The nature of the host's T-lymphocyte population within the intestinal villi following Crypto... more The nature of the host's T-lymphocyte population within the intestinal villi following Cryptosporidium parvum infection was characterized with a bovine model of cryptosporidiosis. In naive animals, infection with C. parvum resulted in substantial increases in the numbers of alpha/beta T cells, both CD4+ (150%) and CD8+ (60%), and of gamma/delta T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to alpha/beta T cells. The number of CD4+ T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD4+ T cells within the intestinal villi. In contrast, the number of CD8+ T cells within the intestinal villi increased following a challenge infection. In addition, the CD8+ T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise c...
resulted in substantial increases in the numbers of a/b T cells, both CD41 (150%) and CD81 (60%),... more resulted in substantial increases in the numbers of a/b T cells, both CD41 (150%) and CD81 (60%), and of g/d T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to a/b T cells. The number of CD41 T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD41 T cells within the intestinal villi. In contrast, the number of CD81 T cells within the intestinal villi increased following a challenge infection. In addition, the CD81 T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise correlation between the accumulation of CD81 T cells and the normal site of parasite development suggests an important role for CD81 T cells in the immune animal. Cryptosporidium parvum infection is now a well-recognized cause of diarrhea in immunolo...
The genome of Cryptosporidium parvum contains a relatively small number of introns, which include... more The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the beta-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the beta-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced beta-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12-72 h), at 48-72 h post-infection unprocessed beta-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing beta-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially ...
Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial s... more Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial species, has been implicated as the causative agent of gangrenous dermatitis. A recombinant noncytolytic C. septicum α toxin (NCAT) peptide was developed for use as a vaccine and demonstrated to be safe at concentrations as high as 1 mg/ml. NCAT, used as a purified antigen, partially purified antigen, or in combination with native antigens, was compared to salt-fractionated α toxin combined with denatured C septicum bacteria (native) in a vaccination trial. Three-day-old poults were placed into one of five groups and received two, 0.2-ml vaccinations 5 wk apart. Subcutaneous challenge with 3.2 x 10(7) log phase C. septicum resulted in 78% to 95% of the vaccinated birds surviving challenge compared to 48% of sham-injected controls. By ELISA analysis on NCAT-coated plates, birds receiving vaccines containing the recombinant NCAT peptide showed significantly higher blood serum antibody conce...
T lymphocytes bearing gamma/delta TCRs are a major population of T cells in neonatal calves and d... more T lymphocytes bearing gamma/delta TCRs are a major population of T cells in neonatal calves and discrete subsets of gamma/delta T cells display tissue-specific accumulation and responsiveness to infection. To enhance our understanding of the immunobiology of gamma/delta T cells, we characterized the gene expression profile of circulating bovine gamma/delta T cells following stimulation with recombinant human IL-2 and ConA. Statistical analysis of microarray data identified 108 genes with significantly altered expression, including four genes associated with apoptosis. Real-time reverse transcription-PCR (RT-PCR) analysis of 15 genes related to apoptotic pathways showed that both the Fas-mediated and the mitochondrial apoptotic pathways were repressed in circulating bovine gamma/delta T cells in response to mitogen activation, indicating that stimulated peripheral bovine gamma/delta T cells are resistant to activation-induced apoptosis.
Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced producti... more Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced production of several skin antimicrobial peptides, but more recent data have called those results into question. To compare the mRNA expression of seven antimicrobial peptide genes in lesional and adjacent nonlesional skin biopsy specimens from dogs with AD with those from normal dogs and from dogs experiencing other inflammatory skin conditions. Normal dogs and patients with AD or other inflammatory skin conditions were enrolled with owner permission and approval of the Institutional Animal Care and Use Committee. Transcripts were measured by quantitative RT-PCR using a standard curve assessment. Normal transcript levels for all seven antimicrobial peptides varied depending on the body site assessed. Transcripts for secretory leukocyte proteinase inhibitor (SLPI) and skin-derived antileucoproteinase (SKALP; also known as elafin) were typically ~10-fold greater in number than transcripts for the canine β-defensins (CBD)-1, -102, -103, -122 and -124. Transcripts for SKALP, SLPI, CBD-1, CBD-103 and CBD-122 were lower in both lesional and adjacent nonlesional skin from dogs with AD in comparison to normal skin. Transcripts were reduced to a similar extent versus normal dogs in skin of dogs with inflammatory skin conditions from both lesional and nonlesional biopsies, except for CBD-122, which was reduced only in lesional skin. Compared with normal dog skin, transcripts for CBD-102 and CBD-124 were unaffected in dogs with AD. Both SKALP and SLPI may be important contributors to skin innate immunity, but their decreased expression in AD patients does not account for increased skin infections compared with other skin conditions.
Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extrac... more Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.
The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and... more The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.
The purpose of the present study was to determine if reverse transcriptase-polymerase chain react... more The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected RNA from as few as 10(3) C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts stored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG RT-PCR may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro.
Cryptosporidium parvum is an obligate intracellular protozoan capable of causing life-threatening... more Cryptosporidium parvum is an obligate intracellular protozoan capable of causing life-threatening diarrhoeal disease in immunocompromised individuals. Efforts to develop novel therapeutic strategies have been hampered by the lack of understanding of the pathogenesis of infection. To better understand the host response to C. parvum infection, gene expression profiles of infected human ileocecal adenocarcinoma cells were analysed by using Affymetrix oligonucleotide microarrays containing probe sets for 12,600 human genes. Statistical analysis of expression data from three independent experiments identified 223 genes whose expression was reproducibly regulated by C. parvum infection at 24 h post-inoculation (125 up-regulated and 98 down-regulated), 13 of which were validated by quantitative reverse transcriptase polymerase chain reaction analysis. This analysis revealed the consistent up-regulation of host heat-shock genes and genes for pro-inflammatory chemokines IL-8, RANTES, and SCYB5. Multiple genes for host actin and tubulin genes were up-regulated whereas genes for actin binding proteins were down-regulated, confirming previous observations of host cytoskeleton rearrangement in response to C. parvum infection. In addition, host genes associated with cell proliferation and apoptosis were differentially regulated, reflecting the complexity of host-parasite interaction. Together, this study demonstrated that C. parvum infection results in significant changes in host biochemical pathways and provides new insights into specific biological processes of infectious disease caused by an intracellular protozoan parasite.
The nature of the host's T-lymphocyte population within the intestinal villi following Crypto... more The nature of the host's T-lymphocyte population within the intestinal villi following Cryptosporidium parvum infection was characterized with a bovine model of cryptosporidiosis. In naive animals, infection with C. parvum resulted in substantial increases in the numbers of alpha/beta T cells, both CD4+ (150%) and CD8+ (60%), and of gamma/delta T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to alpha/beta T cells. The number of CD4+ T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD4+ T cells within the intestinal villi. In contrast, the number of CD8+ T cells within the intestinal villi increased following a challenge infection. In addition, the CD8+ T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise c...
resulted in substantial increases in the numbers of a/b T cells, both CD41 (150%) and CD81 (60%),... more resulted in substantial increases in the numbers of a/b T cells, both CD41 (150%) and CD81 (60%), and of g/d T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to a/b T cells. The number of CD41 T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD41 T cells within the intestinal villi. In contrast, the number of CD81 T cells within the intestinal villi increased following a challenge infection. In addition, the CD81 T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise correlation between the accumulation of CD81 T cells and the normal site of parasite development suggests an important role for CD81 T cells in the immune animal. Cryptosporidium parvum infection is now a well-recognized cause of diarrhea in immunolo...
The genome of Cryptosporidium parvum contains a relatively small number of introns, which include... more The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the beta-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the beta-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced beta-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12-72 h), at 48-72 h post-infection unprocessed beta-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing beta-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially ...
Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial s... more Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial species, has been implicated as the causative agent of gangrenous dermatitis. A recombinant noncytolytic C. septicum α toxin (NCAT) peptide was developed for use as a vaccine and demonstrated to be safe at concentrations as high as 1 mg/ml. NCAT, used as a purified antigen, partially purified antigen, or in combination with native antigens, was compared to salt-fractionated α toxin combined with denatured C septicum bacteria (native) in a vaccination trial. Three-day-old poults were placed into one of five groups and received two, 0.2-ml vaccinations 5 wk apart. Subcutaneous challenge with 3.2 x 10(7) log phase C. septicum resulted in 78% to 95% of the vaccinated birds surviving challenge compared to 48% of sham-injected controls. By ELISA analysis on NCAT-coated plates, birds receiving vaccines containing the recombinant NCAT peptide showed significantly higher blood serum antibody conce...
T lymphocytes bearing gamma/delta TCRs are a major population of T cells in neonatal calves and d... more T lymphocytes bearing gamma/delta TCRs are a major population of T cells in neonatal calves and discrete subsets of gamma/delta T cells display tissue-specific accumulation and responsiveness to infection. To enhance our understanding of the immunobiology of gamma/delta T cells, we characterized the gene expression profile of circulating bovine gamma/delta T cells following stimulation with recombinant human IL-2 and ConA. Statistical analysis of microarray data identified 108 genes with significantly altered expression, including four genes associated with apoptosis. Real-time reverse transcription-PCR (RT-PCR) analysis of 15 genes related to apoptotic pathways showed that both the Fas-mediated and the mitochondrial apoptotic pathways were repressed in circulating bovine gamma/delta T cells in response to mitogen activation, indicating that stimulated peripheral bovine gamma/delta T cells are resistant to activation-induced apoptosis.
Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced producti... more Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced production of several skin antimicrobial peptides, but more recent data have called those results into question. To compare the mRNA expression of seven antimicrobial peptide genes in lesional and adjacent nonlesional skin biopsy specimens from dogs with AD with those from normal dogs and from dogs experiencing other inflammatory skin conditions. Normal dogs and patients with AD or other inflammatory skin conditions were enrolled with owner permission and approval of the Institutional Animal Care and Use Committee. Transcripts were measured by quantitative RT-PCR using a standard curve assessment. Normal transcript levels for all seven antimicrobial peptides varied depending on the body site assessed. Transcripts for secretory leukocyte proteinase inhibitor (SLPI) and skin-derived antileucoproteinase (SKALP; also known as elafin) were typically ~10-fold greater in number than transcripts for the canine β-defensins (CBD)-1, -102, -103, -122 and -124. Transcripts for SKALP, SLPI, CBD-1, CBD-103 and CBD-122 were lower in both lesional and adjacent nonlesional skin from dogs with AD in comparison to normal skin. Transcripts were reduced to a similar extent versus normal dogs in skin of dogs with inflammatory skin conditions from both lesional and nonlesional biopsies, except for CBD-122, which was reduced only in lesional skin. Compared with normal dog skin, transcripts for CBD-102 and CBD-124 were unaffected in dogs with AD. Both SKALP and SLPI may be important contributors to skin innate immunity, but their decreased expression in AD patients does not account for increased skin infections compared with other skin conditions.
Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extrac... more Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.
The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and... more The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.
The purpose of the present study was to determine if reverse transcriptase-polymerase chain react... more The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected RNA from as few as 10(3) C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts stored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG RT-PCR may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro.
Cryptosporidium parvum is an obligate intracellular protozoan capable of causing life-threatening... more Cryptosporidium parvum is an obligate intracellular protozoan capable of causing life-threatening diarrhoeal disease in immunocompromised individuals. Efforts to develop novel therapeutic strategies have been hampered by the lack of understanding of the pathogenesis of infection. To better understand the host response to C. parvum infection, gene expression profiles of infected human ileocecal adenocarcinoma cells were analysed by using Affymetrix oligonucleotide microarrays containing probe sets for 12,600 human genes. Statistical analysis of expression data from three independent experiments identified 223 genes whose expression was reproducibly regulated by C. parvum infection at 24 h post-inoculation (125 up-regulated and 98 down-regulated), 13 of which were validated by quantitative reverse transcriptase polymerase chain reaction analysis. This analysis revealed the consistent up-regulation of host heat-shock genes and genes for pro-inflammatory chemokines IL-8, RANTES, and SCYB5. Multiple genes for host actin and tubulin genes were up-regulated whereas genes for actin binding proteins were down-regulated, confirming previous observations of host cytoskeleton rearrangement in response to C. parvum infection. In addition, host genes associated with cell proliferation and apoptosis were differentially regulated, reflecting the complexity of host-parasite interaction. Together, this study demonstrated that C. parvum infection results in significant changes in host biochemical pathways and provides new insights into specific biological processes of infectious disease caused by an intracellular protozoan parasite.
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Papers by Cheryl Lancto