Cell Culture Immunofluorescence Assay Research Articles

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples.

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.

Effects of Lactobacillus acidophilus and Lactobacillus reuteri cell-free supernatants on Cryptosporidium viability and infectivity in vitro

Protozoan parasites of the genus Cryptosporidium are a leading cause of diarrhea in domestic livestock and humans worldwide with bovine C. parvum and C. hominis being responsible for the vast majority of infections. To date there is no universal treatment for cryptosporidiosis, the disease caused by Cryptosporidium infection, but recent studies in mice suggest probiotic bacteria can reduce the duration and intensity of bovine C. parvum oocyst shedding. The mode of action utilized by these probiotics to exert their effects on bovine C. parvum infection was undetermined and their effect on C. hominis has never been evaluated. The purpose of this study was to determine if the probiotic bacteria, Lactobacillus acidophilus and Lactobacillus reuteri, produce compounds that are detrimental to bovine C. parvum and C. hominis. Cell-free supernatants of L. acidophilus (LA) and L. reuteri (LR) cultures reduced the infectivity of bovine C. parvum and C. hominis in a cell-culture immunofluorescence (CCIF) assay by 21–42% and 30–35%, respectively. A 24-h incubation of bovine C. parvum oocysts in the bacterial cell-free supernatants reduced oocyst viability 40–80% as determined by a flow cytometric assay and oocyst infectivity up to 95% as determined by the CCIF assay. These studies lend support to the hypothesis that production of anti-microbial compounds may be a mechanism by which LA and LR detrimentally affect bovine C. parvum and C. hominis.

Serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures.

A porcine enteric calicivirus-like virus was adapted to serial propagation in primary porcine kidney cell cultures. Attempts to propagate this virus in primary porcine kidney cells in the presence of trypsin or pancreatin or without medium supplementation were unsuccessful. A low-pH medium (pH 6.8) was also ineffective in virus propagation. Successful serial propagation of the virus required the presence of an intestinal-content preparation, derived from uninfected gnotobiotic pigs, in the cell culture medium. The best results were obtained with six-well plate cultures which were centrifuged after virus inoculation. Infected cells were detected by immunofluorescent staining of cell monolayers or detached cells which were harvested by centrifugation. Infected cells were first detected at passage 4 (1% infected cells), and infectivity increased with successive passages, with as many as 80% of the cells infected by passage 16. Extensive cytopathic effects were observed in inoculated cell cultures, but not in uninoculated control cell cultures, at each passage level after passage 13. The infected cells became separated, rounded, and detached, forming holes in the cell monolayer. Only virus particles exhibiting the six-pointed star appearance or stain-filled, cup-shaped depressions characteristic of caliciviruses were detected in inoculated cell culture supernatants by immune electron microscopy. Attempts to determine the titer of the virus by a cell culture immunofluorescence assay or plaque assay were unsuccessful.