Genetics of Dilated Cardiomyopathy: Clinical Implications.

PURPOSE OF REVIEW: This review aims to summarize the current knowledge on the genetic background of dilated cardiomyopathy (DCM), with particular attention to the genotype-phenotype correlations and the possible implications for clinical management. RECENT FINDINGS: Next generation sequencing (NGS) has led to the identification of an increasing number of genes and mutations responsible for DCM. This genetic variability is probably related to the extreme heterogeneity of disease manifestation. Important findings have associated mutations of Lamin A/C (LMNA) and Filamin C (FLNC) to poor prognosis and the propensity to cause an arrhythmic phenotype, respectively. However, a deeper understanding of the genotype-phenotype correlation is necessary, because it could have several implications for the clinical management of the patients. Furthermore, the correct interpretation of pathogenicity of mutations and the clinical impact of genetic testing in DCM patients still represent important fields to be implemented. A pathogenic gene mutation can be identified in almost 40% of DCM patients. The recent discoveries and future research in the field of genotype-phenotype correlation may lead to a more personalized management of the mutation carriers towards the application of precision medicine in DCM.

Trypanosoma cruzi discrete typing units in Chagas disease patients from endemic and non-endemic regions of Argentina.

Genetic diversity of Trypanosoma cruzi may play a role in pathogenesis of Chagas disease forms. Natural populations are classified into 6 Discrete Typing Units (DTUs) Tc I-VI with taxonomical status. This study aimed to identify T. cruzi DTUs in bloodstream and tissue samples of Argentinean patients with Chagas disease. PCR-based strategies allowed DTU identification in 256 clinical samples from 239 Argentinean patients. Tc V prevailed in blood from both asymptomatic and symptomatic cases and Tc I was more frequent in bloodstream, cardiac tissues and chagoma samples from immunosuppressed patients. Tc II and VI were identified in a minority of cases, while Tc III and Tc IV were not detected in the studied population. Interestingly, Tc I and Tc II/VI sequences were amplified from the same skin biopsy slice from a kidney transplant patient suffering Chagas disease reactivation. Further data also revealed the occurrence of mixed DTU populations in the human chronic infection. In conclusion, our findings provide evidence of the complexity of the dynamics of T. cruzi diversity in the natural history of human Chagas disease and allege the pathogenic role of DTUs I, II, V and VI in the studied population.

NIR-labeled nanoparticles engineered for brain targeting: in vivo optical imaging application and fluorescent microscopy evidences.

The presence of the blood-brain barrier (BBB) makes extremely difficult to develop efficacious strategies for targeting contrast agents and delivering drugs inside the Central Nervous System (CNS). To overcome this drawback, several kinds of CNS-targeted nanoparticles (NPs) have been developed. In particular, we proposed poly-lactide-co-glycolide (PLGA) NPs engineered with a simil-opioid glycopeptide (g7), which have already proved to be a promising tool for achieving a successful brain targeting after i.v. administration in rats. In order to obtain CNS-targeted NPs to use for in vivo imaging, we synthesized and administrated in mice PLGA NPs with double coverage: near-infrared (NIR) probe (DY-675) and g7. The optical imaging clearly showed a brain localization of these novel NPs. Thus, a novel kind of NIR-labeled NPs were obtained, providing a new, in vivo detectable nanotechnology tool. Besides, the confocal and fluorescence microscopy evidences allowed to further confirm the ability of g7 to promote not only the rat, but also the mouse BBB crossing.

Elbow Medial Ulnar Collateral Ligament chronic isolated insufficiency: anatomical M-UCL reconstruction technique and clinical experience in a mid-term follow-up.

PURPOSE: This study aims to clinically evaluate, at mid-term follow-up, a group of patients treated by the senior author in the last 6 years with our anatomical double-bundle reconstruction surgical technique for the medial ulnar collateral ligament (M-UCL) insufficiency. METHODS: In this study, we included only patients affected by chronic valgus elbow instability, diagnosed with an accurate clinical evaluation combined with an MRI, without associated fractures that had been surgically treated in the past and without additional instability detected during the first checkup and in the preoperative evaluation under anesthesia. The nine patients enrolled were operated by the senior author between 2011 and 2014 (from 16 to 49 years old at surgery, all amateur sportsmen). The average follow-up is 4 years (47.6 months). The values of the range of movement were recorded and compared. Pain assessment was performed using the VAS scoring system. The recovery of daily activities was evaluated through the validated MEPS and Quick-DASH score scales. All patients underwent an X-ray in two standard projections and a preoperative and follow-up MRI. RESULTS: The recovery of the range of motion was complete in six cases. The remaining three patients had minor loss of extension. None of the patients reported flexion deficits nor pronation-supination at follow-up. All patients achieved subjectively perceived stability and clinically objectified stability at follow-up. Five patients referred a total lack of pain at follow-up. Seven patients achieved full marks in the Mayo Elbow Performance Score and an excellent improvement in the Quick-DASH score. CONCLUSIONS: Excellent functional results indicate that M-UCL isolated reconstruction with autologous hamstrings described in this study is a reliable and replicable technique with a reduced incidence of complications. Resuming sports is consistently successful in our patients.

Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis.

The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm.

Low frequency of detection by nested polymerase chain reaction of enterovirus ribonucleic acid in endomyocardial tissue of patients with idiopathic dilated cardiomyopathy.

OBJECTIVES: The purpose of this study was to determine the prevalence of enteroviral infection in the myocardium of patients with idiopathic dilated cardiomyopathy by using a highly sensitive and specific detection technique. BACKGROUND: Recent molecular studies have suggested that enteroviral persistence (in particular, coxsackieviruses type B) may underlie idiopathic myocarditis and dilated cardiomyopathy. METHODS: The method used to detect enterovirus-specific ribonucleic acids (RNAs) is based on reverse transcription and nested polymerase chain reaction amplification with four pairs of primers from the conserved 5' noncoding region of the enteroviral genome. Several members of the Enterovirus genus are detectable by this assay (coxsackieviruses B1 to B6; polioviruses 1 to 3; echoviruses 9, 19 and 31), with a sensitivity threshold close to the detection of a single molecule of viral RNA in 1 mg of tissue sample. Endomyocardial tissue samples from 84 subjects were analyzed (77 samples obtained from left endomyocardial biopsies, 7 from explanted hearts). The subjects comprised 63 study patients (53 with dilated cardiomyopathy, 3 with idiopathic myocarditis, 1 with right ventricular dysplasia, 1 with restrictive cardiomyopathy, 1 with eosinophilic myocarditis, 1 with primary ventricular fibrillation and 3 with myocarditis of known etiology) and 21 control subjects with other diseases. RESULTS: Positive signals were obtained only in samples from six study patients (four with dilated cardiomyopathy, one with right ventricular dysplasia and one with myocarditis). Samples from control subjects, uninfected rat myocardium and cultured cell lines yielded systematically negative results. Moreover, the nucleotide sequence analysis of the amplification products from patients with positive samples raised doubts about the true positivity of these samples. CONCLUSIONS: This study suggests that the persistence of enteroviral RNA in dilated cardiomyopathy is not a major cause of the disease and that a careful analysis of polymerase chain reaction amplification products is essential in any study in which this technique is pushed to high sensitivity thresholds.

Transduced fibroblasts and metachromatic leukodystrophy lymphocytes transfer arylsulfatase A to myelinating glia and deficient cells in vitro.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease, caused by deficiency of arylsulfatase A (ASA), that manifests primarily in the white matter of the nervous system. Currently, no specific treatment exists that will reverse its fatal outcome. Replacement therapy has been hampered by the blood-brain barrier (BBB). To circumvent this problem we designed an ex vivo gene therapy strategy that includes the retrovirus-mediated ASA transduction of cells, such as activated lymphocytes, that are able to traverse the BBB or other membranes of the CNS. For this purpose, two recombinant retroviruses based on the pLXSN vector were produced, containing the wild-type ASA cDNA or a pseudodeficiency ASA cDNA, which encodes a smaller enzyme with normal activity. After transduction, ASA activity increased more than 100-fold in fibroblasts from an MLD patient. Furthermore, ASA-transduced MLD PBLs expressed 30 times higher ASA activity when compared with control PBLs. Moreover, cell culture experiments demonstrated that transduced fibroblasts could efficiently transfer ASA to deficient cells across a transwell barrier, whereas transduced MLD lymphocytes could transfer ASA to deficient fibroblasts only by direct cell-to-cell contact. Finally, ASA was taken up by normal oligodendrocytes and Schwann cells, the target myelinating glial cells for therapy in MLD. These data suggest possible short-term strategies for transfer of ASA into the CNS via transduced autologous cells while long-term strategies, related to autologous transduced bone marrow transplant, take effect in patients.

Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct Morquio skin fibroblasts in vitro.

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.

Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Intractable fever and cortical neuronal glycogen storage in glycogenosis type 2.

Glycogenosis type 2 is an autosomal recessive glycogen storage disorder caused by deficiency of lysosomal acid alpha-glucosidase. Different phenotypes are recognized. The authors describe two children affected by the late infantile form; both presented terminal hyperthermia not caused by infections. Autopsy performed in one case showed diffuse glycogen storage in the CNS neurons. In light of current interest in enzyme replacement therapy, this finding casts some doubt on how effective enzyme replacement therapy will be unless it can be targeted directly into the CNS.

Sexual functions after high-dose chemotherapy in survivors of germ cell tumors.

We investigated the changes in sexual function in male patients with germ cell tumor continuously disease free after one or two courses of high-dose chemotherapy with hematopoietic stem cell support. A questionnaire was mailed to 35 patients, and 30 patients sent it back. Sexuality was considered a problem by 10 patients (33%), but no patients considered sexuality a major problem. Erection was more difficult to achieve in seven patients (23%) and 10 patients (33%) experienced increased difficulty in maintaining an erection. Eight patients (27%) had the experience of less intensive and less frequent orgasm. In all, 13 patients (43%) thought that both the disease and treatment had worsened their sexual capacity, but 20 patients (67%) were satisfied with their sex life. Most of the patients (63%) considered that insufficient information and counselling had been given by their physicians about the sexual sequelae of therapy. However, the amount of information about the disease and treatment was considered good by 77 and 80% of the patients, respectively. This study shows that 27% of patients were not content with their ability to attain sexual satisfaction due to the illness or its treatment. Communication is an important issue and better information tools could lead to improved compliance in these patients.

Glutathione S-transferase activity in patients with cancer of the digestive tract.

Glutathione S-transferase (GST) and carcinoembryonic antigen were measured in the plasma of 95 patients with neoplasm of digestive tract, in 40 patients suffering from non-neoplastic diseases and in 40 healthy subjects. The mean value of the GST activity was significantly (P < 0.001) elevated in patients with gastric, liver and colorectal cancer (10.4 U/l, 14.1 U/l and 12.3 U/l respectively) as compared with the reference population (3.2 U/l). GST elevations above normal were observed in 26 (90%) patients with gastric cancer, in 18 (100%) with liver cancer and in 25 (89%) with colorectal cancer. Carcinoembryonic antigen appeared less sensitive. In 15 patients the postoperative levels of serum GST were increased after surgery then gradually declined and after 1 month showed a normalization in 10 patients. Our data suggest that GST measurement may be useful as a tumour marker in gastric, liver and colorectal cancer. Moreover the combined determination of GST and other markers increase the sensitivity for cancer detection.

A study of serum glycosidases in cancer.

N-Acetyl-beta-glucosaminidase (NAG) and beta-glucuronidase were measured in the serum of 70 patients with breast and digestive-tract neoplasms and in 70 healthy subjects. The mean value of the NAG activity was significantly (P < 0.001) elevated in patients with gastric, liver and pancreas cancer as compared with the reference population. In patients with liver and pancreas cancer the very high sensitivity contrasted with a low specificity. NAG elevations above normal were observed in 14 (78%) patients with breast cancer, in 11 (100%) with gastric cancer, in 17 (70%) with colorectal cancer, in 8 (100%) with liver cancer and in 9 (100%) with pancreas cancer. In patients with breast and gastric cancer the enzyme shows a good specificity and sufficient sensitivity as a tumor marker. beta-Glucuronidase appeared less sensitive and was significantly elevated (100%) only in patients with pancreas cancer.

Uremic toxins and adenosine deaminase activity.

Predialysis erythrocyte adenosine deaminase activity was depressed in 12 of 20 patients receiving hemodialysis. In contrast, in 17 patients a marked rise in enzyme activity was observed subsequent to dialysis. Six patients had evidence of neuropathy. Predialysis plasma inhibited normal enzyme activity by an average of 36%, but postdialysis plasma had minimal inhibitory effect. A low molecular weight substance or substances, isolated from the dialysate of uremics, inhibited adenosine deaminase activity by 38%. Three patients having the longest courses of dialysis had no neuropathy and showed no depression of activity, with their plasma failing to inhibit normal activity. It is suggested that accumulation of low molecular weight toxins that depress adenosine deaminase activity may lead to myelin sheath degeneration with subsequent neuropathy. By removing the inhibitor, dialysis may permit nerve repair and eventual recovery.

Serum triglyceride levels in uremic patients receiving polyacrylonitrile dialysis.

Fasting levels of serum triglycerides cholesterol and high-density lipoprotein (HDL) were studied in 20 uremic patients with persistent high triglyceride levels receiving polyacrylonitrile dialysis. Up to the sixth month of the trial there was no change in cholesterol and high density lipoprotein; on the contrary, triglyceride levels decreased progressively and reached at the fourth month the normal range. A statistical significative difference (p greater than 0.01) could be detected between cuprophane and polyacrylonitrile dialysis triglyceride levels. A diffusive procedure using polyacrylonitrile is good enough to lower high triglyceride levels, but treatment must be prolonged for four months at least.

Variation of urinary enzymes N-acetyl-beta-glucosaminidase, alanine-aminopeptidase, and lysozyme in patients receiving radio-contrast agents.

A urinary enzyme pattern consisting of two lysosomal enzymes, N-acetyl-beta-glucosaminidase and lysozyme, and one enzyme originating from kidney tubular brush border membrane, alanine-aminopeptidase, were studied in 30 patients undergoing intravenous urography and arteriography. N-acetyl-beta-glucosaminidase and lysozyme showed the greatest diagnostic sensitivity and were still abnormal on the fifth day after the administration of radio contrast agent. The results, which are statistically significant (Student's t test), suggest that radio-contrast agents are potentially nephotoxic.

Diagnostic significance of urinary enzymes: development of a high performance liquid chromatographic method for the measurement of urinary lysozyme.

We describe a new high pressure liquid chromatography (HPLC) method for quantitative determination of urinary lysozyme. The method is simple, reproducible and with detection limit of 0.2 microgram. Before HPLC analysis the urine was purified using a Sep-Pak C18 cartridge. Lysozyme concentration was significantly higher in patients with chronic renal failure than in a control group (p less than 0.001). The concentration of lysozyme in urine is shown to be a sensitive indicator of renal damage.

Chemotherapy with cisplatin, epirubicin, methotrexate in the treatment of locally advanced or metastatic transitional cell cancer of the bladder (TCC).

Forty patients with advanced transitional cell cancer (TCC) of the bladder were treated with cisplatin, epirubicin, methotrexate (PEM, every 3-4 weeks). If creatinine clearance was reduced to 40 ml/min, the usual full doses of cisplatin and methotrexate, 50 mg/m2, were proportionally reduced. 23 patients had full-dose (FD) therapy, 17 had reduced dose (RD) (40-20 mg/m2). Two patients achieved complete response and 17 partial response. The overall response rate was 19/40 (47.5%), 11/23 (48%) for FD and 8/17 (47%) for RD (p = 1.000). 17/40 pts (42.5%) had no-change and 4/40 (10%) had disease progression. The median duration of CR and PR was 32 weeks, range 4-82 (22 weeks, range 12-52 for FD; 32 weeks, range 4-82 for RD, cisplatin p = .7362). The main side effect was vomiting (35/40 pts, 87.5%, 20/23 = 87% for FD, 15/17 = 90% for RD, p = 1.000). Leukopenia was observed in 12 patients (30%, nadir 3,240 range 900-3,800, 6/23 = 26% for FD, 6/17 = 35% for RD, p = .7285), alopecia in 18 patients (45%, 15/23 = 65% for FD, 3/17 = 18% for RD, p = .004). The results of this study show that a dose escalation to 50 mg/m2 for cisplatin, epirubicin and methotrexate in the PEM regimen results in an increase in overall response (OR) (19/40 = 47.5%) with respect to a historical control using the same drugs at doses of 40 mg/m2 (12/35 = 34%). In patients with normal renal function the escalated dose was tolerated without a corresponding increase in toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of uremic middle molecules on in vitro stimulated lymphocytes and interleukin-2 production.

The influence of uremic middle molecules on in vitro lymphocyte blast transformation and interleukin-2 production was studied in 12 patients on long-term hemodialysis and 12 healthy control subjects with normal kidney function. Middle molecules inhibit phytohemoagglutinin induced lymphocyte proliferation in a concentration dependent fashion, achieving more than 50% inhibition of tritiated thymidine incorporation after 72 hrs in culture. In addition, there was less interleukin-2 production in the long-term hemodialysis patients studied compared with healthy control subjects.

Allogeneic gene-modified tumour cells in metastatic kidney cancer. Report II.

In a limited study, comprising only ten patients, we have previously reported that allogeneic irradiated RCC-cell-line cells, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells were used to vaccinate MRCC patients in progression of disease and also receiving IL-2 immunotherapy. The cells, admixed to autologous TC, were administered subcutaneously. We now report an extended study on thirty patients and one hundred thirty-one controls. Patients received 4-20 injections (mean 10 +/- 4), containing an average of 92 x 10(6) +/- 45 x 10(6) ACHN-IL-2 transfected cells (a minimum of 25 x 10(6), and a maximum of 200 x 10(6)). Autologous TC, admixed to allogeneic, were also administered by 4-16 s.c. injections (mean 7 +/- 3), i.e. a total of 12 x 10(6)-160 x 10(6) cells. Vaccination was administered during 73-1451 (307 +/- 316) days, and the follow-up continued for 1122 +/- 1240 days (106-5137). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were noticed. One complete and four partial tumour responses were observed, as well as nine cases of stable disease. Thirteen patients died in the treated group (43%) and 63 (44%) in the control group. Responding patients resumed progression in 4-11 months and died 18 and 36 months after beginning the vaccine therapy. The Gehan Wilcoxon's test showed a significantly (P < 0.01) better survival in the vaccinated patients compared to that of the controls. Thus, we confirm, in an increased number of patients and an extensive follow-up, that our vaccination protocol is safe, devoid of adverse side effects, and promising.