Mast cells, histamine and the pathogenesis of intestinal damage in experimental Trypanosoma brucei brucei infections.

Intestinal damage with increased permeability is a prominent feature of experimental African trypanosomiasis. The possible involvement of mast cells and histamine in the altered gut integrity was investigated, at the level of the jejunum, in BALB/c mice infected with Trypanosoma brucei brucei. Mast cells were studied by selective staining of granule content with Alcian Blue/Safranin and quantitative histology, and histamine concentrations were determined by a fluorimetric method. Mast-cell activation, shown by a marked reduction in the numbers of positive-staining cells seen per villous section, was prominent on days 7 and 14 post-infection (there was, for example, a reduction to 36% of the control value by day 14; P=0.0001). By day 21, however, there were 131% more staining cells per villous section in the infected mice than in the uninfected controls (P=0.003). Histamine levels in homogenates of the jejunal mucosae of the infected mice were found to be significantly elevated at each time-point. The maximum increase was observed on day 14, when the numbers of granulated mast cells were at their lowest, with mean (S.E.) concentrations of 6.744 (0.890) ng/mg tissue for the infected mice and 2.813 (0.321) ng/mg for the uninfected controls (P=0.0008). The jejunal mucosa suffered progressive morphological damage during the infection, with oedema of the lamina propria and villi and disruption of the endothelium. These results indicate that mast cells are involved with the intestinal pathology that develops during experimental African trypanosomiasis.

Activities of glycogen phosphorylase, alanine aminotransferase and aspartate aminotransferase in adult worms of Litomosoides carinii recovered from pyridoxine deficient cotton rats (Sigmodon hispidus).

This paper demonstrates that the activities of glycogen phosphorylase (GP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are reduced in adult worms of the filarial nematode Litomosoides carinii recovered from pyridoxine-deficient cotton rats when compared to worms recovered from pyridoxine-sufficient controls. GP, ALT and AST activities were determined in adult worms L. carinii recovered from cotton rat hosts over a 20-week experimental period. Activities of GP, ALT and AST in the parasite showed a direct correlation with the dietary pyridoxine intake of their host. Throughout the experiment, enzyme activities were significantly lower (P < 0.001) in worms from rats fed a pyridoxine-free diet ad libitum that in worms from rats fed either a stock colony diet, a pyridoxine-free diet ad libitum with daily supplementation of 100 micrograms pyridoxine or limited amounts of pyridoxine-free diet with daily supplementation of 100 micrograms pyridoxine. The lower than normal activity of GP, ALT, AST and other enzymes dependent on the biologically active derivative of pyridoxine, the coenzyme pyridoxal-5-phosphate (PLP), interferes with the protein, carbohydrate and lipid metabolism of L. carinii and may in part cause the reduced establishment, development and growth of the parasite in pyridoxine-deficient hosts.

Trypanosoma brucei products activate components of the reactive response in astrocytes in vitro.

A study was made of the effects of T. b. brucei and the disrupted parasite material on different components of the reactive response of astrocytes in primary cultures prepared from neonatal rats and C6 glioma cells. The effects were compared with lipopolysaccharide (LPS) and fragments of the C6 cells. The disrupted trypanosome material, LPS and C6 fragments caused dose--and time--dependent alterations in morphology of the primary cultures from flat to stellate shape, increases in levels of glial fibrillary acidic protein (GFAp) and enhanced MHC class I and II expression. Exposure to trypanosome material and C6 fragments caused marked increases in phagolysosomes and lysosomes in the primary cultures. The findings demonstrate that T. b. brucei products and astrocyte cell debris are internalized and initiate astrogliosis in vitro.

Effects of physico-chemical treatments on haemagglutination activity of Anopheles gambiae haemolymph and midgut extract.

Anopheles gambiae midgut extracts and haemolymph possessed agglutinins, titre 1:16 to 1:256, against human red blood cells (RBCs). Subjection of both tissues to protein precipitation reagents, organic chemical and selected protease, neuraminidase and other glycosidic hydrolase treatments revealed the haemagglutinins to be protein, most likely glycoprotein, in nature--not lipoprotein, lipid, glycolipid or nucleic acid. An.gambiae agglutinins were thermo-labile > 40 degrees C, affected by freezing and thawing treatments, and contained disulphide and hydrogen bonds on the basis of sensitivity following exposure to dithiothreitol and urea respectively. Optimum haemagglutination depended generally on slightly acid to neutral pH conditions and agglutinin activity was Ca2+ ion, albeit to a lesser extent Mg2+ ion, dependent. The midgut extract agglutinin subunit molecule had a relative molecular weight (M(r)) of 65 kDa whilst that of haemolymph was 40 kDa. This study presents the first report on selected physico-chemical properties, the glycoproteinaceous nature and tentative subunit M(r) of mosquito midgut extract and haemolymph anti-RBC agglutinin(s).

Characteristics of tick, Rhipicephalus appendiculatus, glands distinguished by surface lectin binding.

Incubation of fluorescein-conjugated and biotinylated lectins with Rhipicephalus appendiculatus salivary glands revealed differences between the basal laminae of the haemocoelic surfaces of the three acinal types. There were some similarities in the lectin binding characteristics of the surfaces of type II and type III acini when Con A, PNA and TVA were applied, indicating the presence of galactose, mannose and glucose moieties on the acinal surfaces. The binding of BPA, HPA and HAA lectins, specific for galactosyl and glycosyl ligands, which occurred only on the surface of type III acini, was moderate to intense. The remaining galactose-reactive lectins (GMA, DBA and VVA) also bound only to type III acini and the level of binding was weak to moderate. Although the relationship between the haemocoelic surface of the R. appendiculatus salivary gland acini and Theileria kinetes is not known, the consistent differences in the surface carbohydrate composition of the acini confirm the existence of differences in the basic physiology of the acini which may correlate with the specific susceptibility of the type III acinus to Theileria parva infection.

Salivary gland surface carbohydrate variations in three species of the Anopheles gambiae complex.

Fluorescein isothiocyanate (FITC)-conjugated lectins (agglutinins) were employed as probes to distinguish between the various carbohydrates present on the surface of salivary glands of three species of mosquito of the Anopheles gambiae complex. Of twenty lectins tested, eight (Concanavalin A- Con A, Lathyrus odoratus- LOA, Lens culinaris, Pisum sativum-PSA, Vicia faba- VFA, Triticum vulgaris, Maclura pomifera- MPA and Ulex europaeus) specifically reacted with the salivary gland membrane. Both mannosyl and N-acetylglucosamine moieties were detected in the three Anopheles gambiae sensu stricto strains, the two An. arabiensis strains and a single An. merus strain examined. Variations in the degrees of fluorescent intensities of Con A and MPA in particular suggested interspecies differences in membrane mannosyl and galactosyl residues on the salivary gland lobes of the three mosquito species in this study. Furthermore, intraspecific variations in mannose as indicated by Con A, LOA, PSA and VFA staining were demonstrated between the An. gambiae s.s. strains. The use of either peroxidase-labelled or biotinylated lectins confirmed the binding specificities of the above lectins. The consistent differences observed in lectin binding suggest that variations occur in salivary gland surface carbohydrate residues and that lectins can be used to distinguish between at least some members of the An. gambiae complex.

Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambiae gut extracts and haemolymph.

Lectins that agglutinate red blood cells (RBC) were demonstrated in Anopheles gambiae mosquito haemolymph and gut extracts. No apparent differences in haemagglutinin titres were detected between male and female mosquitoes and overall agglutinin levels were not increased following a bloodmeal. Titres were highest in the haemolymph and midgut extracts versus human AB, horse, chicken and goat RBCs and in hindgut against human AB, chicken and sheep; foregut extract gave relatively low titres. Adsorption of haemolymph and gut extracts with selected RBCs coupled with carbohydrate inhibition and the use of enzyme-treated RBCs revealed the presence of multiple (hetero-) agglutinins. An.gambiae lectins were specific for (1-1)-, (1-4)- or (1-6)-linked glucose based disaccharides, glucose and its (1-2) or (1-3) linkages with fructose and, to a lesser extent, aminated or N-acetylated glucose, or galactose and its deoxy derivatives. This study presents the first report of the occurrence of heterogenous anti-RBC agglutinins in haemolymph and gut extracts of the mosquito An.gambiae, together with the sugar-binding specificities of these lectins.

Identification of midgut trypanolysin and trypanoagglutinin in Glossina palpalis sspp. (Diptera: Glossinidae).

A midgut trypanolysin and an agglutinin from Glossina palpalis subspecies were isolated and partially characterized using anion-exchange chromatography and polyacrylamide gel electrophoresis. FPLC fractions of midgut extracts of Glossina palpalis palpalis caused agglutination and lysis of two trypanosome species (Trypanosoma congolense and Trypanosoma brucei brucei), although Glossina palpalis gambiensis caused only agglutination. The trypanolysin and agglutinin were active only in the posterior midguts, were heat labile above 50 degrees C, had a periodic cycle of 'activity' in response to bloodmeal intake and were not affected by protease inhibitors or trypsin but were inactivated by pronase. The lytic substance contained two proteins with approximate molecular weights (Mr) of 12,000 and 10,000 Da respectively. The agglutinin had an approximate Mr of 67,000 Da. Gamma-irradiation of the two subspecies caused a temporary inhibition of trypanolytic and agglutinin activities in midgut extracts.

The primary immune response of the green toad (Bufo viridis) to challenge with Crithidia fasciculata.

The primary immune response of the green toad (Bufo viridis) following immunization with Crithidia fasciculata choanomastigotes was studied. Lysins, agglutinins, and antibodies detectable by enzyme-linked immunosorbent assay (ELISA) were first detected in the sera of immunized animals one week after injection. The antibody titers increased to significant levels (P less than 0.01) and maximum values were reached seven weeks post-immunization. The stimulated immunoglobulins were antigen-specific, partially heat-labile, sensitive to the reducing agent dithiothreitol, possessed precipitin activity, effectively fixed complement and exhibited an electrophoretic mobility similar to the gamma-globulins of human serum. On this basis, it is probable that the antibody produced during the primary response in green toads is high molecular weight IgM. Increases in serum lysozyme levels paralleled the rise of antibody titers. Overall, the lysozyme concentration increased two-fold compared to the appropriate controls. This is the first report of the immune response in amphibians to experimental injection with protozoan parasites and the use of the ELISA technique to detect antibodies in amphibian sera.

The agglutination of erythrocytes and Leishmania parasites by sandfly gut extracts: evidence for lectin activity.

Lysates of heads, hind- and midguts of male and female Phlebotomus papatasi were found to contain lectins or lectin-like molecules capable of agglutinating human red blood cell types of the ABO(H) system and promastigotes of Leishmania aethiopica, L. major and L. donovani but not L. hertigi hertigi promastigotes or Crithidia fasciculata choanomastigotes. The agglutination of erythrocytes from the human O Rhesus positive blood group by sandfly midgut extracts was inhibited by two disaccharides; trehalose and turanose. This is the first report of haemagglutinating and parasite agglutinating activity in sandflies (Psychodidae).

The effect of dose, route, number of injections and the use of adjuvant on the clearance of and immune response to, haemocyanin following injection into brown trout (Salmo trutta).

The primary and secondary immune responses were investigated in trout injected with haemocyanin (HCN) intramuscularly (IM) or intraperitoneally (IP), with or without adjuvant. HCN was detected in the blood 30 min after injection and clearance times varied after one injection from 8 to 56 days. Fish given two and three injections cleared the antigen faster. Precipitins against HCN were first detected 21 to 30 days after injection and were still present on Day 56. However, antibodies detectable by indirect haemagglutination (IHA) and complement fixation (CFA) were initially demonstrated 7 to 21 days after a single injection and highest titres were reached between 33 and 56 days according to the experimental protocol. In fish given two injections, maximum titres were reached between Days 42 and 56 (IM), and Days 50 and 56 (IP). With three injections, maximum CFA and IHA values occurred on Days 62 and 66 respectively in the case of IP, and on Days 103 and 106 respectively with IM. Overall, higher titres were found with IHA than by CFA.

Lectins and lectin-like molecules in lower plants. II. Freshwater algae.

Freshwater chlamydomonad algae possess flagella surface isoagglutinins of a glycoprotein nature which are involved in gamete recognition and consequent cell fusion. Alternatively, such molecules may have a receptor role in certain Chlamydomonas species. In some freshwater volvocaines, lectin-like substances act as inducers of sexuality. At the moment it is speculative whether these materials can serve as naturally-occurring antibodies to protect algae from environmental antigens.

Lectins and lectin-like molecules in lower plants. I. Marine algae (review).

Marine algae possess agglutinins, generally of unknown carbohydrate specificity, against a diverse array of erythrocytes but in a small number of species these haemagglutinins react with certain human blood group types. In seaweeds, lectins or lectin-like molecules are involved in gamete recognition and consequent reproductive cell fusion. Although some substances produced by algae are antiviral, antifungal, antibacterial, haemolytic and toxic, no similarity to mammalian immunoglobulins either structurally or physico-chemically has been observed for algal agglutinins. Whether such compounds have a defence or immune function and perform an antibody-like role to enable algal species to survive and counteract infections within their environment remains tentative.

Naturally occurring agglutinins against trypanosomatid flagellates in the haemolymph of insects.

In vitro studies of the behaviour of the trypanosomatid flagellates Trypanosoma brucei and Leishmania hertigi in the presence of cell-free haemolymph of locusts, Schistocerca gregaria and cockroaches, Periplaneta americana revealed the presence of parasite agglutinins. The range of normal values of agglutination titres was 2(-4) to 2(-13). Physico-chemical treatment of haemolymph indicated that these agglutinins are protein or glycoprotein in nature and are only partially affected by heat treatment below 65 degrees C, at which temperature incubation of haemolymph for 30 min abrogated all agglutination. Agglutination was not dependent on the presence of Ca2+ or Mg2+. Prior injection of locusts and cockroaches with T. brucei and L. hertigi significantly increased agglutinin titres between Days 4 and 6 in cockroaches (P less than 0.05) and from Days 2 to 4 when L. hertigi was inoculated into locusts. The induced differences in titres observed in locusts infected with T. brucei were not significant. Lysozyme levels were significantly increased after inoculation of T. brucei into cockroaches compared with placebo-inoculated and uninoculated controls. L. hertigi inoculation produced significant increases in lysozyme levels compared with controls between Days 1 and 7 in locusts and 3 to 6 in cockroaches. These studies indicate that, at least in easily manipulated model systems, induced responses to intrahaemocoelic inoculation to trypanosomes and Leishmania can occur. As far as we are aware this is the first report of an induced response of an insect to such important parasites. The possibility that induced responses in natural vector to this parasites occurs requires investigation.

A comparison of selected immunological techniques used to detect anti-leishmanial antibodies in the sera of two reptile species.

European green lizards (Lacerta viridis) and spiny-tailed agamids (Agama caudospinosum) were obtained from areas endemic for human leishmaniasis. Serum antibody titres against Leishmania agamae, a reptilian leishmanial species, in normal lizards and lizards injected with Leishmania agamae promastigotes were measured by 5 immunological methods commonly used in the serodiagnosis of the human and mammalian leishmaniasis viz. immobilisation test (IMM), direct agglutination (DA), complement-fixation test (CFT), indirect haemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA). Correlation coefficients (r) were determined for comparisons between each method and linear regression equations calculated to convert antibody titres by one method to those by another. In each lizard species, the IMM test gave the lowest values while the highest were obtained with ELISA. The highest mean titre obtained by ELISA was between 2 and 10 times that obtained by the other methods for both control and immune sera. The methods of preparing the leishmanial antigen extracts affected the IHA and ELISA titres, while the source of complement was critical in obtaining good CFT values. Correlations ranging from 3% to 77% were found for the control animals but higher values ranging from 65% to 96% were obtained with the immunised lizards. Overall, the best correlation was with IHA and ELISA (r greater than 0.82) and with ELISA values for different antigen preparations compared with each other for both control (r greater than 0.67) and immune (r greater than 0.90) sera. ELISA thus appears the most sensitive method for detection and quantitation of anti-flagellate antibodies in normal lizard serum and for the determination of titres in immune serum. ELISA is the most applicable technique for screening reptiles and other lower vertebrates for anti-parasite immunoglobulins, and for screening potential carriers or reservoirs of infective flagellates in epidemiological studies aimed at disease control, especially in areas where human infections are prevalent.

Responses of European green lizards Lacerta viridis following administration of Leishmania agamae promastigotes.

European green lizards (Lacerta viridis) were injected intraperitoneally, subcutaneously or orally with viable Leishmania agamae promastigotes. Neither promastigotes nor amastigotes were later found in blood and tissue impression smears, or in blood and selected organ cultures. However, by the use of an immunoperoxidase technique, parasite antigens were detected in the liver, stomach, small intestine, kidney, gonad, heart, lung and skin but not in the bone marrow, brain or spleen. Non-precipitating antibodies with beta 2-electrophoretic mobility were induced against L. agamae. They were detected in the sera by enzyme-linked immunosorbent assay 3-7 days post-infection. The titres increased significantly above background levels (P less than 0.001) and reached maxima after 6-7 weeks, with 27 out of 29 lizards producing antibodies. The mean serum protein concentration significantly increased after infection (P less than 0.005) with no significant differences in mean values between male and female animals. Lizard sera separated into 7 components on cellulose acetate membranes with migration rates comparable to albumin, alpha- and beta-globulins of human serum; gamma-globulins were absent. Significant decreases occurred (P less than 0.05) in the albumin fraction, with significant increases in the beta-globulin region of anti-L. agamae sera. C-reactive protein was not detected in either normal or immune lizard sera.

Haemagglutinins and parasite agglutinins in haemolymph and gut of Glossina.

Agglutinating activity was found in the haemolymph and in extracts of midgut and hindgut of Glossina austeni against calf, guinea pig and chicken erythrocytes for the first time. Trypanosoma brucei procyclic forms were also agglutinated by midgut and hindgut extracts, but not by haemolymph. The 3 fractions tested failed to react with erythrocytes of four other vertebrates and other trypanosomatids (Leishmania hertigi and Crithidia fasciculata) examined. The reciprocal titre of agglutinating activity of the 3 fractions against calf, guinea pig and chicken erythrocytes were approximately the same at a dilution of 64. Both midgut and hindgut agglutinins showed higher titres against T. brucei at reciprocal dilutions of 128 and 256 respectively. Sialidase treatment, reduced the agglutinating activity to half its normal range while trypsinisation had no effect. The results of attempts to inhibit the agglutinating activity using a variety of sugars and glycoproteins revealed that midgut and hindgut were specific for D+glucosamine suggesting that midgut and hindgut agglutinin were possibly of the same origin. This is the first report of haemagglutinating and parasite agglutinating activity in Glossina tissues which may play a role not only in insect defence reactions but also in the determination of parasite establishment and infection in vectors.