Lymphocyte Blastogenic Response Research Articles

A MODIFIED METHOD OF IMMUNOLOGICAL DIAGNOSIS OF INTOLERANCE TO ANTI-TUBERCULOSIS DRUGS USING ADDITIONAL STIMULATION OF CULTURED LYMPHOCYTES WITH RECOMBINANT INTERLEUKIN-2

This study is aimed to enhance the immune response to anti-TB drugs (anti-TBD) in vitro of circulating blood lymphocytes from patients with adverse reactions. Methods. The immune response of lymphocytes to first-line anti-TBD was studied in 7 patients with hepatotoxic reactions (test group) and 13 patients without reactions (control group). The modified blast transformation test was used in a culture of lymphocytes in vitro; proliferative activity was increased using recombinant IL-2. Results. The addition of IL-2 to control cultures with no anti-TBD had almost no effect on the blastogenic response of lymphocytes while the response to anti-TBD was increased 1,5–2 times. However, the stimulation index never exceeded 2. The addition of IL-2 to lymphocyte cultures of patients with hepatotoxic reactions caused an increase in the content of blasts to 4.5–5%. Different patients showed immune response simultaneously to 1 or more anti-TBD. In 3 cases the lymphocytes reacted to 3 drug combinations: R+Z+E, H+R+E and R+Z+H. Conclusion. The modified blast transformation test is useful to identify the immune response of lymphocytes to anti-TBD in patients with hepatotoxic reactions.

The administration of diets contaminated with low to intermediate doses of deoxynivalenol and supplemented with antioxidants and binding agents slightly affects the growth, antioxidant status, and vaccine response in weanling pigs.

This study aimed to evaluate the impact of grading levels of deoxynivalenol (DON) in the diet of weaned pigs, as well as the effects of a supplementation with antioxidants (AOX), hydrated sodium calcium aluminosilicates (HSCAS), and their combination on the growth, AOX status, and immune and vaccine responses against the porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). At weaning, 336 piglets were allocated to six dietary treatments according to a randomized complete block design. Treatments were as follows: basal diet (CTRL); basal diet containing DON at 1.2 mg/kg (DON1.2); basal diet containing DON at 2.4 mg/kg (DON2.4); DON2.4 diet + a mix of AOX which included vitamins A and E at 20,000 IU and 200 IU/kg feed respectively, selenized yeast at 0.3 mg/kg, and a grape seed extracts at 100 mg/kg feed (DON2.4 + AOX); DON2.4 diet + the mix of AOX and the modified HSCAS mentioned above (DON2.4 + AOX + HSCAS); DON2.4 + AOX + HSCAS. Pigs were vaccinated against PRRSV and PCV2 at 7 d; on 0, 14, and 35 d, growth performance was recorded, and blood samples were collected in order to evaluate the oxidative status, inflammatory blood markers, lymphocyte blastogenic response, and vaccine antibody response. Increasing intake of DON resulted in a quadratic effect at 35 d in the lymphocyte proliferative response to concanavalin A and PCV2 as well as in the anti-PRRSV antibody response, whereas the catalase activity decreased in DON2.4 pigs compared with the CTRL and DON1.2 groups (P ≤ 0.05). Compared with the DON2.4 diet, the AOX supplementation slightly reduced gain to feed ratio (P = 0.026) and increased the ferric reducing ability of plasma as well as α-tocopherol concentration (P < 0.05), whereas the association of AOX + HSCAS increased the anti-PRRSV IgG (P < 0.05). Furthermore, the HSCAS supplement reduced haptoglobin levels in serum at 14 d compared with the DON2.4 group; however, its concentration decreased in all the experimental treatments from 14 to 35 d and particularly in the DON2.4 + AOX pigs, whereas a different trend was evidenced in the DON2.4 + HSCAS group, where over the same period haptoglobin concentration increased (P < 0.05). Overall, our results show that the addition of AOX and HSCAS in the diet may alleviate the negative effects due to DON contamination on the AOX status and immune response of vaccinated weanling pigs.

Correlation of VEGF with immune suppression involving MDSC, malnutrition, and prognosis in patients with gastric and colorectal cancer.

582 Background: Although a causal relationship for inflammation and immunity of cancer is more widely accepted today, the precise cell mechanisms mediating this relationship have not been elucidated. Vascular endothelial growth factor (VEGF), previously known as vascular permeability factor. 45 kDa protein, belongs to a family of platelet-derived growth factors. VEGF, inflammation-related protein, could contribute to the accumulation of immunosuppressive cells (MDSC, Treg, TAM, and Tie-2-expressingmonocytes) in tumor-bearing hosts through direct or indirect mechanisms. Methods: We tested the serum levels of VEGF by ELISA in 106 patients including 43 with gastric and 63 with colorectal cancer, and they were increased in advanced stages of gastric and colorectal cancer. Production of IL-17, pro-inflammatory cytokine, with a stimulation of PHA was measured by ELISA and MDSC (myeloid-derived suppressor cells), one of major immunosuppressing cells, was measured by flow cytometry (CD11b+CD14-CD33+). Neutrophil to lymphocyte ratio (NLR) and C-reactive protein (CRP) were used as inflammatory markers. Production of IL-12 and SI (stimulation index) of blastogenic response of lymphocytes were used as markers of cell-mediated immune response. Results: The concentrations of VEGF were positively correlated with levels of MDSC, production of IL-17, NLR and CRP, and were inversely correlated with IL-12 production, SI and nutritional markers including prealbumin and retinol binding protein. The patients were both divided into two groups with a serum level of VEGF (330 pg/ml) and OS (overall survival) of patients with stages III and IV gastric or colorectal cancer were both significantly worse in patients with high levels of VEGF than in those with low VEGF although the differences were not significant in patients with stagesⅠand II. Conclusions: The results of the present study suggested that VEGF may have an impact on advancement and progression involving inflammation and serve as useful markers of the immune suppression involving MDSC, malnutrition and poor prognosis in patients with gastric and colorectal cancer.

Correlation between in vitro activity of milk leukocytes with milk constituents in high yielding crossbred cows

The relationship between the in vitro activity of milk leukocytes and milk constituents were studied in 48 high yielding clinically healthy crossbred cows. Milk somatic cell counts (SCC) was negatively correlated with milk lactose, total soid and solid not fat (SNF) content. Milk neutrophils were positively correlated with protein and Cu content and negatively correlated with fat content in milk. However milk lymphocytes were positively correlated with milk fat content and negatively correlated with protein content. Milk macrophages were positively correlated with milk SNF and total solid content. Milk lymphocyte blastogenic response (SI) was significantly positively correlated with milk protein, fat percentage and copper content and negatively correlated with milk lactose, milk total solids, SNF and zinc content. PI of milk neutrophils was positively correlated with zinc content and milk fat percentage and negatively correlated with milk total solid and SNF content. PI of milk macrophages was positively correlated with milk fat content. In the present investigation, immuno-potentiative role of various milk constituents such as milk protein, copper and zinc content on in vitro leukocyte activity was established.

Alteration in the in vitro activity of milk leukocytes during different parity in high yielding cross-bred cows

In vitro activity of milk leukocytes (viz. neutrophils, lymphocytes and macrophages) was evaluated in forty-eight (48) clinically healthy high-yielding cross-bred cows of mid-lactation stage (100–200 days of lactation), divided into four groups namely 1st parity (n = 12), 2nd parity (n = 12), 3rd parity (n = 12) and 4th and above parity (n = 12). Milk samples were taken (250 ml/cow) were taken. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were performed microscopically. In vitro phagocytic index (PI) of milk neutrophils and macrophages was evaluated by colorimetric nitro blue tetrazolium reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (tetrazolium) assay after isolation of the milk leukocytes by density gradient centrifugation. Milk SCC differed significantly (p < 0.01) between different parity. Cows of 4 and above parity showed significantly (p < 0.01) higher milk SCC compared to primiparous cows. There was no significant difference in milk DLC during different parities in high-yielding cross-bred cows. There was a significant (p < 0.01) variation in lymphocyte blastogenesis amongst parity. The highest value of lymphocyte blastogenesis was seen at 3rd parity, whereas lowest value was obtained in the cows of both 1st and 4th or above parity. PI of milk neutrophils did not differ significantly between parity. PI of milk macrophages was significantly (p < 0.01) higher in 3rd parity and lower (p < 0.01) in 1st and 4th parities. The study indicated that depressed activity of milk lymphocytes and macropages was lower and SCC was higher in the cows of 4th and above parity indicating more mammary stress and hence susceptible to udder infection and mastitis. Therefore, better care and managemental interventions should be taken around these periods.

Alteration in the activity of blood and milk leukocytes together with the serum enzyme profile during sub-clinical mastitis in cross-bred cows

In vitro activity of blood and milk leukocytes together with serum enzyme profile during sub-clinical mastitis in crossbred cows were evaluated after collection of blood and milk samples from normal (10) and sub-clinical mastitic (10) cows. Blood total leukocyte counts (TLC) and differential leukocyte counts (DLC) were estimated by standard hematological procedure. Milk somatic cell counts (SCC) was performed microscopically. In vitro phagocytic activity of blood and milk neutrophils was performed by colorimetric nitro blue tetrazolium (NBT) assy and mitogenconcanavalin A (con A) induced blood and milk lymphocyte blastogenic response was evaluated by colorimetric MTT (tetrazolium) assay. Serum total protein and alkaline phosphatase (ALP) were measured by the stand biochemical methods. The alanine amino transferase (ALT) and aspertate amino transferase (AST) activities in serum were estimated by commercially available kit. Milk SCC was significantly higher in sub-clinical mastitic cows. Phagocytic index of both blood and milk neutrophils was significantly lower in sub-clinical mastitic cows than normal animals. Con- A induced blood and milk lymphocyte blastogenic response was significantly loweredin sub-clinical mastitic cows than. Serum albumin, globulin ratio decreased significantly during sub-clinical mastitis. Serum AST and ALP level in sub-clinical mastitic cows was significantly higher. The study indicated decreased blood and milk leukocyte activity and higher AST and ALP during the sub-clinical mastitis which could be used as a diagnostic tool for sub-clinical mastitis.

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows has been studied in 10 high-yielding (milk production: 5000 l per lactation) Karan Fries crossbred (Holstein Friesian × Tharparkar) cows. Milk and blood samples were collected from all the experimental animals. Isolation of milk phagocytes (neutrophils and macrophages) and lymphocytes were done by density gradient centrifugation. In vitro phagocytic index of milk neutrophils and macrophages was performed by colorimetric NBT reductive assay. Mitogen-induced milk lymphocyte blastogenic response was estimated by colorimetric MTT (tetrazolium) assay. Total plasma cortisol and prolactin were estimated by enzyme immune assay. Highest value of plasma cortisol and prolactin was observed at calving which decreased significantly (p < 0.01) on 15th day postpartum for both prolactin and cortisol. Immune activity of milk leukocytes was highest on day 0 colostrum and decreased significantly (p < 0.01) on 7th day postpartum. A significant (p < 0.01) rise of plasma prolactin was observed around 135th and 225th days postpartum, whereas a peak level of plasma cortisol was observed at 105th, 180th, and 270th days postpartum. Phagocytic index of milk neutrophils and macrophages remains almost in a steady state during mid-lactation period (between 100 and 200 days postpartum). A decline in increasing trend of milk phagocytic activity was observed during late lactation. Mitogen-induced milk lymphocyte blastogenic response was highest on day 0 colostrum which decreased significantly (p < 0.01) on 15th day postpartum. Con A-induced milk lymphocyte blastogenic response showed an increasing trend from 120th to 210th days postpartum. Upon correlation study, it showed that the plasma cortisol has a negative effect on milk leukocyte activity, while prolactin has a positive effect, though the effect is lactation stage specific.

Seasonal variation in in vitro immune activity of milk leukocytes in elite and non-elite crossbred cows of Indian sub-tropical semi-arid climate

Immunity of mammary gland in terms of in vitro activity of milk leukocytes has been evaluated during hot-humid, summer, and winter season in elite (n = 10) and non-elite (n = 10) crossbred cows. Milk samples were collected from all the cows throughout the year at 15-day interval. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were evaluated microscopically. Milk neutrophils, macrophages, and lymphocytes were isolated and cultured in vitro. In vitro PI of milk neutrophils and macrophages was evaluated by colorimetric NBT (nitro-blue tetrazolium) reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (thiazolyl blue tetrazolium bromide) assay. Milk SCC was found to be significantly (p < 0.01) higher in elite cows compared to non-elite cows irrespective of season. There was significant (p < 0.05) increase in milk SCC during hot-humid season compared to winter season in both the group of the cows. There was no significant difference between group and season in terms of DLC. In vitro phagocytic index of elite cows was significantly (p < 0.01) higher than non-elite cows. The phagocytic index was significantly (p < 0.01) decreased in summer and hot-humid season compared to winter season in both the group of animals. Macrophages isolated from elite cows having significantly (p < 0.01) lower phagocytic index than non-elite cows which significantly (p < 0.01) decreased during summer and hot-humid season compared to winter. In vitro milk lymphocyte proliferative response was significantly (p < 0.01) lower in elite cows. Activity of B-lymphocytes decreased significantly (p < 0.01) during summer and hot-humid season than winter, but activity of T-lymphocytes remains unaltered during different seasons. In conclusion, the mammary immunity in terms of in vitro activity of milk leukocytes is compromised during summer and hot-humid season in elite crossbred cows; therefore, better care and management should be taken in high-yielding cows during summer and hot-humid season to minimize intramammary infections.

Antigen-specific lymphocyte proliferation as a marker of immune response in guinea pigs with sustained Helicobacter pylori infection.

Helicobacter pylori (H. pylori) bacteria are human pathogens causing symptomatic gastritis, peptic ulcer or gastric cancer. Little is known about the kinetics of immune responses in H. pylori infected patients because the initial moment of infection has not been identified. Various animal models are used to investigate the immune processes related to H. pylori infection. In this study we checked whether H. pylori infection in guinea pigs, mimicking natural H. pylori infection in humans, resulted in the development of specific immune responses to H. pylori antigens by measuring the proliferation of lymphocytes localized in mesenteric lymph nodes, spleen and peripheral blood. The maturity of macrophages and cytokines, delivered by monocyte-macrophage lineage or lymphocytes, were considered as mediators, which might influence the lymphocyte blastogenic response. The obtained results showed the activation of T cells localized in mesenteric lymph nodes by H. pylori antigens in H. pylori infected guinea pigs four weeks postinfection. The blastogenic activity of lymphocytes was shaped by their interaction with antigen presenting cells, which were present in the cell cultures during the whole culture period. Moreover, the balance between cytokines derived from adherent leukocytes including interleukin 8--IL-8 as well as interferon gamma--IFN-γ, and transforming growth factor beta--TGF-β delivered by lymphocytes, was probably important for the successful proliferation of lymphocytes. The H. pylori specific lymphocytes were not propagated in peripheral blood and spleen of H. pylori infected animals. The modulation of immunocompetent cells by H. pylori antigens or their different distribution cannot be excluded.

English

The present study was designed to study cytokine profile and lymphocytic proliferation activity in chicken infectious anaemia virus (CIAV) infected chicks at various intervals of clinically susceptible age. Briefly, ninety specific pathogen free (SPF) chicks were divided into five experimental groups (A to E). Groups A to D were inoculated&nbsp;with 0.5 ml of 104.5TCID50&nbsp;of CIAV intramuscularly at 1, 14, 21 and 28-day-old, respectively, while&nbsp;group E served as uninfected control. Comparative cytokine gene expression levels of interleukin (IL)-1&beta;, IL-10, IL-12&beta; and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured at 4 and 7 days post infection (dpi) by qRT-PCR. Also, lymphocytic proliferation activity was measured at 1, 2 and 3 week post infection (wpi) by lymphocyte transformation test (LTT).&nbsp;The down regulatory changes in cytokines and depressed&nbsp;cell mediated immune (CMI) responses were observed in chicks of all infected groups (A to D), which were significantly&nbsp;(p&lt;0.05)&nbsp;low as compared to control group. Higher intensities and more pronounced effects on cytokine downregulation and blastogenic responses of lymphocytes were seen at younger ages. Apoptosis in thymic DNA as detected by nucleosomal fragmentation was highest on 7 and 10 dpi and lowest on 14 dpi, which correlated with down regulated cytokine profile in all infected groups.&nbsp;The development of clinical signs and lesions along with PCR testing confirmed the establishment of CIAV infection in experimental chicks.&nbsp;Increased apoptosis, decreased PCV, depletion of lymphocytes, down regulated cytokine profile and reduced lymphocyte activity explain involvement of thymic and hematopoietic precursor cells leading to CMI suppression caused by CIAV. Altogether, immunopathological changes were more severe in chicks infected at younger ages as compared to later ages of the clinically susceptible period.However, further studies are suggested regarding the impact of viral load on the various cytokine profiles and revealing more about immunopathogenesis of CIAV both in young and adult birds. &nbsp; Key words:&nbsp;Chicken infectious anemia virus, cell mediated immune response, polymerase chain reaction (PCR), qRT-PCR, cytokine, apoptosis.

Immune response of the mammary gland during different stages of lactation cycle in high versus low yielding Karan Fries crossbred cows

To investigate the magnitude of innate immunity of mammary glands during different stages of the lactation cycle, milk samples were collected from both high (n=10) and low (n=10) yielding Karan Fries (KF) crossbred cows during early, mid and late lactation stages. Milk somatic cells released during different stages of the lactation cycle were measured microscopically. In vitro immune activity of each milk leukocytes (viz. neutrophils, lymphocytes and macrophages) were also evaluated after isolating each of the cells from the total milk somatic cell pellet. Relative mRNA expression profiles of TNF-α, IL-8 and TLR-4 by RT polymerase chain reaction were also studied in total milk somatic cells during various stages of the lactation cycle in high yielding cows, whereas low yielding KF cows were taken as control. The results were analyzed and significance was tested by employing two way ANOVA, and the relative expression ratio of the target genes of high yielding cows was tested and analyzed for significance by Relative Expression Software Tool REST version 2009. Irrespective of the stage of lactation, high yielding animals were having higher (P<0.01) somatic cell counts (SCC) compared to low yielders. Milk SCC was found to be significantly higher (P<0.01) during mid-lactation compared to other stages in high yielding cows. In vitro immune response of milk leukocytes were lower in high yielders (P<0.01) compared to low yielders, irrespective of stage of lactation. In vitro immune response of all the milk leukocytes was found to be lower (P<0.01) in high yielding cows compared to low yielding cows. In vitro phagocytic index (PI) of milk neutrophils was higher (P<0.01) during mid lactation periods in both the groups of cows. PI of milk macrophages was found to be higher (P<0.01) during early lactation period in both high and low yielding cows. LPS induced milk lymphocyte blastogenic response was found to be higher (P<0.01) in early lactation period of low yielders. Abundance of IL-8, TNF-α and TLR-4 transcripts were up-regulated (P<0.01) during mid lactation of high yielders as compared to control cows (low yielding cows). Abundance of TNF-α and TLR-4 transcripts were down-regulated (P<0.01) during late lactation in high yielding cows. This study indicated that innate immune responses of milk somatic cells in KF cows are modulated by both lactation stage and milk yield. It was also found that mammary glands of high yielding cows have better magnitude of innate immune response during mid lactation stage compared to early and late lactation in terms of milk SCC and in vitro immune response of isolated milk leukocytes.

Deactivation of T-2 toxin in broiler ducks by biotransformation

SUMMARY Effects of 2 dietary levels (0.6 and 1.0 ppm) of T-2 toxin, and the possible protective capacity of a mycotoxin-inactivating feed additive (Detoxa Plus) were investigated in growing White Pekin ducklings in a 49-d trial comprising 6 treatment groups of 10 ducks/group. The experimental design consisted of 1 negative and 1 positive control and 4 test groups, as follows: group 1, negative control (no T-2 toxin and no feed additive added to the feeds); group 2, positive control (no T-2 toxin added, but the feeds were supplemented with feed additive at 2 kg/t); group 3, feeds were complemented with 0.6 ppm of purified T-2 toxin (no feed additive added); group 4, feeds were complemented with 0.6 ppm of T-2 toxin and 2 kg/t of the feed additive was provided; group 5, feeds were complemented with 1.0 ppm of T-2 toxin (no feed additive added); group 6, feeds were complemented with 1.0 ppm of T-2 toxin and 2 kg/t of the feed additive was provided. From wk 4 until the end of the trial, the daily BW gain of group 3 ducks was inferior to that of control ducks, and the differences in means were statistically significant (P ≤ 0.05) at wk 4 and 7. The final BW of this group was also significantly lower than that of the control (P ≤ 0.001). The BW gain of ducks in group 5 was also depressed by the toxin treatment. The adverse effect of 0.6 ppm of T-2 toxin was fully counteracted by the feed additive in terms of daily BW gain, cumulative daily BW gain, and BW at exsanguination. The T-2 toxin at both treatment levels depressed the blastogenic response of lymphocytes to nonspecific mitogens (concanavalin A and phytohaemagglutinin), which was counteracted by the feed additive at the lower dietary concentration of T-2 toxin. No such effect was observed with 1.0 ppm of T-2 toxin. Metabolic blood parameters and hematological data showed no consistent treatment effects. We conclude that this feed additive is able to counteract the adverse effects of T-2 toxin at dietary concentrations that might be encountered under field conditions.

Lymphocyte Blastogenic Responses to Food Antigens in Cats Showing Clinical Symptoms of Food Hypersensitivity

Three cats were diagnosed as having food hypersensitivity by food elimination and oral food provocation tests. Twelve allergenic food ingredients were identified by oral food provocation test in the 3 cats. Of the 12 food ingredients, 9 offending food antigens were shown to be positive in a lymphocyte stimulation test; however, none of them were positive in antigen-specific IgE testing, and only four food antigens were positive in intradermal testing. The stimulation indices in the lymphocyte stimulation tests for the 9 food ingredients were found to be decreased after the cats were fed elimination diets. The present study demonstrates that the lymphocyte stimulation test reflects an immunologic reaction involved in food hypersensitivity and can help identify allergenic food ingredients in feline food hypersensitivity.

Effects of yeast cell walls on performance and immune responses of cyclosporine A-treated, immunosuppressed broiler chickens

The present study was conducted to investigate the effects of dietary yeast (Saccharomyces cerevisiae) cell walls (YCW) from the yeast extract industry on performance and immune function of cyclosporine A (CSA)-treated, immunosuppressed broiler chickens. A total of 240 day-old male broilers were allocated randomly into four treatments: (1) non-challenged control; (2) non-challenged control+0·3 % YCW; (3) CSA-challenged group; (4) CSA-challenged+0·3 % YCW. On days 1-4 and 22-25 of age, broilers were subcutaneously injected with CSA or sterile saline. The results showed that supplementation of YCW significantly improved daily weight gain (DWG) during the starter (days 1-21, P < 0·01), finisher (days 22-42, P < 0·01) and overall (days 1-42, P < 0·05) periods compared with the control birds, but had no effect on feed conversion ratio (FCR, P>0·05). Compared with the CSA-treated birds, YCW alleviated the decrease of DWG (P < 0·01) and increase of FCR (P < 0·05) caused by CSA challenge at different periods and cumulatively. On days 21 and 42, YCW mitigated the CSA-induced decrease of peripheral blood lymphocyte blastogenic response (P < 0·01). In addition, YCW improved the relative weights of the bursa of Fabricius (P < 0·01) and thymus (P < 0·01) and up-regulated the splenic expression of pro-inflammatory cytokines IL-1β (P < 0·01) and IL-6 (P < 0·01) on day 42 compared with the CSA-treated birds. These results indicate that YCW supplementation has beneficial effects in attenuating the immunosuppressive effects of CSA challenge, therefore improving growth performance of broiler chickens.

The effect of fermented wheat germ extract on production parameters and immune status of growing pigs

Model experiment was conducted with growing pigs fed diets containing zero or two levels (1 or 2 g/kg) of fermented wheat germ extract (FWGE). In the first part of the experiment (19 days between 49 and 68 days of age) weight gain and feed conversion were tested. The 2 nd part of the experiment (21 days between 68 and 89 days of age) served to measure the effect of FWGE on some compartments of the immune system. In the first part of the experiment supplementation of the starters’ diet with FWGE yielded 7.8 and 14.2% additional daily weight gain in the 1 st and 2 nd experimental groups, respectively (P<0.05). Humoral immune response to purified horse globulin (PHG) antigen was unaffected by the treatment. The blastogenic response of lymphocytes to non specific mitogens (PHA, Con-A, PWM), phagocytic activity and phagocytic index as well as the intradermal PHA test was consistently and significantly enhanced by the treatments.

Immunomodulatory effect of glucan on specific and nonspecific immunity after vaccination in puppies

The objective of the study was to determine the immunostimulatory effect of β-(1,3/1,6)-D-glucan in puppies. The effect exerted on the efficacy of vaccination, especially against canine parvovirus and rabies infection, was studied. The application of vaccine and glucan leads to significant increases in the nonspecific immunological parameters (phagocytic ability of leukocytes, blastogenic response of lymphocytes, metabolic and chemotactic activity of polymorphonuclear cells). The level of antibodies against canine parvovirus (Ab CPV) and rabies infection reached the most statistically significant values on the 28th day after the application of vaccine and a syrup containing β-(1,3/1,6)-D-glucan (Group GV) as compared to the control group (Group V, puppies receiving only vaccine). Dogs without glucan supplementation did not produce such significant levels of antibodies. We can conclude that glucan has relevant immunostimulatory effects in dogs with altered immunity. The glucan product tested in this study (PleraSAN V, PLEURAN, Bratislava, Slovakia) could be used in the small animal clinical practice.

Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment.

A longitudinal investigation has been conducted into the cell-mediated immune responses of onchocerciasis patients after a single-dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection-free control individuals (P less than 0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus-derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin-O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL-1 beta, IL-2 and TNF-alpha in response to OvAg, but higher amounts of IL-4 and interferon-gamma (IFN-gamma) in response to OvAg than control individuals. After ivermectin treatment, the OvAg-induced production of IL-1 beta and TNF-alpha increased significantly 1 and 14 months after treatment. The PHA-induced production of IL-2 and IL-4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg-specific secretion of IL-2, IL-4 and IFN-gamma did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte-subsets in the peripheral blood of untreated patients revealed a relative and absolute (P less than 0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P less than 0.01); likewise there was an absolute increase in T-helper-inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/CD8+ cell ratio (P = 0.001). The expression of the low-affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCR gamma delta + cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.

Lymphocyte blastogenic response to ovalbumin in a model for canine allergy

Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation ( P < 0.05) and borderline significant ( P = 0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture ( P = 0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.

Primate model for onchocerciasis research.

A major impediment to progress in research in onchocerciasis has been the lack of a suitable animal model. However, chimpanzees can be reliably infected by injection of living third-stage larvae of Onchocerca volvulus, and develop a pattern of infection that closely resembles that seen in humans. This includes the formation of nodules by adult worms, the subcutaneous distribution of microfilariae and the apparent lack of development of resistance to infection after repeated larval challenge. Ocular lesions resembling those in humans have not been observed in animals studied so far, but this may reflect in part the low intensity of infection and the limited time of observation. We infected 18 chimpanzees by subcutaneous injection of 250 third-stage larvae of O. volvulus. Six received ivermectin on day 1, another six received ivermectin on day 28 after infection, and six received no drug. Four control animals received no infective larvae and no drug. During the first year after infection, all three infected groups, but not control animals, have shown a clear increase in their lymphocyte blastogenic response to crude O. volvulus antigen in vitro. Antibody responses have also been increasing with time. Further assessment of the cellular and antibody responses in the infected chimpanzees is under way, as is evaluation of the effects of ivermectin on the course of infection.

In vitro and in vivo immunomodulating and immunorestorative effects of Astragalus membranaceus

Astragalus membranaceus is a common traditional Chinese medicinal plant widely used as a tonic to enhance the body's natural defense mechanisms. In this study, bioactive fractions were isolated from the roots of Astragalus membranaceus. One of these fractions, designated as AI, was found to be the most potent with respect to its mitogenicity on murine splenocytes. Effects of AI on both specific and nonspecific immunity in mouse models were examined. Results showed that AI could exhibit mitogenic and co-mitogenic activities on mouse splenocytes, both in vitro and in vivo. Experiments in human cell culture demonstrated that AI was also active on human lymphocytes. It was found that AI was mitogenic to T cell depleted population but virtually inactive on B cell depleted population. Intraperitoneal injection of AI into mice markedly augmented the antibody response to sheep red blood cells. Besides, both the influx of macrophages into the peritoneal cavity and the phagocytic activity of macrophages were found to be enhanced by AI in vivo. On the other hand, AI could significantly increase the interleukin-2 receptor expression on mouse splenocytes in vitro. In terms of immunorestorative activity, it was found that AI could restore the lymphocyte blastogenic response of the older mice to values that are normally found in the younger mice. Moreover, administration of AI in vivo could partially restore the depressed immune functions in tumour-bearing mice and cyclophosphamide-treated mice. Collectively, the results clearly showed that AI could exhibit immunomodulating and immunorestorative effects, both in vitro and in vivo.