Biomedical subjects

M C Healey

Publications and source records attributed to M C Healey.

At least 19 recordsLinked to original sources

PubMed2003

The effect of temperature on swimming performance and oxygen consumption in adult sockeye (Oncorhynchus nerka) and coho (O. kisutch) salmon stocks.

Our knowledge of the swimming capabilities and metabolic rates of adult salmon, and particularly the influence of temperature on them, is extremely limited, and yet this information is critical to understanding the remarkable upstream migrations that these fish can make. To remedy this situation, we examined the effects of temperature on swimming performance and metabolic rates of 107 adult fish taken from three stocks of sockeye salmon Oncorhynchus nerka and one stock of coho salmon O. kisutch at various field and laboratory locations, using large, portable, swim tunnels. The salmon stocks were selected because of differences in their ambient water temperature (ranging from 5 degrees C to 20 degrees C) and the total distance of their in-river migrations (ranging from approximately 100 km for coastal stocks to approximately 1100 km for interior stocks). As anticipated, differences in routine metabolic rate observed among salmon stocks were largely explained by an exponential dependence on ambient water temperature. However, the relationship between water temperature and maximum oxygen consumption (MO2max), i.e. the MO2 measured at the critical swimming speed (Ucrit), revealed temperature optima for MO2max that were stock-specific. These temperature optima were very similar to the average ambient water temperatures for the natal stream of a given stock. Furthermore, at a comparable water temperature, the salmon stocks that experienced a long and energetically costly in-river migration were characterized by a higher MO2max, a higher scope for activity, a higher Ucrit and, in some cases, a higher cost of transport, relative to the coastal salmon stocks that experience a short in-river migration. We conclude that high-caliber respirometry can be performed in a field setting and that stock-specific differences in swimming performance of adult salmon may be important for understanding upstream migration energetics and abilities.

Animal Migration↗

PubMed2003

Excess post-exercise oxygen consumption in adult sockeye (Oncorhynchus nerka) and coho (O. kisutch) salmon following critical speed swimming.

The present study measured the excess post-exercise oxygen cost (EPOC) following tests at critical swimming speed (Ucrit) in three stocks of adult, wild, Pacific salmon (Oncorhynchus sp.) and used EPOC to estimate the time required to return to their routine level of oxygen consumption (recovery time) and the total oxygen cost of swimming to Ucrit. Following exhaustion at Ucrit, recovery time was 42-78 min, depending upon the fish stock. The recovery times are several-fold shorter than previously reported for juvenile, hatchery-raised salmonids. EPOC varied fivefold among the fish stocks, being greatest for Gates Creek sockeye salmon (O. nerka), which was the salmon stock that had the longest in-river migration, experienced the warmest temperature and achieved the highest maximum oxygen consumption compared with the other salmon stocks that were studied. EPOC was related to Ucrit, which in turn was directly influenced by ambient test temperature. The non-aerobic cost of swimming to Ucrit was estimated to add an additional 21.4-50.5% to the oxygen consumption measured at Ucrit. While these non-aerobic contributions to swimming did not affect the minimum cost of transport, they were up to three times higher than the value used previously for an energetic model of salmon migration in the Fraser River, BC, Canada. As such, the underestimate of non-aerobic swimming costs may require a reevaluation of the importance of how in-river barriers like rapids and bypass facilities at dams, and year-to-year changes in river flows and temperatures, affect energy use and hence migration success.

Animal Migration↗

PubMed2001

Infectivity of Cryptosporidium parvum oocysts following cryopreservation.

This study was undertaken to investigate the cryopreservation of Cryptosporidium parvum oocysts. Oocysts purified from mouse feces were suspended in distilled water, 10% glycerin, and 2.5% potassium dichromate. They were stored at -20 C and -80 C for 2, 7, and 30 days, respectively. In addition to the purified oocysts, the feces of C. parvum-infected mice were preserved under the same conditions described above. Purified and fecal oocysts were thawed at 4 C, and their viability was assessed by a nucleic acid stain, excystation test, tissue culture infectivity test, and infectivity to immunosuppressed adult mice. Oocysts purified from fecal material prior to cryopreservation lost most of their viability and all of their infectivity for tissue culture and mice. However, when oocysts were cryopreserved in feces, between 11.7 and 34.0% were judged to be viable and retained their infectivity for mice when stored at -20 C (but not -80 C) for 2, 7, and 30 days. Clearly, fecal material provides a cryoprotective environment for C. parvum oocysts stored at -20 C for at least 30 days.

Animals↗

PubMed2000

Infection of immunosuppressed C57BL/6N adult mice with a single oocyst of Cryptosporidium parvum.

The present study was designed to determine the minimum number of Cryptosporidium parvum oocysts capable of producing patent infections in immunosuppressed C57BL/6N adult mice. Sixty-four female mice were divided into 6 groups of 8 mice each, except group 1 that contained 24 mice. Mice in groups 1-3 were immunosuppressed with dexamethasone and inoculated with 1, 5, and 10 oocysts per mouse, respectively. The accuracy of the inoculum size was microscopically confirmed. Mice in groups 4-6 served as controls: they received either only oocyst inoculation (group 4), or immunosuppression (group 5), or no treatments (group 6). Fecal oocyst shedding was monitored daily for each mouse using an indirect immunofluorescent assay. Parasite colonization in the terminal ileum of each mouse was evaluated histologically. Four of 24 mice in group 1 developed patent infections, with a prepatent period of approximately 6 days. All mice in groups 2 and 3 developed patent infections, with prepatent periods ranging from 4 to 7 days. Mice in groups 4-6 remained uninfected. Parasite colonization was observed in the terminal ilea of all mice in groups 1-3 that shed fecal oocysts. The present study experimentally demonstrates that a single viable oocyst can induce patent C. parvum infections in immunosuppressed C57BL/6N adult mice and indicates that this mouse model could be used for the parasite genotype or isolate cloning.

Animals↗

PubMed1999

A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection.

Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05) C. parvum infection when BFTE cells were pretreated for 60 min at 37 degreesC prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.

Animals↗

PubMed1998

Actin-dependent motility in Cryptosporidium parvum sporozoites.

The present study investigated the role of actin polymerization and myosin motor protein activity in the gliding motility of Cryptosporidium parvum sporozoites. Short motility trails were detected using an indirect immunofluorescent assay (IFA) with a polyclonal antisporozoite antibody following incubation of sporozoites on poly-L-lysine-coated glass slides. Sporozoite motility was blocked following exposure to cytochalasin D, a myosin light-chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapin e, and the myosin ATPase inhibitor 2,3-butanedione monoxime. Sporozoites were observed to form rounded, blunt-ended shapes when exposed to these same inhibitors. Incubation of purified oocysts with these compounds did not significantly inhibit in vitro excystation or subsequent infectivity in cultured epithelial cells. Indirect IFA revealed a uniform distribution of actin protein throughout the body of the sporozoite; immunoelectron microscopy confirmed a diffuse intracellular pattern of gold particles in excysted sporozoites. Collectively, these findings show that sporozoite motility is dependent upon an intact actin-myosin motor system, and the dynamic interaction of F-actin and myosin motor proteins has a further role in maintaining the structural integrity of excysted sporozoites. Further, in vitro excystation and infectivity of C. parvum occurs in the absence of dynamic sporozoite locomotion.

Actins↗

PubMed1997

Synergistic anticryptosporidial potential of the combination alpha-1-antitrypsin and paromomycin.

The combined effect of the serine protease inhibitor alpha-1-antitrypsin (AAT) and the aminoglycoside paromomycin on Cryptosporidium parvum infection in vitro was investigated. AAT and paromomycin were mixed with C. parvum oocysts as either single or combined treatments and used to inoculate epithelial cell cultures. Single- and combined-treatment groups had significantly lower (P < 0.01) parasite numbers than untreated controls. The mean fractional inhibitory concentration indices suggested significant synergistic activity.

Animals↗

PubMed1997

Antagonistic effect of human alpha-1-antitrypsin on excystation of Cryptosporidium parvum oocysts.

This study evaluated the effects of the human serine protease inhibitor alpha-1-antitrypsin (AAT) on in vitro excystation and infectivity of Cryptosporidium parvum. Excystation was monitored at 37 C in RPMI medium in the presence of 0, 100, 500, or 1,000 micrograms/ml AAT. AAT significantly inhibited (P < 0.05) excystation of bleach-decontaminated oocysts in a concentration-dependent manner at incubation intervals from 15 to 90 min but did not alter the excystation dynamics of unbleached oocysts. Bleach-treated oocysts, suspended in RPMI containing 0, 1, 10, 100, 500, or 1,000 micrograms/ml AAT, were used to inoculate bovine fallopian tube epithelial (BFTE) cell monolayers. Alternately, sporozoites, excysted at 37 degrees C and collected by filtration, were used to inoculate BFTE cells under the same conditions. The mean number of parasites counted in AAT-treated, oocyst-inoculated cells was significantly less (P < 0.01) than control mean values at 24 and 48 hr post-inoculation (PI); longer PI intervals (72-96 hr) exhibited a decreased inhibitory effect. AAT did not inhibit parasite infection when cultures were inoculated with C. parvum sporozoites. The findings of this study show that the anticryptosporidial potential of AAT is primarily associated with an antagonistic effect on oocyst excystation.

Animals↗

PubMed1996

Infectivity of preserved Cryptosporidium parvum oocysts for immunosuppressed adult mice.

The present study was undertaken to determine the infectivity of Cryptosporidium parvum oocysts for immunosuppressed adult C57BL/6N mice after the oocysts had been stored from 1-48 months at 4 degrees C in 2.5% potassium dichromate. All mice inoculated with oocysts 1-18 months old developed patent infections, while mice inoculated with older oocysts remained uninfected. The prepatent period was extended from 2 to 6 or 7 days as the storage time for oocysts increased. The finding that C. parvum oocysts remain infective for mice for at least 18 months offers important economic and time-saving advantages for investigators who frequently require large numbers of oocysts that must be painstakingly purified from calf manure.

Animals↗

PubMed1996

Complete development of Cryptosporidium parvum in bovine fallopian tube epithelial cells.

Cryptosporidium parvum is a coccidian parasite responsible for causing protracted and life-threatening diarrheal illness in immunocompromised humans, especially patients with AIDS. The lack of medications effective in treating people suffering from cryptosporidiosis has prompted the development of in vivo and in vitro models for this disease. This study is the first to demonstrate that C. parvum can complete its entire life cycle (from sporozoite to infective oocyst) in a primary culture of bovine fallopian tube epithelial (BFTE) cells. Scanning and transmission electron photomicrographs were used to detail the ultrastructure of individual parasitic stages. Successful infections were produced by inoculating cell cultures with either oocysts or purified sporozoites. Infection of BFTE cells with C. parvum close paralleled in vivo infections with regard to host cell location and chronology of parasite development. Infecting BFTE cells with sporulated oocysts provided a reproducible and quantitative cultivation system with significantly (P < or = 0.001) higher infection rates than in Madin-Darby canine kidney cells. Oocysts produced in BFTE cells were infective for immunosuppressed adult C57BL/6N mice. Cultivation of C. parvum in BFTE cells will facilitate the study of interactions between parasites and host cells as well as provide a reliable system for evaluating anticryptosporidial compound efficacy.

Animals↗

PubMed1996

Primary and secondary infections with Cryptosporidium parvum in immunosuppressed adult mice.

The present study was undertaken to determine if infection of immunocompetent adult C57BL/6N mice with Cryptosporidium parvum would render them more resistant to a challenge infection following immunosuppression with dexamethasone (DEX). Fecal oocyst shedding and parasite colonization of the terminal ilea were greater in immunosuppressed mice than in nonimmunosuppressed mice. Secondary infections with C. parvum resulted in decreased oocyst shedding and reduced parasite colonization compared with primary infections. Flow cytometry revealed fewer splenic B cells but more splenic total T, CD4+ T, CD8+ T cells and macrophages in immunosuppressed mice than in nonimmunosuppressed mice. The CD4+ to CD8+ T cell ratios and blastogenic responses to lipopolysaccharide (LPS), but not to concanavalin A, were decreased in immunosuppressed mice compared with nonimmunosuppressed mice. Blastogenic responses to LPS and percentages of splenic total B cells and macrophages were increased in secondary infections compared to primary infections. Enhanced susceptibility to C. parvum infection in immunosuppressed mice revealed DEX-mediated effects on both cell-mediated and humoral immunity. Our results suggest that increased resistance in immunosuppressed mice to secondary infections with C. parvum may involve increases in B cells and macrophages.

Animals↗

PubMed1996

Interaction of the human serine protease inhibitor alpha-1-antitrypsin with Cryptosporidium parvum.

The protozoan parasite Cryptosporidium parvum was studied for interaction with a human serine protease inhibitor (serpin), alpha-1-antitrypsin (AAT). A C. parvum homogenate (CPH) prepared from oocysts was incubated with purified human AAT and complexes formed between the serpin and CPH were detected using an enzyme-linked immunosorbent assay (ELISA). The optical density read at 450 nm of AAT:CPH reactivity was significantly increased (P < 0.001) relative to CPH in the absence of AAT treatment. Additionally, ELISA reactivity was blocked by incubating AAT with a cognate target enzyme, porcine pancreatic elastase (PPE), prior to treatment of the CPH. Incubation of a partially excysted sample of C. parvum with AAT (37 C x 60 min) demonstrated preferential fluorescence labeling of sporozoites by indirect immunofluorescence assay; AAT complexes were not detected on intact oocysts. Localization of AAT interactions with C. parvum sporozoites was visualized by transmission immunoelectron microscopy. Collectively, these data suggest that C. parvum sporozoites express a protease-like component that is recognized by human AAT. The ability to block ELISA reactivity with PPE suggests that the AAT interactions we detected are functionally similar to the serpin-enzyme complex AAT forms with a protease target.

Animals↗

PubMed1996

Efficacy of serine protease inhibitors against Cryptosporidium parvum infection in a bovine fallopian tube epithelial cell culture system.

The anticryptosporidial potential of the protease inhibitors alpha-1-antitrypsin (AAT), antipain, aprotinin, leupeptin, methoxysuccinyl-ala-ala-pro-valine chloromethylketone (MAAPVCK), soybean trypsin inhibitor (SBTI), and phenylmethylsulfonyl fluoride (PMSF) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system. Protease inhibitor concentrations of 5, 10, 50, 100, and 500 micrograms/ ml (PMSF at 1, 2, and 3 mM) in RPMI medium were mixed with Cryptosporidium parvum oocysts and used to inoculate BFTE cell monolayers. At 24 hr postinoculation (candlejar/37 C), cells were rinsed with RPMI medium, fixed in methanol, and stained with Giemsa. Parasites were enumerated in cell monolayers by brightfield microscopy. The mean number of parasites counted in each protease inhibitor treatment group was expressed as a percentage of the mean number of parasites counted in an infection control group. Leupeptin and SBTI reduced parasite numbers to 40-50% of the control mean at 500 micrograms/ml: AAT, antipain, and aprotinin reduced parasite numbers to 10-15% at the same concentration. PMSF reduced parasite numbers to 40% of the control mean at 3 mM. MAAPVCK did not significantly inhibit cryptosporidial infection. These findings suggest that a protease component of C. parvum may be essential for host cell infection.

Amino Acid Chloromethyl Ketones↗

PubMed1996

Protease activity associated with excystation of Cryptosporidium parvum oocysts.

A Cryptosporidium parvum homogenate (CPH), prepared from partially excysted oocysts, was examined for proteolytic activity capable of hydrolyzing azocasein. Protease activity, measured at pH 7.0, was not detected in fresh oocysts but increased progressively with incubation at 37 C. Activity peaked after 60 min incubation but progressively decreased with extended incubation intervals. Cryptosporidial protease activity was significantly inhibited (P < 0.01) by the serine protease inhibitors phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DIFP), aprotinin, alpha-1-antitrypsin (AAT), and the cysteine protease inhibitor L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64). No single inhibitor completely blocked CPH-associated protease activity; however, the combination of PMSF and E-64 inhibited > 95% of the azocasein hydrolysis measured in untreated control samples. The same group of inhibitors was further evaluated for their ability to inhibit excystation of C. parvum oocysts. PMSF, DIFP, aprotinin, and AAT significantly inhibited (P < 0.05) oocyst excystation at 15-, 30-, and 60-min incubation intervals; E-64 had no significant inhibitory effect on excystation. The results of this study demonstrate proteolytic activity during peak periods of excystation. Further, cryptosporidial protease activity was sensitive to both serine and cysteine protease inhibitors, but only serine protease inhibitors significantly inhibited oocyst excystation. These findings provide preliminary evidence of cryptosporidial proteases of both serine and cysteine protease classes and suggest that serine protease(s) are functionally associated with excystation.

Animals↗

PubMed1995

Effects of dehydroepiandrosterone in immunosuppressed adult mice infected with Cryptosporidium parvum.

Cryptosporidiosis is a diarrheal disease in humans and other animals caused by the coccidian parasite, Cryptosporidium parvum. This study was undertaken to determine the effectiveness of dehydroepiandrosterone (DHEA) in reducing C. parvum infections in immunosuppressed adult C57BL/6N mice and to identify the immunomodulatory effects of DHEA that result in increased resistance to cryptosporidiosis. Dexamethasone-immunosuppressed mice were readily infected with C. parvum following orogastric intubation with 10(6) oocysts/mouse. DHEA treatment of these mice significantly reduced (P < 0.01) both fecal oocyst shedding and parasite colonization of the ilea. Immunosuppressed mice treated with DHEA had more splenic total T cells, CD4+ T cells, and CD8+ T cells than immunosuppressed mice that were not treated, but the differences were not always significant. Moreover, nonimmunosuppressed mice treated with DHEA had significantly more (P < 0.05) splenic total T cells, CD4+ T cells, and total B cells than nonimmunosuppressed mice that did not receive DHEA. Of particular interest was the significantly larger (P < 0.05) number of CD8+ T cells in immunosuppressed, C. parvum-infected, DHEA-treated mice compared with the same mice that were not treated. Up-regulation of the immune system by exogenous DHEA may be useful in the treatment and palliation of cryptosporidiosis.

Animals↗

PubMed1995

Therapeutic efficacy of paromomycin in immunosuppressed adult mice infected with Cryptosporidium parvum.

The intent of this study was to evaluate the therapeutic efficacy of paromomycin in immunosuppressed adult C57BL/6N mice infected with Cryptosporidium parvum. Seven groups of 10 mice/group were used. Groups 1, 2, and 7 served as normal, toxicity, and placebo controls, respectively. Groups 2-7 were immunosuppressed with dexamethasone phosphate administered ad libitum in drinking water. Groups 3-7 were infected with C. parvum on day 7 postimmunosuppression. Groups 3-6 were treated by administering paromomycin per os for 10 consecutive days, beginning on day 10 postinfection, at dosage levels of 0.25, 0.5, 1, and 2 g/kg/day, respectively. Paromomycin was judged to be nontoxic at the dosage levels used. Groups 1 and 2 remained uninfected while groups 3-7 began shedding oocysts by day 3 postinfection. Paromomycin was therapeutically effective against C. parvum at 1 and 2 g/kg/day as determined by significant reductions in fecal oocyst shedding (P < 0.01), parasite colonization (P < 0.05), and villus atrophy (P < 0.05) in the ilea and terminal ilea of infected mice. We conclude that paromomycin may be useful in the treatment and palliation of cryptosporidiosis.

Animals↗

PubMed1994

Patent gut infections in immunosuppressed adult C57BL/6N mice following intraperitoneal injection of Cryptosporidium parvum oocysts.

Two experiments were conducted to determine if intraperitoneal (i.p.) and/or subcutaneous (s.c.) injection of Cryptosporidium parvum oocysts could produce patent gut infections in dexamethasone (DEX) or dexamethasone phosphate (DEXp)-immunosuppressed adult female C57BL/6N mice. The results from experiment 1 were suggestive but did not unequivocally demonstrate that i.p. injection of oocysts could produce patent gut infections in these mice. In experiment 2, all mice were individually caged, immunosuppressed with DEXp (groups 1 and 2) or DEX (groups 3 and 4), and administered C. parvum oocysts either by orogastric intubation (groups 1 and 3), i.p. (group 2), or s.c. (group 4). All but 1 mouse in groups 1 and 3 began shedding oocysts in their feces on day 3 post-infection (PI). Mice in these 2 groups continued to shed oocysts until they were killed on day 17 PI. Mice administered oocysts s.c. did not shed oocysts. In group 2, 1 mouse died, 1 failed to shed oocysts, and 1 began shedding oocysts on day 3 PI. The remaining 5 mice started shedding oocysts on either days 8, 10, or 14 PI and continued to shed oocysts until they were killed. We conclude that immunosuppressed adult C57BL/6N mice administered oocysts i.p. can develop patent gut infections with C. parvum.

Animals↗

PubMed1993

Complete development of Cryptosporidium parvum in a human endometrial carcinoma cell line.

Sporozoites of Cryptosporidium parvum, excysted from oocysts isolated from calves, were applied to monolayers of the human endometrial carcinoma cell line RL95-2. Cells were grown as monolayers in 24-well plates at concentrations ranging from 5 x 10(4) to 1 x 10(5) RL95-2 cells per well. At 1 or 7 days postculturing, C. parvum sporozoites (ranging from 1 x 10(5) to 2 x 10(5) were added to the monolayers of RL95-2 cells. The cells were fixed and stained to estimate the extent of parasite colonization. Light microscopy and electron microscopy confirmed the development and replication of C. parvum within the RL95-2 cells. A standardized and reproducible in vitro culture system for C. parvum is necessary to evaluate therapies against cryptosporidiosis.

Animals↗