Ifeoma Ezeonu

Colonization of fiberglass insulation used in heating, ventilation and air conditioning systems

SummaryThe number of fungal species colonizing thermal and acoustic fiberglass insulations used in heating, ventilation, and air conditioning (HVAC) systems was fewer than that obtained from initial direct culture of these insulations. The colonization, determined by the microscopic observation of conidiophores with conidia, was primarily of acrylic-latex-facing material, but eventually the fungi permeated the fiberglass matrix. Isolates ofAspergillus versicolor were most often obtained from non-challenged insulation, whereasAcremonium obclavatum appeared to be the primary colonizing fungus in high-humidity (>90%) challenge chambers. At a lower humidity (about 70%)Aspergillus flavus was one of the more prominent fungi. Not all duct liner samples were equally susceptible to colonization and duct board appeared relatively resistant to colonization.

Nucleoside transport sites in a cultured human retinal cell line established by SV-40 T antigen gene

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.

Density-Dependent Differentiation in Nontransformed Human Retinal Progenitor Cells in Response to Basic Fibroblast Growth Factor- and Transforming Growth Factor-α

Multipotential retinal precursors give rise to all cell types seen in multilayered retina. The generation of differentiation and diversity of neuronal cell types is determined by both extrinsic regulatory signals and endogenous genetic programs. We have previously reported that cell commitment in human retinal precursor cells (SV-40T) can be modified in response to exogenous growth factors, basic fibroblast growth factor, and transforming growth factor alpha (bFGF and TGFalpha). We report in this study that nontransformed human retinal precursors differentiate into photoreceptors by a cell density-dependent mechanism, and the effects were potentiated by bFGF and TGFalpha alone or in combination. A larger proportion of multipotential precursors plated at a density of 1 x 10(4) cells/cm(2) differentiated into neurons (photoreceptors) compared to cells plated at 3-5 x 10(4)/cm(2) and 1 x 10(5) cells/cm(2) under serum-free conditions and the effects were amplified seven- to eightfold in response to growth factors. Basic fibroblast growth factor (bFGF) and TGFalpha can induce 90% of the cells to assume a photoreceptor phenotype at a lower cell density, compared to only 30 and 25% of the cells acquiring a photoreceptor phenotype at intermediate and higher cell densities. Furthermore, at a lower cell density, 60-70% of the cells incorporate Bromodeoxyuridine (Brdu), suggesting that cells in a cell cycle may make a commitment to a specific fate in response to neurotrophins. Neurons with a photoreceptor phenotype were positive for three different sets of antibodies for rods/cones. Cells also exhibited upregulation of other proteins such as a D4 receptor protein expressed in photoreceptors, protein kinase Calpha (PKCalpha) expressed in rod bipolars and blue cones, and some other neuronal cell types. This was also confirmed by Western blot analysis. Newly derived photoreceptors survive for a few days before significant cell death ensues under serum-free conditions. To summarize, differentiation in precursors is density dependent, and growth factors amplify the effects.

PROTO-ONCOGENE EXPRESSION IN CAMP AND TPA-MEDIATED NEURONAL DIFFERENTIATION IN A HUMAN RETINAL CELL LINE KGLDMSM

PURPOSE A human retinal cell line, KGLDMSM, developed by SV-40T antigen gene transfection, is stable in culture for a long period, unlike the primary cells. The cell line shows some degree of morphological differentiation with limited extension of stublike neurites upon transfer to defined medium. In our effort to explore genes implicated in neuritic extension and neuronal differentiation seen in response to cAMP and TPA, we have analyzed time dependent induction of a variety of proto-oncogenes: c-myc, H-ras, c-ras, and c-fos. METHODS Cells were adapted to grow in defined media and exposed to differentiation inducing agents cAMP, TPA, Retinoic Acid, and sodium butyrata. Cells were assessed for phenotypic changes and altered expression of proto-oncogenes as evaluated by Northern Blot analysis and immunocytochemistry. RESULTS Exposure of the cells to cAMP and TPA induced dramatic changes, with 100% of the cells extending neuritic processes. However, other differentiation inducing agents such as retinoic acid and sodium butyrata failed to elicit any response. We report that agents that promote neuritic extension also induce expression of c-fos. Transcriptional activation of c-fos in response to cAMP (30 min) and TPA (1hr) is also accompanied by expression of fos gene product as evaluated by using fos antibody. No fos expression was seen in uninduced cells. CONCLUSION In retinal cell line KGLDMSM, agents that enhance neuronal differentiation (cAMP, TPA) also induce c-fos expression. Expression of c-fos may be a necessary prerequisite in neuronal differentiation and the established retinal cell line offers an excellent cell model for dissecting the molecular events underlying neuronal differentiation.

Cell Fate Decisions in a Human Retinal Precursor Cell Line: Basic Fibroblast Growth Factor- and Transforming Growth Factor-α-Mediated Differentiation

The purpose of this study was to determine if immortalized human retinal precursor cells could serve as a model to investigate cues that modulate cell fate and differentiation. We investigated the effects of a variety of growth factors broadly but specifically tested the effects of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)a in retinal cell differentiation and commitment. To determine the role of exogenously added growth factors in a human retinal precursor cell line (KGLDMSM), established from a first-trimester retina, cells were adapted to grow in a defined medium and exposed to a variety of trophic factors (epidermal growth factor [EGF], neuron growth factor [NGF], TGFalpha, TGFbeta, acidic FGF, and bFGF). Dose-response curves were developed to arrive at optimal concentrations. The neurotrophic potential of growth factors was determined by 3H-thymidine incorporation and bromodeoxyuridine (BrdU) labeling. The identity of the emerging neuronal phenotypes were determined by phase-contrast microscopy, immunolabeling for the neuron-specific antigens neurofilament protein (NF) and neuron-specific enolases (NSE), and photoreceptor-specific antigens (Rho1D4, 7G6) using immunocytochemistry and Western blot analysis. To identify some of the early response genes (c-fos, c-myc) expressed in response to growth factors, Northern blot analysis was performed. Almost all of the factors tested increased the total number of cells with a neuronal phenotype. Potency of growth factors to generate neurons was TGFalpha > bFGF > EGF > NGF. Both TGFalpha and bFGF, alone or in combination, increased the total number of neurons. Most of the neurons generated were photoreceptors, as depicted by the polarized phenotype, expression of photoreceptor-specific antigens, and processes resembling rudimentary outer segments. The increase in photoreceptor-like neurons is possibly attributable to an increase in numbers rather than greater survival. Additionally, the majority of the photoreceptors generated labeled with BrdU and for photoreceptor-specific antigens, suggesting that an inductive effect of bFGF and TGFalpha could occur in the cell cycle or shortly thereafter. Both bFGF and TGFalpha induced the expression of the early response gene c-fos while not altering the expression of c-actin or c-myc. The emergence of a photoreceptor phenotype was confirmed by both immunocytochemistry and Western blot analysis. The immortalized retinal precursor cell line could prove valuable in determining the role of exogenously added growth factors in retinal development and differentiation. Both bFGF and TGFalpha enhance the photoreceptor phenotype in medium-density cultures under conditions of defined medium. The same was confirmed by phase-contrast microscopy, immunocytochemistry, and Western blot analysis. Furthermore, cell fate determination in cultured precursor cells could occur during the late part of the cell cycle or shortly after completion of cell division. The effects of TGFalpha and bFGF seem to be slightly additive. The cell line will be extremely valuable in studying mechanisms of cell commitment and generation of retinal cell types, which could be tested for their potential for transplantation.

Effects of extracts of fiberglass insulations on the growth of Aspergillus fumigatus and A. versicolor.

Water extracts of thermal and acoustic fiberglass insulations used in the duct work of heating, ventilation and air conditioning (HVAC) systems supported germination of conidia and growth ofAspergillus versicolor (Vuillemin) Tiraboschi 1908–9 andAspergillus fumigatus Fresenius 1863. Urea, formaldehyde and unidentified organics were detected in the extracts. Formaldehyde in concentrations similar to those found in the extracts restricted the growth of both species in enriched media.A. versicolor, the more common species associated with fiberglass insulations, was more resistant to formaldehyde thanA. fumigatus.

Cryptosporidium species and subtypes in diarrheal children and HIV-infected persons in Ebonyi and Nsukka, Nigeria.

INTRODUCTION Cryptosporidiosis is a common disease of children and immune-compromised persons. This study evaluated the diversity and distribution of Cryptosporidium species in diarrheal children and HIV-infected persons on highly active antiretroviral therapy (HAART) and those not on HAART. METHODOLOGY A total of 394 fecal specimens were collected from patients attending clinics in Nsukka and Ebonyi, Nigeria. Detection and identification of Cryptosporidium species were conducted by PCR-RFLP of the small subunit (SSU) rRNA gene, whereas subtyping was done by sequence analysis of the 60 kDa glycoprotein (gp60) gene. RESULTS Twenty-five (6.3%) specimens yielded four Cryptosporidium species, including C. hominis, C. parvum, C. felis, and C. viatorum. C. hominis was the most dominant species with 48.0% occurrence and three identified subtype families: Ia (six specimens), Ib (three specimens), Ie (two specimens), and one un-subtyped species. C. parvum had 44.0% occurrence and two subtype families: IIc (eight specimens) and IIe (three specimens), while C. felis and C. viatorum each had 4.0% occurrence. There were significant differences in Cryptosporidium species distribution between age groups in children and HIV-infected persons, between suburban and urban areas, and between low and high CD4+ cell counts in HIV-infected patients. There were no significant differences in infection rate and species distribution between HIV-infected patients on HAART and those not on HAART. CONCLUSIONS The results from this study show that there is a high diversity of Cryptosporidium spp. in humans in Ebonyi and Nsukka, Nigeria, and that all the C. parvum subtypes identified are most likely anthroponotic in origin.

Prebiotic effects of Vernonia amygdalina and Ocimum gratissimum aqueous leaf extracts in rabbit ( Oryctolagus cuniculus )

The prebiotic potentials of crude extracts of Vernonia amygdalina and Ocimum gratissimum in rabbit ( Oryctolagus cuniculus ) were studied. The aqueous leaf extracts of both plants, taken orally, were evaluated for their effects on the composition and relative abundance of the rabbit gastrointestinal microflora as well as their protective roles against gastrointestinal infection by selected test organisms. The crude extracts were administered to eight-week old rabbits with average weight of 0.85 ± 0.14 kg, at a dosage of 0.1 g/day. Species of Corynebacterium, Enterococcus, Bacteroides, Lactobacillus and Actinomyces were identified as resident bacteria in the rabbit gastrointestinal tract (GIT). Consumption of Vernonia amygdalina extract selectively and significantly (p < 0.05) increased the numbers of Enterococcus and Actinomyces species recovered from the animals faecal specimens, while O. gratissimum increased only the Actinomyces species. In contrast, the numbers of Bacteriodes species were significantly decreased by both extracts. Consumption of V. amygdalina extract also reduced colonization and damage of the animals GIT by Escherichia coli and Staphylococcus aureus as evidenced by restricted shedding of the organisms in faeces and histopathological examination of colon tissues. The results from this study showed that the V. amygdalina leaf extract has prebiotic effects in the rabbit GIT including protection of the animals against some GIT pathogens. Key words: Prebiotics , Vernonia amygdalina, Ocimum gratissimum , gastrointestinal tract, rabbit.

Molecular characterization of Listeria monocytogenes isolated from a ready-to-eat fermented milk and cereal product, Fura-de-Nunu

This study was conducted to determine the occurrence of Listeria (L.) monocytogenes in Fura-de-Nunu, a ready-to-eat (RTE) fermented milk (Nunu) and cereal (Fura) blend, the serogroups as well as the virulence of the isolates. A total of 75 Fura and 75 Nunu samples were examined. Listeria species were isolated on PALCAM medium and Listeria chromogenic agar, and identified phenotypically according to International Standardization Organization (ISO) procedures. Identification of L. monocytogenes, serogrouping and detection of virulence genes were carried out by polymerase chain reaction (PCR). Listeria spp. were recovered from 23 (30.67%) and 41 (54.67%) samples of Fura and Nunu, respectively. The bioloads of Listeria ranged from 103 to 105 CFU/ml. Six presumptive species of Listeria were identified from the samples, with L. monocytogenes accounting for 21.00 and 20.64% of isolates from Fura and Nunu, respectively. Out of the three major serogroups (1/2a, 1/2b and 4b) associated with human disease, only 1/2a and 4b were identified among the isolates. Some of the isolates tested positive for the presence of virulence genes, hlyA and iap. Results from this study show that Fura-de-Nunu, may represent a risk for transmission of listeriosis to consumers. Key words: Listeria monocytogenes, Fura-de-Nunu, fermented milk, ready-to-eat, Listeriosis.