Histochemical studies on the body wall of nematodes: Haemonchus contortus (Rud., 1803) and Xiphinema insigne Loos, 1949

Summary Histochemical studies on the body wall of Haemonchus contortus (Rud.) and Xiphinema insigne Loos have been made. In H. contortus, the cuticle is mainly proteinous in nature. The lipids and PAS-positive materials are only present in cortical layers. In addition, haemoglobin and acid phosphatase are also present. The hypodermis shows the presence of glycogen, lipids, RNA, acid and alkaline phosphatases. The oval dense body is composed of keratinous and collagenous proteins associated with acid mucopolysaccharides. Muscles carry a greater concentration of glycogen granules and phospholipids. In X. insigne, the cuticle is rich in sudanophilic lipids. The cuticle also consists of weakly acidic mucopolysaccharides. Hypodermis and muscles contain lipids and glycogen. In addition, hypodermis also consists of acidic mucopolysaccharides. The functional significance of these components has been fully discussed.     

Glycogen, protein and lipid content of adult Fasciola gigantica (Trematoda).

The concentration of carbohydrates, proteins, and lipids in the whole worm, gut, and body musculature of Fasciola gigantica was investigated from worms collected from cattle slaughtered in the Jos abattoir. Protein accounted for the highest concentration of the constituents constituting 62.59 +/- 0.86% of the dry weight of the fluke. This was followed by lipid with 28.15 +/- 0.86% of the dry weight while carbohydrate (glycogen) was 6.29 +/- 0.11% of the dry weight. Thin-layer chromatography revealed the presence of six distinct bands of neutral lipids, namely: monoglycerides, 1,2-diglycerides, 1,3-diglycerides, free fatty acids, triglycerides and sterol esters from both whole worm homogenate and fractions of the gut and muscle tissues. In the case of phospholipids, lysophosphatidyl choline, sphingomyeline, phosphatidyl choline and phosphatidyl-ethanol amine were recovered. When the free fatty acids were subjected to further fractionation, palmitic, stearic, as well as oleic acids were recovered in appreciable quantities indicating that lipids are being catabolized in these trematodes.     

Molecular modeling: a tool for predicting anthelmintic activity in vivo

The structural and electronic features of the broad-spectrum benzimidazole anthelmintic mebendazole [MBZ, methyl 5-(benzoyl)-benzimidazole-2-carbamate] have been determined using a combination of quantum mechanics, molecular graphics, and molecular modeling techniques. Using conformational analyses and quantum mechanics, we found that the three-dimensional structure and electronic features of MBZ were consistent with those previously reported for highly active broad-spectrum benzimidazole anthelmintics and that in vivo drug efficacy againstHymenolepis diminuta depends upon the orientation of the benzoyl group at position 5 on the heterocyclic ring system, the magnitude of the molecular dipole moment, and the percentage of polar surface area. The chemotherapeutic actions of MBZ onH. diminuta in vivo were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of MBZ 24 h earlier were significantly smaller and contained much less glycogen (as a percentage of the wet weight) than worms from untreated controls. In MBZ-treated worms, protein concentrations rose at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in MBZ-treated worms were considerably lower than the corresponding control values. Differences in the absolute amounts of glycogen between control and drug-treated worms were even more profound. Administration of a curative dose of MBZ to the rat host produced inH. diminuta another change, the onset of which coincided with the gross alterations in worm weight and chemical composition. In vitro studies carried out 24 h after treatment revealed that MBZ-treated worms absorbed and metabolized much smaller quantities of exogenous glucose than did the controls and that the ability of the worm to accumulate glucose against a concentration difference was significantly depressed. A mode of action common to MBZ and other structurally related broad-spectrum benzimidazole anthelmintics is further indicated by the similarity of their biochemical and physiological effects on the tapeworms and their time course of action following their administration to rats infected withH. diminuta.     

Histochemistry of Centroorhynchus falconis (Das, 1950).

With a view to augment the understanding of the animal mucosubstances in general and Acanthocephalan mucosubstances in particular, Acanthocephalan worms of (Centrorhynchus falconis, Das, 1950) were investigated histochemically by employing recent techniques. Variations in the intensity of histochemical reactions in different tissues revealed a heterogenous distribution of mucosubstances. The cuticle contained a mixture of periodate reactive neutral mucosubstances and sulfomucins, whereas the subcuticle contained only glycogen. Retractor muscles contained glycogen together with some acidic mucosubstances which exhibited alcianophilia only at high pH. Cement glands elaborated a mixture of glycogen and galactogen. Histochemical methods revealed two types of acanthors: Some contained only glycogen, whereas others contained glycogen and galactogen.     

Hepatogenic potential of human bone marrow and umbilical cord blood mesenchymal stem cells

Conditions of human BM and umbilical cord blood MSC in vitro differentiation in the hepatogenic direction were studied. Changes in cell morphology, phenotype, acquisition of the capacity to produce albumin and accumulate glycogen, express cytokeratin, alkaline phosphatase, and albumin mRNA indicated that BM and umbilical cord blood MSC differentiated in vitro into immature hepatocyte-like cells.     

Effects of fat soluble vitamins (vitamin A,D3 and E) on axenically in vitro growth ofHymenolepis microstoma

Summary Hymenolepis microstoma has been cultured in vitro from 4 days to 11 days of age using Eagle's medium supplemented with horse serum, lamb liver extract and a vitamin A, D₃ and E mixture in six different concentrations [0.025%, 0.050%, 0.1%, 0.5%, 1% and 2% in the BME (=Eagle's medium)] including a control without the added vitamin mixture.The experiments have shown that growth is inversely related to the concentration of vitamin mixture. In the 2% and 1% concentration of the added vitamin mixture worms show degenerative changes within 24h and 120 h respectively. Although at a concentration of 0.5% of this mixture worms continue to grow until day 11, organogenesis and distribution of calcareous corpuscles in them are severely affected. Worms grown in media containing the lowest three concentrations of the vitamin mixture and in the control medium do not showmarked differences in organogenesis and distribution of calcareous corpuscles. The concentration of neutral lipids in the scolex and parenchyma of worms increases with the increasing concentration of the added vitamin mixture.It is felt that until the culture medium is more defined the requirement of individual fat soluble vitamin is difficult to assess since the control medium used in the experiment probably contained the required amount of these vitamins from the additives — chemical or natural.An abstract of this paper is submitted to the Asian Congress of Parasitology, Bombay (India), 23–26 February, 1978     

Morphological,histochemical, and biochemical studies on the gut ofHaemonchus contortus Rud., 1803

Summary A study has been made on the structure and chemical composition of the gut ofHaemonchus contortus (Rud., 1803). The oesophagus has typically a triradiate, cuticle-lined lumen. The intestinal epithelium is provided with a well-developed brush border which contains periodic acid-Schiffpositive mucoproteins. The intestinal epithelium stores glycogen and lipids. It stains diffusely for phospholipids and general proteins and also for terminal-NH₂ group. The presence of Fe²⁺ and Fe³⁺ containing pigments and activities of acid and alkaline phosphatases, glucose-6-phosphatase, and 5-nucleotidase have been observed in the intestinal epithelium. Biochemically pH optimum for intestinal acid phosphatase has been found to be 4.8. The brush border shows positive reactions for acid phosphatase and glucose-6-phosphatase, and negative reactions for alkaline phosphatase and 5-nucleotidase. The presence of enzymes in the brush border is related to extracellular digestion and absorption of nutrients.     

Chemical composition and histochemistry ofLobatostoma ringens (Trematoda: Aspidogastrea)

Summary The results of analyses of the major chemical constituents ofLobatostoma ringens are as follows: water, 71.6% fresh weight; ash, 4.6% dry weight; protein, 64.3% dry weight; glycogen, 4.7% ethanol-extracted dry weight; lipid, 6.4% fresh weight. Histochemical staining revealed glycogen mainly in the parenchyma and musculature, with smaller deposits in the vitelline cells and spermatozoa; neutral lipid was localized in the gastrodermis and excretory system; and the surfaces of the tegument and gastrodermis were reactive for acidic mucopolysaccharides. Protein synthesis was evident in the tegumental cells, gastrodermis, and vitellaria.     

Histochemical data concerning some metabolic disturbances in acute radiation sickness

Summary Carbohydrate and lipid metabolism, as well as the activity of alkaline glycerophosphatase, cytochrome oxidase, succinic dehydrogenase, and succinic dehydrogenase, were studied histochemically in the liver and lungs of irradiated rabbits. The activity of succinic dehydrogenase and cytochrome oxidase drops considerably in animals with acute radiation, sickness, whereas the activity of alkaline glycerophosphatase is markedly elevated. In addition, a considerable increase of the neutral fat content in the liver, with accumulation of underoxidized products of its splitting in the lungs. The amount of phospholipids is usually markedly reduced in both organs. The amount of glycogen in the liver changes but little; only during the first hours after irradiation and in the, dead animals is a loss of the glycogen store in the liver noted.(Supervisor-Active Member AMN SSSR N.A. Kraevskii)(Presented by Active Member of AMN SSSR N.A. Kraevskii) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 50, No. 7, pp. 58–63, July, 1960     

Histochemical studies of the cestode Diphyllobothrium dendriticum Nitzsch, 1824

Summary Four different types of Diphyllobothrium dendriticum cysts are described.Sections of plerocercoids and sections from the neck region of adult D. dendriticum were investigated for the following non-enzymatic compounds: lipids, carbohydrates, proteins, nucleic acids and calcium.The histochemical tests for enzymes revealed that the enzyme activity generally was low, occurring mostly in the tegument and subtegument with a marked increase of activity in the region of the scolex. The majority of the phosphatase present reacted both at acid and alkaline pH levels and was not inhibited by either KCN or NaF. The adenosine triphosphatase was concentrated mainly in the muscles. The esterase activity was very faint and the acetylcholinesterase occurred mainly in the nerves. Only adult worms gave a reaction for leucine amino-peptidases.The increased enzyme activity in the scolex is related to the close host-parasite interface maintained by the bothria. The low level of activity found generally in the plerocercoids is thought to be a consequence of the low temperatures prevailing in their cold-blooded host.     

Comparative histochemical studies of rats infected with a pathogenic and nonpathogenic trypanosome

Histochemical variations in tissues from rats inoculated with Trypanosoma lewisi and Trypanosoma rhodesiense were investigated. During peak parasitemia, the liver of rats inoculated with T lewisi showed increased glycogen distribution. However, glycogen depletion was noted in the liver and spleen of animals inoculated with living cells of T rhodesiense. Depletion was very apparent from day 4 to day 10. Throughout the period of observation, only a small amount of lipid infiltration occurred in tissues from animals inoculated with both organisms. Protein tests revealed a normal distribution of protein in tissues. Sections of the liver from rats inoculated with T lewisi showed strong alkaline phosphatase activity on days 7, 10, and 13. Alkaline phosphatase activity for T rhodesiense-infected animals was positive for days 4, 7, and 10. Strong positive reactions for acid phosphatase were observed on days 10 and 13 for some tissues (liver, spleen, and kidney) from rats inoculated with T lewisi. On days 4, 7, and 10, intense staining reactions also were observed for livers and spleens of animals inoculated with T rhodesiense. Regardless of tissues observed, histochemical variations were not observed in animals inoculated with the derivatives (ie, metabolic products and homogenates) of T lewisi and T rhodesiense.     

Preliminary characterization of an adult worm “vomit” preparation of <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> and its potential use as antigen for diagnosis

Schistosoma mansoni is a parasitic trematode of the portal–mesenteric veins with a closed-end intestine. Adult worms regurgitate their intestinal content after digestion, together with constituents of the lining gut. Some of these molecules circulate in the blood and are antigenic. We obtain a “vomit” preparation and preliminary evaluate its biochemical composition and antigenic capacity. The “vomit” preparation was obtained after changes in temperature and solutions of incubation of adult worms between 4 and 37°C. Supernatant was assayed for protein, carbohydrate concentration and enzymatic activities associated to the intestine and to the worm tegument. The antigenicity of the product was evaluated using Western blot (WB) analysis against sera of experimentally infected mice, before and after drug cure, sera from people infected with S. mansoni and from individuals infected with other parasitoses. More carbohydrate than protein was detected in the preparations. Cysteine proteinase (CP), N-acetyl-β-d-glucosaminidase and alkaline phosphatase activities were detected. The latter enzyme activity is a marker of the tegument, suggesting that in spite of careful conditions used to avoid the presence of tegumental material, manipulation of the worms always resulted in the release of tegumental molecules. Cationic exchange chromatography was useful to separate various components of this “vomit” preparation, particularly enzymes responsible for CP activity. Two highly immunogenic and specific duplets were observed in the WB analysis, 31/32- and 38/40-kDa components, the former probably referring to the intestinal CPs Sm31/Sm32. None of the two duplets disappeared after successful chemotherapy during the time of evaluation in mice or humans.     

Comparative histochemical observations on the excretory system of helminth parasites

Summary The excretory canals of Ascaridia galli (Nematoda) and the protonephridial ducts of Cotylophoron cotylophorum (Trematoda) and Raillietina cesticillus (Cestoda) have been studied with regard to the histochemical localization of lipids, carbohydrates and hydrolytic enzymes. Distinct excretory organs are absent in the acanthocephalan Centrorhynchus corvi. Triglycerides, phospholipids and lipoproteins are seen in association with the wall of excretory canals of A. galli and R. cesticillus, and phospholipids and lipoproteins at the corresponding site in C. cotylophorum. The physiological significance of lipids in association with excretion of substances has been discussed. Low molecular weight glycogen is present in the lumen of excretory canal of A. galli but not in other worms.The common feature of the excretory canals is the presence of enzyme activities of nonspecific alkaline phosphatase and Mg²⁺-dependent ATPase. Activity of acid phosphatase is seen only in the excretory canals of A. galli. Glucose-6-phosphatase is present in A. galli and C. cotylophorum and absent in R. cesticillus. Weak reaction of 5-nucleotidase is present in the excretory canals of helminth species studied here. The role of these enzymes in transportation of substances across the wall of excretory canals and also in ionic regulation has been discussed in detail.     

Acid, neutral, and alkaline hydrolases in arthritic synovium.

The levels of six lysosomal enzymes (acid phosphatase, beta-acetylglucosaminidase, cathepsin D, beta-galactosidase, arylsulfatase A, and beta-glucuronidase) and four neutral and alkaline hydrolases (esterase, inorganic phyrophosphatase, alkaline phosphatase, and 5'-nucleotidase) were measured in osteoarthritic, rheumatoid and control synovia. All enzyme levels in diseased synovium except esterase values in osteoarthritis were significantly elevated compared with controls. The mean values of the group of acid hydrolases and the group of neutral and alkaline hydrolases in osteoarthritic synovia were 1.9- and 2.0-fold greater than those of control specimens. In rheumatoid synovia, the values were 4.2- and 4.5 fold greater than control for the same enzymes. Levels in rheumatoid synovia were significantly higher than those in osteoarthritic synovia with the exception of 5'-nucleotidase. Only a limited correlation between the extents of inflammation present in the synovia and the levels of a lysosomal marker enzyme (cathepsin D) was observed. These results demonstrate that whatever the mechanism, increased levels of acid hydrolases as well as certain neutral and alkaline hydrolases are present in osteoarthritic and rheumatoid synovia, and these enzymes are probably contained in the synovial lining cells.     

新生儿脐带血白细胞组织化学的观察

用细胞化学方法观察56例,正常新生儿脐带血白细胞的六种生物大分子。实验结果:ALP、POX、糖元和脂类的阳性率分别为78.18％、100％、100％和89.69％;它们的积分分别为137.43、235.84、203.61和164.02。DNA定位于白细胞胞核内,集聚成块,染色明显。RNA在粒细胞和单核细胞中含量偏低,在淋巴细胞胞质内却很丰富。     

Tegument surface membranes of adult Schistosoma mansoni: development of a method for their isolation

Several approaches to surface membrane stripping have been applied to the adult schistosome. Membrane removal was evaluated by the use of different extrinsic and intrinsic markers of which alkaline phosphatase proved to be the most reliable. After initial studies employing incubation of worms in buffer alone, Triton X-100 or freeze/thaw, the last method was chosen for development. The final method applies a single freeze/thaw step to adult worms in balanced salt solution followed by short bursts of agitation on a vortex mixer to release the tegument. Differential and density gradient steps subsequently yield a final membrane pellet enriched over 130 times in surface alkaline phosphatase. The method has been characterized during its development using electron microscopy and enzyme markers for contaminant worm fractions.     

The nutrition of the gill parasitic monogenean Pseudodactylogyrus anguillae

A histological and histochemical study of ingested food material, energy stores and enzymes in the monogenean Pseudodactylogyrus anguillae, parasitizing the gills of the European eel (Anguilla anguilla) is presented. It was found that mucus, epithelial cells and blood from the gills were ingested. Glycogen deposits were small and primarily located in the parenchyma and to a minor extent in the vitellariae. Numerous globules of neutral lipids were found in the vitellariae. A marked esterase activity was found in the gut and a less marked activity in the vitellariae. Acid phosphatase activity was found throughout the body whereas alkaline phosphatase and leucine-aminopeptidase were not detected. Marked activity of succinate dehydrogenase and NADH-diaphorase was found in all cells, indicating a predominantly aerobic metabolism in this monogenean.     

Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.     

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.     

Development,hatching, and infectivity of Echinostoma caproni (Trematoda) eggs,and histologic and histochemical observations on the miracidia

 Light microscopy studies were done on the eggs and miracidia of the intestinal trematode Echinostoma caproni. Eggs were obtained by homogenizing adult worms derived from ICR mice in artificial spring water (ASW). To study egg development and to obtain miracidia, eggs were incubated in ASW in either a light or a dark environment for 10 days at 28° C. Egg development was characterized and staged at 2-day intervals from day 0 to day 10 postembryonation. No significant difference in development was seen in eggs maintained under conditions of light or darkness. Eggs maintained in darkness for 10 days and exposed to incandescent light produced a large synchronous hatch of miracidia within 3 h of exposure to light. As expected, miracidia used to expose Biomphalaria glabrata snails produced patent infections at 5–7 weeks postinfection (p.i.). Embryonated eggs aged from 0 to 10 days that had been fed to B. glabrata snails also produced infections within 4 weeks of infection. Miracidial longevity studies showed that 50 newly hatched miracidia maintained at either 4o, 12o, 22o, or 38° C lived for 6, 28, 14, and 5 h, respectively. Histology studies were done on whole miracidia stained in 0.01% neutral red and in Schneider’s acetocarmine. Histochemistry studies of miracidia stained with alcian blue (pH 2.5) showed the presence of acid mucopolysaccharides in the epidermal plates; miracidia treated with periodic acid-Schiff plus 0.5% malt diastase showed the presence of glycogen in the body and in the epidermal/subepidermal region. Miracidia stained with Lillie’s Oil Red O showed the presence of sparse neutral fat droplets in the body. Received: 15 June 1995 / Accepted: 14 August 1995