Isolation and characterization of two particle-bound oligoaminopeptidases from human meconium that are different from oligoaminopeptidase of adult small intestine

Two oligoaminopeptidases (substrate L-leucyl-beta-naphthylamide) have been separated by ion exchange chromatography after Triton solubilization of meconial particles. The quantitatively major form (oligoaminopeptidase II) has been purified to apparent homogeneity. The two meconial oligoaminopeptidases differ from each other in their polyacrylamide gel electrophoretic mobility and isoelectric points. Both meconial enzymes differ from the adult enzyme by having more acidic isoelectric points and different affinities to Helix pomatia lectin-Sepharose columns. Oligoaminopeptidase II has a faster anodal electrophoretic mobility than the adult enzyme but a similar apparent molecular weight and subunit structure. Extensive neuraminidase digestion of meconial oligoaminopeptidase II does not modify the gel electrophoretic mobility of the enzyme. The charge difference between adult and meconial human brush border oligoaminopeptidases is therefore probably due, at least in part, to differences in the carbohydrate composition of these glycoproteins, and differences in the number of terminal or exposed sialic acid residues do not explain the observed charge differences.     

NT-proBNP, IGF-I and survival in patients with chronic heart failure.

OBJECTIVE: Growth hormone (GH) resistance with a reduction of insulin-like growth factor-I (IGF-I) serum concentrations seems to be implicated in the catabolic process associated with chronic heart failure (CHF). However, data concerning the prognostic value of these alterations in CHF patients without cachexia are scant. In this study, we aimed to determine in CHF patients the prognostic value of IGF-I/GH ratio and its relationships with N-terminal brain natriuretic peptide (NT-proBNP), a known marker of prognosis in these patients. DESIGN: We enrolled 82 non-cachectic patients, mean age 61+/-13 years, with ejection fraction <40% and predischarge New York Heart Association (NYHA) functional classes II-IV. All patients underwent clinical examination, two-dimensional echocardiography and NT-proBNP, GH and IGF-I measurement with log IGF-I/GH ratio calculation. Mortality and clinical status was documented at follow-up (18.4+/-8.1 months). RESULTS: During follow-up 17 patients died of cardiac causes. Non-survivors were at baseline in higher NYHA class (P<0.05) and showed higher values of NT-proBNP (P<0.001) than survivors; differently IGF-I, and log IGF-I/GH ratio were lower (P<0.05). At Cox multivariate analysis, NT-proBNP (P<0.001) and IGF-I/GH ratio (P<0.05) were independent predictors of death. CONCLUSIONS: High NT-proBNP levels and low IGH-I/GH ratio may be useful to stratify CHF patients at higher risk of cardiac death.     

Influence of antiviral therapy on the liver stiffness in chronic HBV hepatitis

Purpose

The aim of this study was to evaluate the effects of antiviral therapy on liver stiffness measurement (LSM).

Methods

Two hundred HBV patients were enrolled from four hospital centers in southern Italy; median age was 50.7 (25–75) males were 68%; 171 patients underwent to liver biopsy and 200 patients had LSM at baseline and 189 at the end of follow-up. One hundred and forty-nine patients were treated with nucleos(t)ide analogs, while 51 patients were untreated. The cutoffs of the LSM, related to the fibrosis stages, were as follows: non-advanced fibrosis ≤ 8.1 kPa and advanced fibrosis ≥ 8.2 Kpa.

Results

At baseline, the median value of LSM was 14.1 kPa for advanced fibrosis/cirrhosis and 6.9 kPa for non-advanced fibrosis. LSM was performed at 24 months from the start of therapy. The treated patients (68% received Entecavir and 32% Tenofovir) showed a decrease in liver stiffness measurement of 1.5 kPa (p < 0.001) in non-advanced fibrosis and of 6 kPa (p < 0.001) in advanced fibrosis/cirrhosis. In the patients not undergoing antiviral treatment, no statistically significant change of the LSM was observed (p = 0.26). A logistic binary regression model showed that the only independent factor associated with a significant change in the LSM was the liver stiffness value at baseline (odd ratio 2.855; 95% CI 1.456–5.788; (p = 0.007).

Conclusion

Long-term antiviral therapy induced a significant reduction of liver stiffness measurement and this result may be related to the reduction of liver fibrosis.

     

Safety and efficacy of ombitasvir/paritaprevir/ritonavir/dasabuvir plus ribavirin in patients over 65 years with HCV genotype 1 cirrhosis

Purpose

To analyse safety and efficacy of treatment based on ombitasvir/paritaprevir/ritonavir/dasabuvir plus ribavirin in the sub-group of GT1 patients older than 65 years.

Methods

We collected data extracted from the ABACUS compassionate-use nationwide Italian programme, in patients with cirrhosis due to hepatitis C virus (HCV) Genotype-1 (GT1) or 4 and at high risk of decompensation. GT1-HCV-infected patients received once-daily ombitasvir/paritaprevir, with the pharmacokinetic enhancer ritonavir (25/150/100 mg) and twice-daily dasabuvir (250 mg) plus Ribavirin (RBV) (OBV/PTV/r?+?DSV?+?RBV) for 12 (GT1b) or 24 (GT1a) weeks. Endpoints were to evaluate safety and efficacy, the latter defined as HCV RNA negative 12 weeks after the end of treatment (SVR12).

Results

Patients who suffered any adverse event (AE) were 74/240 (30.8%); 13/240 (5.4%) discontinued the treatment. A multivariate analysis found albumin <?3.5 g/dL (OR 2.04: 95% CI 1.0–4.2, p?<?0.05) and hypertension (OR 4.6: 95% CI 2.3–9.2, p?<?0.001) as variables independently associated with AE occurrence. The SVR12 was 95% (228/240). Multivariate analysis identified baseline bilirubin <?2 mg/dL (OR 4.9: 95% CI 1.17–20.71, p?=?0.029) as the only variable independently associated with SVR12.

Conclusion

Our findings suggest that OBV/PTV/r?+?DSV?+?RBV is safe and effective in real-life use in patients with compensated cirrhosis, HCV-GT1 infection, and age over 65.

     

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.Natural skeletal muscle consists of terminally differentiated, highly aligned, and contractile myofibers and a population of resident muscle stem cells, known as satellite cells (SCs), that are indispensable for muscle growth (1) and regeneration (2). The ability to create engineered muscle tissues that mimic the structural, functional, and regenerative properties of native muscle would enable design of accurate in vitro models for studies of muscle physiology and development (3, 4) and promote cell-based therapies for muscle injury and disease (5, 6). Pioneering studies of Vandenburgh and coworkers (7) and Dennis and Kosnik (8) were the first to demonstrate in vitro engineering of functional mammalian muscle constructs, followed by other studies reporting that differentiated engineered muscle can survive and vascularize upon implantation in vivo (9–13). Simultaneously, various studies have shown that, compared with differentiated or committed cells, undifferentiated SCs are a more potent myogenic cell source, with the ability to engraft and replenish the host satellite cell pool and support future rounds of muscle regeneration (14–16). Thus, it is likely that, for optimal therapy, engineered muscle tissues should fully recreate the cellular heterogeneity of native muscle and consist of both force-generating, differentiated myofibers and a functioning SC pool to allow further maturation and regeneration in vivo. Additionally, for long-term survival and efficient repair, implanted engineered muscle constructs must rapidly integrate into host vascular system and significantly increase their functional output compared with preimplantation levels.In this study, we used primary rat myogenic cells to engineer skeletal muscle tissues with highly organized architecture and force-generating capacity comparable with those of native muscle. We characterized the temporal dynamics of myogenic processes within engineered muscle and documented the in vitro formation of a homeostatic tissue state with the coexistence of highly contractile muscle fibers and functional satellite cells. To continuously monitor engineered tissue survival, function, and vascularization after implantation, we transduced myogenic cells with GCaMP3, a genetic indicator of intracellular Ca²⁺ concentration used previously in neurobiological (17) and cardiac (18) research, and implanted the muscle constructs in a dorsal skinfold chamber in nude mice. The use of this minimally invasive, in vivo platform allowed us to simultaneously, in real time, quantify and compare changes in Ca²⁺ transient amplitude and vascular density between highly differentiated and undifferentiated engineered muscle implants and to further assess the maintenance of satellite cell pool and enhancement of contractile function relative to those preimplantation. Overall, our studies describe important advances in the field of skeletal muscle tissue engineering and lay the foundation for novel studies of cellular function and signaling in a physiological environment in real time.     

CD1c but neither CD1a nor CD1b molecules are expressed on normal, activated, and malignant human B cells: identification of a new B-cell subset

The CD1 cluster of monoclonal antibodies (MoAbs) CD1a, CD1b, and CD1c, identifies molecules that are differentially expressed on hematopoietic and nonhematopoietic tissues. Our earlier finding that the mantle zone (MZ) but not the germinal center (GC) of normal lymph nodes (LN) is CD1c+, CD1a-, and CD1b- prompted us to further investigate the expression of these molecules on normal, activated, and malignant B cells. We report that blood and spleen contain CD1c+ B cells that account for 49% +/- 20.4% (mean +/- SD) and 50.9% +/- 4.4% of the total B cell population, respectively. CD1a- and CD1b-specific MoAbs are unreactive with both B and T cells; these latter are CD1c- as well. When CD1c+ and CD1c- B cells are activated in vitro, the CD1c molecule is upregulated in the former subset and induced de novo in the latter. Conversely, activated blood T cells remain CD1c-. Neither CD1a nor CD1b molecules are detected on activated T and B lymphocytes. At ultrastructural level, the CD1c+ B cells exhibit distinctive features, namely, condensed chromatin with or without a nucleolus and a unique cluster of cytoplasmic vesicles and organelles; the number of nucleolated cells is higher in the spleen (95%) than in the tonsil (40%) or blood (5%). These findings further confirm the similarity between blood and MZ B cells. The CD1c expression assessed on 27 B-cell chronic lymphocytic leukemias (B-CLL) and 46 B non-Hodgkin's lymphomas (B-NHL) was detected on 41% and 32% of cases, respectively; the latter comprised four follicular and 11 diffuse histotypes. The Burkitt's lymphomas were CD1c-negative. The B-cell neoplasms were all CD1a- and, except for four with a weak cytoplasmic staining, all CD1b- as well. The clear-cut CD1c distribution in normal LN (MZ+, GC-) contrasted with the evidence that some B-NHL cells of GC origin (eg, follicular with predominantly small cleaved cells) were CD1c+. Overall, the finding that CD1c expression is restricted to a fraction of B cells present in lymphoid organs and in peripheral blood indicates that CD1c is a powerful marker for the identification and dissection of B-cell subsets whose functional properties can now be evaluated.     

Improved outcome of Trypanosoma cruzi infection in rats following treatment in early life with suspensions of heat-killed environmental Actinomycetales

The well-established model of Chagas' disease in "l" rats was used to evaluate the effects of three injections of heat-killed Gordonia bronchialis, Rhodococcus coprophilus or saline on Trypanosoma cruzi parasitaemia and acute and chronic myocarditis, sequelae of the infection. Two vaccinating injections were given prior to challenge with T. cruzi, and the third, immunotherapeutic, injection was given 7 days after challenge. Treatment with either actinomycete significantly reduced acute parasitaemia (p<0.04), modified cellular infiltration during acute myocarditis and limited chronic myocarditis (p<0.03) in comparison with the saline-treated control animals. Immunological investigations showed that both bacterial preparations achieved their results through different mechanisms. The relevance of our findings to human Chagas' disease is discussed.     

Resolution of cluster headache after closure of an anterior communicating artery aneurysm: the role of pericarotid sympathetic fibres

We report the case of a patient suffering from migraine without aura since childhood who, at the age of 58 years, developed cluster headache (CH) attacks. This second type of headache was related to an aneurysm of the anterior communicating artery (ACoA) whose bursting caused subarachnoid haemorrhage. The aneurysm's clipping made the cluster headache subside and there was no recurrence for almost four years. However, nine months after haemorrhage, the patient experienced new migraine without aura attacks. As a pathogenetic interpretation of this secondary cluster headache, we discuss the possible role of pericarotid sympathetic nerves in cluster headache attacks. We suggest that the surgical dissection of the pericarotid sympathetic fibres could prevent the onset of the cluster headache attacks by cutting part of the circuit underlying it.     

Regional diastolic function by tissue Doppler echocardiography in systemic sclerosis: correlation with clinical variables

The incidence of left ventricular (LV) diastolic dysfunction is increased in systemic sclerosis (SSc), while systolic dysfunction is present in a small percentage of patients. The aim of this study was to asses the LV “regional” diastolic abnormalities in SSc patients by the mean of Doppler tissue imaging (DTI). Echocardiographic echo-Doppler (DE) and DTI parameters were analyzed for 67 SSc patients: abnormal E/A ratio at DE was detected in 24, while abnormal e/a at DTI was observed in 41. A significant prevalence of DTI diastolic abnormalities in the segments reflecting longitudinal versus those reflecting radial LV motion was found. The segments of the basal regions of LV myocardium were significantly more involved than those of the middle portion. Linear correlation was observed between the extent of the diastolic abnormalities and the duration of disease. Longitudinal myocardial systolic velocities were significantly reduced in patients with abnormal e/a DTI.