phytohemagglutinin-M Research Articles

A Cell-Free Protein Expression System Derived from Human Primary Peripheral Blood Mononuclear Cells.

Historically, some of the first cell-free protein expression systems studied in vitro translation in various human blood cells. However, because of limited knowledge of eukaryotic translation and the advancement of cell line development, interest in these systems decreased. Eukaryotic translation is a complex system of factors that contribute to the overall translation of mRNA to produce proteins. The intracellular translateome of a cell can be modified by various factors and disease states, but it is impossible to individually measure all factors involved when there is no comprehensive understanding of eukaryotic translation. The present work outlines the use of a coupled transcription and translation cell-free protein expression system to produce recombinant proteins derived from human donor peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-M (PHA-M). The methods outlined here could result in tools to aid immunology, gene therapy, cell therapy, and synthetic biology research and provide a convenient and holistic method to study and assess the intracellular translation environment of primary immune cells.

Low-dose endosulfan inhibits proliferation and induces senescence and pro-inflammatory cytokine production in human lymphocytes, preferentially impacting cytotoxic cells

Endosulfan is a DDT-era organochlorine pesticide. Due to past and current environmental contamination, investigation of endosulfan exposure is of current importance. Acute high dose exposure precipitates neural/endocrine system damage, but the effects on the immune system and of lower doses are not well-characterized. Two relatively low concentrations of endosulfan (i.e. 0.1 and 17 µM ENDO) were investigated in an in vitro study using human peripheral blood mononuclear cells (PBMC) to understand effects of relatively low doses (0.1–25.0 µM [≈0.04–10 ppm/40–10,000 ppb]) of ENDO upon normal human T- and B-lymphocytes and NK cells. The study here found that 17 µM ENDO inhibited phytohemagglutinin-M (PHA)-induced human PBMC proliferation. It was also seen that senescence and apoptosis among non-stimulated cells was increased, specifically within CD8 and NK populations, and that CD4:CD8 ratios also were increased. Treatment of non-stimulated PBMC with ENDO led to overall increases in production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, -4, and -6, and decreased production of anti-inflammatory IL-10, suggesting an immunosenescence secretory phenotype. Interestingly, when the cells were pre-stimulated with mitogen (PHA), ENDO became inhibitory against the mitogen-induced proliferation and cytokine formation – with the exception of that of TNFα and IL-6, suggesting differential effects of ENDO on activated cells. Thus, at the organismal level, ENDO might also display differential effects during states of autoimmune disease or chronic viral infection in the exposed host.

Abstract 17107: Distribution of Anti-inflammatory Activity During Remodeling of High Density Lipoprotein: Studies With CSL112

Elevated levels of circulating inflammatory markers predict an unfavorable cardiovascular outcome in acute coronary syndrome patients. CSL112 is human apolipoprotein A-I (apoA-I), reconstituted with phosphatidylcholine to form HDL particles suitable for infusion. Addition of CSL112 to stimulated human whole blood ex vivo strongly reduces pro-inflammatory cytokine production. Infusion of CSL112 into human subjects or addition to human plasma ex vivo causes remodeling of endogenous HDL. Similar remodeling occurs upon incubation of CSL112 with purified HDL3 and results in accumulation of three HDL species: enlarged HDL (HDL2), a smaller, dense species (HDL3c), and lipid-poor apoA-I (pre-β1 HDL). Study aim was to determine the anti-inflammatory activity of remodeled HDL species. CSL112 was incubated with HDL3 and the products of particle remodeling were purified by ultracentrifugation. The inhibitory effects on pro-inflammatory cytokine production were examined using human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin-M (PHA-M) in vitro. Lipid-poor apoA-I, HDL3c as well as parent CSL112 exerted powerful inhibitory effects on secretion of pro-inflammatory mediators (> 89.2% + 4.0% inhibition of TNF-α, IL-1β, IL-6 and Mip-1β); HDL3 and HDL2 were much less effective (< 54.1% + 3.9% inhibition). The extent of inhibition correlated positively with induction of the transcription repressor ATF3, a negative regulator of pro-inflammatory cytokine production, with lipid-poor apoA-I and HDL3c inducing higher protein levels of ATF3 in PHA-stimulated PBMC compared to control medium, HDL3 or HDL2. Anti-inflammatory activity of the remodeled species also correlated with their ability to support cellular cholesterol efflux via the ABCA1 transporter: lipid-poor apoA-I and HDL3c were potent acceptors of cholesterol; HDL2 was inactive. The ability to generate HDL species with high cholesterol efflux and anti-inflammatory activity makes CSL112 a promising candidate for removing cholesterol and reducing inflammation in atherosclerotic plaque, thus reducing the high risk of early recurrent atherothrombotic events following acute MI (AMI). A Phase IIb trial (AEGIS-I; NCT02108262) of CSL112 in AMI patients is ongoing.

Impaired IL-2 expression in latent HIV-1 infection

Regarding the T cell function in HIV-1 infection, activation of T cells is enhanced in acutely HIV-1-infected T cells upon stimuli. However, T cell immune responses underlying the activation of T cell receptor (TCR) signaling molecules and interleukin (IL)-2 production in latently HIV-1-infected cells are poorly understood. The expression and activation of TCR components and its downstream molecules in acutely and latently HIV-1-infected T cells were compared using quantitative reverse transcription polymerase chain reaction (RT–PCR) for mRNA expression and enzyme-linked immunosorbent assay (ELISA) for levels of IL-2 in phytohemagglutinin M (PHA-M). The levels of T cell surface molecules and TCR signaling molecules in latently HIV-1-infected cells were greatly decreased without changes in their mRNA levels. In addition, downstream TCR-signaling molecules in latently HIV-1-infected cells were not activated even in the presence of PHA-M. The phosphorylation of mitogen-activated protein kinases (MAPKs) in the presence of PHA-M was weakly induced in latently HIV-1-infected cells but was greater in acutely HIVNL4-3-infected cells. Finally, the production of IL-2 was significantly decreased in latently HIV-1-infected cells compared with uninfected parent cells. Thus, IL-2-related immunological functions in latently HIV-1-infected T cells were markedly impaired even in the presence of stimuli.

Acute immunomodulatory effects of binge alcohol ingestion

BackgroundBlood alcohol is present in a third of trauma patients and has been associated with organ dysfunction. In both human studies and in animal models, it is clear that alcohol intoxication exerts immunomodulatory effects several hours to days after exposure, when blood alcohol is no longer detectable. The early immunomodulatory effects of alcohol while blood alcohol is still elevated are not well understood. MethodsHuman volunteers achieved binge alcohol intoxication after high-dose alcohol consumption. Blood was collected for analysis prior to alcohol ingestion, and 20 min, 2 h, and 5 h after alcohol ingestion. Flow cytometry was performed on isolated peripheral blood mononuclear cells, and cytokine generation in whole blood was measured by enzyme-linked immunosorbent assay (ELISA) after 24-h stimulation with lipopolysaccharide (LPS) and phytohemagglutinin-M (PHA) stimulation. ResultsAn early pro-inflammatory state was evident at 20 min when blood alcohol levels were ∼130 mg/dL, which was characterized by an increase in total circulating leukocytes, monocytes, and natural killer cells. During this time, a transient increase in LPS-induced tumor necrosis factor (TNF)-α levels and enhanced LPS sensitivity occurred. At 2 and 5 h post-alcohol binge, an anti-inflammatory state was shown with reduced numbers of circulating monocytes and natural killer cells, attenuated LPS-induced interleukin (IL)-1β levels, and a trend toward increased interleukin (IL)-10 levels. ConclusionsA single episode of binge alcohol intoxication exerted effects on the immune system that caused an early and transient pro-inflammatory state followed by an anti-inflammatory state.

Phytohemagglutinin And Light-induced Charge Density Effects On Plasma Membrane Of Plectonema Boryanum

Effects of the plant lectin phytohemagglutinin M (PHA-M) and illumination on the electrophoretic mobility (EPM) and 90°-light scattering of suspension of spheroplasts obtained by lysis of the unicellular diazothrophic cyanobacterium Plectonema boryanum were studied. Cells cultures were grown on media with different iron content: an iron-deficient (denominated as “Fe-starved” culture), an iron-sufficient (“Fesufficient”), and a twentyfold iron excess medium (“Fe-excess”). Lectin addition led to a decrease of the membrane charge density and aggregation of the vesicles. Our results indicate significant influence of the Fe and illumination on the lectin-induced decreasing of EPM and light scattering intensity. *Corresponding author: E-mail: virjird@biofac.uni-sofia.bg ; Tel.: (00 359 2) 2 8167 281 ; Fax: (00 359 2) 2 865 66 41

Urinary biomarkers and cytokine response in recent drinkers compared to non-recent drinkers after binge ethanol dosing and phytohemagglutinin-M (PHA) stimulation

s / Alcohol 47 (2013) 567–576 568 1. Urinary biomarkers and cytokine response in recent drinkers compared to non-recent drinkers after binge ethanol dosing and phytohemagglutinin-M (PHA) stimulation M. Afshar, J. Hasday, G. Netzer, G.S. Smith, Pulmonary and Critical Care Medicine, Shock Trauma and Anesthesiology Research (STAR) – Organized Research Center, University of Maryland Medical Center, Baltimore, MD, USA Ethanol intoxication is present in up to 50% of trauma patients. Severe injury may activate the systemic inflammatory response syndrome (SIRS). How binge ethanol affects patients with SIRS is unclear. We performed a pilot study evaluating binge ethanol consumption prior to ex-vivo endotoxin challenge and its effects on immune responsiveness. Urinary EtG (Ethyl Glucuronide) and EtS (Ethyl Sulfate) were used to compare subjects consuming ethanol 24–48 h prior to ethanol dosing (recent drinkers) to those who did not (non-recent drinkers). Ten healthy human volunteers characterized as low to moderate drinkers provided urine samples prior to and 24 h after ethanol dosing (vodka drink over 20 min: males 0.89 g/kg, females 0.81 g/kg). Blood alcohol content (BAC) and heparinized blood was collected prior to dosing, 20 min, 2 h, and 5 h after dosing. Aliquots of blood were diluted 1:1 RPMI 1640 medium and incubated for 24 h with 0.6–6% PHA. Culture supernatants were collected by centrifugation and cytokine levels were measured by ELISA. Four subjects were recent drinkers and had elevated pre-dosing EtG and EtS levels (23,921 35,178 ng/ml and 4418 6262 ng/ml), and the 6 non-recent drinkers had no measurable levels. The mean peak BAC was 142 36 mg/ dl. No difference in BAC between recent and non-recent drinkers was found at the three sampling points. No differences in cytokine expressions by drinking type were found in PHA-stimulated blood prior to binge ethanol dosing. Similar elevations of PHA-stimulated IFN-g, and IL-8 levels occurred after binge ethanol dosing. IL-2 expression was greater in recent drinkers after binge (p 1⁄4 0.03) compared to non-recent drinkers. This suggests that recent drinkers of ethanol may have a modulated immune response after binging compared to non-recent drinkers. EtG and EtS are useful to identify recent drinkers who have no detectable BAC. Studies investigating the immunomodulatory effects of ethanol exposure after injury or SIRS should further delineate exposure to include recent drinkers utilizing EtG and EtS. 2. Role of microRNA-155 in alcoholic liver disease S. Bala, T. Csak, J. Zatsiorsky, D. Catalano, K. Kodys, G. Szabo, Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA MicroRNAs (miRNAs) are small non-coding RNAs that emerged as new crucial regulatory players of all cellular processes. miRNA-155 is a multifunctional miRNA that regulates various immune and non-immune biological processes. Previously, we showed the induction of miR-155 in alcoholic liver disease (ALD) where it contributes to inflammation via regulating TNF alpha. To further elucidate its biological role in ALD, we employed miR-155 deficient (KO) mice. Female wild type (C57/BL6J) or miR-155 KO or TLR4 KO mice (n 1⁄4 8–10) were fed the Lieber-DeCarli diet containing either ethanol or control diet for 5 weeks. Our results indicate that miR-155 KO mice are protective from alcohol-induced liver inflammation and gut permeability. A significant attenuation in liver TNF alpha, IL-1beta and MCP1 (mRNA and protein) was found in alcohol-fed miR-155 KO mice compared to wild type mice treated with or without LPS (0.5 mg/ kg for 3 h prior to conclusion of the experiment). Surprisingly, there was no increase in plasma endotoxin levels in miR-155 deficient mice after chronic alcohol feeding, indicative of intact gut epithelium. Further, a partial, but significant decrease in fat accumulation was observed in alcohol-fed miR-155 KO mice compared to wild type mice. Alcohol feeding resulted in a significant induction of oxidative stress (measured by TBAR assay) in wild type mice and this increase was prevented in miR-155 KO mice. Interestingly, alcohol-induced decrease in hepatic miR-122 levels observed in wild type mice was prevented in miR-155 KO mice, suggesting miR-155 plays a causal role in alcoholic liver injury. Our results also indicate that alcohol induces miR-155 via TLR4 pathway as TLR4 KO mice in contrast to wild type mice showed no increase in miR-155 after alcohol feeding. In summary, our results suggest that miR-155 plays an essential role in the pathogenesis of ALD and therapeutic inhibition of this miRNA might be an attractive strategy to ameliorate ALD (Supported by NIH AA020744 [GS]). 3. Chronic plus binge ethanol feeding synergistically induces neutrophil infiltration and liver injury: A critical role for E-selectin A. Bertola, O. Park, B. Gao, Laboratory of Liver Diseases, NIAAA, NIH,

Visualization of immunoreactive growth hormone in cultured peripheral bovine lymphocytes

Growth hormone (GH) has been shown to be released by immune cells in vitro. Thus, the intracellular confinement of GH immunoreactivity was investigated in cultured bovine lymphocytes using con-focal microscopy. Peripheral blood lymphocytes from cows in early pregnancy (10–20days post insemination; pi) or during mid-pregnancy (day 110–140 pi) were harvested and cultured for 48h in presence of phytohemagglutinin-M (PHA-M) or served as controls. Thereafter, immunocytochemistry was conducted using a homologous GH-antibody. Double staining (GH-antibody and directly DYE 549 labeled CD3-antibody) was performed to classify the cells. Con-focal laser scanning was applied verifying the immunofluorescence labeling. Interestingly, the presence of GH immunoreactivity in the cytoplasm, which indicates GH synthesis, was restricted to small cells. Whereas, few T-like cells revealed surface bound GH. Lowest immunoreactivity, concerning the number of the total labeled cells as well as the intensity of labeling was recorded in early pregnancy. Stimulation with PHA-M enhanced total labeled cells in early pregnancy. In contrast, PHA-M had no such effects in mid-pregnancy. The results confirm the specific regulation of synthesis of lymphocytic GH during pregnancy in the cow. The identification of cells producing GH and the elucidation of the mechanisms underlying the expression of GH in larger number of cells during mid-pregnancy than in the early pregnancy need further investigations.

Alterations in T-lymphocyte sub-set profiles and cytokine secretion by PBMC of systemic lupus erythematosus patients upon in vitro exposure to organochlorine pesticides

Chronic exposure to organochlorine pesticides (OCP) has been suspected of causing immunoregulatory abnormalities that eventually lead to development and progression of Systemic Lupus Erythematosus (SLE), but the role of these non-genetic stimuli has remained poorly understood. The objectives of the study were to quantify the levels of different OCP residues in the blood of SLE patients and to study the effects of in vitro treatment of peripheral blood mononuclear cells (PBMC) from these patients and healthy controls with OCP. Levels of different OCP residues in the blood were measured by gas-liquid chromatography. Isolated PBMC were treated in vitro with hexachlorocyclohexane (HCH), o,p’-dichlorodiphenyltrichloroethane (DDT), or phytohemagglutinin-M (PHA-M) for 72 h, then stained with different dye-labeled monoclonal antibodies to analyze alterations in T-lymphocytes using flow cytometry. Levels of different TH1 and TH2 cytokines were also estimated by ELISA. Significantly higher levels of p,p’-DDE and β-HCH were detected in the blood of SLE patients than in healthy controls. HCH exposure markedly increased the percentages of CD3+CD4+ T-lymphocytes and expression of CD45RO+ on CD4+ and CD8+ T-lymphocytes, but decreased CD4+CD25+ T-lymphocytes in SLE patients. DDT exposure increased the percentages of CD3+CD4+ T-lymphocytes and decreased those of CD4+CD25+ T-lymphocytes in SLE patients as compared to healthy controls. No significant responsiveness of patient PBMC to PHA-M stimulation was observed indicating suppression of T-lymphocytes by these OCP. Further, both HCH and DDT decreased the levels of IL-2 and IFNγ but had no effect on IL-4 levels in SLE patients. DDT also increased significantly the levels of IL-10 in patients. It is likely that higher levels and prolonged durations of exposure to HCH and DDT may significantly influence T-lymphocyte sub-sets and cytokine expression in vivo that could lead to the development or exacerbation of SLE.

Telomerase activity in peripheral blood mononuclear cells from senile patients with pneumonia

To investigate the changes of the activity of telomerase in peripheral blood mononuclear cells (PBMCs) from senile patients with pneumonia, the telomerase activity was examined before and after the stimulation of phytohemagglutinin-M (PHA-M) in PBMCs from 10 control subjects (group A), 12 non-senile patients with pneumonia (group B) and 9 senile patients with pneumonia (group C). Also observed was the proliferative response of these PBMCs to PHA-M. The results showed that, both with or without the stimulation of PHA-M, the values of telomerase activity in PBMCs from group C patients (A values: pre-stimulation, 0.43 +/- 0.04; post-stimulation, 0.63 +/- 0. 03) were significantly lower than those in PBMCs from both group A patients (A values: pre-stimulation, 0.65 +/- 0.05; post-stimulation, 1.26 +/- 0.13; P<0.001, respectively) and group B patients (A values: pre-stimulation, 0.63 +/- 0.03; post-stimulation, 0.93 +/- 0.03; P<0.05, respectively). The results of MTT test showed that the proliferative activity of PBMCs in group C patients (A value: 0.35 +/- 0.03) was also significantly lower than that in group A patients (A value: 0. 55 +/- 0.04; P<0.05) and group B patients (A value: 0.46 +/- 0.03; P<0.05). These results indicate that the telomerase activity decreases in senile patients with pneumonia, which may be one of the mechanisms for the weakened immune function in those patients.

Immunomodulatory compounds from Pestalotiopsis leucothës, an endophytic fungus from Tripterygium wilfordii

The immunomodulatory effects of three compounds designated BS, GS, and YS produced by Pestalotiopsis leucothës, an endophytic fungus isolated from Tripterygium wilfordii, were evaluated. The 50% inhibition concentration (IC 50) value of BS in the proliferative assay with various stimulating agents such as phytohemagglutinin-M (PHA-M), phorbol myristate acetate (PMA)/ionomycin, mixed lymphocyte reaction (MLR) and poke weed mitogen (PWM) was 0.35, 1.6, 0.8 and 5.4 μg/ml, respectively. In addition, BS significantly inhibited the production of cytokines such as interleukin (IL)-1β, IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, by peripheral blood mononuclear cells (PBMNC) and soluble IL-2 receptor expression at concentrations greater than 1 μg/ml. Inhibition of PHA stimulated PBMNC proliferation and IL-2 and sIL-2R production by BS indicates that it is a T-cell specific immunosuppressant. However, BS also moderately inhibited immunoglobulin (Ig) G and M at concentrations greater than 1 μg/ml suggesting that it also has B cell immunosuppressive effects. YS was 10% less active than BS in all assay systems. In contrast, GS exhibited both suppression and enhancement of PBMNC proliferation in the presence of various stimulants. However, GS inhibited PWM stimulated PBMNC proliferation and IL-4 and IgG and IgM production at concentrations above 1 μg/ml. All three fungal compounds altered the percentage of T-lymphocyte subpopulations only at high concentrations. Cell viability was not affected at the immunosuppressive concentrations of these compounds. In conclusion, work from our laboratory has identified three potentially potent immunomodulatory compounds from P. leucothës. These compounds have variable effects on T- and B-cells and monocytes. They may partially explain the immunosuppressive activity of T. wilfordii. In addition, they may represent a new source of immunomodulatory compounds for the treatment of human immune mediated diseases.

The modulator role of the hypothalamic paraventricular nucleus on immune responsiveness

Role of the paraventricular nucleus of the hypothalamus (PVH) upon immune modulation was studied by either mechanically destroying the PVH (PVHL) or by isolating the PVH (PVHI) with a knife-cut. PVHL or PVHI manipulations induced significant leukopenia characterized by a decrease in the number of neutrophils and lymphocytes two weeks post surgery. The numbers of circulating monocytes and eosinophils were not affected by PVH interventions. In addition, PVHL and PVHI were also associated with a reduction, relative to controls, in the phagocytosis by neutrophils and an increase in blastic transformation of T lymphocytes induced by phytohemagglutinin-M (PHA-M). Antibody titers rose against sheep red blood cells (SRBC) after either PVHL or PVHI were reduced. The magnitude of the SRBC antibody reduction after PVH manipulations was similar to that observed in rats that received a peripheral chemical sympathectomy two hrs prior immunization. Comparison of thyroid hormones blood levels two weeks after PVHL or PVHI revealed significant reductions in comparison with sham-operated group (SO), whereas blood corticosterone was not significantly altered. In summary, we provide evidence that lesion or isolation of the PVH selectively reduces circulating white blood cells and the primary immune response, while it enhances the cell-mediated immune function. Taken together our data showed that PVH modulates immune functions by altering both the peripheral sympathetic tone and thyroid hormone secretion.

Adrenal steroids in the blood and urine and the immune status of women with age-associated disorders of glucose tolerance

Dehydroepiandrosterone (DHA), hydrocortisone, and insulin are naturally occurring substances influencing the immune functions. The present paper is devoted to their combined effects on the mitogen-stimulated proliferation of lymphocytes (MSPL) with phytohemagglutinin-M (PHA-M) and Pokeweed mitogen used as stimulants. A positive correlation between blood insulin and urinary DHA levels (r=0.534; p0.05) has been revealed. The level of DHA secretion was in a negative correlation with the values of MSPL stimulated by PHA-M (r=-0.651; p0.01). Hence, age-associated hyperinsulinemia may be regarded as an important regulator of both, adrenal androgen production and the cellular immunity status.

Antigens of Lyme disease of spirochaeteBorrelia burgdorferiinhibits antigen or mitogen-induced lymphocyte proliferation

Modulation of cellular immune responses by the spirochaete Borrelia burgdorferi, the bacteria that causes Lyme disease, was demonstrated. When cultured in the presence of sonicated Borrelia preparation (Bb), the mitogen- or antigen-stimulated proliferative responses of normal lymphocytes were consistently lowered. Bb caused the greatest reduction in Concanavalin A (ConA) or antigen-stimulated proliferation, where almost 100% reduction in proliferation could be achieved. Bb also reduced phytohemagglutinin-M (PHA) or pokeweed mitogen (PWM)-stimulated peripheral blood lymphocyte (PBL) proliferation, with the PWM proliferation being the least affected. This regulatory activity was not due to toxicity and was determined to be caused by Bb protein antigens. The degree of the proliferation reduction was directly proportional to both Bb quantity and length of exposure to lymphocytes. IL-2 production was significantly reduced from Bb-exposed lymphocytes. The entry of lymphocytes into the proliferating phases of the cell cycle was also shown to be blocked. These results have demonstrated an immune suppressive mechanism of B. burgdorferi. The magnitude of host immune responses may be dependent on the degree of suppression which is related to the spirochaete quantity and their length of presence in the host.

Research Note: T-Cell Activity of White Leghorn Chickens Selected for High and Low Antibody Responses to Sheep Erythrocytes

Mitogen responses of peripheral blood lymphocytes (PBL) and the cell cycle analysis of T-cells were examined for lines of chickens selected for high (HA) and low (LA) antibody response to SRBC. At 6 wk of age, within each line, blood samples and thymic tissue were collected from chicks representing the B13B13, B13B21, B21B13, and B21B21 genotypes. Although no influence of Ea-B haplotype on T-cell activity was detected in either line, there were line differences in phytohemagglutinin-M (PHA-M) and concanavalin A (Con A) mitogen responses and the percentage G0/G1 and S phase T-cells. The HA PBL had greater in vitro PHA-M and Con A responses, and the cell cycle analysis of T-cells revealed a greater percentage of cells in S phase for Line HA chicks than for LA chicks. There were significantly more resting cells in the G0/G1 phase of LA than HA chicks. Although there was no Ea-B haplotype effect observed on T-cells from either line, generalized selection for high or low antibody response did result in divergent T-cell activity.

Immunity to Pasteurella multocida in Protein-Deficient Chickens

Studies were conducted to determine the effects of dietary protein restriction on the humoral immunity (HI) and cell-mediated immunity (CMT) of chickens. New Hampshire chickens were separated into two dietaiy treatment groups: basal, containing 3,200 kcal/kg and 21% protein; or protein restricted (PR), containing 3,200 kcal/kg and 7% protein. In studies involving HI, half of the birds in each dietary treatment were vaccinated against fowl cholera at 4 and 8 wk of age. Blood samples were collected weekly beginning at 4 wk of age. Overall, unvaccinated birds had lower titers than vaccinated birds and PR groups generally showed lower titers than basal groups. All birds were challenged by palatine cleft inoculation of live, virulent Strain X-73 of Pasteurella multocida. The vaccinated PR group survived live challenge as well as the vaccinated basal group, but all unvaccinated birds died as a result of the challenge, regardless of antibody titer.In studies involving CMI, half of the birds in each dietary treatment were vaccinated at 5 wk of age. At 2 to 3 wk postvaccination, representative birds from each treatment were bled for total and differential blood counts. Also, birds were sacrificed and spleen cells collected. Cells were cultured in Roswell Park Memorial Institute (RPMI) medium with phytohemagglutinin-M (PHA-M), sonicated P. multocida (X-73), or RPMI only. The PR birds had significantly decreased numbers of lymphocytes, as well as an overall decrease in total white blood cell counts. Within dietary treatments, vaccinated birds tended to have higher numbers of white blood cells, but (he differences were only significant for the PR groups in Experiment 4. The PR diet suppressed proliferation of splenic cells stimulated with PHA-M, and vaccination significantly increased lymphocyte proliferation in response to sonicated P. multocida, Strain X-73.

Elevated sister chromatid exchange rate in lymphocytes of the European eel (Anguilla anguilla) with cauliflower tumor

Sister chromatid exchange (SCE) frequency and chromosomal aberration were studied in cultured lymphocytes from the European eel (Anguilla anguilla) with cauliflower tumors. SCE was significantly increased in the affected fishes compared with normal controls, whereas chromosomal aberration did not. In comparison to normal cells, the lymphocytes responding to phytohemagglutinin M (PHA-M) in cultures from tumorous eels showed no evidence of cell cycle retardation. A possible mechanism for elevation of SCE and the significance of this finding relation to the etiology were discussed.

Lymphocyte response to plant mitogens

Cell-mediated immune response was measured by the uptake of tritiated thymidine by lymphocytes cultured in serum-free medium in the presence and absence of different doses of phytohemagglutinin-M (PHA). Four groups were studied: (i) healthy women (controls); (ii) pregnant women; (iii) patients with carcinoma of the cervix; (iv) patients with ovarian carcinoma. The cancer patients were studied before and after treatment which consisted of radiotherapy and/or chemotherapy. No significant difference in response was found in the controls in terms of the response to the different doses of PHA or between the controls and pregnant women. When the pregnant women were grouped according to trimester dates, variability in response was observed which was dependent on the dose of PHA used. However, there was a decreased response in the second trimester relative to the first and third trimester at different doses of PHA. The lymphocyte response of the cancer patients was depressed only at 5 μg/ml of PHA. The response before treatment was decreased relative to the response after treatment. After treatment the response of the patients with ovarian carcinoma was similar to the control value.

Lymphocyte response to plant mitogens: I. The distribution of lymphocyte response to various doses of phytohemagglutinin-M in pregnant women and women with carcinoma of the cervix or ovary

Cell-mediated immune response was measured by the uptake of tritiated thymidine by lymphocytes cultured in serum-free medium in the presence and absence of different doses of phytohemagglutinin-M (PHA-M). Four groups were studied: (i) healthy women (controls), (ii) pregnant women, (iii) patients with carcinoma of the cervix, (iv) patients with ovarian carcinoma. The patients were studied before and after treatment which consisted of radiotherapy and/or chemotherapy. It was observed that the log index of lymphocyte response to the different doses of PHA-M could be segregated into log index <1.0, ⩾1.0 <2.0, ⩾ 2.0 < 3.0. In the control group there was a decrease in the frequency of the lymphocyte response in the <1.0 log index range as the concentration of PHA-M was increased. This decrease in frequency in the lower range was accompanied by an increase in frequencies in the higher range. Although there were similarities in the distribution of the lymphocyte response to PHA-M in the pregnant women and women with carcinoma of the cervix and ovary, it was possible to differentiate between the groups on the basis of the lymphocyte response to the different doses of PHA-M.

The Qa-1 antigenic system. Relation of Qa-1 phenotypes to lymphocyte sets, mitogen responses, and immune functions.

The antiserum (B6 X A-Tlab) anti-A (Tlaa) defines several TL antigens expressed exclusively on thymocytes. When reacted with peripheral lymphocytes, the same antiserum defines another antigenic system, provisionally termed Qa-1. The genotypic disparity distinguishing the recipients and donors in this immunization comprises a section of chromosome 17 extending from a crossover point between H-2D and Tla to a presently unmarked point beyond Tla. Therefore although Qa-1 may constitute a single cell surface component, it is equally probable that the Qa-1 system defines two or more cell surface components determined by genes in this region, each of which may be expressed on a different cell set. Cytotoxicity assays indicate that Qa-1 antigen is expressed on Lyt-1 cells and Lyt-123 cells, and may serve to subclassify these two cell sets; it is not known whether Qa-1+ cells may occur within the small Lyt-23 set. There may be also be a cell set with the phenotype Thy-1--:Qa-1+. Another distinctive feature of the Qa-1 system is the characteristic profile of responses to mitogens exhibited by spleen cell populations from which Qa-1+ cells have been eliminated; in conventional assay of [3H]thymidine incorporation the response to lipopolysaccharide was essentially unchanged, the response to phytohemagglutinin M (PHA-M) was virtually abolished, and the response to concanavalin A (Con A) was reduced by 40%. The third distinctive feature of the Qa-1 system is the characteristic profile of changes which elimination of Qa-1+ cells produces in tests of immune function in vitro: (a) proliferation, measured by [3H]thymidine incorporation, in mixed lymphocyte culture (MLC) with major histocompatibility complex (MHC)-incompatible stimulator cells, was not affected. (b) in tests of cell-mediated cytotoxicity (CMC) of MHC-incompatible target cells, neither the generation nor the effector functions of cytotoxic lymphocytes was affected, implying that Lyt-23 prekiller and killer cells are Qa-1--. (c) primary and secondary responses to SRBC were considerably augmented, suggesting that Qa-1+ cells may be responsible for suppression in this test system. (d) accordingly the suppression of the anti-sheep erythrocyte (SRBC) response normally engendered in spleen cells by culture with SRBC was profoundly reduced by elimination of Qa-1+ cells, either before or after culture. (e) the suppression of the anti-SRBC response normally engendered in spleen cells cultured with Con A was reduced by removal of Qa-1+ cells before but not after culture with Con A. Although analysis is as yet far from complete, the Qa-1 system should already be of considerable value because it distinguishes a population of lymphocytes that is not defined by any other antigenic system, according to three criteria: (a) representation of Qa-1 cells among T-cell sets defined by Lyt phenotypes, (b) the profile of responses to mitogens exhibited by lymphocyte populations depleted of Qa-1+ cells, and (c) the profile of immune responses of lymphocyte populations depleted of Qa-1+ cells.