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.

Sex-related variation in migration phenology in relation to sexual dimorphism: a test of competing hypotheses for the evolution of protandry.

Timing of arrival/emergence to the breeding grounds is under contrasting natural and sexual selection pressures. Because of differences in sex roles and physiology, the balance between these pressures on either sex may differ, leading to earlier male (protandry) or female (protogyny) arrival. We test several competing hypotheses for the evolution of protandry using migration data for 22 bird species, including for the first time several monochromatic ones where sexual selection is supposedly less intense. Across species, protandry positively covaried with sexual size dimorphism but not with dichromatism. Within species, there was weak evidence that males migrate earlier because, being larger, they are less susceptible to adverse conditions. Our results do not support the 'rank advantage' and the 'differential susceptibility' hypotheses, nor the 'mate opportunity' hypothesis, which predicts covariation of protandry with dichromatism. Conversely, they are compatible with 'mate choice' arguments, whereby females use condition-dependent arrival date to assess mate quality.

Administration of sulpiride or domperidone for advancing the first ovulation in deep anestrous mares.

Since results with using sulpiride and domperidone are conflicting and since both have not been tested at the same time, the aim of this study was to compare the efficacy of these substances for the induction of ovulation in deep anestrous mares in the same experimental conditions and to determine their fertility after artificial insemination (AI) at the induced estrus. Twenty-six non-pregnant, non-lactating standardbred anestrous mares were randomly assigned to three groups and treated daily for 25 days (from February 3rd to February 28th) with either sulpiride (1mg/kg of body weight im SID, n=10), or domperidone (1mg/kg po SID, n=10); 6 animals were used as control. The beginning of the transition period and the first ovulation were hastened in sulpiride (16.4+/-0.8 days) but not in domperidone (46.0+/-3.3 days) treated mares (P<0.05). The diameter of the largest follicle was affected by treatment, time and interaction of treatment-by-day (P<0.05) and significantly increased in the sulpiride group (P<0.05). Although a main effect of treatment on plasma LH concentration was not observed (P=0.06), time and interaction of treatment-by-day were statistically significant (P<0.05). The interval from the beginning of treatment to first ovulation was shorter (P<0.05) in the sulpiride group (36.9+/-2.5 days) than in the domperidone (74.7+/-3.3 days) and control (81.4+/-3.1) groups. The establishment of pregnancy was significantly (P<0.05) hastened in sulpiride (61.0+/-35.2 days) but not in domperidone (83.0+/-44.0 days) treated mares. Treated mares not pregnant after the first AI, showed normal estrous cycles with regular interovulatory intervals (P>0.05). It was concluded that sulpiride is effective in advancing the beginning of transition period and the first ovulation whereas domperidone is successful only in some mares.

[Medical legal aspects of the requirement to report noise-induced hearing loss notified to INAIL to the judicial authorities]. / Alcune riflessioni medico legali sull'obbligo di informativa all'autorità giudiziaria delle ipoacusie da rumore segnalate all'INAIL.

BACKGROUND: Law 689/81 redefined how personal lesions could be prosecuted by means of explicit mention of occupational diseases among the type of offences subject to mandatory reporting. The high prevalence of noise-induced hearing loss (NIHL) among occupational diseases has monopolized attention towards identification of a method that can define the penal limits of this occupational disease; however, up to now no single univocal approach exists. For this reason operators in this field are perplexed as to the requirement of reporting judicial authorities (J.A.). On the other hand, the great changes that have occurred in compensation of occupational diseases by INAIL (sentence 179/88 of the Constitutional Court) and the evaluation of the same in terms of biological impairment (Law D.Lgs. 38/00 and Law D.M. 12.7.00) have led to an ample and accurately assessed protection against, work-related hearing loss. OBJECTIVES AND METHODS: From this perspective the authors analysed 52 cases of NIHL reported to INAIL. They compared the assessments made according to Law D.M. 12.7.00 and the guidelines for reporting to the J.A. according to four different methods generally used in the criminal field: Benciolini, Merluzzi, SIMLII guide lines and SIO guidelines. By stressing the need for a preliminary qualitative evaluation of NIHL in the penal report, the authors. restricted the analysis to the quantitative aspect with technically compatible graphs. RESULTS: Processing the data resulting from application of the different methods, led to the assumption that audiometric graphs that showed a percentage of biological impairment according to Law D.M. 12.7.00 higher than 2.40% must always be reported to the JA. For audiometric graphs that show impairment of less than 0.5% recommendations to report tare rather sporadic. For the graphs with intermediate values recommendations to report to the J.A, which are always present in at least one of the methods, are not constant, and in particular there is no linear correlation between the percent grading of biological impairment and the recommendation to report; this is probably due to a difference in concept of the various methods which reflects on the respective scale of values. CONCLUSIONS: On the basis of these results the authors suggest that reporting to the judicial authority can be recommended for all those cases whose quantification, according Marello's schedule, is higher than 0.5%, as these cases can, according to the penal code, supplement assessment of impairment.

Long-term azithromycin in cystic fibrosis: another possible mechanism of action?

Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are not completely understood. Besides its antiinflammatory and antimicrobial activities, one possibility could be the overexpression induction of the multidrug resistance-associated protein (MRP), which could affect chloride transport, thus overcoming the ion transport defect of cystic fibrosis. Seven patients were evaluated before and after 4 weeks of azithromycin treatment (500 mg once daily). Ion transport was studied in vivo by measuring nasal potential difference (NPD). MRP mRNA expression was studied in nasal cells by an internal standard-based semiquantitative RT-PCR assay. NPD was consistent with cystic fibrosis before treatment. After azithromycin treatment, sodium transport was still impaired, whereas a significant increase in chloride conductance was observed (p = 0.03). A significant direct correlation was found between MRP mRNA expression levels and NPD chloride response after azithromycin treatment (p = 0.04, r = 0.78). In conclusion, azithromycin may induce MRP overexpression and restore chloride conductance in the airways of cystic fibrosis patients. These findings suggest a new potential role of azithromycin in the treatment of cystic fibrosis pulmonary disease, i.e. the possibility to upregulate proteins whose function may, at least in part, compensate for the basic defect of cystic fibrosis.

Note: strain sensitivity comparison between fiber Bragg gratings inscribed on 125 and 80 micron cladding diameter fibers, case study on the solidification monitoring of a photo-curable resin.

The influence of fiber Bragg grating diameter when measuring strain is investigated and quantified. Two fiber Bragg gratings with bare cladding diameter of 125 µm and 80 µm are produced by excimer laser irradiation through a phase mask, and are used to simultaneously monitor the Bragg wavelength shift due to the strain produced by the solidification of a photo-curable resin during light exposure. It is found that the ratio of the measured strains in the two fiber Bragg gratings is close to the inverse ratio of the fiber's cladding diameter. These results represent a direct simultaneous comparison between 125 µm and 80 µm diameter fiber Bragg grating strain sensors, and demonstrate the feasibility of strain measurements in photo-curable resins using bare 80 µm cladding diameter fiber Bragg gratings with an increased sensitivity and spatial resolution compared with standard 125 µm diameter fiber Bragg gratings.

Inhibition of MDR1 gene expression by antimessenger oligonucleotides lowers multiple drug resistance.

The multiple drug resistance of neoplastic cells is mediated by overexpression of the human MDR1 gene, which encodes the transmembrane efflux pump P-glycoprotein. In both cell lines and human tumors the MDR phenotype closely correlates with MDR1 mRNA and P-glycoprotein levels. Reversion of the MDR phenotype was attempted in human colorectal adenocarcinoma doxorubicin (Dx)-resistant cells (Lo Vo/Dx) by long-term administration of an equimolecular mixture of three unmodified ODNs (18mer) targeted to adjacent binding sites of the MDR1 mRNA and carried by a synthetic cationic lipid (DOTAP). Three different experimental parameters were used to evaluate the antimessenger agent's effectiveness in comparison with a random sequence ODN: the level of cell resistance to Dx; the level of P-glycoprotein (determined by flow cytometry); the level of MDR1 mRNA (determined by quantitative RT-PCR). Experimental data indicate that the level of both the MDR1 mRNA and the P-glycoprotein is reduced by approximately 50% by treatment of Lo Vo/Dx cells with a 10 microM total concentration of the aODN mixture every 24 h for 15 days. In agreement with these findings, sensitivity to Dx of the antimessenger agent-treated Lo Vo/Dx cells was almost doubled in comparison with random sequence ODN-treated controls.

Marked variation of thymidylate synthase and folylpolyglutamate synthetase gene expression in human colorectal tumors.

Patients with advanced colorectal cancer are currently being treated with 5-fluorouracil (5-FU)-based chemotherapy. A growing number of patients with resectable disease receive adjuvant therapy with 5-FU/levamisole (LEV) or 5-FU/folinic acid (LV). However, many patients still fail on these treatments, due to occurrence of natural or acquired tumor resistance. Among clinically relevant mechanisms of resistance to fluoropyrimidines, increased expression of thymidylate synthase (TS) has been emphasized. Another potentially relevant mechanism involves a decrease in folylpolyglutamate synthetase (FPGS) expression. To establish the value of these genes as prognostic factors and predictors of the outcome of 5-FU-based chemotherapy in colorectal cancer, we measured their expression in colorectal tumors from patients undergoing surgery and postoperative chemotherapy and compared it with that in normal colonic mucosa. This was done by a semi quantitative, nonradioisotopic polymerase chain reaction (PCR) method using beta-actin as an internal standard and expressed as a TS/beta-actin or a FPGS/beta-actin mRNA ratio. In tumor samples from 21 colorectal cancer patients, TS gene expression varied 118-fold. The median TS/beta-actin ratio was, in fact, 41.36 x 10(-3) (range 2.49 x 10(-3) to 294.54 x 10(-3)). Little variation in TS gene expression was observed in corresponding normal colic mucosa; the TS/beta-actin gene ratio was lower (median 26.16 x 10(-3); range 8.49 x 10(-3) to 69.49 x 10(-3)). Among tumor explants from 20 patients, FPGS expression varied over 161-fold. A similar marked variation was also observed in normal colonic mucosal samples (over 185-fold). Overall and disease-free survival data suggest an inverse association between the level of tumor TS and FPGS expression and clinical prognosis. The availability of this sensitive and accurate assay for gene expression should now make it possible to extend these laboratory/clinical correlations to larger populations.

Expression of tissue-type plasminogen activator, plasminogen activator inhibitor and von Willebrand factor in the supernatant of endothelial cell cultures in response to the seeding of adenocarcinoma cell line HRT-18.

Plasminogen activator inhibitor (PAI-1), tissue type plasminogen activator (tPA) and von Willebrand factor (vWF) concentrations were measured by ELISA in the supernatant of the following cultures: endothelial cells from human umbilical vein (HUVEC); human colon cancer cells (HRT-18); and co-culture cells of HUVEC + HRT-18. No measurable amount of the three substances was found in the supernatant of HRT-18 cell culture. Compared to the value in the HUVEC supernatant, in the UVEC/HRT-18 co-cultures, tPA concentration was significantly lower (P = 0.0047), PAI-1 significantly higher (P = 0.026) and vWF also significantly higher (P = 0.0048). These data indicate that HRT-18 tumor cells do not produce tPA, PAI-1 and vWF; however, these tumor cells induce endothelial cells to change the production of these substances. As a consequence, the interaction between tumor and endothelial cells in vivo may lead to depression of fibrinolysis and enhancement of platelet adhesion.

Plasmatic parameters of coagulation activation in thrombotic microangiopathy.

Coagulation activation and fibrinolysis parameters were studied in eleven cases of thrombotic microangiopathy concerning eight adult patients. In addition to routine coagulation tests, antithrombin III, von Willebrand factor (vWF), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), D-dimer (DD), and plasminogen activator inhibitor type 1 (PAI-1) were measured in the plasma at the time of diagnosis and as soon as remission was achieved after therapy with plasma exchange and Iloprost. In the acute phase all patients showed normal aPTT, normal or slightly prolonged prothrombin time, normal or enhanced plasma levels of fibrinogen and antithrombin III, at variance with results in patients affected by disseminated intravascular coagulation. Mean F1+2, TAT, DD, vWF and PAI-1 were elevated in the acute phase, but decreased significantly in the early phase of remission. Our data provide evidence of increased thrombin generation rate which takes place in the acute phase of the disease and does not result in consumption coagulopathy, due to appropriate inhibition by antithrombin III; blood coagulation activation promptly decreased as soon as remission was achieved. Cross-linked fibrin deposition together with PAI-1 may consolidate the platelet plug, eventually resulting in microvascular occlusion and ischemia.

Detection of minimal but significant amount of von Willebrand factor in human omentum mesothelial cell cultures.

The presence of von Willebrand factor (vWF) in human mesothelial cells is a controversial issue. The aim of this paper was to investigate the presence of vWF in human mesothelial cell cultures by means of multiple specific techniques and to compare the amount of vWF to that in endothelial cell cultures. Morphological evidence that vWF is present in the cell cytoplasm in human omentum mesothelial cells (HOMC) has been obtained by vWF staining by means of anti-vWF antibodies and immunofluorescence. The vWF concentration value (measured by ELISA) was 0.02 ng/mL in the supernatant of HOMC (160,000 ng/mL cells/mL after 12-48 hours) and between 0.08-0.12 ng/mL in the supernatant of endothelial cell cultures (160,000 cells/mL). Ethidium-bromide staining of PCR products recorded the typical vWF signal both in the endothelial and in the mesothelial cell cultures, although it was noticeably more intense in the endothelial cell culture. These data show that minimal but significant amounts of vWF are present in human mesothelial cell cultures.

Von Willebrand's factor mediates the adherence of human tumoral cells to human endothelial cells and ticlopidine interferes with this effect.

The aim of this study was to explore whether von Willebrand's factor (vWF) plays a role in the adhesion of human colon tumor cells to human endothelial cells in our coculture system. Cell colony density was evaluated basally (endothelial plus colon tumor cells) and following the addition of: purified vWF, vWF plus vWF-blocking antibodies, antibodies against various integrins and adhesion molecules (alpha2 b integrin, beta1 integrin, beta3 integrin, intercellular adhesion molecule-I, intercellular adhesion molecule-II, vitronectin receptor CD61 CD51, laminin alpha6/beta4 receptor), and various drugs inhibiting the hemostatic system (ticlopidine, heparin, acetyl salicylic acid [ASA], defibrotide, indobuphen, dipyridamole, sulfinpyrazone). Furthermore, vWF concentration was measured in the supernatant fluid of the coculture system basally and following the addition of the above-listed drugs. Cell colony density (as measured by light absorption) increased by 33% following the addition of vWF and returned to a value similar to the basal level with antibodies against vWF, while it did not change significantly following the addition of antibodies against the other integrins or adhesion molecules tested. The same parameter was reduced by 35% following the addition of ticlopidine, while it showed a smaller or no change with the other drugs tested. Similarly, vWF concentration in the cell coculture supernatant showed the greatest reduction (from 0.22 to 0.11 mg/mL) following the addition of ticlopidine. These data suggest that vWF mediates the adherence of human tumor cells to human endothelial cells and that ticlopidine interferes with this effect.

Atherosclerosis and cancer: common pathways on the vascular endothelium.

This article reviews experimental and clinical data on atherosclerosis and cancer showing common pathogenic mechanisms. It is suggested that common pathways follow dysfunction of the vascular endothelium. The activation of the haemostatic system and the overexpression of cytokines and adhesion molecules by the endothelial cells represent important features of this dysfunction. These mechanisms can be responsible for progression of both diseases and explain the higher incidence of thromboembolic events in cancer patients, the occurrence of similar laboratory findings and the effect of many drugs on the course of the two diseases. Our article confirms that atherosclerosis and cancer share common mechanisms, and we hope it will stimulate further clinical trials on the use of drugs active on the haemostatic system in cancer patients.

Uremic medium increases cytokine-induced PAI-1 secretion by cultured endothelial cells.

The overall fibrinolytic activity is depressed in patients with chronic renal failure where a prothrombotic state is described, thereby enhancing the risk of vascular occlusive events. The mechanism responsible for fibrinolysis derangement has not yet been elucidated. To evaluate the effect of the uremic environment on the fibrinolytic activity of endothelial cells, we studied plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) production by human umbilical vein endothelial cells (HUVEC) in culture, exposed either to uremic or normal sera, before and after cytokine stimulation. Twenty uremics were studied: 11 were on conservative dietary treatment and nine were on maintenance hemodialysis. Eight healthy subjects served as controls. Before cytokine stimulation, no difference in the HUVEC supernatant concentration of t-PA and PAI-1 was found among the groups studied. After stimulation with interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha, the HUVEC supernatant levels of PAI-1 in the uremics were higher than in the controls, whereas the supernatant levels of t-PA did not differ. Our data provide evidence that uremic serum, in concert with IL-1 or TNF-alpha, can enhance PAI-1 secretion by endothelial cells, thereby depressing the fibrinolytic system. This impaired endothelial fibrinolytic response to hypercoagulation could favor vascular events, which are the major cause of morbidity and mortality in patients with chronic uremia.

Bisegmentectomy 5-8 for the removal of tumors of the 8th hepatic segment--a technical note.

Isolated resection of the 8th segment is a technical challenge. The deep location of the afferent portal pedicle mandates the performance of a wedge resection that leaves a deep and narrow wound in which hemostasis is difficult to achieve. Furthermore the relationships with the middle and right hepatic veins jeopardize the transparenchymal approach. For the removal of tumors located in the 8th segment, we propose a combined resection of the 8th and 5th segments, whose pedicles arise from the anterior right pedicle of the portal vein. The technique permits a safe liver resection and ensures a satisfactory surgical margin.

Recognition of HLA class II molecules by antipeptide antibodies elicited by synthetic peptides selected from regions of HLA-DP antigens.

Repeated immunizations of rabbits with chemically synthesized peptides from selected regions of HLA-DP histocompatibility antigens resulted in the production of specific antibodies that were then isolated from the immune sera by chromatography on Sepharose-peptide immunoadsorbents. The purified antibodies, when tested with an enzyme-linked immunosorbent assay, specifically bound to the inciting fragments; moreover, two of them recognized glycoproteins extracted by nonionic detergents from human chronic lymphocytic leukemia cells, as revealed by binding assays. The results suggest that amino acid stretches 51-61 of the alpha chain and 80-90 of the beta chain of HLA-DP histocompatibility antigens are likely exposed on the surface of the protein molecule. The specific recognition of DP regions is strongly suggested by the difference in the binding of those antibodies to soluble membrane proteins, as compared to the binding of monomorphic anti-Class II monoclonal antibodies to the same antigens.

Oligodendrocyte-derived J1-160/180 extracellular matrix glycoproteins are adhesive or repulsive depending on the partner cell type and time of interaction.

We have studied the functional involvement of J1-160 and J1-180 in the interaction between oligodendrocytes and neurons, astrocytes, or L cells in short- and long-term adhesion assays using monoclonal antibodies directed against topographically distinct epitopes on the molecules. Whereas antibodies to mouse liver membranes and monoclonal antibody 597 do not interfere with neuron-oligodendrocyte or astrocyte-oligodendrocyte adhesion after 30 min of coculture, antibodies 596, 619, and 620 interfere with astrocyte to oligodendrocyte and neuron to oligodendrocyte adhesion. The adhesion of L cells to oligodendrocytes is not affected by the antibodies. When neurons or astrocytes are cultured on oligodendrocytes for more than 30 min, monoclonal antibody 619 continues to reduce adhesion of astrocytes to oligodendrocytes after 1 and 2 h. However, during this time period the antibody affects neuron to oligodendrocyte adhesion in a different manner. It does not interfere with adhesion of neurons to oligodendrocytes at 1 h and enhances the adhesion of neurons to oligodendrocytes after 2 h of coculture. After 6 and 24 h of coculture, antibody 619 does not affect the adhesion of neurons or astrocytes to oligodendrocytes, suggesting that other adhesive mechanisms are predominant at later times of interaction. At all times studied, neurons and astrocytes adhered well to the oligodendrocytes. To study the influence of the J1 molecules on neuronal interactions in the absence of other oligodendrocyte-derived cell surface components, purified J1-160 was coated as a substrate and neuron attachment was measured as a function of time. Two hours after plating neurons adhered well to J1-160, as they did to laminin, while cell detachment was subsequently observed from J1-160, but not from laminin. These results implicate J1-160 and J1-180 in a recognition process between oligodendrocytes and neurons or astrocytes, but not fibroblasts. This recognition process appears to merge into adhesion or stabilization of cell contacts for astrocytes and destabilization of cell interactions or repulsion for neurons. It is likely that these two opposite effects in cell behavior elicited by the J1 molecules result from differential intracellular responses to a cell surface trigger possibly mediated by different cell surface receptors and/or different consequences in intracellular signaling networks.

Generation of rabbit antipeptide antibodies to HLA-class II antigens by the use of synthetic peptides.

A group of eight synthetic peptides, corresponding in sequence to selected regions of HLA-DQ histocompatibility antigens, was used for rabbit immunization to examine their antigenicity and for localizing exposed regions in the native glycoproteins. Those antibodies were then tested in their ability to recognize the HLA-DQ alloantigens. Seven peptides elicited rabbit antibodies, four of which reacted with human glycoproteins prepared from chronic lymphocytic leukaemia cells. The results indicate that sequence stretches 63 to 79 and probably 82 to 93 of the beta chain correspond to exposed regions in DQw1, DQw2 and DQw3 molecules. However, the specificity of those antipeptide antibodies was low, due to extensive crossreactions with amino acid sequencies of high homology occurring in DQ alloantigens.