.5 to 1 mM final concentration) to mitogen-stimulated B cells led to a marked increase in IgM and a modest increase in IgG secretion. When mixed with conditioned medium, 8MGuo acted synergistically in stimulating secretion of both isotypes, arguing that 8MGuo has both B cell-differentiating factor-mu (BCDF-mu) and BCDF-gamma activity. 8MGuo had no IL 2-like activity when tested on a mouse IL 2-dependent cell line, and no IL 1-like activity on addition to mouse thymocytes with or without submitogenic doses of lectin. However, when added to cultures of murine allogeneic cells in which the stimulating cell populations had been heat-inactivated, 8MGuo induced the generation of specific allogeneic cytotoxic T lymphocytes. Together, these results suggest that a simple derivatized nucleoside can induce both T and B cell differentiation without concomitant proliferation, and thus represent a unique probe for studying events in lymphocyte differentiation.

Murine natural killer cells express the Ly24 (Pgp-1) marker on their surface.

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24- T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24- T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK1.1- strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.

Lymphokine-activated killer (LAK) cells. III. Characterization of LAK precursors and susceptible target cells within the murine thymus.

In our search for a biologic role for lymphokine-activated killers (LAK), we examined their generation from murine thymocytes. Normal adult thymocytes were capable of generating LAK upon culture with relatively large doses (500 to 1000 U/ml) of interleukin 2. Normal thymocytes were fractionated into four subsets by virtue of their co-expression of the Lyt-2 and L3T4 markers: Lyt-2+ L3T4+ (2+4+); 2+4-; 2-4+; and 2-4-. None of these subsets had any natural killer activity. Upon examining the ability of these subsets to generate LAK, it was found that the 2-4- subset was the most potent and required the smallest relative amounts of interleukin 2. In addition to lysing tumor cells, thymus-derived LAK were capable of killing "fresh" 2+4+ thymocytes. Fresh 2+4-, 2-4+, 2-4-, and cortisone-resistant thymocytes were resistant to lysis by LAK. Upon mitogen stimulation, however, the cortisone-resistant thymocytes and 2+4- thymocytes became LAK-susceptible. These data demonstrate a possible mechanism for the elimination of the 2+4+ thymocyte subset which is generally believed to be a "dead-end" population. Moreover, these data suggest a possible biologic role for LAK in the process of thymocyte maturation and intrathymic selection.

Lymphokine-activated killer cells. VII. IL-4 induces an NK1.1+CD8 alpha+beta- TCR-alpha beta B220+ lymphokine-activated killer subset.

Murine IL-2-induced lymphokine-activated killers (LAK) can be divided into two mutually exclusive subsets: NK1.1+CD8- and NK1.1-CD8+. We have previously established that the lytically active subset of each of these two populations expressed B220, as determined by the mAb 6B2, on its surface. In this study, we examined the lytically active subsets induced by IL-4. We found that, similar to IL-2, the lytically active IL-4-LAK expressed B220 on their surface. Similar to IL-2-LAK, IL-4-LAK cultures also contained NK1.1+CD8- B220+, and NK1.1-CD8+B220+ subsets. IL-4-LAK cultures, however, contained two novel and unexpected lymphocyte subsets determined by their surface markers to be either: i) NK1.1+CD8 alpha+beta-B220+, or ii) NK1.1-CD8 alpha+beta-B220+. These subsets were not derived from NK1.1+ or CD8+ precursors but were induced from a CD4-CD8-NK1.1-Slg- splenocyte subpopulation. Most of the CD8 alpha+beta- cells seen in the IL-4 cultures expressed TCR-alpha beta with little or no TCR-gamma delta detected on their surface. Similar to IL-2-LAK, the IL-4-LAK subsets appeared to display a bias as to their susceptible target cells with the NK1.1-CD8 alpha+beta+ subset being most potent against trinitrophenyl-modified autologous lymphoblasts (2,4,6-trinitrobenzene sulfonic acid (TNBS)-self). The NK1.1+CD8 alpha-beta- effectors were most potent against YAC-1 and CL27A with little activity against TNBS-self. All three targets were susceptible to lysis by the NK1.1+CD8 alpha+beta- subset. These findings document the characteristics of a novel lymphocyte subset, derived from splenic precursors, which shares certain features with cells previously thought to be present exclusively in intraepithelial lymphocytes.

Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation.

Guanine ribonucleosides, substituted at the C8 position with either a bromine or a thiol group, have recently been shown to regulate several immunologic responses. We have previously shown that 8-mercaptoguanosine (8MG) can replace the requirement for cytokines in the generation of MHC-restricted CTL. In this paper, we examined the ability of 8MG to induce MHC-nonrestricted killer cells. We found that 8MG did not induce significant lytic activity from normal resting lymphocytes. However, 8MG was able to synergize with minimal amounts of IL-2 in inducing lytic activity similar to lymphokine-activated killers (LAK) in that both NK-sensitive and NK-resistant tumor cells were killed. Both the precursors and effectors of 8MG-LAK activity were similar to NK cells and were CD4- CD8- asialo-GM1+ NK1.1+. Similar to IL-2-induced LAK, 8MG-LAK were B220+. 8MG appeared to "stage" these precursor lymphocytes to become more responsive to IL-2 because optimal induction of 8MG-LAK required preincubation with 8MG before the addition of IL-2. This "staging" appeared to be due to the release of a "second signal" since it was readily inhibited by cyclosporine A. Anti-IFN-alpha beta was as efficient as cyclosporine A in inhibiting 8MG-LAK generation, whereas anti-IFN-gamma and anti-IL-1 did not exhibit significant inhibition. These findings suggest that 8MG can be of possible utility as an IL-2-sparing agent in LAK generation from NK cells.

Lymphokine-activated killer (LAK) cells. IV. Characterization of murine LAK effector subpopulations.

The precursors of murine lymphokine-activated killers (LAK) can be divided into two major subsets: NK-like (CD8-, NK1.1+, asialo GM1+) and T-like (CD8+, NK1.1-, asialo GM1+). LAK effectors have generally been characterized as being either CD8+ or NK1.1+. In this study, we divided each of these effector subsets further by virtue of their expression of B220 (as defined by the mAb 6B2) and Ly-24 (Pgp-1). Freshly obtained CD8+ and NK1.1+ cells were found, by fluorescence analysis, to be B220-. Lytically active LAK effector subsets were either CD8+ B220+ Ly-24+ or NK1.1+ B220+ Ly-24+. Most interestingly, a distinct NK1.1+ B220- Ly-24+ subset existed but had minimal lytic activity, suggesting that only a subset of NK cells is capable of acquiring the broad lytic activity of LAK. The acquisition of the B220 marker by the CD8+ subset closely paralleled its expression of lytic activity. However, classical MHC-restricted CD8+ CTL were Ly-24+ but remained B220- suggesting that the acquisition of the B220 marker, as defined by the 6B2 mAb, is not merely the result of cellular differentiation but may serve as a marker of MHC-nonrestricted killers. Three "classes" of target cells were examined for their susceptibility to lysis by the LAK effector subsets: YAC-1 (NK sensitive), CL27A (NK resistant), and autologous lymphoblasts that have been modified with 2, 4, 6-trinitrobenzene sulfonic acid. YAC-1 was lysed exclusively by the NK1.1+ B220+ Ly-24+ subset, 2,4,6-trinitrobenzene sulfonic acid-self was lysed exclusively by the CD8+ B220+ Ly-24+ subset whereas CL27A was lysed by both subsets. This pattern of lysis was confirmed by the in vivo depletion of NK1.1+ cells. It, therefore, appears that LAK effector subsets may be more selective in their lytic repertoire than previously thought.

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.