Decreasing trend of drug-resistant TB in Italy.

BACKGROUND: TB caused by rifampicin-resistant (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis strains is a major concern to TB control globally. However, in the European Union, MDR-TB notifications among all bacteriologically confirmed TB cases with available drug susceptibility testing (DST) results decreased over the last years.METHODS: We conducted a retrospective analysis on DST results reported from 2011 to 2020 by 46 laboratories in 19 out of 20 regions in Italy in order to evaluate resistance trends to first- and second-line drugs in MDR/RR-TB strains isolated from Italian-born persons (IBPs) and foreign-born persons (FBPs).RESULTS: Of 23,972 M. tuberculosis strains examined (15,519 from FBPs and 8,453 from IBPs), MDR-TB decreased from 3.2% in 2011 to 2.2% in 2020. High MDR/RR-TB rates occurred mostly in FBPs from former Soviet Union countries. In 2017, a MDR/RR-TB increase was detected in FBPs from sub-Saharan Africa. MDR-TB strains showed consistent increase in resistance to pyrazinamide (PZA), slight increase in resistance to fluoroquinolones and a decrease in resistance to other drugs.CONCLUSION: While MDR/RR-TB cases slightly decreased, a worrisome increase of resistance to PZA and fluoroquinolones among MDR/RR-TB patients was seen. This implies that a fast and efficient diagnosis aligned with therapy is crucial for TB control.

Activity of drugs against dormant Mycobacterium tuberculosis.

OBJECTIVE/BACKGROUND: Heterogeneous mixtures of cellular and caseous granulomas coexist in the lungs of tuberculosis (TB) patients, with Mycobacterium tuberculosis (Mtb) existing from actively replicating (AR) to dormant, nonreplicating (NR) stages. Within cellular granulomas, the pH is estimated to be less than 6, whereas in the necrotic centres of hypoxic, cholesterol/triacylglycerol-rich, caseous granulomas, the pH varies between 7.2 and 7.4. To combat TB, we should kill both AR and NR stages of Mtb. Dormant Mtb remodels lipids of its cell wall, and so lipophilic drugs may be active against NR Mtb living in caseous, lipid-rich, granulomas. Lipophilicity is expressed as logP, that is, the logarithm of the partition coefficient (P) ratio Poctanol/Pwater. In this study, the activity of lipophilic drugs (logP>0) and hydrophilic drugs (logP⩽0) against AR and NR Mtb was measured in hypoxic conditions under acidic and slightly alkaline pHs. METHODS: The activity of drugs was determined against AR Mtb (5-day-old aerobic cells: A5) and NR Mtb (12- and 19-day-old hypoxic cells: H12 and H19) in a Wayne dormancy model of Mtb H37Rv at pH 5.8, to mimic the environment of cellular granulomas. Furthermore, AR and NR bacilli were grown for 40days in Wayne models at pH 6.6, 7.0, 7.4, and 7.6, to set up conditions mimicking the caseous granulomas (hypoxia+slightly alkaline pH), to measure drug activity against NR cells. Mtb viability was determined by colony-forming unit (CFU) counts. RESULTS: At pH 5.8, lipophilic drugs (rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide: logP⩾2.14) reduced CFU of all cells (H12, H19, and A5) by ⩾2log10. Among hydrophilic drugs (isoniazid, pyrazinamide, ethambutol, amikacin, moxifloxacin, metronidazole: logP⩽0.01), none reduced H12 and H19 CFUs by ⩾2log10, with the exception of metronidazole. When Mtb was grown at different pHs the following Mtb growth was noted: at pH 6.6, AR cells grew fluently while NR cells grew less, with a CFU increase up to Day 15, followed by a drop to Day 40. AR and NR Mtb grown at pH 7.0, 7.4, and 7.6 showed up to 1 log10 CFU lower than their growth at pH 6.6. The pHs of all AR cultures tended to reach pH 7.2-7.4 on Day 40. The pHs of all NR cultures remained stable at their initial values (6.6, 7.0, 7.4, and 7.6) up to Day 40. The activity of drugs against H12 and H19 cells was tested in hypoxic conditions at a slightly alkaline pH. Under these conditions, some lipophilic drugs were more active (>5 log CFU decrease after 21days of exposure) against H12 and H19 cells than clofazimine, nitazoxanide, isoniazid, pyrazinamide, amikacin (<1 log CFU decrease after 21days of exposure). Testing of other drugs is in progress. CONCLUSION: Lipophilic drugs were more active than hydrophilic agents against dormant Mtb in hypoxic conditions at pH 5.8. The Wayne model under slightly alkaline conditions was set up, and in hypoxic conditions at a slightly alkaline pH some lipophilic drugs were more active than other drugs against NR Mtb. Overall, these models can be useful for testing drug activity against dormant Mtb under conditions mimicking the environments of cellular and caseous granulomas.

Pyrazinamide susceptibility testing: proposed new standard with the BACTECTM MGITTM 960 system.

The susceptibility of 253 Mycobacterium tuberculosis complex isolates to pyrazinamide (PZA) was assessed using the BACTECTM MGITTM 960 (M960) system. Resistant strains underwent paired repeat testing using 1) a critical concentration of 200 g/ml (PZA-200), and 2) a reduced inoculum of 0.25 ml. They were also examined using the BACTEC 460 (B460) reference method and investigated for pncA mutations. On M960, 37 isolates were resistant. In the PZA-200 assay, 20 of these were resistant and 17 susceptible, while 18 were resistant and 19 susceptible with reduced inoculum. The B460 assay and pncA sequencing confirmed results with reduced inoculum.

Magnesium deficiency modulates hepatic lipogenesis and apolipoprotein gene expression in the rat.

The present study was performed to determine the effects of magnesium (Mg) deficiency upon plasma lipoproteins and hepatic apolipoprotein gene expression in the rat. The most obvious effect of Mg-deficiency on plasma lipids is a marked increase in post-prandial triacylglycerol concentration. This increased triglyceridemia persists in fasted rats. Density gradient ultracentrifugation analysis revealed marked alterations in the distribution of plasma lipoproteins in Mg-deficient rats. An increase in triacylglycerol-rich lipoproteins (TGRLP) was associated with a significant increase in plasma apolipoprotein B (apo B) concentration and was accompanied by selective accumulation of apo B-48. A decrease in high-density lipoproteins (HDL) was accompanied by a corresponding decrease in plasma apo E concentration and a concordant decrease in hepatic apo E mRNA abundance and biosynthesis. Hepatic apo B-100 synthesis was reduced by over 75% in Mg-deficient animals despite an increase in hepatic apo B mRNA abundance. However, this change in hepatic apo B gene expression was not associated with alterations in posttranscriptional apo B mRNA editing. These changes in apolipoprotein gene expression were associated with increased hepatic lipogenesis, despite the observation that net triacylglycerol secretion rates were not different between Mg-deficient and control animals. Taken together, the data demonstrate a complex pattern of alterations in hepatic lipid metabolism and apolipoprotein gene expression in the Mg-deficient rat and suggest a defect in the catabolism rather than secretion of TGLRP as the major factor underlying the altered plasma lipoprotein profile.

Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (apobec-1) ameliorates hypercholesterolemia in LDL receptor-deficient rabbits.

Apolipoprotein (apo) B48, a protein contained in intestinally derived lipoprotein particles, is synthesized by post-transcriptional editing of apoB100 mRNA. This reaction is mediated by an enzyme complex that includes the catalytic subunit, apobec-1. The liver of most mammals, by contrast, contains only unedited apoB mRNA and secretes apoB100, the major protein component of plasma low-density lipoprotein (LDL). Because rabbits, like humans, fail to edit hepatic apoB100 mRNA, we introduced a recombinant adenovirus encoding apobec-1 into the livers of LDL receptor-defective rabbits to determine the impact on lipoprotein metabolism of hepatic apoB48 secretion. Transgene expression was mainly confined to the liver and was sustained for up to 3 weeks following virus administration, as evidenced by the presence of apobec-1 mRNA and the ability of hepatic S100 extracts to edit a synthetic apoB RNA template in vitro. The transient induction of hepatic apoB mRNA editing accompanied alterations in very-low-density lipoprotein (VLDL) size, the presence of apoB48 in fractions spanning the VLDL and LDL range, and modest reductions in total plasma cholesterol levels.

Heat-shock induced protein modifications and modulation of enzyme activities.

Upon heat stress, the cell physiology is profoundly altered. The extent of the alterations depends on the severity of the stress and may lead to cell death. The heat shock response is an array of metabolic changes characterized by the impairment of major cellular functions and by an adaptative reprogramming of the cell metabolism. The enhanced synthesis of the HSPs is a spectacular manifestation of this reprogramming. Numerous post translational modifications of proteins occur in response to heat stress and can be related to altered cellular functions. Some proteins are heat-denatured and temporarily inactivated. Heat-denaturation is reversible, chaperones may contribute to the repair. The extent of heat-denaturation depends on the cell metabolism: (a) it is attenuated in thermotolerant cells or in cells overexpressing the appropriate chaperones (b) it is enhanced in energy-deprived cells. Covalent modifications may also rapidly alter protein function. Changes in protein glycosylation, methylation, acetylation, farnesylation, ubiquitination have been found to occur during stress. But protein phosphorylation is the most studied modification. Several protein kinase cascades are activated, among which the various mitogen activated protein kinase (MAP kinase) cascades which are also triggered by a wide range of stimuli. As a possible consequence, stress modifies the phosphorylation status and the activity of components from the transcriptional and translational apparatuses. The same kinases also target key enzymes of the cellular metabolism. Protein denaturation results in constitutive hsp titration, this titration is a signal to trigger the heat-shock gene transcription and to activate some of the protein kinase cascades.

A new set of monoclonal antibodies against acute lymphoblastic leukemia.

Six monoclonal antibodies produced by immunization of Balb/c mice with common acute lymphoblastic leukemia (cALL) cells were tested against various types of normal and malignant tissues. ALB1 and ALB2 are directed to the cALL antigen (CALLA gp100); ALB6 recognizes a determinant of p24; ALB7, ALB8 and ALB9 have a pattern of reactivity similar to Ba1. None of these antibodies specifically identify cALL but they should be useful tools for diagnosis or depletion of bone marrow in autologous therapy in transplantation. In addition, the example of ALB6 which acts as a platelet aggregating agent, suggests that the study of other cell systems expressing the antigens associated with cALL may shed light on the function of these antigens and subsequently on the physiopathology of the leukemic cells.

Increased hepatic synthesis and accumulation of plasma apolipoprotein B100 in copper-deficient rats does not result from modification in apolipoprotein B mRNA editing.

Experimentally induced copper deficiency in the rat results in increased plasma apolipoprotein B100 (apo B100) concentration in association with increased hepatic apo B100 synthesis. This enhancement of apo B100 synthesis and plasma accumulation accounts for the rise of plasma low density lipoprotein in these animals. In the present study, we have investigated if the selective increase in hepatic apo B100 synthesis is accounted for by changes in apo B mRNA editing. Reverse transcription coupled with polymerase chain reaction amplification and primer extension analysis of apo B cDNA revealed no differences in apo B mRNA editing in either the liver or small intestine between control and copper-deficient rats. We speculate that the increase in apo B100 synthesis in the liver of copper-deficient rats reflects posttranslational alterations in gene expression accompanying changes in very low density lipoprotein assembly and secretion.

Late results of reconstructive surgery for renovascular hypertension.

In order to evaluate the late results of reconstructive surgery for renovascular hypertension, a review was made on a series of 120 consecutive patients who underwent operations over a 11 year period. There were 82 males (68.3%) and 38 females (31.7%) with a mean age of 48.4 years. Renal artery by-pass grafts were used in 90% (120/133), a thromboendarterectomy in 5.2% (7/133), and other surgical procedures were performed in 4.8% (6/133). Associated vascular procedures were performed in 38.3% (46/120) of patients. Operative mortality was 2.5% (3/120) overall; there was no mortality in the isolated renal artery reconstructions. There was a clinical success (after a mean follow-up of 48 months) in 80.4% of patients. The most important factors influencing clinical result after renal revascularization were: a generalized atherosclerosis (p less than 0.05), duration of hypertension (p less than 0.01) and the early post-operative response of the blood pressure (p less than 0.01). The overall five- and ten-year actuarial survival probabilities were 85 and 68%, respectively. The most common causes of death were myocardial infarction, stroke and cancer. Cox regression analysis for variables influencing survival indicated that persistence of severe hypertension was the major determinant of late survival (p less than 0.05). Hypertension in females is better tolerated, while younger patients appear to have better results and late survival after surgical treatment.

[Treatment of pancreatic pseudocysts]. / Traitement des pseudokystes du pancréas.

Based on the results of treatment of a personal series of 13 cases of pseudocyst of the pancreas, between 1974 and the present day, it is suggested that the choice of therapy should be surgical. An internal shunt is preferred for pseudocysts as a result of acute pancreatitis or injury, whereas a wider cysto-wirsung jejunostomy is recommended for cysts developing during the course of chronic pancreatitis. These proposals follow analysis of immediate and long-term (mean: 51 months) follow-up, on the basis of mortality, morbidity, pain symptoms, malabsorption and postoperative diabetes.

REPR and complementation factor(s) interact to modulate rat apolipoprotein B mRNA editing in response to alterations in cellular cholesterol flux.

Apolipoprotein B (apoB) mRNA editing is a post-transcriptional cytidine deamination involving several protein factor(s), one of which has recently been cloned. We have examined the effects of alterations in cellular cholesterol flux in the rat liver and small intestine as a means of dissecting the physiologic mechanisms regulating apoB mRNA editing, both in vivo and in isolated S-100 extracts. Hepatic cholesteryl ester accumulation was produced by feeding rats a high cholesterol diet, alone, or in combination with either ethinyl estradiol treatment, or after induction of hypothyroidism. Endogenous hepatic apoB mRNA editing decreased in parallel with the increase in cellular cholesteryl ester content (r = -0.948, P < 0.001). None of these conditions altered endogenous intestinal apoB mRNA editing. Hepatic S-100 extracts demonstrated decreased in vitro apoB RNA editing activity, in parallel with the changes observed in vivo. By contrast, the activity of intestinal S-100 extracts demonstrated a paradoxical increase in hypothyroid rats and a similar, paradoxical decrease in hyperthyroid rats, when compared to controls. Hepatic REPR mRNA, quantitated by RNase protection assay, showed a 25-50% decrease in cholesterol-fed rats. The editing activity of hepatic S-100 extracts prepared from cholesterol-fed, hypothyroid rats was restored to control levels with REPR supplementation but not with chicken intestinal S-100 extracts, suggesting that changes in REPR, but not complementation activity, may play a critical role in the regulation of apoB mRNA editing in rat liver. By contrast, the editing activity of intestinal S-100 extracts prepared from hyperthyroid animals was unaltered by supplementation with REPR, but was restored to control levels after the addition of chicken intestinal S-100 extracts. Taken together, the data suggest that tissue-specific factors regulate apoB mRNA editing in the rat and that the complex interplay of REPR and complementation factor(s) may be modulated in response to alterations in cholesterol flux, in vivo.

Interferon-beta strong cytopathic effect on human papillomavirus type 16-immortalized HPK-IA cell line, unexpectedly not shared by interferon-alpha.

We report a novel, unusually severe cytopathic effect of interferon-beta (IFN-beta). Data concerning antibody neutralization, induction and recovery time course, CPE50 dose, impact on oxidative metabolic activity and 1D SDS-PAGE total cellular protein analysis are provided for preliminary characterization. This cytopathic effect appears to be linked to human papillomavirus type 16 (HPV-16) genome presence as it is markedly evident in the HPV-16-immortalized HPK-IA cell line, but is not induced in diploid keratinocytes. It is also not induced in highly malignant SiHa cells suggesting that it also requires a fairly conserved phenotype. This effect is unexpectedly not shared by IFN-alpha pointing to a discrimination between IFN-alpha and -beta signal despite the well-known sharing of a common receptor. It remains to be clarified whether this divergence, undetectable in other cellular systems, represents a direct effect of viral presence or a non-specific consequence of cellular homoeostatic disregulation induced by the papillomavirus genome.

An improved reverse transcription-polymerase chain reaction method to study apolipoprotein gene expression in Caco-2 cells.

We report a method for the detection and analysis of apolipoprotein B mRNA using the thermostable enzyme rTth to perform coupled reverse transcription-polymerase chain reaction (RT-PCR) amplification. This method, which is at least a 100-fold more sensitive than traditional RT-PCR, was used to examine elements of apolipoprotein B (apoB) gene expression in Caco-2 cells. A region of apoB mRNA spanning the edited site was amplified from pre- and postconfluent Caco-2 cells both under different growth conditions and following alterations in exogenous lipid flux to determine changes in posttranscriptional editing. Apolipoprotein A-IV (apoA-IV) mRNA levels were examined in the same samples. The results suggest that apoB mRNA editing increases in Caco-2 cells during growth but this response is more variable than previously reported. Additionally, evidence was found for differential editing of the 14 kb and 7 kb transcripts. By contrast, there was a consistent growth-related increase in apoA-IV mRNA abundance. Neither apoB mRNA editing nor apoA-IV mRNA abundance was modulated in postconfluent cells in response to different combinations of exogenous lipid. This method should facilitate the study of apolipoprotein gene expression in Caco-2 cells and other situations where the target RNA is limited either as a result of low abundance or limiting tissue sample size.

Tumour necrosis factor-alpha gene polymorphism: implications in kidney transplantation.

In this study we have analysed the TNFA biallelic polymorphism at the -308 position, in 169 kidney recipients that received the graft in a single Italian transplantation facility and we have then correlated the TNFA genotypes with the post-transplant outcome. To assess the cytokine genotypes, a polymerase chain reaction-sequence specific primer (PCR-SSP) methodology has been utilised. By the analysis of the different genotypes, the corresponding TNF-alpha phenotypes and the level of the TNF-alpha production, were deduced: the TNF(*)1/TNF(*)1 genotype gives a low TNF-alpha production level, TNF(*)1/TNF(*) 2 and TNF(*)2/TNF(*)2 genotypes give a high TNF-alpha production level. Out of the one hundred and sixty-nine patients studied, one hundred and twenty-one recipients (72%) had a low TNF-alpha producer phenotype, whereas forty-eight (28%) had a high TNF-alpha producer phenotype. These frequencies were not statistically different from those of the control group. The incidence of acute rejection episodes, vascular damage (grade III degrees of Banff classification), and serum creatinine levels at 1 month, were significantly greater in high TNF-alpha producers (P=0. 048, 0.031 and 0.007 respectively). The logistical regression model indicated that only the high producer genotype and donor age were significantly and independently correlated with acute graft failure (P=0.02 and P=0.013 respectively). This analysis shows that recipient TNFA polymorphism, previously associated with differential production TNF-alpha by in vitro studies could be related to the clinical outcome of kidney transplantation.

Nuclear factors bind to a conserved DNA element that modulates transcription of Anopheles gambiae trypsin genes.

The Anopheles gambiae trypsin family consists of seven genes that are transcribed in the gut of female mosquitoes in a temporal coordinated and mutually exclusive manner, suggesting the involvement of a complex transcription regulatory mechanism. We identified a highly conserved 12-nucleotide motif present in all A. gambiae and Anopheles stephensi trypsin promoters. We investigated the role of this putative trypsin regulatory element (PTRE) in controlling the transcription of the trypsin genes. Gel shift experiments demonstrated that nuclear proteins of A. gambiae cell lines formed two distinct complexes with probes encompassing the PTRE sequence. Mapping of the binding sites revealed that one of the complex has the specificity of a GATA transcription factor. Promoter constructs containing mutations in the PTRE sequence that selectively abolished the binding of either one or both complexes exerted opposite effects on the transcriptional activity of trypsin promoters in A. gambiae and Aedes aegypti cell lines. In addition, the expression of a novel GATA gene was highly enriched in A. gambiae guts. Taken together our data prove that factors binding to the PTRE region are key regulatory elements possibly involved in the blood meal-induced repression and activation of transcription in early and late trypsin genes.

Tissue-specific, developmental and nutritional regulation of the gene encoding the catalytic subunit of the rat apolipoprotein B mRNA editing enzyme: functional role in the modulation of apoB mRNA editing.

Apolipoprotein B (apoB) mRNA editing, a posttranscriptional site-specific cytidine deamination reaction, is mediated by a protein complex of which the catalytic component (REPR) has recently been cloned. REPR mRNA was demonstrated by RNase protection at highest abundance in small intestine and colon but the transcript was detectable in all tissues examined including kidney, spleen, lung, liver, and ovary. ApoB mRNA was found predominantly in the liver and small intestine but low levels were detected in all adult tissues examined and found to be variably (29-86% TAA) edited. In addition, S100 extracts prepared from spleen and kidney were competent to edit an apoB RNA template in vitro, suggesting that the entire apoB mRNA editing complex is present and functionally active in these tissues. In situ hybridization demonstrated REPR mRNA to be distributed along the entire villus-crypt axis, while apoB mRNA distribution did not extend into the crypts. In the liver, both apoB and REPR mRNA were detected in all cells of the hepatic lobule without an apparent gradient of expression. REPR mRNA was found in the red pulp of the spleen and in the superficial crypt cells of the colon. This distribution of REPR mRNA was recapitulated by immunocytochemical localization of the protein within these tissues. Finally, the developmental and nutritional modulation of REPR was examined in relation to endogenous apoB mRNA editing. Small intestinal apoB mRNA editing was found to undergo a developmentally regulated increase beginning at gestational day 20, preceding a developmental increase in REPR mRNA abundance. Additionally, hepatic and kidney apoB mRNA editing both revealed a temporal dissociation from alterations in REPR mRNA abundance. By contrast, adult rats subjected to fasting and refeeding a high carbohydrate diet, demonstrated concordant modulation of endogenous apoB mRNA editing and REPR mRNA abundance (r = 0.92, P < 0.001). Taken together, the data demonstrate that REPR and other components of the rat apoB mRNA editing complex are widely distributed and undergo distinct developmental and metabolic regulation that interact to regulate apoB mRNA editing in a tissue-specific manner.

The transcriptional inhibitors, actinomycin D and alpha-amanitin, activate the HIV-1 promoter and favor phosphorylation of the RNA polymerase II C-terminal domain.

Actinomycin D and alpha-amanitin are commonly used to inhibit transcription. Unexpectedly, however, the transcription of the human immunodeficiency virus (HIV-1) long terminal repeats (LTR) is shown to be activated at the level of elongation, in human and murine cells exposed to these drugs, whereas the Rous sarcoma virus LTR, the human cytomegalovirus immediate early gene (CMV), and the HSP70 promoters are repressed. Activation of the HIV LTR is independent of the NFkappaB and TAR sequences and coincides with an enhanced average phosphorylation of the C-terminal domain (CTD) from the largest subunit of RNA polymerase II. Both the HIV-1 LTR activation and the bulk CTD phosphorylation enhancement are prevented by several CTD kinase inhibitors, including 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. The efficacies of the various compounds to block CTD phosphorylation and transcription in vivo correlate with their capacities to inhibit the CDK9/PITALRE kinase in vitro. Hence, the positive transcription elongation factor, P-TEFb, is likely to contribute to the average CTD phosphorylation in vivo and to the activation of the HIV-1 LTR induced by actinomycin D.