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.

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.

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.

Potential use of polymeric nanoparticles for drug delivery across the blood-brain barrier.

Nanomedicine is certainly one of the scientific and technological challenges of the coming years. In particular, biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted delivery of different agents, including recombinant proteins, plasmid DNA, and low molecular weight compounds. PLGA NPs present some very attractive properties such as biodegradability and biocompatibility, protection of drug from degradation, possibility of sustained release, and the possibility to modify surface properties to target nanoparticles to specific organs or cells. Moreover, PLGA NPs have received the FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, thus reducing the time for human clinical applications. This review in particular deals on surface modification of PLGA NPs and their possibility of clinical applications, including treatment for brain pathologies such as brain tumors and Lysosomal Storage Disorders with neurological involvement. Since a great number of pharmacologically active molecules are not able to cross the Blood-Brain Barrier (BBB) and reach the Central Nervous System (CNS), new brain targeted polymeric PLGA NPs modified with glycopeptides (g7- NPs) have been recently produced. In this review several in vivo biodistribution studies and pharmacological proof-of evidence of brain delivery of model drugs are reported, demonstrating the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. Moreover, another relevant development of NPs surface engineering was achieved by conjugating to the surface of g7-NPs, some specific and selective antibodies to drive NPs directly to a specific cell type once inside the CNS parenchyma.

Novel formulation of a double threshold algorithm for the estimation of muscle activation intervals designed for variable SNR environments.

The aim of this work is the development of an improved formulation of the double threshold algorithm for sEMG onset-offset detection presented by Bonato and co-workers. The original formulation, which keeps the threshold fixed, suffers from performance degradation whenever the SNR changes during the analysis. The novel approach is designed to be adaptive to SNR changes in either burst or inter-burst zones of sEMG signals recorded in static and dynamic conditions. The detection parameters (i.e. detection and false alarm probabilities) are updated on the basis of an on-line estimation of the SNR. The proposed formulation has been assessed on both simulated and real sEMG data. For constant SNR the performance of the original formulation is confirmed (for SNR > 8 dB, bias and standard deviation less than 10 and 15 ms, respectively; detection percentage higher than 95%), while the novel implementation performs better with time-varying SNR (for SNR varying in the range 10-25 dB the standard approach detection percentage decreases at 50%). Detection on signals recorded during isometric contractions at different force levels confirms the performance on simulated signals (StD = 134 ms; FP = 22%, and StD = 42 ms; FP = 2%, respectively for standard and novel implementation calculated as average on five experimental trials). The pseudo real-time detection allowed by this formulation can be profitably exploited by biofeedback applications based on myoelectric information.

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.

A SNR-independent formulation of a double threshold algorithm for the estimation of muscle activation intervals.

The aim of this work is to propose an improvement to the double threshold algorithm for muscular activation intervals estimation developed by Bonato and his co-workers. The proposed method has been designed in order to be adaptive also when the Signal to Noise ratio (SNR) of the sEMG signal changes during the trial, by re-evaluating the parameters of the algorithm according to the estimated local SNR and the desired detection and false alarm probabilities. This novel implementation is also suitable for working in pseudo real-time since it can give information on burst estimation shortly after the end of the current muscular activity. The proposed method was tested on simulated signals taking into account changes in the SNR during the trial, and results were compared with those obtained with the classical implementation of the algorithm.

Detection of tremor bursts from the sEMG signal: an optimization procedure for different detection methods.

Two different detection techniques for EMG burst detection are here used to reveal tremor in both a set of synthetic data and in a small sample of experimental trials. An optimization procedure that employs the minimization of a cost function to provide the parameter set characterizing the two techniques is here presented and its performance assessed. The results obtained with the optimization procedure are satisfactory and suitable for practical use: the values for both bias and standard deviation in the estimation of both onset and offset time instants are lower than 10 ms, and the sensitivity and positive predictive value in the detection of tremor bursts are > 96% for SNR levels higher than 6 dB.

Multimodal BCI-mediated FES suppression of pathological tremor.

Tremor constitutes the most common movement disorder; in fact 14.5% of population between 50 to 89 years old suffers from it. Moreover, 65% of patients with upper limb tremor report disability when performing their activities of daily living (ADL). Unfortunately, 25% of patients do not respond to drugs or neurosurgery. In this regard, TREMOR project proposes functional compensation of upper limb tremors with a soft wearable robot that applies biomechanical loads through functional electrical stimulation (FES) of muscles. This wearable robot is driven by a Brain Neural Computer Interface (BNCI). This paper presents a multimodal BCI to assess generation, transmission and execution of both volitional and tremorous movements based on electroencephalography (EEG), electromyography (EMG) and inertial sensors (IMUs). These signals are combined to obtain: 1) the intention to perform a voluntary movement from cortical activity (EEG), 2) tremor onset, and an estimation of tremor frequency from muscle activation (EMG), and 3) instantaneous tremor amplitude and frequency from kinematic measurements (IMUs). Integration of this information will provide control signals to drive the FES-based wearable robot.

Two novel cosegregating mutations in tRNAMet and COX III, in a patient with exercise intolerance and autoimmune polyendocrinopathy.

We report a 12-year-old patient with growth retardation, exercise intolerance, lactic acidosis (increasing after exercise) and autoimmune polyendocrinopathy type 2. Muscle biopsy shows abundant COX-negative fibers, subsarcolemmal mitochondrial aggregates and markedly reduced activities of all respiratory chain complexes. Genetic analysis identified two new cosegregating mutations in Met-tRNA (m.4415A>G) and Cox III (m.9922A>C), located in highly conserved regions of MtDNA. Both the mutations are heteroplasmics in multiple patients' tissues. Single-muscle fiber analysis showed significantly higher levels of both the mutations in COX-negative than in normal fibers. In addition, a possible link between the mitochondrial dysfunction and the autoimmune disease is suggested.

Two novel POLG mutations causing hepatic mitochondrial DNA depletion with recurrent hypoketotic hypoglycaemia and fatal liver dysfunction.

BACKGROUND: Inherited mtDNA depletion syndromes (MDS) are a group of severe mitochondrial disorders resulting from defects in nucleus-encoded factors and often associated with severe or fatal liver failure. PATIENT: In this article, we describe the case of an 18-month-old patient with recurrent hypoketotic hypoglycaemia and fatal hepatic dysfunction with liver mtDNA depletion. METHODS: The assessment of mtDNA copy number was performed on leucocytes, liver and muscle biopsy by Quantitative Real Time PCR and total RNA from liver biopsy was used as a template to amplify the cDNA of the POLG1 gene. RESULTS: Sequence analysis identified two previously undescribed mutations (1868T>G and 2263A>G) located in the gene coding the catalytic subunit of mitochondrial DNA polymerase gamma (POLG), predicting an L623W and K755E amino acid change, respectively. Both mutations were located in the highly conserved linker region of the protein and were absent in more than 200 healthy unrelated control subjects. The identification of these two mutations allowed us to perform genetic counselling and prenatal diagnosis. CONCLUSION: Our data further expand the spectrum of POLG1 gene mutations and the unique phenotype reported (late onset isolated liver disease without lactic acidosis) increase the variability of clinical presentations associated with mutations in this gene.

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.

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.

Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

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.

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.

Determination of ethyl-p-hydroxybenzoate in sow pancreatic juice by reversed-phase high-performance liquid chromatography.

We have developed a high-performance liquid chromatographic-UV-Vis-diode-array detection (HPLC-DAD) method for the determination of ethyl-p-hydroxybenzoate, a hydrolytic degradation product of the synthetic protease inhibitor, gabexate-mesilate ethyl-p-(6-guanidinohexanoyloxy) benzoate methanesulfonate (GM) (FOY) in sow pancreatic juice. Methyl-p-hydroxybenzoate (I) was used as the internal standard. The pancreatic juice was deproteinised by acetonitrile and the analytes were chromatographed on a reversed-phase C18 LC column using the gradient elution method. The mobile phase consisted of a solution of 0.017 M orthophosphoric acid and another solution of acetonitrile-water (80:20, v/v). The wavelength of detection was 237 nm. The limit of quantification of the method was 0.20 microM at a 9:1 signal-to-noise ratio. The overall intra- and inter-day accuracy (relative error, RE) ranged from 14.2 to 8.3% and from 13.3 to 9.8, respectively. The overall intra- and inter-day precision (relative standard deviation, RSD) ranged from 7.6 to 2.62% and from 6.7 to 3.1%, respectively. The method proved to be sensitive, specific, accurate and precise and was successfully used to determine the ethyl-p-hydroxybenzoate (II) in sow pancreatic juice.

[Oxidative stress in kidney failure: an experimental study]. / Stress ossidativo nell'insufficienza renale: uno studio sperimentale.

We studied the activity of some enzymes directly involved in the endogenous antioxidative defense system: glutathione-peroxidase (GPX), glutathione s-transferase (GST) and superoxide dismutase (SOD). We have investigated the effects of selenium and vitamin E diet supplementation, in form of selenium-vitamin E enriched yeast, in Wistar rats that were undergone to surgical right nephrectomy and 30 minutes of hypoxia. Blood samples were tested for several parameters as glucose, cholesterol, etc. to assess the general health conditions. The protocol consisted of 3 groups of 25 Wistar rats: a control group, a pre-fed group and a post-fed group. The results showed a significative difference in the behaviour of azotemia, proteins and cholesterol. In the control group the activity rapidly increased, then the values decreased slowly and differently for each substance. The pre and post-fed group showed a pronounced increase after 48 h but the normal values are reached more rapidly. We observed an increase in the activity of the GPX and GST after surgical operation and ischemia, but the GPX in pre-fed group reached the normal value before the other groups.

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.