Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization.

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.

Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry.

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest.

Prognosis of intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer by immunological urinary measurements: statistically weighted syndrome analysis.

PURPOSE: The goal of this research was to discover new biological indicators in urine which could be used for short-term prognosis of local Bacillus Calmette-Guerin (BCG) therapy outcome in patients with superficial bladder cancer. PATIENTS AND METHODS: We measured and statistically evaluated soluble immunological molecules in urine from bladder cancer patients (n = 34) receiving BCG intravesically. Urine was collected following each of 6 weekly treatments, processed and assayed. The data base included measurements of interleukin-1 (IL-2, IL-4, IL-6, IL-10, IL-12, soluble intercellular adhesion molecule-1 (sICAM-1), tumour necrosis factor-alpha (TNF alpha), soluble CD14 (sCD14), interferon-gamma (IFN gamma), GM-CSF, volume of urine and its pH. The clinical response was evaluated by urine histology and random quadrant biopsy 3 months after the start of therapy. Patients were divided into 2 groups, with good and poor therapeutic effect. The initial complete response rate was 62% (21/34). The data base was analyzed using traditional multivariate statistical methods and a pattern recognition method which deals with combinatorial-statistical analysis (statistically weighted syndromes (SWS) method) of the gradated features. The SWS method is capable of identifying robust patterns in small "fuzzy" sets with high dimensional objects and some missing values. RESULTS: Only one parameter gave significant differences at p < 0.05, GM-CSF at instillation 6. Repeated measurement analysis of variance, backward stepwise multiple logistic regression and linear discriminant analysis failed to show any significance. However, significant differences in the structure of correlation between features in the groups with and without therapeutic effect were observed and four highly informative variables (the masses of sICAM-1, TNF alpha, sCD14 and pH) relating to 5th-6th installations were selected by SWS. These features provided accurate individual prediction of therapeutic outcome for all our patients. Cross-validation analysis and computer simulation showed the statistically significant stability of the prediction. CONCLUSION: We have selected a set of urinary variables that could be considered as a perspective combination of indicators (syndromes) of outcome of pre-operation BCG therapy of patients with superficial bladder cancer. A larger patient database will provide testing and evaluation of the biological and clinical significance of selected features. The computational syndrome-disease approach should be applicable for the solution of decision-making problems for management of cancer.

[Effect of the spread of the process and treatment on the phenotype of peripheral blood lymphocytes in patients with prostatic cancer]. / Vliianie rasprostranennosti protsessa i lecheniia na fenotip limfotsitov perifericheskoi krovi bol'nykh rakom predstatel'noi zhelezy.

A wide panel of monoclonal antibodies to differentiation antigens has been originally used to determine phenotype of peripheral blood lymphocytes in patients with prostatic cancer (PC). Patients with local PC exhibited marked lymphocytopenia existent in vertually all populations and subpopulations. In generalized PC the above changes are more pronounced. Treatment of PC adds to immunodepression observed in this disease. Thus, immunotherapy as an adjuvant modality of PC treatment is quite appropriate.

[Role of immune disorders in the pathogenesis of suppurative inflammatory diseases of the adnexa uteri]. / Rol' immunnykh narushenii v patogeneze gnoinykh vospalitel'nykh zabolevanii pridatkov matki.

Clinical and immunological studies were carried out in 60 patients with tubal pyoinflammatory diseases and in 20 healthy controls of a reproductive age. The immunity status was studied using IKO monoclonal antibodies to differentiation antigens of peripheral blood mononuclears. An acute purulent process was found to be characterized by an adaptive increase in the count of mononuclears carrying the markers of B lymphocytes (CD22, IgM mu-chain), of activated cells (CD38, HLA-D(r)), and of the total number of positive correlations between immunocompetent cells. A persistent course of tubal inflammation is connected with hyperproduction of antibodies and immune complexes, increased expression of HLA-Dr antigens in combination with congenital or acquired insufficiency of T-helper/inductors and suppressors/cytotoxic T-cells. An important factor in the development of a chronic suppurative process is triggering of immunocomplex mechanisms associated with fixation of IgG, IgM, and the complement in the walls of small vessels in the uterine tubes, with a high level of circulating immune complexes, and appearance of plasma cells containing IgG and IgM in the tissues of uterine tubes.

[The effect of indomethacin and leukinferon on the level of blood plasma thromboxane B2, platelet aggregation and the immune system of patients with endometrial cancer in the perioperative period]. / Vliianie indometatsina i leikinferona na uroven' tromboksana B2 v plasme krovi, agregatsiiu trombotsitov i immunnuiu sistemy bol'nykh rakom endometriia v perioperatsionnom periode.

The perioperative influence of leukinferon, indomethacin and their combination on the blood plasma level of thromboxane B2 (TxB2), platelet aggregation ability and humoral and cellular immunity has been assessed in 40 endometrial cancer patients. It has been found that the perioperative use of indomethacin diminishes the blood plasma level of TxB2 and platelet aggregation ability. Leukinferon did not affect substantially the parameters. However, the combination of leukinferon with indomethacin causes a more stable reduction in platelet aggregation and TxB2 level than the use of indomethacin alone. The use of these drugs and their combination prevented postoperative immune suppression in the endometrial cancer patients. However, leukinferon alone or its combination with indomethacin were more effective than the use of indomethacin alone. Possible mechanisms of indomethacin and leukinferon effect on tumor cell metabolism of arachidonic acid and possible role of eicosanoids in the pathogenetic mechanisms of tumor growth and metastasis dissemination are discussed.

[The immune status of patients with lymphoproliferative diseases of the skin and Kaposi's sarcoma]. / Izuchenie immunnogo statusa u bol'nykh limfoproliferativnymi zabolevaniiami kozhi i sarkomoi Kaposhi.

Manifest disorders of cellular and humoral immunity are detectable with the use of immunologic tests in patients with mycosis fungoides, skin reticulosis, and Kaposi's sarcoma. The pattern of changes in Kaposi's sarcoma differs from that in malignant lymphomas of the skin. The detected immune status disorders necessitate the use of immunomodulators in combined therapy of such patients.

[The effect of therapy on the immunological indices in patients with skin lymphomas and Kaposi's sarcoma]. / Vliianie terapii na immunologicheskie pokazateli u bol'nykh limfomami kozhi i sarkomoi Kaposhi.

Manifest changes in the immunity status are detected in lymphoproliferative diseases of the skin and in Kaposi's sarcoma. Therapy results in elevation of the initially lowered levels of T mu- and T gamma-cells, this being a favorable result. Scintillation of T mu- and T gamma-cells may be used to monitor the efficacy of therapy in such patients.

[Disorders of immune homeostasis in patients with tumors of the gastrointestinal tract]. / Narushenie immunnogo gomeostaza u bol'nykh s opukholiami zheludochno-kishechnogo trakta.

To study immune homeostasis status and to improve the diagnostic value of immunological tests in healthy donors and patients with benign and malignant gastrointestinal tumors, 13 clinico-immunological parameters were identified and measured simultaneously in most of the examinees. A new method of analysis of clinico-immunological information based on stage-by-stage visual evaluation of data shown on dispersion programs and identification of areas corresponding to "normal" immune homeostasis is suggested. Data obtained simultaneously from blastogenic reaction of lymphocytes, cytotoxic test for levels of natural killers in blood and tests for determining subpopulational levels of T-lymphocytes were shown to be diagnostically valuable.