[Fecal microbiota transplantation for graft-versus-host disease in children and adults: methods, clinical effects, safety].

AIM: Was to evaluate clinical efficacy, adverse events and changes in the gut microbiome after fecal microbiota transplantation (FMT) in patients with gastrointestinal (GI) form of graft-versus-host disease (GVHD). MATERIALS AND METHODS: The prospective single-center study in R.M. Gorbacheva institute included 27 patients with GI GVHD after allogeneic stem cell transplantation. 19 patients received FMT, 8 patients received placebo. Clinical scales for GI autoimmune diseases were used to evaluate response. Microbiome alterations were assessed with multiplex PCR. RESULTS: After FMT higher overall bacterial mass (р=0.00088), higher bacterial numbers ofBifidobacteriumspp. (р=0.021),Escherichia coli(р=0.049) andBacteroides fragilisgr. (р=0.000043) compared to placebo group. Also higher bacterial mass was observed in patients with clinical response (р=0.0057). The bacterial mass after procedure in non-responders was compared to the placebo group (р=0.31). Partial response of GVHD was achieved faster in the FMT group compared to placebo (median 4 days vs 48 days,p=0.014). Complete response was observed in 8 (42%), 14 (74%) and 16 (84%) at 30, 60 and 90 days respectively, while in the placebo group only 0%, 1 (13%) and 4 (50%) achieved complete response at the same time points. The incidence and severity of adverse events was comparable between FMT and the placebo group. CONCLUSION: FMT in patients with refractory GI GVHD was associated with favorable clinical outcomes and recovery in certain marker bacterial populations. Multiplex PCR can be used to assess an engraftment of a donor microbiota. FMT in GI GVHD was not associated with life-threatening adverse events, but further studies are required to validate clinical efficacy.

[The diagnostic role of chemokines and their receptors in chronic hepatitis C].

The chronic hepatitis C is characterized by the increase of inflammatory disorders and progression of fibrosis of liver The corresponding immunologic mechanisms of hepatic lesions are still undiscovered. The actual review presents the analysis of scientific publications and genuine research data concerning the role of chemokines in pathogenesis of chronic hepatitis C. The chemokines are small cationic proteins enhancing transit and precipitation of migrating cells (leucocytes mainly) in tissues and organs. The significant role of chemokines in tissue homeostasis, in case of inflammation, wound healing and cell proliferation is demonstrated. The particular kinds of chemokines are produced by different types of cells and impact target cells through their specific receptors. According the data of various studies, chemokines and chemokine receptors of CC-families and CXC-families are involved in fibrosing processes and anti-inflammatory activation of hepatic-biliary system under chronic hepatitis C. The diversity of producers and targets of chemokines in liver is very pronounced: hepatocytes, stellar cells, endothelium cells, macrophages (Kupffer cells), dendritic cells, lymphocytes and monocytes. The review considers pathogenesis of chronic hepatitis C from the standpoint of participation of chemokines and chemokine receptors at different stages of cellular transit. The most important cellpopulations involved into pathologic changes under chronic hepatitis C are characterized. The decrease of expression of such gens as CCR1, CCR2, CCR3, and CCR5 in blood leucocytes deserves additional studies to establish their diagnostic values as a marker of disorders of immune system in patients with chronic hepatitis C.

[Clinical significance of functional variants of matrix metalloproteinase genes in thyroid cancer].

Under analysis were results of genetic investigations of 105 patients with thyroid cancer (TC) treated from 2004 through 2007 in the St. Petersburg City Center of surgery and oncology of the endocrine system organs. The patients' age was from 17 to 80 years (mean age 51.6 +/- 1.9 years). The influence of functional variants of matrix metalloproteinase genes in TC was determined. A reliable correlation was noted between certain gene markers and TC patients' age. An association was revealed between matrix metalloproteinase-3 5A (more active allele) and size of the tumor. A reliably decreased frequency of hyperactive genotype MMP-1 2G was detected in a group of women with metastatic forms of papillary cancer as compared with patients without metastases. It was shown that MMP genes could be a substantial factor of slowing down the rate of malignant growth and invasive properties of cancer of the thyroid gland.

Expression of mRNA for chemokines and chemokine receptors in tissues of the myometrium and uterine leiomyoma.

Tissues samples of leiomyoma and myometrium obtained intraoperatively were analyzed. For evaluation of the synthesis of MIP-lalpha, MIP-1beta, RANTES, eotaxin, eotaxin-2, interleukin-8, CCR1, CCR3, CCR5, CXCR1, and CXCR2, mRNA isolated from tissues samples of leiomyoma and myometrium was subjected to reverse transcription-PCR and assayed by a semiquantitative method (relative to beta-actin). The content of eotaxin, MIP-1alpha, MIP-1beta, and CCR5 mRNA in leiomyoma tissue was lower than in the myometrium. The concentration of MIP-1beta, CCR5, and eotaxin mRNA in common leiomyoma was much lower than in the myometrium. Eotaxin mRNA expression in myometrial tissue of patients with single nodes was much higher than in those with multiple nodes. Moreover, expression of eotaxin mRNA in common leiomyoma was higher than in proliferating leiomyoma. The concentration of mRNA for interleukin-8 in leiomyoma tissue, as well as the content of mRNA for MIP-1alpha and CCR3 in myometrial tissue increased in patients with submucosal nodes (as distinct from nodes of another location). A direct correlation was revealed between the size of the uterus and concentration of mRNA for interleukin-8 and MIP-1beta in myometrial tissue. The concentration of mRNA for MIP-1alpha and MIP-1beta in leiomyoma tissue negatively correlated with the size of the uterus (maximum size of the node) and duration of leiomyoma, respectively. Our results indicate that chemokines play an important role in the pathogenesis of uterine leiomyoma.

[Detection of infectious agents in heart valves during endocarditis using PCR technique].

Infectious endocarditis can be caused by various microorganisms. Diagnostics of local infection by microbiological methods is not always effective. For that reason we performed a study aimed for direct detection of potential infectious agents by polymerase chain reaction in patients' heart valve tissue. DNA of infectious agents was revealed in 72% of heart valve tissue samples from patients with septic endocarditis; in studied samples, along with bacterial DNA, herpesviruses' DNA was detected. Obtained results confirm the presence of infection, which allows to perform specific diagnostics of infectious complications after implantation of prosthetic cardiac valves.

[PCR detection of periodontal bacteria and Streptococcus mutans in the biological samples from the oral cavity].

The paper describes an optimized technique for DNA isolation from the Streptococcus mutans cultures and from the clinical samples including proteinase K treatment and detergent lysis, followed by sorbent-based enrichment. This technique was employed for isolating DNA from the periodontal bacteria Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Bacteroides forsythus. A multiplex PCR technique was adapted for large-scale studies of clinical samples. An original PCR method was developed for the semiquantitative detection of Str. mutans, showing the sensitivity of 100 genome copies per reaction. Extensive studies of approximately 2,000 individuals have demonstrated that these methods are applicable to the study of the dental and gingival microflora in the biological sample from the oral cavity of persons with dental diseases from various age groups.

Bacterial markers of periodontal diseases and their practical significance in dentistry.

The incidence and prevalence of Actinobacillus actinomycetemcomitans, Porphyronmonas gingivalis, and Tannerella forsythensis in specimens of subgingival dental deposit were evaluated in 495 residents of St. Petersburg aged 6-82 years. The microorganisms were detected by gene-specific PCR of 16S rDNA. In accordance with age-specific increase in the incidence of gingival diseases, the percentage of samples containing T. forsythensis and P. gingivalis was significantly higher in adult and elderly patients in comparison with adolescents. The presence of T. forsythensis significantly correlated with the presence of gingivitis and dental deposit. In addition, the incidence of T. forsythensis was significantly higher in tobacco smokers. These results attest to a relationship between T. forsythensis infection and more frequent periodontal diseases associated with aging and tobacco smoking.

[The combained effects of the long-term gamma-irradiation and heavy metall ions on the haematopoietic system of rats].

The combiened effects of different dose rates (0.625 microGy/s - 1.1 mGy/s) of gamma-irradiation and of cuprum and of cadmium ions on the haematopoietic system of rats were studied. It was found that only low dose rates (0.625-10 microGy/s, summary doses 0.5-2.0 Gy) of gamma-irradiation yields in the increasing proliferative activity of bone marrow. The number of myelocariocytos in S-phase was increased at 1.5-1.8 times. In case of the treatment with both cadmium chloride and radiation the changes in proliferative activity of bone marrow are completely due to the radiation factor. Combination of cuprum acetate and ionizing radiation induce opposite effects providing formal normalization of the haematopoietic characteristic of bone marrow up to 3, 6 and 12 months after the end of the radiation and the chemical exposure of the animal.

[Oncologic risk factors in papillomavirus infection]. / Faktory onkologicheskogo riska pri papillomavirusnoi infektsii.

Cytological screening is essential for adequate diagnosis of gynecological cancer. Moreover, every effort should be made to identify such risks of cancer as viruses (HPV, Herpes simplex, cytomegalovirus, Epstein-Barr virus, etc.). When high risk HPV-16 and HPV-18 are detected, diagnostic strategies should include assay of blood prolactin and prolactin/TTH ratio. Virus-related tumors and various risk groups may be studied using HPV genotyping detection of high-risk alleles of certain genes. Hence, some HPV gene variants and gene polymorphism can be investigated as potential risk factors in women.

[Increased apoptosis of peripheral blood leukocytes correlates with leukopenia after high-dose chemotherapy]. / Usilinie apoptoza leikotsitov perifericheskoi krovi v sviazi s razvitiem leikopenii posle intensivnoi khimioterapii.

Time-related changes in apoptotic leukocyte numbers were studied in peripheral blood sampled from 12 cancer patients before and after myeloablative chemotherapy (MC); six of them received hemopoietic cell grafts. Apoptotic leukocytes were counted in fresh and ex vivo incubated blood samples. Nuclear chromatin autolysis was registered in supravital Acridin Orange-stained granulocytes and lymphocytes. The levels of apoptosis in fresh peripheral blood were under 1.5%. Incubation for another 3 h revealed considerable numbers of apoptosis-prone leukocytes both in untreated patients and healthy donors. High-dose chemotherapy with busulphan, cyclophosphamide or alkeran was followed by a sharp increase in apoptotic cell counts which peaked on days 7-9, before leukopenia started on days 13-15 at the earliest. It is suggested that apoptosis of mature leukocytes is a major cause of leukopenia development induced by chemotherapy. The simple technique of apoptosis-prone cell detection in whole blood may be effectively used in serial screening of hematological side-effects of chemotherapy.

[A trial of using allogeneic bone marrow transplantation in children with different hematologic neoplastic diseases]. / Opyt primeneniia allogennoi transplantatsii kostnogo mozga u detei s razlichnymi onkogematologicheskimi zabolevaniiami.

AIM: To define optimal time for transplantation of bone marrow (TBM) in children with hematological malignancies. MATERIALS AND METHODS: 20 allogenic TBMs were performed in children with acute myeloblastic leukemia (6 patients, 2 of them in recurrence), acute lymphoblastic leukemia (7 patients, 4 of them in recurrence), chronic myeloid leukemia (CML) in a chronic stage (3 patients), severe aplastic anemia (3 patients), generalized neuroblastoma (1 patient). Pretransplantation preparation included cyclophosphamide and busulphane or cyclophosphamide, busulphane and vepezide. The graft-versus-host reaction (GVHR) was prevented with cyclosporin A plus methotrexate or cyclosporin A plus urbazone. Engrafting was recognized by change of karyotype and blood group. RESULTS: From 13 children with acute leukemia subjected to TBM in a complete remission 4(33%) are alive, 5 died within 100 days after TBM (TBM was made in recurrence in 4 children), 3 patients died of recurrence 12 months after TBM. One patient with CML and one with severe aplastic anemia remain in remission. The main complications and causes of death in early posttransplantation period were hemorrhagic syndrome, infectious complications, GVHR. According to a one-year follow-up, the recurrent disease caused death most frequently. CONCLUSION: Positive result of TBM is related to the disease stage at transplantation.

[Cerebrospinal fluid sorption in patients with heroin addiction]. / Likvorosorbtsiia u bol'nykh geroinovoi narkomaniei.

The sorption of cerebrospinal fluid (CSF) was attempted as a special detoxifying procedure in a group of sixty heroine addicts. CSF contents of cells, total protein, nucleic acids and interleukin-1 (IL-1), as well as acridine orange (AO) binding to CSF cells were determined before and after the procedure. The treatment provided immediate clinical improvement for 70% of the patients. Clinical effect was accompanied by decreased of CSF cells, diminished nucleic acids and protein amounts, along with increased DNA-AO binding and accumulation of IL-l. These data are interpreted in terms of intensive apoptosis of CSF cells and increased acute phase of aseptic inflammation-like events induced by the procedure of liquor sorption.

Ultraviolet irradiation induces multiple DNA double-strand breaks and apoptosis in normal granulocytes and chronic myeloid leukaemia blasts.

Major leucocyte subpopulations were isolated from peripheral blood of healthy donors, and patients with chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL). In vitro UV irradiation was performed at the wavelength of 257nm (UVC band). DNA double-stranded breaks (DNAdsbs) were detected immediately after UV-irradiation, by means of agarose gel electrophoresis. Cell viability was estimated after 18h in culture, as relative numbers of residual non-apoptotic cells. Evaluation of the dose-response curves revealed that normal CLL lymphoid cells showed only moderate damage after UV-irradiation, as assessed by DNAdsbs and cell viability criteria. However, normal granulocytes and myeloid blasts from CML patients expressed a sharp increase in DNAdsbs, even at lower doses of UV-radiation. UV-induced amplification of endogenous oxidative systems (e.g. NADPH-dependent oxidase) is suggested as a probable reason for enhanced DNA breakage and apoptosis in cells of the granulocytic lineage.

Early post-radiation changes of red blood cell shape in rats.

Scanning electron microscopy (SEM) of red blood cells in whole blood samples from rats was performed following acute gamma-irradiation of animals with 0.25 to 1 Gy. Increased incidence of echinocytosis was observed and found to be dose- and time-dependent. At a higher radiation dose (1 Gy), echinocytosis was revealed within 5 minutes after treatment and persisted up to 3 weeks. The data demonstrate the applicability of SEM for detecting minimal radiation-induced lesions of red blood cells.

Apoptosis and red blood cell echinocytosis: common features.

Apoptosis of nucleated blood cells induced by oxidants and/or reactive oxygen species is accompanied by the typical membrane pathology. Meanwhile, red blood cell (RBC) membrane is a popular object for studying appropriate cytotoxic effects. Scanning electron microscopy provides a reliable tool for detecting the oxidative changes in RBC shape and size. Transition of normal discoid erythrocytes to crenated forms (echinocytes) is often induced by the same factors which cause apoptosis of blood cells, e.g., ionizing radiation and other reactive oxygen intermediate-inducing agents, exogenous oxidants, in vitro aging conditions, cytosolic calcium increase, etc. Moreover, the biochemical membrane alterations in oxidant-induced echinocytosis is strongly reminiscent of the changes associated with apoptosis, e.g., cell shrinkage, lipid oxidation, energy depletion and loss of transmembrane lipid asymmetry. Hence, characteristic changes in cell shape in oxidant-treated RBCs are of value for interpreting the membrane alterations occurring in leukocyte apoptosis.