Monitoring environmental Aspergillus spp. contamination and meteorological factors in a haematological unit.

The opportunistic pathogens belonging to the Aspergillus genus are present in almost all seasons of the year, and their concentration is related to meteorological conditions. The high density of Aspergillus spp. conidia in a haematological hospital ward may be a significant risk factor for developing invasive fungal diseases in immunocompromised patients. Aim of the present study was to evaluate the variability of airborne Aspergillus spp. conidia contamination in a Haematological Unit (HU) within a period of 16 months in relation with some meteorological parameters. An environmental Aspergillus surveillance was conducted in the HU in four rooms and their bathrooms, in the corridor and in three external sites using an agar impact sampler. During each sampling, temperature and relative humidity at each site were recorded and current wind speed and rainfall events were taken from the official weather service. Aspergillus spp. conidia concentration differed significantly across the sampling sites. Internal Aspergillus spp. loads were significantly dependent on temperature, internal relative humidity and rain. External conidia concentrations were significantly influenced by outdoor temperature and relative humidity. A suitable indicator was introduced to evaluate the seasonal distribution of Aspergillus spp. conidia in the sampling sites, and a significant dependence on this indicator was observed inside the HU. Seventeen different fungal species belonging to the Aspergillus genus were detected during the sampling period. Aspergillus fumigatus was the most frequently isolated species and its distribution depended significantly on the seasonal indicator both inside and outside the hospital ward.

Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens.

In this work, two biosurfactant-producing strains, Bacillus subtilis and Bacillus licheniformis, have been characterized. Both strains were able to grow at high salinity conditions and produce biosurfactants up to 10% NaCl. Both extracted-enriched biosurfactants showed good surface tension reduction of water, from 72 to 26-30 mN/m, low critical micelle concentration, and high resistance to pH and salinity. The potential of the two lipopeptide biosurfactants at inhibiting biofilm adhesion of pathogenic bacteria was demonstrated by using the MBEC device. The two biosurfactants showed interesting specific anti-adhesion activity being able to inhibit selectively biofilm formation of two pathogenic strains. In particular, Escherichia coli CFT073 and Staphylococcus aureus ATCC 29213 biofilm formation was decreased of 97% and 90%, respectively. The V9T14 biosurfactant active on the Gram-negative strain was ineffective against the Gram-positive and the opposite for the V19T21. This activity was observed either by coating the polystyrene surface or by adding the biosurfactant to the inoculum. Two fractions from each purified biosurfactant, obtained by flash chromatography, fractions (I) and (II), showed that fraction (II), belonging to fengycin-like family, was responsible for the anti-adhesion activity against biofilm of both strains.

The assessment of airborne bacterial contamination in three composting plants revealed site-related biological hazard and seasonal variations.

AIMS: The purpose of this study was to evaluate the degree of bacterial contamination generated by three Italian composting plants (1, 2 and 3) in two different seasons and to assess the health risk for the employees. METHODS AND RESULTS: Aerosols samples were collected with an agar impact sampler. Several plant sites and external upwind and downwind controls were examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram-negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to isolate pathogenic bacteria. The levels of total mesophilic and thermophilic micro-organisms ranged between 33 and >40,000 CFU m(-3) in plant 1, 39 and 18,700 CFU m(-3) in plant 2 and 261 and 6278 CFU m(-3) in plant 3. Strains of Escherichia coli, Staphylococcus aureus and Clostridium perfringens were also found. CONCLUSIONS: The plants monitored in this study have proved to be sources of aerosolized bacteria. The activities involving mechanical movement of the composting mass and the indoor activities were of greatest potential risk. In all the studied plants, a statistically significant dependence was found between the bacterial contamination and the season for some or almost all the analysed parameters, but a clear seasonal trend could not be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides broad evidence of bacterial aerosol dispersion and site-related biological hazards that may be useful to the regional government to implement regulations on worker safety in composting plants.

Influence of biotic and abiotic factors on human pathogens in a finished compost.

The role of indigenous microflora of a finished compost, defined NK12, on the growth suppression of pathogens under different moisture and temperature storages was investigated. Total count of mesophilic and thermophilic bacteria was evaluated by the most probable number method and growth of seeded Salmonella arizonae 3924 serogroup B and enteropathogenic Escherichia coli 84 M in NK12 at different moisture temperature conditions was monitored. Results on sterile and non-sterile NK12 were compared. In all tested experimental conditions, the NK12 indigenous microflora was stable and biologically active. S. arizonae 3924 and E. coli 84 M grew rapidly in sterilized NK12 at different moistures and storage temperatures, and their growth was suppressed in non-sterilized NK12. Pathogens inactivation was lower when compost was stored at 40% and 80% humidity and at 37 degrees C. Our results show that the major role in the pathogens suppression was played by the indigenous microflora of the finished compost, although physical factors too influenced the growth phenomenon.

Interferons inhibit onset of murine cytomegalovirus immediate-early gene transcription.

Treatment of NIH 3T3 fibroblasts with interferon-alpha or interferon-gamma (IFN-alpha or IFN-gamma) significantly reduced murine cytomegalovirus (MCMV) replication. Determination of viral DNA in the nuclei of the infected cells before onset of DNA replication demonstrated that virus uptake, transport to the nucleus, and DNA stability were not decreased. Analysis of the virus specified mRNAs soon after infection revealed that in the cells exposed to IFNs expression of the immediate early (IE) genes was strongly reduced. Nuclear run-off transcription analysis showed that this inhibition is due to significant reduction of IE gene transcription rates following IFN treatment. Since transcription of the MCMV IE region is regulated by a strong enhancer element, a construct containing the chloramphenicol acetyltransferase (CAT) reporter gene, driven by an 1.2 kb segment spanning the enhancer and IE1/3 promoter region of the IE transcription unit, was transfected into NIH 3T3 cells. Treatment with IFN-alpha or IFN-gamma after transfection strongly reduced CAT activity compared to untreated controls. In an attempt to define a negative IFN-responsive element in the IE enhancer, a series of deletion mutants driving the CAT reporter gene were transfected into NIH 3T3 cells that were then treated with IFN-alpha. With the sole exception of the construct containing the minimal MCMV IE1/3 promoter (-102 to the cap site), all other deletion mutants were strongly down-regulated by IFN-alpha-treatment. Taken as a whole, these results suggest that IFNs inhibit MCMV replication by impairing the transcription of the IE transcription units, and that this negative regulation is carried out by sequences scattered throughout the IE enhancer region.

Effect of interferon-alpha on immediate early gene expression of murine cytomegalovirus.

Interferon-alpha (IFN-alpha) significantly reduced the replication of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts derived from the susceptible mouse strain C3H/HeJ. When infectious virus production was measured, a strong decrease in virus titer was observed in IFN-treated cells at a multiplicity of infection (moi) of 1 and 0.5 pfu/cell. Analysis of virus-specified mRNAs by Northern blot assay revealed that IFN-alpha had a significant effect on the expression of viral mRNAs at 48h. In particular, the mRNAs of the major immediate early (IE) transcription units, IE1, IE2, and IE3, were impaired by IFN-alpha. In addition, decrease of IE1 mRNA synthesis was accompanied by a reduction of the major IE product (pp89), as revealed by Western blot assay. These results suggest that IFN-alpha may inhibit MCMV replication by directly impairing IE gene transcription.

Effects of interferon alpha on murine cytomegalovirus replication.

The present study examined the protective effect of IFN-alpha against mouse cytomegalovirus (MCMV) infection in embryo fibroblasts (MEF) of genetically resistant (C3H/HeJ) and susceptible (C57BL/6) mouse strains. At a M.O.I. of 1 IFN-alpha was protective in C3H/HeJ-MEF but not in C57BL/6-MEF. Dot-blot analysis during MCMV replication in C3H/HeJ-MEF showed that IFN-alpha pretreatment reduced the steady state level of immediate early and late mRNAs but partially reduced early gene expression.

Characterization of the nuclear factors involved in 202 gene induction by IFN-alpha in NIH-3T3 cells.

When treated with IFN-alpha, NIH-3T3 cells express after a few hours high levels of the mouse 202 gene mRNA. This activation takes place at the transcriptional level as shown by nuclear "run on" assay. For this purpose a fragment of 806 base-pairs (the b fragment), spanning the 5'-flanking region of the 202 gene, was linked to the reporter CAT gene and transiently transfected into mouse NIH 3T3. The data suggest that the b fragment is sufficient to confer transcriptional inducibility upon IFN stimulation and can account in large part for the response of the 202 gene. Binding assays, using a 40-bp probe derived from the IFN-stimulated response element (ISRE) comprised in the b fragment, demonstrated the presence of two DNA-binding proteins. One of these, defined as complex A, was inducible upon IFN treatment, whereas the other, defined as complex B, was constitutively present regardless of IFN treatment. The IFN-alpha-induced complex A appears to have the necessary characteristics to be the transcriptional activator of the 202 gene: it requires the same nucleotides for binding as are required for IFN-dependent gene activation and is dependent on IFN-alpha treatment.

Activation of interferon-inducible genes in vivo by synthetic double-stranded RNA, poly rI:rC.

We analyzed the effects of synthetic dsRNA (poly rI:rC) treatment on the expression in vivo of two interferon (IFN)-inducible genes. DBA/2 mice were injected i.p. with poly rI:rC and its effect on the levels of the following mRNAs was determined; 202, 2'-5' oligoadenylate synthetase (2-5A synthetase) and beta-actin. After poly rI:rC treatment the levels of the 202 and 2-5A synthetase mRNAs in the spleen and in bone marrow peaked between 12 and 24 h and decreased thereafter, whereas beta-actin levels remained unchained unchanged. Pretreatment of DBA/2 mice with sheep anti-murine IFN-alpha/beta antibodies before rI:rC injection strongly diminished the induction of 202 mRNA indicating that IFN-alpha/beta mediated this induction.

In-vitro interaction of cephalosporin and the immune system.

In this study we have evaluated the effects of an antibiotic, such as cefonicid, on some immune reactivities in vitro. The following immune parameters have been analyzed: i) interferon-gamma and interleukin-2 production and ii) lymphocyte proliferation following mitogen stimulation in the presence of cefonicid concentrations ranging from 50 to 500 microg/ml. No. decrease in lymphokine production in the presence of cefonicid concentrations up to 250 microg/ml was observed between untreated or antibiotic-treated lymphocyte cultures. When lymphocyte proliferation upon Con A stimulation, as evaluated by 3H-dHtd incorporation, was measured in the presence of cefonicid, no differences were observed with untreated controls. These results demonstrate that the in vitro interaction of cefonicid with the immune system does not lead to a decrease of the immune response.

The role of interferons in the resistance to murine cytomegalovirus.

The protective effect of IFN alpha/beta or IFN-gamma against MCMV infection in mouse embryo fibroblasts (MEF) of genetically resistant and susceptible strains have been examined. For this purpose, MEF derived from Balb/c, C3H, DBA2, C57BL6 mouse strains were used. Cells were pretreated for 24 hrs with either IFN alpha/beta or IFN-gamma and subsequently infected with MCMV at different M.O.I. No difference in susceptibility to MCMV was observed between untreated Balb/c, C3H, C57 and DBA2-MEF when infected at a M.O.I. of 5 whereas at a M.O.I. of 1 or 0.5 untreated DBA2-MEF displayed the highest susceptibility to viral infection. Pretreatment of MEF with IFN alpha/beta showed that the degree of protection to MCMV was dependent on the M.O.I. used. At a M.O.I. of 5, IFN alpha/beta did not inhibit viral replication in any MEF tested, whereas at a M.O.I. of 1 or 0.5 was protective in C3H-MEF and to a lesser extent in Balb/c-MEF. No protection was observed in DBA2 and C57-MEF. Different results were observed when MEF were pretreated with IFN-gamma. C57-MEF were protected when infected at a M.O.I. of 5 or 1; no protection was observed at a M.O.I. of 0.5. Balb/c-MEF were protected only at a M.O.I. of 1. No protection was observed in C3H and DBA2-MEF.

Production of sheep polyclonal antibodies against the 2'-5' oligoadenylate synthetases.

A 20-amino acid peptide corresponding to a common part of the 40 and 46 kD forms of human 2'-5' oligoadenylate synthetase was coupled to keyole lymphet haemocyanin (KLH) and used as immunogen in sheep. After a cycle of four immunizations, immunoglobulins able to recognize the 20-amino acid peptide as evaluated in ELISA assays were purified by an immunoadsorbent with the peptide immobilized on Sepharose CL-4B and used in Western blot employing a secondary anti-sheep antibodies and iodinated protein A as indicator system. Results obtained using extracts from human and mouse cells treated for 15 hr with IFN-alpha as antigen demonstrated that the anti-peptide antibodies recognize several forms of the 2'-5' oligoadenylate synthetase enzyme complex. These antibodies therefore represent a useful tool for monitoring the induction of the above enzymes.

Regulation of gene expression by interferons.

IFNs are a family of proteins produced by various cells following stimulation by biological or synthetic inducers. The interaction with the membrane receptor is followed by the activation of the expression of particular genes that are responsible for their antiviral, antiproliferative and immunomodulatory properties. Initiation of transcription is an early and critical event in the control of eukaryotic gene expression. In this review we will briefly discuss the mechanisms exploited by IFNs to control at transcriptional level the expression of inducible genes. In particular we will focus on some characteristics if cis-acting DNA elements that are located upstream from the initiation site for RNA transcription and of nuclear trans-acting factors that are required for modulation of gene expression by IFNs.

Secretory hydrolases of Trichomonas vaginalis.

Supernatants of three Trichomonas vaginalis strains obtained from culture or after contact and cultivation with WISH cells were assayed to characterize the nature of the factor/s responsible for the cytotoxic activity on WISH cell monolayers. This factor, mainly secreted from the live, exponentially grown parasite, with a molecular weight higher than 10,000 daltons and heat-unstable, does not appear to be a protease. In its place, we demonstrated that the presence of some hydrolases (particularly acid phosphatase and beta-N-acetylglucosaminidase) was related to the cytotoxic activity of T. vaginalis strains. Hence, some hydrolytic enzymes may indicate the pathogenetic role of a T. vaginalis strain.

[Macrophage activation and lymphokine production in the mouse by lymphocytes sensitized against Trichomonas vaginalis]. / Attivazione macrofagica e produzione di linfochine nel topo da parte di linfociti sensibilizzati verso Trichomonas vaginalis.

In this study we have examined whether resident peritoneal macrophages could be activated in vitro by immune T lymphocytes obtained from mice which were immunized with live Trichomonas vaginalis (T. vaginalis). T lymphocytes obtained from mice previously sensitized against T. vaginalis, showed a significant proliferative response when cultured in vitro in the presence of T. vaginalis. Resident peritoneal macrophages obtained from untreated syngenic Balb/c mice revealed an increasing cytotoxic activity against the protozoa when seeded with increasing concentrations of purified immune T lymphocytes and a constant number of H3 thymidine-labeled T. vaginalis. This cytotoxicity was detectable after 24 h of culture and peaked after 48 h. Supernatants obtained from cocultures of macrophages, immune T lymphocytes and T. vaginalis contained gamma-Interferon and TNF alpha/beta.

A new childhood T-cell lymphoma established in nude mice and in vitro.

A T-lymphoma cell line was established from a lymph node biopsy of a boy currently alive in complete remission. Neoplastic cells from this biopsy did not grow in vitro, whereas they formed a progressively growing s.c. tumor in splenectomized and sublethally irradiated nude mice and became serially transplantable in splenectomized and sublethally irradiated nude mice with a stable latency time. After the fourth transplant, cells were stored in liquid nitrogen and referred to as ST-4 cells. ST-4 cells display a membrane phenotype and a karyotype similar to that of the biopsy cells. After thawing, ST-4 cells grow both in splenectomized and sublethally irradiated nude mice and in vitro. They do not secrete interferon or interleukin 2, do not have natural killer activity, and do not respond to mitogen or alloantigen stimulation. The stable features of these T-lymphoma cells and the availability of normal autologous lymphocytes from the patient make this in vivo system quite unique and of importance for studies in tumor immunotherapy.

Influence of gender in pathogenesis of trichomoniasis in congenitally athymic (nude) mice.

Evaluation of abscesses appearing in male and female euthymic and athymic (nude) Balb/c mice after subcutaneous injection of Trichomonas vaginalis in the dorsal region showed that females were more susceptible than males. Female euthymic mice, however, were more susceptible than male athymic mice, and splenectomised athymic males were more susceptible than non-splenectomised athymic males. F1 female athymic mice were the most susceptible, as their abscesses reached a peak size five days earlier than those of athymic homozygous females. F1 male athymic mice, though slightly more susceptible than athymic homozygous males, did not develop abscesses that were similar in size. These results suggest that resistance or susceptibility to T vaginalis infection depends on the gender of the host and on thymus dependent cellular populations.

Use of a crude extract or a purified antigen of Trichomonas vaginalis for the detection of secretory antibodies by ELISA.

A crude aqueous extract (T2) or a gel-filtration purified antigen of T. vaginalis was utilized to detect secretory antibodies in cervico-vaginal secretions by ELISA. The test was developed under optimum conditions using a rabbit anti-Trichomonas serum. Conditions established for monitoring antibodies to trichomonas in immunized rabbits were equally effective for human secretions. The crude extract was capable of assessing secretory antibodies in 45% of women with acute trichomoniasis and 43% of healthy women. The purified antigen showed a marked antigenic activity and compared to the crude antigen, testing 6 samples from infected women, detected a false positive and reduced the O.D. value of another sample by 1/4.

[Cefonicid toxicity. I. Effects on the reactivity of the specific immune system]. / Analisi della tossicita' del cefonicid. I. Effetti sulla reattivita' del sistema immunitario specifico.

The degree of toxicity of the antibiotic Cefonicid on the cellular reactivity of the immune system was evaluated. The effects on some lymphokine (IL-2 and IFN-gamma) production and the degree of proliferation of splenic lymphocytes following mitogen stimulation have been considered. Our results show that Cefonicid does not impair the immune response, except at very high doses (500 micrograms/ml).

[DNA probes in the diagnosis of infectious diseases]. / Le sonde di DNA nella diagnosi delle malattie infettive.

With the recent and rapid developments in recombinant DNA technology it has become much easier to use nucleic acid probes in diagnosis of infectious diseases. In this review some parameters like designing of probes, selection of tags, hybridization reaction conditions and in situ hybridization have been discussed. In addition, some examples have been reported which employ techniques of dot-blot hybridization and in situ hybridization for the diagnosis of latent cytomegalovirus infection.