Susceptibility to Cryptosporidium parvum infections in cytokine- and chemokine-receptor knockout mice.

To determine the role of cytokines and a chemokine receptor in the susceptibility to, and outcome of, infection, 4 different knockout mice (IL-4, IL-10, IL-12, and CCR5) were infected with Cryptosporidium parvum and monitored for infection intensity by collection of fecal pellets from individual mice. Because adult immunocompetent mice are refractory to infection, wild-type mice on the same background as the knockout mice (C57BL/6) were used as a negative control. No infection was detected over a 4-wk time period in IL-4, IL-10, and CCR5 knockout mice inoculated with 106 oocysts.

Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells.

Cryptosporidiosis and the challenges of chemotherapy.

Cryptosporidium parvum is a protozoan parasite that infects the epithelial cells of the small intestine causing diarrheal illness in humans. Cryptosporidium has a worldwide distribution and is considered an emerging zoonosis. Despite intensive efforts to develop workable experimental models, and the evaluation of over 200 chemotherapeutic agents, adequate therapies to clear the host of these parasites are still lacking.

Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast.

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast.

Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene.

Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice.

Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines.

Target cells promote the development and functional maturation of neurons derived from a sympathetic precursor cell line.

The role of target interactions in the development and functional maturation of peripheral neurons was investigated using an immortalized sympathetic precursor cell line. bMAH cells underwent neuronal differentiation in response to neurotrophic factors, but maintained an immature neuronal phenotype characterized by small cell bodies and continued cell division. Co-culture with cardiac myocytes, a target of sympathetic innervation, promoted the appearance of large-diameter postmitotic bMAH neurons.

Human T and B cell immunoreactivity to a recombinant 23-kDa Cryptosporidium parvum antigen.

Cryptosporidial infection in humans results in parasite-specific IgG, IgM, and IgA antibody responses, but little is known of the cell-mediated immune responses to cryptosporidial antigens. In a convenience sample of 35 Haitian residents, there was a high level of cryptosporidial exposure (>90%) as determined by immunoblot reactivity of serum against cryptosporidial antigens. An attempt was made to determine if there was a relationship between antibody and T cell-mediated responses to recombinant Cp23 antigen and how this correlated with reactivity to crude sporozoite antigen preparations (SAg).

A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice.

In the present study, we focused on a 23-kDa antigen, Cp23, which has been shown to be a major target of humoral immune responses in Cryptosporidium parvum infections and is present in both the sporozoite and merozoite stages. Recombinant Cp23 antigen was shown to stimulate a specific proliferative response by splenocytes and mesenteric lymph node cells from infected interferon gamma knockout BALB/c mice. Cp23 stimulation also induced TNF-alpha, IL-2, and IL-5 mRNA production by spleen cells from infected animals.

Cytokine expression and specific lymphocyte proliferation in two strains of Cryptosporidium parvum-infected gamma-interferon knockout mice.

Differences in the immune response between 2 strains of interferon-gamma knockout mice (BALB/c-GKO and C57BL/6-GKO) infected with Cryptosporidium parvum were examined because the course of infection among these 2 strains is markedly different. Infection of the BALB/c-GKO with C. parvum (2 X 10(6) oocysts/mouse) resulted in slight weight loss, oocyst shedding, and recovery from infection by 2 wk postinfection (PI).

Nerve growth factor collaborates with myocyte-derived factors to promote development of presynaptic sites in cultured sympathetic neurons.

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones.

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus.

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.

Scanning mutagenesis of Mcm1: residues required for DNA binding, DNA bending, and transcriptional activation by a MADS-box protein.

MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending.

Lipoprotein lipase, a key role in atherosclerosis?

Lipoprotein lipase (LPL) plays a central role in lipid metabolism and transport by catalysing the hydrolysis of triacylglycerol-rich lipoproteins. The importance of LPL expressed by the adipose tissue and muscles in the provision of non-esterified fatty acids and 2-monoacylglycerol for tissue utilisation is well established. However, recent studies on LPL expressed by cells of the vascular wall, particularly macrophages, have identified additional actions of the enzyme that contribute to the promotion of foam cell formation and atherosclerosis.

Characterization of an alpha-tubulin gene of Cryptosporidium parvum.

A gene encoding an alpha-tubulin of Cryptosporidium parvum was isolated and characterized. It had no introns, and encoded a 441-amino acid protein whose predicted ORF represented a typical alpha-tubulin protein with a MW of 50.5 kDa.

Characterization of experimental Cryptosporidium parvum infection in IFN-gamma knockout mice.

Severe cryptosporidial infections were produced in gamma interferon (IFN-gamma) knockout mice. Mean oocyst shedding increased from 332 to 30,717 oocysts/100 microliters of faecal suspension between day 4 and 9 after administration of 1 x 10(5) oocysts/mouse. No significant differences in oocyst shedding were observed in mice after being inoculated with 1 x 10(5), 1 x 10(4) or 1 x 10(3) oocysts/mouse (P > 0.

Susceptibility differences to Cryptosporidium parvum infection in two strains of gamma interferon knockout mice.

Differences in susceptibility to cryptosporidial infections were investigated between 2 strains of gamma interferon knockout (GKO) mice. Male C57BL/6J-Ifg and BALB/c-Ifg (GKO) mice, ages 8-10 wk, were inoculated with infectious oocysts at various doses. C57BL/6J-Ifg mice developed overwhelming infections and died 9-12 days after infection.

Characterization of OXA-9, a beta-lactamase encoded by the multiresistance transposon Tn1331.

The enzyme OXA-9, an oxacillinase-carbenicillinase, is encoded by the blaOXA-9 gene which was originally found within the structure of Tn1331, a multiresistance transposon first isolated from a clinical Klebsiella pneumoniae strain. Studies to characterize OXA-9 demonstrated that it has a pI of 6.9 and the optimal pH for enzyme activity was between 7.