Correlation between tcdB gene PCR cycle threshold and severe Clostridium difficile disease.

A retrospective study, including all samples tested for Clostridium difficile from 2015 to 2018, was conducted. 222 and 199 patients were respectively classified as having a mild/moderate or severe disease. A CT ≤ 26 was significantly associated with severe disease. Furthermore, low CT values were significantly associated to older patients and leukocytosis.

Invasive Candidiasis in Brescia, Italy: Analysis of Species Distribution and Antifungal Susceptibilities During Seven Years.

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a large teaching hospital in Brescia, Italy, and the in vitro antifungal susceptibility of isolates. We analyzed 196 isolates causing fungemia in patients admitted in our hospital, between January 2009 and December 2015. Strains were identified by VITEK 2 and MALDI-TOF MS. MICs were determined by Sensititre Yeast OneTM. The resistance was defined by using the revised CLSI breakpoints/epidemiological cutoff values to assign susceptibility or wild type to systemic antifungal agents. Most infections were caused by Candida albicans (60%), Candida parapsilosis (15%), Candida glabrata (12%) and Candida tropicalis (6%). The susceptibility rate for fluconazole was 96.5%. Non-Candida species isolates exhibited full susceptibilities to echinocandins according to CLSI breakpoints. Amphotericin B demonstrated excellent activity against all Candida species. Local epidemiological and antifungal susceptibility studies are necessary in order to improve empirical treatment guidelines.

Emergence of the first levofloxacin-resistant strains of Streptococcus agalactiae isolated in Italy.

Of 901 group B streptococcus strains analyzed, 13 (1.4%) were resistant to levofloxacin (MICs of >32 µg/ml for seven isolates, 2 µg/ml for four isolates, and 1.5 µg/ml for four isolates). Mutations in the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV were identified. A double mutation involving the Ser-81 change to Leu for gyrA and the Ser-79 change to Phe or to Tyr for parC was linked to a high level of fluoroquinolone resistance. In addition, two other mutational positions in parC were observed, resulting in an Asp-83-to-Tyr substitution and an Asp-83-to-Asn substitution. Different mutations were also observed in gyrB, with unknown significance. Most levofloxacin-resistant GBS strains were of serotype Ib and belonged to sequence type 19 (ST19) and clonal complex 19 (CC-19). Most of them exhibited the epsilon gene.

Lymphatic endothelial cells derived from metastatic and non-metastatic lymph nodes of human colorectal cancer reveal phenotypic differences in culture.

Colorectal cancer is one of the most frequent causes of death in Western countries. Most patients develop metastasis traveling through the lymphatic system, and regional lymph node metastasis is considered a marker for dissemination, increased stage, and worse prognosis. Despite rapid advances in tumor biology, the processes that underpin lymphatic invasion and lymph node metastasis remain poorly understood. The aim of this study was to establish an easy protocol for isolation of pure tumor lymphatic endothelial cells derived from lymph nodes to study differences compared with normal endothelial cells of uninvolved tissue from the same patients. Cells were isolated with very high purity via magnetic cell sorting and express the specific lymphatic markers Prox-1 and Lyve-1. They show differences in expression of adhesion molecules, chemokines, and growth factor secretion, and capability to form capillaries when seeded on basal membrane, thereby, revealing important differences between the two cell type. These cultures may provide a promising platform for the comparative analysis of both cell types at the molecular and biological level and to optimize treatment strategies.

LRP1/CD91 is up-regulated in monocytes from patients with haemophilia A: a single-centre analysis.

The low-density lipoprotein receptor-related protein 1 (LRP1) is an ubiquitously expressed endocytic receptor that, among its several functions, is involved in the catabolism of coagulation factor VIII (FVIII) and in the regulation of its plasma concentrations. Although LRP1/CD91 polymorphisms have been associated with increased FVIII levels and a consequent thrombotic risk, no data are available on LRP1/CD91 expression in patients with inherited FVIII deficiency. With the aim of elucidating this issue, 45 consecutive patients with haemophilia A (HA) (18 severe, 5 moderate and 22 mild HA) were enrolled in this cross-sectional, single-centre survey. The LRP1/CD91 mean fluorescence intensity (MFI) in monocytes from HA patients was significantly higher than that detected in 90 healthy blood donors (105 vs. 67, P < 0.001). This over-expression was independent of hepatitis C virus infection status and varied according to the severity of the haemophilia, being higher in patients with more severe FVIII deficiency. In conclusion, our study documents for the first time that LRP1/CD91 is over-expressed on monocytes from HA patients, with the intensity of expression varying according to the severity of the FVIII deficiency. Further studies are needed to assess the clinical implications of these findings.

Contact investigation based on serial interferon-gamma release assays (IGRA) in children from the hematology-oncology ward after exposure to a patient with pulmonary tuberculosis.

BACKGROUND: Interferon-gamma release assays (IGRAs) have high specificity and sensitivity for the diagnosis of tuberculosis (TB) infection. However, their role as a screening tool in children with immunodeficiency disorders is still unclear. In the present study, we performed a contact investigation using serial IGRAs on children with immunodeficiency conditions exposed to a contagious TB patient. METHODS: Children who were exposed to a contagious TB case underwent serial QuantiFERON(®) TB Gold In-Tube (QFT-GIT) and T-SPOT(®).TB (T-SPOT) testing. RESULTS: Eighteen children were tested. At the first testing, only two children (11 %) were positive to T-SPOT. Indeterminate results were more frequent with QFT-GIT (35 %) than with T-SPOT (12 %). In the multivariable analysis, a statistically significant association of lymphocyte count <500 cells/mm(3) (p < 0.00005) and low age (p = 0.03) with indeterminate results for the QFT-GIT test but not for T-SPOT (p = 0.10 and p = 0.88, respectively) was found. At the end of October 2012, 15 of the 18 children were alive and none developed active TB disease. CONCLUSION: T-SPOT provided more determinate results and was less influenced by low age and lymphocytopenia than QFT-GIT in this population of immunodeficient children. These findings suggest that T-SPOT is a more accurate test for the identification of TB infection in young children with lymphocytopenia and should be preferred to QFT-GIT under such specific conditions.

Risk of congenital toxoplasmosis in women with low or indeterminate anti-Toxoplasma IgG avidity index in the first trimester of pregnancy: an observational retrospective study.

OBJECTIVES: Congenital toxoplasmosis (CT) affects one to ten fetuses per 10 000 live newborns in western countries. Without knowing pre-conception serostatus, it is hard to date the infection when anti-Toxoplasma IgG and IgM antibodies are positive at first screening. Although a high IgG avidity index (AI) in the first trimester excludes CT, the same cannot be said of intermediate and low AI. The aim of this study was to estimate the risk of CT when intermediate or low AI is detected in the first trimester of pregnancy. METHODS: Our observational retrospective study enrolled women with positive anti-Toxoplasma IgG and IgM, and low/intermediate AI in the first trimester of gestation seen at two reference centres in northern Italy between 2006 and 2015. All women received spiramycin. When requested by women, a sample of fluid obtained through amniocentesis was tested with a commercial real-time PCR. CT was defined by positive PCR result confirmed on aborted materials or by newborn follow up. RESULTS: Overall, 778 first-trimester pregnant women were included; AI was low in 532/778 (68%) and intermediate in 246/778 (32%). Amniocenteses were performed in 528/778 (67.9%), with no fetal loss. In all, 19/778 (2.4%) miscarriages and 15/778 (1.9%) pregnancy terminations were recorded; 9/778 (1.6%) were lost to follow up. In two women, PCR on amniotic fluid was positive, but CT was confirmed in only 1/747 cases (0.13%, 95% CI 0.02%-0.75%). CONCLUSION: In our study, the risk of CT was much lower than anticipated. These data must be considered when counselling these women.

Characterization of the paradoxical growth hormone inhibitory effect of galanin in acromegaly.

Galanin is a 29-amino acid straight-chain biologically active peptide which has been found to decrease circulating GH levels in some acromegalic patients, whereas is able to increase GH secretion in normal subjects. The aim of our study was to ascertain the incidence, entity and mechanism of the paradoxical GH inhibitory effect of galanin in acromegaly also looking at possible correlations between the GH responses to galanin and the main clinical and biochemical features of the patients. Finally, the effects of either successful or unsuccessful neurosurgical intervention on the GH inhibitory effect of galanin in acromegaly were investigated. A series of 23 consecutive patients with active acromegaly seen at the Endocrine Section of the Department of Internal Medicine of the University of Brescia (Italy) between 1991 and 1994 was examined. The acromegalic patients were subdivided in group 1 (i.e. patients who were 1) untreated, 2) evaluated before surgery, and 3) not cured after surgery and radiotherapy) and group 2 (i.e. surgically cured). All patients were submitted at least once to the following biochemical and radiological evaluations: 1) baseline serum insulin-like growth factor-I and PRL samples, 2) iv infusion of synthetic porcine galanin (500 micrograms in 100 mL saline) from -10 to 30 min, 3) iv bolus injection of TRH (200 micrograms) at time zero, 4) oral glucose tolerance test (75 g glucose, orally) at time zero, and 5) magnetic resonance of the pituitary sella. Adenomatous tissue obtained during neurosurgery in four patients was cultured in vitro, and the effect of the addition of galanin in the culture medium on GH secretion was tested. During galanin infusion in 19 of 21 group 1 patients, serum GH levels were lower with respect to baseline (range of GH decrease, -6.2 to -85.4% with respect to basal levels). During galanin infusion, no reductions in GH levels were observed in the acromegalic patients cured after neurosurgery (group 2); on the contrary, 6 of 7 patients displayed a normal stimulatory response to galanin (range of GH increase, +120-1533.3% of the basal level). A significant correlation between the percent decrease in GH levels after galanin treatment and the percent increase after TRH was found in group 1 patients (r = -0.783; P < 0.05). In three of the four adenomas examined, galanin determined a clear decrease in GH secretion (mean nadir, 63.3 +/- 12% of the baseline secretion rate). In conclusion, we demonstrated that the large majority of numerous patients with active acromegaly show a decrease in serum GH levels after galanin administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of the expression of IFN-gamma in lymphocytes using a monoclonal antibody and flow cytometry.

A stable hybridoma cell line secreting specific antibodies against human interferon-gamma (IFN-gamma) and designated IGMB-14 has been established. It belongs to the IgG1, kappa subclass and it reacts in Western blot with the 28 kDa and 56 kDa polypeptides present in two different affinity purified IFN-gamma preparations. Peripheral blood mononuclear cells (PBMC) from a healthy individual, stimulated in vitro by PHA, were analysed for IFN-gamma production both when viable and following fixation. The presence of cytoplasmic or surface IFN-gamma was visualized by an indirect immunofluorescence assay using monoclonal antibody (MAb) IGMB-14 and a single laser FACS-III fluorescence-activated cell sorter. The staining permitted the detection of newly synthesized cytoplasmic IFN-gamma molecules in lymphocytes at day 1 after PHA stimulation and surface IFN-gamma at day 2. IFN-gamma was expressed on almost all the CD4+ lymphocytes as shown by a double staining technique. The specificity of the reaction was confirmed by Western blots and abolishing IFN-gamma staining by pretreatment of MAb IGMB-14 with IFN-gamma. The presence of surface IFN-gamma was also visualized on freshly isolated PBMC from two patients suffering from measles and AIDS but not on PBMC from a healthy individual. The experiments showed that this immunofluorescent method is useful for the detection, enumeration, and phenotypic characterization of IFN-gamma-producing cells in vitro and, in addition, for evaluating the presence of PBMC expressing IFN-gamma on their surface during a viral disease.

Interferon-gamma is associated with the surface of the human immunodeficiency virus and binds to the gag gene product p17.

Purified HIV-1 antigen preparations produced in cell culture were found to contain interferon-gamma (IFN-gamma). Electron microscopic examination of HIV-1 released by H9 cells, a cell line found to produce IFN-gamma, showed the presence of this molecule on the surface of the virus particle. The HIV-1 protein p17 was found to bind IFN-gamma by a solid-phase radioimmunoassay. The specificity of the reaction was confirmed by Western blot analysis. This finding opens new questions about the biologic role of IFN-gamma itself and of its interaction with HIV.

Inhibitory effect of papaverine on HIV replication in vitro.

The ability of papaverine to inhibit human immunodeficiency virus (HIV) replication in H9 cell line and in peripheral blood mononuclear cell (PBMC) culture was examined. HIV-infected H9 cells were exposed to different concentrations of papaverine for 20 days. Reverse transcriptase (RT) activity and the presence of p24 in the supernatant were determined to assess the level of viral replication in treated and control cultures. The most effective concentration of papaverine in the culture medium was 10 micrograms/ml, a dose that did not significantly affect cell proliferation. At this drug concentration the treatment resulted in no RT activity or p24 expression in the supernatant and no virus antigen detection at the cellular level as demonstrated by Western blot (WB) analysis. The activity of the drug occurred in a short period of time (60 hours) as shown by radioimmunoprecipitation (RIP) assay and affected the synthesis of the env precursor protein gp160. The drug was also effective in inhibiting HIV replication in PBMC cultures and influenced specific viral markers, namely, RT and p24. Evidence of the efficacy of papaverine treatment was enforced by the finding in the treated PBMC cultures, compared with the untreated ones, of a reduced percentage of cells forming syncitia and of the inhibition of the virus-induced decrease in the number of cells. When an equal number of virus-infected H9 cells exposed or unexposed to papaverine was analyzed for HIV-specific proteins, a marked decrease in the expression of the viral proteins was observed in the treated cultures. At the same time, one cellular protein of molecular weight 69,000 was not inhibited by papaverine. This may indicate that, at least for one protein, synthesis may not be affected by the drug. Our data suggest that papaverine merits attention as a possible nontoxic candidate for the treatment of HIV infection.

Aspects of molecular interaction between HIV p17 and human gamma interferon.

We describe the specific interaction between high-purity recombinant human immunodeficiency virus (HIV) type 1 p17 and human gamma interferon (hIFN-gamma) proteins. This interaction was found to be dose dependent and to involve conformational epitopes on both sides. Specificity was confirmed by competition ELISA, using monoclonal antibodies (MAbs) to hIFN-gamma as specific reagents. By competition experiments we also identified the epitope(s) on the hIFN-gamma molecule involved in p17 binding, very close to the receptor binding site. The kinetic constants were determined by surface plasmon resonance (SPR) analysis. The affinity constant (KA) of the complex was 2.78 x 10(8) M-1, that is, the ratio between a low dissociation rate constant (Koff)(1 x 10(-5)sec-1) and a high association rate constant (Kon) (3 x 10(3) M-1sec-1). However, p17 did not displace the binding of hIFN-gamma to its cellular receptor, nor did it interfere with the capability of the lymphokine to induce de novo expression of HLA-DR antigens on human monocytic cells or to inhibit the proliferation of tumor cells.

Inhibitory effects of galanin on growth hormone (GH) release in cultured GH-secreting adenoma cells: comparative study with octreotide, GH-releasing hormone, and thyrotropin-releasing hormone.

The aim of the present study was to characterize in a large series (N = 12) of cultured somatotrope adenomas the in vitro effects of the neuropeptide galanin on growth hormone (GH) secretion. This was contrasted with two peptides known to be GH secretagogues (GH-releasing hormone [GHRH] and thyrotropin-releasing hormone [TRH]) and a peptide with a known GH-inhibitory effect (the somatostatin analog octreotide). Groups of three wells were incubated for 4 hours with growth medium alone (control incubation), galanin, GHRH(1-29)NH2, TRH, or octreotide. Galanin and octreotide were applied at concentrations of 0.1, 1, and 10 mumol/L, and GHRH and TRH at concentrations of 0.01, 0.1, and 1 mumol/L. Galanin was able to inhibit GH release in nine of 12 cultured somatotrope adenoma cells. This inhibitory effect was clearly dose-dependent in five adenomas. Overall, the mean GH nadir after galanin was -36.1% in nine responder adenoma cultures versus control wells. Octreotide inhibited GH release in five of eight cultured somatotrope adenoma cells. The mean GH nadir after octreotide was -32.7% in five responder adenoma cultures compared with control wells. GHRH and TRH were able to stimulate GH release, respectively, in seven of 11 and in six of seven cultured somatotrope adenoma cells. The mean GH peaks after either GHRH or TRH in responder adenoma cultures were, respectively, +71.5% and +143.7% compared with levels in the control wells. In conclusion, the consistency and potency of the in vitro GH-inhibitory effect of galanin in a large series of somatotrope adenomas are at least similar to those of the most effective available GH-lowering agent, the somatostatin analog octreotide.

Effects of the selective estrogen receptor modulator LY117018 on growth hormone secretion: In vitro studies.

Sex steroids play an important role in modulating pulsatile growth hormone (GH) release, acting at both hypothalamic and pituitary level in both humans and experimental animals. Selective estrogen receptor modulators (SERMs) act as either estrogen receptor agonists or antagonists in a tissue-selective manner. In postmenopausal women, serum GH levels correlate positively with endogenous estradiol levels and insulin-like grwoth factor-I (IGF-I) is positively related to bone mineral density (BMD) at the spine and hip. The aim of the present study was to evaluate, for the first time, the direct effect of LY117018, an analog of raloxifene, on GH secretion from both human and rodent pituitary cells in vitro. Our results demonstrated that pharmacological concentrations of the raloxifene analog LY117018 can stimulate GH secretion through a direct action on the pituitary. LY117018 also showed an estrogen-like activity, inducing the proliferation of rat pituitary GH-secreting adenomatous cells (GH1).

Hexarelin, a novel GHRP-6 analog, stimulates growth hormone (GH) release in a GH-secreting rat cell line (GH1) insensitive to GH-releasing hormone.

Previous studies demonstrated that GHRP-6 has modest GH-releasing activity in primary pituitary cell monolayer cultures. However, the effects of this peptide have always been tested on cells very sensitive to GHRH. We have previously reported that GHRH is unable to stimulate GH secretion in the GH1 rat tumor cell line. The aim of the study was to assess for the first time the effect on GH secretion of the GHRP-6 analog, hexarelin, in the GH1 cells; moreover, we investigated the potential involvement of GHRH in the effects of hexarelin in the GH1 rat cell line. The GHRP-6 analog hexarelin (0.01-1 microM) significantly stimulated GH release in both normal and GH1 rat cells. The greatest GH-releasing effect of hexarelin was observed with the 1 microM dose both in GH1 (155+/-25% vs. control wells) and in normal rat pituitary cells (185+/-23% vs. control wells). GHRH significantly stimulated GH secretion in normal rat somatotrophs (3-fold increase). In this latter cell model, GHRH and hexarelin were demonstrated to have additive stimulatory effects on GH secretion. Conversely, GHRH did not affect hexarelin-stimulated GH release in GH1 cells at any of the doses used. Finally, 8Br-cAMP significantly stimulated GH secretion in both normal rat and GH1 cells. These results provide in vitro evidence that non-GHRH-mediated pathways for GHRP action exist. Moreover, the observation that cells not sensitive to GHRH can be significantly stimulated by hexarelin strongly suggests that GHRPs and GHRH have two distinct sites and modes of action at the pituitary level.

Antibiotic resistances and plasmids in Staphylococcus aureus from Italian hospitals.

A total of 473 Staphylococcus aureus isolates from six Italian hospitals was examined for susceptibility to several antimicrobial agents and for plasmid content. Methicillin-resistant S. aureus (MRSA) were characterised by a plasmid of mol. wt (10(6)) 18-22 or 25 that carried the determinants for penicillinase production, resistance to cadmium ions and resistance to tetracycline. MRSA isolates usually harboured other smaller plasmids of mol. wt (10(6)) 2.8, 2.6 and 1.65 that encoded resistance to tetracycline, chloramphenicol and erythromycin, respectively, and cryptic plasmids of mol. wt (10(6)) c. 2 and 1 were found frequently. Methicillin-sensitive S. aureus (MSSA) that produced penicillinase often carried plasmids of mol. wt (10(6)) 11 or 13. No particular difference was found in plasmid patterns of strains from the various sources. Analysis of plasmids by EcoRI digestion showed that plasmids of similar mol. wt and phenotypic characteristics may have different restriction patterns, but often share one or more fragments in common.

Direct enzyme immunoassay for detection of specific IgG antibody to rubella virus by use of labelled antigen.

A new solid phase enzyme immunoassay (EIA) for detection of rubella-specific immunoglobulin G (IgG) antibody was developed. The test uses polystyrene microtiter strips coated with rabbit anti-human IgG immunoglobulins as the solid phase and an enzyme-labelled semipurified rubella antigen as indicator. The direct EIA was compared with hemagglutination inhibition (HI), single radial hemolysis (SRH), radioimmunoassay (RIA) and time-resolved fluoroimmunoassay (TR-FIA) using 52 serum specimens from patients with remote rubella infection. The overall agreement of direct EIA with HI was 96.1%, with SRH and RIA 98.1% and with TR-FIA 100%. The linear regression coefficient varied from 0.77 to 0.91, the best being obtained with direct EIA and SRH. The direct EIA was also suitable for diagnosis of acute infections, as a significant increase in antibody levels was detected in all paired specimens tested from patients with acute rubella infection. The sensitivity and were comparable to those of the assays employed. An advantage of the present assay is that the same method and same labelled antigen can be used to test for different classes of antibody using simply a solid phase with capture antibodies of different chain specificity.

Liquid competition radioimmunoassay for the detection and quantitation of the HIV p24.

Productive infection of permissive cell cultures by HIV has been detected by different assays of which the measurement of reverse transcriptase (RT) activity has been considered highly specific and sensitive. Here we describe the production and characterization of a mouse hybridoma cell line, MB12, secreting monoclonal antibodies to HIV p24, the major core protein, and the use of this monoclonal antibody to develop a type specific indirect liquid competitive radioimmunoassay (RIA) capable of providing earlier detection of the replicating virus than the RT assay. This assay also provides a quantitative analysis of HIV p24, which can be used to study the viral replication in permissive cell cultures. The ease of methodology and the adaptability of the competitive RIA to various assay conditions make this immunoassay suitable for the study of HIV expression in infected cell cultures.

Inhibitory effect of galanin on growth hormone release from rat pituitary tumor cells (GH1) in culture.

The growth hormone (GH) releasing effect of GH-releasing hormone (GHRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, was investigated in cultured rat pituitary tumor cells (GH1) as compared to normal rat somatotrophs. GHRH stimulated dose-dependently GH secretion in normal somatotrophs but did not affect GH secretion in GH1 cells. Galanin (1-10 microM) stimulated GH release in a concentration-dependent manner, but with lower potency as compared to GHRH, in normal rat pituitaries but was inhibitory in rat GH1 cells. The results of this study indicate that while galanin has the ability to stimulate GH release from dispersed pituitary cells of normal rats it has potent direct inhibitory effects on GH release from tumor rat cells.