Short communication: Seroprevalence of paratuberculosis in Italian water buffaloes (Bubalus bubalis) in the region of Campania.

Paratuberculosis is a chronic enteric disease affecting virtually all ruminants, but only anecdotal information is currently available about the occurrence of this disease in water buffaloes (Bubalus bubalis). We carried out a survey study aimed at determining the prevalence of paratuberculosis in 2 provinces in the region of Campania, Italy, where about half of all Italian buffaloes are reared. From May 2017 to December 2018, we collected 201,175 individual serum samples from 995 buffalo herds. The sera were collected from animals over 24 mo old and were tested using a commercial ELISA test. The herd-level apparent prevalence result was 54.7%, and the animal-level apparent prevalence was 1.8%. The herd-level true prevalence was estimated using a Bayesian approach, demonstrating a high herd-level prevalence of paratuberculosis in water buffaloes from the Campania area. These findings suggest that the urgent adoption of paratuberculosis herd-control programs for water buffaloes in this area would be beneficial.

Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing.

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2-1.3 × 105 somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 102 cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.

Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants.

AIMS: To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis-infected dairy herds. METHODS AND RESULTS: Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one-stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two-stage plant. CONCLUSIONS: Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two-stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.

Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

AIMS: The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. METHODS AND RESULTS: MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). CONCLUSIONS: The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex.

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy.

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated.

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds.

The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC50 and MIC90, respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to both compounds. The MIC50 and MIC90 of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae, respectively. The MIC50 and MIC90 of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae, respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC90, whereas an 8-fold increase of the MIC90 was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti-Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows.

Simultaneous identification by multiplex PCR of major Prototheca spp. isolated from bovine and buffalo intramammary infection and bulk tank.

UNLABELLED: Bovine mastitis caused by Prototheca spp. infection is increasing worldwide, therefore becoming more relevant to the dairy industry. Almost all Prototheca isolates from bovine mammary protothecosis came from P. zopfii genotype 2, with a lower prevalence of infection due to P. blaschkeae and rarely to P. wickerhamii. In this study, we report the development of two multiplex PCR assays able to discriminate among the three species responsible for bovine intramammary infection (IMI). Our assay is based on the specific amplification of new DNA target from mitochondria and chloroplasts partial sequences, of different Prototheca isolates. Both methods were set up using reference strains belonging to all Prototheca species and validated by the analysis of 93 isolates from bovine and buffalo IMI and bulk tank milk samples. The investigation involves 70 isolates from North, 13 from Central and 10 from South Italian regions. Isolates from bovine were most commonly identified as P. zopfii genotype 2, and only in one case as P. blaschkeae, whereas isolates from buffaloes belonged both to P. zopfii genotype 2 and P. wickerhamii. These findings proved the suitability of our multiplex PCRs as a rapid test to discriminate among pathogenic Prototheca strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports PCR assays based on novel Prototheca spp. mitochondrial and chloroplastic target sequences. The multiplex PCR protocol described in this study is useful for rapid simultaneous detection of P. zopfii, P. wickerhamii and P. blaschkeae.

A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0-24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤ 2.0%) up to a maximum of 100% of herds with a within-herd AP ≥ 8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate control measures aimed at reducing the prevalence of infection in dairy herds and milk contamination.

Technical note: Identification of Prototheca species from bovine milk samples by PCR-single strand conformation polymorphism.

We report the development of a PCR-single strand conformation polymorphism (SSCP) method to identify Prototheca spp. responsible for bovine mastitis: P. zopfii and P. blaschkeae. The method was set up using reference strains belonging to P. zopfii genotype 1, P. zopfii genotype 2, and P. blaschkeae as target species and P. stagnora, and P. ulmea as negative controls. The assay was applied on 50 isolates of Prototheca spp. isolated from bovine mastitic milk or bulk-tank milk samples, and all isolates were identified as P. zopfii genotype 2. We conclude that the described PCR-SSCP approach is accurate, inexpensive, and highly suitable for the identification of P. zopfii genotype 2 on field isolates but also directly on milk, if preceded by a specific DNA extraction method.

A rapid real-time PCR/DNA resolution melting method to identify Prototheca species.

AIM: The study describes the development of a simple and rapid tool to identify yeast-like microalgae belonging to the genus Prototheca. METHODS AND RESULTS: The method, based on two-step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non-pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype-specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. CONCLUSIONS: The assay two-step Real Time PCR is accurate, robust, cost-effective and faster than auxonographical, biochemical or conventional molecular biology methods. SIGNIFICANCE AND IMPACT OF THE STUDY: the rapid and high throughout two-step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.

Transmission patterns of a Mycobacterium avium subsp. paratuberculosis clone within a single heard investigated by Whole Genome Sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale.

Molecular characterization of Prototheca strains isolated from Italian dairy herds.

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.

Evaluation of Mycobacterium avium subsp. paratuberculosis survival during the manufacturing process of Italian raw milk hard cheeses (Parmigiano Reggiano and Grana Padano).

Mycobacterium avium subsp. paratuberculosis (MAP), the agent of paratuberculosis in ruminants, is suspected to be involved in the aetiology of some human diseases. Notably, the consumption of milk and dairy products is considered to be the main route of human exposure to MAP because of its ability to survive during pasteurization and manufacturing processes. The aim of this study was to investigate, through a microbiological challenge test, the survival of MAP during the manufacturing and ripening period of two Italian hard cheeses, Parmigiano Reggiano and Grana Padano, made from raw bovine milk. The challenge test was performed in two different phases: the creaming phase and the manufacturing phase. The creaming phase, which is the first step of cheese production, was reproduced in the laboratory employing raw cow's milk spiked with a MAP reference strain at a final concentration of 5.58 log10 CFU/mL. After the creaming at 18 °C and 27 °C for 12 h, a decrease of 0.80 log10 and 0.77 log10 was observed in partially skimmed milk, respectively. In the second phase, two batches of raw cow's milk (1000 L each) were inoculated with MAP reference and wild strains, respectively. Then, the entire manufacturing process for Parmigiano Reggiano and Grana Padano, both of Protective Designation of Origin (PDO), was reproduced in an experimental cheese factory, starting from a concentration in milk of 5.19 ±â€¯0.01 and 5.28 ±â€¯0.08 log10 CFU/mL of MAP reference and wild strains, respectively. Heating the curd at 53 °C for 20 min did not affect MAP survival, however a significant decrease (p < 0.05) in MAP viability was observed during the moulding phase and after salting in brine, regarding the wild strains and the reference strain, respectively. In addition, a significant decrease was observed during the ripening period, at which time the MAP concentration dropped below the limit of detection from the second and the third month of ripening, for the wild and reference strains, respectively. Taking into account the poor data availability about MAP survival in hard cheeses, this study may improve the knowledge regarding the effect of the cheese manufacturing process on the MAP dynamics, supporting also the safety of traditional raw milk hard cheeses.

Review article: hepatic steatosis and insulin resistance.

Hepatic steatosis may be both an adaptive phenomenon and an example of lipotoxicity. Its prevalence ranks in the same order of magnitude of insulin resistance in the general population. Studies support the finding that hepatic steatosis is secondary to insulin resistance and not vice versa. A steatotic liver will further contribute to the development of insulin resistance through impaired clearance of insulin from the portal blood, creating a vicious cycle. Insulin resistance is the leading force in the pathogenesis and natural history of non-alcoholic fatty liver disease. Dysfunction of energetic homeostasis and the interaction of adiponectin, leptin and tumour necrosis factor-alpha are key events in the pathogenesis of steatosis and insulin resistance. Insulin resistance represents the frame within which hepatic and extrahepatic non-alcoholic fatty liver disease-related clinical manifestations are to be anticipated and interpreted.

Review article: the metabolic syndrome and non-alcoholic fatty liver disease.

Metabolic syndrome represents a common risk factor for premature cardiovascular disease and cancer whose core cluster includes diabetes, hypertension, dyslipidaemia and obesity. The liver is a target organ in metabolic syndrome patients in which it manifests itself with non-alcoholic fatty liver disease spanning steatosis through hepatocellular carcinoma via steatohepatitis and cirrhosis. Given that metabolic syndrome and non-alcoholic fatty liver disease affect the same insulin-resistant patients, not unexpectedly, there are amazing similarities between metabolic syndrome and non-alcoholic fatty liver disease in terms of prevalence, pathogenesis, clinical features and outcome. The available drug weaponry for metabolic syndrome includes aspirin, metformin, peroxisome proliferator-activated receptor agonists, statins, ACE (angiotensin I-converting enzyme) inhibitors and sartans, which are potentially or clinically useful also to the non-alcoholic fatty liver disease patient. Studies are needed to highlight the grey areas in this topic. Issues to be addressed include: diagnostic criteria for metabolic syndrome; nomenclature of non-alcoholic fatty liver disease; enlargement of the clinical spectrum and characterization of the prognosis of insulin resistance-related diseases; evaluation of the most specific clinical predictors of metabolic syndrome/non-alcoholic fatty liver disease and assessment of their variability over the time; characterization of the importance of new risk factors for metabolic syndrome with regard to the development and progression of non-alcoholic fatty liver disease.

Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant.

Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013-March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170-230 MAP cells/g (62-115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended.

First outbreak of bovine mastitis caused by Prototheca blaschkeae.

The most important animal disease caused by yeast-like algae belonging to the genus Prototheca is bovine mastitis. Although the infection can be caused by both Prototheca zopfii genotype 2 and Prototheca blaschkeae, the bulk of prevalence of bovine protothecal mastitis has been so far attributed to the former, being P. blaschkeae only sporadically isolated. However, we report here the first outbreak of bovine mastitis caused by P. blaschkeae in an Italian dairy herd. One hundred and four individual milk samples, three bulk tank milk and 16 environmental samples within the herd were screened for the presence of Prototheca: five, one and four positive samples, were respectively observed. Molecular analysis revealed that, with the sole exception of one environmental isolate belonging to P. zopfii genotype 2, all Prototheca strains were identified as P. blaschkeae. Our results might suggest that even P. blaschkeae can induce mastitis outbreaks, while it is not clear if the higher incidence of P. zopfii genotype 2 as causative agent of protothecal mastitis could reflect an intrinsic higher pathogenicity or it could be simply the consequence of its, so far observed, higher diffusion in worldwide dairy herd ecosystems.

Genetic polymorphisms in non-alcoholic fatty liver disease: interleukin-6-174G/C polymorphism is associated with non-alcoholic steatohepatitis.

BACKGROUND: Environmental and genetic factors play a role in the pathogenesis and natural history of non-alcoholic fatty liver disease (NAFLD). METHODS: In 114 subjects with NAFLD we report the prevalence and correlation with clinical parameters of three polymorphisms: interleukin-6 (-174G/C), plasma cell differentiation antigen (K121Q) and microsomal transfer protein (-493G/T). In 59 biopsied patients with NAFLD the polymorphisms were also related to histological features. RESULTS: IL-6 -174C variant was more prevalent (p<0.01) in NAFLD compared to controls. In the NAFLD group, C carriers had higher HOMA-IR and fasting insulin than G carriers (p<0.05). The prevalence of IL-6/C variant was higher (83%) in biopsied than in not biopsied subjects (66%) (p<0.05). In biopsied subjects, C carriers had higher HOMA and fasting insulin (p<0.05) compared than those with G allele. The prevalence of IL-6 -174G/C polymorphism was significantly higher in NASH than in NAFLD (p=0.048). At logistic regression analysis IL-6 -174C was an independent predictor of both NAFLD (OR 4.116, C.I. 1.126-15.048) and NASH (OR 7.035, C.I. 1.167-42.394). Conversely, the distribution of PC-1 and MTP polymorphisms was not significantly different compared to the control group, nor associated with clinical or histological characteristics. CONCLUSIONS: Our data suggest that IL-6 -174C genetic polymorphisms, involved in inflammation and insulin resistance, are associated with NASH. These data may contribute to the understanding of the genetic susceptibility to NAFLD.

Age-related changes in bile acid synthesis and hepatic nuclear receptor expression.

BACKGROUND: Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7alpha-hydroxylase and related nuclear receptor expression in human livers. DESIGN: Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7alpha-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. RESULTS: Ageing was inversely correlated with serum 7alpha-hydroxy-4-cholesten-3-one and with cholesterol 7alpha-hydroxylase mRNA levels (r = -0.44 and r = -0.45 on a semi-log scale, respectively, P < 0.05). Among different nuclear factors, cholesterol 7alpha-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 (r = 0.55 on a log scale, P < 0.05); hepatocyte nuclear factor-4 levels were also inversely correlated with age (r = -0.64 on a semi-log scale, P < 0.05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7alpha-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7alpha-hydroxylase was observed. CONCLUSIONS: Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7alpha-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism.