The role of altered immune function during the dry period in promoting the development of subclinical ketosis in early lactation.

Subclinical ketosis (SCK) may impair white blood cell (WBC) function and thus contribute to the risk of disease postpartum. This preliminary study investigated changes occurring in the immune system before disease onset to elucidate their role in the occurrence of SCK. A group of 13 Holstein dairy cows were housed in tie-stalls and retrospectively divided into 2 groups based on their levels of ß-hydroxybutyrate (BHB) measured in plasma between calving day and 35 d from calving (DFC). Levels of BHB <1.4 mmol/L were found in 7 cows (control cows, CTR group) and levels >1.4 mmol/L were found in 6 cows at ≥1 of 6 time points considered (cows with SCK, KET group). From -48 to 35 DFC, body condition score, body weight, dry matter intake, rumination time, and milk yield were measured, and blood samples were collected regularly to assess the hematochemical profile and test the WBC function by ex vivo challenge assays. Data were submitted for ANOVA testing using a mixed model for repeated measurements that included health status and time and their interactions as fixed effects. Compared with CTR cows, KET cows had more pronounced activation of the immune system (higher plasma concentrations of proinflammatory cytokines, myeloperoxidase, and oxidant species, and greater IFN-γ responses to Mycobacterium avium), higher blood concentrations of γ-glutamyl transferase, and lower plasma concentrations of minerals before calving. Higher levels of nonesterified fatty acids, BHB, and glucose were detected in KET cows than in CTR cows during the dry period. The effect observed during the dry period was associated with a reduced dry matter intake, reduced plasma glucose, and increased fat mobilization (further increases in nonesterified fatty acids and BHB) during early lactation. A reduced milk yield was also detected in KET cows compared with CTR. The KET cows had an accentuated acute-phase response after calving (with greater concentrations of positive acute-phase proteins and lower concentrations of retinol than CTR cows) and impaired liver function (higher blood concentrations of glutamate-oxaloacetate transaminase and bilirubin). The WBC of the KET cows, compared with CTR cows, had a reduced response to an ex vivo stimulation assay, with lower production of proinflammatory cytokines and greater production of lactate. These alterations in the WBC could have been driven by the combined actions of metabolites related to the mobilization of lipids and the occurrence of a transient unresponsive state against stimulation aimed at preventing excessive inflammation. The associations identified here in a small number of cows in one herd should be investigated in larger studies.

Reverse transcription real-time PCR for detection of porcine interferon α and ß genes.

A few studies provided convincing evidence of constitutive expression of type I interferons (IFNs) in humans and mice, and of the steady-state role of these cytokines under health conditions. These results were later confirmed in pigs, too. In line with this tenet, low levels of IFN-α/ß can be detected in swine tissues in the absence of any specific inducer. These studies are compounded by the utmost complexity of type I IFNs (including among others 17 IFN-α genes in pigs), which demands proper research tools. This prompted us to analyse the available protocols and to develop a relevant, robust, reverse transcription (RT) real-time polymerase chain reaction (PCR) detection system for the amplification of porcine IFN-α/ß genes. The adopted test procedure is user-friendly and provides the complete panel of gene expression of one subject in a microtitre plate. Also, a proper use of PCR fluorochromes (SYBR(®) versus EvaGreen(®) supermix) enables users to adopt proper test protocols in case of low-expression porcine IFN-α genes. This is accounted for by the much higher sensitivity of the test protocol with EvaGreen(®) supermix. Interestingly, IFN-ß showed the highest frequency of constitutive expression, in agreement with its definition of 'immediate early' gene in both humans and mice. Results indicate that the outlined procedure can detect both constitutively expressed and virus-induced IFN-α/ß genes, as well as the impact of environmental, non-infectious stressors on the previous profile of constitutive expression.

Impact of cadmium exposure on swine enterocytes.

We tested cadmium (Cd2+) effects on porcine IPEC-J2 cells, which represent an in vitro model of the interaction between intestinal cells and both infectious and non-infectious stressors. Accordingly, we investigated the effects of low (2 µM) to moderate (20 µM) concentrations of Cd2+, in terms of pro-inflammatory gene expression and protein release, as well as of infectivity in a Salmonella typhimurium penetration model. Our data showed a significant (P < .001) increase of intracellular Cd2+ after 3, 6 and 24 h of exposure with respect to levels at 1 h. These data showed the ability of IPEC-J2 to absorb Cd2+ as a function of both time and concentration. Also, the absorption of this heavy metal was related to a significant modulation of important pro-inflammatory messengers. In particular, down-regulation of IL-8 was associated with a significant decrease of Salmonella typhimurium ability to penetrate into IPEC-J2 cells, in agreement with a previous study in which an anti-IL 8 antibody could significantly inhibit Salmonella penetration into the same cells (Razzuoli et al., 2017). This finding demonstrates the ability of Cd2+ to affect the outcome of an important host-pathogen relationship. In conclusion, our study highlighted the ability of an environmental pollutant like Cd2+ to modulate innate immune responses in terms of chemokine release and gene expression, and susceptibility to microbial infections.

Archaeometric researches on the provenance of Mediterranean Archaic Phoenician and Punic pottery.

The aim of this study is to setup a first chemical database that could represent the starting point for a reliable classification method to discriminate between Archaic Phoenician and Punic pottery on the base of their chemical data. This database up to now can discriminate between several different areas of production and provenance and can be applied also to unknown ceramic samples of comparable age and production areas. More than 100 ceramic fragments were involved in this research, coming from various archaeological sites having a crucial importance in the context of the Phoenician and Punic settlement in central and western Mediterranean: Carthage (Tunisia), Toscanos (South Andalusia, Spain), Sulci, Monte Sirai, Othoca, Tharros (Sardinia, Italy) and Pithecusa (Campania, Italy). Since long-time archaeologists hypothesised that Mediterranean Archaic Phoenician and Punic pottery had mainly a local or just a regional diffusion, with the exception of some particular class like transport amphorae. To verify the pottery provenance, statistical analyses were carried out to define the existence of different ceramic compositional groups characterised by a local origin or imported from other sites. The existing literature data are now supplemented by new archaeometric investigations both on Archaic Phoenician ceramics and clayey raw materials from Sardinia. Therefore, diffractometric analyses, optical microscopy observations and X-ray fluorescence analyses were performed to identify the mineralogical and chemical composition of Othoca ceramics and clayey raw material. The obtained results were then compared with own literature data concerning Phoenician and Punic pottery in order to find features related to the different ceramic productions and their provenance. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were also performed on the chemical compositional data in order to discriminate ceramic groups. A very complex situation was found: imported ceramics coming from Carthage, with a large-scale distribution, were found together with a predominant local production pottery. The archaeometric results demonstrate that historical and typological approach has to be supported by scientific analyses to better understand local or Mediterranean exchanges.

Low-dose interferon-alpha treatment for feline immunodeficiency virus infection.

Feline immunodeficiency virus sustains an AIDS-like syndrome in cats, which is considered a relevant model for human AIDS. Under precise enrolment requirements, 30 naturally infected cats showing overt disease were included in a trial of low-dose, oral human interferon-alpha treatment. Twenty-four of them received 10 IU/Kg of human interferon-alpha and 6 placebo only on a daily basis under veterinary supervision. The low-dose human interferon-alpha treatment significantly prolonged the survival of virus-infected cats (p<0.01) and brought to a rapid improvement of disease conditions in the infected hosts. Amelioration of clinical conditions was neither correlated with plasma viremia, nor with proviral load in leukocytes. A good survival of CD4+ T cells and a slow increase of CD8+ T cells were also observed in human interferon-alpha-treated cats. Interestingly, the improvement of the total leukocyte counts showed a much stronger correlation with the recovery from serious opportunistic infections. As shown in other models of low-dose interferon-alpha treatment, there was a rapid regression of overt immunopathological conditions in virus-infected cats. This hints at a major role of interferon-alpha in the control circuits of inflammatory cytokines, which was probably the very foundation of the improved clinical score and survival despite the unabated persistence of virus and virus-infected cells.

Disease risk assessment by clinical immunology analyses in periparturient dairy cows.

A disease prediction system was investigated in a case-control study in the dry period of high-yielding dairy cows. Blood samples of 75 cows from 26 herds were collected before calving between -23 and -33 days (T1) and also between -2 and -6 days (T2) to investigate a panel of clinical immunology and chemistry parameters. Cows with abnormal serum lysozyme and interleukin-6 concentrations showed a greater disease prevalence until the 60th day in milk compared with non-responder cows (P<0.05 and lower at T1). Differences in disease prevalence were observed on the basis of T1 data, and also by combining the results at T1 and T2. The other laboratory parameters under study were not predictive of a disease risk. Results indicate that environmental stressors in the dry period may cause a negative imprinting of the innate immune response, underlying predisposition to later disease occurrence.

Trabecular bone score in healthy ageing.

OBJECTIVE: The main aim of this work was to report on trabecular bone score (TBS) by dual-energy X-ray absorptiometry (DXA) of healthy Italian subjects to be used as a reference standard for future study in clinical and research settings. The secondary aim was to investigate the link between TBS and conventional parameters of bone and body composition by DXA. METHODS: 250 individuals of 5 age bands (spanning from 18 to 70 years of age, equally distributed for both age and sex) were prospectively recruited. A lumbar spine (LS) DXA scan (Lunar iDXA™; GE Healthcare, Madison, WI) was acquired for each subject and then analysed with the latest version of TBS iNsight v. 2.1 (Med-Imaps, Pessac, France) software. LS bone mineral density (LS BMD), Z-score, T-score and TBS values were collected. Pearson's test was used to investigate the correlations between TBS and LS BMD and the influence of age, body mass index (BMI) and body composition on these parameters. RESULTS: A significant decrease of TBS and LS BMD was observed with ageing in both males (TBS mean values from 1.486 to 1.374; LS BMD mean values from 1.219 to 1.187) and females (TBS mean values from 1.464 to 1.306; LS BMD mean values from 1.154 to 1.116). No statistically significant difference was achieved among males and females of the same age group for both TBS and LS BMD, with the exception of the fifth age group. A significant correlation was found between LS BMD and TBS values in both sexes (r = 0.555-0.655, p < 0.0001). BMI influenced LS BMD but not TBS. TBS values were inversely correlated with some fat mass parameters, in particular with visceral adipose tissue (in males: r = -0.332, p < 0.001; in females: r = -0.348, p < 0.0001). No significant correlation was found between TBS and total lean mass, opposite to LS BMD (in males: r = 0.418; p < 0.0001; in females: r = -0.235; p < 0.001). CONCLUSION: This report is an attempt to start building a database for healthy Italian people providing age- and sex-specific reference curves for TBS. This could help clinicians to improve patient management in the detection of impaired bone mineral status and to monitor bone changes. ADVANCES IN KNOWLEDGE: The study reports TBS values of a selectively enrolled Italian healthy population, ranging from younger to older ages and including males as a reference standard. Moreover, links between body composition and TBS are explored.

Role of a distinct population of bovine gamma delta T cells in the immune response to viral agents.

A distinct population of bovine gamma delta T cells was isolated from peripheral blood mononuclear cells of foot-and-mouth disease (FMD)-vaccinated cattle; these lymphocytes were shown to exert a natural killer-like activity against cells infected by different viruses. The antiviral activity was dependent upon cognate recognition of target cells and could operate by both cytostatic and cytotoxic mechanisms. Among these, secretion of a serine esterase was shown after binding to target cells. This population of bovine gamma delta T cells is recognized by murine monoclonal antibodies 1E7, 5D4, and 6F9, raised in our laboratory. To define an in vivo antiviral role, four heifers were infected with a strain of bovid herpesvirus 1 by the intranasal/intravaginal routes and contact exposure. The prevalence of 1E7+/5D4+ cells among peripheral blood lymphocytes increased dramatically in the first days after infection; the same held true for in-contact cattle, albeit with a different time kinetics. In another experiment, colonization of mucosae was demonstrated by immunoperoxidase staining on tongue and palate sections of healthy cattle. The infiltration of gamma delta T cells altogether in the palate mucosa was much more accentuated in foot-and-mouth disease-vaccinated, as compared to nonvaccinated, control calves.

Prognostic factors and survival in non-Hodgkin's lymphomas: the experience of the Istituto Oncologico Romagnolo (IOR).

In an attempt to evaluate natural history, prognostic factors and survival, the data of 340 patients with NHL were collected. 267 patients were evaluable for the analysis of prognostic factors and survival. The tumor samples were reviewed and reclassified according to the Kiel classification. At completion, 180 patients were affected by low-grade (LG)-NHL and 87 patients had high-grade (HG)-NHL. Numerous potential prognostic factors were analysed in univariate and multivariate analyses. Globally 154 patients (57.4%) obtained complete remission (CR) and 65 patients (24.3%) partial remission (PR). The response rate was similar in LG and HG-NHL groups. 5-years survival was 52% for all patients (53% in LG-NHL and 44% in HG-NHL). Median survival was 62 months in LG-NHL and 38 months in HG-NHL (p = n.s.). At the univariate analysis overall survival (OS) in LG-NHL was favourably influenced by age < 65 years (p = 0.004), performance status > 80 (p < 0.02), early clinical stage (p < 0.001), absence of systemic symptoms (p < 0.001), low serum LDH (p < 0.001) and achievement of CR (p < 0.001), while in the HG-NHL only by age (p = 0.005) and achievement of CR (p < 0.001). The multivariate analysis showed early clinical stage, low serum LDH, absence of systemic symptoms and achievement of CR as independent prognostic factors in LG-NHL and only achievement of CR in HG-NHL.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological activities of interleukin-2 and gamma-interferon in bovine T lymphocyte conditioned media.

Interleukin 2 and gamma-interferon were revealed in cultures of Concanavalin A-activated bovine mononuclear cells from peripheral blood leukocytes and their kinetics of production were described. Cyclosporin A could dramatically inhibit the synthesis of interleukin 2, whereas it did not affect the interleukin 2-dependent proliferation of bovine T blasts. Bovine T lymphocyte conditioned media (TLCM) containing gamma-interferon increased the expression of beta-2 microglobulin in human HEL (Human Embryo Lung) 299 cells and exerted a potent anti-proliferative effect upon bovine Aubek cells. The beta-2 microglobulin modulating activity of bovine gamma-interferon was exerted at very low concentrations, which showed no detectable antiviral activity on human cells.

Human lymphoblastoid interferon as vaccine adjuvant in cattle.

Human lymphoblastoid interferon from Namalwa cells was purified for clinical use by ethanol fractionation, and used as adjuvant of an inactivated Bovid Herpesvirus 1 vaccine in calves. In agreement with other in vitro and in vivo models, low and high interferon doses were shown to increase and depress the specific antibody response, respectively. The low, effective interferon dose (100 International Units/kg) also reduced the variability of antibody titres after the first vaccine injection. This latter dose had apparently no influence on the regulatory T cell circuits, as opposed to the other doses under study.

Target recognition by bovine mononuclear, MHC-unrestricted cytotoxic cells.

A population of bovine non B/non T, cytotoxic lymphocytes with natural killer activity against virus-infected and non-infected embryonic kidney cells was functionally characterized. The data obtained in experiments of flow cytometry and immuno-peroxidase staining show that a CD2-, CD4-, CD8-, TcR gamma delta-, CD3+, CD45+, FcR+ lymphoid killer cell does exist within bovine peripheral blood leucocytes. This population can detect the down-regulation of class I MHC antigens or the expression of embryonic forms thereof, as shown by experiments of 17-hour 51Cr release and binding to target cells. This model was tested in vitro in experiments on virus-infected bovine kidney cells. The emerging picture was substantially in agreement with the "missing self" theory as a possible option for target cell recognition. In this respect, the profound alteration of MHC Class I expression could represent a major early event, recognized on virus-infected cells by the immune system.

Use of a macrophage cell line for rapid detection of Mycobacterium bovis in diagnostic samples.

Mycobacterium bovis isolation on bacteriological media from suspected cases of bovine tuberculosis (TB) demands laborious and time-consuming procedures. Even polymerase chain reaction (PCR) and radiometric analyses are secondary procedures and not alternatives to bacteriological procedures. Therefore, there is a need to develop new techniques aimed at rapid M. bovis detection in diagnostic samples. The human macrophage cell line THP-1 was thus investigated in experiments of M. bovis propagation and isolation from reference lymph node suspensions. THP-1 cells were shown to support a high-titered propagation within 48h of minute amounts of both M. bovis BCG and fully pathogenic M. bovis strain 503. A semi-nested PCR for TB-complex-specific insertion sequence IS6110 revealed M. bovis infection in THP-1 cells. The same was true of a flow cytometry (FC) assay for expression of M. bovis chaperonin 10 in infected cells. The reduced time for isolation and identification of M. bovis (48-72h) and the consistency of the test results make the use of macrophage cell cultures attractive and cost-effective for veterinary laboratories involved in TB surveillance.

Induction and characterization of swine beta interferon.

Newcastle Disease Virus (NDV) strain "H" and Polyinosinic-Polycytidylic acid (Poly I:C) were used for interferon (IFN) induction in secondary pig kidney cells. A functional IFN system was detected and characterized. A wide similarity with the correspondent human and bovine systems was appreciated, with particular regard to the kinetics of synthesis. A glycosylated protein was essential for activity in bovine cells, but not in swine cells. Poly I:C proved to be a very weak inducer, even in conditions which promote IFN synthesis in other cell substrata. beta IFN from secondary pig kidney cells was very effective against Swine Vesicular Disease Virus (SVDV), whereas no activity was detected against porcine Rotavirus; Aujeszky's disease virus, BUK strain, proved to be of intermediate sensitivity. The results of these latter experiments are discussed, with regard to the cells used and to the IFN sensitivity of the tested viruses.

Primary lymphoma of the central nervous system.

Primary lymphoma of the central nervous system (CNS) represents a pathology that is no longer considered rare, also in the light of its high correlation with the human immunodeficiency virus (HIV) syndrome reported recently. Often the correct diagnosis of the disease is difficult to reach, owing to the wide spectrum of non-lymphoma pathologies from which it should be differentiated and the invasiveness of some diagnostic techniques. The biologic aggressiveness of the neoplasm often makes a combined radio-chemotherapeutic approach necessary. In contrast, surgical resection does not seem to provide any significant benefit. The clinical experience reported here, together with a review of the most recent literature, lead the authors to suggest the opportunity of treating primary lymphoma of the CNS with the most active and modern chemotherapeutic protocols in association with traditional treatments to obtain an improvement in overall survival.

Combination chemotherapy in advanced ovarian carcinoma.

Ovarian carcinoma is the fifth most common cause of death among women in western countries. It is often diagnosed in an advanced stage (FIGO Stage III and IV) and requires effective chemotherapy as first-line treatment. The advent of cis-platin combined with adriamycin and cyclophosphamide has remarkably increased the response rate in advanced disease. The authors report 31 cases of epithelial ovarian neoplasia, without prior chemotherapy, treated with cis-platin, adriamycin and cyclophosphamide (PAC I). Of the 30 evaluable patients, 15 had clinical complete remissions (cCR = 50%), 10 clinical partial remissions (cPR = 33%) and 5 no response (NR = 17%). The total response (cCR + cPR) was equal to 83%. Twelve of the 15 patients in cCR underwent second-look laparotomy; in 8 of these cases, histologic and cytologic confirmation of CR was obtained. PAC I was found to be a highly effective therapeutic regimen with moderate toxicity. The individual toxicity reported was gastroenteric (nausea and vomiting), but transitory. No chronic toxic side-effects from cisplatin or adriamycin were noted. However, more definitive results must be obtained to verify its impact on the prolongation of survival.

Evaluation of the specificity of the gamma-interferon test in Italian bovine tuberculosis-free herds.

We investigated the specificity of the gamma-interferon test for bovine tuberculosis (TB) in 1,557 cattle in 30 paratuberculosis-free and officially certified TB-free dairy herds, located in three provinces of the Lombardy Region in Northern Italy. The TB-free status of the herds under examination was further confirmed by the tuberculin skin test, by an antibody assay and by post mortem examination of animals culled from the herds during the study period. The specificity of the gamma-interferon tests after a single test and a double sampling scheme were 88.8% and 95.4%, respectively. After a single test, 11.7% of dubious reactors were also detected, while most cattle (47.4%) were shown to be avian reactors, probably due to contamination from infected birds and/or forage. There was strong evidence that the specificity of the test could be related to the animals' interaction with environmental mycobacteria and/or ageing. To reduce the percentage of nonspecific bovine reactors under alleged TB-free conditions, test procedures might involve the use of more specific antigens and/or different reaction thresholds.

[Pulmonary metastasis from an eccrine carcinoma: thoracic perfusion with the aorto-caval stop-flow technique. Description of a clinical case]. / Metastasi polmonari da carcinoma eccrino: perfusione toracica con tecnica dello stop-flow aorto-cavale. Descrizione di un caso clinico.

A case of a 64-year-old man with eccrine carcinoma arising from hand skin is reported. At the time of diagnosis he showed bilateral pneumonic metastases. Although the patient underwent two systemic chemotherapy lines, he showed further progressive disease of the lung. For this reason a third chemotherapy line was started through thoracic stop-flow infusion. In this way, a five month stable disease had been achieved. The patient died 7 months later for progressive disease. The rarity of this disease, the uncertain treatment, the feasibility and efficacy of thoracic stop-flow infusion are underlined and further studies are suggested.

The wall paintings in the Oratorio of San Giovanni Battista in Urbino: a study finalized to a correct conservation project.

The walls of the Oratorio of San Giovanni Battista in Urbino are decorated with outstanding mural paintings dating back to the 15th century. Due to degradation processes that have occurred in the past years, such paintings require a conservative restoration project. In order to evaluate reasons of the decay, some scientific studies have been performed. They consist of macroscopic observations and chemical (EDS), morphological (SEM) and mineralogical (XRD) analyses of samples both from the original preparatory layers under the painted layers and from the restored plasters at the surbase of the wall. In addition, environmental studies have been performed to verify microclimatic conditions of the church in which the mural paintings are located. Finally, a conservative restoration project was proposed.