Резюме
Зборовська О.В.¹, Прейс Н.І.² Зміни загального імунного статусу при дифузному та ускладненому діабетичному макулярному набряку різних видів у хворих на цукровий діабет ІІ типу після проведення імунокорекції.
Діабетичний макулярний набряк є найбільш розповсюдженою причиною зниження зору у пацієнтів з ЦД ІІ типу. Він може супроводжувати будь-яку стадію захворювання і в середньому зустрічається в 10 % пацієнтів з ЦД. Ключовою ланкою патогенезу ДМН є накопичення рідини в екстрацелюлярному просторі сітківки внаслідок дисбалансу гемато-ретинального бар’єра. Основними принципами лікування діабетичної ретинопатії на сучасному етапі вважаються стабільна компенсація цукрового діабету, нормалізація артеріального тиску та лікування ураженої сітківки. Всім пацієнтам проводились загальноофтальмологічні обстеження, ОКТ, ФАГ та імунологічні дослідження. В дослідженні приймали участь хворі цукровим діабетом  ІІ типу з діабетичним макулярним набряком різних видів. Всього 64 пацієнта (128 очей), з них 28 жінок ( 43,8%) і 36 чоловіків, ( 56,3%), середній вік котрих складав 58 років (SD 10), від 24 до 78 років. Досліджували дифузний (оклюзійний та неолюзійний) та ускладнений (оклюзійний та неолюзійний) ДМН. Всього з дифузним оклюзійним ДМН 33 випадки, дифузним неоклюзійним ДМН 35 випадків, ускладненим оклюзійним 28 випадків, ускладненим неоклюзійним 32 випадки. Статистична обробка даних проводилась в програмі Statistica 10.0. Після лікування з використанням комбінації препаратів циклоферону та цитофлавіну була виявлена позитивна динаміка з боку показників імунного статусу у вигляді зниження рівня відносного відсоткового значення рівня Т-лімфоцитів - СD-3 при дифузному та ускладненому ДМН різних видів, зниження рівня сенсибілізації до АГ- сітківки при дифузному та ускладненому (оклюзійному та неоклюзійному виді) ДМН (p=0,0001). Щодо показників гуморального імунітету, спостерігається тенденція до зниження концентрації імуноглобулінів класу A, G, M при дифузному та ускладненому ДМН різних видів. Отримані дані дозволяють вважати доцільним і патогенетично обґрунтованим використання цитофлавіну та циклоферону в комплексній терапії хворих на ЦД ІІ типу при дифузному та ускладненому (оклюзійному та неоклюзійному) ДМН.
Ключові слова: діабетичний макулярний набряк, імунологічний статус, клітинний імунітет, гуморальний імунітет.
Резюме
Зборовская А.В., Прейс Н.И. Изменения общего иммунного статуса при диффузном и осложненном диабетическом макулярном отека различных видов у больных сахарным диабетом II типа после проведения необходимой иммунокоррекции.
Диабетический макулярный отек является наиболее распространенной причиной снижения зрения у пациентов с СД II типа. Он может сопровождать любую стадию заболевания и в среднем встречается в 10% пациентов с сахарным диабетом. Ключевым звеном патогенеза ДМО является накопление жидкости в экстрацеллюлярном пространстве сетчатки вследствие дисбаланса гемато-ретинального барьера. Основными принципами лечения диабетической ретинопатии на современном этапе считаются стабильная компенсация сахарного диабета, нормализация артериального давления и лечения и осложненный (оклюзионный и неоклюзионный) ДМО. Всего с диффузным оклюзионным ДМО 33 случая, диффузным неоклюзионным ДМО 35 случаев, осложненным оклюзионным 28 случаев, осложненным неоклюзионным ДМО 32 случая. Статистическая обработка данных проводилась в программе Statistica 10.0. После лечения с использованием комбинации препаратов циклоферона и цитофлавина была выявлена положительная динамика со стороны показателей иммунного статуса в виде снижения уровня относительного процентного значения уровня содержания Т-лимфоцитов - CD-3 при диффузном и осложненном ДМО различных видов, снижение уровня сенсибилизации к АГ-сетчатки при диффузном и осложненном (оклюзионном и неоклюзионном виде) ДМО (p = 0,0001). Относительно показателей гуморального иммунитета наблюдается тенденция к снижению концентрации иммуноглобулинов класса A, G, M при диффузном и осложненном ДМО различных видов. Полученные данные позволяют считать целесообразным и патогенетически обоснованным использование цитофлавина и циклоферона в комплексной терапии у больных СД II типа при диффузном и осложненном (оклюзионном и неоклюзионном) ДМО.
Ключевые слова: диабетический макулярный отек, иммунологический статус, клеточный иммунитет, гуморальный иммунитет.
Summary
Zborovska O.V., Preys N.I. Change of general immune status in type 2 diabetes patients with various types of diffuse and complicated diabetic macular edema after immune correcting therapy.
Diabetic macular edema is the most common cause of impaired vision in patients with type II diabetes. It can accompany any stage of the disease and occurs on average in 10% of diabetes patients. A key element of the DME pathogenesis is the accumulation of fluid in the extracellular space due to imbalance of the blood-retinal barrier. The main principles of treatment of diabetic retinopathy at the contemporary stage include stable compensation of diabetes, normalization of blood pressure and treatment of the affected retina. All the patients underwent general ophthalmic examination, fluorescent angiography, optical coherent tomography and immunological tests. The study involved type 2 diabetes patients with various types of diabetic macular edema and enrolled the total of 64 patients (128 eyes), including 28 females (43.8%) and 36 males (56.3%) aged from 24 to 78 - 58 (SD 10) on average. The study embraced cases of diffuse (occlusive and non-occlusive) and complicated (occlusive and non-occlusive) DME. There were 33 cases of diffuse occlusive DME, 35 cases of diffuse non-occlusive DME, 28 cases of complicated occlusive DME and 32 cases of complicated non-occlusive DME. Statistical analysis was performed in the environment of Statistica 10.0. Treatment with the combination of cycloferon and cytoflavin resulted in a favourable trend on the part of the immune status indicators in the form of lower relative percentage levels of T-lymphocytes CD-3 in diffuse and complicated DME cases, reduced sensitization to S-antigen for diffuse and complicated (occlusive and non-occlusive) DME (p = 0.0001). Regarding humoral immunity indicators, there is a tendency towards reduced concentration of A, G, M immunoglobulins for various types of diffuse and complicated DME. These data suggest that the use of cytoflavin and cycloferon in the treatment of type II diabetes patients with diffuse and complicated (occlusive and non-occlusion) DME is viable and pathogenetically substantiated.
Key words: diabetic macular edema, immune status, cellular immunity, humoral immunity.
Рецензент: д.мед.н., проф. А.М. Петруня

УДК 617.736-005:616.379-008.64-097-092.18

ДУ ‹‹Інститут очних хвороб і тканинної терапії ім. В.П. Філатова НАМН України›› (Одеса)

"Институт глазных болезней и тканевой терапии им. В.П.Филатова НАМН Украины" (Одесса)

SI “The Filatov Institute of Eye Diseases and Tissue Therapy of the National Academy of Medical Sciences of Ukraine”  (Odessa)

¹filatoveye@mail.ru

²nataliya_@bk.ru

Introduction. According to the WHO, the global number of diabetes (D) patients totals over 150 millions. This figure is growing exponentially all over the world with increasing incidence of diabetic complications.

Manifestations of diabetes on the fundus include diabetic retinopathy (DR) and diabetic macular edema (DME). It is DME that is the major cause of visual impairment in patients with type 2 diabetes. DME can develop at any stage of the disease and occurs in 10% of D patients on average. DME frequency correlated with D duration – after 20 years of the disease DME is diagnosed in 28% of the cases. Risk factors of DME development include inadequate glycemic control (high glycohemoglobin levels), diabetes duration, arterial hypertension, dislipidemy and proteinuria [6]. The essential link in DME pathogenesis is accumulation of liquid in the extracellular space of the retina due to the imbalanced blood-retinal barrier (GRB) [2,3,5]. Annually, the number of the patients gains 5-7%, doubles every 12-15 years and is expected to reach 300 millions by 2025. According to the Early Treatment Diabetic Retinopathy Study, macular edema is a serious health problem. In the developed countries it is a prevailing cause of blindness in people aged 20-64 [11].

In case of DR, prognosis for eyesight in patients with t2 D is worsened by DME development, which is diagnosed in 32.8% of cases. Prompt DME treatment allows preservation of high visual acuity and quality of life in 25% of patients [1].

V.F.Ekgardt studied systemic and local immunity by А, М, G immunoglobulins (Ig) levels and С3 and С4 complement components in the blood serum and tears in DR cases. The researcher showed that systemic immunity imbalance is characterized by activated humoral immune response and depressed cellular immune response. Simultaneously with systemic immune disorders at the early stages of diabetic retinopathy, activation of local immunity was detected, which manifested itself by increased levels of A and G immunoglobulins in patients' tears. Further DR development is triggered and promoted by local immune processes in the retina caused by disturbed GRB. The study revealed the activated immune-competent cells to interact with neuroglial and endothelial retinal cells and to produce lots of vasoproliferative factors that favour the growth of newly formed imperfect blood vessels on the retina, glial-fibrous growths and retinal haemorrhages, intensify retinal capillaries occlusion and exert neurotoxic effects.  In case of ischemic maculopathy, capillary occlusion causes deep intra-retinal haemorrhages, retinal edema and retinal ischemia [4].

The main contemporary principles of diabetic retinopathy therapy include stable compensation of diabetes, normalized blood pressure and treatment of the affected retina   [10].

The widely-used method is intra-vitreous administration of prolonged corticosteroid, triamcinolone acetate. Along with sufficient clinical efficacy, this method is accompanied by a high rate of complications, including such serious ones as elevated intraocular pressure and increased risk of cataract and endophthalmitis [12].

Laser coagulation is known to remain one of the leading therapies for treating macular edema. However, in spite of the adequate laser therapy, in certain patients DME keeps progressing. Laser treatment is aimed at eliminating only those manifestations and complications of diabetic maculopathy that are directly behind impaired vision; metabolic and haemocirculatory disorders playing a major role in DME pathogenesis are not corrected [2].

Until recently, focal laser coagulation and grid laser coagulation of the macular were popular DME treatment methods. As it was shown by the Early Treatment Diabetic Retinopathy Study (ETDRS) data published in 1985, laser coagulation enabled a slow-down in vision deterioration in DME patients in the experimental group as compared with the control patients who were simply observed [7,10].

N.V. Pasechnikova forwarded a mathematical model of thermal processes occurring in the tissues of the fundus during thermal coagulation, which is important for better insight into a true nature of laser effect on the retina and choroid. Comprehension of these processes makes it possible to effect a desired influence which is most optimal in every particular case by varying the laser parameters. Visualization of laser treatment allows minimizing the damage caused by laser radiation, thereby lessening long-term consequences and lowering the degree of destructive effect of laser intervention. According to the developed mathematical model, a high-frequency laser impulse train with a certain energy and exposure is more promising than constant impulse of light energy. Using a train of impulses allows decreasing their energy and, consequently, reducing tissue damage [8].

On the other hand, laser coagulation is known to be a destructive treatment method which causes damage to the retinal tissue. Moreover, a number of scientists showed that some types of edema, and first of all diffuse and cystic types are refractory to laser action [9].

Owing to the enhancement of diagnostic techniques, there has been achieved a remarkable progress in managing complications of diabetes. Still, search for drug therapies which would eliminate edema quickly and effectively remains an important task.

Objectives: The study aims to explore the immune status and level of sensitization to retinal antigen (S-antigen) in t2 diabetes patients with various types of diabetic macular edema after the administered therapy and to estimate its efficacy.

Materials and methods: The study involved type 2 diabetes patients with various types of diabetic macular edema and enrolled the total of 64 patients (128 eyes), including 28 females (43.8%) and 36 males (56.3%) aged from 24 to 78 - 58 (SD 10) on average. The average length of type 2 D was 9.3 years (SD 2.8), the minimum length being 6 years and the maximum 19 respectively. Visual acuity before the treatment fell into the range from 0.01 to 0.6, namely from 0.01 to 0.1 in 67 cases (52.3 %); from 0.1 to 0.2 in 24 cases (18.8%); from 0.2 to 0.3 in 18 cases (14.1%); from 0.3 to 0.4 in 10 cases (7.8%); from 0.4 to 0.5 in 6 cases (4.7%); from 0.5 to 0.6 in 3 cases (2.3%).

All the patients underwent a complex ophthalmologic examination, including visometry, refractometry, biomicroophthalmoscopy, optical coherent tomography, fluorescent angiography, as well as immunological tests included in the research plan. The study embraced cases of diffuse (occlusive and non-occlusive) and complicated (occlusive and non-occlusive) DME. There were 33 cases of diffuse occlusive DME, 35 cases of diffuse non-occlusive DME, 28 cases of complicated occlusive DME and 32 cases of complicated non-occlusive DME.

Analysis of OCT data relied on Macular Thickness Tabular protocol. For differentiating DME types, all the patients underwent fluorescent angiography. Immunological tests were conducted at the institute laboratory using monoclonal antibodies СD-3, СD-4 and СD-8, which evaluated phagocytic activity of the neutrophils, А,G,М immunoglobulins level by Mancini's technique and sensitization to retinal AG.

 We proposed using cytoflavin and cycloferon for complex treatment of type 2 D with various types of DME. The patients were divided into two groups – Group I including non-occlusive cases of diffuse and complicated DME and Group II made up of occlusive cases of diffuse and complicated DME. In the complex treatment of DME, Group I patients were prescribed cycloferon, whereas Group II patients were given both cycloferon and cytoflavin.

 Cycloferon is a low-molecular interferon inducer, which underlies its broad spectrum of biological activity – antivirus, immune-modulating, anti-inflammatory, anti-proliferative and antitumor effects.

The drug induces high titres of alpha-, beta-, gamma-interferon in the organs and tissues which contain lymphoid elements (the intestinal mucosa, spleen, liver, lungs) and penetrates the blood-brain barrier.

The immune-modulating effect of cycloferon manifests itself in activation of phagocytosis, natural killer cells, cytotoxic T-lymphocytes and correction of the immune status for immune-deficient conditions of various origins. The course of treatment includes 10 intra-muscular injections according to the base scheme with the single dose of 2.5 g.

 Pharmacological effects of cytoflavin stem from the complex action of substances which form components of the drug, namely 1 ml of the solution comprises succinic acid 100 mg, niacinamide 10 mg, inosine 20 mg, riboflavin mononucleotide (riboflavin) 2 mg and such excipients as N-methylglucamine (meglumine), sodium hydroxide and water for injection. The drug stimulates breathing and energy production in the cells, improves oxygen uptake by the tissues, and restores scavenging enzymes activity. It activates intracellular protein synthesis, promotes disposal of glucose and aliphatic acids and re-synthesis of γ-aminobutyric acid (GABA) in the neurons by GABA shunt.

Cytoflavin improves coronary and cerebral blood circulation and activates metabolic processes in the central nervous system. Due to administration of cytoflavin within the first 12 hours from the stroke, ischemic and necrotic processes in the affected area take a favourable course resulting in the reduced focus. All the occlusive DME patients were administered cytoflavin by intravenous drop infusion of the medication diluted with 100 – 200 ml of 5 – 10% glucose solution or 0.9% sodium chloride solution in the dose of 10 ml q.d. for 10 days.

Both drugs were approved for clinical use in ophthalmologic practice and registered in Ukraine under Ukrainian Ministry of Health Order No758 of 16/11/2006, Registration Certificate  No UA/5449/01/01 for cytoflavin and Ukrainian Ministry of Health Order No85 of 27/02/2006, Registration Certificate No П.05.03/06972 for cycloferon.

Experimental data were processed using Statistica 10.0. Descriptive statistics relied on the arithmetic mean, 95% confidence interval, median and mean square error (± m). The comparative study used single-factor analysis of variance with further application of Scheffe's multiple comparison procedure.

Results and discussion. In all the patients with various types of DME prior to the treatment the clinical examination revealed edema of the macular area, presence of hard and soft exudates as well as of spot and linear hemorrhages in the peripheral retina and along the blood vessel branches. Biomicroscopy showed the optical section of the retina to be non-uniformly enlarged. The recommended drugs were well-tolerated by the patients, causing no allergic reactions, complications or any other adverse effects. After 20-day treatment the patients reported subjective improvement of vision. Objectively, ophthalmoscopy showed reduced macular edema, resolution of the soft exudates and haemorrhages in the peripheral retina.

The treatment by the above-specified combination of cycloferon and cytoflavin resulted in the positive dynamics of the immune status indicators, namely in reduced relative percentage of T-lymphocytes СD-3 for diffuse and complicated DME of various types, which was recorded at (61±0.9)% before the treatment and (59.3±0.8)% after it (p=0.0001) for non-occlusive DME types. The similar tendency was observed for occlusive types of DME: (59.6±1.3)% and (57.8 ±1.1)%, respectively, (p=0.0001) (Fig.1).

Fig.1 Relative percentage of T-lymphocytes СD-3 for diffuse and complicated (occlusive and non-occlusive) DME before and after treatment; 1- diffuse non-occlusive and complicated non-occlusive DME; 2- diffuse occlusive and complicated occlusive DME.

At the same time, the administered therapy effected normalization of immunoglobulin A level in the serum for different DME types, which is shown in Fig.2.  This value was found to be (3.12 ±0.15) g/l before the treatment and (2.95 ±0.2) g/l after it (p=0.0001) for non-occlusive DME cases, and (2.95 ±0.2) g/l and (2.79 ±0.18) g/l (p= 0.005) for occlusive types of complicated and diffuse macular edema, respectively.

Fig.2 Ig A levels for diffuse and complicated (occlusive and non-occlusive) DME before and after treatment; 1- diffuse non-occlusive and complicated non-occlusive DME; 2- diffuse occlusive and complicated occlusive DME.

Reduced autoimmune responses are confirmed by changes in the percentage value of sensitization to S-antigen for diffuse and complicated (occlusive and non-occlusive) DME, which was found to be 8.1 (SD 4.1) before the treatment and 7.3 (SD 1.4) after it for diffuse non-occlusive DME;  up to 10.0 (SD 5.41) and 7.2 (SD 2.0) for diffuse occlusive DME, respectively. In case of complicated occlusive DME, the therapy resulted in the lowering of sensitization to S-antigen from 12.8 (SD 4.19) to 6.5 (SD 1.1), and for complicated occlusive DME from 14.8 (SD 4.6) to 6.4 (SD 0.8) after the imuune correction, respectively, which is a statistically significant change (p=0.0001). After the immune-correcting therapy, degree of sensitization to S-antigen reaches the normal level for all types of diffuse and complicated DME, which is shown in Fig.3.

Fig.3 Percentage of sensitization to S-antigen for diffuse and complicated (occlusive and non-occlusive) DME before and after treatment.

Analysis of immune status dynamics for various DME types reveals reduced absolute and relative number of T-helpers CD-4 and cytotoxic T-lymphocytes CD-8 due to the administered treatment. Owing to the lowered CD-4 levels, a slight increase of immunoregulatory index is observed after the therapy. Relative and absolute phagocytic activity of the neutrophils falls down. Regarding humoral immunity, there is a tendency towards reduced levels of A, G, M immunoglobulins when cycloferon and cytoflavin are included into the comprehensive therapy for various types of diffuse and complicated DME. Table 1 illustrates the dynamics of change of immunological indices in patients with different DME types.

Table 1.

Dynamics of change of immunological indices in type 2 diabetes patients with DME in Group I and Group II; 1- patients with diffuse non-occlusive and complicated non-occlusive DME; 2- patients with diffuse occlusive and complicated occlusive DME

Conclusions

1. Use of cycloferon and cytoflavin in the complex therapy for patients with diffuse and complicated (occlusive and non-occlusive) DME promoted improvement of disease pattern, namely macular edema reduction, faster resolution of haemo- and plasmorrhagias and improved visual acuity as reported by the patients after the administered treatment.
2. Including the proposed drugs into the comprehensive therapy for patients with diffuse and complicated DME (occlusive and non-occlusive types) resulted in the normalized systemic immunity in the form of statistically significant reduction of relative percentage of T-lymphocytes CD-3 (p=0.0001), normalized immunoglobulin A level (p=0.0001) and reduced sensitization to retinal antigen.

The data obtained make it possible to consider use of cycloferon and cytoflavin in the complex therapy for type 2 diabetes patients with diffuse and complicated (occlusive and non-occlusive) DME to be viable and pathogenetically substantiated.

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