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Aims: To evaluate the safety and efficacy of various doses of recombinant glucagon-like peptide-1 (7-36) amide (rGLP-1) administered subcutaneously (s. c.) via bolus injection or continuous infusion to lower fasting serum glucose (FSG) levels in subjects with type 2 diabetes treated by diet, hypoglycemic drugs, or insulin injection., Methods: rGLP-1 was administered s. c. to 40 type 2 diabetics currently treated by diet, sulfonylurea (SU), metformin, or insulin in a double-blind, placebo-controlled, cross-over trial; preexisting treatments were continued during the study. In the bolus injection protocol, 32 subjects (8 from each of the 4 treatment groups) received 0.0, 0.5, 1.0, and 1.5 nmol rGLP-1/kg per injection (two injections, two hours apart, beginning one hour after the evening meal) in a randomized order on separate days. In the continuous s. c. infusion protocol, 40 subjects received rGLP-1 at 0.0, 1.5, 2.5, 3.5, and 4.5 pmol/kg/min for 10-12 hours overnight starting one hour after the evening meal. Fasting bloods were taken the morning after for glucose, insulin, and glucagon measurements., Results: In the diet, SU, and metformin cohorts, bolus rGLP-1 injections produced modest reductions in mean FSG levels, averaging 17.4 mg/dl (7.3-27.5; 95 % CI) at the highest dose (p < 0.001 vs. placebo). Reductions in FSG levels were greater by continuous infusion at up to 30.3 mg/dl (18.8 - 41.8; 95 % CI; p < 0.001 vs. placebo). The greatest reduction in mean FSG occurred in the SU cohort (up to 43.9 mg/dl, 24.7 - 63.1; 95 % CI; p < 0.001). rGLP-1 infusions resulted in significant increases in fasting plasma insulin and decreases in fasting plasma glucagon levels. There were no serious adverse events; GI-related symptoms were dose-related and more commonly associated with injections., Conclusions: rGLP-1 (7-36) amide dose-dependently lowered FSG in a broad spectrum of type 2 diabetics when added to their existing treatment. Subcutaneous infusion was more effective than injection, and the combination with SU was more effective than with metformin.

Background: Mutation or deregulation of certain cellular genes (protooncogenes) results in expression of proteins that appear to promote malignant transformation. Human non-small cell lung cancer has been documented to express many such oncogenes including c-myc, bcl-2, and mutant p53. Antisense oligodeoxyribonucleotides (ASODN) complementary to these oncogenes were tested on three non-small cell lung cancer cell lines for their efficacy in inhibiting cellular proliferation and oncoprotein expression., Methods: Established non-small cell lung cancer cell lines A427, SKMES-1, and A549 were grown in the presence of ASODNs complementary to messenger RNA of c-myc, bcl-2, p53, or controls at 1 mumol/L or 10 mumol/L concentrations for 4 or 10 days. Cellular proliferation was measured by tritiated thymidine uptake. Flow cytometry was used to quantitate oncoprotein expression. Intranuclear ASODN uptake was documented by fluoresceine-tagged ASODNs., Results: Fluoresceine-tagged ASODNs were readily taken up by all cell lines. c-myc, as well as bcl-2 and p53 ASODNs, were found to inhibit proliferation of all cell lines significantly compared with controls, most notably in line A549 (40.1% +/- 7.1% of control, p = 0.000 with c-myc ASODN). Antisense c-myc reduced c-myc protein by as much as 71.3% in A427, although protein levels were only minimally reduced in the viable cells of the other lines., Conclusions: c-myc ASODNs inhibit proliferation of non-small cell lung cancer cell lines as well as reduce c-myc protein expression. Antisense bcl-2 and p53 also cause similar growth inhibition. These results suggest a critical role for activation of these oncogenes in the growth of cultured lung cancer cells. Furthermore, the efficacy and rapid cellular uptake of ASODNs support the potential role of antisense targeting of oncogene expression for pharmacologic control of non-small cell lung cancer.

A 2184-nucleotide (nt) sequence of the p53 gene in Rhesus monkey (Macaca mulatta) was determined in order to facilitate the use of the Rhesus as an animal model in the testing of novel antisense oligodeoxyribonucleotides for a variety of human cancers, including acute myelogenous leukemia (AML). Within overlapping regions, we found greater than 95% identity between the Rhesus and human p53 sequences, and greater than 98% identity between Rhesus and African green monkey p53 sequences. The deduced amino acid (aa) sequence of the p53 protein is highly conserved between human and Rhesus monkey, with only 18 minor differences in 393 aa.

Background: Little is understood about reticular erythematous mucinosis and Jessner's lymphocytic infiltrate of skin., Objective: Our purpose was to define reticular erythematous mucinosis and Jessner's lymphocytic infiltrate of skin further with focus on immunologic studies., Methods: In patients with reticular erythematous mucinosis and Jessner's lymphocytic infiltrate of skin, we measured circulating immune complexes before, during, and after therapy. We examined natural killer cells in a functional assay; we performed direct immunofluorescence and T- and B-cell marker studies in skin biopsy specimens., Results: The infiltrate in reticular erythematous mucinosis is composed of helper T cells. Circulating immune complexes are increased in both reticular erythematous mucinosis and Jessner's lymphocytic infiltrate of skin and decrease with hydroxychloroquine therapy and clinical clearing. Natural killer cell function is decreased in reticular erythematous mucinosis and Jessner's lymphocytic infiltrate of skin., Conclusion: Changes in circulating immune complex titers accompanying therapy with hydroxychloroquine and clinical clearing, with recurrence of the condition and increase in circulating immune complexes on discontinuation of treatment, point to a possible relation between these events.

Human peripheral blood mononuclear cells were incubated at 37 degrees C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p less than 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both less than 5%). When bilirubin-treated cells were stained with fluorescein-labeled anti-CD14 antibody, the CD14 positive cell population can be fractionated without interference of autofluorescence derived from intracellular bilirubin. These results suggest that flow cytometric analysis of bilirubin-treated cells can quantitate intracellular bilirubin, and that bilirubin does not interfere with analysis of surface antigens.

Decreased immune responses have been observed in hyperbilirubinemic patients. This study investigates bilirubin transport into human peripheral blood mononuclear cells (PBMNCs). In vitro incubation of PBMNCs at 37 degrees C with 0-12 mg/dl bilirubin in solution with a fixed bovine serum albumin (BSA) concentration (3.0 g/dl) resulted in a dose-dependent increase of intracellular bilirubin in both monocytes and lymphocytes. Bilirubin uptake in monocytes was significantly higher (up to 2.7 times) than in lymphocytes under the same culture conditions. When PBMNCs were incubated with varying concentrations of bilirubin (0-16 mg/dl) in fixed BSA (3.0 g/dl) solution or at a fixed bilirubin/albumin molar ratio (0.4), the initial velocity of uptake in both cell fractions was proportional to the free (unbound to albumin) bilirubin concentration rather than the total bilirubin concentration. Bilirubin uptake by both cell fractions was significantly inhibited by treatment with metabolic inhibitors. Bilirubin uptake by monocytes continued to increase in parallel with incubation temperature from 0 degrees C to 40 degrees C, whereas uptake by lymphocytes reached a maximal level at 20 degrees C and remained constant thereafter. These results suggest that monocytes and lymphocytes incorporate bilirubin in proportion to the free bilirubin concentration and this function may rely on different energy-dependent mechanisms.

Phenol sulfotransferases (PSTs) catalyze the sulfate conjugation of catecholamines and a variety of phenolic compounds. Thermolabile and thermostable forms of PST exist in human tissue. Blood component thermostable PST activities have proved useful as measures of the enzyme activities in other tissues such as the liver. The most thoroughly studied blood component is the human platelet, which contains both thermolabile and thermostable PST activities. Partial localization of PST activity in blood has been characterized only for thermolabile PST. We performed the studies reported here to define the cellular and subcellular localization of both thermolabile and thermostable PST activities in blood elements. Blood samples from four adults were pooled and aliquots for platelet studies were anticoagulated with ethylenediaminetetraacetic acid. Aliquots for studies of granulocytes, mononuclear cells, and erythrocytes were defibrinated to avoid platelet contamination and were separated through Ficoll-Hypaque gradients. Cytosol thermolabile PST activities assayed with dopamine as the substrate and expressed as a percent of the total thermolabile PST activity per milliliter of whole blood were as follows: platelets, 97%; granulocytes, 0.6%; mononuclear cells, 0.7%; and erythrocytes, 0.4%. Cytosol thermostable PST activities measured with p-nitrophenol were as follows: platelets, 77% of the total activity; granulocytes, 19%; mononuclear cells, 1.2%; and erythrocytes, 0.5%. Plasma and membrane-bound activities were less than 2.3% of total activities for each form. Because granulocyte thermostable PST was present in an amount greater than expected, it was further characterized. The Michaelis-Menten constant values for p-nitrophenol and 3'-phosphoadenosine-5'-phosphosulfate were 1.13 mumol/L and 0.6 mumol/L, respectively. The pH optimum of 6.6, a 50% inhibitory concentration for 2,6-dichloro-4-nitrophenol of 1.0 mumol/L, and retention of 56% of activity after preincubation at 45 degrees C for 15 minutes were the same for the granulocytes as for platelet thermostable PST. In summary, our study confirms and extends our knowledge of localization of blood thermolabile PST. Our data define for the first time the localization of blood thermostable PST and highlight the substantial contribution of granulocyte thermostable PST activity. Granulocytes represent an easily obtained nucleated cell for the study of human thermostable PST.

Conventional therapy of pancreatic exocrine cancer is disappointing. The poor prognosis of the disease challenges development of novel therapeutic strategies. We report the results of clinical trials of the monoclonal antibody (Mab) 17-1A in patients with histologically verified unresectable pancreatic exocrine cancer. No antitumor response was seen in 18 patients treated with Mab 17-1A (500 mg) admixed with 10(9) autologous mononuclear cells, and 81% of the patients developed antimouse antibody response. Combination of recombinant gamma interferon and Mab 17-1A mixed with autologous mononuclear white cells resulted in complete response of 4-mo duration in 1 out of 25 evaluable patients and unusually stable disease from 4 to 48+ mo in another 6 patients. High intermittent doses of infused Mab 17-1A did not show any objective antitumor response and caused serious anaphylaxis in two of the patients in the trial. Because examination of six pancreatic adenocarcinoma cell lines with different doses of Mab 17-1A and IL-2 failed to augment lytic activity of mononuclear effector cells against all cancer cell lines tested, there seemed to be no rationale for pursuing clinical studies with IL-2 and Mab 17-1A in either the murine or chimeric form. Attractive therapeutic approaches include active immunotherapy with immunization using idiotypic antibodies or targeted toxicity with the use of radioimmunoconjugates, particularly 125I-labeled chimeric Mab 17-1A.

Thirty patients with advanced measurable pancreatic adenocarcinoma were entered onto a phase II trial with recombinant interferon gamma (Biogen, Cambridge, MA; 10(6) U/m2 daily for 4 days) and monoclonal antibody (Mab) 17-1A (Centocor, Malvern, PA; 150 mg in autologous leukocytes on days 2, 3, and 4 following interferon infusion). The effect of a single interferon gamma treatment on natural and antibody-dependent cellular cytotoxicity (ADCC), Fc receptor occupancy by antibody, and human leukocyte antigen-DR (HLA-DR) expression on monocytes and lymphocytes was also studied. Toxicity was modest and generally limited to grade I to II fever, nausea and vomiting, and hepatotoxicity. Five patients were considered to be nonassessable for response. Of the 25 assessable patients, one objective response (complete remission for a duration of 4 months) was observed. Stable disease for 2 months or greater was noted in nine patients. The median survival for the group was 5 months. Analysis of cytotoxicity data obtained prior to treatment showed reduced natural cytotoxic activity in these patients compared with normal volunteers. A significant improvement in natural cytotoxic activity to normal levels occurred within 24 hours following the interferon gamma infusion. This was also associated with augmented antibody-dependent cellular cytotoxicity. Although HLA-DR expression was not increased on either monocytes or lymphocytes, an increased capacity of both lymphocytes and monocytes to bind Mab 17-1A was observed. In all in vitro assays of ADCC, the presence of antibody excess was associated with improved cytolytic activity. In spite of the favorable modulation of cytolytic activity and improved ability of effector cells to bind Mab, we failed to demonstrated adequate clinical efficacy in the treatment of patients with pancreatic adenocarcinoma using this dose and schedule of interferon gamma and Mab 17-1A. Future trials will focus on alternate schedules of Mab 17-1A with the hope of improving tumor antigen saturation and circulating levels of infused antibody.

In vitro natural killer (NK) activity expressed by blood mononuclear cells from patients with chronic granulomatous disease of childhood (CGD) was equivalent to that expressed by cells from normal, healthy volunteers. Because neutrophils and monocytes from these same donors exhibited extremely depressed oxidative functions, our data could be interpreted to show that a) NK cells derived from a unique and separate cellular lineage unaffected by the disease-related oxidative defect, or b) the in vitro cytolytic mechanism(s) of NK cells were not dependent on oxygen metabolites. These hypotheses were examined by using as NK effector cells large granular lymphocytes (LGL) from healthy donors whose monocytes and neutrophils had normal oxidative functions. Such functions were measured in the nitroblue tetrazolium dye reduction assay, which is a qualitative measurement of superoxide anion production; by reduction of ferric cytochrome c, a more specific and quantitative measurement of superoxide anion production; and in the luminol-enhanced chemiluminescence assay, an extremely sensitive measure of several reactive oxygen radicals, including superoxide anion, hydroxyl radical, and singlet oxygen. Whereas monocytes and neutrophils from healthy donors were readily stimulated with zymosan or phorbol myristate acetate (PMA) in each of these assays. LGL produced no detectable amounts of oxygen metabolites when co-incubated either with K562 erythroleukemia cells, PMA, E. coli endotoxin, or the calcium ionophore A23187. Thus, because NK cell activity is normal in CGD patients with major oxidative defects, and because no reactive oxygen metabolites could be detected in LGL that simultaneously exhibited potent NK activity, we conclude that in vitro NK activity by human mononuclear cells involves a lytic mechanism(s) independent of oxygen metabolites.

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.

Members of the genus Leishmania are important intracellular pathogens that produce either cutaneous, mucocutaneous, or visceral disease in many areas of the world. In humans as well as in other mammals, the parasite is inoculated through the skin as a flagellated, extracellular promastigote by its arthropod vector, the sandfly. Once in its mammalian host, the promastigote converts to its amastigote stage, which lacks an exteriorized flagellum and is found solely within mononuclear phagocytes during established infection. In vitro, human monocyte-derived macrophages and peritoneal macrophages from several species of rodents can ingest both promastigotes and amastigotes, and they can permit intracellular multiplication of amastigotes only. Although serum factors may play a role in the pathogenesis of the disease and in protection against reinfection, the resolution of leishmaniasis is dependent primarily on cell-mediated immune responses. There appears to be a complicated interplay between cell-mediated helper and suppressor activities. The outcome of infection in each type of leishmaniasis depends on the complex and intriguing interaction of virulence factors inherent in the parasite and genetically determined host defense mechanisms.

Lymphocytes with Fc receptors (FcR) for IgG active in natural cytotoxicity and antibody-dependent cellular cytotoxicity were separated into sheep erythrocyte rosetting (E+) and nonrosetting (E-) fractions, and examined for reactivity with the OK panel of hybridoma-produced monoclonal antibodies. Few cells in either the E+ FcR+ or the E- FcR+ fraction reacted with seven antibodies used to define T cells in various stages of differentiation (OK3, OKT4, OKT5, OKT6, OKT8, OKT9, OKT10). Neither fraction expressed an Ia-like antigen (detected by OKI1), but both were highly reactive with OKM1, an antibody that reacts with monocytes and granulocytes. Incubation of these cytotoxic effector cells with OKM1 plus complement abolished all cytotoxic reactivity, but incubation with a pan-T cell antibody (OKT3) plus complement had no significant effect. These cells were not monocyte precursors, because they could not be induced in vitro to develop macrophage characteristics. The data indicate that most cytotoxic effector cells in natural cytotoxicity and antibody-dependent cellular cytotoxicity are not in the T cell lineage, but have a myeloid origin.

The authors developed 52 permanent lymphoid cell cultures from various human sources, mainly from neoplastic lymphoproliferative disease. These cultures are reviewed against the background of similar cultures obtained in several other laboratories. The peculiar features of these cultures are: 1) the phenomenon of lymphoblastoid transformation; 2) the production of both globulins and mediators of delayed hypersensitivity; 3) the presence of herpes type virus as related to etiology or autoimmunity; and 4) cytotoxicity and other assays for the demonstration of tumor-specific antigens.

Platelet-free cellular elements and non-hemolyzed, chemically unaltered serum are important research components of the cellular immunology laboratory. Both can be recovered from the same peripheral blood sample if it is properly defibrinated. The numbers of cells recovered from heparinized aliquots of blood from healthy donors were not significantly different from the numbers of mononuclear leukocytes, polymorphonuclear leukocytes, and erythrocytes recovered from blood samples which had been mechanically defibrinated in vitro with a stationary, cone-shaped 'TP'-like device which we here describe. Compared with serums obtained from clotted blood, or from blood defibrinated by using glass beads, we found that serums from blood defibrinated with the 'TP'-like device had the lowest detectable levels of hemoglobin, free DNA, or LDH. Serums from TP-defibrinated blood were not different from clotted serum samples with regard to the function of the classical complement pathway, the alternative complement pathway, C4 hemolytic activity, and most serum chemistries. Use of the TP-defibrinator in immunology laboratories is an ideal way to prepare blood for rapid isolation of cellular elements and non-hemolyzed serum from the same sample.

A role for the dietary trace mineral element selenium in the reduction of cancer incidence has been documented in numerous epidemiological and experimental studies. The precise mechanism of this antitumor effect is not well understood, but published data suggest that both inhibition of tumor cell growth and enhancement of host immunity are likely to be involved. In this study we report that selenium at physiologic concentrations can inhibit human lymphocyte proliferation in response to irradiated tumor cells in mixed lymphocyte/tumor cell cultures (MLTC). In addition, we demonstrate that the various lymphocyte functional activities generated in these cultures exhibit different levels of sensitivity to the effects of selenium. The generation of suppressor-cell activity in MLTC was strongly inhibited by the presence of physiologic levels of selenium, while the development of cytotoxic T-lymphocyte activity in identical cultures was not affected by selenium. Production of interleukin-2 in these cultures showed an intermediate sensitivity to the effects of selenium. Thus, selenium appears to be capable of selectively regulating the generation of functional lymphocyte subsets in vitro. Such selective regulation could explain the published effects of selenium on immunity and would be consistent with a role for immunity in the observed reduction of cancer incidence associated with elevated selenium intake.

An inverse correlation between cancer incidence and dietary intake of the trace mineral element selenium has been well established in epidemiological and experimental studies. The mechanisms for this chemoprotective effect are unresolved. Much attention has been focused on the antiproliferative effects of selenium on various normal and neoplastic cell types. However, dietary selenium supplementation can also enhance the expression of various humoral and cellular immune responses. In examining the effects of dietary selenium on cell-mediated immunity in mice, we observed that selenium supplementation caused the enhanced expression of spontaneous natural killer (NK) cytotoxicity in spleen cells and of specific cytotoxic T-lymphocyte (CTL) cytotoxicity in peritoneal exudate cells (PEC). NK activity of spleen-cell suspensions from selenium-supplemented mice increased an average of 70% over that of the control group (basal diet). Cytotoxic activity of PEC from mice injected with tumors intraperitoneally peaked earlier in selenium-supplemented animals, and the appearance of cells staining positively for Thy 1.2 surface antigen in selenium-supplemented animals also preceded the values observed in control animals. We propose here that enhancement of in vivo cytotoxic mechanisms, is likely to act synergistically with tumor growth inhibition in the reduction of tumor incidence associated with selenium intake.

The influx of neutrophils into the alveolar structures can be induced by stimulation of the resident lung phagocyte, the alveolar macrophage, to release a potent neutrophil chemoattractant(s). We hypothesized that the fifth component of complement (C5) on the cell surface may be required for activation of the alveolar macrophage to release neutrophil chemotactic activity. C5 was identified on guinea pig alveolar macrophages by epifluorescent microscopy, flow cytometry, and enzyme-linked immunoabsorbent assay of eluted macrophages. When cultured for 4 h with stimuli that induce the release of chemotactic activity or for 24 h without added stimuli, purified Fab' fragments of a goat anti-C5 antibody significantly inhibited the ability of macrophages to release chemotactic activity as determined by a blindwell chamber method (p less than 0.001, all comparisons). This inhibition of chemotactic activity was not detected when anti-C5 antibody was added after the culture period. In contrast, anti-C3 antibody had no inhibitory effect at 4 h or at 24 h (p greater than 0.2, all comparisons). Partial characterization of released chemotactic activity revealed it was of low molecular weight, partially lipid soluble, and not inhibited by C5a chemotactic factor inactivator. These studies suggest that C5 may have a regulatory role in the release of chemotactic activity by alveolar macrophages.

Virulent Bordetella organisms produce an adenylate cyclase which is extracytoplasmic in location, activated by the eukaryotic regulatory protein, calmodulin, and able to act as a toxin, promoting cyclic AMP accumulation in target mammalian cells. Initial steps in purification of this novel adenylate cyclase toxin reveal two forms: one which possesses only enzymatic adenylate cyclase activity, but has no effect on intact target cells; and the other which has both enzymatic and intoxicating activity. These data suggest that this toxin may conform to the A/B model for bacterial toxins. A variety of mammalian cell types can be affected by the adenylate cyclase toxin, including neutrophils, macrophages, monocytes, lymphocytes, lymphoma cells, and pituitary cells. Although the consequence of intoxication in many cells is inhibition of normal function, the enhancement of pituitary hormone secretion by this toxin suggests that its biological effects are the result of cAMP accumulation. These data confirm the hypothesis that Bordetella adenylate cyclase is, indeed, a toxin and illustrate its role as a novel research probe.

A chemoprotective role for dietary selenium in malignancy has been well documented in numerous epidemiological and experimental studies. The precise mechanisms of this relationship are not understood, but may be related to observations that selenium can inhibit the proliferation of various normal and neoplastic cells, both in vivo and in vitro. In this study, we present evidence that selenium at physiologic concentrations can effectively inhibit the overall proliferation of human lymphocyte populations in response to various immune stimuli in vitro, including mixed lymphocyte response and response to soluble antigen (tetanus toxoid). This inhibition was reversible, indicating that selenium was not toxic to the lymphocytes at these concentrations. Preliminary data from our laboratory indicate that the antiproliferative effects of selenium may be specific for certain lymphocyte subsets. Similar modulation of immune responses in vivo could enhance various humoral and cellular immune mechanisms. Together with published evidence that selenium can inhibit tumor cell proliferation, these data may help to explain the decreased incidence of cancer associated with elevated selenium intake.

The usefulness of sonicated Leishmania braziliensis panamensis promastigotes for vaccination was evaluated in Mystromys albacaudatus, the African white-tailed rat. Thirty-two animals received three intradermal injections of 2 x 10(6) sonicated promastigotes derived from L. braziliensis panamensis at weekly intervals. One month after completion of the immunization schedule, the experimental group of animals was challenged in vivo with 2 x 10(6) live L. braziliensis panamensis promastogotes. At the same time, a matched group of 40 control animals was similarly challenged. Within 2 months, 35 of the 40 animals (87.5%) the control group developed ulcers, while only 14 of 32 previously vaccinated animals (43.7%) developed ulcers at the site of challenge within the same time period. The remaining 18 vaccinated animals (56.2%) remain free from ulcers 7 months after challenge. When lymphocytes from the spleens of vaccinated and control animals were challenged in vitro with antigen derived from sonicates of varying numbers of promastigotes, only cells from immunized animals responded vigorously to the antigenic challenge, a response which was not enhanced by the addition of immune antiserum to the reaction.

When Fc receptors (FcR) on normal human peripheral blood lymphocytes were induced to modulate by overnight (18 h) incubation in the presence of soluble or particulate immune complexes, the natural killer (NK) activity of the effector lymphocyte suspension, as measured against the K562 erythroleukemia cell line, was significantly, but only partially, inhibited. The NK activity which remained was always strong, and was not significantly inhibited by inclusion of antigen-antibody complexes in the cytotoxicity assay, nor was it further depleted by adsorbing the modulated cells on plastic surfaces coated with immobilized antigen-antibody complexes. Antibody-dependent cell-mediated cytotoxicity (ADCC) against rabbit antibody-sensitized Chang liver cells was totally abrogated following the modulation process, and could not be restored by exposure of modulated effector cells to trypsin, indicating that the FcR had actually been shed and were not merely being blocked with immune complexes. Although freshly isolated peripheral blood lymphocytes active in natural (or "spontaneous") cytotoxicity have been shown to bear FcR, our data indicate that NK activity against the K562 cell line can be effectively mediated by NK cells which have lost their FcR. This supports the concept that NK activity against K562 is independent of FcR, and, therefore, of IgG.

Using a series of techniques to identify and deplete various peripheral blood lymphocyte subpopulations, we studied the cytotoxic reactivity of normal individuals against the myeloid cell line K-562 in a 4-hr 51chromium-release assay. Depletion of lymphocytes bearing complement receptors had a variable, usually negligible effect on cytotoxicity. In contrast, depletion of lymphocytes bearing Fc receptors abrogated target cell lysis. Separation of lymphocytes with high-affinity binding of sheep red blood cells (SRBC) evidenced by rosette formation at 29 degrees C yielded a population of rosette-forming cells containing few cytotoxic cells, whereas separation of total E-RFC under optimal rosetting conditions produced a rosette fraction containing a major proportion of the effector cells. These data indicate that the cytotoxic lymphocyte in this system is Fc receptor positive, largely complement receptor negative, and may possess low density or low affinity receptors for SRBC.

A simple procedure is described which permits rapid layering of large volumes of diluted blood onto Ficoll-Hypaque gradients with no requirement for specialized syringes, cannulas, pipettes, or support racks. In use for several years, the method is as good as, or better than, other techniques.

Selenium is a trace mineral and a required nutrient for animals and humans. Selenium intake appears to be inversely correlated with the risk of developing cancer. Since immunological effects of selenium have been described we studied the capacity of selenium to modify the lupus-like disease of NZB/NZW female mice. Our data indicate that selenium supplementation (sodium selenite 4 parts per million in the drinking water) significantly improves survival in these autoimmune mice: mean survival 55.6 +/- 4.6 weeks (mean +/- s.e.) for treated mice versus 36.1 +/- 1.9 weeks for controls (P less than 0.04). Additionally, selenium supplemented mice had significantly higher natural killer cell activity (P less than 0.001). However, no obvious effects of selenium supplementation on autoantibody production were observed.