Tissue Culture Assay Research Articles

Dual regulation by the Hunchback gradient in the Drosophila embryo

The regulation of segmentation gene expression is investigated by computational modeling using quantitative expression data. Previous tissue culture assays and transgene analyses raised the possibility that Hunchback (Hb) might function as both an activator and repressor of transcription. At low concentrations, Hb activates gene expression, whereas at high concentrations it mediates repression. Under the same experimental conditions, transcription factors encoded by other gap genes appear to function as dedicated repressors. Models based on dual regulation suggest that the Hb gradient can be sufficient for establishing the initial Kruppel (Kr) expression pattern in central regions of the precellular embryo. The subsequent refinement of the Kr pattern depends on the combination of Hb and the Giant (Gt) repressor. The dual-regulation models developed for Kr also explain some of the properties of the even-skipped (eve) stripe 3+7 enhancer. Computational simulations suggest that repression results from the dimerization of Hb monomers on the DNA template.

What can embryos teach us about communication?

Multicellular organisms coordinate their growth, development and homeostasis through cell-cell communication mediated by a relatively small number of extracellular signaling molecules, often referred to as growth factors. Nowadays, it is difficult to open a new volume of a developmental biology

Differential susceptibility of blue catfish, Ictalurus furcatus (Valenciennes), channel catfish, I. punctatus (Rafinesque), and blue × channel catfish hybrids to channel catfish virus

Channel catfish virus (CCV), also known as ictalurid herpesvirus-1 (IHV-1), primarily affects juvenile channel catfish, Ictalurus punctatus (Rafinesque), that are less than 6 months old and was first reported by Fijan (1968). CCV outbreaks can be sporadic, and are usually associated with fry and fingerlings when the water temperature is above 25 C (Plumb 1978). The virus has been reported to be transmitted vertically (Wise, Harrell, Busch & Boyle 1988) and horizontally (reviewed in Plumb 1978). The external signs of CCV disease (CCVD) include exophthalmia, distended abdomen and haemorrhages at the bases of fins. The trunk kidney may exhibit oedema and necrosis, and this tissue is commonly used to confirm the presence of the virus using a tissue culture assay (reviewed in Wolf 1988). In addition, the virus appears to maintain a latent state in leucocytes (Bowser, Munson, Jarboe, Francis-Floyd & Waterstrat 1985), which raises the possibility that latent CCV infection may alter the immune response to other pathogens. It has been over 30 years since it was determined that different strains of catfish exhibited differential resistance to CCV when the virus was mixed with their feed (Plumb, Green, Smitherman & Pardue 1975). A subsequent study by Plumb & Chappell (1978) examined the relative susceptibility of blue catfish, I. furcatus (Valenciennes), and reciprocal blue · channel hybrids to CCV. Since Plumb s (1978) study, there have been no reports on the relative susceptibility of blue catfish or hybrids to CCV. The present study was conducted to determine the relative susceptibility of four different groups of fish: blue catfish, a blue · channel hybrid, a group of channel catfish obtained from 10 farms in the Mississippi Delta, hereafter referred to as the industry pool (IP), and a new strain of catfish produced by the Catfish Genetics Research Unit of USDA at Stoneville, MS (USDA 102 · 103). For each strain of fish, nine replicate tanks were stocked with 40 fish per tank; eight tanks were used for virus challenge while the remaining tank was used as an uninfected control. Fish were placed in 38 L tanks that were filled to 11 L and had a flowthrough rate of 1.8 L min and allowed to acclimatize for 8 days. Fish were fed to satiation twice per day beginning the day after stocking and feeding continued throughout the course of the study. The blue catfish (avg. wt. 3.45 0.18 g) used in this study were of the D and B strain. Fry from seven different spawns were pooled and raised communally in tanks until used in the challenge. The hybrid catfish (avg. wt. 4.10 0.21 g) were produced by crossing female USDA 103 strain channel catfish and D and B strain blue catfish. Twelve hybrid spawns were pooled and used in this Journal of Fish Diseases 2008, 31, 77–79

Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

From January to December 1998, nasopharyngeal aspirates were obtained from 482 children with acute respiratory infections attended in emergence department and wards of a teaching hospital in the city of Salvador, Brazil. The samples were tested for the presence of adenovirus by isolation in tissue culture and indirect immunofluorescence assay. Eleven adenoviruses were detected by both methods in the same clinical samples. Infections by adenovirus were observed during seven months of the year without association with rainy season. Genome analysis was performed on these 11 isolates. Species C was represented by serotypes 1, 2 and 5. Within species B, only serotype 7 (Ad7) was detected. Two genomic variants of Ad1, two variants of Ad2, one of Ad5, and one of Ad7 (7h) were identified. This is the first study of molecular epidemiology of adenovirus associated to acute respiratory infections in children living in Northeast Brazil, and contributes to a better understanding of adenovirus infections in the country.

Infection and Vertical Transmission of Kamiti River Virus in Laboratory BredAedes aegyptiMosquitoes

Kamiti river virus (KRV) is an insect-only Flavivirus that was isolated from field-collected Ae. macintoshi mosquitoes in 1999, and is closely related to cell fusing agent virus. Both of these viruses belong to the family Flaviviridae, which also contains other viruses of medical importance, such as yellow fever virus, West Nile virus and dengue. Because Ae. macintoshi is the only known natural host to KRV, the main objective of this study was to establish the possibility that other mosquito hosts of the virus exist, by determining its ability to infect Ae. aegypti mosquitoes under laboratory conditions. The study also sought to determine the rates of infection and, subsequently, vertical transmission as a possible means of its maintenance and propagation in nature, given that it neither grows in vertebrate cells or mice. The mosquitoes were infected by the virus either as larvae or adults. Virus assay was done by re-isolation in tissue culture and indirect immunofluoresce assay methods. KRV infected Ae. aegypti mosquitoes, with the observed rates as high as 74 to 96 %. The virus was also transmitted vertically in these mosquitoes. Vertical transmission rates of 3.90 % were observed for the 2nd and 3rd ovarian cycles combined. These results suggest that Ae. aegypti mosquitoes are likely to be infected with KRV in nature, and that vertical transmission is the natural means by which it is maintained and propagated in this host, and possibly others.

Etiology of Acute Diarrhea in Children and Adults in Tunis, Tunisia, with Emphasis on Diarrheagenic Escherichia coli: Prevalence, Phenotyping, and Molecular Epidemiology

A total of 271 stool specimens were collected from children (diarrheagenic, n = 115 and control, n = 54) and adults (diarrheagenic, n = 73 and control, n = 29) from Tunis, Tunisia, and processed to detect bacterial enteropathogens, parasites, and viruses. Diarrheagenic Escherichia coli (DEC) were identified by their virulence genes (polymerase chain reaction) and adherence patterns (tissue culture assays). The most frequently isolated enteric pathogens from diarrheagenic children were enterotoxigenic E. coli (ETEC, 32.3%), enteroaggregative E. coli (EAEC, 11.3%), enteroinvasive E. coli (EIEC, (11.3%), adenovirus (10.4%), enterohemorrhagic E. coli (EHEC, 10.4%), and Salmonella spp. (9.5%). For children in the control group, ETEC (37%), EAEC (15%), EHEC (11.1%), and typical enteropathogenic E. coli (EPEC, 11.1%) were the most common enteric pathogens. In adults in the diarrheagenic group, Salmonella spp. (34.2%), ETEC (12.3%), adenovirus (7%), and Shigella spp. (4%) were the most common enteric pathogens. In adults in the control group, ETEC (31%) was the most common enteric pathogen. Multiple pathogens were recovered from 22% of the diarrheagenic children and 7% of the diarrheagenic adults. Escherichia coli strains showed high resistance rates to tetracycline, streptomycin, and beta-lactams. The most frequent combinations were ETEC-rotavirus and ETEC-adenovirus. Pulsed-field gel electrophoresis for DEC indicated a large number of DEC clones (five major clones) persistent in the community reservoir for a considerable period of time that caused diarrhea in the population. This suggests the confluence of small epidemics by clonally related DEC strains circulating in this region.

The Insulin-Like Growth Factor System Modulates Retinal Pigment Epithelial Cell Tractional Force Generation

The goal of this study was to determine the influence, if any, of the insulin-like growth factors (IGFs) on retinal pigment epithelial (RPE) cell tractional force generation and the contributions of vitreous insulin-like growth factor-binding proteins (IGFBPs) toward control of growth factor activity. IGF effects on RPE were evaluated in tissue culture assays that involved incubation on three-dimensional collagen matrices with responses measured as progressive reduction in matrix thickness. IGFBP effects were evaluated by using the same system, exposing cells to a non-IGFBP-binding growth factor analogue (R(3)IGF-I) or IGFBPs alone or in combination with native growth factors. RPE cells generated tractional forces in response to IGF-I and -II with IGF-I being the more potent stimulus. Differential RPE responses to R(3)IGF-I reflected minor amounts of endogenous IGFBP production. IGFBP-2, -3, and -5 were effective inhibitors of both ligands, whereas IGFBP-6 reduced cell responses to IGF-II only. IGFBP-direct effects on the cells were binding-protein-specific, in that IGFBP-1 had detectable stimulatory effects, and IGFBP-3, -4, -5, and -6 inhibited RPE responses. IGF-I and -II are potent promoters of RPE cell tractional force generation in vitro. The effects of the six high-affinity IGFBPs on RPE responses are generally inhibitory and protein-specific. IGF ligands and binding proteins are known to be present in the vitreous, the environment that drives RPE responses in proliferative vitreoretinopathy (PVR), suggesting that the IGF system plays a potentially important role in the pathophysiology of this fibrocontractive disease.

From MicroRNA to Carcinogenesis

Misregulation of microRNA (miRNA) function has been implicated in cancer. However, the precise role of miRNAs in tumorigenesis has been unclear. High mobility group A2 protein (Hmga2) is a small, non-histone, chromatin-associated protein found in a number of benign and malignant tumors, where the gene is often truncated at the 3′ end. Mayr et al . now show that it is the loss of the noncoding 3′ untranslated region of the Hmga2 messenger RNA, and specifically of regulator sites for the let-7 miRNA, that causes the overexpression of Hmga2, and that this overexpression contributes to the progression of carcinogenesis both in a tissue culture assay and in nude mice. C. Mayr, M. T. Hemann, D. P. Bartel, Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 315 , 1576-1579 (2007). [Abstract] [Full Text]

Interactions of DNR1 with the apoptotic machinery ofDrosophilamelanogaster

Caspases are crucial activators of apoptosis and NF-kappaB signaling in vertebrates and invertebrates. In Drosophila, the caspase-9 counterpart Dronc is essential for most apoptotic death, whereas the caspase-8 homolog Dredd activates NF-kappaB signaling in response to gram-negative bacterial infection. The mechanics of caspase regulation are conserved and include the activities of a family of inhibitor of apoptosis (IAP) proteins. The RING-domain-bearing protein Defense repressor 1 (Dnr1), blocks ectopic Dredd-mediated induction of an NF-kappaB reporter in the Drosophila S2 cell line. In this study, we present novel data indicating that Dnr1 impacts on Dronc-dependent regulation of the apoptotic program. We show that depletion of Dnr1 results in elevated Dronc protein levels, which translates to increased caspase activation and activity upon induction of apoptosis. Conversely, we demonstrate that overexpression of Dnr1 blocks apoptotic caspase activity and prevents induction of apoptosis in tissue culture assays. Furthermore, we show that Dnr1 overexpression significantly reduces Dronc protein levels and identify the domains of Dnr1 necessary for these effects. From these data, we propose that Dnr1 inhibits initiator caspases in S2 cells.

Use of intravenous immunoglobulin for the treatment of severe clostridium difficile colitis

Background: Clostridium difficile is a gram-positive, anaerobic, spore-forming, rod-shaped bacterium responsible for most of the hospital-acquired diarrhea in developed countries. The organism received its name because it was difficult to isolate and grow in culture. Infections in the elderly have been associated with significant morbidity and mortality as well as prolonged hospitalization. Case summary: A 72-year-old white male presented with a 5-day history of abdominal pain, nausea, and severe diarrhea but no fever or chills. He had had recent chemotherapy for Merkel cell carcinoma of the right ear. Medical history included hypothyroidism for 10 years and non-Hodgkin's lymphoma in remission for 4 years after a stem cell transplant. The patient was receiving oral vancomycin, levofloxacin, thyroxine, and esomeprazole. He had severe infection secondary to chemotherapy for Merkel cell carcinoma; in addition, he had failed to respond to metronidazole and vancomycin treatment, with the resulting development of colon dilatation and hypoalbuminemia. Colonoscopy showed severe ulceration with inflammation suggestive of severe persistent colitis. At that point, the patient was given 1 dose of IV immunoglobulin (IVIG) 400 mg/kg; vancomycin treatment was continued. Two days after IVIG therapy, the patient's diarrhea improved, with complete resolution after 6 days; bowel dilatation resolved completely after 7 days; and oral intake improved after 2 days. The patient continued on a tapering dose of vancomycin for 6 weeks. He was discharged home and had no recurrence despite antibiotic use for pseudomonas and staphylococcus bacteremia. Conclusions: Severe C difficile colitis has been reported more frequently in the literature recently, especially in elderly patients. Tissue culture assay is the best diagnostic test to detect the cytotoxin; enzyme immunoassay is the test used in most hospitals, but it has a sensitivity of only −75%. Treatment options remain limited to eradicate this serious infection. Antibiotic therapy, infection control measures, and early diagnosis are essential components of successful outcome for this disease. This patient's infection resolved with the addition of IVIG with no recurrence, suggesting the possible benefit of this treatment in certain patients with severe colitis who do not respond to standard therapy.

Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes.

Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans.

Two Novel Members of the ABLIM Protein Family, ABLIM-2 and -3, Associate with STARS and Directly Bind F-actin

In addition to regulating cell motility, contractility, and cytokinesis, the actin cytoskeleton plays a critical role in the regulation of transcription and gene expression. We have previously identified a novel muscle-specific actin-binding protein, STARS (striated muscle activator of Rho signaling), which directly binds actin and stimulates serum-response factor (SRF)-dependent transcription. To further dissect the STARS/SRF pathway, we performed a yeast two-hybrid screen of a skeletal muscle cDNA library using STARS as bait, and we identified two novel members of the ABLIM protein family, ABLIM-2 and -3, as STARS-interacting proteins. ABLIM-1, which is expressed in retina, brain, and muscle tissue, has been postulated to function as a tumor suppressor. ABLIM-2 and -3 display distinct tissue-specific expression patterns with the highest expression levels in muscle and neuronal tissue. Moreover, these novel ABLIM proteins strongly bind F-actin, are localized to actin stress fibers, and synergistically enhance STARS-dependent activation of SRF. Conversely, knockdown of endogenous ABLIM expression utilizing small interfering RNA significantly blunted SRF-dependent transcription in C2C12 skeletal muscle cells. These findings suggest that the members of the novel ABLIM protein family may serve as a scaffold for signaling modules of the actin cytoskeleton and thereby modulate transcription.

Curcumin Suppresses AP1 Transcription Factor-dependent Differentiation and Activates Apoptosis in Human Epidermal Keratinocytes

The diet-derived cancer preventive agent, curcumin, inhibits skin cancer cell proliferation and tumor formation. However, its effect on normal human keratinocyte differentiation, proliferation, and apoptosis has not been adequately studied. Involucrin (hINV) is a marker of keratinocyte differentiation and a useful model for the study of chemopreventive agent action. We show that curcumin suppresses the differentiation agent-dependent activation of hINV gene expression and that an AP1 transcription factor DNA binding site in the hINV gene is required for this regulation. A protein kinase C, Ras, MEKK1, MEK3 signaling cascade controls hINV expression by regulating AP1 factor level. Curcumin treatment inhibits the novel protein kinase C-, Ras-, and MEKK1-dependent activation of hINV promoter activity and reduces the differentiation agent-dependent increase in AP1 factor level and DNA binding. This reduction requires proteasome function. In addition, curcumin treatment reduces cell number, which is associated with a reduced cyclin and cdk1 levels. Curcumin treatment also suppresses the Bcl-xL level, leading to reduced mitochondrial membrane potential and increased cleavage of procaspases and poly(ADP-ribose) polymerase. These studies provide important insights regarding the mechanism whereby curcumin acts as a chemopreventive agent in normal human epidermis.

Requirement of Pygopus 2 in breast cancer.

The development of novel therapeutic strategies for breast cancer requires the identification of molecular targets involved in malignancy. Human Pygopus (Pygo)-1 and -2 are recently discovered components of the Wnt signaling pathway required for beta-Catenin/Tcf dependent transcription in embryos and colorectal cancer cells, but the role of these proteins in malignant cell growth and survival has not yet been determined. We report the expression and requirement for proliferation of hPygo2 in breast cancer cells. hPygo2 protein was overexpressed in malignant breast tumors and in the nuclei of five breast cancer cell lines, but was not expressed in the nuclei of non-malignant breast cells. Phosphorothioated antisense oligonucleotides were used to specifically knockdown expression hPygo2 in Mcf-7 and MDA-MB-468 cell lines. hPygo2 was required for the growth, in tissue culture and anchorage-independent assays, of both cell lines and for the expression of the Wnt target gene Cyclin D1. We conclude that hPygo2 is highly expressed in, and required for the growth of breast carcinoma cells.

Chorionic Gonadotropin: A Narrative of Its Identification and Origin and the Role of Georgeanna Seegar Jones

The following article on the history of human chorionic gonadotropin is interesting on several levels. I hope that you will enjoy the first-hand description of the events surrounding the discovery of site of origin of this important pregnancy-related hormone as told by Dr. Howard Jones Jr., husband of the late Georgeanna Seegar Jones. They were both editors of the SURVEY for more than 25 years. In today's world of microarray and PCR technology it is fun to hear about the progress of science using tissue culture and mouse assays. But this story is also about the progress we have made on the human level. At a time when well over 50% of the residents in Obstetrics and Gynecology are women, it is astounding to think that in 1938 Mom's name would have been listed as "G. Emory Seegar" on this important paper because it was felt that the paper would not be accepted for publication if one of the authors was a woman!

Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal–amoeboid–like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.

The Meckel–Gruber Syndrome proteins MKS1 and meckelin interact and are required for primary cilium formation

Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal malformation syndrome characterized by renal cystic dysplasia, central nervous system malformations (typically, posterior occipital encephalocele), and hepatic developmental defects. Two MKS genes, MKS1 and MKS3, have been identified recently. The present study describes the cellular, sub-cellular and functional characterization of the novel proteins, MKS1 and meckelin, encoded by these genes. In situ hybridization studies for MKS3 in early human embryos showed transcript localizations in agreement with the tissue phenotype of MKS patients. Both MKS proteins predominantly localized to epithelial cells, including proximal renal tubules and biliary epithelial cells. MKS1 localized to basal bodies, while meckelin localized both to the primary cilium and to the plasma membrane in ciliated cell-lines and primary cells. Meckelin protein with the Q376P missense mutation was unable to localize at the cell membrane. siRNA-mediated reduction of Mks1 and Mks3 expression in a ciliated epithelial cell-line blocked centriole migration to the apical membrane and consequent formation of the primary cilium. Co-immunoprecipitation experiments show that wild-type meckelin and MKS1 interact and, in three-dimensional tissue culture assays, epithelial branching morphogenesis was severely impaired. These results suggest that MKS proteins mediate a fundamental developmental stage of ciliary formation and epithelial morphogenesis.

Klebsiella oxytocaas a Causative Organism of Antibiotic-Associated Hemorrhagic Colitis

Antibiotic-associated hemorrhagic colitis is a distinct form of antibiotic-associated colitis in which Clostridium difficile is absent. Although the cause is not known, previous reports have suggested a role of Klebsiella oxytoca. We studied 22 consecutive patients who had suspected antibiotic-associated colitis and who were negative for C. difficile. Patients underwent diagnostic colonoscopy, and among those who received a diagnosis of antibiotic-associated hemorrhagic colitis, stool samples were cultured for K. oxytoca. We isolated K. oxytoca strains and tested them for cytotoxin production using a tissue-culture assay. In addition, we also cultured stool samples obtained from 385 healthy subjects for K. oxytoca. An in vivo animal model for antibiotic-associated hemorrhagic colitis was established with the use of Sprague-Dawley rats. Of the 22 patients, 6 had findings on colonoscopy that were consistent with the diagnosis of antibiotic-associated hemorrhagic colitis, and 5 of these 6 patients had positive cultures for K. oxytoca. No other common enteric pathogens were found in the five patients. Before the onset of colitis, all five were receiving penicillins, and two were also taking nonsteroidal antiinflammatory drugs (NSAIDs). All isolated K. oxytoca strains produced cytotoxin. K. oxytoca was found in 1.6% of the healthy subjects. In the animal model, K. oxytoca was found only in the colon of rats that received amoxicillin-clavulanate in addition to being inoculated with K. oxytoca. In these rats, infection with K. oxytoca induced a right-sided hemorrhagic colitis that was not observed in uninfected animals that received amoxicillin-clavulanate, indomethacin (an NSAID), or both. Our fulfillment of Koch's postulates for cytotoxin-producing K. oxytoca suggests that it is the causative organism in at least some cases of antibiotic-associated hemorrhagic colitis. Infection with K. oxytoca should be considered in patients with antibiotic-associated colitis who are negative for C. difficile.

Polyamine Analogs with Xylene Rings Induce Antizyme Frameshifting, Reduce ODC Activity, and Deplete Cellular Polyamines

Numerous studies have correlated elevated polyamine levels with abnormal or rapid cell growth. One therapeutic strategy to treat diseases with increased cellular proliferation rates, most obviously cancer, has been to identify compounds which lower cellular polyamine levels. An ideal target for this strategy is the protein antizyme-a negative regulator of polyamine biosynthesis and import, and a positive regulator of polyamine export. In this study, we have optimized two tissue-culture assays in 96-well format, to allow the rapid screening of a 750-member polyamine analog library for compounds which induce antizyme frameshifting and fail to substitute for the natural polyamines in growth. Five analogs (MQTPA1-5) containing xylene (1,4-dimethyl benzene) were found to be equal to or better than spermidine at stimulating antizyme frameshifting and were inefficient at rescuing cell growth following polyamine depletion. These compounds were further characterized for effects on natural polyamine levels and enzymes involved in polyamine metabolism. Finally, direct measurements of antizyme induction in cells treated with two of the lead compounds revealed an 8- to 15-fold increase in antizyme protein over untreated cells. The impact of the xylene moiety and the distance between the positively charged amino groups on antizyme frameshifting and cell growth are discussed.

Potential prophylactic value of bovine colostrum in necrotizing enterocolitis in neonates: anin vitrostudy on bacterial attachment, antibody levels and cytokine production

Necrotizing enterocolitis (NEC) is an important disease of low birth-weight neonates. The immaturity of the gut mucosa may result in close contact between the host epithelium and microorganisms which are normally confined to the gut lumen. Damage of the mucosa due to endotoxin, cytokine production or other factors is believed to then occur. The aim of this study was to determine whether spray-dried bovine colostrum demonstrated potential in vitro as a prophylactic for NEC. Antiadherence was measured using a tissue culture assay and antibody levels against Enterobacteriaceae were determined by ELISA. The effect of bovine colostrum on the production of cytokines implicated in NEC was determined by a multiplex bead assay. Enterobacter cloacae, Klebsiella oxytoca, Escherichia coli, Serratia marcescens and Klebsiella pneumoniae ssp. pneumoniae were common in both NEC positive and NEC negative infants and IgA and IgG1 antibodies to these species were present in the bovine colostrum. Pretreatment with bovine colostrum produced a significant decrease (P<0.001) in attachment of bacteria to HT-29 cells. Bovine colostrum significantly increased the production of IL-8 in HT-29 cells and IL-8, IL-6 and TNF-alpha in THP-1 cells (P<0.001). The potential of bovine colostrum to increase the production of inflammatory mediators could limit its usefulness.