The influence of protein supplementation on the immune response to Haemonchus contortus

A major protective mechanism in lambs against the abomasal parasite Ostertagia (Teladorsagia) circumcincta appears to be the immunoglubulin (Ig)A-mediated suppression of worm growth and fecundity. The present study indicates that IgA may play a similar role in the control of another abomasal parasite Haemonchus contortus. Hampshire Down lambs were offered one of two diets: (i) a basal diet and (ii) a diet supplemented with additional protein. Lambs were then 'trickle' infected with H. contortus and killed 10 weeks after the start of infection. Those lambs on the supplemented diet had shorter adult worms and produced significantly more antiparasite IgA. There was a significant association between reduced female adult worm length and increased IgA against third-stage larvae. Most of the difference between the two groups in worm length could be accounted for by differences in IgA responses. Therefore, IgA may be the major mechanism controlling fecundity of H. contortus and the magnitude of the IgA response is influenced by the quality of the diet.     

Isotype-specific antibody responses to Haemonchus contortus in genetically resistant sheep

The kinetics of anti-Haemonchus antibody responses in serum and faecal extracts of pasture-reared, genetically resistant and random-bred sheep infected with Haemonchus contortus were examined using an isotype-specific ELISA. Anti-Haemonchus antibodies of IgA, IgG1, IgG2 and IgM isotypes were detected in serum and faecal extracts of both resistant and random-bred sheep after challenge infection. Serum IgG1 and IgA levels in resistant sheep were significantly higher than in random-bred sheep between 10 and 31 days after infection. However, there were no differences in IgG2 and IgM antibody responses between the two genotypes. Faecal antibody responses to H. contortus showed a clear genetic effect with resistant sheep exhibiting higher IgA levels throughout infection and higher IgG1 levels between 24 and 31 days after infection. Furthermore, serum IgG1 and IgA, and faecal IgA responses were negatively correlated with faecal egg counts in both genotypes on 17, 24 and 31 days after infection. Together, these results are taken to indicate that anti-parasite IgA and IgG1 antibodies may play an important role in genetically determined resistance of sheep to haemonchosis     

Vaccination of Manchego lambs against Haemonchus contortus with a somatic fraction (p26/23) of adult parasites

A low molecular weight fraction from adult Haemonchus contortus containing two peptides (p26/23) was used to vaccinate Manchego female lambs between 3.5 and 5 months of age. Immunizing injections were given three times on days 0, 14 and 28 of the experiment. On day 43, lambs were challenged with 400 third stage larvae/kg live weight. Vaccination induced a lengthening of prepatent periods, significant reduction (> 60%) in mean faecal egg counts and smaller variations in packed cell volume values. At necropsy, average worm burden in the vaccinated lambs was significantly lower (61.6%) than that found in unvaccinated challenged animals. A clear correlation was found between protection and serum antibody response in immunized lambs.     

Immune response of Clun Forest sheep to vaccination with membrane glycoproteins from Haemonchus contortus

Clun Forest lambs were injected with a fraction containing integral membrane glycoproteins derived from the intestinal microvilli of Haemonchus contortus in two equal doses of 0.5, 5, 50 or 500 micrograms protein and then challenged with 10,000 infective larvae. The time-course of serum specific antibody responses were determined. Compared to the adjuvant control group, the animals injected with 1000 micrograms protein were better than 84% protected and those injected with 100 micrograms, better than 95% protected by all criteria. For these two groups parasite egg output was reduced 99% on average over the patent period. Two of the animals injected with 10 micrograms protein were partially protected, with 86% reduction in egg output. Two animals in the group injected with 1 microgram protein also showed partial protection. Antibody level correlated with protection.     

Mechanisms of immunity to Haemonchus contortus infection in sheep

In two separate experiments, sheep were immunized by nine to 12 weekly immunizing infections with 4000 Haemonchus contortus third stage larva (L3), drenched with anthelminthics and maintained free of H. contortus infection for a further 12 weeks. The anamnestic cellular immune responses in both the abomasal lymph node (ALN) and mucosa of the immunized sheep were examined 3 and 5 days post challenge with 50 000 H. contortus L3. Sheep in the two experiments clearly segregated out in two distinct groups, one in which challenge larvae were obviously present in the tissues of all 12 sheep at 3 and 5 days post challenge while no challenge larvae were detected in tissues of seven of the eight sheep in the other group. In sheep in which no tissue larvae were detected, very few changes were noted in either the ALN or mucosa. In contrast, dramatic changes were observed in the cellular profiles of the ALN and mucosa after challenge infection in sheep in which larvae were observed in the abomasal tissues. In the ALN, these changes were characterized by an increase in the relative percentage of gammadelta-TCR+ T cells and B cells and an increase in the proportion of CD4+ T cells coexpressing the activation markers MHC class II and CD25. In the abomasal mucosa, an increase in the number of infiltrating CD4+ and gammadelta-TCR+ T cells and B cells was observed by 3 days postinfection and these levels were further increased at 5 days postinfection. This infiltration of the abomasal mucosa by lymphocytes was accompanied by a dramatic increase in the number of infiltrating eosinophils, which were often in intimate association with the surface of H. contortus larvae. None of these changes occurred in the mucosa of the sheep that showed no sign of challenge larvae in the tissues; however, a transient increase in gammadelta T cells in the ALN and a drop in intraepithelial globule leucocytes were uniquely observed in these sheep at 5 days post challenge. These results suggest that two different types of immune responses can be generated after challenge infection of immunized sheep, one where tissue larvae are excluded from their tissue niche as observed previously and which is associated with changes in globular leucocyte population but no mobilization of the local immune system. In contrast, when challenge larvae reach their tissue niche, dramatic changes in the local immune system occur, including a pronounced infiltration of eosinophils. These two immune mechanisms may be associated with the rapid and delayed rejection of parasite infections in immune sheep.     

Protective studies in sheep immunized with cuticular collagen proteins and peptides of Haemonchus contortus

Twenty-eight nonsibling sheep aged approximately 12 months and raised in a helminth-free environment were used in two protection studies. Immunizations were conducted by two intramuscular injections 30 days apart with a synthetic 18AA cuticle collagen peptide and native cuticle collagens derived from the third- and fourth-stage larvae of Haemonchus contortus. Ten days following the last immunization, the sheep were each given 500 infective H. contortus larvae per day for five consecutive days by intraruminal injection. Both collagen materials induced antibodies reactive with cuticle collagens; however, neither induced reproducible protection to H. contortus infections in vaccinated/infected sheep. In the most extensive test, there were no statistical differences in mean faecal worm egg count for 56 days post worm challenge, in mean numbers of H. contortus and female fecundity ratios at necropsy of immunized and unimmunized sheep. Failure to reproducibly immunize sheep with cuticle collagens may be due to the inability of antibodies or host immune cells to reach the collagen epitopes in the nematode cuticle without prior surface coat removal as postulated in human nematode studies.     

Effects of tannic acid on Haemonchus contortus larvae viability and immune responses of sheep white blood cells in vitro

Direct inhibitory effects of tannic acid on Haemonchus contortus viability were studied in vitro using the larval migration inhibition (LMI) assay. Sheep white blood cells (WBC) were preincubated with 5 and 50 μg/mL tannic acid or not followed by whole H. contortus antigen (WHA). Cells were harvested at 24 h post‐incubation to test host immune responses. Concentrations of 50, 100, 500, 1000, 3000 and 5000 μg/mL tannic acid inhibited larvae migration by 19·8, 42·4, 46·3, 92·0, 93·7 and 100%, respectively, within 96 h post‐incubation (P < 0·001). The relative mRNA levels of interferon (IFN)‐γ, interleukin (IL)‐2, IL‐4 and IL‐10 were increased by WHA stimulation without tannic acid. However, the increased effects on IFN‐γ and IL‐2 were inhibited by tannic acid preincubation (P < 0·001), while the increases in IL‐4 and IL‐10 were greatly enhanced by tannic acid preincubation (P < 0·001). Changes in protein levels of all cytokines essentially paralleled the changes in their corresponding mRNA levels. In conclusion, tannic acid is directly harmful to larvae in a dose‐ and time‐dependent manner and modulates immune responses of sheep WBC stimulated by H. contortus antigen by inhibiting Th1 cytokines and increasing Th2 cytokine expression in vitro.     

Increased susceptibility to Haemonchus contortus infection by interleukin-5 modulation of eosinophil responses in sheep

Eosinophils are prominent effector cells in immune responses against gastrointestinal nematode infections in ruminants, but their in vivo role has been hard to establish in large animals. Interleukin-5 is a key cytokine in the induction and stimulation of anti-parasitic eosinophil responses. This study attempted to modulate the eosinophil response in sheep through vaccination with recombinant interleukin-5 (rIL-5) and determine the effect on subsequent Haemonchus contortus infection. Nematode-resistant Canaria Hair Breed (CHB) sheep vaccinated with rIL-5 in Quil-A adjuvant, had lower blood eosinophil counts and higher mean worm burdens than control sheep vaccinated with Quil-A adjuvant alone. In addition, adult worms in IL-5-vaccinated sheep were significantly longer with higher eggs in utero in female worms, supporting an active role of eosinophils against adult parasites in CHB sheep. These results confirm that eosinophils can play a direct role in effective control of H contortus infection in sheep and offer a new approach to study immune responses in ruminants.     

Cytokine and antioxidant gene profiles from peripheral blood mononuclear cells of Pelibuey lambs after Haemonchus contortus infection

The expression profiles of cytokines and antioxidant genes were determined from an experimental infection with H. contortus in Pelibuey lambs. The infection was followed for 34 days (d) to determine the number of eggs per gram (epg) and the packed cell volume (PCV). Differential white cell counts and expression profile estimations of IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐13, FCεR1A, GPX and SOD1 were determined at 0 hour, 4 hours, 2 days and 14 days post‐infection (PI) in infected and control groups. Comparison of the fold change between 0 and 4‐hours, 4‐hours and 2‐days and 2‐ and 14‐days periods was performed. Significant differences (P<.05) between epg (>2000) and PCV (>30%) were determined after 21 days and were also observed with regard to monocyte and lymphocyte cells after 2 and 7 days PI. At 0 hour and 14 days PI, the GPX and IL‐2 genes showed a 0.37‐ and 0.49‐fold decrease in expression, respectively. In contrast, upregulation was observed at 4 hours of IL‐8 (2.58) and FCεR1A (2.71), at 2 days for IL‐4 (2.14) and IL‐8 (4.02) and at 14 days for IL‐2 (0.41), IL‐10 (2.35) and FCεR1A (2.28). The comparison between the intervals of infection showed high expression values against H. contortus infection in Pelibuey sheep after the 2nd period of PI involving a dichotomy T cells.     

Early IL‐4 gene expression in abomasum is associated with resistance to Haemonchus contortus in hair and wool sheep breeds

Early immune events associated with reduced larval burden remain unclear in parasite‐resistant breeds of sheep. Therefore, our objective was to determine breed differences in immune‐related gene expression following infection with H. contortus. Gene expression in abomasal tissue and mucosa and in abomasal lymph nodes (ALN) was measured in 24 St. Croix (hair) lambs and 24 Dorset x (Finn–Rambouillet) (wool) lambs at 0 (uninfected), 3, 5 and 7 days after infection with 10 000 L3 H. contortus larvae. Expression of IL‐4 in abomasal mucosa was detected on day 3 and increased to day 7 in hair lambs, but was not detectable in wool lambs. Genes that recruit neutrophils (CXCL1) and macrophages (MCP1) were upregulated in abomasal mucosa of hair lambs. Genes associated with alternative macrophage activation (ARG‐1) and eosinophil activation (Gal‐14) were also upregulated in the abomasal mucosa of hair lambs. Tissue remodeling genes (MMP13, PDGF) and tumour necrosis factor alpha (TNF‐α) and MCP1 were upregulated in abomasal tissue of wool lambs; these lambs also had greater expression of forkhead box P3 in ALN. These data indicate a role for early IL‐4 expression locally and demonstrate potential downregulation of immunity in wool sheep that could facilitate establishment of H. contortus.     

Immune response to the filaria Litomosoides sigmodontis in susceptible and resistant mice

Comparisons were made between the immune responses evoked during the course of chronic and patent infections of Litomosoides sigmodontis in susceptible BALB/c mice and non-patent infections in resistant B10.D2 mice. Early antigen specific responses of spleen cells were weak in both mouse strains. However, by day 58 post infection a strong Th2 response, as determined by production of IL-4, IL-5 and IL-10, was observed in BALB/c mice but not in B10.D2 mice. Antibody responses seemed to appear sooner in B10.D2 than in BALB/c mice, and these differentially recognised two antigens of 15 kD and 80 kD.     

Evaluation of immunization with gut membrane glycoproteins of Ostertagia ostertagi against homologous challenge in calves and against Haemonchus contortus in sheep

Peanut and ConA lectins were used as ligands to isolate glycoproteins from detergent extracts of adult Ostertagia ostertagi membranes. As judged by their profiles following SDS-PAGE, these fractions closely resembled the equivalents from Haemonchus contortus which are derived from the nematode intestinal cell microvillar membranes and which are highly protective when used as antigens. Groups of calves were immunized with the peanut and ConA binding fractions of Ostertagia, either as separate or pooled antigens mixed with QuilA as adjuvant. All calves, including controls immunized with adjuvant only, were challenged with a single dose of infective Ostertagia larvae and faecal egg counts were monitored for 5 weeks. In two experiments where the antigen fractions were pooled, moderate (30-50%), but statistically significant reductions in egg output were observed, but the number of worms was not diminished. No significant protection was observed in a third trial where groups of calves were immunized with peanut or ConA binding proteins given separately. Two further trials were conducted in sheep immunized with the same Ostertagia fractions but challenged with Haemonchus. Irrespective of whether they were administered separately or together, the Ostertagia antigens cross protected efficiently against Haemonchus reducing egg counts by between 81% and 97% and worm numbers by between 57% and 84%.     

Kinetics of the local cellular response in the gastric lymph of immune and susceptible sheep to infection with Teladorsagia circumcincta

Groups of yearling sheep were trickle infected with Teladorsagia circumcincta for 8 weeks, then the infection cleared with anthelmintic and both these animals and a group of parasite naïve sheep were challenged with 50 000 infective T. circumcincta larvae. The previously infected sheep demonstrated acquired immunity to the parasite, manifested by reduced worm burdens which were evident as early as 2 days after challenge. Cannulation of the common efferent gastric lymph duct allowed the kinetics of their local cell traffic to be monitored, and the phenotype of these lymphocytes was analysed. A blast cell response, consisting of both T and B lymphocytes, was observed in both groups of sheep, however this occurred more rapidly in the previously infected, immune animals. CD4⁺, CD8⁺ and CD25⁺ blast cell output peaked at day 3 in the previously infected animals, whereas CD21⁺ blast cell output peaked slightly later at day 5. In the control group the peak output of all phenotypes of blast cells occurred more slowly, peaking 10 days after infection.     

Role of Innate Immunity against Human Papillomavirus (HPV) Infections and Effect of Adjuvants in Promoting Specific Immune Response

During the early stages of human papillomavirus (HPV) infections, the innate immune system creates a pro-inflammatory microenvironment by recruiting innate immune cells to eliminate the infected cells, initiating an effective acquired immune response. However, HPV exhibits a wide range of strategies for evading immune-surveillance, generating an anti-inflammatory microenvironment. The administration of new adjuvants, such as TLR (Toll-like receptors) agonists and alpha-galactosylceramide, has been demonstrated to reverse the anti-inflammatory microenvironment by down-regulating a number of adhesion molecules and chemo-attractants and activating keratinocytes, dendritic (DC), Langerhans (LC), natural killer (NK) or natural killer T (NKT) cells; thus, promoting a strong specific cytotoxic T cell response. Therefore, these adjuvants show promise for the treatment of HPV generated lesions and may be useful to elucidate the unknown roles of immune cells in the natural history of HPV infection. This review focuses on HPV immune evasion mechanisms and on the proposed response of the innate immune system, suggesting a role for the surrounding pro-inflammatory microenvironment and the NK and NKT cells in the clearance of HPV infections.     

Immune response to minor variant antigen types (VATs) in a mixed VAT infection of the African trypanosomes

Summary It has been shown that soon after the onset of acute infection with Trypanosoma brucei gambiense , mice are able to detect immunologically small numbers of minor variant antigen types (VATs) within the population. However, in more longstanding infections, considerably larger populations of minor VATs are required to stimulate an effective immune response. As a result, larger populations of minor VATs evade immune detection and, following a decrease in parasitaemia, become part of the relapse population. We hypothesize that the development of immunosuppression increases the effectiveness of antigenic variation as an escape mechanism.     

Molecular cloning of PEPCK and stress response of black porgy (Acanthopagrus schlegeli) to increased temperature in freshwater and seawater

Stress responses to increased temperature in black porgy reared in freshwater (FBP) and seawater (SBP) were examined via endocrinological and blood physiological methods. A rise in temperature increased plasma cortisol levels, which were significantly higher in FBP compared to SBP. The stimulated expression of phosphoenolpyruvate carboxykinase (PEPCK) mRNA in liver might result from the high cortisol level, and this explains the observed higher plasma glucose levels in FBP versus SBP. Full-length cDNA sequence for PEPCK was determined by 3' and 5' RACE procedures. PEPCK cDNA clone was found to contain 2563 nucleotides including an open reading frame that encodes 624 amino acids. While aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of FBP increased with temperature, there was no change in SBP. In FBP, T(3) were 2.3+/-0.3 ng/ml at 20 degrees C and significantly decreased to 1.0+/-0.3 ng/ml at 30 degrees C. On the other hand, in SBP, it were 3.1+/-0.5 ng/ml at 20 degrees C but significantly increased to 5.2+/-0.4 ng/ml at 30 degrees C. When comparing osmolality at the temperature of 30 degrees C and of 20 degrees C, the difference was found to be greater for FBP than SBP. Accordingly, the results suggest that FBP suffers greater stress than SBP with increased temperature, and provide stress responses and osmoregulatory abilities against stressors in black porgy that could differ depending on salinities.     

Early changes in the arachidonic acid metabolism of HeLa cells in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and related compounds

Summary In order to search for possible mediators involved in the transient radiomimetic effectiveness of TPA and related compounds early changes in the AA metabolism of HeLa cells prelabeled with 1-¹⁴C-AA have been analyzed. Maximum release of AA with different concentrations of TPA (3×10^-9 to 3×10^-5 M) was observed after 2–3 h treatment in the presence of 10% calf serum. Released AA was reincorporated by the cells after that period, a phenomenon which was largely abolished or delayed by cycloheximide. Reincorporation of released AA was observed in the presence of 10% fresh serum as well as with 0.5% BSA, and appears to be due to an induction of responsible enzyme(s) by the phorbol ester. The earliest metabolites of AA produced via the cyclooxygenase such as PGE₂ and PGF₂ and via lipoxygenases such as 12-, and 15-hydroxyeicosatetraenoic acids appear in small amounts and after later time points. AA release exhibited a pluriphasic dose response to TPA with maxima at 3×10^-8 M and 10^-5 M. Comparative dose response measurements with respect to AA release were established using various promoting skin mitogens which exhibited the following order of potency: TPA > teleocidin RPA > mezerein EPA>4-O-Me-TPA. For reasons discussed it appears unlikely that AA, Prostaglandins, or hydroxyeicosatetraenoic acid products play a significant role as mediators of the radiomimetic effects of TPA in G₂ of the cell cycle.Abbreviations AA arachidonic acid - BSA bovine serum albumin - EPA 12-O-ethacrynylphorbol-13-acetate - 4-O-Me-TPA 4-O-methyl-TPA - PGE prostaglandin E₂ - RPA 12-O-retinoylphorbol-13-acetate - TPA 12-O-tetradecanoylphorbol-13-acetate Dedicated to Professor Dr. E. Hecker on the occasion of his 60th birthday     

Interferon alfa-2c in chronic myelogenous leukemia (CML): Hematologic,cytogenetic and molecular-genetic response of patients with chronic phase CML previously resistant to therapy with interferon gamma

Summary Alpha- and gamma-interferons have been shown to actively suppress hematopoiesis in patients in the chronic phase of chronic myelogenous leukemia in vitro and in vivo. Since both interferons act through different receptors on their hematopoietic target cells, they are expected to be capable of independently inhibiting abnormal blood cell development in patients with chronic myelogenous leukemia. We have utilized recombinant human interferon alfa-2c to treate 11 patients with Philadelphia chromosome positive chronic myelogenous leukemia in chronic phase, who were resistant to previous interferon gamma therapy. Ten of the patients were evaluable for hematologic, cytogenetic and molecular-genetic response following interferon alfa-2c therapy for 6 to 30 months. In 5 patients, IFN alfa-2c treatment failed due to lack of hematologic response. A complete hematologic or partial hematologic response was achieved in the remaining 5 patients. Three of these experienced cytogenetic improvement with reappearence of 100% diploid hematopoietic cells and disappearence of c-abl/bcr rearrangement in one patient. In two patients interferon alfa-2c did not prevent transformation of the disease into an accelerated state or blast crisis, respectively. We conclude that recombinant human interferon alfa-2c may also control hematopoiesis in interferon-gamma resistant chronic myelogenous leukemia patients, although the long-term response will need to be elucidated in further studies.