ПОДЖЕЛУДОЧНАЯ ЖЕЛЕЗА НОВОРОЖДЕННЫХ КРОЛИКОВ КАК ИСТОЧНИК ПРОГЕНИТОРНЫХ КЛЕТОК

The newborn rabbit pancreas was used as a source of ductal epithelial cells. Our approach includes the cultiva- tion of pancreatic tissue microfragments without preliminary islet and duct isolation. Under ordinary conditions we found the epithelial pancreatic cell monolayer. The cultivated monolayer cells were shown by immunofluo- rescence to express special ductal cytokeratin 19 and were insulin- and glucagon-negative for 7–15 days. Поджелудочная железа новорожденных кроликов была использована в качестве источника получения протоковых клеток. Эти клетки, обладая свойствами прогениторных, сохраняют способность к диффе- ренцировке in vivo и in vitro и могут служить дополнительным альтернативным источником островко- вых клеток для трансплантации больным сахарным диабетом. Наш метод основан на культивировании микрофрагментов панкреатической ткани без предварительного выделения островков и отдельных про- токов. В стандартных условиях путем подбора режима культивирования удалось получить стабильное формирование монослоя протокового эпителия, клетки которого активно экспрессировали специфиче- ский маркер цитокератин 19.

УСКОРЕННОЕ ГУМОРАЛЬНОЕ ОТТОРЖЕНИЕ АЛЛОТРАНСПЛАНТАТА СЕРДЦА

In the article there is described the case of the accelerated humoral (antibody-mediated) rejection of the cardiac allograft with morphologic and immunohistochemical details revealed at the investigation of the endomyocardial biopsy and myocardial autopsy. В статье приводится случай ускоренного отторжения аллотрансплантата сердца с подробным описанием морфологических и иммуногистохимических особенностей, выявленных при исследовании эндомиокардиального биоптата и миокарда аллотрансплантата, удаленного при аутопсии.

НАРУШЕНИЕ МАКРОМОЛЕКУЛЯРНОЙ СТРУКТУРЫ КАРДИОМИОЦИТОВ АЛЛОТРАНСПЛАНТАТА СЕРДЦА КАК ПРИЗНАК ХРОНИЧЕСКОГО ОТТОРЖЕНИЯ

Chronic rejection, especially cardiac allograft vasculopathy, is a major limiting factor for long-term transplant survival. This process affects not only the blood vessels, but also cardiomyocytes. However, there are extremely few reports on the evaluation of their macromolecular structure state. The aim of the study was to evaluate the structural proteins of cardiomyocytes (actin, myosin, troponin I, titin, desmin, vinculin) of heart allografts in different periods after the operation (from 6 days to 15 years). Major changes of macromolecular structure were revealed in late period after transplantation (6 months – 15 years). The contribution of humoral immune response in the process of chronic cardiac allograft rejection was observed: in eight of twelve recipients episodes of acute humoral rejection had been repeatedly registered; disorders of the expression of 5 proteins out of 6 characterized were found in recipients with recurrent and persistent antibody-mediated rejection. Хроническое отторжение, и прежде всего болезнь коронарных артерий, является основным лимитирую- щим фактором длительной функции аллотрансплантата сердца. Данный процесс затрагивает не только со- суды, изменениям также подвержены и кардиомиоциты. Однако сообщений, касающихся оценки состоя- ния их макромолекулярной структуры, крайне мало. Целью нашей работы явилось исследование состояния структурных белков кардиомиоцитов (актин, миозин, тропонин I, титин, десмин, винкулин) аллотрансплан- тата сердца в разные периоды после операции (от 6 дней до 15 лет). Основные изменения макромолекуляр- ной структуры выявлены в подгруппе позднего периода (6 мес.–15 лет). Продемонстрирован вклад гумо- рального звена иммунного ответа в процесс хронического отторжения аллотрансплантата сердца: у восьми из двенадцати реципиентов данной подгруппы неоднократно регистрировали эпизоды острого антитело- опосредованного отторжения; нарушения экспрессии пяти белков из шести охарактеризованных были об- наружены у реципиентов с возвратным и персистирующим антителоопосредованным отторжением.

EMPLOYMENT OF A «SIDE POPULATION» APPROACH TO STEM CELL ISOLATION IN NORMAL AND TUMOR TISSUES

Abstract. A combination of fluorescent staining with Hoechst 33342 dye, and flow cytometry of murine bone marrow cells may be used for separation of a side population (SP), which is highly enriched for hematopoietic stem cells capable of long-term hematopoietic reconstitution in lethally irradiated recipients. Recently, this approach was also applied to analysis of SP cells in several types of non-hematopoietic tissues, and malignant tumours. In spite of yet poor definition of phenotype and functional potency of SP cells from various tissues, the method of SP isolation may be a useful tool for analysis and pre-enrichment of stem cell-like cells of different origin. Present review article presents a brief description of Hoechst 33342-staining approach, and of recent reports concerning SP studies in various normal and malignant tissues. (Med. Immunol., vol. 10, N 4-5, pp 319-326)

Combination of chronic myocarditis and progressive coronary artery disease: differential diagnosis and stepwise treatment

Aim. To assess the differential diagnosis in a patient with a combination of coronary artery disease and myocarditis and the results of stepwise treatment (including immunosuppressive therapy (IST), and coronary stenting).Material and methods. A 56-year-old female patient with hypertension, obesity (body mass index, 31,6 kg/m2), diabetes and psoriasis developed shortness of breath after a respiratory viral infection. Primary echocardiography revealed left heart dilatation, ejection fraction (EF) of 21%. Coronary angiography revealed anterior descending artery stenosis of 75%, circumflex artery — 80%, right coronary artery (RCA) — 70%. RCA stenting was performed and cardiovascular and diuretic therapy was started. However, shortness of breath and low exercise tolerance persisted.Results. In the blood test, anti-endothelial cell antibodies were 1:320, anticardiomyocyte and anti-smooth muscle antibodies — 1:80, anti-cardiac conduction system fibers — 1:320 (N≤1:40). During myocardial perfusion scintigraphy with computed tomography, an uneven distribution of the indicator was noted. Signs of myocardial scarring and indications for further revascularization were not revealed. Cardiac magnetic resonance imaging confirmed a decrease in left ventricular (LV) contractility (LVEF 37%) and moderate dilatation. Biopsy was not performed due to dual antiplatelet therapy. The condition is regarded as infectious-immune myocarditis. IST was started with azathioprine 150 mg/day. We noted dyspnea relief and a stable increase in LVEF to 50-52%. The clinical course was complicated by sick sinus syndrome with pauses up to 6 seconds and presyncope; a pacemaker was implanted. After 5 years from the onset of IST, dyspnea episodes reappeared without exacerbation of myocarditis. As their cause, ischemia was diagnosed due to the progression of coronary atherosclerosis. Symptoms regressed after repeated coronary stenting.Conclusion. The presence of moderate coronary atherosclerosis without signs of ischemia and myocardial infarction should not be considered as the only cause of severe systolic myocardial dysfunction. Diagnosis and treatment of myocarditis in combination with coronary artery disease is carried out according to the standard principles and can improve LV systolic function and control the heart failure symptoms

Treatment efficacy of arrhythmias and dilated cardiomyopathy syndrome of immune-inflammatory nature using plasmapheresis

Aim. To study the efficacy of plasmapheresis as the main type of pathogenic treatment or in combination with immunosuppressive therapy in patients with dilated cardiomyopathy (DCMP) and arrhythmias of immune-inflammatory nature.Material and methods. The main group included 20 patients with arrhythmic myocarditis (with premature supraventricular / ventricular contraction >3000/day, n=3/8, atrial fibrillation (AF) n=9) and 14 patients with DCMP syndrome (enddiastolic volume (EDV) left ventricle (LV) 6,3±0,6 cm, ejection fraction (EF) 33,5±8,1%). The inclusion criterion was an increase of at least 2 types of anti-cardiac antibodies titers ≥ twice. Myocarditis is diagnosed using myocardial biopsy, magnetic resonance imaging, multispiral computed tomography, scintigraphy, coronary angiography. We used a course of discrete plasmapheresis. The comparison group included 26 patients with an arrhythmic myocarditis and 19 with DCMP syndrome (EDV 6,6±0,8 cm, EF 32,6±7,3%), which plasmapheresis was not used. Dynamics was assessed at 6 and 12 months.Results. In groups of patients with arrhythmias and DCMP, a significant decrease in anti-cardiac antibodies titers was observed immediately after plasmapheresis and in control studies (p<0,05). In patients with arrhythmias, a health-promoting effect (a decrease in the number of premature contraction and a frequency of atrial fibrillation ≥75%) was observed in 65% of the main group and 58% of the comparison group. Predictor of plasmapheresis efficiency was a titer of specific antinuclear factor ≥1: 40 (sensitivity — 92,3%, specificity — 71,4%, AUC — 0,813, p<0,05). Methylprednisolone was prescribed to 45% of patients in the main group and 73% to patients in the comparison group (p>0,05) at a dose of 8 [4; 16] and 16 [10; 24] mg per day, respectively, p>0,05. In patients with DCMP in the main group, a significant increase in EF (p<0,05) (up to 41,4±8,2% and 46,3±12,7% vs 39,1±13,7% and 37,2±10,7% in the comparison group) and the distance of 6-minute walking test was obtained. A good effect (increase in EF by 10% or more) was noted in 50% of the main group and 32% of the comparison group. The predictor of plasmapheresis efficacy was systolic pressure in the pulmonary artery ≥28,5 mm Hg. (sensitivity — 100%, specificity — 71,4%, AUC — 0,893, p<0,05). In the main group, methylprednisolone was assigned to 43% of patients, in the comparison group — 89%, p<0,05. The average doses of methylprednisolone in the main group were significantly lower than in the comparison group (8 [8; 17,25] vs 16 [13; 28] mg per day, p<0,05).Conclusion. Positive clinical response to plasmapheresis was noted in 65% of patients with arrhythmias and in 50% of patients with DCMP of immune-inflammatory nature. In patients with different types of myocarditis, plasmapheresis increases the efficacy of antiarrhythmic and immunosuppressive therapy

COVID-19 Pneumonia in Patients with Chronic Myocarditis (Recurrent Infectious Immune): Specifics of the Diseases Course, the Role of Basic Therapy (Part 1)

Patients with chronic myocarditis have a high risk of an unfavorable course of the novel coronavirus disease (COVID-19) due to the ability of the SARS-Cov-2 virus to independently cause acute myocarditis, to have a direct and cytokine-mediated cytopathic effect on the myocardium, as well as immunosuppressive therapy. At the same time, the features of the interaction of chronic myocarditis and COVID-19 have not been studied. The article describes a 31-year-old patient with a 10-year history of chronic recurrent infectious-immune myocarditis, who was on long-term immunosuppressive therapy (methylprednisolone and azathioprine in the past, then hydroxychloroquine). In May 2020, a serologically confirmed COVID-19 diagnosis was made. There were risk factors for the unfavorable course of coronavirus infection: heart failure and a history of persistent atrial fibrillation, male sex. Basic therapy with hydroxychloroquine (with an increase in its dose to 800-400 mg/day), ceftriaxone, and levofloxacin was carried out. The severity of pneumonia was moderate, despite febrile fever and severe intoxication. No relapses of arrhythmias, respiratory or heart failure were observed. Minimal laboratory (some increase in anticardial antibody titers) and echocardiographic signs of exacerbation of myocarditis without an increase in troponin T levels were revealed, which quickly regressed. It can be assumed that the maintenance immunosuppressive therapy of myocarditis with hydroxychloroquine had a positive effect on the course of coronavirus pneumonia and made it possible to avoid recurrence of myocarditis. Further study of the features of the course of the pre-existing myocarditis and pneumonia in COVID-19 is necessary

THE PANCREAS OF NEWBORN RABBITS AS A SOURCE OF PROGENITOR CELLS

The newborn rabbit pancreas was used as a source of ductal epithelial cells. Our approach includes the cultiva- tion of pancreatic tissue microfragments without preliminary islet and duct isolation. Under ordinary conditions we found the epithelial pancreatic cell monolayer. The cultivated monolayer cells were shown by immunofluo- rescence to express special ductal cytokeratin 19 and were insulin- and glucagon-negative for 7–15 days

COVID-19 Pneumonia in Patients with Chronic Myocarditis (HBV-Associated with InfarctLike Debute): Specifics of the Diseases Course, the Role of the Basic Therapy (Part II)

Chronic infectious-immune myocarditis of severe course can potentially be considered as a factor that aggravates the course of new coronavirus disease (COVID-19) and increases the risk of adverse outcomes. The interaction of chronic myocarditis and COVID-19 during long-term immunosuppressive therapy has not been studied. We present a description of a 35-year-old female patient with chronic infectious-immune myocarditis (morphologically confirmed, with a history of infarction-like onset and thromboembolic complications), who had continuous immunosuppressive therapy with methylprednisolone and mycophenolate mofetil. The patient also received new oral anticoagulants and tenofovir (for chronic HBV infection). COVID-19 (SARS-Cov-2 RNA+) was diagnosed in May 2020. Risk factors for the adverse course of coronavirus infection included severe obesity, heart failure, and life-threatening ventricular arrhythmias. Correction of immunosuppressive therapy (withdrawal of the cytostatic agent, administration of hydroxychloroquine) and therapy with levofloxacin, an interleukin-17 inhibitor (netakimab) were performed. The severity of pneumonia and respiratory failure was moderate despite high fever and high levels of inflammatory markers in the blood (including interleukin-6). Signs of exacerbation of myocarditis, increased levels of troponin T and anticardial antibodies (compared with the initial ones) were not found. It can be assumed that supportive immunosuppressive therapy for myocarditis has a positive effect on the course of coronavirus pneumonia and avoids exacerbation of myocarditis. Careful continuation of immunosuppressive therapy with temporary withdrawal of aggressive cytostatics can be recommended in chronic myocarditis. Further study of the features of the course of previous myocarditis and COVID-19 pneumonia is necessary