Evaluation of isavuconazole MIC strips for susceptibility testing of Aspergillus and Scedosporium species.

Isavuconazole is a new triazole with an expanded-spectrum and potent activity against moulds and yeasts. It has been authorized for use in adults for the treatment of invasive aspergillosis and for mucormycosis. The only commercially available isavuconazole susceptibility test is the minimum inhibitory concentration (MIC) strip isavuconazole test. The objective of this study was to assess the in vitro activity of isavuconazole using gradient concentration MIC strips, compared with the EUCAST broth microdilution reference method. A total of 147 clinically relevant fungal isolates comprising 120 Aspergillus sp. and 27 Scedosporium apiospermum complex were tested for susceptibility to isavuconazole using the EUCAST broth microdilution method and by the MIC strip isavuconazole test. The percent essential agreement between the two methods was calculated within a 1-fold dilution. The geometric means for the MICs using the EUCAST reference methods and the strip test were respectively: 0.60 mg/l and 0.65 mg/l for A. fumigatus, 0.70 mg/l and 0.77 mg/l for A. flavus, 1.50 mg/l and 1.25 mg/l for A. niger, 0.41 mg/l and 0.38 mg/l for A. terreus, 1.22 mg/l and 1.08 mg/l for S. apiospermum complex. The isavuconazole MIC strips showed good agreement with the EUCAST reference method. Isavuconazole MIC strips could be useful for susceptibility testing of Aspergillus sp. and S. apiospermum complex.

Hyperbaric oxygen therapy (HBOT) and peridontal health.

This study has tested the effects of hyperbaric oxygen in periodontal structures in agreement with the theories supported by literature research. Eight patients, from 30 to 50 years-of-age, were tested with pure oxygen inhalation, at the 2.5 ATA absolute pressure. Main approved tests of periodontal health were evaluated before and after HBOT’s cycles. The results in all patients treated with HBOT, have founded clear improvement of clinical and instrumental parameters.

Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells.

The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth.

Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism.

Gemcitabine (GEM, 2',2'-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl-L-cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells.

Antimicrobial resistance pattern of methicillin-resistant Staphylococcus aureus in the food industry.

There is increasing concern about the impact on public health of methicillin-resistant Staphylococcus aureus (MRSA) associated with animal food products. MRSA remains a serious problem because of the high incidence and multidrug resistance of the strains, even for strains isolated from foods, food environments and food handlers. The objectives of this study are: (i) to evaluate the susceptibility of S. aureus strains isolated from food, food handlers and food-processing environments to 14 antibiotics currently used in veterinary and human therapy; (ii) to assess the presence of the mecA gene. A total of 1007 samples were collected from food, food handlers, and environments and were analyzed for the presence of S. aureus. S. aureus was present in 165 of the 1007 samples. A total of 157 isolates were methicillin-susceptible S. aureus (MSSA) and 8 isolates were MRSA. In particular, out of 8 MRSA strains detected, 4 strains harboured the mecA gene. All MRSA strains were resistant to at least one of the tested antibiotics and 6 strains demonstrated multi-resistance. Considering the high level of resistances in S. aureus and the isolation of MRSA strains, the surveillance of antimicrobial resistance and the spreading of this pathogen is of crucial importance in the food production chain. These data are useful in improving background data on antimicrobial resistance of S. aureus isolated from food, processing environments and food handlers, supporting the prudent use of antibiotics and the development of international control programs.

Surveillance of human papilloma virus infection and cervical cancer in kidney transplant recipients: preliminary data.

INTRODUCTION: Development of cancer after transplantation has rapidly became one of the leading causes of death in kidney transplant recipients with functioning grafts. Anogenital malignant neoplasms may occur with a 14-fold increased incidence, and human papilloma virus (HPV) infection has been recently identified as the leading cause of cervical carcinoma. We report the preliminary findings of a prospective study that evaluated the incidence of HPV infection and cervical carcinoma in a population of kidney transplant recipients. PATIENTS AND METHODS: The study included 35 female recipients of a deceased donor kidney with at least 6 months of follow-up. All patients underwent a cervicovaginal brushing, an HPV DNA test, and a Papanicolaou test. RESULTS: Twenty-two patients (62.8%) were positive for HPV DNA. Thirteen of 22 HPV DNA-positive recipients (59%) demonstrated a high-risk HPV genotype. No cytologic anomalies were detected in Papanicolaou smears. CONCLUSIONS: These preliminary data demonstrated a high incidence of HPV infection in renal transplant recipients. Most of our recipients exhibited a high-risk HPV genotype, which suggests higher aggressiveness of such infection in immunosuppressed patients. The HPV test is useful to monitor patients at higher risk of anogenital malignant neoplasms by identifying the cytologic anomalies at an earlier stage. This ongoing study will investigate the rate of progression of HPV infection and the clinical patterns of HPV-positive cytologic anomalies in renal transplant recipients.

Intracellular zinc increase inhibits p53(-/-) pancreatic adenocarcinoma cell growth by ROS/AIF-mediated apoptosis.

We show that treatment with non-toxic doses of zinc in association to the ionophore compound pyrrolidine dithiocarbamate (PDTC) inhibits p53(-/-) pancreatic cancer cell growth much more efficiently than gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Both the metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and the radical scavenger N-acetyl-l-cysteine are able to recover cell growth inhibition by Zn/PDTC, demonstrating that this effect depends on the increased levels of intracellular zinc and of reactive oxygen species (ROS). Zn/PDTC treatment induces a strong apoptotic cell death that is associated to ROS-dependent nuclear translocation of the mitochondrial factor AIF, but not to the regulation of apoptotic genes and caspase activation. Primary fibroblasts are more resistant than pancreatic cancer cells to Zn/PDTC treatment and exhibit a lower basal and Zn/PDTC-induced enhancement of intracellular zinc. We show that Zn/PDTC induces p53 proteasomal degradation and that the proteasome inhibitor MG132 further increases fibroblast growth inhibition by Zn/PDTC, suggesting that p53 degradation plays an important role in fibroblast resistance to Zn/PDTC.

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes.

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.

Inducible nitric oxide synthase expression elicited in the mouse brain by inflammatory mediators circulating in the cerebrospinal fluid.

Expression of inducible nitric oxide synthase (iNOS) protein was studied in the brain after intracerebroventricular injections of interferon (IFN)-gamma, and IFN-gamma combined with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha, compared to ovalbumin as control. Wild-type mice and mice with targeted deletion of the IFN-gamma receptor gene were used. Findings based on iNOS immunoreactivity were evaluated at 1, 2, 4 and 7 days post-injection, using also quantitative image analysis and double labeling with glial cell markers. IFN-gamma administration induced iNOS immmunostaining in activated microglia and macrophages in the parenchyma surrounding the ventricular system, several cortical fields and fiber tracts. IFN-gamma-elicited iNOS immunoreactivity was down-regulated after 1 day. The number of iNOS-immunopositive cells was significantly enhanced by co-administration of LPS or TNF-alpha; IFN-gamma+TNF-alpha injections also resulted in longer persistence of iNOS immunoreactivity. No immunopositive cells were seen in the brain of IFN-gamma receptor knockout mice after IFN-gamma administration; very few immunostained macrophages were detected in these cases, mostly around the injection needle track, after co-administration of LPS or TNF-alpha. Western blot analysis confirmed a marked iNOS induction in the brain of wild-type mice 24 h after IFN-gamma+LPS injections. The findings show that inflammatory mediators circulating in the cerebrospinal fluid induce in vivo iNOS in the brain with topographical selectivity and temporal regulation. The data also demonstrate that the signaling cascade activated by IFN-gamma binding to its receptor is critical for iNOS induction, and the synergistic action of LPS and TNF-alpha as iNOS inducers in brain cells is largely mediated by the receptor-regulated action of IFN-gamma.

[Clinical and sperm follow-up after subinguinal varicocelectomy]. / Follow-up clinico e spermatico dopo varicocelectomia subinguinale.

In order to evaluate the sperm output and the adverse-side-effects after subinguinal varicoceloctomy, a follow-up study of 16 months was performed on 196 selected patients (aged from 22 to 43 years) affected by left varicocele (VR). In the pre-treatment, both Doppler ultrasonography and didymo-epididymal ultrasonography allowed to distinguish two homogeneous patient groups: group A (no. = 136), including patients affected by VR alone and, group B (n. = 60), including patients with VR combined to coincidental didymo-epididymal morphological abnormalities, DEMA). These DEMA lesions (testis size < 12 ml, epididymides abnormalities: increased head- > or = 12 mm- and/or tail- > or = 6 mm-diameter, multiple microcysts, large idrocele) were omolaterally to VR in 30/60 (50%), eterolaterally in 19/60 (31.7%) or bilaterally in 11/60 (18.3%). During sperm follow-up, group A patients showed both a significant temporal change (p < 0.01 ANOVA) of all sperm parameters studied (sperm density, total sperm count, motility and morphology) from month 8 onward and sperm values significantly higher than found in group B patients. On the contrary, the sperm parameters of group B patients did not change significantly during the follow-up observations. As far as the varicocelectomy-mediated clinical symptoms, some patients complained early and transiently (on 1-2-4 weeks following varicocelectomy) the following symptoms: didymal pain (1.5%), didymo-epididymal pain (4.1%) and parasthesiaes on the anterior-medial side of the left thigh (4.1%) or scrotal (3.1%); only four patients (2%) complained permanent paresthesiaes on the anterior-medial side of the left thigh. Furthermore, the clinical follow-up also revealed a low rate of complications: persistent VR (3.6%), hydrocele (1.5%), intrascrotal venous ecstasies (6.1%), epididymitis (0.5%). Some morpho-structural abnormalities at US scans were transient (1-2 weeks): scrotal oedema (6.1%), orchitis (2%), orchi-epididymitis (1%). Subinguinal varicocelectomy performed on large population demonstrated a significant improvement of the sperm output from month 8th onward in patients with VR alone, while sperm parameters did not show any significant change in patients with VR plus coincidental DEMA. This surgical technique also demonstrated safety since both low rates of symptoms and (transient) complications were registered.

Cell-specific differences in the regulation of IL-6 expression by PMA.

We have studied the regulation of IL-6 expression in human blood monocytes and lymphocytes. LPS and IFN-gamma induced IL-6 gene expression with a similar qualitative profile in both cell types. Treatment of monocytes and lymphocytes with PMA resulted, instead, in different effects: monocytes accumulated IL-6 and its message, while lymphocytes were inhibited either in the absence or the presence of LPS and IFN-gamma. These results suggest that the signal transduction pathways triggered by LPS and IFN-gamma are similar in both cell types, while PMA may activate a tissue-specific pathway which leads to opposite responses.

Lithostathine messenger RNA expression in different types of chronic pancreatitis.

Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous tissue from pancreatic cancer subjects. However, about 70% of the pancreatic cancer subjects showed lithostathine and chymotrypsinogen mRNA levels comparable to those of chronic pancreatitis patients. These results indicate that the decrease in the level of mRNA is not specific to lithostathine and it is unrelated to the presence of pancreatic stones.

[A facilitated technique for donor hepatectomy: experimental study in the pig]. / Proposta di una tecnica facilitata per il prelievo di fegato: studio sperimentale nel suino.

A facilitated technique of porcine donor hepatectomy is reported underlying the possibility to make an easy procedure without in situ portal perfusion, so other abdominal organs supplied by superior mesenteric system can be harvested at the same time. The viability of those grafts procured without in situ portal perfusion has been compared with an other group of livers harvested with in situ aortic and portal flushing. The evaluation of the histology and early graft function two hours after riperfusion has shown no differences between both groups.

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action.

Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.

Protein synthesis inhibitors cycloheximide and anisomycin induce interleukin-6 gene expression and activate transcription factor NF-kappaB.

In two human cell lines, MDA-MB-231 and HeLa, the inducible expression of the interleukin-6 (IL-6) gene by two protein synthesis inhibitors, cycloheximide and anisomycin, was compared with the induction by the most potent physiological inducer of IL-6 described to date, interleukin-1beta (IL-1beta). In cycloheximide or anisomycin treated cells, the accumulation of the IL-6 message and the activation of transcription factors required for IL-6 gene expression occurs at an extent similar to that obtained with IL-1beta. Furthermore, IL-6 mRNA accumulation stimulated by cycloheximide or anisomycin is almost completely inhibited in the presence of actinomycin D, indicating that this effect occurs mainly through the activation of the transcriptional machinery. These data indicate that transcriptional induction of the IL-6 gene by inhibitors of protein synthesis is triggered by the same nuclear signals as other inducers.

[Role of hyperbaric oxygen in the treatment of necrotizing infections of the soft tissues]. / Ruolo dell'ossigeno iperbarico nel trattamento delle infezioni necrotizzanti dei tessuti molli.

The Authors report their experience in the treatment of soft tissue necrotizing infections. The correct "timing" of the association of surgery, antibiotics and hyperbaric oxygen therapy is stressed. The good clinical results obtained in the patients examined confirm the usefulness of an early treatment modulated according to the needs of each single patient.

Nuclear factor kappa B (NF-kappa B), nuclear factor interleukin-6 (NFIL-6 or C/EBP beta) and nuclear factor interleukin-6 beta (NFIL6-beta or C/EBP delta) are not sufficient to activate the endogenous interleukin-6 gene in the human breast carcinoma cell line MCF-7. Comparative analysis with MDA-MB-231 cells, an interleukin-6-expressing human breast carcinoma cell line.

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted IL-6 and mRNA which, as expected, are dramatically enhanced following induction by either IL-1 beta or tumor necrosis factor-alpha (TNF-alpha). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1 beta in spite of the fact that stimulation by TNF-alpha alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-kappa B, NFIL6 and NFIL6 beta, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA-MB-231 cells. Furthermore, transfection of a recombinant plasmid carrying the IL-6 promoter linked to a luciferase reporter gene shows that both cell lines are able to drive IL-1 beta or TNF-alpha activation of this construction in a very similar manner. Finally, when MCF-7 cells were treated with IL-1 beta or TNF-alpha in the presence of cycloheximide, transcription of IL-6 mRNA from the endogenous IL-6 gene was observed. These data suggest that a mechanism of IL-6 gene repression is active in MCF-7 cells.

Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species.

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants. In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species. We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.