Five-year surveillance of human tuberculosis caused by Mycobacterium bovis in Bologna, Italy: an underestimated problem.

Human tuberculosis (TB) caused by Mycobacterium bovis surveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused by M. bovis over a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles of M. bovis strains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused by M. bovis was 1·76%, significantly associated to extra-pulmonary localization (P = 0·004) and to being elderly (P < 0·001) and Italy-born (P = 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that most M. bovis strains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases of Mycobacterium bovis BCG infection were also characterized. In conclusion, the rate of human TB caused by M. bovis was not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.

Investigating the Origin of Mycobacterium chimaera Contamination in Heater-Cooler Units: Integrated Analysis with Fourier Transform Infrared Spectroscopy and Whole-Genome Sequencing.

Mycobacterium chimaera is ubiquitously spread in the environment, including factory and hospital water systems. Invasive cases of M. chimaera infection have been associated with aerosols produced by the use of heater-cooler units (HCU) during cardiac surgery. The aim of this study was to evaluate for the first time the performance of IR-Biotyper system on a large number of M. chimaera isolates collected from longitudinal environmental HCUs samples and water sources from hospitals located in three Italian provinces. In addition, IR-Biotyper results were compared with whole-genome sequencing (WGS) analysis, the reference method for molecular epidemiology, to investigate the origin of M. chimaera contamination of HCUs. From November 2018 to May 2021, 417 water samples from 52 HCUs (Stockert 3T, n = 41 and HCU40, n = 11) and 23 hospital taps (used to fill the HCU tanks) were concentrated, decontaminated, and cultured for M. chimaera. Positive cultures (n = 53) were purified by agar plate subcultures and analyzed by IR-Biotyper platform and Ion Torrent sequencing system. IR-Biotyper spectra results were analyzed using a statistical approach of dimensionality reduction by linear discriminant analysis (LDA), generating three separate clusters of M. chimaera, ascribable to each hospital. Furthermore, the only M. chimaera-positive sample from tap water clustered with the isolates from the HCUs of the same hospital, confirming that the plumbing system could represent the source of HCU contamination and, potentially, of patient infection. According to the genome-based phylogenies and following the classification proposed by van Ingen and collaborators in 2017, three distinct M. chimaera groups appear to have contaminated the HCU water systems: subgroups 1.1, 2.1, and branch 2. Most of the strains isolated from HCUs at the same hospital share a highly similar genetic profile. The nonrandom distribution obtained with WGS and IR-Biotyper leads to the hypothesis that M. chimaera subtypes circulating in the local plumbing colonize HCUs through the absolute filter, in addition with the current hypothesis that contamination occurs at the HCU production site. This opens the possibility that other medical equipment, such as endoscope reprocessing device or hemodialysis systems, could be contaminated by M. chimaera. IMPORTANCE Our manuscript focuses on interventions to reduce waterborne disease transmission, improve sanitation, and control infection. Sanitary water can be contaminated by nontuberculous Mycobacteria, including M. chimaera, a causative agent of invasive infections in immunocompromised patients. We found highly similar genetic and phenotypic profiles of M. chimaera isolated from heater-cooler units (HCU) used during surgery to thermo-regulate patients' body temperature, and from the same hospital tap water. These results lead to the hypothesis that M. chimaera subtypes circulating in the local plumbing colonize HCUs through the absolute filter, adding to the current hypothesis that contamination occurs at the HCU production site. In addition, this opens the possibility that other medical equipment using sanitized water, such as endoscope reprocessing devices or hemodialysis systems, could be contaminated by nontuberculous Mycobacteria, suggesting the need for environmental surveillance and associated control measures.

Transanal haemorrhoidal artery echodoppler ligation and anopexy (THD) is effective for II and III degree haemorrhoids: a prospective multicentric study.

AIM: We report a multicentric prospective study which aimed to evaluate Doppler-assisted ligation of the terminal haemorrhoidal arteries (THD) for II and III degree haemorrhoids. METHOD: A total of 112 patients from five colorectal units, including 81 men, mean age 48 +/- 13 years, with II degree (39) and III degree (73) haemorrhoids were treated by Doppler-guided transanal de-arterialization and anopexy using a new device (THD). RESULTS: The mean operative time was 33.9 +/- 8.8 minutes, and the mean number of ligatures applied was 7.2 +/- 1.5. Postoperatively, 72% of patients did not need analgesics and the other 28% used nonsteroidal antiinflammatory drugs 1-3 times/day for less than 2 days. All the patients were operated as a day case. Early postoperative complications included haemorrhoidal thrombosis (2 patients), bleeding (1) treated by haemostatic suture, dysuria (6) and acute urinary retention (1). After a mean follow-up of 15.6 +/- 6.5 months (range 6-32), 2/105 (20.9%) patients complained of minor bleeding, while mild pain was still present in 4/51 patients (7.8%). There were no statistically significant differences in the sample population regarding the gender or stage of the disease. Tenesmus was cured in 15/17 patients, dyschaezia in 20/22 patients and mucous soiling in 10/10 patients. No new cases of altered defaecation or faecal incontinence were recorded. Overall, 85.7% of patients were cured and 7.1% improved. Residual haemorrhoids were treated by elastic band ligation in nine (8%) patients and by surgical excision in further five patients (4.5%). CONCLUSION: Doppler-assisted ligation of the terminal branches of the haemorrhoidal arteries for II and III degree haemorrhoids is highly effective and painless. Complications are few and the technique can be performed as a day case.

TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2.

OBJECTIVE: The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat. METHODS: HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors. RESULTS: We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted. CONCLUSIONS: The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.

Human cytomegalovirus product UL44 downregulates the transactivation of HIV-1 long terminal repeat.

OBJECTIVE: Human cytomegalovirus (HCMV) is often isolated from HIV-1-infected patients and the two viruses can infect the same cell type giving rise to direct bidirectional interactions. Whereas the long terminal repeat (LTR) transactivation ability of HCMV immediate early gene (IE1/IE2) is well documented, no information is available on the possible role of other HCMV proteins. In this study, the activity of ppUL44, an early DNA-binding protein, on HIV LTR transactivation was investigated. METHODS: HIV LTR transactivation by ppUL44 in presence or absence of HIV-1 Tat and HCMV IE1/IE2 was determined in J-Jhan and U973 cells through transient transfection experiments with a series of different expression vectors. Some experiments were also performed on U373-MG astrocytoma cells permanently transfected with UL44 or with another HCMV gene used as a control (UL55). RESULTS: The basal transactivation activity of the HIV LTR was not influenced by the presence of ppUL44. On the contrary, the transactivation observed in the presence of Tat, IE1/IE2 or both factors in synergy was strongly downregulated by ppUL44 in a dose-dependent manner. Deletion constructs of ppUL44 demonstrated that the region of the molecule responsible for the inhibition of the LTR is located within the last 114 amino acids at the carboxyl-terminal region. CONCLUSIONS: The results obtained indicate that within the last 114 amino acids of ppUL44 there is a domain that has a negative effect on the ability of HIV-1 LTR to be activated by both its autologous transactivator Tat and the heterologous transactivator HCMV IE1/IE2 functioning individually or synergistically.

Intracellular nucleotides of lymphocytes and granulocytes from normal ageing subjects.

Impaired lymphocyte and granulocyte function in the aged may, in part, reflect intrinsic aged-related biochemical alterations. In this study we compared the ribonucleotide contents of lymphocytes and granulocytes from young and old subjects evaluated by means of an HPLC-anion exchange method. We found that in general both populations from old subjects present higher levels of the various nucleotides, in particular: ATP, UDP, CTP, UDP-glucose in granulocytes, AMP, CTP, UDP-N-acetylglucosamine, UDP-glucose in lymphocytes. These data suggest that these molecules accumulate in aged subjects because of altered biochemical pathways. The increased pool of UDP-sugars, in particular, could be due to a depressed activity of some glycosyltransferases which therefore fail to glycosylate some plasma membrane cell proteins, thus accounting for their functional impairment.

IgG subclass distribution of anti-HBs antibodies following vaccination with cDNA HBsAg.

Serum samples were obtained during follow-up of nine young adults vaccinated over 1 year with cDNA hepatitis B antigen. The absolute amounts of anti-HBs IgG subclass antibodies present in the sera were determined by comparing the optical densities (OD) obtained using an antigen-specific ELISA with those obtained by serial dilutions of known amounts of human IgG1-4. The calibration curves for each IgG subclass were corrected for the corresponding coating efficiency. Our data suggest that HBs antibody responses of vaccinated subjects occur in all IgG subclasses but IgG1 and IgG2 are the major subclasses involved.

Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry.

A general titration calorimetry method is described that can be used to determine the affinity of tight binding interactions with proteins. The method is based on the thermodynamic linkage between ligand binding and coupled protonation reactions. The protons linked to a given ligand-binding reaction are measured by titration calorimetry, and integration of the resulting data set yields the pH dependence of the binding affinity based on thermodynamic relationships developed elsewhere. When the pH dependence of the binding affinity is combined with the absolute affinity determined independently at a pH at which the affinity can be conveniently measured, the absolute binding affinity over the entire pH range is determined. The method is well suited for determining high-affinity binding interactions of protein antigens with antibodies, but is applicable to any macromolecular ligand-binding reaction that is coupled to protonation.

Albumin gene expression in adenocarcinomas with hepatoid differentiation.

Two cases of hepatoid adenocarcinomas were studied with an in situ hybridization technique (ISH) using a RNA probe for human albumin mRNA. In case 1 the urinary bladder of a 67-year-old woman was affected; in case 2 the tumour was located in the gastric antrum of an 80-year-old woman. In neither case had alpha fetoprotein (AFP) been determined preoperatively. Histologically these cases showed adenocarcinomatous features intermingled with hepatoid areas. These latter areas were characterized by cords of polygonal cells, each with an oval nucleus and prominent nucleoli, separated by a fine network of sinusoids. In the hepatoid areas the immunohistochemical profile was similar to that observed in hepatocellular carcinomas, in that the tumour cells were positive with AFP, alpha-1-antitrypsin (A1AAT) and albumin antisera and there was a canalicular type of reactivity with polyclonal anti-CEA (pCEA) antibody. ISH revealed albumin mRNA in virtually all hepatoid cells in case 1, and in about 50% of those in case 2. In addition, in case 2 occasional cells in the adenocarcinomatous areas showed albumin transcripts of ISH. Our findings confirm that ISH for albumin mRNA probe is a valuable method of establishing hepatocellular differentiation, and that hepatoid adenocarcinomas are tumours with true extrahepatic hepatocellular differentiation.

Standardized reporting of histological diagnoses for non-neoplastic liver conditions in needle biopsies.

The importance of standardizing surgical pathology reports is emerging from the literature. The use of checklists has recently been proposed for diagnosing the major tumour types, but no attention has been given to non-neoplastic conditions. In this paper a checklist for standard reports of liver needle biopsies for non-neoplastic conditions is presented.

Identification of mitochondria in liver biopsies. A study by immunohistochemistry, immunogold and Western blot analysis.

Hepatocytes are rich in mitochondria, which play an important role in hepatic metabolism. In certain pathologic conditions (most often alcoholic liver disease) mitochondria became enlarged; nevertheless, even in these conditions they are hardly detectable on light microscopy. Recently an antimitochondrial antibody (mAM), which recognizes a 60-kDa protein, has been characterized. The purpose of the present study was to study immunoreactivity of this antibody in a series of liver biopsies. We studied 146 liver biopsies using an mAM. In 8 cases an ultrastructural study was also done, and in 2 cases Western blot analysis was performed. Cases were divided as follows: alcoholic liver disease (ALD, 31); steatosis (8); nonalcoholic steatohepatitis (NASH, 1); hepatitis C virus (HCV)-related hepatitis (83); hepatitis B virus (HBV)-related hepatitis (6); primary biliary cirrhosis (1); sclerosing cholangitis (1); haemosiderosis (1); sarcoidosis (1); alpha-1-antitrypsin deficiency (1); nonspecific findings (12). All the patients were investigated for alcohol or drug abuse, pharmacological treatment, hyperlipidaemia, hypercholesterolaemia and diabetes. Immunoreactivity was diffuse in cases of ALD, NASH and steatosis, and in patients with drug abuse. Electron microscopic immunogold and Western blot analysis confirmed that in the conditions examined the protein recognized by the mAM showed greater expression. Immunohistochemical staining was helpful in demonstrating a toxic or a metabolic insult even in cases in which the histological picture was blurred by viral infection.

Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies.

We isolated and characterized from a lambda gt11 expression library clones expressing portions of human cytomegalovirus (HCMV)-p52. This nonstructural viral protein is encoded by UL44 and is known to be one of the best IgM reactive antigens. The reactivity of these clones was studied with human antibody and the gene fragment coding for the most immune-reactive portion of p52 (aa 202-434) was cloned in a prokaryotic expression vector, pROS, which overexpresses the antigen as a fusion protein to a truncated molecule of beta-galactosidase.

Treatment of chronic erosive gastritis: a double-blind trial of pirenzepine and cimetidine.

In a double-blind study, 59 patients with chronic erosive gastritis received 50 mg of pirenzepine twice daily and 55 patients received 400 mg of cimetidine twice daily for six weeks. In both groups, days of pain, of heartburn, and of nausea per week were significantly reduced during treatment (P less than 0.01). After six weeks, 64% of the pirenzepine group and 62% of the cimetidine group were free of symptoms and endoscopy revealed healing of lesions in 78% and 80%, respectively. Differences between groups were not significant.

Correlation between histologic staging, hepatitis C virus genotypes and clinical features in HCV chronic hepatitis: evidence of a new pattern.

Genome heterogeneity may be related to the wide variability of clinical and pathological features in hepatitis C virus (HCV)-related chronic liver disease. This paper addresses the possible association between HCV subtypes and clinical and histological features of chronically infected patients. Sixty-eight consecutive liver biopsies of chronic hepatitis constituted the basis of the study. HCV genotyping was performed on frozen tissue. Grading of necroinflammatory activity and staging of fibrosis were histologically assessed. Serologic HCV-RNA and liver function were assessed at the same time. All information was compared with clinical data including age, sex, HCV serology, and probable data and route of infection. Two cases were excluded as inadequate tissue was available. Five cases were negative to HCV-RNA in both serum and tissue. In 61 cases HCV RNA was present at the same time in serum and liver tissue. Forty-four patients were men (72%) and 17 (28%) were women. Two peaks of age were observed: 1 in the 4th decade of life, the 2nd in the 7th. The 2 groups had different HCV genotypes. Patients with genotypes 1b (mean age 50.7 years), 2c (mean age 61.3 years), and a subgroup of coinfections (mean age 60 years) were older than patients with genotypes 1a (mean age 35.5 years), 3 (mean age 36 years), and a subgroup of coinfections (mean age 33 years). Patients with genotypes 1b, 2, or 2c and a subgroup of coinfections more frequently had a history of blood transfusion and or surgical intervention dating up to 49 years previously. Patients with HCV 1a, 3, and a subgroup of coinfections frequently admitted a period of intravenous drug abuse. Patients with advanced liver disease, i.e., severe fibrosis and cirrhosis, showed the same 2 peaks of incidence: in the 4th and 7th decades of life, the first group mainly comprising patients with HCV types 1a and 3, the second, patients with HCV types 1b and 2c. Both these groups shared a clinical history of a long-standing infection. Two profiles of patients emerged. The largest group was composed of elderly patients, infected by HCV genotypes 1b or 2c, with a history of blood transfusion and/or surgery, presenting an advanced stage of liver disease (namely, severe fibrosis or cirrhosis). The second group was composed of younger patients, mainly in the 4th decade of life, infected by HCV types 3 or 1a, often presenting with chronic hepatitis in the stage of severe fibrosis or cirrhosis. The latter could be the profile of HCV infection in the near future.

A combination of pirenzepine and cimetidine: a new approach to treatment of duodenal ulcer in "non-responders".

Treatment of peptic ulcer in "non-responders" with the combination of pirenzepine and cimetidine was evaluated in a longterm study. Patients who did not respond to two months of treatment, first with cimetidine (1 g/day), and then pirenzepine (150 mg/day), were given one of these two compounds alone for 6 months or a combination at lower doses for 24 months. The combination of pirenzepine (75 mg/day) and cimetidine (400 mg/day) was successful in a high percentage of cases, and showed fewer side effects than in the single treatment groups. The effectiveness of the combination treatment might be due to its more significant anti-secretory effects.

Esaprazole in the treatment of gastric ulcer: preliminary clinical trial.

The activity and tolerability of esaprazole administered once a day at doses of 900 and 1350 mg/day in the treatment of gastric ulcer were evaluated in a preliminary study carried out in 47 patients. The results failed to show any difference between the two doses in terms of percentage of ulcer healing after 4 weeks (43% and 45% respectively) or 8 weeks (86% and 82%), or in the course of symptoms and signs, which improved rapidly. The safety of esaprazole was good, no modifications of laboratory parameters being observed. Although the study involved only a small number of patients, the findings obtained indicate the potential usefulness of esaprazole in the treatment of gastric ulcer.

Detection and consequences of recombinant protein isoforms: implications for biological potency.

Protein isoforms commonly occur in nature and in products produced by recombinant DNA technologies. Some isoforms may be generated as a consequence of the manufacturing process. Bioassay methods may not be sufficiently sensitive enough or specific enough to determine the biological effect of having a new or increased level of a particular isoform. Sophisticated analytical techniques exist for the structural characterization of protein isoforms. The biological implications of these isoforms are not easy to determine. Additional "Biological Characterization" work may need to be done to evaluate the biological consequences of isoforms. An understanding of the product's mechanism of action and the disease mechanism of action is essential for a thorough evaluation.