Genetic detection and characterization of emerging HoBi-like viruses in archival foetal bovine serum batches.

Bovine viral diarrhea viruses (BVDV) are members of the Pestivirus genus within the family Flaviviridae. Based on antigenic and nucleotide differences, BVDV are classified into two recognized species, BVDV-1 and BVDV-2. More recently, a new putative pestivirus species, tentatively called "HoBi-like", has been associated with bovine viral diarrhea. HoBi-like viruses were first identified in fetal bovine serum (FBS) imported from Brazil. Subsequently, a number of HoBi-like viruses have been detected as contaminants in FBS or cell culture and in live ruminants. To further investigate the possible pestivirus contamination in commercially available FBS batches, 26 batches of FBS with various countries of origin, were tested in this study for the presence of bovine pestiviruses. All the 26 batches were positive by RT-PCR for at least one species of bovine pestiviruses. HoBi-like viruses were detected in 15 batches. Analysis of the 5'UTR and N(pro) sequences of 15 newly identified HoBi-like viruses combined with analysis of additional sequences from GenBank, identified 4 genetic groups tentatively named 3a-3d. The current study confirmed the presence of the emerging HoBi-like viruses in FBS products labeled with different geographic origins. This finding has obvious implications for the safety of biological products, such cell lines and vaccines.

Phylogenetic analysis of small ruminant lentivirus (SRLV) in Italian flocks reveals the existence of novel genetic subtypes.

In order to investigate the genetic heterogeneity of small ruminant lentivirus (SRLV) isolates in Italy, 55 clinical samples collected between 1998 and 2010 were analysed. The phylogenetic study was based on analysis of gag-pol sequences. Our findings revealed that the SRLVs belonged to the subtype A9 (n = 3, sheep), B1 (n = 5, goat), B2 (n = 3, sheep) and E2 (n = 5, goat). Interestingly, 39 isolates from both sheep and goat, significantly differed from all the other SRLVs previously described and formed two separate clusters within genotypes A and B tentatively named A11 (n = 27, goat and sheep) and B3 (n = 12, goat and sheep), which have never been shown before. These results revealed a marked diversity among Italian field SRLV strains which might reflect the absence of any systematic control measures.

Improved Strategy for Molecular Characterization of African Swine Fever Viruses from Sardinia, Based on Analysis of p30, CD2V and I73R/I329L Variable Regions.

African swine fever virus (ASFV) is the aetiological agent of a highly lethal haemorrhagic disease affecting pigs that inflicts significant economic damage on the swine industry. ASF is present in many African countries, in several eastern and central European countries and in Sardinia (Italy). Sequence analyses of variable genomic regions have been extensively used for molecular epidemiological studies of ASFV isolates. Previous sequencing data of genes that codify for viral protein p54, p72 and the central variable region (CVR) within the B602L gene revealed that Sardinian isolates show a very low level of variability. To achieve a finer level of discrimination among such closely related viruses, in this study, we have chosen three different genome regions to investigate the within-genotype relationships and to provide a more accurate assessment of the origin of outbreaks. The analysis of p30 and I73R/I329L sequences obtained from ASFV collected in Sardinia over a 13-years period confirms a remarkable genetic stability in these regions. The sequence comparison of the protein encoded by the EP402R gene (CD2v), carried out on various strains from 1978 to 2014, revealed a temporal subdivision of Sardinian viruses into two subgroups: one group includes the historical isolates from 1978 to 1990, and the second one is comprised of the viruses collected from 1990 until 2014. These data, together with those obtained from CVR within the B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72 genotype I, but have undergone genetic variations in two different regions of the genome since 1990. We proposed the cytoplasmic region of CD2v protein as a new genetic marker that could be use to analyse ASFVs from different locations to track virus spread. Our study reaffirms the need to analyse other genome regions in order to improve the molecular characterization of ASFV.

Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins.

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity.

Interleukin-1 and interleukin-6 differentially regulate the accumulation of newly synthesized extracellular matrix components and the cytokine release by developing chick embryo skin fibroblasts.

In the present study, we demonstrate that both interleukin-1 (IL-1) and interleukin-6 (IL-6) induced a significant decrease in glycosaminoglycan (GAG) synthesis and, more strikingly, secretion by 7 and 13 day-old chick embryo skin fibroblasts. We demonstrated that interleukin treatment also inhibited the synthesis of collagenase-digestible proteins (type I collagen). In addition, tissue culture supernatants (conditioned media, CM) were tested for reactivity for IL specific ELISAs and for their ability to stimulate proliferative responses in mouse thymocytes and hybridoma cells. Our findings demonstrate that chick embryo skin fibroblasts spontaneously produce IL-1 and, in even greater amounts, IL-6. Highest levels of interleukin secretion were found in the CM of 13 day-old fibroblasts and the IL-1 beta isoform was predominant over IL-1 alpha. Pretreatment of the fibroblasts with either IL-1 or IL-6 increased the secretion of both cytokines. Increased IL-1 levels were correlated with enhanced IL-1 bioactivity in the CM of IL-6 treated fibroblasts. By contrast, the raised concentrations of IL-1 in the CM of IL-1 treated cells and IL-6 in the CM of IL-1 or IL-6 treated fibroblasts failed to translate into augmented bioactivity. These observations, taken together, indicated that IL-1 and IL-6 are able to regulate the synthesis and secretion of ECM macromolecules of developing connective tissues and the cytokine release by chick embryo skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study.

Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.

Interleukin pattern of Apert fibroblasts in vitro.

The phenotype of cultured fibroblasts from patients affected by Apert's syndrome, a rare connective disorder, differs from that of normal cells in its extracellular matrix macromolecule composition (glycosaminoglycans, collagens and fibronectin) and is further modulated by treatment with interleukins (ILs). As the mechanisms responsible for the changes are unknown, we used our recently described model system for Apert periosteal fibroblasts to ascertain whether the pattern of ILs they secrete into the medium is comparable to that of normal fibroblasts. The results obtained by enzyme-linked immunosorbent assay (ELISA) show that the levels of interleukin-1 (IL-1) and interleukin-6 (IL-6) were lower in Apert than in normal media, whereas levels of IL-1 receptor antagonist (IL-1ra), the natural inhibitor of IL-1, were markedly higher. IL-1 specific bio-activity on thymocyte proliferation was also decreased in Apert supernatants. As we provided also evidence that active transforming growth factor beta (TGFbeta1), an IL-1 antagonist, was not secreted in greater amount in Apert media with respect to normals, the enhancement of IL-1ra appeared critical in down-regulating IL-1. Northern blot analysis of cytokine mRNA revealed no detectable IL-1 or IL-6 gene expression in normal fibroblasts, but high amounts of IL-6 mRNA transcripts in Apert cells. As the increased IL-6 gene expression did not translate into a parallel increase of secreted IL-6, the control of IL-6 secretion may be mainly post-transcriptional. Furthermore, the result that a treatment of the cultures with IL-1ra was able to induce a decrease of IL-6 secretion, suggests that the observed decreased secretion of IL-6 may be due to the autocrine action of overproduction of IL-1ra. The observed imbalance in the production of ILs which we show for the first time suggests ILs may be the natural autocrine regulators of ECM production in Apert fibroblasts. We hypothesize that in vitro differences previously reported in fibroblast phenotypes and several clinical features of Apert's syndrome may correlate with different cytokine patterns.

Cell uptake and DNA adduct formation of the 2-amino-3 methyl-imidazo (4,5-f) quinoline in human enterocytes and chick embryo liver cells.

The compound 2-amino-3-methyl-imidazo(4,5-f)quinoline (IQ) is a powerful mutagenic compound that can induce tumours in different rat and murine target organs (liver, forestomach and small and large bowel). The ability of mutagenic IQ to form adducts to human embryonic enterocyte DNA (intestine 407 cell lines ATCC) has been studied, considering the DNA extracted from the cells and DNA of the in vitro-cultured cells. The activation with the rat hepatic microsomial fraction S9 plays an important role not only in DNA binding, but also in the uptake of IQ by the enterocytes. In the presence of S9 mix, the DNA adduct formation increases with the incubation time; in the absence of metabolic activation, the binding does not occur. Parallel experiments were carried out for comparison on chick embryo hepatocytes; for these cells, the metabolic activation with S9 mix is not as critical as for enterocytes in IQ uptake and DNA binding.

Antimicrobial susceptibility of potentially pathogenic halophilic vibrios isolated from seafood.

Susceptibility patterns to 27 antimicrobial agents and beta-lactamase production were investigated in potentially pathogenic halophilic vibrios from seafood. The effect of salinity on the response to the drugs in vitro was also studied. All isolates were uniformly sensitive to choramphenicol, imipenem, meropenem but resistant to lincomycin. All were highly sensitive to oxolinic acid, trimethoprim-sulphamethoxazole, doxycycline, flumequine, cefotaxime, nalidixic acid and ciprofloxacin. Some strains of V. harveyi, V. alginolyticus and V. parahaemolyticus apparently had mechanisms of resistance to several beta-lactam antibiotics other than by the production of beta-lactamases. Sixty-nine strains produced penicillinase but a low correlation between beta-lactamase activity and resistance to beta-lactam antibiotics was noted. The salt concentration affected the in vitro susceptibility of halophilic vibrios and the effect of salinity depended on both the individual strains and the antimicrobial tested.

Chondroitin sulphates and embryonic chick skin fibroblast proliferation.

We have investigated the action of sulphated glycosaminoglycans (GAGs) upon primary 11-day chick embryo skin fibroblasts cultured for various periods of time in the presence or absence of fetal calf serum (FCS). Chondroitin 4- and 6-sulphate (CS) added to serum supplemented medium provoked a decrease in DNA synthesis both in sparse and crowded cultures only at high concentrations (250 micrograms/ml). Synthesis of endocellular and secreted proteins was not affected. In contrast, CS administered to fibroblasts for 24 h in serum-free medium stimulated DNA synthesis. We postulate that the CS stimulating effect seen in serum-free cultures might be antagonized by peptide regulators of cell growth normally present in serum.

Genetic detection and characterization of atypical bovine pestiviruses in foetal bovine sera claimed to be of Australian origin.

Two European laboratories independently detected atypical bovine pestiviral nucleic acids in three commercial batches of foetal bovine serum (FBS) that was claimed by the producers to be of Australian origin. Additional batches of FBS were obtained directly from Australia to exclude possible contamination of the Australian FBS with that of South American origin during manufacturing/packaging in European countries. RT-PCR amplification of partial 5'untranslated region and the complete N(pro) gene yielded a specific band with expected size, which was confirmed by DNA sequencing. Bayesian analysis of sequence data demonstrated a closer phylogenetic relation of the newly detected atypical bovine pestiviruses to those of South American origin, which were related to the recognized bovine pestivirus species, bovine viral diarrhoea virus. Taken together, the results indicated the presence of atypical bovine pestiviruses in the Australian FBS, and most likely in Australian Continent.

Seroconversion against SU5 derived synthetic peptides in sheep experimentally infected with different SRLV genotypes.

Synthetic peptides were generated, corresponding to SU5 domain of envelope glycoprotein of Italian SRLV isolates It-561 and It-Pi1, belonging respectively to MVV- and CAEV-like genotypes. The peptides, encompassing an N-terminal variable and a C-terminal conserved antibody-binding site, were used in an ELISA assay to analyse the sera of two groups of sheep experimentally infected with these isolates. The kinetics and specificity of the humoral response to the homologous and heterologous antigen and the affinity maturation of the sera were evaluated. Seroconversion occurred between week 3 and 8. The response to SU5 antigen was mostly type-specific. The few broadly reacting sera may reflect the production of antibodies directed to the SU5 constant antibody-binding site. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies. This study suggests constant monitoring of the circulating viral variants to develop a panel of diagnostic peptides representative of local genotypes.

Genetic characterization of a caprine pestivirus as the first member of a putative novel pestivirus subgroup.

Currently, the genus Pestivirus comprises four approved species, namely bovine viral diarrhoea viruses 1 and 2 (BVDV-1, BVDV-2), classical swine fever virus and border disease virus (BDV). Recently, three major genotypes have been identified within the species BDV and termed as subgroups BDV-1, BDV-2 and BDV-3. Here, an isolate from animals in a herd showing BD-like syndromes, which occurred in central Italy was analysed. A reverse transcriptase polymerase chain reaction was performed using primers that specifically amplify a fragment of the 5'-non-coding region (5'-NCR) from BDV. Both the 5'-NCR fragment and the entire Npro gene were sequenced and used for genetic typing. The 5'-NCR sequence revealed that the newly isolated Pestivirus could be allocated to the BDV species. Interestingly, the Npro sequence of this virus isolate significantly differed from all the ovine pestiviruses previously described, providing evidence for the presence of an additional subgroup within the species BDV.

Early induction of interleukin-12 by human monocytes exposed to Cryptococcus neoformans mannoproteins.

Interleukin-12 (IL-12) production by human monocytes stimulated with mannoproteins (MPs) of Cryptococcus neoformans was investigated. The results reported show that secreted or cell-associated MPs induce an early and significant production of IL-12. MPs show different capabilities to quantitatively affect IL-12 production; MP2, an 8. 2-kDa MP purified from the culture supernatant of C. neoformans, appears to be the most potent stimulator. Cytochalasin B inhibits both internalization and IL-12 induction by MP. In addition, a drastic reduction of IL-12 was observed when monocytes were cultured in the absence of normal human serum or treated with soluble mannan. Early production of IL-12 promotes early secretion of gamma interferon by T cells but does not influence the magnitude of the MP-induced lymphoproliferative response. Overall our results identify cryptococcal antigens responsible for rapid and potent induction of IL-12 in monocytes. MPs appear to regulate IL-12 secretion by internalization via the endocytic pathway and by interaction with monocyte receptors or serum factors.

Role of growth factors on extracellular matrix production by chick embryo fibroblasts in vitro. Antagonist effect of TGF-beta through the control of IL-1 and IL-1Ra secretion.

Growth factors and extracellular matrix (ECM) macromolecules create specific environments that regulate cell proliferation and differentiation during embryonic development. This study had two main aims: (1) to characterize the phenotype of 7- and 13-day-old chick embryo fibroblasts treated with interleukin 1 (IL-1) and interleukin 6 (IL-6) evaluating in particular the neosynthesis of the total proteins and of the fibronectin (FN); (2) to examine the mechanisms through which transforming growth factor beta (TGF-beta) antagonizes the action of IL-1. The results show that protein neosynthesis and secretion is modulated by treatment with IL-1 and IL-6 only in 7-day-old fibroblasts. IL-1 favours cellular accumulation, while IL-6 promotes secretion more. FN, isolated by affinity chromatography and analysed by SDS-PAGE and flurography, is produced in greater concentrations by 7-day-old fibroblasts. Treatment with IL-1 promotes the accumulation of FN in the cellular compartment both at 7 and 13 days, while it stimulates the secretion only in 7-day-old fibroblasts. IL-6 only has a stimulating effect on the processes of accumulation and secretion on 13-day-old fibroblasts. The authors show in addition, that TFG-beta blocks the IL-1 induced inhibitory effect on glycosaminoglycan (GAG) and collagen production, evaluated with [3H]glucosamine and [3H]proline incorporation studies, respectively. The effect is evident when TGF-beta is added either before of with the cytokine. Analysis in cell culture supernatants, using specific ELISA kit and the study of the proliferative responses in mouse thymocytes, showed that TGF-beta induces its antagonist effects through a downregulation of IL-1 and/or an upregulation of IL-1 receptor antagonist protein (IL-1Ra). Considered as a whole the data indicate that the ILs and TGF-beta are involved in the physiological age-dependent accumulation of ECM and that the balance between the secreted cytokine network and IL-1Ra amount, may represent a homeostatic mechanism aimed at controlling normal embyrogenesis.

Effects of interleukins on Crouzon fibroblast phenotype in vitro. Release of cytokines and IL-6 mRNA expression.

In Crouzon's syndrome, the cranial morphogenic processes are altered due to the early fusion of the facial and basal cranial bones. Evidence that Crouzon fibroblasts have an altered phenotype which is modulated by interleukin 1 (IL-1) and interleukin 6 (IL-6) is provided. [3H]Thymidine incorporation and cell count studies showed that Crouzon fibroblasts have an accelerated proliferation rate. [3H]Glucosamine incorporation studies, followed by chromatography analysis of culture medium samples, revealed that the various classes of glycosaminoglycans (GAG) secreted into the medium were differently distributed in Crouzon and normal fibroblasts. Crouzon fibroblasts secreted less total GAG, particularly hyaluronic acid (HA) and heparan sulfate (HS), but a relatively greater quantity of chondroitin sulfate. Type I and III collagen were raised in Crouzon fibroblast medium whereas the concentration of fibronectin was lower than in normal cells. Interleukin treatment induced changes in cell growth and neosynthesis of extracellular matrix macromolecules. Both IL-1 and IL-6 stimulated proliferation of Crouzon fibroblasts, whereas only IL-6 increased [3H]thymidine incorporation in normal cells. IL-1 provided a drop in HA and a rise in GAG sulfates in normal fibroblasts, but caused an opposite effect in Crouzon fibroblasts. Type I collagen and fibronectin secretions are differently modulated by the cytokines in the two populations. Moreover, level of IL-1 able to stimulate [3H] thymidine incorporation into mouse thymocytes, and level of IL-1 receptor antagonist (IL-1ra), as determined by ELISA, were higher in pathological than in normal fibroblasts. Also evidence that levels of IL-1 alpha and IL-6 proteins measured by ELISA and also IL-6 mRNA expression are enhanced in Crouzon fibroblasts is provided. These novel data suggested that, in Crouzon's syndrome, the modifications in the relative concentrations of the extracellular matrix (ECM) components associated with the abnormal cytokine networks may alter the balance of the microenvironment where the morphogenic events take place.

Internalization of Candida albicans and cytoskeletal organization in macrophages and fibroblasts treated with concanavalin A.

This paper investigates the ability of macrophages and of non-typically phagocitic cells such as fibroblasts to internalize 51Cr-labelled C. albicans in presence or in absence of lectin concanavalin A (Con A). The results obtained demonstrate that fibroblasts are also able to internalize C. albicans and that this property is potentiated by the presence of Con A. Lectin modifies only the phenotype of the fibroblast, which, poorly attached to the substrate, is globular in shape. Despite reduced cellular spreading, phagocytosis is stimulated by the lectin. In both cell populations, changes in the organization of some cytoskeletal proteins such as tubulin, actin and alpha-actinin are evident during the C. albicans infection; such rearrangements are more evident and longlasting in the fibroblasts treated with Con A.

Glycosaminoglycan metabolism and cytokine release in normal and otosclerotic human bone cells interleukin-1 treated.

Glycosaminoglycans (GAGs), normal components of the extracellular matrix (ECM), and the glycosidases, that degrade them, play a key role in the bone remodelling process. The effects of interleukin-1 alpha (IL-1 alpha) on GAG metabolism in normal and otosclerotic human bone cells as well as its capacity to modulate IL-1 alpha, IL-1 beta and IL-6 secretion in both populations was analyzed. The amount of radiolabeled GAGs was lower in otosclerotic than in normal bone cells. IL-1 alpha reduced newly synthesized cellular and extracellular GAGs in normal cells, but only those of the cellular compartment in otosclerotic bone cells. It depressed heparan sulphate (HS) more in normal cells and chondroitin sulphate (CS) more in otosclerotic bone cells. The HA/total sulphated GAG ratio was shifted in favour of the latter in otosclerotic cells, whereas the opposite effect was seen after IL-1 alpha treatment. There was little difference in the beta-D-glucuronidase levels of the normal and pathological cells, while beta-N-acetyl-D-glucosaminidase was significantly increased in otosclerotic bone cells. As the activity of neither enzyme was modified by treatment with IL-1 alpha, the cytokine seems to exert its influences on GAG synthesis rather than on the degradation process. IL-1 alpha, IL-1 beta and IL-6 secretion was markedly higher in otosclerotic cells. IL-1 alpha modulated the secretion of each interleukin differently, thus resulting in a cytokine cascade that may act in autocrine/paracrine manner on target cells. The authors suggest that changes in the cytokine network may have a specific, yet still unknown, role during normal and pathological osteogenesis.

Phenotype of in vitro human otosclerotic cells and its modulation by TGF beta.

A study was carried out to obtain a more detailed picture of the phenotypes of human otosclerotic and normal bone cells and to analyse the response of both populations to treatment with TGF beta 1. Total collagen synthesis was found to be decreased, but fibronectin secretion increased in otosclerotic with respect to normal cells. Although overall glycosaminoglycan (GAG) synthesis was lower in otosclerotic cells, the sulphated GAG to hyaluronic acid (HA) ratio was higher, in particular there was greater expression of chondroitin (CS) and dermatan sulphates (DS). TGF beta 1 induced a more marked increase in collagen and fibronectin release and greater production of sulphated GAGs as DS and heparan sulphate (HS) in the otosclerotic cells. The fact that the phenotype of the otosclerotic cells differed from that of the normal cells and could be modified by TGF beta 1 treatment, suggests that TGF beta 1 is implicated in the pathogenesis of otosclerosis.

Collagen synthesis and cell growth in chick embryo fibroblasts: influence of colchicine, cytochalasin B and concanavalin A.

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases alpha 2 more than a1 chain production. 3H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.