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Non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) are closely associated, and liver fibrosis has been related to macrovascular complications. We examined whether liver fibrosis, diagnosed by FibroScanWe recruited 394 outpatients with T2DM attending five Italian diabetes centres who underwent liver ultrasonography (US), FibroScanSteatosis by US was present in 89%. Almost all patients (96%) were on hypoglycaemic drugs, 58% had at least one chronic vascular complication, 19% a macrovascular complication (prior myocardial infarction and/or ischaemic stroke) and 33% a microvascular one (26% chronic kidney disease [CKD]; 16% retinopathy; 6% neuropathy). In all, 171 (72%) patients had CAP ≥ 248dB/m (ie hepatic steatosis), whereas 83 (21%) patients had LSM ≥ 7.0/6.2 kPa (M/XL probes) (significant liver fibrosis). CAP was not associated with any macro/microvascular complications, whereas LSM ≥ 7.0/6.2 kPa was independently associated with prior cardiovascular disease (adjusted OR 3.3, 95%CI 1.2-8.8; P = .02) and presence of microvascular complications (adjusted OR 4.2, 95%CI 1.5-11.4; P = .005), mainly CKD (adjusted OR 3.6, 95%CI 1.3-10.1; P = .01) and retinopathy (adjusted OR 3.7, CI 95% 1.2-11.9; P = .02). Neither diabetes duration nor haemoglobin A1c differed according to CAP or LSM values.Significant fibrosis, detected by FibroScan

Background The metabolic syndrome is an independent risk factor for ischaemic heart disease. Patients with systemic lupus eriythematosus have un increased prevalence of the metabolic syndrome.1 There are no controlled studies of the metabolic syndrome in patients with SSc. Objectives To compare the prevalence of the metabolic syndrome in patients with SSc and controls and to evaluate its relationship to activity and severity of disease. Methods Methods: 50 consecutive patients with SSc (45 female, median age 48; range 20–72) and 50 controls (patients whit fibromyalgia) (45 female, median age 46; range 25–72) were studied. The prevalence of the metabolic syndrome was compared in patients and controls using the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATPIII): central obesity: waist. 102 cm in men and. 88 cm in women;2 hypertriglyceridaemia:>150 mg/dl;3 low HDL, 40 mg/dl in men and, 50 mg/dl in women;4 high blood pressure:>130/85 mm Hg or use of drugs for high blood pressure; and5 high fasting glucose >110 mg/dl, and associations with activity and severity of the disease were examined. Results The metabolic syndrome was present in 24% of patients and in 30% of controls subjects (p>0.05). Among patients with SSc, the metabolic syndrome was significantly associated with higher value of ESR (ESR >30) (5/7 vs 4/34;p=0.027). Neither disease activity nor severity scores were associated with the metabolic syndrome Conclusions Patients with SSc have a lower prevalence of the NCEP-ATPIII-defined metabolic syndrome than controls. This result may help to justify the coronary prevalence in SSc patients similar to that of the general population.2–3 References [1] Bengtsson AA, et al. PLoS One2016Jul;21;11(7). [2] Hattema ME, et al. Rheumatology2008;45:578–83. [3] Akram MR, et al. Rheumatology2006;45:1395–8. Disclosure of Interest None declared

Objectives The aim of the present study was to evaluate the body composition by bioelectrical impedance analysis (BIA) and to assess the nutritional status by BMI and possible correlations with epidemiological and clinical characteristics in patients with SSc Methods Malnutrition was defined as BMI 10% in combination with a fat-free mass index (FFMI) 70 years). Body composition was assessed in 40 patients (female) (age mean ±sd: 45.2±12.2) and 20 healthy controls (age mean ±sd 41.5±5.3) with BIA (Akern, Italy) and BMI. The manufacturer’s equation and the Geneva equation were used to estimate fat mass (FFM). In addition, correlations with disease activity, gastrointestinal severity, disease subset, autoantibody profile, skin score were evaluated. Results Malnutrition was found in 10% of patients vs 8% of healty controls; and low FFMI in 30% of patients vs 10 5 of HC. Bioimpedentiometry showed a Fat Free Mass (FFM) (metabolically active component of fat free mass) reduced in patients compared to controls (46.8±7.6 vs 53.6±6.3 respectively; p=0.01). Furthermore, with the same instrument a lower basal metabolic rate was found in patients compared to controls: 1462±145 vs 1720±169 calories (p=0.001). The correlations between FFM and basal metabolism with the clinical features of the patients were not statistically significant Conclusions This study confrim the study of Spanjer MJ et al1 and shows a relatively low prevalence of malnutrition in comparison with other studies, but a high prevalence of low FFMI, underlining the necessity of measuring body composition in SSc patients with a standardised and validated method. Furthermore, Caporali et al,2 have shown an alteration of the nutritional status of patients of SSc probably related to a gastro-intestinal commitment. In our patients despite the presence of an apparent good nutritional status the use of bioimpedanceometry revealed a different body composition, a lower share of muscle mass, in patients compared to controls, related, in part, to musculoskeletal involvement by systemic sclerosis (increase in muscle catabolism and/or poor nutrient supply due to malabsorption phenomena). The early detection of such alterations could be useful to insert subjects at risk in physical rehabilitation programs. References [1] Spanjer MJet al, Rheumatology2017 [2] Caporaliet al; Clin Nutr2012 Disclosure of Interest None declared

Introduction: An innovative method, known as sonoporation, was used to induce the expression of silenced genes, such as (but not restricted to) TRAIL and p53, in liver cancer cells (HepG2). The principal aim of the present study was the reactivation of silenced apoptotic pathways in liver cancer models, by using diagnostic synovial microbubble as plasmid gene delivery tools in combination with epigenetic treatments. Material and methods: HepG2 cells were used as a liver cancer model. Microbubbles (Sonovue®) were chosen as gene deliver system in combination with the sonoporation approach. Plasmid pEGFP-TRAIL and pEGFP-p53 were selected and propagated in Escherichia coli grown in LB broth, in order to obtain the necessary amount. Results: Sonoporation was induced by using transducer (Sonitron 2000) and, among the several conditions tested, 3 MHz, 51% Duty Cycle, and 5 W/cm2, 30 s resulted as the best parameters. Data collected showed a dose dependent effect in terms of output energy. A transfection efficacy of 30 – 50% was achieved and recombinant gene expression induced apoptotic effects. In order to increase efficacy, we used the histone deacetylase inhibitor (HDACi, entinostat) MS-275, able to activate TRAIL and thus inducing a stronger pro-apoptotic effect in combination with TRAILgene re-expression. Conclusion: For the first time, it was shown the possibility to induce the exogenous expression of the pro-apoptotic gene TRAIL and p53 in a liver cancer HepG2 cells via a sonoporation procedure. The epigenetic treatment using HDACi was able to increase the pro-apoptotic effects of the gene therapy.

Chronic hepatitis C virus (HCV), particularly genotype 1, is associated with insulin resistance (IR) and diabetes. This study evaluated the impact of HCV clearance by all-oral direct-acting antiviral treatments on IR and glycemic control.Included in this prospective case-control study were 133 consecutive HCV-genotype 1 patients with advance liver fibrosis (F3-F4) without type 2 diabetes. Sixty eight were treated with direct-acting antiviral and 65 were untreated. Liver fibrosis was assessed by transient elastography. Pre-treatment, end-treatment, and 3 months post-treatment withdrawal IR homeostasis was assessed by homeostatic model assessment (HOMA)-IR, HOMA-S, and HOMA-B.At baseline, treated, and untreated patients showed similar liver fibrosis levels, HOMA-IR was 4.90 ± 4.62 and 4.64 ± 5.62, respectively. HOMA-IR correlated with HCV RNA levels. At the end of treatment, all patients cleared HCV RNA, regardless of liver fibrosis and body mass index, and a reduction in HOMA-IR at 2.42 ± 1.85 was showed (P 0.001); in addition, increased insulin sensitivity, decreased insulin secretion, reduction of serum glucose, and insulin levels were observed. Data were confirmed 3 months after treatment withdrawal in the 65 patients who cleared HCV. No variation occurred in untreated patients. Overall, 76.5% of sustained virologic response patients showed IR improvements, of which 41.2% normalized IR. Improvement of IR was strictly associated with HCV clearance; however, patients with the highest levels of fibrosis remain associated with some degree of IR.The data underline a role of HCV in development of IR and that viral eradication reverses IR and improves glycemic control and this could prevent IR-related clinical manifestations and complications.

We conducted a long-term prospective study of 89 cancer survivor children who had acquired hepatitis B virus (HBV) and/or hepatitis C virus (HCV) during treatment for neoplasia, the aim being to evaluate the natural history of the diseases and the effect of interferon (IFN) treatment. Patients were followed up for a median period of 13 years (range, 8 to 20); 46 were infected by HBV, 11 by HCV, and 32 coinfected by HBV and HCV. A spontaneous clearance of hepatitis B surface antigen (HBsAg) occurred more frequently in coinfected patients (19%) than in the HBV-infected (2%; P = .004), with an annual seroconversion rate of 2.1% and 0.2%, respectively (P= .008). Loss of hepatitis Be antigen (HBeAg) occurred in 44% of coinfected and in 28% of HBV-infected patients. Clearance of serum HCV-RNA was observed in 34% and 9%, respectively, of coinfected and HCV-infected patients. Seventeen HBV-infected, 4 HCV-infected, and 16 coinfected patients received -IFN treatment. In the HBV group, 6 patients (35%) cleared serum HBV DNA and seroconverted to anti-HBe; in the HCV-group, none cleared HCV-RNA. In the coinfected group, 1 patient cleared both HBV DNA and HCV-RNA, 6 patients cleared serum HCV-RNA alone, and 1 only HBV DNA and HBeAg. Overall, the diseases showed a mild histological course with no evidence of liver cirrhosis. A reciprocal interference on viral replication between HBV and HCV may occur in coinfected patients. Treatment seems to be effective for selected cases and is justified in view of the uncertain prognosis of the disease in these patients.

HCV genotypes 2- or 3-infected patients with a rapid virological response (RVR) to therapy with pegylated interferon and ribavirins who have a low viral load, noncirrhotic and nonobese may be considered for a shorter course of treatment. However, no studies have assessed host-viral factors associated with relapse in genotype 2 and 3 separately. Accordingly, we assessed whether 12 weeks of pegylated interferon and ribavirin was an optimized regimen for treatment of HCV genotype 2 and 3 with positive predictors of response. Power and sample size were a priori calculated and 96 consecutive chronic hepatitis C patients (53, genotype 2 and 43, genotype 3) without cirrhosis who were not obese and who achieved a RVR to therapy with peg-IFN-α-2a and ribavirin were enrolled. Fibrosis, steatosis, homeostatic model assessment-insulin resistance and HCV RNA were predefined variables to be evaluated in relapse. An intention-to-treat analysis was performed. SVR rates were 98% and 84% for genotype 2 and 3, respectively. Analysis of genotype 3 patients who had relapse showed a negative correlation with steatosis (P0.0001) and HCV RNA (P0.015). Multivariate analysis showed that steatosis was the independent predictor of relapse (OR, 0.988; 95% CI, 0.981-0.993; P0.001). Genotype 3 patients with steatosis had a relapse rate of 36.4% and 15.8% in those with high and low viral load, respectively, whereas there was no relapse in those without steatosis. In conclusion, a 12-week course of therapy is sufficient for patients without cirrhosis, not obese and infected with HCV genotype 2 achieve a RVR. This is not the case for genotype 3. Steatosis is the independent predictor of relapse. New therapeutic strategies are necessary for this subgroup of HCV genotype 3.

The intracellular activity of streptomycin, gentamicin, and netilmicin on Escherichia coli phagocytosed by murine peritoneal macrophages was studied using a sensitive and standardized method. Intracellular activity of streptomycin and gentamicin at therapeutic concentrations was seen after 1 h of incubation of antibiotics with macrophages containing phagocytosed bacteria, whilst for netilmicin a significant intracellular activity was observed only after 3 h exposure. The activity of these antibiotics against intraphagocytic bacteria was significantly lower than that observed against extracellular bacteria. Sub-inhibitory concentrations of streptomycin were active against intracellular E. coli. Streptomycin was also active for a phagocytosed streptomycin-resistant strain of E. coli, but this activity was eliminated when the O2-dependent killing mechanisms of macrophages were inhibited by sodium fluoride. The data demonstrate that aminoglycosides may exert a dose-dependent intraphagocytic activity against E. coli that correlates with the time of incubation of the antibiotics with infected macrophages. Streptomycin appears to be the most effective agent followed by gentamicin and the least active was netilmicin. In the case of streptomycin, the intraphagocytic activity seems to be due, at least in part, to the stimulation of O2-dependent cellular microbactericidal mechanisms.

Pegylated-interferon-alpha (peg-IFN-alpha) is the mainstay of treatment for chronic hepatitis C (CHC). Treatment is often complicated by neutropaenia due to inhibition of haematopoiesis. However, there are no data on the kinetics of granulocyte-colony stimulating factor (G-CSF), a major neutrophil growth factor, in this setting. We therefore evaluated G-CSF synthesis in CHC patients on peg-IFN-alpha treatment.A total of 40 CHC patients were studied. None had pre-existing haematological disorders, or hepatitis B virus or HIV coinfection. For controls, 30 healthy subjects were used. Laboratory examinations, including liver function tests, were performed at baseline and monthly over treatment and follow-up. Serum G-CSF was measured in all patients and controls at baseline and in a subgroup of 20 CHC patients also at weeks 2, 4, 24, 48 and 72 after treatment start.CHC patients had a significantly lower pre-treatment neutrophil count (3,256 +/- 1,197 versus 3,804 +/- 859; P = 0.03). Notwithstanding, they showed lower baseline G-CSF serum levels than healthy controls (16.1 +/- 6.2 versus 19.4 +/- 7.5; P = 0.048). Consistently, baseline G-CSF levels were poorly correlated with the neutrophil count in CHC patients (r = -0.2; P = 0.2). Moreover, serum G-CSF levels did not increase in any of the 20 CHC patients during peg-IFN-alpha treatment, despite declining neutrophil counts.The lower neutrophil counts observed in CHC might be related to an absolute deficiency in G-CSF production. In the human model of neutropaenia induced by peg-IFN-alpha, we show that endogenous G-CSF levels are not physiologically up-regulated to overcome the decline in neutrophil counts. Our study provides a rationale for the evaluation of recombinant human G-CSF treatment in peg-IFN-alpha-induced neutropaenia.

The role of steatosis in the progression of liver damage in chronic hepatitis C (CHC) was studied. Enrolled were 180 consecutive liver biopsy-proven CHC patients and 41 additional subjects with a known duration of infection. We evaluated the histological activity index (HAI), grade of fibrosis and steatosis, body mass index (BMI; kg/m(2)), distribution of body fat, HCV genotype, and levels of HCV RNA. Eighty six (48%) patients showed steatosis, and a higher prevalence was observed in genotype 3a infection (P.01). A correlation between the grade of steatosis and fibrosis was observed (P.001). Fibrosis was also associated with age (P.001). After adjusting for age, the association between steatosis and fibrosis remained significant. The grade of steatosis also correlated with the HAI (P.007) with a significant increase in periportal necrosis. No relation was found between steatosis and age, gender, iron storage, or levels of HCV RNA. Patients with a high grade of steatosis (30%) showed higher serum levels of gamma-GT and ALT (P.001). Overall, steatosis was not significantly associated to BMI. Analysis by single genotype showed a significant association between the grade of steatosis and BMI in type 1 infection r =.689; P.001) and with levels of HCV RNA in type 3a infection r =.786; P.001). Visceral fat distribution rather than BMI proved to be associated with steatosis (P.001). Data obtained from patients with a known date of infection confirmed that steatosis grades 3-4 were associated with a higher annual rate of fibrosis progression, and showed that alcohol and steatosis act together in increasing fibrosis (P.05). Our data indicate that steatosis is an important cofactor in increasing liver necroinflammatory activity and in accelerating fibrosis in CHC. Visceral obesity and genotype 3a play a role in the development of steatosis.

The role of HCV RNA levels and host factors in the severity of liver injury was studied. Enrolled were 298 consecutive liver biopsy-proven chronic hepatitis (CH) C patients (179 men; median age: 52 years, range 19-68; CH, 198; cirrhosis, 100) and 18 chronic hepatitis C with normal ALT. HCV genotypes were: 1a, 4.3%; 1b, 53%; 2a/c, 28%; 3a, 7%; 4, 1.3%, and mixed 6.4%. Serum HCV RNA levels were similar for all genotypes (median: 2.8 x 10(6) eq/ml; range0.2-69). In patients with chronic hepatitis without cirrhosis, the serum HCV RNA levels reflected the grade of liver necroinflammatory activity (R = 0.45; P0.001) and the stage of fibrosis (R = 0.51; P0.001), regardless of age, gender, HCV genotype, hepatic steatosis, and hepatic iron overload. Patients with high serum HCV RNA levels (or =3 x 10(6) eq/ml) had higher ALT values (P0.002) than those with lower HCV RNA levels. Patients with normal ALT showed low HCV RNA levels (median: 0.82 x 10(6) eq/ml) and histological features of minimal or mild chronic hepatitis. Cirrhotic patients showed significantly lower levels of viremia than those with chronic hepatitis with a similar HAI. The data of a subgroup of 62 patients with an established time of infection showed that for a similar duration of disease, patients with serum HCV RNA levelsor =3 x 10(6) eq/ml had a significantly higher fibrosis score than those with lower levels. HAI and fibrosis score were significantly higher in patients with HCV RNA levelsor =3 x 10(6) eq/ml and grade 3-4 steatosis than those with lower HCV RNA levels and steatosis grades. The data indicate that the liver damage is correlated with the HCV RNA levels and that a high viral load acts together with steatosis in accelerating the progression of liver injury.

Pulmonary nocardiosis is an infrequent but insidious disease in transplant patients. It has occurred in our centre in 3 out of 233 heart-transplant recipients since 1988. Common clinical features were mild symptoms and a severe nodular lung involvement. Early diagnosis was based upon cultures of bronchoalveolar lavage or fine-needle aspirate specimens of the lung lesions. Susceptibility studies and tests of antibiotic synergism guided the therapy. Two patients were treated with a combination of piperacillin-tazobactam and ciprofloxacin, and one with imipenem and amikacin, for 3-4 wk followed by a 3-month course of trimethoprim-sulphamethoxazole. The nocardial disease was successfully treated in the 3 patients; however, one died of subsequent invasive pulmonary aspergillosis. In the absence of consensus on the length of therapy, this experience suggests that a synergistic combination of a beta-lactam/beta-lactamase inhibitor with ciprofloxacin or amikacin followed by a short course of trimethoprim-sulphamethoxazole may be effective in eradicating nocardial disease and may reduce the need for long-term treatment.

Symptomatic patients with sick sinus syndrome (SSS) are candidates for permanent pacemaker implantation. Single-chamber atrial (AAI) or dual-chamber (DDD) pacing rather than single-chamber ventricular (VVI) pacing is the recommended pacing mode, with rate response (-R) if appropriate [1]. Most retrospective studies have demonstrated that mortality is greater in VVI paced patients and that atrial-based pacing is hemodynamically superior and associated with a lower rate of atrial fibrillation (AF) and thromboembolic complications [2–6]. Other observational studies comparing physiological (AAI or DDD) with VVI pacing, while they confirmed that VVI pacing was an independent predictor of AF and stroke, did not identify any difference in mortality and heart failure incidence between the two groups [7–9].

In the medical environment there are several sources of possible magnetic interference, such as magnetic resonance imaging (MRI), clinical radiation therapy, electrocautery, and lithotripsy. Certain types of electrical equipment may interfere with automatic implantable cardioverter defibrillator (AICD) and/or pacemaker performance. The reason why this occurs is that AICDs and pacemakers are designed to sense the electrical activity of the heart. In the event a particular piece of equipment should mimic the activity of the heart, the pulse generator may be affected.

To evaluate the safety and efficacy of a new algorithm for automatic mode switching (AMS) from DDD-DDDR to DDIR, 26 patients, 16 females and 10 males, mean age 73 +/- 6 years of age, affected by sinus node disease, chronotropic incompetence, and recurrent paroxysmal atrial fibrillation (PAF) received the Medtronic Thera DR pacemaker. The device continuously calculates, in ms, the running average of the intrinsic atrial rate (MAR) and compares the current atrial interval (CAI) with the stored MAR. When the CAI is greater than the MAR it increases by 8 ms, and when the CAI is less than the MAR, it decreases by 23 ms. When MAR < or = 330 ms (182 beats/min), tachycardia is detected and AMS is activated. All patients had clinical evaluation, 12-lead ECG, Holter monitoring, and exercise testing after implantation and every 3 months for 1 year. The results were compared with the data stored in the pacemaker memory: AMS episodes number; the histogram of the last 14 episodes; and atrial electrogram recording. Twenty-two Holter recordings in 13 patients showed PAF and in all of them AMS occurred simultaneously. AMS lasted between 10 seconds and 20 hours, and MAR ranged from 195-400 beats/min. No episode of PAF and no AMS were recorded in 39 Holter recordings in 22 patients. Appropriate AMS was confirmed in five patients by stored atrial electrogram and in nine by 12-lead ECG and pacemaker event markers. Mean atrial sensing was 2.13 +/- 1.04 mV during PAF and 3.18 +/- 1.46 mV during sinus rhythm. No PAF episode and no AMS were recorded during exercise testing. In conclusion, this new algorithm was very reliable, sensitive, and specific.

To evaluate the role played by hepatitis C virus (HCV) in mixed cryoglobulinemia in subjects with chronic hepatitis, we studied 72 consecutive patients: 43 had HCV-related chronic hepatitis, 19 HBV-related chronic hepatitis, and 10 chronic hepatitis of other etiology. We used second generation ELISA and RIBA to test for mixed cryoglobulinemia and anti-HCV antibodies in both serum and cryoprecipitates; HCV RNA were evaluated by 'nested' PCR. Serum levels of rheumatoid factor and complementemia were also determined. The immunoglobulins in the cryoimmunoprecipitate were characterized by immunofixation electrophoresis. Cryoglobulinemia was present in 47% of the patients with chronic hepatitis C but in none of the sera of patients with HBV-related chronic hepatitis nor in those with chronic hepatitis of non-viral etiology. Type II mixed cryoglobulinemia was observed in 45% of the cases, and type III in 55%. HCV RNA and anti-HCV antibodies were present in all the cryoimmunoprecipitates. Ninety-five percent of the cryoglobulinemic patients had serum rheumatoid factor and 80% of them had low serum levels of C4. Our data indicate that mixed cryoglobulinemia is frequently associated with HCV-related chronic hepatitis, and that HCV and anti-HCV antibodies play an essential role in the development of mixed cryoglobulinemia.

The therapeutic index of either taurocholate (TC) or tauroursodeoxycholate (TUDC) administration in the treatment of drug-induced cholestasis was evaluated in perfused rat liver using a dose-response study. During estradiol-17-beta-glucuronide cholestasis, TC was more effective than TUDC in ameliorating bile flow but showed a low margin of safety since high doses caused additional toxicity. In contrast, TUDC ameliorated cholestasis even at very high doses with no adverse effects. In the model of chlorpromazine cholestasis, TC infusion did not correct but rather aggravated cholestasis, whereas TUDC at nonsaturating doses reversed the cholestasis and only at very high doses caused some toxicity. TUDC shows a good therapeutic index and may be employed with a reasonable margin of safety in the treatment of drug cholestasis.

The efficacy and safety of sequential treatment with prednisone and interferon was evaluated in a randomized, controlled study on 43 children with biopsy proven HBsAg/HBeAg/hepatitis B virus-DNA positive, anti-delta negative, chronic hepatitis (34 chronic persistent hepatitis, 9 chronic active hepatitis). Patients received either a 1-month course of prednisone (0.6 to 0.3 mg/kg per day) followed by interferon alfa-2a (3 MU/m 2 , thrice weekly, for 12 months; 22 patients) or no treatment (21 patients). At the end of the study (20 months), clearance of hepatitis B virus-DNA and HBeAg seroconversion were observed in nine (41%) of the patients treated with prednisone and interferon and in two (9.5%) of the untreated controls ( p =0.020). Two of the treated patients who lost HBeAg, also cleared HBsAg. In the treated group, 13 (59%) patients had stable normal levels of alanine aminotransferase on their last examination. The baseline serum level of hepatitis B virus-DNA was an important predictor of response. In fact, HBeAg clearance was observed in 75% of patients with a baseline hepatitis B virus-DNA level lower than 100 pg/ml and in none with a level above 100 pg/ml. We suggest that combined treatment with prednisone followed by alfa-interferon may be safe and effective in inducing a stable clearance of HBeAg and, in some cases, of HBsAg in children with chronic hepatitis B and with a low level of viral replication. For children with high levels of viral replication, this regimen seems to be ineffective.

Cyclosporin A prevents visceralization of Leishmania major infection of BALB/c mice (N. C. Behforouz, C. D. Wenger, and B. A. Mathison, J. Immunol. 136:3067-3075, 1986; W. Solbach, K. Forberg, E. Kammerer, C. Bogdan, and M. Rollinghoff, J. Immunol. 134:702-707, 1986). We report that cyclosporin A exacerbates disseminated leishmaniasis caused by L. donovani in C57BL/10 mice. Normal mice challenged with 5 x 10(6) amastigotes intravenously cleared the infection within several months by spontaneous acquisition of cell-mediated immunity. In contrast, cyclosporin A administered daily intraperitoneally at a dose of 1.25 mg per mouse prevented development of curative immunity and converted C57BL/10 (curing) mice to a noncuring phenotype. A rationale for the contrasting effects of cyclosporin A in the two murine models of leishmaniasis is provided.

The therapeutic efficacy of CP-46,665-1, a synthetic lipoidal amine with proven immunomodulatory and anti-tumor properties, in combination with chemotherapy was evaluated in L. donovani-infected C57Bl/6 mice. Immunostimulation and drug treatment resulted in a 10-fold lesser infection level than in untreated mice, while animals treated with Glucantime® alone exhibited only a modest amelioration of the infection. We also studied the capacity of CP-elicited peritoneal macrophages of C57Bl/6 mice cultured alone or in combination with Glucantime® and/or lymphokine to eliminate intracellular L. donovani amastigotes. When CP-elicited cells were incubated with Glucantime®, they exhibited a significantly higher killing potential than did drug treated thioglycollate-elicited cells. CP-macrophages stimulated with lymphokine alone or in combination with antimonial drug, killed amastigotes more rapidly and efficiently than similarly treated thioglycollate-elicited macrophages. In vivo and in vitro results of this study show that a combined regimen of immunostimulation with CP and antimonial drug is more effective in treatment of L. donovani infection than either treatment alone.

A regimen of immunostimulation with 6-0-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine, a lipophilic analog of muramyl dipeptide, combined with antimonial drug therapy was evaluated in the treatment of visceral leishmaniasis of mice and hamsters. The combined treatment was found to be more effective in the elimination of Leishmania donovani amastigotes from infected tissue macrophages than was either of the two treatments applied individually. In mice, it was found that immunostimulation of animals prophylactically, therapeutically, or both enhanced the effects of the antimonial drug (Glucantime) administered more than 1 week after a challenge of BALB/c and C57BL/6 mice. The superiority of the combined treatment of the parasite infection was demonstrable in both short-term (14 days) and long-term (40 to 45 days) infections of the two inbred strains of mice. The combined therapy was also effective in preventing the lethal course of leishmaniasis in hamsters which succumb to disseminated disease in the absence of therapeutic intervention. The efficacy of this dual approach to the therapy of disseminated leishmaniasis of experimental animals holds promise for similar application in the treatment of similarly afflicted human populations.

The prognostic value of induction of ventricular tachycardia (VT) by programmed electrical stimulation (PES) was analyzed in 123 patients: 64 (Group I) with spontaneous recurrent VT and 59 (Group II) without a history of serious arrhythmias. Thirty-three patients with spontaneous VT underwent coronary and left ventricular angiography to compare electrical instability with the presence of ventricular disfunction and/or the extent of coronary artery disease (CAD). PES reproducibly induced VT in 49/64 patients with spontaneous VT (sensitivity = 77%) and in 6/59 patients without VT (specificity = 90%). Twenty-two patients (66%) had ventricular disfunction defined by an ejection fraction of less than or equal to 40% or regional wall motion abnormalities. Only 4 patients (33%) had proximal 3-vessel CAD. The mean follow-up period was 16 +/- 12 months. Eight of Group I patients died suddenly and 24 had recurrent symptomatic VT. Three of Group I patients died (1 cardiac failure, 2 non-cardiac deaths), all the survivors were free of serious arrhythmias. In Group I patients mortality was correlated with: recent anterior myocardial infarction, inducible sustained VT with PES, ejection fraction less than or equal to 0.40, ventricular ipoasynergy and or at least one coronary stenosis greater than or equal to 70%. This study suggests that inducible VT is a marker of the risk of sudden death. Electrical instability may occur independent from the etiology of cardiopathy, ventricular disfunction and extent of CAD, but these parameters are correlated to global and sudden mortality in the group of patients with spontaneous VT.

The influence of pretreatment of Escherichia coli and Staphylococcus aureus with sub-MICs of the new beta-lactam antibiotic imipenem on phagocytosis and killing by murine peritoneal macrophages and the susceptibility of these organisms to serum bactericidal activity were studied. The effects of imipenem, a round form inducer in gram-negative rods, and piperacillin, a filamentous form inducer, were compared. Bacteria grown in the presence of sub-MICs of imipenem or piperacillin were incubated for 30 min with macrophage monolayers in the absence of antibiotic. Phagocytosis, killing, and survival within macrophages were evaluated by microbiological and fluorescence microscope assays. Bacteria grown in the presence of a sub-MIC of imipenem were phagocytized and killed in numbers significantly higher than untreated or piperacillin-treated bacteria were. Intracellular bacteria pretreated with a sub-MIC of imipenem were also readily killed by lymphokine-activated macrophages. Prior treatment with a sub-MIC of imipenem resulted in an increased susceptibility of E. coli but not S. aureus to the bactericidal activity of immune serum. Imipenem treatment and immune serum acted synergistically to enhance phagocytosis and killing. The data indicate that exposure of E. coli and S. aureus to a sub-MIC of imipenem enhances the susceptibility of these potential pathogens to cellular and humoral host defense mechanisms.

Sixteen monoclonal antibodies (MAbs) directed against toxic shock syndrome toxin-1 (TSST-1) were generated by immunization of mice with purified TSST-1 and subsequent fusion of spleen cells with myeloma cells. Antibody-producing clones, identified by an enzyme-linked immunosorbent assay, were maintained as ascites tumors, and MAbs were purified by protein A chromatography. High-titered clones were further characterized and tested for the ability to neutralize several biological activities of TSST-1. The MAbs, which are of several immunoglobulin subtypes, reacted specifically with purified TSST-1 and TSST-1 present in Staphylococcus aureus culture supernatants. Three MAbs neutralized TSST-1-induced mitogenesis in a dose-dependent manner. Three of eight MAbs tested were able to neutralize induction by TSST-1 of interleukin-1 production by human monocytes. One neutralizing MAb, 8-5-7, was tested for the ability to protect rabbits from a constant infusion of TSST-1. Rabbits given the MAb had an attenuated clinical illness and were protected from the hypocalcemia, lipemia, and hepatic and renal insufficiency seen in control rabbits. Six of seven control rabbits died, compared with only one of seven rabbits treated with MAb 8-5-7. These experiments suggest that MAb 8-5-7 is directed against an antigenic determinant critical to the toxicity of TSST-1 and that the MAbs should be useful as probes in structure-function analyses of the TSST-1 molecule.

An anti-TSST-1-specific monoclonal antibody (MAb 8-5-7) was tested for its protective capacity in a rabbit infection model to toxic shock syndrome (TSS). The challenge strain of Staphylococcus aureus (RN4710), which contained a plasmid encoding TSS toxin-1, was introduced into previously implanted chambers. Purified monoclonal antibody (1.25 mg of immunoglobulin G) administered parenterally 1 day before and 1 day after initiation of infection provided complete protection against the TSS-like syndrome and the mortality which occurred in unprotected rabbits.

Estradiol-17β-D-glucuronide (E-17G), a metabolite of natural estrogen, is a potent cholestatic agent in vivo. We, therefore, studied the mechanisms of E-17G cholestasis using in vitro perfused rat liver system. Furthermore, since it has been postulated that sodium taurocholate (TC) may interfere with either uptake or biliary excretion of other steroid agents, we tested whether E-17G cholestasis could be modified by TC administration. During a constant infusion of TC at a physiological rate (0.50 μ mole per min), a dose-dependent decrease of bile flow was observed after E-17G addition from 1.5 to 5 x 10−5M. E-17G decreased bile acid excretory rate but not bile acid concentration in bile. In separate experiments, TC was infused at different rates (0, 0.25, 0.50, and 0.75 μ mole per min) into the perfusate over the entire experimental period, and E-17G was added at 1.75 x 10−5M. In this setting, E-17G cholestasis was diminished by increasing TC infusion rate and was prevented by TC at 0.75 μ mole per min. Infusion of sodium dehydrocholate (0.75 μ mole per min), a nonmicelle-forming bile acid, did not prevent E-17G cholestasis. During E-17G cholestasis, an increased biliary permeability to 14C-sucrose was observed. This effect was also prevented by TC, but not by sodium dehydrocholate which was infused at 0.75 μ mole per min. The perfusate disappearance curves of 3H-E-17G at the different TC infusion rates showed no changes in the initial uptake phase, but a profound dose-dependent difference in the excretory phase. The amount of E-17G left in perfusate at the end of experiments was 34% when no TC was infused and decreased to 28%; 22 and 7% at 0.25; 0.50 and 0.75 nmole per min TC rate, respectively. Measurement of 3H-E-17G excreted in bile as well as its biliary concentration in the four TC groups showed that TC significantly increased E-17G excretion. Small amounts of E-17G were recovered in liver plasma membranes. However, these amounts were directly proportional to changes of bile flow induced by estrogen at different TC infusion rates. In these same liver membrane preparations, neither Na+,K+− nor Mg++−ATPases were altered. The data suggest that alterations of canalicular membrane permeability may be critical in the development of E-17G cholestasis. Prevention of E-17G cholestasis by TC infusion may occur through increased micellar solubilization and facilitated biliary excretion of the estrogen thus avoiding its membrane-altering action.

To investigate the mechanisms of erythromycin cholestasis, the effects of erythromycin estolate (EE) on the excretory function of the isolated perfused rat liver and on liver plasma membrane (LM) preparations were studied and compared to those of erythromycin base (EB) and lauryl sulfate (LS), added alone or in combination. EE (at 125 to 200 microM) caused dose-dependent reductions of bile and perfusate flows, bile acid (BA) excretion, and biliary BA concentration. The alterations of the excretory function were only in part due to the decreased perfusate flow. In contrast, both 200 and 300 microM concentrations of EB elicited similar choleretic responses, which were presumably related to the osmotic activity of the drug excreted in the bile. LS did not affect hepatic excretory functions. However, the simultaneous addition of EB and LS resulted in a rate of bile flow lower than that observed with EB alone. EE, but not EB, increased canalicular permeability to (/sup 14/C)sucrose as measured by bile to plasma (B:P) ratio. Neither drugs altered (/sup 14/C)erythritol B:P ratio. In LM preparations both Na+,K+- and Mg2+-ATPase activities were inhibited in a dose-dependent manner by EE, but not by EB. The data suggest that EE could affect bile flow bymore » inhibiting cotransport of Na+ and BA and by altering LM permeability and support the view that the effect of erythromycins on the liver may be related to their surface activity.« less

The purpose of this investigation was to evaluate the role of IL28-B polymorphism in the clearance of hepatitis C virus (HCV) in chronic hepatitis B virus (HBV)/HCV coinfection during a long-term follow-up. Thirty-four consecutive patients with HBV surface antigen (HBsAg)-positive/anti-HCV-positive chronic hepatitis were retrospectively enrolled at their first liver biopsy (LB). For all patients, a documented clinical, serological and virological follow-up of at least 3 years (range 3-16 years) after LB and a sample of whole blood for genetic evaluation were available. Of the 24 patients with detectable serum HBV-DNA and HCV-RNA at their first observation, three cleared both HBV-DNA and HCV-RNA, 12 HCV-RNA and five HBV-DNA. Of the seven HBV DNA-positive/HCV RNA-negative patients at enrolment, three cleared HBV-DNA and one remained HBV DNA-positive and became HCV RNA-positive. All three HBV DNA-negative/HCV RNA-positive patients remained unchanged. Compared with the 12 patients with HCV persistence, the 15 patients who cleared HCV were younger, had lower serum alanine aminotransferase (ALT), HCV load, and histological activity index (HAI) and fibrosis score, more frequently had IL28-B CC variant, had been receiving an interferon-based treatment and less frequently cleared serum HBV-DNA. To investigate the relationship between the IL28-B variants and clearance of HCV, excluding the confounding effect of interferon-based treatment, the Mantel-Haenszel test was used, which indicated an association between HCV clearance and IL28-B variants (p=0.009). In chronic HBV/HCV coinfection, a long-term follow-up showed a frequent spontaneous or treatment-related clearance of active replication of one or both viruses and identified the IL28-B CC genotype as an independent predictor of HCV clearance. © 2013 Springer-Verlag.