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Vitamin E and selenium are two components which contribute to the antioxidant potential of plasma and tissues. In the present study we aimed to define the type of tissue toxicity deriving from chronic deficiency of either vitamin E or selenium and to evaluate the reliability of peripheral markers of tissue toxicity in these conditions. We studied rats fed a vitamin E or selenium-deficient diet for 3 or 7 months and a selenium-supplemented diet. The effectiveness of the dietary treatment was confirmed by measuring vitamin E and selenium in plasma. Heart and kidney malondialdehyde (MDA), a typical product of lipid peroxidation, was significantly increased after the 3-month diet in both vitamin E- and selenium-deficient rats. The iron-binding capacity of plasma, an activity ascribed to plasma transferrin, was reduced in selenium-deficient and increased in selenium-supplemented animals. In red cells globular resistance (resistance to osmotic haemolysis) was low in vitamin E- and selenium-deficient, but high in selenium-supplemented animals. Glutathione peroxidase was also increased in selenium-supplemented rats. Platelet count did not differ from controls in any of the three conditions studied. Platelet MDA formation induced by arachidonic acid was raised in both selenium-deficient and, particularly, vitamin E-deficient groups. This can be regarded as a peripheral marker of reduced antioxidant defence at tissue level.

Adriamycin induces a nephrotic syndrome in rats characterized by severe ultrastructural changes of visceral epithelial cells similar to those observed in puromycin aminonucleoside (PA) nephrosis and in human 'minimal changes' glomerulopathy. Since steroids have been shown to be effective in human 'minimal changes' glomerulopathy and in PA nephrosis, we undertook the present study to assess whether steroids had a therapeutic effect on adriamycin nephrosis. Groups of rats injected with different doses of adriamycin were subsequently treated with prednisolone. No significant differences were observed in proteinuria and in ultrastructural findings between the control and the steroid-injected animals. This study suggests that the mechanism underlying adriamycin-induced nephrotic syndrome might be different from that responsible for PA nephrosis or human 'minimal changes' glomerulopathy.

A survey network for congenital toxoplasmosis (TOXO-NET) was set up in December 1996 in Piedmont (Italy). Participants were asked to classify the infections in pregnant mothers and newborns by the criteria of the European Network on Congenital Toxoplasmosis published by Lebech in 1996. Because the IgG Avidity test is largely employed as a 2nd level test in toxoplasmosis diagnosis and it could be helpful to date infection, the co-ordinators of TOXO-NET suggested including it in the "case definition" of "probable" infection and "unlikely" infection. 117 cases of toxoplasmosis in pregnancy divided into the risk categories under Lebech's criteria were re-examined using the "new" case definitions. 77 out of 117 (65.8%) Toxoplasma gondii infections during pregnancy could be defined with only one serum sample using the IgG Avidity test. The IgG Avidity test proved a useful method to classify the Toxoplasma gondii infections in pregnancy, especially when we had only one serum sample.

We have investigated the relevance of some laboratory tests of platelet function in predicting conditions of thrombotic tendency. For this purpose, we studied platelet survival, platelet aggregation in response to different stimuli, TxB2 and 6-keto-PGF1 alpha production in serum of rats bearing a nephrotic syndrome induced by adriamycin. These animals show a heavy predisposition to the development of both arterial and venous thrombosis. The mean survival time was normal in nephrotic rats in comparison to controls. As to aggregation tests, a lower aggregating response was found in ADR-treated rats using ADP or collagen as stimulating agents. With arachidonic acid (AA) we observed similar aggregating responses at lower AA concentrations, whereas at higher AA concentrations a significantly lower response was found in nephrotic rats, despite their higher TxB2 production. Also TxB2 and 6-keto-PGF1 alpha levels in serum of nephrotic rats were significantly higher than in controls. No consistent differences were found in PGI2-activity generated by vessels of control or nephrotic rats. These data show that platelet function may appear normal or even impaired in rats with a markedly increased thrombotic tendency. On the other hand, the significance of high TxB2 levels in connection with mechanisms leading to thrombus formation remains a controversial issue.

IDN5109 is a new taxane, derived from 14beta-hydroxy-10-deacetylbaccatin III, selected for its lack of cross-resistance in tumor cell lines expressing the multidrug resistant phenotype. Because, unlike paclitaxel, IDN5109 is a poor substrate for P-glycoprotein, we hypothesized that IDN5109 given p.o. could improve bioavailability compared with paclitaxel. Here, we studied the p.o. and i.v. pharmacokinetics of IDN5109 together with its antitumor activity. Using a high-performance liquid chromatography method, the bioavailability of IDN5109 was determined to be 48% after oral delivery. IDN5109 given p.o. was highly active against the two human ovarian carcinoma xenografts 1A9 and HOC18 (90-100% tumor regressions) and showed significant activity on the paclitaxel-resistant MNB-PTX1 xenograft (10% tumor regressions). The p.o. administration was as active as the i.v. route at doses reflecting the pharmacokinetic data. IDN5109 is the first taxane with good oral bioavailability and potent antitumor activity and represents a potential candidate for clinical investigation.

International audience; Consumption of flavonoid-rich foods and beverages is thought to reduce the risk of cardiovascular diseases. Whereas the biological activities of flavonoids have been characterized in vitro, there are no clear experimental data demonstrating that chronic dietary intake and intestinal absorption of flavonoids actually protects the heart against ischemia-reperfusion injury. We tested whether long-term consumption of specific flavonoids (anthocyanins) included in normal food could render the heart of rats more resistant to myocardial infarction. Maize kernels that differed specifically in their accumulation of anthocyanins were used to prepare rodent food in which anthocyanins were either present or absent. Male Wistar rats were fed the anthocyanin-rich (ACN-rich) or the anthocyanin-free (ACN-free) diet for a period of 8 wk. Anthocyanins were significantly absorbed and detected in the blood and urine of only rats fed the ACN-rich diet. In Langendorff preparations, the hearts of rats fed the ACN-rich diet were more resistant to regional ischemia and reperfusion insult. Moreover, on an in vivo model of coronary occlusion and reperfusion, infarct size was reduced in rats that ate the ACN-rich diet than in those that consumed the ACN-free diet (P < 0.01). Cardioprotection was associated with increased myocardial glutathione levels, suggesting that dietary anthocyanins might modulate cardiac antioxidant defenses. Our findings suggest important potential health benefits of foods rich in anthocyanins and emphasize the need to develop anthocyanin-rich functional foods with protective activities for promoting human health.

Cathelicidins are antimicrobial peptides, stored by mammalian leukocytes, showing an antimicrobial activity against bacteria, fungi, protozoa and enveloped viruses. In accordance with other authors, we reported in a previous study that the protegrin-1 (PG-1), at 80 microg mL(-1), inhibited the in vitro growth of Chlamydia trachomatis serovars D, H and L2; however, we observed an increased infectivity of some animal chlamydial species after their treatment with the same PG-1 concentration. In this study, the treatment of LLC-MK2 cells with PG-1 before chlamydial infection resulted in an increased infectivity of Chlamydophila abortus probably due to their easier entry into the host cells, whereas no increase in S26/3 infectivity was detected in LLC-MK2 cells treated with PG-1 postchlamydial infection.

Nine Chlamydia suis isolates, obtained from pigs with conjunctivitis, were molecularly characterized by ompA sequencing and their in vitro susceptibility to six cathelicidin peptides (SMAP-29, BAC-7, BMAP-27, BMAP-27, BMAP-28, PG-1, LL-37) determined in cell culture. SMAP-29 was the most active peptide, reducing the intracellular inclusion number by ≥50% at a concentration of 10 μg/ml (3 μM) in six of the nine isolates tested. Three molecularly identical isolates were insensitive at a concentration as high as 80 μg/ml (25 μM). Of the remaining cathelicidin peptides tested, BAC-7 and BMAP-27 were active against six C. suis isolates at a concentration of 80 μg/ml (25 and 26 μM, respectively). Cathelicidins LL-37 and PG-1 did not show any anti-chlamydial activity at 80 μg/ml.

Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8+ T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8+ T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts., MPS-I patients receiving enzyme replacement therapy develop an anti-IDUA immune response that may affect ex vivo gene therapy. Squeri et al. show, in the MPS-I murine model, that pre-existing IDUA-specific CD8+ T cell response impairs LV-transduced HSC engraftment and that the efficacy of ex vivo gene therapy is rescued by lympho-depleting drugs.

Until recently, Chlamydia psittaci was considered to be the only etiological agent of avian chlamydiosis, but two new avian species, Chlamydia gallinacea and Chlamydia avium, have recently been described in poultry and pigeons or psittacine birds, respectively. The aim of this study was to explore the occurrence of C. psittaci and C. gallinacea in backyard chickens in Italy. Cloacal swabs were taken from 160 asymptomatic chickens reared in 16 backyard farms. Samples were tested for C. psittaci and C. gallinacea by specific real-time polymerase chain reaction assays, with 24 (15%) of the 160 chickens resulting positive for C. gallinacea. To attempt chlamydial isolation, new samples were obtained from two farms harboring a high prevalence (60% and 70%, respectively) of C. gallinacea-positive chickens. In total, eight C. gallinacea and one C. psittaci isolates were successfully recovered from 13 chickens. C. gallinacea was confirmed to be the endemic chlamydial species in chickens, with a high ompA intraspecies diversity. The presence of viable C. psittaci and C. gallinacea demonstrated by isolation from chickens in backyard farms poses a potential public health problem.

A mouse model for Chlamydia suis genital infection was developed. Ninety-nine mice were randomly divided into three groups and intravaginally inoculated with chlamydia: 45 mice (group 1) received C. suis purified elementary bodies (EBs), 27 (group 2) were inoculated with C. trachomatis genotype E EBs and 27 mice (group 3) with C. trachomatis genotype F EBs. Additionally, 10 mice were used as a negative control. At seven days post-infection (dpi) secretory anti-C. suis IgA were recovered from vaginal swabs of all C. suis inoculated mice. Chlamydia suis was isolated from 93, 84, 71 and 33% vaginal swabs at 3, 5, 7 and 12 dpi. Chlamydia trachomatis genotype E and F were isolated from 100% vaginal swabs up to 7 dpi and from 61 and 72%, respectively, at 12 dpi. Viable C. suis and C. trachomatis organisms were isolated from uterus and tubes up to 16 and 28 dpi, respectively. The results of the present study show the susceptibility of mice to intravaginal inoculation with C. suis. A more rapid course and resolution of C. suis infection, in comparison to C. trachomatis, was highlighted. The mouse model could be useful for comparative investigations involving C. suis and C. trachomatis species.

Chlamydia pneumoniae and human cytomegalovirus (HCMV) are intracellular pathogens able to infect hepatocytes, causing an increase in serum triglycerides and cholesterol levels due to the production of inflammatory cytokines. We investigated whether these pathogens could interfere with cholesterol metabolism by affecting activity of hepatic cholesterol 7α-hydroxylase ( CYP7A1 ) promoter. CYP7A1 is the rate-limiting enzyme responsible for conversion of cholesterol to bile acids, which represents the main route of cholesterol catabolism. A straightforward dual-reporter bioluminescent assay was developed to simultaneously monitor CYP7A1 transcriptional regulation and cell viability in infected human hepatoblastoma HepG2 cells. C. pneumoniae and HCMV infection significantly decreased CYP7A1 promoter activity in a dose-dependent manner, with maximal inhibitions of 33 ± 10% and 32 ± 4%, respectively, at a multiplicity of infection of 1. To support in vitro experiments, serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose levels were also measured in Balb/c mice infected with C. pneumoniae. Serum cholesterol and triglycerides also increased in infected mice compared with controls. Although further investigation is required, this work presents the first experimental evidence that C. pneumoniae and HCMV inhibit CYP7A1 gene transcription in the cultured human hepatoblastoma cell line.

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

A large evidence-based review on the effects of a moderate consumption of beer on human health has been conducted by an international panel of experts who reached a full consensus on the present document. Low-moderate (up to 1 drink per day in women, up to 2 in men), non-bingeing beer consumption, reduces the risk of cardiovascular disease. This effect is similar to that of wine, at comparable alcohol amounts. Epidemiological studies suggest that moderate consumption of either beer or wine may confer greater cardiovascular protection than spirits. Although specific data on beer are not conclusive, observational studies seem to indicate that low-moderate alcohol consumption is associated with a reduced risk of developing neurodegenerative disease. There is no evidence that beer drinking is different from other types of alcoholic beverages in respect to risk for some cancers. Evidence consistently suggests a J-shaped relationship between alcohol consumption (including beer) and all-cause mortality, with lower risk for moderate alcohol consumers than for abstainers or heavy drinkers. Unless they are at high risk for alcohol-related cancers or alcohol dependency, there is no reason to discourage healthy adults who are already regular light-moderate beer consumers from continuing. Consumption of beer, at any dosage, is not recommended for children, adolescents, pregnant women, individuals at risk to develop alcoholism, those with cardiomyopathy, cardiac arrhythmias, depression, liver and pancreatic diseases, or anyone engaged in actions that require concentration, skill or coordination. In conclusion, although heavy and excessive beer consumption exerts deleterious effects on the human body, with increased disease risks on many organs and is associated to significant social problems such as addiction, accidents, violence and crime, data reported in this document show evidence for no harm of moderate beer consumption for major chronic conditions and some benefit against cardiovascular disease.

We used serology to estimate the prevalence of exposure to chlamydiae in Italian populations of wild boars (Sus scrofa). Sera from 173 hunter-killed wild boars harvested during the 2006-2009 hunting seasons in three Italian regions were tested for antibodies to Chlamydia suis, Chlamydophila pecorum, Chlamydophila abortus, and Chlamydophila psittaci by the microimmunofluorescence test. Antibody titers to chlamydiae ≥1:32 were detected in 110 of the 173 samples tested (63.6%). Specific reactivity could be assessed only in 44 sera with antibody titers to C. suis that were two- to threefold higher than antibody titers against the other chlamydial species; the other 66 sera had similar reactivity against all the chlamydia species tested. Antibody to C. suis was detected in sera from wild boar populations with rare or no known contact with domestic pigs. These results suggest that the wild boar could be a chlamydia reservoir and may acquire chlamydiae independent of contacts with the domestic pig.

Despite several decades of intensive studies, no vaccines againstChlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4+Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4+Th1 cells both in chlamydia-infected mice and in human patients with diagnosedC. trachomatisgenital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 fromC. trachomatishuman isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate againstC. trachomatisinfection.

We characterized 29 unrelated patients presenting with the severe form of Pompe disease (Glycogen Storage Disease Type II, acid maltase deficiency) and identified 26 pathogenic mutations divided over 28 different genotypes. Among the eight new mutations, five were exonic point mutations (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A) leading to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y); two were intronic point mutations (c.-32-3C>A and c.1636+5G>C) affecting mRNA processing; one was a single base deletion (c.742delC) generating a truncated protein (p.L248PfsX20). A comprehensive evaluation, based on different methodological approaches, confirmed the detrimental effect of the eight mutations on the protein and its function. Structural alterations potentially induced by the five missense mutations were also predicted through visual inspection of the atomic model of the GAA protein, in terms of both function and spatial orientation of specific residues as well as disturbance generated by amino acid substitutions. Although the remarkable heterogeneity of the mutational spectrum in Pompe disease was already known, our data demonstrate and confirm the power of molecular and functional analysis in predicting the natural course of Pompe disease.

AVIAN chlamydiosis is primarily caused by the intracellular bacterium Chlamydia psittaci , belonging to the Chlamydiaceae family. Depending on the species and age of the bird and the virulence of the infectious bacterial strain, avian chlamydiosis can be subclinical or characterised by respiratory, digestive, or systemic disorders (Knittler and others 2014). Seven C. psittaci outer-membrane protein A ( omp A) genotypes (A-F and E/B), have been initially detected in birds. All these genotypes can be transmitted to humans by contact with contaminated faeces or feathers or by inhalation of an infectious aerosol, causing a mild flu-like illness or severe atypical pneumonia. Recently, six additional C. psittaci omp A genotypes, all occurring in wildlife birds, have been proposed (Sachse and others 2008). Recent studies suggested that more chlamydial agents, beyond C. psittaci, can be involved in avian chlamydiosis. In this respect, Chlamydia abortus , Chlamydia pecorum, Chlamydia trachomatis and Chlamydia pneumoniae have been detected in birds (Pantchev and others 2009, Sachse and others 2012, Frutos and others 2015). Recently, two new bacterial species belonging to the Chlamydiaceae family have been described: Chlamydia avium from pigeons and psittacine birds and Chlamydia gallinacea from poultry (Sachse and others 2014). In addition, a novel candidate species, named Chlamydia ibidis, …

We investigated the Chlamydia spp. occurrence in Eurasian Collared Doves (Streptopelia decaocto) from urban and suburban areas in northern Italy. Among 76 doves screened, prevalence of Chlamydia spp. was 61%. Chlamydia psittaci genotype E was identified in 33 of the 46 positive samples. The multilocus sequence typing pattern of one highly positive sample showed a new allelic combination. The same molecular features were observed in a C. psittaci strain subsequently isolated from a live dove. Our results reveal a high C. psittaci prevalence in S. decaocto. The spread of this zoonotic pathogen from collared doves to other birds or humans seems to be a potential risk.

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.

The aim of this study was to investigate Chlamydia suis in a pig farm with an outbreak of conjunctivitis and diarrhea. Eye swabs and pooled fecal samples were investigated for the presence of C. suis by real-time PCR and ArrayTube microarray. Samples positive for C. suis by ArrayTube microarray assay were further tested for the presence of the tet(C) resistance gene by PCR. In the first examination, C. suis was identified in 12 six-week-old pigs showing conjunctivitis. Of these, the tet(C) gene-coding region was amplified in one pooled fecal sample and one eye swab, respectively. After oral treatment with tetracycline, clinical symptoms disappeared. Subsequently, all eye swabs investigated from 10 healthy pigs were positive for C. suis and the tet(C) gene-coding region. The present study reports rapid selection for tetracycline-resistant C. suis after antibiotic treatment.

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.

Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

The modification of infectivity of different animal chlamydial species after their in vitro treatment with 10 and 80 µg/ml of PG-1 peptide, was studied. PG-1 at 10 µg/ml concentration had neither inhibitory nor increasing effects on the infectivity of all Chlamydia tested, whereas the incubation of 80 µg/ml of PG-1 with C. suis and Cp. pecorum isolates resulted in a twofold increase in the number of inclusions; a five- to tenfold increase in chlamydial infectivity was observed when Cp. psittaci and Cp. abortus strains were tested with the concentration of 80 µg/ml of PG-1 peptide. On the contrary, C. muridarum strain and Cp.felis isolates did not show any increase in the number of inclusions when incubated with PG-1 at a 80 µg/ml concentration. The different effect of PG-1 on the animals strains of chlamydiae could be due to the unique characteristics in the membrane structure of each strain.

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.

Summary Background: [18F]-FDG is a widely used tracer for the non-invasive evaluation of hypermetabolic processes like cancer and inflammation. However, [18F]-FDG is considered inaccurate for the diagnosis of urinary tract and genital infections because of its urinary excretion. Since the 1970s, Gallium scintigraphy is a well established test that has been used for the evaluation of inflammation and infection in human patients. Aim: The aim of this study was to assess the feasibility of 68Ga-Chloride small animal PET for the analysis of an animal model of genital infection, induced after the vaginal inoculum of Chlamydia muridarum. Material and methods: Thirty mice were infected by placing 15 μl of sucrose phosphate glutamic acid (SPG) 107 inclusion forming units of C. muridarum into the vaginal vault. As controls of inflammation, three animals were challenged with 15 μl of SPG and one healthy animal was used to assess the tracer biodistribution. Four animals died during the experiment. Eleven animals were evaluated with 68Ga-Chloride small animal PET (GE, eXplore Vista) 3–5, 10–12, 17–19 days after infection, as well as three controls of inflammation and one healthy animal. Infection was monitored by obtaining cervical-vaginal swabs from all the animals on the day of each PET procedure. Moreover, five groups of three animals each were killed at 6, 13, 20, 27 and 34 days after infection were studied. Results: 68Ga-PET turned out positive in all the infected animals, concordantly to data obtained by the cervical swabs and by the ex vivo analysis. The tumour-to-background ratio (TBR) decreased over time as the inflammation tended to naturally extinguish. The controls showed a slightly increased uptake of tracer due to the aseptic inflammation caused by SPG and frequent cervical swabs. The healthy control did not show any pelvic uptake. Conclusion: 68Ga-Chloride is a promising tracer for the assessment of genital infection in a mouse animal model.

Feral pigeons (Columba livia domestica) which thrive in most European towns and cities are commonly infected with the zoonotic bacterium Chlamydophila psittaci, the agent of psittacosis (also known as ornithosis) in humans. A number of surveys carried out in the last thirty years across Europe allowed to detect high seropositivity values and high percentages of infection in feral pigeon populations. Overall, considering data from 11 European countries, seropositivity values to C. psittaci in the sampled populations ranged from 19.4 to 95.6 %. In most surveys, antibodies were detected with the complement fixation test in a percentage of samples from 19.4 to 66.3 %, with a median of 46.1 %. An indirect immunofluorescence test and an ELISA were employed less frequently, and allowed to detect higher percentages of seropositivity (23.7 – 67.7 % and 56 – 95.6 %, respectively). Attempts to grow C. psittaci in cell cultures or in embryonated chicken eggs were successful in 2 – 42.3 % and 0 – 57.1 % of samples respectively, antigen detection methods were positive in 2.3 – 40% of samples, while conventional PCR and real-time PCR with different genomic targets detected the organism in 3.4 – 50 % of samples. Twenty-five C. psittaci strains were typed as genotype B (n=14), E (n=10) and E/B (n=1). The huge increase of feral pigeon populations in Europe is a major cause of concern for the detrimental effect of pigeon droppings to environmental hygiene, in addition to the extensive damage due to the fouling of buildings and monuments. The most important pathogenic organism transmissible from feral pigeons to humans is C. psittaci, with 101 cases of disease reported in the literature. Exposure to C. psittaci-contaminated dust, direct contact with pigeons through handling and, to a lesser extent, through pigeon feeding have been identified as hazardous exposures in more than half cases of human disease, while loose or transient contacts with feral pigeons have been referred in about 40 % cases. Education initiatives as to the communication of a health risk deriving from contact with pigeons and pigeon excreta should primarily be targeted to workers that may be exposed to C. psittaci-contaminated dust, such as demolition/construction labourers. Recommendations to this category of workers include wearing protective clothes with hood, boots, gloves and air face filter masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections over time should also be considered in these workers. Children should be warned not to handle sick or dead pigeons and immunocompromised individuals should be educated to carefully limit their contact with feral pigeons. The culling of pigeons by shooting or poisoning is both unethical and useless as the place of the killed birds in the population is quickly filled by new juveniles or by birds immigrating from neighbouring areas. Pigeon-deterring systems such as nets and plastic or metal spikes applied to buildings and monuments will prevent their fouling, and the administration of contraceptive drugs may allow to resize the pigeon populations, but the measure that will ultimately lead to their permanent reduction and to establish healthy sustainable populations is the restriction of their indiscriminate feeding by pigeon lovers. The building of dovecotes and artificial breeding facilities should be considered for providing shelter and a balanced diet to the birds, as well as a chance of interaction for pigeon lovers in a hygienically-controlled environment.

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C. pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes. METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-alpha by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzyme-linked immunosorbent assay. RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20. The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture. Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-alpha levels. CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.

The TprI antigen of Treponema pallidum subsp. pallidum is a putative virulence factor predicted to be located in the outer membrane of the syphilis spirochete. In this study, we analyzed the immune response against TprI and its subunits in sera collected both from rabbits experimentally infected with the Nichols strain and from patients with syphilis, showing a different pattern of reactivity toward the antigen in these two groups of samples. The protective ability of recombinant TprI and its hypothetical outer membrane location were also investigated. Although no rabbit was protected after challenge, immunoelectron microscopy results, to be further investigated, were compatible with the outer membrane location of the antigen.

In this study the in vitro neutralising activity of 71 pig sera resulted positive by MIF technique against Chlamydia suis has been evaluated: these samples were collected from 2004 to 2006 from pigs with clinical signs of conjunctivitis and/or reproductive disorders, reared in two different farms in Emilia-Romagna (Northern Italy). Neutralisation reactions using purified EBs of Chlamydophila abortus, Chlamydophila psittaci and Chlamydophila pecorum were performed to control the specificity of neutralising antibodies to C. suis. Twenty serum samples randomly collected in Emilia-Romagna in a farm from pigs without signs of conjunctivitis and reproductive problems were tested for the presence of neutralising antibodies, as a control. Neutralising antibodies specific to C. suis were present in 57 out of the 71 (80×3 per cent) pig sera resulted positive by MIF with titres ranging between 32 to 2048. The presence of neutralising antibodies was significantly correlated (p < 0×001) with MIF titres. Pig sera neutralising to C. suis EBs, tested against the EBs of Cp. psittaci, Cp. abortus and Cp. pecorum, did not show any neutralising activity against these three different chlamydial species. Control sera collected from pigs without clinical signs of conjunctivitis or reproductive disorders presented no neutralising activity to C. suis.

Chlamydophila felis is thought to be an important ocular pathogen in cats. Conjunctivitis caused by C felis has been reported in many countries by several authors (Moraillon 1988, Wills and others 1988, Hanselaer and others 1989, Pointon and others 1991, Gruffydd-Jones and others 1995, Pudjiatmoko and others 1996, McDonald and others 1998, Sykes and others 1999, Cai and others 2002). In Italy, previous serological investigations using the complement fixation test and ELISA showed seroprevalence rates for chlamydiosis ranging from 2 to 13 per cent in populations of free-living feral cats (Piccoli and Capelli 1991, D’Amore and others 1997) and from 10 to 15 per cent in household cats (Ferrari and others 1992). These seroprevalences were lower than those found in other countries during surveys of feral and household cats using the homologous indirect immunofluorescence antibody (IFA) test (Wills and others 1988, Pudjiatmoko and others 1996, Yan and others 2000). This difference may be ascribable to a number of factors, such as limited circulation of the pathogen in the Italian feline population, the different types and sizes of populations examined, and the types of serological test used. This short communication describes a study to test this last hypothesis by determining the prevalence of antibodies to C felis in sera from cats registered at a serum collection bank by a homologous IFA test. A total of 430 serum samples, collected between October 1997 and November 2000 in the Veneto region of northern Italy, were examined; there were 254 samples from household cats and 176 from feral cats, which belonged to two catteries and 15 colonies of free-living cats. The free-living feral cats were blood sampled during a programme of neutering carried out by veterinarians of the public veterinary service. The IFA test was performed using as antigen the purified elementary bodies of a chlamydial isolate from a conjunctival swab taken from a cat kept in a cattery in Bologna, in the Emilia-Romagna region of northern Italy (Di Francesco and others 2004). The elementary bodies were purified by sucrose density-gradient ultracentrifugation by the method described by Fukushi and Hirai (1988). The presence of C felis antibodies was assessed using a fluorescein-conjugated goat anticat immunoglobulin G serum (Euroclone). The IFA test was carried out according to the method of Wills and others (1988). Sera were screened at 1:16 and 1:32 dilutions. Sera that fluoresced an apple-green colour at 1:32 dilution were considered to be positive, and were tested by serial dilution to determine the antibody titre. The reciprocal of the highest serum dilution showing apparent fluorescence was considered to be the antibody titre of that sample. The results of the IFA test for C felis antibodies are shown in Table 1. Overall, 130 (30·2 per cent) of the 430 samples tested positive, 54 (21·3 per cent) of the 254 household cats, 58 (64·0 per cent) of the 90 cattery cats and 18 (21·0 per cent) of the 86 free-living feral cats testing positive. The seroprevalence rates in the two catteries were 68·0 per cent and 36·0 per cent. Seropositive animals were found in six of the 15 feral populations investigated. Antibody titres ranged from 32 to 2048 in the household and cattery cats, and from 128 to 2048 in the free-living cats. When the seroprevalences were compared by the chisquared test, the seroprevalence was significantly higher in the cats kept at the two catteries than in the free-living feral cats (χ2=33·94; P=0·001) and the household cats (χ2=56·44; P=0·001). Additionally, the seroprevalences in the free-living and household cats were virtually identical. This may be due to the epidemiological features of C felis, the transmission of which requires close contact between infected and healthy animals. This appears to be more frequent within closed feline communities, such as residential and boarding catteries and breeding centres, where unavoidable contact among animals, within a restricted space, favours the spread of infection. Among the 15 free-living feral populations tested, the infection appeared to be endemic in six colonies and absent in nine. The lower distribution of C felis might be due to the fact that these are free feline communities in which the cats are not forced to have contact with one another. With regard to the household cats which tested positive for antibodies to C felis, they had come from catteries or pet shops and lived in a semi-free condition, implying that they were able to have contact with other cats. In all the categories of cats tested, it was not possible to assess whether there was any correlation between the cats’ antibody status and the presence of clinical signs suggestive of chlamydiosis, because the collection of data had been focused on other pathologies. A few of the cats had antibody titres at or above 1024, comparable with those found in other investigations of cats with active chlamydial infection (Wills and others 1988). Lower antibody titres may only be suggestive of a contact with C felis, but may not be indicative of when infection occurred, because cats experimentally infected with chlamydiae showed high antibody titres which persisted for more than a year after the onset of the infection (Wills 1986). In conclusion, the results of the IFA test showed that there was a high seroprevalence for chlamydiosis in cats in the area surveyed, especially in closed feline populations, and confirmed that the homologous IFA test is a reliable tool for the serological diagnosis of feline chlamydiosis. Veterinary Record (2004) 155, 399-400

Cancer Procoagulant (CP), a cysteine proteinase which triggers blood coagulation by directly activating Factor X (FX) in the absence of Factor VII (F VII), has recently been isolated from rabbit V2 carcinoma and biochemically characterized. We have studied the procoagulant activity of tissue extracts from 4 murine experimental tumors in order to define whether or not a F VII-independent activity with cysteine proteinase characteristics was present. The tumors studied were: Lewis lung carcinoma (3LL), B16 melanoma (B16), JW sarcoma (JWS) and the M4 variant of the mFS6 fibrosarcoma (M4). Extracts from 3LL, B16 and JWS tumor initiated coagulation in both the presence and absence of F VII, their procoagulant activity was sensitive to iodoacetamide (1 mM) and mercury chloride (0.1 mM). The procoagulant of M4 extract was dependent on the presence of F VII and was not significantly affected by the cysteine proteinase inhibitors. An Ouchterlony double immunodiffusion study showed immunological cross-reactivity of all but M4 extracts to a polyclonal antibody to purified CP. The present study suggests that the procoagulant(s) present in the murine tumors 3LL, B16 and JWS are enzymatically and immunologically indistinguishable from cancer procoagulant of the rabbit V2 carcinoma.

Factors contributing to the antioxidant power of plasma may play a role in the control of arachidonic acid (AA) metabolism. The purpose of this study was to evaluate the possible effects of vitamin E deficiency on platelet and vascular prostaglandin synthesis. CD-COBS male rats were fed for 7 mo a diet containing either 1 or 75 mg/kg vitamin E. At the end of this period, serum levels of vitamin E were 0.4 +/- 0.1 and 10 +/- 0.6 micrograms/ml in the two groups, respectively. Malondialdehyde (MDA) generation by AA was significantly higher in platelet-rich plasma from vitamin E-deficient rats. Thromboxane B2 (TxB2) in serum from vitamin E-deficient rats increased about 16 times compared with controls, whereas 6-keto-PGF1 alpha levels increased only 3 times. The ratio between TxB2 and 6-keto-PGF1 alpha, increased therefore manyfold in vitamin E-deficient animals. MDA formation and [14C]AA metabolism in washed platelets were similar in the two groups of animals. No significant difference was found in the PGI2 (prostacyclin) antiaggregating activity released from the aortic rings resuspended in buffer. In contrast, the capacity of plasma to stimulate PGI2 activity from "exhausted" aortic rings (prostacyclin-stimulating factor) was significantly reduced in vitamin E-deficient animals. In conclusion, vitamin E deficiency induces an unbalanced plasma regulation of AA metabolism. This results in an excessive production of platelet TxA2 compared with vascular PGI2 generation. On the other hand, vitamin E deficiency does not seem to affect directly the enzymatic pathways of AA metabolism.

Antimicrobials are often used for prophylaxis and therapy of chlamydial infections. The large use of tetracycline has encouraged the selection of resistant organisms. The aim of this study was to evaluate the sensitivity to tetracycline of some Chlamydia suis strains isolated in Italy. The sensitivity of 14 C. suis isolates to doxycycline was tested and compared with the susceptibility to the same drug of the urethral Italian isolate GO86 of C. trachomatis serovar D, the reference strain 6BC of C. psittaci and three isolates of C. abortus, C. pecorum and C. felis, respectively. C. suis isolates were tested for the presence of the tet(C) resistance gene by a PCR assay amplifying a 525 base pair product of the tet(C) gene-coding region. As far as sensitivity to doxycycline is concerned, in comparison to the five strains of C. trachomatis, C. psittaci, C. abortus, C. pecorum and C. felis which were sensitive to doxycycline with MIC and MBC ranging from 0×03 to 0×125 µg/ml, respectively, the MIC and MBC values of doxycycline ranged from 4 to 8 µg/ml, respectively, in most swine isolates tested. All the C. suis isolates carried an identical nucleotide sequence that showed 100 per cent homology with those of the structural gene tet(C) described by Dugan and others (Gene Bank Accession AY428551). None of the other five chlamydial tetracycline sensitive strains carried the resistance gene tet(C).

Anticoagulants of the coumarin type have long been reported to inhibit metastasis growth in experimental animals; however, the mechanisms of such effects has not been clarified. Systemic anticoagulation per se does not appear to account completely for such metastasis growth depression. More recent information gathered on a cell procoagulant activity, which is vitamin K-dependent, could probably supply a fresh insight into this problem. Indeed, vitamin K deficiency induced either dietarily or pharmacologically by warfarin, does inhibit the activity of a cysteine protease with direct factor-X-activating properties. This protease is only present in warfarin-sensitive tumors. The correlation of this activity with cancer cell invasiveness is supported by experimental data in metastatic variants and, lately, also by the observation of markedly higher cancer procoagulant activity in extracts from metastases than from primary human melanomas.