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Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Summary Intestinal contents of newborn and young germ-free minipigs and germ-free rats were investigated for the following biochemical parameters – conversion of cholesterol to coprostanol, degradation of β-aspartylglycine, level of tryptic activity, formation of urobilinogen and the profile of short-chain fatty acids. Additionally, germ-free minipigs and germ-free rats were monoassociated with non-pathogenic strains of Escherichia coli and were investigated for the same biochemical parameters. The conversion of cholesterol to coprostanol, degradation of β-aspartylglycine, tryptic activity and the short-chain fatty acid profile were similar to those found in previous studies in germ-free animals. Slightly higher amounts of urobilinogen than in the other species investigated so far were found in samples from germ-free and monoassociated minipigs. Except for the total amount of short-chain fatty acids in rats, monoassociation with E. coli did not alter any of the parameters either in the minipigs or in the rats.

The influence of zinc bacitracin (ZB) and of Bacillus licheniformis on host microbial-related functions in young piglets was investigated by applying the concept of microflora-associated characteristics. Six biochemical parameters were determined before and after weaning in faecal samples from piglets in four litters having access to a diet containing ZB, to a diet containing B. licheniformis, to a diet with both additives, or to a diet with no additives, from 3 weeks of age. Statistically significant differences were found in three of the intestinal functions investigated: formation of short-chain fatty acids (at 7 and 10 weeks of age), degradation of mucin (at 7 and 10 weeks of age) and conversion of bilirubin to urobilins (at 7 weeks of age). We also found age-dependent influences on the formation of short-chain fatty acids, on conversion of cholesterol to coprostanol and on conversion of bilirubin to urobilins. We conclude that a functional approach is appropriate for measuring exogenous influence(s) on the microbial intestinal metabolisms in weaned piglets.

Intestinal microbial functions reflect cross-talk between a host and its flora, and external factors may influence these functions. The aim of this investigation was to follow the development of six biochemical microbial-related functions of piglets, raised outdoors (OPs) or indoors (IPs), from birth to slaughter age. The following parameters (microflora-associated characteristic; MAC) were consecutively measured at five different ages: production of short-chain fatty acids (SCFA), conversion of cholesterol to coprostanol and of bilirubin to urobilinogens, inactivation of trypsin, degradation of beta-aspartylglycine and of mucin. Additionally, four parameters (production of SCFA. conversion of cholesterol to coprostanol, inactivation of trypsin, degradation of beta-aspartylglycine) were investigated in faecal samples from germ-free minipigs. The differences in MAC patterns between OPs and IPs were most pronounced at 20 days of age. Differences were found in the total amount of SCFAs, proportions of the acetic, propionic and butyric acids, conversion of bilirubin to urobilinogens, degradation of faecal tryptic activity and degradation of mucin. The values found in the minipigs were within the range of a germ-free animal characteristic (GAC) pattern. Our results show that environmental factors influence the development of some intestinal microbial functions in pigs.

The intestinal flora may act on cholesterol metabolism either, directly by converting the molecule to coprostanol, or indirectly, by influencing upon intestinal bile acid metabolism, mainly by deconjugation. In the present study, sixteen probiotic strains belonging to the genera: Bifidobacterium , Enterococcus , Lactobacillus and Streptococcus were investigated for possible direct action on cholesterol metabolism in vitro and in vivo in ex-germfree mice monoassociated with each probiotic. Additionally, deconjugation of taurodeoxycholic acid (TDC) and of glycodeoxycholic acid (GDC) was investigated in vitro as an indirect effect upon cholesterol metabolism, using the same strains. Gas-liquid chromatography and thin-layer chromatography were used. None of the probiotic strains, was able to transform cholesterol to coprostanol in vitro or in vivo . Of the total probiotics analyzed, B. bifidum B12, L. acidophilus La5, L. acidophilus ATCC4356, L. fermentum ATCC14931, L. plantarum 299v, L. rhamnosus ATCC7469 and E. faecium were able to deconjugate both TDC and GDC in vitro . We conclude that the formation of coprostanol does not account for the supposed cholesterol-lowering effect of the probiotics tested. As some of the probiotics were able to deconjugate TDC and/or GDC in vitro , this microbial function should be studied more extensively in vivo . Keywords: germfree, monoassociated, probiotics, cholesterol, coprostanol, bile acid, taurodeoxycholic acid, glycodeoxycholic acid.

The establishment ofβ-glucuronidase activity was investigated in intestinal contents from gnotobiotic mice monoassociated either with Lactobacillus rhamnosus ATCC 53103 (LGG) or with Escherichia coli X7 for different periods of time. Additionally, the enzyme activity was investigated after the animals had been conventionalized. Neither the monoassociated animals nor the conventionalized animals had reached normal levels ofβ-glucuronidase activity after the colonization period (3 and 7 days, or 7 and 14 days, respectively). A great inter-individual variation of the enzyme activity was observed in the group that was conventionalized for longer (14 days), mainly in the caecum. These results indicate a very slow establishment of theβ-glucuronidase activity in the intestinal tract of mice after exposure to either individual bacterial strains or exclusively to the environment.Key words:β-glucuronidase, intestinal microbial enzymes, germ-free mice, gnotobiotic mice, monoassociation

It is generally accepted that gastrointestinal short-chain fatty acids (SCFAs) - acetic, propionic and butyric acid - are mostly derived from carbohydrates, while iso-butyric and iso-valeric acids are from protein sources.We have investigated the faecal SCFAs and the correlation between the branched-chain fatty acids iso-butyric and iso-valeric in humans, rats, horses and pigs. The subjects were of different ages, fed on different diets and housed in different environments. High differences in the total output of SCFAs were observed within and between species. Despite these differences, a remarkable correlation between the iso-butyric and the iso-valeric acids was found. The fact that the correlation is strong irrespective of species, age, diet and living conditions indicates a common source actually reaching the hindgut. We hypothesize that this source is intestinal sloughed cells. Key words: short-chain fatty acids, iso-butyric acid, iso-valeric acid, diet, environment

A probiotic bacterial strain, Bacillus licheniformis NCTC 13123, has been mono-associated with germ-free mice. After colonization, the following biochemical microflora-associated characteristics (MACs) were analyzed in the large intestinal contents: excretion of short-chain fatty acids (SCFAs), inactivation of tryptic activity, degradation ofβ-aspartylglycine, breakdown of mucin, conversion of cholesterol to coprostanol and transformation of bilirubin to urobilins. An additional MAC, b-glucuronidase activity, was tested in vitro.β-Glucuronidase activity was not expressed by the bacterium. The amount of total SCFAs was lower than that previously found in germ-free mice. Utilization of individual SCFAs rather than production by the strain may be assumed. The other MACs remained similar to the basal values in germfree mice. We conclude that B. licheniformis NCTC 13123 is not able to alter any of the microbial functions investigated in vitro or in the gastrointestinal tract of germ-free mice. Whether and to what extent the strain has the capability to interfere with these functions in the presence of other micro-organisms remains to be investigated. Key wordis : Bacillus licheniforms, probiotics, microflora-associated characteristics, germ-free mice.