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The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.

Efalizumab is an mAb directed against CD11a, a molecule involved in T-cell activation and extravasation from blood into tissue. Ten patients with severe atopic dermatitis were treated with efalizumab for 84 days, and peripheral blood mononuclear cells were analyzed for expression of activation and adhesion markers. Efalizumab treatment led to decreases in CD11a mean fluorescence intensity (MFI) on naive, central memory, and effector memory CD4+ and CD8+ T cell subsets. MFI for CD18 was decreased in both CD4+ and CD8+ T cells. Percentages of cells positive for cutaneous lymphocyte antigen (CLA) were increased fourfold in all CD4+ and CD8+ T cell subsets. Increases in the percentages of CD4+ and CD8+ T cells expressing beta7 and CD49d were also observed. No significant changes were observed in the percentages of CD4+ and CD8+ T cells that produced either IFN-gamma or IL-4. In summary, efalizumab treatment resulted in (i) decreases in CD11a and CD18 expression in all circulating T-cell subsets and (ii) increases in the percentages of blood T cells expressing tissue homing markers (CLA, beta7, CD49d). These data suggest that blockade of T-cell extravasation into tissue is the major pathway by which efalizumab leads to improvement in cutaneous inflammation.

Background: Patients with B-cell chronic lymphocytic leukemia (B-CLL) often demonstrate variable responses to similar treatments. It would be highly desirable to develop a personalized therapeutic strategy for selection of appropriate drugs or regimens based on the drug sensitivity profiles of leukemic cells from individuals., Methods: We applied a multiparameter flow cytometric drug cytotoxicity assay to evaluate drug effects specifically on B-CLL cells from 43 individuals after leukemic cells were incubated in vitro with fludarabine, chlorambucil, cladribine, or prednisolone., Results: We demonstrated that different B-CLL cell populations from 43 individuals showed a marked variability in drug sensitivity. In vitro resistance to fludarabine was greatest in B-CLL cells with deletions of p53, a cytogenetic abnormality that is almost invariably associated with a poor therapeutic response clinically., Conclusions: In vitro drug sensitivity profiles analyzed by a multiparameter flow cytometric cytotoxicity assay may serve as a tool to facilitate individualized selection of appropriate drugs for treatment in B-CLL. Prospective trials will be needed to validate the clinical utility of this flow cytometric cytotoxicity assay., (Copyright 2007 Clinical Cytometry Society.)

This manuscript describes a new technique for the microbiological cloning of chlamydia-infected cells using a fluorescence activated cell sorter (FACS). The approach exploits chlamydial acquisition of the fluorescent, Golgi-specific, stain 6-((N-7-(-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosine (C6-NBD-cer). This fluorescent lipid is delivered from the Golgi apparatus to the chlamydial inclusion membrane and then to the developmental forms within the inclusion in living, infected cells. Labeling with C6-NBD-cer results in easily identifiable chlamydial inclusions that can then be analyzed and sorted by FACS. This technique was used successfully to sort individual chlamydia-infected cells into individual wells of a culture dish and, in this experimental system, resulted in the isolation of cloned chlamydial isolates. FACS-based sorting was used to isolate clonal populations of prototype strains from Chlamydia trachomatis, C. caviae and C. suis. Recent clinical isolates were also successfully cloned using FACS. The procedure is simple and rapid, with single cloning cycles being completed 24 h post-culture of a sample. It is anticipated that FACS-based sorting of live chlamydia-infected cells will be a significant technical tool for the isolation of clonal populations of any chlamydial strain.

The Columbia basin subpopulation of pygmy rabbit Brachylagus idahoensis was listed as endangered by the United States Fish and Wildlife Service in November 2001, and no pygmy rabbits have been seen in the wild since spring 2002. Captive propagation efforts have attempted to increase population size in preparation for reintroduction of animals into central Washington. Disseminated mycobacteriosis due to Mycobacterium avium has been the most common cause of death of adult captive pygmy rabbits. Between June 2002 and September 2004, mycobacteriosis was diagnosed in 28 captive adult pygmy rabbits (representing 29% of the captive population), in contrast to 18 adult pygmy rabbits dying of all other causes in the same time period. Antemortem and postmortem medical records were evaluated retrospectively to describe the clinical course of mycobacteriosis in pygmy rabbits, physical examination findings, and diagnostic test results in the diagnosis of mycobacteriosis in pygmy rabbits. Various treatment protocols, possible risk factors for mortality, and recommendations for prevention of mycobacteriosis were evaluated also. Compromised cell-mediated immunity appears to be the best explanation at this time for the observed high morbidity and mortality from mycobacterial infections in pygmy rabbits.

Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the lambda1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/lambda1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward lambda1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/lambda1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/lambda1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.

Since Zap-70 expression in chronic lymphocytic leukemia (CLL) cells correlates with a lack of somatic mutation of the immunoglobulin variable heavy chain (IgVH) genes, it has been proposed as a surrogate marker for disease prognosis. However, published studies of Zap-70 expression have used different commercial antibodies and analytic strategies. This study was undertaken to determine if any strategy was broadly applicable in a clinical flow cytometry laboratory. Expression of Zap-70 was determined in 37 CLL patients using four different commercial antibodies. T, NK, and CLL cells were identified by immunophenotyping along with Zap-70 expression. Data was analyzed in terms of both percent of CLL cells expressing Zap-70 and the ratio of Zap-70 expression in CLL cells compared to that in T + NK cells. Three Zap-70 antibodies showed wide ranges of Zap-70 expression as a percentage of tumor cells, while a fourth gave consistently elevated results. Comparing the percent Zap-70 expression with any two antibodies gave poor correlations (r(2) = 0.45-0.63). Our results indicated that the previous analytical strategies were not reproducible. A ratio metric is proposed, which gave better correlations (r(2) as high as 0.95) and would allow separation of CLL patients with elevated or decreased Zap-70 expression., ((c) 2005 Wiley-Liss, Inc.)

Background: Patients with Glanzmann thrombasthenia (GT) have normal platelet counts but abnormal platelet aggregation and carry the risk of life-threatening bleeding. We report three patients who received bone marrow transplantation (BMT) for type I GT and discuss the risk and management of anti-platelet antibodies., Patients and Results: Diagnosis of GT was made through abnormal platelet aggregation studies or the absence of GPIIb/IIIa by flow cytometry. All patients had severe bleeding requiring multiple red blood cell transfusions. One patient received an unrelated donor transplant and two received matched sibling donor transplants following conditioning therapy with busulfan, cyclophosphamide, and fludarabine. Two patients developed an anti-platelet antibody, treated in one with intravenous immune globulin (IVIG). Engraftment of white blood cells and platelets was achieved on day +13 to +14 and +17 to +25, respectively. Complete donor chimerism and GPIIb/IIIa+ platelets are sustained at +22 to +30 months post transplant., Conclusions: In summary, patients with GT and history of severe hemorrhage can be cured with BMT, but the presence of anti-platelet antibodies should be sought and platelet transfusions minimized prior to transplant. IVIG may be helpful in cases of refractory immune thrombocytopenia related to anti-platelet antibodies. Improvement in transplant-related complications with current transplant regimens allows consideration of BMT for life-threatening non-malignant disorders such as GT., (2005 Wiley-Liss, Inc.)

Background: Obesity affects the regulation of immune and inflammatory responses. This study characterizes differences in peripheral blood lymphocyte phenotype in obese humans., Methods: Frequencies of lymphocyte subsets among peripheral blood mononuclear cells were compared between 10 obese (BMI > or = 35) and 10 lean subjects, as determined by antibodies directed against cluster differentiation (CD) markers., Results: Obese patients demonstrated an increased frequency of CD3+CD4+ T-cells (mean difference 12%, P=0.004), a decreased frequency of CD3+CD8+ T-cells (mean difference 9.4%, P=0.016) and an increased frequency of CD3+CD8+CD95+ T-cells (mean difference 13.3%, P=0.032). No other differences among T-cell or monocyte subsets were noted., Conclusions: Obesity is associated with alterations in frequencies of peripheral CD4+ and CD8+ T-cells and aberrations in the expression of CD95 among CD8+ T-cells. These data suggest both CD4+ and CD8+ T-cell compartments, as well as the regulation of CD95 expression on CD8+ T-cells, as targets for further study into obesity's effects on the immune system.

IPEX syndrome is a genetic autoimmune disease characterized by immune-mediated polyendocrinopathy, enteropathy, and X-linked inheritance. We describe a case of IPEX in which lymphocyte phenotypes were assessed at birth, before initiation of Cyclosporin A therapy, and at frequent intervals to 18 months of age. We performed flow cytometry for lymphocyte subtypes and for activation markers (HLA-DR, CD25, and CD69 or CD71). The ratios of both T to B cells and CD4+ to CD8+ cells were elevated at birth, but CD4+ cells were not activated. HLA-DR+ and CD25+ activated T-cells increased in association with two episodes of clinical deterioration: colitis and the onset of type I diabetes mellitus. These results indicate that measures of activation, particularly HLA-DR+ and CD25+ frequency, correlate well with the development of early active disease and may presage clinical episodes. Continuous maintenance of immunosuppression, once started, appears critical for prevention of permanent tissue damage.

Multiple sclerosis (MS) is a debilitating neurological disease characterized by a progressive loss of motor and sensory function, eventually leading to paralysis and death. The primary cause of neurological impairment is demyelination of the central nervous system (CNS) caused by an inflammatory autoimmune response. Previous studies have shown that the severity of MS is reduced during pregnancy, suggesting that the increased level of sex hormones may reduce the autoimmune response. Recently, we have shown that estrogen treatment confers protection from experimental autoimmune encephalomyelitis (EAE), which is an animal model for MS. However, the cellular basis of estrogen's action remains unknown. In the current study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells in spleen and CNS, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45(dim)) and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA-4, an extracellular protein involved in cellular migration. Several lines of evidence suggest that these novel cells, defined as CD45(dim)VLA-4(+) cells, may play a role in the protective effects of estrogen in EAE. Injection of purified CD45(dim)VLA-4(+) cells conferred protection from spontaneous EAE (Sp-EAE). In contrast, injection of CD45(high)VLA-4(+) cells exacerbated the disease course. CD45(dim)VLA-4(+) cells also suppressed antigen-specific proliferation of primed lymphocytes in coculture. A better understanding of how CD45(dim)VLA-4(+) cells suppress the harmful immune response of EAE may help in explaining the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases., (Copyright 2004 Wiley-Liss, Inc.)

The scurfy mutant mouse is the genetic and phenotypic equivalent of the single-gene human autoimmune disease immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). The scurfy mutation disrupts the Foxp3 gene, a putative master switch for T regulatory cell development. Bone marrow transplant without conditioning was previously reported to be ineffective in scurfy mice, yet clinical remission occurs in transplanted human IPEX patients despite limited donor engraftment. In view of this contradiction, we sought to validate scurfy as a model for studying the pathogenesis and treatment of human IPEX, in particular the phenomenon of dominant immune regulation. One half of scurfy mice given bone marrow transplants after sublethal irradiation recovered and survived long-term with donor chimerism ranging from 1.7% to 50%. Early transfer of 2 x 107 normal T cell-enriched splenocytes also prevented or limited disease and permitted long-term survival. Donor T cells in rescued mice made up 3-5% of lymphocytes and became highly enriched for CD25+ T cells over time. Transfer of 106 CD4+ CD25+ sorted T cells showed some beneficial effect, while CD4+ CD25- cells did not. Thus, both partial bone marrow transplant and T-enriched splenocyte transfer are effective treatments for scurfy. These results indicate that scurfy results from a lack of cells with dominant immune regulatory capacity, possibly T regulatory cells. The potency of small numbers of normal cells indicates that IPEX may be a feasible target for gene therapy.

Objective: To determine the effect of hypnotic-guided imagery on immune function and psychological parameters in patients being treated for Stage I or II breast cancer., Methods: To determine the effects of hypnotic-guided imagery on immune function and psychological parameters, the following study was undertaken. Psychological profiles, natural killer (NK) cell number and activity were measured at baseline, after the 8-week imagery training program and at the 3-month follow-up., Results: There were significant increases in improvement in depression (P<.04) and increase in absolute number of NK cells, but these were not maintained at the 3-month follow-up. Hypnotic-guided imagery did cause some transient changes in psychological well-being and immune parameters. However, these changes were not retained after the treatment ended., Conclusions: Many studies during the last 15 years have demonstrated interactions between the central nervous and the immune systems. While a negative effect of stress on immune responses has been demonstrated, there have also been published reports that psychological treatments can positively alter the immune system. However, given the complexities of immune system kinetics, the transient nature of any psychological effect and the insensitivity of immune assays, our study indicates that there is a role for hypnotic-guided imagery as an adjuvant therapy.

Cells from individuals with Fanconi anemia (FA) arrest excessively in the G2/M cell cycle compartment after exposure to low doses of DNA cross-linking agents. The relationship of this abnormality to the fundamental genetic defect in such cells is unknown, but many investigators have speculated that the various FA genes directly regulate cell cycle checkpoints. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FAC) functions to control a cell cycle checkpoint and that cells from group C patients (FA[C]) have abnormalities of cell cycle regulation directly related to the genetic mutation. We found that retroviral transduction of FA(C) lymphoblasts with wild-type FAC cDNA resulted in normalization of the cell cycle response to low-dose mitomycin C (MMC). However, when DNA damage was quantified in terms of cytogenetic damage or cellular cytotoxicity, we found similar degrees of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells as well as in normal lymphoblasts. Similar results were obtained using isogenic pairs of uncorrected, FAC- or mock-corrected (neo only) FA(C) cell lines. To test the function of other checkpoints we examined the effects of hydroxyurea (HU) and ionizing radiation on cell cycle kinetics of FA(C) and normal lymphoblasts as well as with isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C) cell lines. In all cases the cell cycle response of FA(C) and normal lymphoblasts to these two agents were identical. Based on these studies we conclude that the aberrant G2/M arrest that typifies the response of FA(C) cells to low doses of cross-linking agents does not represent an abnormal cell cycle response but instead represents a normal cellular response to the excessive DNA damage that results in FA(C) cells following exposure to low doses of cross-linking agents.

Human immunodeficiency virus (HIV)-1 infection is associated with the development of aggressive extranodal B-cell non-Hodgkin's lymphomas. Using microvascular endothelial cell (MVEC)-enriched bone marrow stromal cultures, HIV infection of stromal MVECs from lymphoma patients induced the outgrowth of malignant B cells. MVECs were the only HIV-infected cells in the stroma, and purified brain MVECs also induced a phenotype supportive of neoplastic B-cell attachment and proliferation. HIV infection of MVECs stimulated surface expression of CD40 and allowed preferential induction of the vascular cell adhesion molecule VCAM-1 after CD40 triggering. B-lymphoma cells expressed the CD40 ligand (CD40L), and blocking of CD40-CD40L interactions between HIV-infected MVECs and B-lymphoma cells inhibited B-cell attachment and proliferation. These observations suggest that HIV promotes B-lymphoma cell growth through facilitating attachment of lymphoma cells to HIV-infected MVECs and represent a novel mechanism through which viruses may induce malignancies.

Interleukin-12 (IL-12) is a heterodimeric cytokine implicated in the early differentiation of naive T-lymphocytes into the Th1 subset. IL-12 is important for induction of the cellular immune response against viruses, intracellular parasites and neoplasms. Its role in alloresponsiveness has not been fully elucidated. Preliminary data in the literature point toward the prevalence of Th1 lymphocytes in processes of allograft rejection. In attempt to further investigate the expression of this cytokine during episodes of cellular rejection of renal allografts, we searched for IL-12 message in human kidney allograft biopsies using the reverse transcriptase-polymerase chain reaction technique. Twenty-three allograft core biopsies from 19 patients were obtained percutaneously for clinical indications in 18 cases, and as part of an investigational protocol in five cases. A portion of the tissue was used for RNA extraction using the guanidium-thiocyanide phenol-chloroform method. Histology was performed on the remaining core material. Ten mg of total RNA were used for reverse transcription. PCR of the c-DNAs was done for 40 cycles using primers for the p40 subunit of IL-12 and GAPDH which was used as a control. PCR products were photographed after electrophoresis, transferred to a nylon membrane and hybridized with a radiolabelled cloned human IL-12 p40 1 kb c-DNA fragment. Autoradiographies were developed after 20-min exposure. All samples were run in triplicate. IL-12 p40 m-RNA was expressed in all 17 biopsies showing acute cellular rejection as well as in all three biopsies showing focal interstitial fibrosis. No message was found in the presence of normal allograft histology. This is the first in vivo report of IL-12 p40 subunit m-RNA expression during renal allograft rejection in humans. The role of this Th1 cytokine in the alloresponse deserves further investigation.

To investigate TCR V alpha gene expression in the Lewis rat model of experimental autoimmune encephalomyelitis, we obtained V alpha chain sequences from two V beta8.2+-encephalitogenic, BP72-89-specific T cell clones. Two different V alpha genes, a V alpha2 gene and a V alpha23 gene, are utilized, but both were found to contain an asparagine repeat (Asn3+) sequence present in the V alpha CDR3 region. This Asn3+ motif is also present in the previously reported sequence of a BP68-88-specific hybridoma, 510, which utilizes a different V alpha2 gene family member. In further experiments, spinal cord T cells were isolated at the onset of basic protein (BP)-induced disease and sorted for the OX-40 activation marker, which we have previously used to enrich for specifically activated T cells. Analysis of V alpha expression in the OX-40+ population revealed the biased use of three V alpha genes, V alpha1, V alpha2, and V alpha23. The Asn3+ motif was present in the V alpha CDR3 region of V alpha1, V alpha2, and V alpha23 cDNA derived from OX-40+ spinal cord T cells but found to be generally absent in the OX-40- spinal cord population. Since these Asn3+ motif-bearing V alpha chain sequences are nearly identical to those utilized by the BP-specific encephalitogenic clones described, it is likely that these V alpha sequences are derived from disease-associated T cells in the spinal cord. Thus, we demonstrate that the Asn3+ V alpha CDR3 motif is strongly associated with experimental autoimmune encephalomyelitis in the Lewis rat and propose that it plays a role in TCR recognition of a specific BP peptide/MHC complex.

Objective: Several small short-term intervention studies have suggested that beta-carotene supplementation in HIV-infected patients can increase the number of various immune cells including CD4 cells. This prospective double-blinded study was designed to investigate whether beta-carotene supplementation would result in this immuno-enhancement in a larger number of patients over a longer time period., Methods: HIV-positive patients were randomly assigned to receive either 60 mg beta-carotene orally three times daily or a matched placebo. In addition, all patients received a multivitamin supplement. Patients were evaluated at baseline, 1 month, and 3 months for T-cell quantitative subsets, natural killer cells, HIV p24 antigen, beta-carotene levels, complete blood counts and chemistry batteries. Body weights and Karnofsky scores were evaluated at each visit., Results: Seventy-two patients signed informed consent forms and entered the study. Except for serum beta-carotene concentration, there were no statistically significant differences (P < 0.05) between the treatment (60 mg beta-carotene three times daily and multivitamins) and placebo (placebo and multivitamins) groups at baseline or after either 1 or 3 months of treatment., Discussion: Earlier studies suggesting that beta-carotene supplementation increased levels of immune cells in HIV-infected patients were not replicated in this study. The addition of a multivitamin supplement to both arms of this study may have masked any difference between the two groups. However, on the basis of the results of this study, we would not recommend supplementation with high doses of beta-carotene for HIV-infected patients.

Objective: To determine the immunocytologic characteristics of the various subsets of nonseptic olecranon bursal fluid mononuclear cells., Methods: Twenty consecutive patients with culture negative olecranon bursitis had immunocytochemical and flow cytometric analysis performed using a panel of monoclonal antibodies to determine lymphocyte and monocyte/macrophage population subtypes and proportions., Results: In traumatic bursitis (n = 9), the mean (+/- SD) white blood cell (WBC) count/mm3 was 1,368 +/- 1,559; WBC mononuclear differential count was 29.5 +/- 19% lymphocytes and 55 +/- 26% monocyte/macrophages. In idiopathic bursitis (n = 11), the mean WBC/mm3 was 376 +/- 515; WBC mononuclear differential count was 28.5 +/- 16% lymphocytes and 52 +/- 27% monocytes/macrophages. Flow cytometry revealed 88% CD2+ T lymphocytes in the lymphocyte population, 3% B lymphocytes and CD4/CD8 T cell mean ratio of 2.5 +/- 1.5 for traumatic bursitis and 88% CD2+ T lymphocytes, 4% B lymphocytes and CD4/Cd8 ratio of 1.4 +/- 0.6 for idiopathic bursitis (p < 0.05, t test). Both groups contained increased proportions of lymphocyte subtypes expressing activation markers: CD25+, CD26+ and HLA DR+ compared to normal peripheral blood. In traumatic bursitis, the mean percent of CD14+ cells (monocyte/macrophages) was 62 +/- 24; in idiopathic bursitis, the mean percent was 51 +/- 28. The vast majority expressed high levels of HLA-DR indicating activation., Conclusion: We observed a preponderance of activated T cell subpopulations and monocyte/macrophages suggesting an immunologic role for these cell populations in the development and perpetuation of nonseptic bursitis.

The peripheral T cell repertoire is shaped by positive and negative selection. These intrathymic events are dependent on the direct interaction of MHC and TCR molecules. Inasmuch as one possible mechanism for HLA-linked disease involves the role that these molecules play in shaping the peripheral T cell repertoire, an understanding of how stable the repertoire remains is an important question that will influence future studies. The purpose of this study was to analyze the stability of the T cell repertoire in monozygotic twins. To investigate this question the percentage of CD4 and CD8 T cells expressing TCR V beta gene products was determined for seven sets of healthy monozygotic twins ages 2 through 44. V beta expression was determined by three-color flow cytometric analysis using antibodies to V beta-5.1, -5.2, -5.3, -6.7, -8, and -12. The percentage of CD4 cells expressing each V beta gene was highly concordant between twins. In contrast, differences were noted for V beta expression within the CD8 subset. This was especially marked when sets of twins were studied (n = 3) where one individual had an underlying disease. Although expression in the CD4 subset was again concordant, significant differences were noted within the CD8 subset compared to the healthy twin. These data indicate that in both health and disease, the CD4 T cell repertoire is tightly regulated although often sizable differences have developed in the CD8 compartment.

To test growth effects of angiotensin II (ANG II) in porcine vascular smooth muscle cells (VSMC) and potential ANG II synergy with epidermal growth factor (EGF), we exposed subconfluent, near-quiescent porcine aortic VSMC to ANG II, EGF, or ANG II + EGF (each 10(-9) M) in Dulbecco's modified Eagle's-Ham's F-12 medium with insulin + 0.4% fetal calf serum (FCS) selected for minimal ANG II-degrading capacity. Cell number and DNA and protein synthesis (by [3H]-thymidine and [35S]methionine incorporation, respectively) were determined serially over 1-6 days. ANG II alone induced an early 20% increase and then a plateau in cell number over the 0.4% FCS control (P < 0.01; n = 8), thus without sustained increase in proliferation rate. Yet ANG II alone did not increase fractional DNA or protein synthesis (each as cpm/10(3) cells) and, by flow cytometry, reduced S phase fraction without increase in cell size. EGF alone induced brisk DNA synthesis yet minimal cell division over days 0-4, thus late-cycle arrest. ANG II + EGF, despite no increase in fractional DNA or protein synthesis rates over EGF alone, induced significant indomethacin-resistant dose-dependent (P < 0.001) increase in cell proliferation rate over EGF alone with a median effective dose of 5 x 10(-10) M ANG II, thus proliferative synergy. We propose that 1) ANG II induces a subpopulation of cells arrested in or beyond S phase to proceed through mitosis but does not influence G1 traversal or S phase entry and 2) ANG II + EGF achieve proliferative synergy by complementary actions at sequential cell cycle loci, with EGF supporting progression from G0/G1 to S phase and ANG II inducing completion of mitosis by cells already in or beyond S phase ("late-cycle completion").

Dysplastic nevi (DN), described in 1978, have been associated with increased risk of melanoma, but the role of DN as precursors of melanoma is still controversial. Recent studies have shown that DN are very common in the general population, bringing into question this purported association. Numerous investigations have attempted to correlate the presence of DN in individuals with specific phenotypic and genotypic features, including the presence of abnormal DNA content. Because the occurrence of such abnormal DNA stemlines in neoplasms may be associated with malignant behavior, we studied 38 biopsies from 19 patients that histologically fulfilled criteria for DN in order to ascertain characteristics of DNA content. Nuclear suspensions made from paraffin-embedded tissue were evaluated by both flow cytometry and image analysis techniques. All cases demonstrated diploid populations by both DNA measurement methods. Our results contradict previous reports of aneuploid populations in these melanocytic lesions.

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

Anti-idiotypic anti-DNA antibodies (anti-anti-DNA) have previously been described in both patients with systemic lupus erythematosus and healthy individuals. Jerne's hypothesis predicts that such antibodies would bear a paratope reactive with non-sequence specific DNA binding proteins. Here we have explored the notion of a molecular mimicry between anti-anti-DNA antibodies and antibodies to a previously described 28-29 kD cell surface DNA binding molecule. It was shown that affinity purified anti-anti-DNA antibodies inhibit the binding of DNA to cells and that MoAb to the 28-29 kD receptor react with anti-DNA antibodies. These findings indicate that a subset of anti-anti-DNA antibodies are idiotypically related to antibodies reactive with a cell surface DNA binding molecule. It is hypothesized that anti-DNA antibodies may arise when a convergence of genetic and environmental influences favours an unrestrained anti-idiotypic response to cell surface DNA binding molecule(s).

The molecular basis for the cellular interaction of DNA and nucleosomes and the physiological consequences of this binding were examined. Both DNA and nucleosomes were demonstrated to bind specifically to the surface of human peripheral blood mononuclear cells and the murine T cell line S49. Western blots of S49 cell membranes, using probes of biotin-labeled DNA and nucleosomes, showed reactivity at 29 and 69 kDa. Functionally, the interaction of DNA and nucleosomes with murine spleen cells stimulated the release of significant amounts of IL-6 activity. There is evidence that nucleosomes, a product of apoptosis, are the major component of circulating DNA found in the plasma of patients with systemic lupus erythematosus (SLE). The interaction of nucleosomes with cell-surface DNA binding molecules may have physiological relevance to some of the immune aberrations observed in patients with SLE.

Autoimmunity to a 28-29-kDa cell-surface DNA-binding molecule has previously been described in patients with systemic lupus erythematosus and related autoimmune diseases. This report describes experiments that implicate a similar antigen-antibody system in the evolution of autoimmunity in lupus-prone mice. DNA binding to murine spleen cells was found to be a saturable phenomenon that was inhibited by excess cold DNA and trypsinization. The role of autoimmunity to murine cell-surface DNA-binding molecules in lupus-prone mice (MRL lpr/lpr, MRL +/+, BXSB) was compared to normal mice (BALB/c, C3H.SW) by means of an assay that measured the inhibition of cell-surface DNA binding. Only sera from lupus strains had inhibitory activity and this component was shown to be an IgM autoantibody. Furthermore, we isolated a spontaneously occurring IgM monoclonal antibody from the spleen of an MRL/lpr mouse, which inhibited DNA binding to mouse cells. Time-course studies indicated that young female MRL/lpr mice lacked detectable activity against cell-surface DNA-binding molecules; however, by 8-10 weeks maximal inhibitory activity was observed. This response occurred prior to the development of significant antinuclear antibody activity. With the appearance of overt disease and anti-DNA antibodies, inhibition of DNA-binding activity became undetectable. These findings mirror previous studies on autoimmunity to a cell-surface DNA-binding molecule on human leucocytes, but have the added advantage of permitting the study of the temporal evolution of this inhibitory activity in relation to disease expression.

To characterize growth effects of epidermal growth factor (EGF) in subconfluent quiescent porcine aortic vascular smooth muscle cells (VSMC), we measured DNA and protein synthesis by [3H]thymidine (Thd) and [35S]methionine (Met) incorporation, respectively, and cell proliferation rates over 0-6 days in Dulbecco's modified Eagle's-Ham's F-12 media containing 0.4% fetal calf serum (FCS) and insulin. EGF induced dose-dependent [3H]Thd uptake (P less than 0.001); after 10(-9) M EGF, DNA synthesis rate peaked at 24 h, averaging 77% of the response to 10% FCS, and then declined steeply with nadir at 48-60 h. Unexpectedly, EGF failed to induce cell proliferation in the first 4 days, leaving this initial burst of DNA synthesis (12-60 h) uncoupled from cell division. A second lesser but sustained phase of increased DNA synthesis, apparent by day 3-4, was associated with a small increase in cell number on day 6 (P less than 0.05). The early unsustained burst of DNA synthesis reflects EGF's potent mitogenic efficacy for DNA synthesis (G1- to S-phase traversal), probably acting on a subset of cells partially synchronized initially at an EGF-responsive G0/G1 locus; the minimal cell division despite brisk DNA synthesis documents EGF's limited efficacy for (or inhibition of) late cell-cycle events required for completion of mitosis. Late cell-cycle processes are thus rate limiting. EGF also increased protein synthetic rate over control (P less than 0.03) but to a lesser degree (P less than 0.01) than 10% FCS. Indomethacin (10(-6) M) did not alter DNA or proliferative responses to 10(-9) M EGF but transiently augmented EGF-induced protein synthesis (P less than 0.025) at 24 h only.(ABSTRACT TRUNCATED AT 250 WORDS)

A patient with high levels of serum rheumatoid factor and an open lung biopsy which showed high-grade interstitial pneumonia with large numbers of lymphocytes and plasmocytes had intense gallium uptake in the lungs. Lymphocytes and/or plasmocytes might be responsible for the gallium uptake even though neutrophils are usually credited with high-level uptake. Differential cell counts demonstrated plasmocyte and lymphocyte preponderance, but neutrophil paucity. In vitro cell cultures of purified neutrophils, monocytes, leukemic plasmocytes, and resting and stimulated lymphocytes with 67Ga showed that plasmocytes take up comparatively low levels of 67Ga, but that activated lymphocytes take up levels that approach neutrophils. It is probable that both rheumatoid lung plasmocytes and activated lymphocytes are responsible for the pulmonary 67Ga concentration in this patient.

Previous experiments have established the presence of a 30-kD DNA binding protein on the surface of human leukocytes. Herein we report that selected sera from patients with systemic lupus erythematosus (SLE) and MCTD are reactive with a 28-30 kD protein on immunoblots of peripheral blood mononuclear cells (PBMC) cell membrane preparations; the reactivity is abolished by prior incubation of the blot with DNA. Antibodies eluted from the 28-30 kD strip inhibited the binding of 3H. DNA to human PBMC. An immunomatrix of 28-30 kD reactive immunoglobulins was able to extract a 29-kD DNA binding protein from a PBMC cell membrane preparation. Flow cytometry experiments confirmed the cell surface IgG reactivity of sera with T lymphocytes. Additional experiments indicated that cell surface IgG binding was not due to antibodies binding to cell surface DNA, DNA anti-DNA immune complexes reacting with a DNA binding protein, anti-histone antibodies or anti-Sm antibodies. It is hypothesized that this autoimmune response could be one component of an idiotypic network involving anti-DNA antibodies.

Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) beta-gene products as part of the alpha beta heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR V beta-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain V beta genes were consistently overrepresented in the CD4 population (V beta-5.1, -6.7a, and -18); some were skewed to the CD8 population (V beta-14) while others showed variable patterns (V beta-12 and -17). Deletion of entire V beta gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.

This study was undertaken to delineate the elements of the interleukin-2 (IL-2) system in premature newborns by quantitating IL-2 production and IL-2 receptor (IL-2R) expression and density in cord blood mononuclear cells (CBMC) from 19 premature (estimated gestational age = 27-36 weeks, mean = 33.7), but otherwise healthy, infants and 25 term newborns. Phytohemagglutinin (PHA)-induced IL-2 production by premature CBMC was significantly greater (p less than 0.05) than that produced by term newborn CBMC with a mean value of 52.0 mu/ml for prematures versus 18.2 mu/ml for terms. The mean percentage of CBMC expressing PHA-induced cell-surface IL-2R was 23.4 and 23.0% while the IL-2R density per cell was 4.72 x 10(4) and 6.93 x 10(4) for premature and term newborns, respectively. PHA-induced proliferation was similar in both groups. These results show a significantly increased PHA-induced IL-2 production, but similar IL-2R expression, in CBMC from premature as compared to term newborns, demonstrating that the preterm newborn possesses a competent IL-2 system at birth.

In 1973 Frøland and Natvig reported that 14.5% of human blood lymphocytes expressed distinctive Fc receptors for IgG (FcR) and that this subset lacked surface receptors believed to be characteristic for T and B cells. They named this subset 'third population' cells. Although these cells have been extensively studied since that time, whether they are a single unique population or immatureforms of other mononuclear populations is controversial. Their lineage is unknown. They have morphologic featur s that distinguish them from other mononuclear cells, but studies with monoclonal antibodies have revealed that they share surface markers andfunctions with T cells, monocytes and even granulocytes. Subsets of these cells are the effectors of natural and antibody-dependent cell killing, regulators of T-cell proliferation and suppressors of immunoglobulin synthesis. Here, David Horwitz and Antony Bakke discuss the present understanding of these cells, which they call 'L cells' because of the characteristic temperature-labile attachment of IgG molecules to their Fc receptors., (Copyright © 1984. Published by Elsevier B.V.)

Dictyostelium chromatin has been purified from nuclei in high yield by differential centrifugation and nuclease cleaving. Its chemical composition has been assayed, and its histones have been analyzed by gel electrophoresis, peptide fingerprints, amino acid composition, and ion-exchange chromatography. The mass ratios of DNA/RNA/histone/nonhistone are 1.0:0.18:0.98:1.02. There are four histones including one unusual histone, H7, which is the most abundant histone in the slime mold. The H4-like protein is the most conserved protein, while the other histones show both similarities and differences with mammalian histones.

Peripheral blood T lymphocyte subsets were measured by flow cytometry in 122 patients with leprosy, in 23 normal controls, and in 27 patients with systemic lupus erythematosus (SLE). Active lepromatous patients not in reaction showed a significant lymphopenia and a significant proportionate reduction in the number of OKT3-positive (pan T), OKT4-positive (helper/inducer), and OKT8-positive (suppressor/cytotoxic) cells, but no alteration in distribution as judged by percentage and no abnormality in the helper: suppressor ratio. Borderline lepromatous subjects not in reaction had a significant selective deficiency in the number of cells of the OKT4-positive subset, with a significant but secondary lymphopenia and OKT3-positive cytopenia, a pattern similar to that found in SLE patients. Patients undergoing reversal reactions had a selective deficiency in the OKT4-positive subset in both absolute numbers and as a percentage of total lymphocytes, and a secondary deficiency in the percentage of OKT3-positive cells. No abnormalities were demonstrated in patients with active erythema nodosum leprosum, lepromatous patients with long-term treatment, and untreated or treated borderline tuberculoid patients.

This report describes the use of a fluorescent activated cell sorter (FACS) in combination with an enzyme immunosorbent assay (EIA). This combination of techniques expands the versatility of the flow cytometer. It introduces a new set of fluorescent enzyme products for antigen detection. These highly substantive fluorescent compounds permit the flow cytometer to quantify cell-EIA reactions and also to delineate subpopulations of cells with different quantities of surface antigens. Because the FEIA product is colored, as well as fluorescent, a simple light microscope may also be used to define the distribution of the label on the cell surface. These techniques have been applied to the examination of antigens on human erythrocytes, human T cell lymphoma cells (H9), and to surface markers on the tissue culture cell line K562.

The active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), suppresses in vitro immunoglobulin (Ig) production by activated peripheral blood mononuclear cells (PBM) from normal human subjects by inhibiting T helper/inducer TH cell activity. Normal PBM were fractionated into B, TH and T suppressor/cytotoxic (Ts) cells by fluorescence-activated cell sorting techniques. The resultant subsets were activated with mitogens and were cultured in the presence or absence of a receptor-saturating concentration of 1,25-(OH)2-D3. The sterol reduced [3H]thymidine incorporation in TH cells by 56%, with no effect on Ts or B cells. When 1,25-(OH)2-D3-treated TH cells were co-cultured with untreated B cells and culture supernatants assayed for Ig production, 1,25-(OH)2-D3 abrogated the inducing effect of TH cells on Ig synthesis by B cells. There was no inhibitory effect of the sterol on Ts or B cell activity. In addition, 1,25-(OH)2-D3 produced a dramatic inhibition of interleukin 2 (IL 2) production by activated PBM, but did not inhibit IL 2 receptor generation by these cells. Other vitamin D metabolites tested did not produce this effect. These results suggest that the TH lymphocyte is the specific cellular target for the immunoinhibitory effects of 1,25-(OH)2-D3.

To study T lymphocytes in erythema nodosum leprosum (ENL), monoclonal antibodies were used to identify T-lymphocyte subpopulations in the blood and skin lesions of patients with ENL and patients with nonreactional lepromatous leprosy. The blood of nonreactional lepromatous patients had a lymphopenia and a proportionate reduction in pan T cells, helper-inducer, and suppressor-cytotoxic subsets, but a normal helper-suppressor ratio, as compared with controls. Patients with ENL did not differ significantly from the controls. In skin lesions, an admixture of helper and suppressor phenotypes among foamy histiocytes was found. The ENL tissue had more numerous cells of the helper-inducer phenotype and fewer of the suppressor-cytotoxic phenotype, as compared with nonreaction lepromatous tissues. In 22 patients with simultaneous examination of tissue and blood T-cell subsets, there was no correlation between tissue and blood helper-suppressor ratios, indicating that some sort of selection process brings lymphocytes into tissues from peripheral blood.

Lymphocytes displaying iC3b (Type 3) complement receptors (CR3) were quantified by flow cytometry in patients with systemic lupus erythematosus. The percentages and absolute numbers were compared to age and sex matched controls. Total CR3+ lymphocytes identified by the monoclonal antibodies OKM1 or Leu 15 were significantly decreased in patients with symptomatic arthritis, serositis or vasculitis and those with lupus nephritis, whereas values for CR3+ lymphocytes in patients with inactive disease were similar to normal donors. The phenotype of CR3+ lymphocytes was markedly different in patients with active SLE. In normals granular lymphocytes bearing Fc receptors for IgG (L cells) comprised two-thirds of CR3+ lymphocytes. However, in SLE this subset was reduced to 20% and there was a corresponding increase in CR3+ lymphocytes co-expressing the T3 marker. Percentages of CR3 T4+ but not CR3+ T8+ lymphocytes were significantly increased in SLE. Although patients with active disease were lymphopenic, absolute numbers of CR3+ lymphocytes co-expressing T cell markers were similar to normal controls. Since L cells are non-specific suppressors of Ig production, the reduction of this subset along with the increase in CR3 T4+ cells could contribute to unregulated antibody production characteristic of SLE.

Interleukin-2 (IL-2) production in vitro is depressed in systemic lupus erythematosus (SLE) patients. It is not known whether this abnormality is caused by a defect in the producer lymphocytes or by excessive suppression. We report that removal of OKT8 (Leu 2a)+ cells increased the IL-2 production by in vitro-stimulated lymphocytes to normal or above normal levels in 19 of 21 SLE patients. This increase was more apparent in those patients with clinically inactive disease and/or receiving less than 7.5 mg of prednisone. Removal of OKT8+ cells from normals did not significantly increase IL-2 activity. SLE, but not normal, OKT8+ cells decreased IL-2 production when added back to autologous OKT8-depleted cells. In some experiments, OKT8+ cells from normal donors also suppressed IL-2 production in SLE. This result suggests that the defect in IL-2 production is complex and may involve multiple cell interactions. Three lines of evidence suggest that the SLE OKT8+ cells actively inhibit the production of IL-2 rather than passively absorb this lymphokine: (a) only 3.2% of SLE lymphocytes expressed IL-2 receptors as detected with anti-Tac; (b) freshly prepared SLE lymphocytes did not absorb IL-2; and (c) cell-free supernatants from SLE OKT8+ cells inhibited IL-2 production, but not IL-2 activity. Double-labeling studies by flow cytometry revealed that 19.3% of SLE OKT8+ cells were also Ia-positive, and approximately 33% co-expressed the natural killer cell marker, HNK-1 (Leu 7). Removal of Leu 7+ cells also significantly elevated IL-2 production in SLE. These studies suggest that one or more circulating mononuclear cell subsets in SLE patients can suppress IL-2 production and that one subset may possibly belong to a non-T, non-B "third mononuclear population."

Patients with very active rheumatoid arthritis that was being treated only with nonsteroidal antiinflammatory drugs had increased numbers of peripheral blood OKM1+ lymphocytes. In 3 patients, 90 degrees light scatter analysis revealed a double lymphocyte peak. When sorted, the high scatter peak contained a large percentage of granular lymphocytes. Patients with mild-to-moderately active rheumatoid arthritis had normal levels of OKM1+ lymphocytes, but when the drugs were discontinued, the activity of the disease and the numbers of OKM1+ cells increased. Administration of piroxicam was associated with clinical improvement and a decrease in levels of OKM1+ cells. OKM1+ granular lymphocytes are increased in some rheumatoid arthritis patients, and their numbers may correlate with clinical disease activity and/or therapy.

Friend erythroleukemic cells can be induced to differentiate by chemicals such as dimethyl sulfoxide and hexamethylene bisacetamide (HMBA) in a dose-dependent manner. Others have shown that dexamethasone and other steroid hormones can inhibit the differentiation induced by dimethyl sulfoxide. We show here that dexamethasone has a dual mode of action on the HMBA-induced differentiation of a Friend cell line, DS19. At concentrations above 10(-10) M, dexamethasone inhibits the HMBA-induced differentiation in DS19 cells. We further found that as the concentration of HMBA is reduced, the amount of dexamethasone required to inhibit differentiation increases. Such inhibition seems to act primarily by decreasing the probability that a cell will become committed to differentiate. Besides, dexamethasone does not inhibit hemoglobin synthesis of cells which are committed in its absence. In addition, we show that sensitivity to the inhibition by dexamethasone is inversely related to the inducibility of cell populations. The second action of dexamethasone, observed at concentrations between 10(-10) and 10(-13) M, is to increase the proportion of hemoglobin-containing cells relative to that for cells cultured in its absence. The degree of this synergistic effect for induced differentiation is inversely related to the level of induction in the absence of dexamethasone and thus is best observed in the cells cultured at low levels of HMBA. We present evidence that this synergistic effect may be due to an increased viability of the induced cells and possibly an increase in the proliferative capacity of these cells.

DNA measurement by flow cytometry has been demonstrated to be a potentially useful technic in the diagnosis of bladder cancer by detecting neoplastic cells in bladder washings and urine specimens. The authors' goal was to develop a simple and practical method utilizing the new generation of cytofluorographs designed for use in the clinical laboratory. This method combined direct fixation with cell lysis yielding fixed intact nuclei. Following RNase and pepsin digestion, the nuclei were separated from debris and aggregates on a sucrose barrier, stained with ethidium bromide, and analyzed with an argon laser analytic cytofluorograph. Urines and bladder washings from 14 patients with positive urinary cytology and histologically diagnosed bladder cancers were compared with specimens from patients without urothelial malignancies. DNA histograms clearly delineated aneuploid from diploid populations and often identified S, G2M, and G1 phase nuclei. Aneuploid populations have been detected in all tumor specimens with positive cytologies studied to date.

The comparative cytotoxic specificities of freshly isolated human adherent and nonadherent blood mononuclear cells were examined against seven established target cell lines in 4 and 18 hr chromium release assays. The relative sensitivity of each target cell line to the cytotoxic effects of both adherent and nonadherent effector cells in cultures was identical. Moreover, the relative enhancing effects of interferon on cytotoxicity by both effector cell types were also identical. These adherent cell preparations were contaminated with up to 6% NK cells, as demonstrated by OKM1 staining and flow microfluorometry. These NK cells were loosely adherent and could be removed by vigorous wash procedures. The remaining tightly adherent monocytes also had the capacity to kill K562 cells and Chang cells, but these cytotoxic effects could not be increased by interferon. Enhancement by lactoferrin, however, was consistently observed. Treatment of mononuclear cells with Leu-lla, a monoclonal antibody that reacts with all NK cells, also abolished the enhancing effects of interferon, but not lactoferrin. These studies suggest that caution must be exercised in attributing all cytotoxic activities in adherent cell preparations to monocytes, and that lactoferrin and interferon can be used as functional probes to detect two distinct blood mononuclear cell subsets with natural cytotoxicity.

The objective of this study was to define some causes of the immunologic impairment characteristic of systemic lupus erythematosus. Blood mononuclear cells from 19 patients were stimulated to produce interleukin 1 and interleukin 2 and compared with controls. A severe defect in both IL 1 and IL 2 activity was observed. The low cytokine levels did not correlate with clinical or serologic activity of disease. These defects could not be explained by concurrent corticosteroid therapy. There was no correlation between a lower number of OKT4+ cells observed in these patients and the levels of IL 2 production, nor did removal of monocytes bring IL 2 to normal. Impaired IL 2 production could not be restored to normal by IL 1. The observed deficiency in these regulatory cytokines may therefore be a primary defect that is important in the pathogenesis of this disorder.

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.