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Biomedical subjects

A Reddy

Publications and source records attributed to A Reddy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Maintenance of Helisoma trivolvis naturally infected with Echinostoma trivolvis in spring water at 4 degrees C for 300 days.

Helisoma trivolvis (Pennsylvania strain) snails naturally infected with Echinostoma trivolvis larvae were maintained for 10 months (300 days) at 4 degrees C in artificial spring water to determine the effects of storage on parasite survival. Three of 20 snails were alive at 10 months and when isolated in spring water at 22-24 degrees C released active cercariae. The mean number of cercariae released in 2 h from each stored snail was significantly less than that from freshly collected snails. Infectivity of cercariae from stored hosts to experimentally infected, laboratory-raised Helisoma trivolvis (Colorado strain) snails was significantly less than that of cercariae from fresh hosts based on cyst recoveries in the Colorado strain of H. trivolvis within 24 h p.i. There was no significant difference in the redial number or the number of cercariae per redia in stored versus fresh snails. In conclusion, some reduction in cercarial emergence and decrease in cercarial infectivity to a second intermediate snail host occurred following storage of H. trivolvis naturally infected with E. trivolvis in ASW at 4 degrees C for 10 months.

Animals↗
PubMed1998

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity.

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.

Amino Acid Sequence↗
PubMed1997

A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells.

Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell-depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-alpha, IL-1beta, IL-6, and interferon-alpha. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction-stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.

Cell Differentiation↗
PubMed1997

Dendritic cells as immunogens for human CTL responses.

The cellular requirements for generating potent human CD8+ CTLs to influenza A virus in vitro have been defined. Furthermore, we have developed improved methods for generating large numbers of DCs from non-proliferating progenitors. These developments have enabled the design of new strategies to elicit CTLs in vivo. For example, together with IL-12, antigen-pulsed DCs may be a useful approach for boosting CTL responses against infectious agents and malignancies. Our results also reopen the potential use of inactivated virus preparations as immunogens for CTL responses.

Blood Cells↗
PubMed1997

Maintenance of the life cycle of Echinostoma trivolvis (Trematoda) in dexamethasone-treated ICR mice and laboratory-raised Helisoma trivolvis (Gastropoda).

Echinostoma trivolvis is a ubiquitous 37-collar-spined echinostome found in aquatic birds and mammals and in the planorbid snail Helisoma trivolvis. This echinostome has not been cycled continuously in the laboratory. The present report provides details on the continuous life cycle of E. trivolvis in dexamethasone-treated ICR mice and laboratory-raised H. trivolvis snails. Previous attempts to obtain patent adult of E. trivolvis in mice hosts failed because of worm injection within 2 weeks of infection. ICR mice infected with encysted metacercariae and injected with 2 mg/kg dexamethasone daily for 28 days yielded gravid worms that produced 250-500 eggs/worms at 21 and 28 days postinfection (p.i.). Miracidia derived from these eggs or eggs containing fully developed miracidia were capable of infecting 3- to 5-mm shell-diameter, laboratory-reared H. trivolvis snails. These snails released cercariae by 35 days p.i. Cercariae encysted in the kidney-pericardium of the snails. Encysted metacercariae could be excysted in vitro in an alkaline trypsin-bile salts medium or in vivo when fed to domestic chicks.

Animals↗
PubMed1997

Chemoattraction and penetration of Echinostoma trivolvis and E. caproni cercariae in the presence of Biomphalaria glabrata, Helisoma trivolvis, and Lymnaea elodes dialysate.

A petri-dish bioassay was used to study the chemoattraction and penetration of the cercariae of Echinostoma trivolvis and E. caproni in the presence of snail dialysates from Helisoma trivolvis (Pennsylvania and Colorado strains). Biomphalaria glabrata, and Lynmaea elodes. Significant chemoattraction was seen with E. trivolvis cercariae in the presence of all snail dialysates released from nonperforated dialysis sacs with a molecular-weight exclusion of 12,000. Under the same conditions, E. caproni was significantly attracted to B. glabrata and H. trivolvis (CO strain) but not to L. elodes or H. trivolvis (PA strain). Dialysis sacs were perforated with needles to allow the release of snail substances of all molecular weights into the bioassay. Cercariae of both species were significantly attracted to all snail dialysates released from perforated sacs. Moreover, cercariae entered these sacs and penetrated the snails, and 24 h later the percentage of cysts per snail species ranged from 70% to 83% for E. trivolvis and from 73% to 93% for E. caproni. Dialysates released from intact sacs were extracted in choloroform-methanol (2:1) to obtain hydrophilic and lipophilic fractions. When these extracts were placed on agar plugs in the bioassay, the lipophilic fraction, but not the hydrophilic fraction, was mainly chemoattractive.

Animals↗
PubMed1997

Effects of diet on the lipid composition of Echinostoma caproni (Trematoda) in ICR mice.

High-performance thin-layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in the intestinal trematode Echinostoma caproni from experimentally infected ICR mice fed a high-fat diet (hen's egg yolk) as compared with worms from mice fed a standard laboratory diet. Worms were removed from the hosts at 2, 3, and 4 weeks postinfection (p.i.). Analysis by TLC-densitometry showed significantly greater amounts of triacylglycerols and free sterols at 2, 3, and 4 weeks p.i. in worms from mice on the high-fat diet as compared with worms from mice on the standard laboratory diet. Significantly greater amounts of phosphatidylcholine and phosphatidylethanolamine were found in worms from mice on the high-fat diet as compared with worms from those on the standard diet at 2 weeks p.i. but not at 3 and 4 weeks p.i. The results of this study suggest that the host diet influences the lipid content of E. caproni adults.

Animals↗
PubMed1997

Experimental infection of Rana pipiens tadpoles with Echinostoma trivolvis cercariae.

Studies were done on laboratory-raised Rana pipiens tadpoles experimentally infected with Echinostoma trivolvis cercariae. Tadpoles exposed individually to 250 cercariae died within 24 h. They were edematous at death and their kidneys were heavily infected with metacercarial cysts. Of 20 tadpoles exposed to 100 cercariae each, 9 survived the infection, and their growth was compared for 4 weeks postinfection (p.i.) with that of 20 control tadpoles that had not been exposed to cercariae. There was a significant weekly decline in the total length and body weight of the infected versus control tadpoles. Surviving tadpoles retained their metacercarial infections in the kidneys following metamorphosis to frogs. Following exposure of tadpoles to cercariae, cercarial bodies were first seen in the kidneys by 0.5 h p.i. Metacercariae that were molding their inner and outer cyst walls were first seen at 2.3 h, and by 8.5 h the inner and outer cyst walls were clearly defined. Domestic chicks exposed to cysts aged 2.5 and 4.0 h did not become infected, whereas ovigerous adults of E. trivolvis were recovered from chicks fed 12-h-old cysts. Cercariae aged 6 to 8 h were more infective to tadpoles than were either 1- or 20-h-old cercariae. The E. trivolvis-R pipiens tadpole model is suitable for the study of host-parasite relationships of echinostome larvae in a cold-blooded vertebrate host.

Animals↗
PubMed1997

Infectivity and growth of Echinostoma revolutum (Froelich, 1802) in the domestic chick.

Infectivity and growth studies in domestic chicks were carried out on a strain of Echinostoma revolutum isolated from Lymnaea elodes snails in Indiana, U.S.A. Of 21 chicks, each fed 40 +/- 10 cysts of Echinostoma revolutum, 16 (64%) were infected with a total of 269 (32%) worms from approximately 840 cysts. Worms were found only in the ceca and rectum at 2-14 days p.i. In vivo excysted metacercariae were obtained in the lower ileum and ceca at 4 h p.i. Excysted metacercariae averaged 0.2 mm in length and 0.02 mm2 in body area. Worm length averaged 1.3 mm on day 6, 2.3 mm on day 8 and 3.6 mm on day 14. Mean body area averaged 0.29 mm2 on day 6, 0.62 mm2 on day 8 and 1.93 mm2 on day 14. Worms first became ovigerous on day 12. Growth of E. revolutum in the chick was delayed compared to previous findings on E. trivolvis, a closely related species of 37-collar-spined echinostome in the E. revolutum complex.

Animals↗
PubMed1997

Low molecular weight hydrophilic chemicals that attract Echinostoma trivolvis and E. caproni cercariae.

A Petri dish bioassay was used to study chemoattraction of the cercariae of Echinostoma trivolvis and E. caproni in the presence of various bench chemicals prepared as 5 mM solutions in agar. The design compared the attraction of cercariae migrating toward the plugs impregnated with chemicals compared to unimpregnated plugs (controls). Cercariae of both species were significantly attracted to an array of amino acids and to some carbohydrates. Cercariae of E. trivolvis but not E. caproni were attracted to the peptide glutathione. Cercariae of E. trivolvis and E. caproni were repelled from valeric and butyric acids, respectively. Cercariae of E. caproni but not E. trivolvis were repelled from dextrose. Although broad specificity exists for echinostome cercarial penetration in snail intermediate hosts, results of these studies suggest that there are specific differences in cercarial chemoattractants between E. trivolvis and E. caproni.

Amino Acids↗
PubMed1997

Comparative studies on excystation and morphological features of the metacercariae of Echinostoma paraensei and E. caproni.

Studies were done to determine specific differences between the metacercariae of Echinostoma paraensei and E. caproni. The percentage of excystation of E. caproni in an alkaline trypsin-bile salts medium was about 6x greater than that of E. paraensei. The body and acetabular length of excysted metacercariae of E. caproni were significantly greater (Student's t-test, P < 0.05) than those of E. paraensei. The length and width of encysted metacercariae of E. caproni were significantly greater than those of E. paraensei. The number of excretory concretions in both encysted and excysted metacercariae of E. caproni was significantly greater than that of E. paraensei. In conclusion, in vitro excystation data and morphometric light microscopic studies were useful in determining specific differences in the metacercariae of these 2 closely related allopatric species of 37-collar-spined Echinostoma.

Animals↗
PubMed1996

IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro.

CD8+ cytolytic T lymphocytes (CTLs) are important mediators for resistance to infections and malignant diseases. IL-12 enhances proliferative and cytolytic responses by killer cells, but its function in the generation of human antiviral CD8+ T cell responses has not been defined. We therefore evaluated the role of IL-12 in the generation of CTLs to influenza-infected dendritic cells. IL-12 was not detectable in supernatants of infected-dendritic cells, or during CTL generation. Furthermore, anti-IL-12 antibody did not block CTL generation. However, exogenous IL-12 (30-300 pg/ml) enhanced CD8+ T cell proliferative and cytolytic responses. The effect was greatest in individuals with weak reactivity to influenza virus or at antigen-presenting cell (APC):T cell ratios of 1:100 or less. IL-12 augmented interferon-gamma production during CTL generation. The CTL enhancing effects of the cytokine, however, could not be blocked by neutralizing anti-interferon-gamma antibody. Together with IL-12, antigen-pulsed dendritic cells may be a useful approach for boosting CTL responses against infectious agents and malignancies.

Dendritic Cells↗
PubMed1996

Studies on identifying the catalytic role of Glu-204 in the active site of yeast invertase.

In a previous study on yeast invertase (Reddy, A., and Maley, F. (1990) J. Biol. Chem. 265, 10817-10820), we identified Asp-23 through the procedures of affinity labeling and site-directed mutagenesis as a catalytic nucleophile. In the present study we undertook to determine other residues involved in the catalytic process. Earlier studies suggested histidine as a potential proton donor in the hydrolysis of sucrose, but by mutagenizing each of the enzyme's four histidines this amino acid was eliminated from consideration. Another candidate appeared to be cysteine, since iodine at about a 2-fold molar excess inactivated invertase by modifying both of the enzyme's cysteine residues. Dithiothreitol treatment restored the sulfhydryl groups and enzyme activity. Replacement of each of the cysteines with alanines revealed that C108A invertase retained full activity whereas C205A was reduced about 4-fold in its kcat. A comparison of the amino acid sequences of fructosylhydrolases revealed a conserved region coincident with Glu-204/Cys-205. Mutagenizing Glu-204 to Ala resulted in a 3, 000-fold reduction in the kcat of invertase indicating that Glu-204 plays a major role in catalysis. Based on these findings, a mechanism is proposed for the hydrolysis of sucrose which involves Asp-23 as a nucleophile and Glu-204 as an acid/base catalyst.

Amino Acid Sequence↗
PubMed1996

Egg laying in vitro of Echinostoma caproni (Trematoda) in nutritive and nonnutritive media.

Egg laying in vitro was studied in Echinostoma caproni adults placed in 10 ml of nutritive or nonnutritive media for 48 h in petri-dish cultures maintained at 37 degrees C in an atmosphere containing 7.6% CO2. Maximal egg laying occurred within 24 h in the defined medium RPMI 1640. Egg laying was significantly greater in this medium than in McCoy's or Locke's solution. Eggs released into the RPMI medium were capable of producing miracidia that were infective to Biomphalaria glabrata snails. Fried and Huffman (1996) referred to a technique used to obtain eggs of Echinostoma caproni in the defined medium RPMI 1640, but details of the study were not given. No information is available on egg laying of echinostomes in vitro. Such information could contribute to a better understanding of egg release in digeneans and would also be helpful in the acquisition of eggs for biology and chemistry studies. Current techniques used to obtain echinostome eggs involve worm homogenization, teasing of eggs from the worms' uteri, or recovery of eggs from feces (see Idris and Fried 1996 for details). The purpose of this communication is to report on an efficient procedure for the acquisition of eggs of E. caproni after the placement of adult worms in the defined medium RPMI 1640. E. caproni adults were grown in ICR mice for either 17 (young worms) or 112 days (old worms) as described previously (Ursone and Fried 1995a). Worms were removed from the small intestines and rinsed rapidly in three changes of sterile Locke's solution containing penicillin (200 IU/ml) and streptomycin (200 micrograms/ml; Fried and Contos 1973). Worms were placed in culture media within 30 min of their removal from hosts. Nutritive media consisted of RPMI 1640 and McCoy's medium (Sigma, St. Louis, Mo.). Non-nutritive media consisted of Locke's or Locke's 1:1 (Ursone and Fried 1995b). All media contained antibiotics as described above.

Animals↗
PubMed1996

Effects of high fat diets on worm recovery, growth and distribution of Echinostoma caproni in ICR mice.

The effects of high fat diets (HFDs) on the host-parasite relationship of Echinostoma caproni in ICR mice were studied. Two HFDs were used, one consisting of a customized high fat diet (CHFD) with 45% lipid in the form of cottonseed oil, and the second consisting of an egg yolk diet (EYD) from domestic hens' eggs, with 31% lipid. Controls for both diets consisted of mice fed a standard laboratory diet with 5% lipid. Mice were each fed 25 cysts of E. caproni by stomach tube and necropsied 2, 3 and 4 weeks postinfection (PI). The weight of mice on the CHFD declined significantly compared to mice on the control diet, but there was no significant decline in weight of mice fed EYD compared to the controls. Worm recoveries from mice on both HFDs were significantly less than those from control hosts. There was a significant decline in worm dry weight, body area and uterine egg counts in worms on HFD compared to those on the control diet. Worms from hosts on HFD were located more anteriad in the gut than those recovered from mice on the control diet.

Animals↗
PubMed1996

In vitro studies on intraspecific and interspecific chemical attraction in daughter rediae of Echinostoma trivolvis and E. caproni.

In vitro pairing and aggregation studies on daughter rediae of Echinostoma trivolvis and E. caproni were done at 22 degrees C in a Petri dish bioassay containing an agar substratum and a Locke's solution overlay. Pairing or aggregation was considered positive when rediae were in contact or within 1 mm of each other. Intraspecific and interspecific pairing or aggregation occurred in the bioassay when rediae were initially placed 5 or 10 mm apart. Movement of a single redia in the bioassay to a dialysis sac containing 1-10 rediae showed that intraspecific and interspecific pairing occurred in the absence of redial tactile stimulation. Movements of single rediae in the bioassay to agar plugs impregnated with redial excretory/secretory (ES) products occurred. The lipophilic fraction of the ES products was significantly more attractive than the hydrophilic fraction. The significance of redial chemical communication is not clear.

Animals↗