Lymphocyte Blastogenic Response Research Articles

Immune response and growth of stressed weanling pigs fed diets supplemented with organic or inorganic forms of chromium.

A 2 x 4 factorial arrangement of treatments was used in a randomized complete block designed study to determine the effects of chromium level and source on growth and immune response of stressed and non-stressed 3-wk-old crossbred weanling pigs (BW was 6.35 kg). Factors included 1) immune stress or control and 2) no supplemental Cr or .2 ppm of supplemental Cr from either CrCl3, Cr-picolinate, or Cr-nicotinic acid complex. The basal diet was a corn-soybean meal-whey diet containing 1.2% lysine. Escherichia coli lipopolysaccharide (LPS) was the stress-inducing agent and was injected on d 7, 10, and 13 of the experiment. Immune challenge with LPS resulted in reduced gain (P < .05) and feed intake (P < .10). Supplementation with Cr was not effective in alleviating the depression in growth due to LPS. However, supplementation of control pigs with Cr tended to improve (P < .10) gain and feed intake. In vitro cellular immune response as measured by a lymphocyte blastogenesis assay was increased (P < .10) in pigs fed supplemental Cr from CrCl3, or Cr-picolinate. Antibody response to sheep red blood cells tended to be increased (P < .10) in pigs supplemented with Cr-nicotinic acid, but antibody response to ovalbumin was decreased (P < .05) in pigs supplemented with organic forms of Cr. At the end of the study, effects of Cr supplementation on lymphocyte proliferative response were investigated before and after ACTH administration. Injections of ACTH resulted in increased (P < .001) serum cortisol levels and increased lymphocyte proliferation. Supplementation of Cr did not affect lymphocyte blastogenic response before or after ACTH injection (P > .10). These data suggest that Cr supplementation was not beneficial during immune stress in pigs.

Primary and Secondary Toxoplasma gondii Infection in Normal and Feline Immunodeficiency Virus-Infected Cats

Normal and asymptomatic feline immunodeficiency virus (FIV)-infected adult cats were inoculated orally with Toxoplasma gondii tissue cysts to assess differences in clinical disease, T. gondii serologic test results, hematologic results, and oocyst shedding. There was no difference between FIV-naive and FIV-infected cats in terms of clinical illness and duration of oocyst shedding following primary exposure. Both groups of cats developed significant decreases in neutrophil counts following primary inoculation with T. gondii; FIV-infected cats that were neutropenic prior to inoculation with T. gondii developed the most profound decreases in neutrophil numbers. Both FIV-naive and FIV-infected cats became lymphopenic during acute T. gondii infection; however, only FIV-naive cats developed lymphocytosis in the recovery stage. FIV-infected cats had lower total CD4+ and higher total CD8+ T-lymphocyte counts than FIV-naive cats prior to inoculation with T. gondii, but changes in these lymphocyte subsets were similar between groups of cats during the first several weeks after inoculation. Toxoplasma gondii infection had neither an ameliorating nor enhancing effect on T-lymphocyte subset abnormalities in FIV-infected cats during acute or chronic infection. Both groups of cats developed comparable levels of T. gondii-specific IgM and IgG antibodies and T. gondii antigen-specific lymphocyte blastogenic responses following primary inoculation. Both groups of cats were fed T. gondii tissue cysts 66 wk following primary exposure and both groups were solidly immune as evidenced by a lack of oocyst shedding and only minor changes in IgM but not IgG antibodies.

Blastogenic response of lymphocytes from foals infected with Rhodococcus equi.

The blastogenic response of lymphocytes from 16 newborn foals naturally infected with Rhodococcus equi was investigated, in order to evaluate the relationship between R. equi infection and depressed host response. Naturally infected foals showed evidence of R. equi infection at 5-6 weeks of age, as determined by clinical, haematological, bacteriological and serological methods. The blastogenic response of lymphocytes against phytohaemagglutinin was significantly depressed (stimulation index < 1.80; P < 0.01, P < 0.05) in R. equi-infected foals at 5-6 weeks of age compared with those of control foals. Serum IgG concentration decreased rapidly after foals reached 1 week of age, and minimum levels of IgG were observed at 5-7 weeks of age in R. equi-infected foals. This study suggests that the onset of R. equi infection may be associated with the depressed immune function of naturally infected foals during the first 5-6 weeks after birth.

Colorimetric Assay of Equine Peripheral Lymphocyte Blastogenesis Using MTT.

A rapid colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was applied to evaluate equine lymphocyte blastogenic response to mitogens. The optimum concentrations of concanavalin A (Con A), phytohemagglutinin-P (PHA) and pokeweed mitogen (PWM) were 5 μg/ml, 2.5 μg/ml, and a 1:80 dilution, respectively, when lymphocytes were cultured at a concentration of 1 × 106 cells/ml for 5 days in a 96-well microplate. Significant correlations between the results obtained by the MTT assay and the 3H-thymidine incorporation assay were observed. The correlation coefficients were 0.727 (n=68), 0.692 (n=57), and 0.681 (n=44), when lymphocytes were stimulated with Con A, PHA, and PWM, respectively. The MTT assay may be simple and reliable for studying equine cellular immune functions and a suitable alternative to conventional assay systems using radioactive materials.

Route of BCG administration in possums affects protection against bovine tuberculosis

The Australian brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis in New Zealand. Control of bovine tuberculosis in farmed animals requires measures to reduce the transmission of M. bovis from wildlife. Possums were vaccinated with BCG intranasally by aerosol spray, orally or subcutaneously to compare the efficacy of these three routes on protection against challenge with virulent M. bovis. Possums vaccinated with BCG by the intranasal or subcutaneous routes had a marked reduction in severity of disease compared to possums which had been unvaccinated or orally vaccinated. The severity of the disease was assessed by changes in body weight and pathology. BCG vaccination by all three routes resulted in reduced dissemination of M. bovis to the spleen and liver following challenge. Intranasal and oral BCG vaccination induced lower mean peripheral blood lymphocyte blastogenic responses to bovine PPD than subcutaneous vaccination, indicating that these responses did not correlate well with protection from the disease. Given a suitable delivery system, aerosol vaccination of possums, used in conjunction with other control measures, may be a suitable method of reducing the spread of M. bovis from wildlife to domestic animals.

Membrane-bound/soluble IL-2 receptor (IL-2R) and levels of IL-1 alpha, IL-2, and IL-6 in the serum and in the PBMC culture supernatants from 17 patients with hematological malignancies.

The present study investigated the peripheral blood mononuclear cells (PBMC) blastic responses to PHA, PHA plus recombinant IL-2 (rIL-2) and rIL-2 alone; the expression of membrane-bound IL-2R on PHA-stimulated PBMC; and the levels of IL-1 alpha, IL-2, IL-6, and sIL-2R in serum and in culture supernatants from PHA-stimulated PBMC in 17 patients with with non-Hodgkin's lymphoma (NHL), 4 with Hodgkin's lymphoma (HL), 5 with Hairy cell leukemia, 1 with chronic myelogenous leukemia, and 1 with chronic lymphocytic leukemia. The patients with HL and NHL with active disease (AD) were separated from those in clinical remission. The patients with AD were studied at diagnosis (obviously before therapy) and the patients in clinical remission were out of therapy since at least 6 mo. The lymphocyte blastogenic response to PHA was significantly lower in patients with HL and NHL with AD than in the control group. The response to rIL-2 alone was in the same range in the control group and in HL and NHL AD patients. By adding rIL-2 to PHA there was an increase of the blastogenic response of the same patients. The percentage of CD25 expressed on PHA-stimulated lymphocytes from patients with HL and NHL AD and from normal subjects is in the same range. Serum levels of IL-2, IL-6, and sIL-2R were significantly higher in HL and NHL AD patients than in controls as well as in all other hematological malignancies. Supernatants derived from PHA-stimulated PBMC were assessed for the presence of cytokines and sIL-2R by ELISA. The levels of IL-2, IL-6, and sIL-2R were significantly lower in HL and NHL AD patients than in controls as well as in all other hematological malignancies.

Successful Immunization Against Acanthamoeba Keratitis in a Pig Model

The feasibility of inducing protective immunity to Acanthamoeba keratitis was tested in a pig model. Experiments were designed to determine if ocular infection with Acanthamoeba trophozoites would elicit protection against reinfection. Additional experiments examined whether injection of parasite antigens either intramuscularly, subconjunctivally, or by both routes would induce immunity. Therefore, four groups of animals were examined: (a) pigs that had resolved a primary corneal infection with Acanthamoeba; (b) pigs immunized intramuscularly; (c) pigs immunized subconjunctivally; and (d) pigs immunized intramuscularly and subconjunctivally. Animals were subsequently challenged with parasite-laden soft contact lenses and observed clinically for the appearance of Acanthamoeba keratitis. Acanthamoeba-specific serum antibody titers and blastogenic responses of peripheral blood lymphocytes were determined weekly. The results indicated that intramuscular injection of Acanthamoeba antigens failed to protect against ocular infection even though hosts developed high titers of IgG antibodies and displayed lymphocyte blastogenic responses to parasite antigens. Ocular infection alone failed to stimulate immunity in any of the animals. By contrast, 50% of the hosts immunized subconjunctivally were protected against corneal disease, and 100% of the animals immunized by a combination of intramuscular and subconjunctival administration of parasite antigens were completely protected against two separate ocular challenges with infectious parasites. Protection did not correlate with either IgG antibody titers or blastogenic potentials of peripheral blood lymphocytes. Interestingly, ocular infection alone failed to stimulate immunity to subsequent ocular challenge with infectious parasites. Thus, administration of parasite antigen via the subconjunctival route can protect against Acanthamoeba keratitis.

Mitogenic Responses of the Head-Associated Lymphoid Tissues of the Chicken

A blastogenesis microassay employing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was adapted to measure blastogenic responses of lymphocytes from the chicken's head-associated lymphoid tissues (i.e., the harderian gland and conjunctiva-associated lymphoid tissue) to T- and B-cell mitogens. Lymphocytes isolated from peripheral blood, spleen, and the harderian gland had highly significant (P < 0.01) responses to T- and B-cell mitogens compared with control lymphocytes cultured without mitogens. Cultured lymphocytes obtained from the harderian gland had highly significant mitogenic responses to the T-cell mitogen concanavalin A (25 to 100 micrograms/ml) and to the B-cell mitogen Salmonella typhimurium lipopolysaccharide (1.25 to 5.0 micrograms/ml) compared with the control lymphocytes. Mitogenic responses of cultured lymphocytes obtained from the conjunctiva-associated lymphoid tissue could not be measured within the given parameters of the blastogenesis microassay. This was primarily due to the low yield of lymphocytes, which proved to be a limiting factor. The ability of the MTT blastogenesis microassay to detect blastogenic responses of the harderian gland to mitogens may be indicative of its usefulness for measuring cell-mediated immunity responses to other antigens.

Effectiveness of BCG vaccination in protecting possums against bovine tuberculosis

Three groups of eight possums were vaccinated with BCG Pasteur by the subcutaneous, intratracheal or intragastric routes, with a fourth group serving as unvaccinated controls. Forty-two days after the start of vaccination, five possums from each group were challenged intratracheally with virulent Mycobacterium bovis. The vaccination by the subcutaneous or intratracheal routes resulted in a marked reduction in the severity of disease compared with the unvaccinated animals or the animals vaccinated intragastrically. The severity of the disease was assessed by changes in bodyweight, pathological changes in the lungs and bronchial nodes and the number of acid-fast bacilli in the lesions. Before the challenge, lymphocyte blastogenic responses to bovine ppd were observed in the eight animals vaccinated subcutaneously and in two of the animals vaccinated intratracheally.

LACK OF CORRELATION BETWEEN DEFECTIVE CELL-MEDIATED-IMMUNITY AND LEVELS OF SECRETED OR CIRCULATING CYTOKINES IN A STUDY OF 90 CANCER-PATIENTS

In this study we examined the levels of IL-1 alpha, IL-1 beta, IL-2, IL-6, TNF-alpha and sIL-2R in the sera and culture supernatants of PHA-stimulated lymphocytes from a series of 90 cancer patients. The expression of the IL-2R p55 chain (alpha subunit) on PHA-stimulated lymphocytes was also evaluated together with the blastogenic response of peripheral blood mononuclear cells (PBMC) to PHA, PHA plus rIL-2 and rIL-2 alone. Ninety cancer patients (70 men and 20 women; mean age 57.8 years, range 27-80) with advanced solid malignancies at different sites were studied. The lymphocyte blastogenic response to PHA was significantly lower in cancer patients than in normal individuals. The proliferative response to rIL-2 alone was also significantly depressed in cancer patients. The frequency of CD25(+) PHA-stimulated lymphocytes from cancer patients was not significantly different from that of the control group. The serum values for IL-1 alpha, IL-1 beta, IL-6 and sIL-2R were significantly higher in cancer patients than in controls, while the serum level of IL-2 was within the normal range. The levels of sIL-2R released in the supernatant of PHA-stimulated PBMC of cancer patients were significantly lower than those of the control group. However, the levels of IL-1 alpha, IL-1 beta, IL-2, IL-6 and TNF-alpha, in the supernatants of PHA-stimulated PBMC of cancer patients were in the same range as those of the control group. These results suggest that the observed immune-deficiency in cancer patients cannot be explained on the basis of a defective production of key immunoregulatory cytokines since the lymphocytes from cancer patients produced physiological amounts of cytokines. We suggest that the observed defective cell-mediated immunity may be due to a defect in transmembrane signalling by the cytokines.

Effect of climatic stress on the immunological reactivity of weaned pigs

Weaned pigs exposed daily to either unpredictable draught (experiment 1) or intermittent unpredictable draught (experiment 2) showed different lymphocyte blastogenic responses after mitogenic stimulation with phytohaemagglutinin (PHA) and concanavalin A (ConA). In both experiments PHA skin test responses were lower for draught exposed pigs than for control pigs and leucocyte numbers or profiles were altered compared to those of control pigs. Superoxide production and chemiluminescence of porcine granulocytes were similar for draught exposed and control animals. Furthermore, serum globulin content did not differ significantly between pigs in the experimental and control room. The strong increase in serum gamma-globulin after the Aujeszky Disease Virus (ADV)-challenge was the same for draught exposed and control pigs. The same held for the lymphocyte blastogenic response with ADV protein as antigenic stimulus. The present study shows the effects of climatic stress on immunological reactivity, which may reflect a homeostatic disturbance of the pig's immune system elicited by exposure to unpredictable draught.

Immunological Parameters of Periparturient Holstein Cattle: Genetic Variation1,2

The genetic variability of blood neutrophil functions, lymphocyte blastogenic responses to mitogens, serum Ig concentrations, and serum complement and conglutinin activities was investigated from 35 d prepartum to 35 d postpartum for 137 Holstein cows. Periparturient cows experience an immunosuppression of various immuno-logic parameters at calving. Heritability estimates were obtained before, during, and after the episodes of immunosuppression. Significant genetic variability occurred in the periparturient changes for total number of neutrophils, neutrophil chemokinesis, assays of the neutrophil respiratory burst associated with phagocytosis (cytochrome c reduction, chemiluminescence, and iodination), serum concentrations of IgG1, IgG2, and IgM, and serum hemolytic complement activity. This variability implies that immune profiles could be used for the selection of cattle with improved innate immune response without adverse effects on milk productivity. These results should be considered tentative, however, because the number of observations included in the data were limited.

The effect of immunization of pigs with Ascaris suum cuticle components on the development of resistance to parenteral migration during a challenge infection

The development of immunity to Ascaris suum was studied in pigs immunized with isolated cuticle fragments from A. suum second and third stage larvae (L2/L3) and adult worms, and compared with other methods that stimulate a strong protective response in pigs. A significant protective response was seen in animals immunized with isolated cuticle fragments from A. suum L2/L3 and adults, but it was less than that seen in animals inoculated with UV-irradiated eggs or naturally exposed to eggs on a dirt lot. Significant IgG responses to 2-mercaptoethanol (2ME)-soluble cuticle components were seen in all groups, but the level of the antibody response did not relate to protection. Group differences in antibody and lymphocyte blastogenic responses to cuticle proteins indicated quantitative and qualitative stage specific differences in 2ME-soluble and insoluble cuticular proteins. Intestinal immunity was notably absent from cuticle immunized pigs because a marked liver white spot response was observed following the challenge inoculation. Thus, cuticle fragments from larval and adult A. suum are capable of inducing a protective response to larval migration; however, the development of intestinal immunity is not a direct function of exposure to these antigens.

Impaired cell-mediated immunity to cytomegalovirus (CMV) in leukemic children with prolonged CMV viruria

To determine the role of cell-mediated immunity (CMI) to cytomegalovirus (CMV) in leukemic children after CMV infection, CMI to CMV antigen was studied using CMV-specific lymphocyte blastogenic responses (LBR) and interferon (IFN) production. Four children, who continuously secreted CMV in urine more than 2 years after symptomatic CMV infection (CMV disease) (group 1), showed impaired LBR to CMV antigen, though they had normal LBR to phytohemagglutinin (PHA) and concanavalin A (Con A). Impairment of LBR either to AD-169 strains or autologous and heterologus wild strains was observed. IFN production was not detected in three of four children. Six leukemic children, who had no viruria after cessation of CMV disease (group 2), showed good responses to CMV antigens. IFN was detected in all six children in group 2. Eight leukemic children, who were seropositive to CMV and the onset of leukemia (group 3), showed good responses to CMV antigens and IFN production. These results suggest that impaired cell-mediated immunity to CMV antigen might contribute to prolonged excretion of CMV in urine in leukemic children.

Stimulation of macrophage oxygen free radical production and lymphocyte blastogenic response by immunization with porins.

Porins were prepared from smooth strain of Salmonella typhi 0-901 and chemotype rough mutant of S. typhimurium Ra-30. Porins could significantly stimulate the immune systems of mice. Immunization of mice with the porins provoked synthesis of anti-porin antibodies. Macrophages from the immunized mice showed increased capacity to generate oxygen free radicals, and lymphocytes from these mice showed proliferative response to the porins. Thus porins may play a role in providing protection from salmonellosis by stimulating the antibody production and increasing the capacity of macrophages to generate oxygen free radicals along with stimulation of lymphocytes.

The behavioural, endocrine and immune responses of sheep to isolation

AbstractTwo groups of five sheep (7 months of age) were moved and isolated in pens which did not allow visual and tactile contact with other sheep for 24 h. They were then moved back to their original pens where visual and tactile contact was possible. After 24 h the procedure was repeated seven times for one group (group 8-1) and thirteen times for the other group (group 14-1). One group (control) of five sheep remained in pens where visual and tactile contact was possible. When isolated the lambs spent more time standing still in an alert posture, less time eating and resting, and vocalized more than control lambs. The heart rate of the lambs increased when they were moved between pens and during isolation. The plasma concentration of cortisol was significantly increased after 1·5 h and 3 h of isolation. The plasma concentration of prolactin was increased after 1·5 h of isolation. After 3 h of isolation the number of neutrophils in the blood was increased and the numbers of T-lymphocytes (CD2), T-helper-lymphocytes (CD4) and yd-lymphocytes (T19) were decreased. After 24 h of isolation the lymphocyte blastogenic response to Con A was lower and the numbers of T-lymphocytes and T-helper-lymphocytes were still less than those in control lambs. Although there were still behavioural changes when the lambs were isolated for the seventh time, no cortisol, prolactin and leucocyte changes were found. These results suggest that stressors similar to isolation, which can occur during normal management practice, may elicit short-term leucocyte changes in lambs.

Immunomodulative effects of Chinese herbs in mice treated with anti-tumor agent cyclophosphamide

Extracts of Chinese herbs were administered with antitumor agent, cyclophosphamide (CY), and their effects on macrophages and lymphocytes were studied. Number of peritoneal macrophages significantly decreased and their chemotactic activity was suppressed by treatment with CY. Blastogenic responsiveness to Concanavalin A and NK cell activity of spleen lymphocytes were suppressed significantly in CY-treated mice. Extracts of Lithospermi radix, Astragali radix and Glycyrrhizae radix showed protective effects on immunosuppressive mice. The number of macrophages, chemotactic activity of macrophages and blastogenic response of lymphocytes were recovered to the same or more than that of normal levels. An extract of Ginseng radix showed protective effects on the number and functions of macrophages by treatment with CY but did not show any effects on the lymphocytic blastogenesis. On the contrary it showed a strong inhibitory effect on the NK cell activity. These results suggest that Chinese herbs could modulate cellular immune response, especially in the activation of macrophages and splenic lymphocytes.

The role of cell-mediated immunity in chickens inoculated with the cell-associated vaccine of attenuated infectious laryngotracheitis virus.

The cell-mediated immune responses of the chickens inoculated with the cell-associated (CA) ILT vaccine were studied. Lymphocyte blastogenic response was tested by MTT assay with spleen, thymus, bursa of Fabricius and peripheral blood lymphocytes of the vaccinated chickens. The blastogenic response of peripheral blood and spleen lymphocytes was enhanced by a T-cell mitogen. The CA vaccine induced an immunity satisfactorily in bursectomized chickens. To study the role of immune cells in the vaccinated chickens, the bursectomized chickens were inoculated intravenously with the splenic, thymic, bursal and peripheral blood lymphocytes of the vaccinated chickens. Protective effects were shown in the chickens received the splenic cells and peripheral blood lymphocytes, indicating that these play an important role in eliciting the protective effects of the CA vaccine. From these results, it was found that the immune mechanism with CA vaccine against ILT involves mainly cell-mediated immunity.

Involvement of Nitric Oxide in the Blastogenic Response Deficiency in Splenocytes From Spontaneously Hypertensive Rats

It has been shown that spontaneously hypertensive rats (SHR) exhibit some abnormalities in their immune system. These include a reduced delayed hypersensitivity response, a reduction in the number of rosette-forming cells and a decreased lymphocyte blastogenic response. In this study, we further investigated the blastogenic responses of splenocytes, thymocytes, and T-enriched lymphocytes from SHR. In SHR splenocytes, the blastogenic responses to concanavalin A (Con A), phytohemagglutinin (PHA), interleukin-2 (IL-2), and phorbol 12,13-dibutyrate (PDB) plus ionomycin were significantly reduced compared with those from Wistar-Kyoto rats (WKY). In SHR thymocytes and T-enriched lymphocytes, the blastogenic responses to these activators were the same as in WKY rats. The IL-2 production by SHR splenocytes was similar to that of WKY. To elucidate the possible mechanism responsible for the blastogenic defects in SHR splenocytes, the involvement of the nitric oxide (NO) synthetic pathway was studied. The inhibition of NO synthesis by NG-monomethyl-L-arginine (L-NMMA) corrected the defect in SHR splenocytes. L-NMMA had no effect on the splenocytes, thymocytes, or macrophage-depleted splenocytes from WKY or on thymocytes or macrophage-depleted splenocytes from SHR. The removal of macrophages from SHR splenocytes also corrected the blastogenic defect in these cells. Furthermore, the NO synthesis in Con A stimulated SHR splenocyte culture medium was statistically significantly higher than that in WKY. These results suggested that overproduction of nitric oxide by SHR macrophages may be responsible for the SHR splenocyte blastogenic defect.

Effect of bovine immunodeficiency-like virus infection on immune function in experimentally infected cattle

Bovine immunodeficiency-like virus (BIV) is a bovine lentivirus that has antigenic and genetic homology with the human immunodeficiency virus. Little work has been reported on the effect of BIV infection on bovine immune function. This study was designed to evaluate lymphocyte blastogenesis, mononuclear cell subset numbers, neutrophil function, hematology, and clinical signs in three groups of cattle. These groups were evaluated at 0–2 months post inoculation (PI, Group 1), 4–5 months PI (Group 2), or 19–27 months PI (Group 3). BIV infected animals were inoculated with the R-29 isolate of BIV in tissue culture cells, peripheral blood mononuclear cells from a R-29 infected calf, or a molecular clone of the R-29 isolate. Most inoculated animals seroconverted to BIV by Western immunoblot. BIV was reisolated from most of the animals inoculated. BIV infection was associated with an increase in the lymphocyte blastogenic response to the mitogen phytohemagglutinin in Groups 2 and 3. Neutrophil antibody dependent cell mediated cytotoxicity and neutrophil iodination were decreased ( P<0.05) in BIV infected cattle (Groups 2 and 3 and Group 3, respectively). All animals were clinically normal during the evaluation periods. Notable differences were not observed in the other assessments performed. Work with additional BIV isolates and over longer time frames is warranted.