In vitro analysis of the effect of supplementation with activated charcoal on the equine hindgut.

The present study uses in vitro analytical techniques to investigate the effect of activated charcoal on the microbial community of the equine hindgut and the metabolites they produce. Incubations were performed in Wheaton bottles using a 50 ml incubation of a high-energy feed or a low-energy feed, plus bottles with no added food source, together with five levels of activated charcoal (0, 10, 25, 50 or 100 mg per bottle) and fecal samples as a bacterial inoculum. Using this method the rate of gas production, volatile fatty acid and ammonia concentrations, and pH values were analyzed and found to vary depending on the addition of feed, but the activated charcoal had no effect (P>0.05) on any of these. It is already believed that the effect of activated charcoal as a control for toxic substances is at its highest in the foregut or midgut of animals, and therefore should have little impact on the hindgut. The data presented here suggest that if any of the activated charcoal does reach the hindgut, then it has no significant impact on the microbial community present, nor on the major metabolites produced, and so should not have a detrimental effect on the principal site of fermentation in the horse.

Microbial diversity in the digestive tract of two different breeds of sheep.

AIMS: This work aims to determine the factors which play a role in establishing the microbial population throughout the digestive tract in ruminants and is necessary to enhance our understanding of microbial establishment and activity. METHODS AND RESULTS: This study used Terminal Restriction Fragment Length Polymorphism (TRFLP) to investigate the microbial profiles of 11 regions of the digestive tract of two breeds of sheep (Beulah and Suffolk). TRFLP data revealed that the regions of the digestive tract were highly significantly different in terms of the composition of the bacterial communities within three distinct clusters of bacterial colonization (foregut, midgut and hindgut). The data also show that breed was a significant factor in the establishment of the bacterial component of the microbial community, but that no difference was detected between ciliated protozoal populations. CONCLUSIONS: We infer that not only are the different regions of the tract important in determining the composition of the microbial communities in the sheep, but so too is the breed of the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that a difference has been detected in the digestive microbial population of two different breeds of sheep.

Early morning cortisol response and emotional processing in adults exposed to postnatal depression in infancy.

Emotional processing and cortisol were investigated in non-depressed young adults whose mothers experienced PND. PND-exposed participants (n=11) had higher waking salivary cortisol and slower performance on an emotional categorization task than controls (n=15). This supports the hypothesis that early exposure to maternal depression is associated with characteristics reminiscent of vulnerability to depression.

Kaposi's sarcoma: a model of both malignancy and chronic inflammation.

Kaposi's sarcoma (KS) is a complex cancer characterized by angioproliferative multifocal tumors of the skin, mucosa and viscera. KS lesions are comprised of both distinctive spindle cells of endothelial origin and a variable inflammatory infiltrate. There are four different epidemiological forms of KS: classic (sporadic), African (endemic), AIDS-associated (epidemic), and immunosupression-associated (iatrogenic). Although these various forms of KS have different environmental and immunological components, the development of each depends upon infection with Kaposi's sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8). KSHV encodes an arsenal of gene products that induce cellular proliferation, transformation, cell signaling, cytokine production, immune evasion, antiapoptosis and angiogenesis. Yet, KSHV alone is insufficient to give rise to KS. The exact origin of the tumor cell (spindle cell), which is generally agreed to be a type of endothelial cell, remains elusive. Current evidence supports their derivation from lymphatic endothelium. However, both lymphatic and vascular endothelial cell types can be infected by KSHV in vitro, and recent studies suggest that this virus may reprogram the target cell, thus masking the cell's true origin. It is also possible that the original target cell is an uncommitted progenitor. In addition to the potentially neoplastic spindle cells, the KS lesion also contains dendritic cells, macrophages, plasma cells and lymphocytes. The presence of this admixed immune infiltrate has led to the suggestion that KS may result from reactive hyperproliferation induced by chronic inflammation, and that it is therefore not a true neoplasm. This review details the data that support KS as a model of both oncogenesis and chronic inflammation.

Measurement of the resonant d(mu)t molecular formation rate in solid HD.

Measurements of muon-catalyzed dt fusion ( d(mu)t-->4He + n + mu(-)) in solid HD have been performed. The theory describing the energy dependent resonant molecular formation rate for the reaction (mu)t + HD-->[(d(mu)t)pee](*) is compared to experimental results in a pure solid HD target. Constraints on the rates are inferred through the use of a Monte Carlo model developed specifically for the experiment. From the time-of-flight analysis of fusion events in 16 and 37 microg x cm(-2) targets, an average formation rate consistent with 0.897+/-(0.046)(stat)+/-(0.166)(syst) times the theoretical prediction was obtained.

Efficient human immunodeficiency virus-based vector transduction of unstimulated human mobilized peripheral blood CD34+ cells in the SCID-hu Thy/Liv model of human T cell lymphopoiesis.

The methods available to efficiently transduce human CD34(+) hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they fully retain their engraftment potential and maintain high levels of transgene expression in vivo, have been unsatisfactory. The current murine retrovirus-based gene transfer systems require dividing cells for efficient transduction, and therefore the target HSCs must be activated ex vivo by cytokines to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for efficient gene transfer to human HSCs in the absence of any ex vivo cytokine stimulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ability to transduce unstimulated human CD34(+) HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expresses the enhanced green fluorescent protein (EGFP) for evaluating transduction efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-MULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mouse phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34(+) cells as measured by EGFP expression. When these cells were injected into the human fetal thymus implants of irradiated SCID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In contrast, the MULV-based vectors were effective at transducing HSCs only in the presence of cytokines. Our results demonstrate that the improved HIV-based gene transfer system can effectively transduce unstimulated human CD34(+) HSCs, which can then differentiate into thymocytes and provide long-term transgene expression in vivo.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to moloney murine leukemia virus-based vectors.

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34(+) cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin(-) bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Infectivity enhancement by HIV-1 Nef is dependent on the pathway of virus entry: implications for HIV-based gene transfer systems.

Retroviruses have been extensively used in the development of gene transfer systems. Recently, there has been a great deal of interest in the use of lentiviruses for gene transfer because they infect nondividing cells. Human immunodeficiency virus (HIV) has been the lentivirus most often used for this purpose, but its genomic complexity and limited tropism present some challenges to the establishment of efficient gene transfer systems. In this paper we present data showing intrinsic differences between the infectivity of wild-type HIV and HIV particles pseudotyped with heterologous envelope glycoproteins. Interestingly, HIV pseudotypes with envelope glycoproteins from the amphotropic murine leukemia virus or the vesicular stomatitis virus (VSV) are 3 and 40 times more infectious than wild-type HIV, respectively. In addition, we show that the reliance on Nef expression for maximal infectivity of HIV particles is dependent on the path of virus entry. The dependence on Nef for higher infectivity is greater for amphotropic pseudotypes and wild-type HIV than for VSV-G pseudotypes. We conclude that VSV-G pseudotypes of HIV vectors are an excellent choice for gene transfer purposes and Nef-mediated viral infectivity enhancement is affected by virus entry pathway.

The amphotropic murine leukemia virus receptor gene encodes a 71-kilodalton protein that is induced by phosphate depletion.

The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.

Open reading frame 26 of human herpesvirus 8 encodes a tetradecanoyl phorbol acetate- and butyrate-inducible 32-kilodalton protein expressed in a body cavity-based lymphoma cell line.

DNA sequences corresponding to a novel herpesvirus (human herpesvirus 8 [HHV8]) are associated with Kaposi's sarcoma (KS), Castleman's disease, and body cavity-based lymphomas (BCBL). Studies of a BCBL-derived cell line suggest a direct correlation between seropositivity against antigens specifically present in such lines and the development of KS. We have generated recombinant proteins corresponding to open reading frame (ORF) 26 of HHV8 and have produced affinity-purified antibodies. Using these antibodies, we studied the expression of HHV8 ORF26 in a BCBL-derived cell line and found that it encodes a cytoplasmic protein whose expression is induced 16-fold by treatment with phorbol ester or sodium butyrate. This protein induction correlates with a significant induction of viral RNA transcripts. Interestingly, under our experimental conditions minimal increases in viral DNA were observed. No antibodies to the ORF26 protein of HHV8 were found in the sera from two human immunodeficiency virus-positive patients with KS as determined by immunoprecipitation analysis. However, antibodies in the sera from the two KS patients immunoprecipitated a 34-kDa protein found in extracts from induced BCBL1 cells that was not recognized by the control sera.

Immortalization of primary epithelial cells by E1A 12S requires late, second exon-encoded functions in addition to complex formation with pRB and p300.

Immortalization of primary cells is an early and important event in multistep tumorigenesis and is itself a multistep process. Adenovirus E1A 12S encodes an oncoprotein that can rescue cells from senescence and overcome apoptosis, leading to their immortalization. Five regions of 12S, located in both exons, are required for immortalization. Two regions in the first exon are necessary to activate the cell cycle, increase the number of population doublings, and overcome the M1 stage of mortality. However, extension of life span requires overcoming crisis or M2, which can be accomplished by the expression of the second exon. Several cellular proteins associate with the peptide encoded by the first exon of 12S including pRB, p107, p130, and p300. The importance of pRB-E1A and p300-E1A complexes in transformation is well established; however, their roles in 12S-mediated immortalization remain undefined. Results obtained from the present study using a panel of second exon immortalization-defective mutants demonstrate that formation of pRB-E1A and p300-E1A complexes is insufficient for immortalization of primary cells. We further demonstrate that the expression levels of another tumor suppressor protein, p53, also do not correlate with the inability of the mutants to immortalize. Thus, mutations in the second exon of 12S do not affect the early steps in the immortalization pathway. The second exon mutants are defective in performing a late function in immortalization, involving the reactivation of the cell cycle, indicating that it is a crucial event in immortalization.

Structural limitations of the Ad5 E1A 12S nuclear localization signal.

The Ad5 E1A 12S gene encodes an oncoprotein with the ability to immortalize and cooperate with other viral or cellular oncoproteins to transform primary epithelial cells. The immortalizing function is dependent on the protein's efficient localization to the nucleus. A five amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro, has been identified at the extreme COOH-terminus. This signal is necessary but not sufficient for efficient nuclear localization. A mutational analysis has been undertaken to further characterize the 12S NLS. The individual amino acids of the signal appear to have varying functional relevance. The lysine residue (a.a. 239) and the first arginine residue (a.a. 240) are the most critical. Changing the second arginine (a.a. 242) to threonine or either proline (a.a. 241 or 243) to alanine marginally diminishes signal function. Replacing the 12S NLS with the SV40 large T antigen (LT) NLS does not measurably affect the protein's nuclear localization. Sequences directly upstream of the NLS have a significant role in the proper localization of the 12S protein as illustrated by inefficiently localized mutants that have deletions of these sequences. Analyses of these mutants using a monoclonal antibody that recognizes the COOH-terminal four amino acids of the NLS have revealed that their signals are probably masked. To further investigate the importance of protein context in signal function, several NLS insertion mutants were constructed. Two regions in the first exon with predicted high surface probabilities and no known functions were chosen as sites for NLS insertions. Neither a wild-type 12S- nor a SV40 LT-NLS was functional in any of the new locations, indicating that for 12S, positioning of the NLS in the protein is critical.

Efficient nuclear localization and immortalizing ability, two functions dependent on the adenovirus type 5 (Ad5) E1A second exon, are necessary for cotransformation with Ad5 E1B but not with T24ras.

Expression of adenovirus type 5 E1A 12S is sufficient to immortalize primary baby rat kidney cells, but another viral or cellular oncogene, such as E1B or T24ras, is necessary for complete transformation. The regions of 12S sufficient for T24ras cotransformation have been well characterized and are located in the first exon. The second exon is dispensable for ras cotransformation, although it contains a region which appears to modulate the transforming phenotype. The same 12S first exon regions important in ras transformation are also necessary for E1B transformation. Analysis of an extensive series of second exon deletion and amino acid point mutations demonstrated that mutations affecting either the efficient nuclear localization and/or the immortalizing ability of the 12S protein also prevented cooperation with E1B. In general, the entire C-terminal half of 12S, including the nuclear localization signal, was necessary for efficient cotransformation with E1B. In addition to the differences between T24ras and E1B regarding 12S regions necessary for cotransformation, the characteristics of E1B-cotransformed foci differed from those of T24ras. The E1B foci took longer to appear and had a much slower growth rate. No hypertransformed foci were produced with E1B cotransfections, and established E1A-E1B lines exhibited minimal growth in soft agar compared with that of E1A-T24ras lines.

[Snake bites in children: antivenom early reaction frequency in patients pretreated with histamine antagonists H1 and H2 and hydrocortisone]. / Envenenamento ofídico em crianças: frequência de reações precoces ao antiveneno em pacientes que receberam pré-tratamento com antagonistas H1 e H2 da histamina e hidrocortisona.

Type and frequency of early reactions (ER) were studied in 24 children aging 2-14 years victims of snake bites who received pretreatment with histamine antagonists H1 (dextrochlorfeniramine) and H2 (cimetidine or ranitidine) and hydrocortisone from 1989 to 1993. None of them had atopy nor received any type of anti-venoms(AV) and antitoxins before. Of 24 children, 15 received bothropic AV (ER in 5), 7 crotalic AV (ER in 5), 1 crotalic plus crotalic-bothropic AV, and 1 elapidic AV (ER in 1). In 3 children severe early reactions were observed and they were classified as severe crotalic accident. Results suggest that pre-treatment did not offer safety protection at the appearance of early reactions.

Efficient nuclear localization of the Ad5 E1A 12S protein is necessary for immortalization but not cotransformation of primary epithelial cells.

The Ad5 E1A 12S gene encodes a nuclear protein that can immortalize and cooperate with other oncoproteins to transform primary epithelial cells. Expression of the first exon is necessary for the activation of cellular proliferation, immortalization, and transformation. The second exon is necessary for the maintenance of cellular proliferation, immortalization, and the induction of an epithelial cell growth factor but is dispensable for cotransformation with T24 ras. Expression of the second exon is also necessary for efficient nuclear localization of the 12S polypeptide. A five-amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro is encoded by the last 15 nucleotides of the second exon. To determine the role of subcellular localization in immortalization and transformation, a mutational analysis of the second exon and the 12S NLS was undertaken. The results from our analysis indicate that regions of the second exon outside the NLS affect nuclear localization. These regions necessary for efficient nuclear localization of the 12S protein are coincident with regions necessary for immortalization. In contrast, these regions are dispensable for cotransformation with T24 ras. The importance of the positively charged amino acids of the NLS in signal function and immortalization is varied. Lys 239 is the most critical residue, followed by Arg 240 and Arg 242, respectively. These data indicate that efficient nuclear localization of 12S is necessary for immortalization but not cotransformation with T24 ras.

Immortalization of primary epithelial cells requires first- and second-exon functions of adenovirus type 5 12S.

Immortalization of primary cells is a multistep process. The adenovirus E1A 12S gene product is a member of the class of oncoproteins that have the ability to establish primary cells as cell lines in culture. It is encoded by two exons. Extensive mutational analysis demonstrates that four regions of the E1A 12S gene, encoded by both exons, are necessary for immortalization of primary epithelial cells. Expression of two regions is necessary to activate quiescent cells into the cell cycle but is unable to extend the life span of these cells in culture and thus cannot immortalize them. These regions are encoded by the first exon. A third first-exon region, for which no function has yet been identified, is also required. These three regions are also required for 12S to cooperate with an activated ras gene to bring about tumorigenic transformation. The fourth region is required to maintain the cells in a proliferative mode, extend their life span in culture, and induce an autocrine growth factor. These functions are encoded by the second exon. The cells immortalized by wild-type 12S and immortalization-competent mutants retain their epithelial morphology and expression of keratin and vimentin intermediate filament proteins.

Modulation of transformation of primary epithelial cells by the second exon of the Ad5 E1A12S gene.

Expression of the Ad5 E1A first exon is necessary and sufficient to cooperate with an activated RAS oncogene to transform primary epithelial cells. The second exon, although necessary for immortalization and induction of an epithelial cell growth factor, is not essential for co-transformation with T24 RAS. To determine whether the second exon has a role in the cooperation of E1A with an activated RAS gene, we have performed an extensive mutational analysis of this region. All of the deletion and point mutants that we have generated and analyzed retained the ability to enable the transformants to grow in serum-free media and in soft agar. A region in the C-terminus of the E1A polypeptide encoded by nucleotides 1437-1488 appears to modulate the level of transformation. Co-transfections of T24 RAS and E1A genes with mutations that bring about specific amino acid substitutions or deletions in the C-terminus result in enhanced transformation. There is an increase in the number of transformed foci and they appear earlier. A single amino acid change can bring about this phenotype, which is dominant over wild type. Thus, it seems that expression of the wild-type second exon retards or suppresses transformation. The hypertransforming phenotype does not correlate with any differences in the expression of the mutated E1A or the co-cotransfected RAS gene. The C-terminus encodes a nuclear localization signal for E1A, however the subcellular localization of the mutant polypeptides does not affect their co-transforming ability.

Photosensitizers in dermatology.

Systemic injection of hematoporphyrin derivative (HpD) in contribution with visible light (red or blue-green) delivered by laser was used to treat a patient with psoriasis. The psoriatic lesions responded vigorously to laser treatments, forming eschars by 1 week post irradiation. In contrast, only minimal erythema was observed in the noninvolved, clinically normal appearing skin. Two approaches for localized HpD administration were investigated in the guinea-pig and minipig models as a means of achieving local photodynamic effects. Intracutaneous injection of HpD produced localized cutaneous photosensitization with either UVA or red light. Azone increased percutaneous penetration of HpD in human skin in vitro. Topical application of HpD and irradiation with UVA produced localized cutaneous photosensitivity and inhibition of epidermal DNA synthesis.