Features of effector lymphocyte subsets in patients with uveal melanoma in recurrent and chronic herpesvirus infection

The aim of the study is to conduct a comparative analysis of percentages for peripheral blood effector lymphocyte subsets in patients with uveal melanoma manifested by recurrent and chronic herpesvirus infection. There were 141 subjects enrolled: 70 patients with uveal melanoma, 38 patients with corneal ulcers and involvement of the uveal tract as well as 33 healthy donors. Immunophenotyping was performed by using laser flow cytometry with panel of monoclonal antibodies to differentiate lymphocyte subpopulations. IgM and IgG antibodies to herpesvirus infections were determined by using enzyme-linked immunosorbent assay on an automatic ELISA analyzer Lazurit (USA) with diagnostic kits of CJSC “Vector-Best” (Koltsovo). The data obtained showed that the absolute number of blood lymphocytes (CD45+) in patients with uveal melanoma did not differ from those in healthy donors. In contrast, patients with corneal ulcers and involvement of the uveal tract had this parameter increased. A decreased relative and absolute count of T cells (CD3+) in uveal melanoma, but increased absolute CD3+ number in inflammation was observed. No difference in relative and absolute content of the CD3+CD4+ helper/inducer subpopulation in patients with recurrent herpesvirus infections was found. Corneal ulcers in cancer patients revealed significantly increased absolute level of CD3+CD4+ helpers/inductor cells. Chronic herpesvirus infection in uveal melanoma patients showed increased relative and absolute number of cytotoxic T lymphocytes (CD3+CD8+). Recurrent herpesvirus infection was featured with decreased relative number of T lymphocytes (CD3+CD8+), upon inflammation, there was noted increased absolute and decreased relative number compared with healthy subjects. Double positive T cells increased in tumor and inflammation. B lymphocytes (CD19+) increased in melanoma and inflammation. The relative number of blood natural killer cells (CD16+CD56+) in uveal melanoma increased upon recurrent infection. Inflammation was coupled to decreased relative level of natural killer cells (CD16+CD56+). Melanoma showed no changes in CD4+/ CD8+ ratio; upon inflammation, its increase was noted in acute and chronic herpesvirus infections (p < 0.05). The suppression of the immune system in uveal melanoma, restricting antiviral defense, was revealed. The data obtained seem to be important for development of personalized approaches to prognosis and treatment of patients with uveal melanoma

Features of systemic cytokine production in Behcet's disease associated with uveitis without ocular lesions

Behcet's disease (BD) is a systemic disease underlyed by chronic vasculitis. Hyperactivity of innate and adaptive immunity plays important role in its pathogenesis. Uveitis occurs in 30-70% of the patients, often recurring and reducing visual function. The objective of our work was to study the features of systemic production of immune mediators in BD patients, depending on presence and activity of uveitis. 116 BD patients were divided into 3 groups: (1) 41 patients with active uveitis (UA), (2) 64 subjects with uveitis remission (UR), (3) 11 uveitis-free BD patients (WU). Control group (CG) comprised 34 conditionally healthy people. Detection rate (%) and contents (pg/ml) were measured for IL-1β IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-13, IL-18, IFNγ, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin, СXCL1/GRO-α, CXCL8/IL-8, CXCL10/IP-10, CXCL12/SDF-1α, GM-CSF, TNFα in blood serum by means of multiplex analysis using MAGPIX analyzer (Luminex Corp., USA), Procarta Plex “Human Th1/Th2&Chemokine Panel 20 plex” kits (Bioscience, Austria). TGF-P1, TGF-P2 levels were assayed by ELISA-test (“Vfector-Best”). All the BD patients showed high detection rates of CXCL1/GRO-α (but not its level) in comparison with CG. Detection rate and levels of IL-6, IL-8 were increased in 1st and 2nd BD groups, compared to CG. In UR, unlike UA and WU groups, IL-4 was detected more often than in CG. WU patients showed increased detection rate of only CXCL1/GRO -α. When compared with UA, WU patients had lower serum concentrations of IFNγ, MCP-1, IP-10, MIP-1a, SDF-1α, TGF-β1; UR patients also showed decreased serum levels of IL-18, Eotaxin, GRO-α, RANTES, TGF-β2. Our results indicate the importance of angiogenic and proinflammatory chemokines and cytokines in pathogenesis of BD uveitis, as well as imbalanced production of various immunomediators. Higher detection rates and levels of IL-6 and IL-8 in UA and UR patients may result from weak persistent intraocular inflammation, even upon relief of clinical symptoms, thus, probably, requiring therapeutic correction

EFFECT OF HUMAN HERPES VIRUS REACTIVATION ON SYSTEMIC CYTOKINE PRODUCTION IN PATIENTS WITH BEHCET’S DISEASE AND UVEITIS

Behcet’s disease (BD) is a systemic autoinflammatory-autoimmune disease (chronic systemic vasculitis) of unknown etiology, almost 70% of patients develop uveitis. BD pathogenesis is complex, human herpesviruses (HHV) play an important role among infectious trigger factors. Ability of herpesviruses to modulate cytokine production and evade host’s immune response is known.Aim of the study was to assess the effect of Herpes simplex virus type 1, Herpes simplex virus type 2, Cytomegalovirus, Epstein-Barr virus on systemic levels of chemokines, pro- and anti-inflammatory cytokines in BD with and without uveitis. Serum samples were collected from 116 BD patients chronically infected with HHV and examined in ELISA-test for markers of HHV reactivation (IgG-antibodies to immediate early HSV antigens 1, 2 and CMV, early EBV antigen). Concentration of IL-1β, IFNγ, MCP-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12p70, IL-13, IL-18, TNFα, GMCSF, Eotaxin, GRO-α, IP-10, MIP-1α, MIP-1β, SDF-1α, RANTES detected in multiplex analysis. TGF-β1, TGF-β2 were measured in ELISA-test. Depending on presence and activity of uveitis 3 groups of patients with BD were identified: group 1 – active uveitis, group 2 – remission of uveitis, group 3 – BD without ocular manifestations. After serological study 2 subgroups were highlighted in each group: a) patients with antibody markers of reactivation of at least one HHV, b) patients chronically infected with HHV, without serological signs of reactivation. Mean level and detection rate of cytokines and chemokines in patients with active uveitis (1a, 1b) and in remission (2a, 2b) were compared with patients without eye damage (3a, 3b). Chronic HHV infection (subgroup “b”) was compared with reactivation (subgroup “a”). A significant increase of MCP-1/ CCL2, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, IP-10, SDF-1α chemokines in serum, as well as IFNγ, TGF-β1, and TGF-β2 was observed in patients with uveitis (regardless of their activity) and HHV reactivation compared to patients without uveitis. Our data indicate that systemic production of cytokines and chemokines in BD patients and uveitis could be affected by the activity of chronic herpesvirus infections, and the greatest changes are related to chemokines

Применение ингибиторов фактора некроза опухоли α у женщин с анкилозирующим спондилитом во время беременности

Objective: to present our own experience of tumor necrosis factor α (TNFα) inhibitors (iTNFα) usage during pregnancy in women with ankylosing spondylitis (AS), to assess AS activity and outcomes of gestation.Patients and methods. A prospective observation of 55 pregnant women with AS who met the modified New York criteria of 1984. Fifty-six pregnancies were followed. The average age of the patients was 31.7±4.7 years, the duration of the disease was 132.2±85.4 months. The median BASDAI for pregnancy trimesters was 2.4 [1.2; 4.4], 2.7 [1.4; 4.2] and 2.2 [1.5; 4.0], respectively. 14 women received iTNFα 3 months before pregnancy.Results and discussion. In the first trimester, TNFα was used in 9 (16.1%) patients, in the second – in 9 (16.1%) and in the third – in 5 (9.3%); the median BASDAI for trimesters was 2.3 [1.0; 3.7], 3.4 [1.2; 3.5], 3.0 [0.8; 3.4], respectively. All patients who discontinued iTNFα just before or in early pregnancy had indications for resuming therapy in the second half of gestation. Cancellation of iTNFα at the end of the second trimester was not a risk factor for high activity in the third trimester. There was 1 adverse pregnancy outcome. In other cases, childbirth occurred at 38.9±1.4 weeks, newborns' body weight was 3273.1±435.6 g.Conclusion. Women with AS who plan a pregnancy should be prescribed drugs with the maximum allowed duration of use during gestation. Cancellation of iTNFα before and in early pregnancy is a risk factor for high AS activity, while renewal of iTNFα therapy during pregnancy is not always effective.Цель исследования – представить собственный опыт применения ингибиторов фактора некроза опухоли (иФНОα) во время беременности у женщин с анкилозирующим спондилитом (АС), оценить активность АС и исходы гестации.Пациенты и методы. Проведено проспективное наблюдение 55 беременных с АС, соответствовавших модифицированным Нью-Йоркским критериям 1984 г. Прослежено 56 беременностей. Средний возраст пациенток составлял 31,7±4,7 года, продолжительность болезни – 132,2±85,4 мес. Медиана BASDAI по триместрам беременности – 2,4 [1,2; 4,4], 2,7 [1,4; 4,2] и 2,2 [1,5; 4,0] соответственно. ИФНОα за 3 мес до наступления беременности получали 14 женщин.Результаты и обсуждение. В I триместре иФНОα применяли 9 (16,1%) пациенток, во II – 9 (16,1%) и в III – 5 (9,3%); медиана BASDAI по триместрам составляла 2,3 [1,0; 3,7], 3,4 [1,2; 3,5], 3,0 [0,8; 3,4] соответственно. Все пациентки, отменившие иФНОα накануне или на ранних сроках беременности, имели показания к возобновлению терапии во второй половине гестации. Отмена иФНОα в конце II триместра не являлась фактором риска высокой активности в III триместре. Имел место 1 неблагоприятный исход беременности. В остальных случаях роды произошли на сроке 38,9±1,4 нед, масса тела новорожденных – 3273,1±435,6 г.Заключение. Женщинам с АС, планирующим беременность, необходимо назначать препараты с максимально разрешенным сроком применения во время гестации. Отмена иФНОα перед беременностью и на ее ранних сроках – фактор риска высокой активности АС, при этом возобновление терапии иФНОα во время беременности не всегда эффективно

Боль в спине и оценка активности анкилозирующего спондилита на фоне гестации: симптомы, отражающие обострение заболевания, и беременность

Objective: to study the dynamics of back pain severity and the frequency of its individual components, characterizing the inflammatory and mechanical rhythm, on the background of gestation in women with ankylosing spondylitis (AS) and compare them with manifestations of dorsopathy in healthy pregnant women; to determine the information value of BASDAI during pregnancy, i.e. to identify its components with the optimal sensitivity and specificity ratio for assessing AS activity during gestation.Patients and methods. The main group consisted of 49 pregnant women with AS who met the modified New York criteria of 1984. The average age of patients was 31.7±4.9 years, the average duration of the disease was 134.4±85.8 months. BASDAI for trimesters of pregnancy was: 2.3 [1.2; 4.4], 2.8 [1.4; 4.2] and 2.2 [1.6; 4.0]. The control group included 51 pregnant women with back pain associated with gestation, without rheumatic diseases. The average age was 28.0±4.4 years. The intensity of back pain was assessed on a numerical rating scale. ROC-analysis was performed with calculation of the area under the curve (AUC) of each component of BASDAI in each pregnancy trimester.Results and discussion. More than 80% of pregnant women with AS experienced back pain, while the intensity of general back pain did not differ from that in the control group. Night back pain with improvement on awakening in the main group was present in 70%, 58% and 68.8% of women, respectively, in the I-III trimesters; its intensity was 3 [1; 5], 3.5 [3; 6] and 3.4 [2; 5] respectively; the frequency and severity of night pain were higher than in the control group. The number of women with improvement after exercise in the II and III trimesters did not differ in both groups. In the second half of pregnancy, 40% of AS patients noted improvement during rest, 52.1% – increased pain after exercise; the frequency of mechanical rhythm pain elements remained lower than in the control group. The AUC value for BASDAI in the first trimester was 0.74; AUC of all BASDAI components was >0.5. In the II and III trimesters, the AUC values for fatigue and back pain were 0.8.Conclusion. During pregnancy the vast majority of women with AS experience back pain, the nature of which changes in the second half of gestation. Night pain that improves on awakening reflects AS activity and is not related to pregnancy. The BASDAI components of severity and duration of morning stiffness have the highest classification value during pregnancy. Цель исследования – изучить динамику выраженности боли в спине и частоту ее отдельных компонентов, характеризующих воспалительный и механический ритм, на фоне гестации у женщин с анкилозирующим спондилитом (АС) и сравнить их с проявлениями дорсопатии у здоровых беременных; определить информативность BASDAI во время беременности, т. е. выявить его компоненты с оптимальным соотношением чувствительности и специфичности для оценки активности АС при гестации.Пациенты и методы. Основную группу составили 49 беременных с АС, соответствовавших модифицированным Нью-Йоркским критериям 1984 г. Средний возраст пациенток – 31,7±4,9 года, средняя продолжительность болезни – 134,4±85,8 мес. BASDAI по триместрам беременности равнялся: 2,3 [1,2; 4,4], 2,8 [1,4; 4,2] и 2,2 [1,6; 4,0]. В контрольную группу вошла 51 беременная с болью в спине, связанной с гестацией, без ревматических заболеваний. Средний возраст – 28,0±4,4 года. Интенсивность боли в спине оценивали по числовой рейтинговой шкале. Выполнен ROC-анализ с вычислением площади под кривой (AUC) каждого компонента BASDAI в каждом триместре беременности.Результаты и обсуждение. Более 80% беременных с АС испытывали боль в спине, при этом интенсивность общей боли в спине не отличалась от таковой в контрольной группе. Ночная боль в спине с улучшением при пробуждении в основной группе имелась у 70, 58 и 68,8% женщин соответственно в I–III триместрах; ее интенсивность составила 3 [1; 5], 3,5 [3; 6] и 3,4 [2; 5] соответственно; частота и выраженность ночной боли были выше, чем в контрольной группе. Количество женщин с улучшением после выполнения физических упражнений во II и III триместрах не различалось в обеих группах. Во второй половине беременности 40% больных АС отметили улучшение во время отдыха, 52,1% – усиление боли после физических упражнений; частота элементов боли механического ритма оставалась ниже, чем в контрольной группе. Значение AUC для BASDAI в I триместре составляло 0,74; AUC всех компонентов BASDAI была >0,5. Во II и III триместрах значения AUC для утомляемости и боли в спине оказались 0,8.Заключение. Подавляющее большинство женщин с АС во время беременности испытывают боль в спине, характер которой меняется во второй половине гестации. Ночная боль с улучшением при пробуждении отражает активность АС и не связана с беременностью. Наиболее высокой классификационной ценностью при беременности обладают такие компоненты BASDAI, как выраженность и длительность утренней скованности

The Effectiveness of PCR in Diagnosis of Fungal Keratitis

Fungi implicated in mycotic keratitis include different species. Conventional methods for the diagnosis of fungal keratitis include staining of corneal scarpings, culture medium (Sabouraud agar) for isolating fungi.Purpose. To evaluate the effectiveness of polymerase chain reaction (PCR) for the detection of fungal etiology in comparison with the conventional diagnostic methods in cases with suspected fungal corneal ulcer.Patients and methods. Seven patients with severe corneal ulcers with more than 3 weeks duration. Corneal scarpings and corneal buttons from seven patients who had undergone therapeutic keratoplasty were used for microbiological and PCR analysis. PCR diagnostic kits for the differential detection of Candida albicans DNA and total fungi DNA (DNA Fungi), which allows to identify most pathogenic fungi without determining their species were used. Microbiological methods: microscopy of gramstained smears, culture techniques, including selective for fungi agar Saburo with chloramphenicol.Results. PCR: Fragments of all corneas removed from keratoplasty (6 patients) revealed fungal-common DNA (Fungi DNA) and did not detect Candida albicans DNA, which correlated with sowing results on Saburo medium (mold fungi found in 5 of 6 corneas). Fungi DNA was also detected in the corneal scraps taken prior to surgery; however, growth of fungi during sowing on various nutrient media was not found.Conclusion. Corneal fungal ulcers are a serious disease, often leading to visual disability. The rapid determination of etiology and the correct choice of therapy determines the outcomes of the disease. The advantage of PCR over the culture method: the speed of obtaining results (4 hours instead of 3–7 days); high sensitivity, which allows detecting fungi not only in the tissue of the removed cornea, but also in scrapes from the cornea ulcer of patients who previously received antifungal therapy. The presence of commercial kits for differential detection of fungal-common DNA and DNA of Candida albicans extends the possibilities of PCR in the screening diagnosis of fungal keratitis and the selection of drugs before determining the type of pathogen

The Effectiveness of Laboratory Methods for the Early Differential Diagnosis of Ophthalmohlamidiosis and Conjunctival Lymphoma (Clinical Cases)

Conjunctival lymphomas are predominantly extranodal B-cell non-Hodgkin’s lymphomas (NHL), most of them are MALT-lymphomas originating from the mucosa-associated lymphoid tissue. Manifestations of the conjunctival NHL are very diverse, and often imitate the appearance of other ocular diseases, which makes their clinical diagnosis difficult and significantly lengthens the time from the first visit to the ophthalmologist until the diagnosis is verified. The article presents the case histories of two young patients who were treated for a long time at the place of residence as acute chlamydial conjunctivitis (in one case — 3, in the other — almost 5 months). A comprehensive laboratory study to detect specific blood antibodies, DNA of the pathogen in conjunctival scrapings and the cytological picture of the conjunctiva did not confirm the chlamydial etiology of the process. A pronounced lymphoid reaction mainly due to small lymphoid cells was found in both patients in scrapings from the conjunctiva of the eyelid, which was the basis for referring patients for consultation to oncologist. Histological examination of conjunctival biopsy specimens also revealed proliferation of lymphoid tissue: patients with suspected MALT lymphoma were referred to onco-hematologists. A PCR analysis of the biopsy material revealed HHV-8 DNA in one patient and Epstein-Barr (EBV) DNA in another, although no pathogen genomes were detected in the conjunctiva scrapings. Immunohistochemical analysis in one patient confirmed the conjunctival MALT-lymphoma, in another one diagnosed hyperplasia of the conjunctival mucous-associated lymphoid tissue, caused by prolonged antigenic stimulation (EBV DNA was detected in the biopsy). The follicular appearance of the lymphocyte hyperplasia in conjunctiva may imitate the clinical picture of infectious diseases. For differential diagnosis of chlamydial conjunctivitis and MALTlymphoma, especially in young patients with refractory follicular conjunctivitis, it is advisable to include a set of serological, molecular biology and cytological methods