Infection with Eimeria tenella: modulation of lymphocyte blastogenesis by specific antigen, and evidence for immunodepression

The blastogenic effects of specific parasite antigen and of mitogens on the lymphocytes of chickens infected with Eimeria tenella were examined. Lymphocytes from infected chickens were stimulated to divide when cultured with parasite antigen, but their responses to the T-cell mitogen, phytohemagglutinin (PHA), were depressed throughout the period of infection. Responses to the B-cell mitogen, lipopolysaccharide (LPS), were depressed during the first week of infection but enhanced in the second week. The inclusion of plasma samples from infected chickens in the culture medium depressed the responses of normal spleen lymphocytes to PHA, suggesting that soluble suppressor factors are generated during infection.     

Infection with Eimeria tenella: Modulation of Lymphocyte Blastogenesis by Specific Antigen,and Evidence for Immunodepression1

ABSTRACT. The blastogenic effects of specific parasite antigen and of mitogens on the lymphocytes of chickens infected with Eimeria tenella were examined. Lymphocytes from infected chickens were stimulated to divide when cultured with parasite antigen, but their responses to the T-cell mitogen, phytohemagglutinin (PHA), were depressed throughout the period of infection. Responses to the B-cell mitogen, lipopolysaccharide (LPS), were depressed during the first week of infection but enhanced in the second week. The inclusion of plasma samples from infected chickens in the culture medium depressed the responses of normal spleen lymphocytes to PHA, suggesting that soluble suppressor factors are generated during infection.     

Stimulation of the cellular immune response to lymphocytic choriomeningitis (LCM) virus infection in suckling mice

Death occurred earlier and its rate was higher in one-week-old mice treated with phytohaemagglutinin (PHA) and subsequently inoculated intracerebrally with LCM virus than in their virus infected but untreated littermates. Thus PHA treatment contributed to the outcome of LCM virus infection in the form of lethal meningitis. The course of LCM virus infection in 1-week-old PHA treated mice was similar as in the untreated 2-week-old mice. This indicates that PHA treatment accelerated the development of cell mediated immunological capacity in suckling mice.     

Combined phytohaemagglutinin and dianhydrodulcitol treatment of lymphocytic choriomeningitis virus infection in mice.

The course of intracerebral lymphocytic choriomeningitis (LCM) virus infection was studied in mice treated simultaneously with dianhydrodulcitol (DAD) and phytohaemagglutinin (PHA). Earlier experiments revealed that DAD decreased and PHA enhanced the cellular immune response of mice to LCM virus infection. On applying the treatments simultaneously they inhibited each other and neither the decreasing effect of DAD nor the enhancing effect of PHA on the cellular immune response could be observed.     

Immunocompetence and helminth community of the white-toothed shrew, <Emphasis Type="Italic">Crocidura russula</Emphasis> from the Montseny Natural Park,Spain

Host immunocompetence assessed by spleen size and response to phytohaemagglutinin (PHA) injection may give some indications on the control of parasite infection and on host mediation effect, through immunity, on parasite community structure. We investigated the helminth community and immunocompetence of the white-toothed shrew in a small area to test the relationship between immunocompetence and intensity of helminth infection. At the proximate level and if spleen mass and PHA response reflect the level of immunocompetence, we expected that individuals with a large spleen or a high PHA response should harbour a lower parasite load than individuals with relatively small spleen or low PHA response. In addition, we predicted that the structure of the helminth community should be mediated by the host’s immune defence. Spleen mass was linked to helminth infection. Nematodes and cestodes were negatively associated within hosts. PHA response was not related to total helminth intensity of infection but was negatively related to cestode intensity and positively to nematode intensity. This result suggests either a differential modulating effect on immunity by the two groups of worms or the existence of an antagonistic association between nematodes and cestodes mediated by the immune response of the host.     

Dipetalonema viteae: Response of spleen cells in experimental mouse filariasis to mitogens and antigens

Balb/c mice were infected by transplanting 3, 5, 10, or 20 female adult Dipetalonema viteae under the dorsal skin. The microfilaremias resulting from infections with 3 or 5 adult worms were of lesser magnitude and of shorter duration than those produced in infections with 10 or 20 worms. Spleen cells taken from these mice at various intervals after infection were assayed in vitro for their ability to respond to phytohemagglutinin (PHA) or lipopolysaccharide (LPS). There was no depression in the response to LPS or to PHA in mice given infections of 3 or 5 D. viteae adult worms. In contrast, the response to PHA was significantly depressed in groups receiving 10 or 20 adult female worms 12 days after infection and by Day 25, the depression was severe. Thereafter the PHA responsiveness recovered gradually to reach control values on Day 60. In mice transplanted with 10 or 20 D. viteae adult worms there was no significant depression in the response to LPS at any time during the infection, but the response was increased slightly sporadically during infection. These results indicate that in mice, this infection causes an initial suppression in the function of PHA-sensitive T cells but has little effect on the B cells which respond to LPS. A factor present in serum taken on Day 25 from mice infected with 10 or 20 adult worms inhibited the proliferative response to PHA by spleen cells from normal mice. The recovery of PHA responsiveness in mice given the heavier infections coincided with death of the adult worms, but mitogen reactivity and microfilaremia were unrelated. Antigens from male or female worms induced cell division in spleen cells taken from infected mice after microfilaremia had ceased whether they were implanted with 3 or 10 adult worms.     

Blastogenesis of splenic lymphocytes to specific antigens and PHA in Paragonimus westermani infected mice]

Paragonimus westermani is a common fluke in Korea. The present study aimed to observe the cell mediated immune response in experimental paragonimiasis of mice. The mouse (BALB/c) was orally inoculated with 40 metacercariae of P. westermani from Cambaroides similis. During the infection (1, 2, 4, 6 weeks) of mouse, blastogenic response of splenic lymphocytes to P. westermani adult antigen, metacercaria antigen, and PHA were observed. Sera from infected and noninfected mice added to normal mouse splenic lymphocytes with or without PHA. The blastogenic response of splenic lymphocytes to PHA was reduced after 1 week of infection. However after 6 weeks of infection, the response was restored to the control level. The blastogenic response of splenic lymphocytes to P. westermani adult or metacercaria antigen increased significantly on 1 week after infection, and maintained up to 6 weeks after infection. The response of non-infected mice was suppressed by addition of the infected mouse serum. The present results suggested that cellular immunity was involved in P. westermani infected mice and that P. westermani anti-serum inhibited proliferation of T lymphocytes.     

Interleukin 2 deficiency in murine Leishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin

This study examined the kinetics and mechanisms of depressed spleen cell responses to phytohemagglutinin (PHA) that occur during Leishmania donovani infection of BALB/c mice. In co-culture experiments, neither spleen cells from infected animals nor parasite-infected macrophages suppressed PHA responses of normal spleen cells. In addition, parasite-mediated suppression of PHA-stimulated spleen cell proliferation could not be demonstrated. Mice with 2 wk of infection did manifest an impairment in spleen cell production of interleukin 2 (IL 2) and by 8 wk IL 2 activity in supernatants from these cells was reduced by approximately 95%. This finding was not explained by an alteration in the kinetics of IL 2 production. Furthermore, diminished IL 2 activity in supernatants of PHA-activated spleen cells from infected animals was not caused by suppressive factors in these fluids as shown by their inability to suppress IL 2 stimulation of IL 2-dependent T cells. When spleen cells from mice with 8 wk of infection were cultured with PHA and supplemented with exogenous IL 2, there was an approximately 48% increase in mitogenesis. These data indicate that abnormal PHA-induced spleen cell activation in BALB/c mice with L. donovani infection is associated with impaired production of IL 2. In addition, the observation that supplementation of spleen cells from infected mice with IL 2 resulted in partial reconstitution of the PHA response is consistent with a defect in IL 2 responsiveness.     

Continuous Production of Long-Side-Chain Poly-beta-Hydroxyalkanoates by Pseudomonas oleovorans

Shake flask experiments showed that Pseudomonas oleovorans began to be growth inhibited at 4.65 g of sodium octanoate liter, with total inhibition at 6 g liter. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter in the feed, the maximum specific growth rate was 0.51 h, and the maximum specific rate of poly-beta-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein h at a dilution rate (D) of 0.25 h. When the specific growth rate (mu) was <0.3 h, the PHA composition was relatively constant with a C(4)/C(6)/C(8)/C(10) ratio of 0.1:1.7:20.7:1.0. At mu > 0.3 h, a decrease in the percentage of C(8) with a concomitant increase in C(10) monomers as mu increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At D = 0.24 h and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate consumed, the yield of PHA was 0.085 g of PHA g of octanoate used, and 7.9 g of octanoate was consumed for each gram of NH(4) supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass h. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.     

Effects of carotenoids, immune activation and immune suppression on the intensity of chronic coccidiosis in greenfinches

Allocation trade-offs of carotenoids between their use in the immune system and production of integumentary colouration have been suggested as a proximate mechanism maintaining honesty of signal traits. We tested how dietary carotenoid supplementation, immune activation and immune suppression affect intensity of coccidian infection in captive greenfinches Carduelis chloris, a passerine with carotenoid-based plumage. Immune activation with phytohaemagglutinin (PHA) decreased body mass among birds not supplemented with lutein, while among the carotenoid-fed birds, PHA had no effect on mass dynamics. Immune suppression with dexamethasone (DEX) induced loss of body mass and reduced the swelling response to PHA. DEX and PHA increased the concentration of circulating heterophils. Lutein supplementation increased plasma carotenoid levels but had no effect on the swelling response induced by PHA. PHA and DEX treatments did not affect plasma carotenoids. Immune stimulation by PHA suppressed the infection, but only among carotenoid-supplemented birds. Priming of the immune system can thus aid in suppressing chronic infection but only when sufficient amount of carotenoids is available. Our experiment shows the importance of carotenoids in immune response, but also the complicated nature of this impact, which could be the reason for inconsistent results in studies investigating the immunomodulatory effects of carotenoids. The findings about involvement of carotenoids in modulation of an immune response against coccidiosis suggest that carotenoid-based ornaments may honestly signal individuals’ ability to manage chronic infections.     

In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate (PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity

In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G, showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of accumulated PHA ranged over 107–126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA biosynthesis. Electronic Publication     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of cell-mediated immunity.

To assess the effects of the selective T-cell mitogens concanavalin A (Con A) and phytohemagglutinin P (PHA) on cell-mediated immunity (CMI), the mitogens were injected before, with, or after intravenous (iv) challenge of mice with Listeria monocytogenes. Mitogenic treatment differentially influenced the CMI response to Listeria. Con A enhanced listericidal activity when given before or with Listeria challenge, but Con A suppressed the CMI response when given after infection with Listeria. In contrast, PHA enhanced listericidal activity at all intervals. Since Con A, but not PHA, affected the growth of Listeria in the spleens of mice 24 and 48 hr after infection, Con A was shown to have an immediate effect on the development of listericidal activity and PHA was shown to have a delayed effect. In addition, Con A induction of immediate nonspecific listericidal activity was short-lived, while PHA induced a longer-lasting effect on resistance to Listeria. The mitogen-induced effects in the CMI response to Listeria were shown to be dependent upon the activities of activated T cells. The enhancement and suppression of listericidal activity appears to result from the activation of different T-cell subpopulations, known to be stimulated preferentially by Con A or PHA.     

Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies

Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.     

Abnormalities in lymphocyte proliferation in classical and atypical measles infection

Impaired in vitro lymphocyte responses to PHA stimulation have been observed in seven patients with acute classical measles infection, and three patients with atypical measles. The impairment is evident only at suboptimal concentrations of PHA and disappears in convalescence. Spontaneous DNA synthesis in peripheral leukocytes was elevated in both types of patients, but the classical measles patients demonstrated a delayed return to normal values as compared to the atypical measles group. We failed to demonstrate a serum inhibitory factor as an explanation for the impaired PHA responses of lymphocytes in these patients.     

Specific character of bacterial biodegradation of polyhydroxyalkanoates with different chemical structure in soil

The study addresses the influence of the physicochemical properties of the reserve cellular macromolecules (polyhydroxyalkanoates, PHA) with different chemical composition on their biodegradation in the agro-transformed field soil of the Siberian region (Krasnoyarsk Territory, Russia). It was shown that the degradation of the PHA samples depends on the degree of polymer crystallinity (C_x). For the first time, it was shown that the range of PHA-degrading microorganisms differs for each of PHA types. The study defines the primary degraders specific to each PHA type and common to all types of examined polymers.     

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates)

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Serologic diagnosis of intestinal yersiniosis. The design of a stable erythrocyte preparation for the indirect hemagglutination reaction

Nine types of erythrocyte diagnostica of serovars O3 and O9, differing in the methods of obtaining sensitins and the physical state of erythrocytes, were put on trial. The preparations were used for the titration of hyperimmune sera and blood sera obtained from about 500 healthy persons, 300 patients with Yersinia enteric infection and 300 patients with other diseases. Freeze-dried diagnostica, when compared with liquid ones, were found to be less sensitive, but more stable and specific. Sensitins isolated by the methods of Westphal ad Boivin showed the highest degree of specificity. The authors believe freeze-dried sheep red blood with activated Boivin's antigen adsorbed onto them to be the optimal preparation for use in the passive hemagglutination (PHA) test. The preparation was found to retain its serological activity for as long as 2-3 years. The titer 1:160 (1:200) in the PHA test is recommended as the minimal diagnostic indicator. Erythrocyte diagnosticum is more sensitive, specific and stable than bacterial one. Since 1984 dried Yersinia erythrocyte diagnostica (serovars O3 and O9) have gone into quantity production at the Leningrad Research Institute for Vaccines and Sera.     

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.     

Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral responses during the course of infection.

The kinetics of various specific and nonspecific immunologic responses were examined in BALB/c mice infected with 17X nonlethal Plasmodium berghei yoelii (a self-limiting infection). The sequence of events after infection was characterized by rapid sensitization of splenic T cells to malaria antigen and polyclonal B cell activation, followed by a period of depressed splenic proliferative responses in vitro to mitogens (PHA and LPS) and malaria (specific) antigen. At the same time, suppressed primary in vitro splenic PFC responses to trinitrophenyl-aminoethylcarbamylmethyl-Ficoll (TNP-F) were seen. This suppression was an active process requiring adherent cells. During this period, levels of antimalarial antibody also increased exponentially. As the infection was cleared, splenic malaria antigen-specific proliferative responses were again observed and splenic PFC and in vitro mitogen responses returned to preinfection levels after variable periods of time. Both splenic proliferative responses to malaria antigen and antimalarial antibody responses remained persistently elevated. In addition, some responses were examined in mice infected with 17X lethal P.b. yoelii (a fatal infection); in comparison to the early responses of mice infected with the nonlethal substrain, there was a decrease and delay in the development of a splenic T cell response to malaria antigen and a blunted antimalarial antibody response.