In Vivo DYSF Gene Viral Delivery Provides a Histoprotective Effect in Skeletal Muscle Tissue in Dysferlin-Deficient Mice.

We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 µl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.

[Metastatic basal cell carcinoma with a SMO gene mutation. A case report]. / Metastaticheskaya forma bazal'no-kletochnoi kartsinomy s mutatsiei v gene SMO. Opisanie nablyudeniya.

The authors describe a clinical case of a patient with metastatic basal cell carcinoma and secondary resistance to vismodegib. A SMO G416E mutation of unknown clinical significance was found in the gene encoding the transmembrane receptor protein Smoothened (SMO) protein, which suggests a defect in the Sonic Hedgehog (SH) pathway and may cause tumor resistance to the prescribed drug. Comprehensive genomic profiling and in silico analysis have been used to diagnose the tumor.

First experience of hematopoietic stem cell transplantation treatment of Shwachman-Diamond syndrome using unaffected HLA-matched sibling donor produced through preimplantation HLA typing.

The only proven cure for Shwachman-Diamond syndrome (SDS) bone marrow failure is allogeneic hematopoietic stem cell transplantation (HSCT). However HSCT with donors other than HLA-identical siblings is associated with high mortality and unfavorable prognosis. This paper presents the first experience of HSCT treatment of SDS using an unaffected HLA-identical sibling produced through preimplantation genetic diagnosis (PGD). The patient was a 6-year-old blood transfusion-dependent SDS baby girl with secondary myelodysplastic syndrome, for whom no HLA-identical donor was available. As a result of PGD, two unaffected HLA matched embryos were identified; one of them was randomly selected for transfer, resulting in a clinical pregnancy and birth of an apparently healthy child. The patient underwent allogeneic transplantation of cord blood hematopoietic stem cells, together with bone marrow from this sibling, resulting in complete hemopoietic recovery. The patient was no longer transfusion-dependent and had normal blood values 160 days after transplantation.

[Five-year results of treating patients with chronic lower limb ischaemia by means of gene engineering]. / Pyatiletnie rezul'taty lecheniya bol'nykh khronicheskoi ishemiei nizhnikh konechnostei s ispol'zovaniem gennoi terapii.

The authors share their experience in comprehensive conservative treatment of patients presenting with chronic lower limb ischaemia (CLLI) associated with atherosclerosis of peripheral arteries by means of the first Russian registered gene therapeutic agent "Neovasculgen" (plasmid with the vegf165 gene), analysing the long-term outcomes of treating a total of 45 patients with stage II and III CLLI according to the classification of Pokrovsky-Fontain. The patients were followed up for 5 years. Efficacy of treatment was assessed by registering the dynamics of the pain-free walking distance (PFWD), linear blood velocity (LBV), ankle-brachial index (ABI), as well as the limb salvage rate and survival of patients. All patients showed good tolerance of treatment, with neither side effects nor complications noted. Clinical improvement in stage IIB CLLI was observed in 91% of patients with complete stabilization of the clinical course during 5 years. The limb salvage rate in this group amounted to 95%, with the survival rate equalling 82%. In patients with stage III CLLI, improvement was noted in 78% of cases, manifesting itself by a decrease of its degree to stage IIB (44.4%) and to stage IIA (33.3%). Progression of CLLI followed by amputation was registered in 22% of cases, with the survival rate of 78%. Hence, the use of a single course of combined treatment including the gene therapeutic agent "Neovasculgen" in patients with stage II and III CLLI resulted in a persistent positive effect in a considerable majority of patients in the remote period of not less than 5 years.

World's First Clinical Case of Gene-Activated Bone Substitute Application.

Treatment of patients with large bone defects is a complex clinical problem. We have initiated the first clinical study of a gene-activated bone substitute composed of the collagen-hydroxyapatite scaffold and plasmid DNA encoding vascular endothelial growth factor. The first patient with two nonunions of previously reconstructed mandible was enrolled into the study. Scar tissues were excised; bone defects (5-14 mm) between the mandibular fragments and nonvascularized rib-bone autograft were filled in with the gene-activated bone substitute. No adverse events were observed during 12 months of follow-up. In 3 months, the average density of newly formed tissues within the implantation zone was 402.21 ± 84.40 and 447.68 ± 106.75 HU in the frontal and distal regions, respectively, which correlated with the density of spongy bone. Complete distal bone defect repair with vestibular and lingual cortical plates formation was observed in 6 and 12 months after surgery; thereby the posterior nonunion was successfully eliminated. However, there was partial resorption of the proximal edge of the autograft entailed to relapse of the anterior nonunion. Thus, the first clinical data on the safety and efficacy of the gene-activated bone substitute were obtained. Given a high complexity of the clinical situation the treatment, results might be considered as promising. NCT02293031.

[PAD prevalence in Russian Federation]. / Rasprostranennost' simptomnoi ishemii nizhnikh konechnostei v Rossiiskoi Federatsii.

BACKGROUND: Currently there is no data on peripheral arterial disease prevalence in Russia. AIM: To estimate prevalence of patients with symptomatic low limb ischemia (LLI) in Russian Federation on 2014. PATIENTS AND METHODS: «League for patient' protection¼ had sent questionnaires for all regional health departments asking to name prevalence of ICD-10 codes I70.2 (atherosclerosis of arteries of extremities) and I73.1 (thromboangiitis obliterans (Buerger)) on 2014. The figures were listed separately for ischemia I-IIa and III-IV (critical limb ischemia, CLI) (Pokrovsky, 1976). Also were mentioned major amputations and mortality. RESULTS: Total number of received questionnaires was 44, we've got information from all RF' federal regions with a population of appr. 71 million people. There were 174 125 cases of symptomatic LLI. Prevalence of symptomatic LLI among Russian population aged more than 40 years was 0.51%. Prevalence of intermittent claudication was 0.36% and of critical limb ischemia - 0.13%. Rate of major amputations was 6.9%. CONCLUSION: World PAD prevalence among people older than 25 years is 8.3%. The same figure in our study was only 0.5%. This is due to the fact that we considered only symptomatic LLI and only with codes I70.2 and I73.1. Prevalence of symptomatic patients among all PAD patients is 10-35%. So the real number of PAD patients in Russian might be 1.5-5%. We can conclude that PAD is still unrevealed in Russia. Another conclusion could be that the treatment results are less than satisfactory. Finally there is urgent necessity for special PAD register in RF.

[Remote results of treatment of patients with chronic lower-limb ischaemia by means of indirect revascularization and gene therapy].

Presented herein are comparative remote results of combined surgical treatment of 121 patients with stage IIB-III lower limb chronic ischaemia (LLCI) by means of indirect revascularization (lumbar sympathectomy--LSE and revascularizing osteotrepanation of the tibial bone--ROT) and gene therapy using the first registered Russian gene therapeutic agent Neovasculgen®. Depending on the LLCI degree and the method of treatment, during 3 years we assessed such parameters as the limb salvage rate, pain-free walk distance (PFWD), ankle-brachial index (ABI) and linear blood velocity (LBV). An increase in the PFWD in patients with initial stage IIB LLCI in the group of gene therapy was considerably higher than in other types of treatment (p=0.0001-0.0004). Using indirect methods of revascularization was accompanied and followed by less positive alterations in the PFWD values which by the end of the third year of follow up were observed to decrease. The values of PFWD after ROT at 2 and 3 years were higher than after LSE (p=0.006). During the first year of follow up the highest increment of the ABI was observed after ROT. At two years, the ABI values after ROT and gene therapy became equal. The worst result during 3 years as compared with other methods of treatment was demonstrated by LSE (p=0.006). Changes in ABI after gene therapy were statistically significant at all terms of follow up (p=0.008-0.02). There were no limb amputations in the remote period of follow up in patients with the initial stage IIB of the disease. Patients with initial stage III LLCI also showed a considerably better result by the increment of increased PFWD after gene-therapeutic treatment (p=0.001-0.0005). A small increment of the PFWD after LSE maintained during 1 year and after LSE during 2 years. The ABI values in all periods of follow up were higher after gene therapy (p=0.01-0.003). During the 2- and 3-year period the increment of this parameter after ROT was more significant than after LSE (p=0.046-0.05). Changes in the ABI after gene therapy at all terms also turned out to be more substantial (p=0.03-0.008). An increase in the LBV during the first and second years after ROT turned out more significant as compared to LSE (p=0.05). The limb salvage rate in patients with initial stage III LLCI during the whole period after gene therapy amounted to 78%, after ROT being 54% and after LSE equalling 45%.

Ordinary and Activated Bone Grafts: Applied Classification and the Main Features.

Bone grafts are medical devices that are in high demand in clinical practice for substitution of bone defects and recovery of atrophic bone regions. Based on the analysis of the modern groups of bone grafts, the particularities of their composition, the mechanisms of their biological effects, and their therapeutic indications, applicable classification was proposed that separates the bone substitutes into "ordinary" and "activated." The main differential criterion is the presence of biologically active components in the material that are standardized by qualitative and quantitative parameters: growth factors, cells, or gene constructions encoding growth factors. The pronounced osteoinductive and (or) osteogenic properties of activated osteoplastic materials allow drawing upon their efficacy in the substitution of large bone defects.

[Efficacy of using VEGF165 gene in comprehensive treatment of patients with stage 2A-3 lower limb chronic ischaemia].

The authors share their experience in treating a total of 100 patients presenting with stage 2A-3 chronic lower limb ischaemia according to the classification of Pokrovsky-Fontain (the clinical group was composed of 75 patients and the control group comprised 25 subjects), in whom it was impossible to perform surgical revascularization. The clinical group patients received in addition to the conventional vascular therapy local intramuscular injections of Neovasculogen (plasmid genetic construction containing human gene VEGF165) at a course dose of 2.4 mg. The results were assessed after 1 year. It was shown that administration of this gene therapeutic agent is safe with no local or systemic allergic reactions and free form neoplastic processes. Efficacy of treatment was assessed by registering the pain-free walking distance (PFWD), transcutaneous oxygen tension (TCPO2), linear velocity of blood flow, ankle-brachial index (ABI), angiography, and by means of SF-36 questionnaire. It was determined that after 12 months the statistical significance of intergroup and intragroup differences was reached for PFWD (increment 167.2%), TCPO2 (increment 20.4%). The highest clinical response for the PFWD was registered in patients with stage 3 of the disease (547.5%), as well as in those with multi-storey vascular lesions (269.1%). The obtained findings make it possible to consider gene therapy with Neovasculogen as an efficient component of comprehensive treatment of this cohort of patients.

[Therapeutic angiogenesis in treatment of patients with chronic obliterating diseases of lower limb arteries].

The authors share their experience gained in realization of the concept of "therapeutic angiogenesis" by means of the first Russian gene therapeutic agent Neovasculogen in comprehensive treatment of patients with stage II-III chronic obliterating disease of lower limb arteries (CODLLA) according to the Pokrovsky-Fontain classification. A total of 45 patients were treated. The findings were assessed and analyzed depending on the stage of chronic lower limb ischaemia. The clinical results including such measures such as the pain-free walk (PFW), ankle/brachial index (ABI), transcutaneously determined oxygen tension (TDOT), linear blood velocity (LBV) were followed up for 2 years. All patients were found to have good tolerance and at terms up to 2 years, and no side effect or complications were revealed. Patients with stage IIB of the disease after one year demonstrated statistically significant results suggesting improved blood circulation in the ischemized lower limbs after using the agent according to the indices of PFW, TDOT, LBV (p<0.05). In stage III of the disease? significant results were observed for PFD and TODT (p<0.05). Considerable part of patients with stage IIB and III CODDLLA (84.4%) showed increased physical capabilities for ambulation at the expense of considerably increased PFW (p<0.01). It was observed that using the agent was enough for obtaining a persistent satisfactory result preserving for not less than one year, with 50% of patients from stage IIB and 16.7% from stage III transferred to stage IIA of the disease. The effect of treatment persisted during the second year of follow up.

[Worldwide experience and recent trends in gene therapy of ischaemic diseases].

Fifteen odd years have passed since the first application of a gene-therapeutic modality in clinical practice for treatment of lower-limb chronic ischaemia. Over this time, vast experience has been gained worldwide, with not less than one thousand patients treated by gene-based therapies, thus making it possible to generalise the published findings of these clinical trials. Resulting from such an analysis, it should be recognized that the least dangerous gene therapeutic modalities available so far are plasmid ones, with the most efficient being those containing the gene of vascular endothelial growth factor VEGF(165). The most convincing results were obtained while treating chronic ischemia of the lower extremities, whereas gene-based therapy used for treatment of coronary artery disease failed to have yielded, as of yet, clear cut positive results.

[The laparoscopic adrenalectomy in children].

22 children with tumors or other surgical diseases of the adrenal glands were operated on laparoscopically. Children aged from 5 months to 17 years. Operation lengths varied from 45 to 190 minutes. There were no lethal outcomes, conversions and serious complications of the procedure. Authors consider the laparoscopic adrenalectomy to be the safe and widely appropriate method for the use in juvenile cathegory of patients.

[Optimization of laparoscopic splenectomy at children].

Treatment results of 130 patients aged 1 to 16 years with benign hematological diseases (congenital microspheric anemia--93 patients, thalassemia--18, severe idiopathic thrombocytopenic purpura--19) who has undergone laparoscopic cholecystectomy due to concomitant cholelithiasis. Optimization of surgical techniques permitted to achieve excellent functional and cosmetic results, to reduce operation time to 35-110 min (64.67+/-10.07 min on average), volume of hemorrhage to 30-110 ml (62.91+/-14.46 ml on average), to avoid intraoperative and postoperative complications. It is concluded that laparoscopy is the method of choice for the treatment of children with benign hematological diseases required splenectomy despite the age of child, spleen size, including the children with severe disorders of hemostasis system.

[Life quality of children with hematological diseases after laparoscopic splenectomy].

Problems of assessment of life quality of children with benign hematological diseases (hereditary microspheric anemia, thalassemia, idiopathic thrombocytopenic purpura) after laparoscopic cholecystectomy are discussed. The standard SF-36 scale was used before and after endoscopic surgery. It is demonstrated that after reasonable laparoscopic splenectomy almost all parameters of life quality are improved. Improvement of physical parameters argues for small surgical trauma and slight postoperative period after laparoscopic splenectomy.

[Effect of use of bone marrow cells in therapy for experimental tuberculosis].

The authors studied the efficiency of use of bone marrow cells in the treatment of experimental tuberculosis. Bone marrow cell transplantation to H37Rv tuberculosis-infected H37Rv mice was shown to prolong the life span in the animals as compared to untreated animals. Examination of humoral immunity indicated that administration of allogenic bone marrow cells resulted in the nonspecific polyisotypic stimulation of antituberculosis antibodies, which is essential in producing the protective humoral background. A more significant generation of IgG2a antibodies than that of IgG1 antibodies was also found in therapy with bone marrow cells, which pointed to the fact that there was a Th1 response that is obviously protective in tuberculosis. The high level of IgG2a antibodies correlated with the high specific cellular immune response estimated by the delayed hypersensitivity reaction.

[Laparoscopic operation for Hirschsprung's disease in children].

Experience in the treatment of 5 children aged 6 months to 14 years with different types of aganglionosis of the colon using laparoscopic endorectal bringing down of the colon (like Coabe surgery) is analyzed. In all the cases this method permitted mobilization and resection of the intestine (to 60 cm), bringing down intact intestine on the perineum with simultaneous coloanal anastomosis. Duration of the surgery ranged from 120 to 240 min, postoperative hospital stay did not exceed 8 days. Functional and cosmetic results were excellent. The method is characterized by combination of advantages of classical Coabe operation with minimal surgical trauma of laparoscopic mobilization of the colon.