Biological microchip for a simultaneous quantitative immunoassay of tumor markers in human serum.

The biochip was constructed for simultaneous assay of total and free prostate-specific antigen, alpha-fetoprotein, cancer embryonic antigen, human chorionic gonadotropin, and neuron-specific enolase. These biochips represent an array of gel elements with covalently immobilized proteins. The major analytic characteristics of the developed method were obtained. It was shown that the results of simultaneous assay of six tumor markers in blood serum well correlated with routine measurement of each marker using enzyme immunoassay kits. This approach allowed us to reveal the hook effect of high concentrations during biochip assay, which prevents distortion of the diagnostic picture at high concentration of the analyte in the sample.

[Aseptic meningitis season caused by enteroviruses in 2007: molecular-epidemiological aspects].

AIM: Analysis of manifestation of epidemic process of aseptic meningitis and causes of its activation using molecular genetic methods. MATERIALS AND METHODS: Samples of feces and CSF, nasopharyngeal swabs from 296 patients with aseptic meningitis (AM), as well as 240 samples of drinking water and 6 samples of lake water were studied. Epidemiologic analysis, isolation of enteroviruses in Hep-2 and RD cell cultures, RT-PCR, partial sequencing of 5'NTR and genome region coding VP1 were performed. RESULTS: Marked rise of AM caused by enteroviruses in Nizhny Novgorod during 2001 - 2007 was demonstrated. From August to October 2007, enteroviruses were detected in 93.8% of patients with AM (22.5 per thousand). Seasonal rise of incidence was determined by 9 serotypes of enteroviruses: E30 - 26 cases (53.1%), E7 - 7 (14.3%), E18 - 5 (10.3%), E13 - 3 (6.1%), E9 - 2 (4.0%), CB5 - 3 (6.1%),CA1 - 1 (2.0%), CA9 - 1 (2.0%), CA13 - 1 (2.0%). Serotype E30, represented by two subtypes, dominated. Dominating subtype E30-N1 was closely related with E30 strains isolated in 1994 - 2001 in Europe. Subtype E30-N2 was genetically related with Asian strains isolated in 2000 - 2006. RNA of E7, E9, E13, E18, CB5 viruses and dominating subtype E30-N1 were detected in nasopharyngeal swabs from patients with AM, which can explain rapid and wide spread of these viruses in susceptible population by aspiration route of transmission. CONCLUSION: Increased incidence of AM in Nizhny Novgorod in 2007 was caused by variant of E30 virus, which was genetically related with strains isolated in European countries in 1997.

[Development of a biochip for quantitative determination of two forms of prostate specific antigen using internal calibration curve].

Three-dimensional gel-based microchip allowing simultaneous quantitative detection of total (PSAtot) and free (PSAfree) forms of prostate specific antigen in human serum (in a format "one patient-one biochip") was developed. A method, which doesn't require preliminary construction of calibration curves when performing an assay, was applied for quantitative determination of PSAtot and PSAfree. Gel elements with immobilized antigen (PSA) in different concentration, forming an internal calibration curve, were included in a structure of the microchip, in addition to the elements with immobilized antibodies specific against PSAtot and PSAfree. The specialized software "ImaGelAssay" was used for data processing and interpretation. The sensitivity of the assay performed on biochips was 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. Variation coefficient for the measurements inside one series of microchips didn't exceed 10%. Correlation coefficient between the results of measurements in human sera obtained on biochips and by the standard ELISA method was 0.988 for PSAtot and 0.987 for PSAfree.

[Medical waste: the problem of handling].

The paper provides the environmental and hygienic characteristics of medical waste, the necessity of introducing a new system of collection, storage, transportation, and removal of waste from health care facilities. In accordance with SanPiN 2.1.7.728-99 (subparagraph 8.7.9.3a), an enterprise for thermal decontamination of hazardous waste should be necessarily set up with the purpose of possible removal of classes B and C garbage to the polygons, which will secure medical waste to man and the environment.

[The role of adhesion molecules (ICAM-1 and E-selectin) in development of diabetic microangiopathies]. / Rol' molekul adgezii (ICAM-1 i E-selektin) v razvitii diabeticheskikh mikroangiopatii.

AIM: To examine the expression of the intercellular adhesion molecule-1 (ICAM-1, CD54) on the surface of leukocytes and a soluble E-selectin (CD62) level in patients with diabetes mellitus type 1 (DM1) at various stages of diabetic nephropathy and retinopathy. MATERIAL AND METHODS: 42 patients with newly diagnosed and long-standing DM1 with presence or absence of microangiopathy (diabetic nephropathy and retinopathy) were examined. Routine laboratory, ophthalmologic tests, a special immunological test with ICAM-1 expression and soluble E-selectin (sES) examination were performed. RESULTS: The tests show increased ICAM-1 expression and sES level in all the patients vs controls. In newly diagnosed DM1 a high sES level was observed. In long-standing DM1 and absence of microangiopathy a sES level was also high. In patients with neovascularisation and proteinuria the sES level was two times higher than that in other subgroups. We also found an increased percentage of ICAM-1, CD54-positive lymphocytes and granulocytes in patients with microalbuminuria and especially with proteinuria and neovascularization. There were no correlations of ICAM-1 and sES levels with sex, DM duration, cholesterol and triglycerides. CONCLUSION: The findings show endothelium and leukocytes activation at early disease stages. Enhanced endothelium and leukocytes activation is associated with late complications.

[Current problems of occupational health in polyurethane production]. / Aktual'nye aspekty gigieny truda v proizvodstvakh poliuretanov.

Prospective production of polyurethane is not safe for humans. This production is characterized by possible workers' exposure to chemical hazards mixture. The work safety includes cautionary and current sanitary supervision, implantation of sanitary recommendations concerning individual protective means also.

[Gas chromatography in detection of toluene diisocyanate in biological medium]. / Gazokhromatograficheskoe opredelinie toluilendinzotsianata v biosredakh.

The authors suggested a method of gas chromatography for detection of toluylene diisocyanate (TDI) in biologic material: liver, lungs, spleen, brain, kidney. The method includes TDI extraction from tissues by solvent, subsequent concentration and the extract chromatography by a device with electron capture detector. The method was tested in white rats.

[Pathogenesis of tolylene diisocyanate intoxication]. / O patogeneze intoksikatsii toluilendiizotsianatom.

Experimental and clinical investigations to evaluate immunity, lipid peroxidation (LPO), and antioxidative defense (AOD) under the action of tolylene diisocyanate at the level of MAC and 10 MAC were conducted for further studies of the agent's pathogenetic mechanisms. The experimental investigations showed the predominant activation of cellular and humoral immunities, as well as the suppression of nonspecific defence at 10 MAC, enhanced LPO, and phasic changes in AOD. Most profound immunological changes were observed in those having as long as 5 years of length of service.

Special differentiation requirements of original and enriched secondary cytotoxic T lymphocyte precursors (pCTL-2 memory cell) specific for the class I histocompatibility molecule.

The in vivo-induced pCTL-2 cells of the L3T4- Lyt-2+ phenotype specific for the H-2Kb molecule are converted into effector CTLs in mixed lymphocyte culture (MLC) in the presence of heat-treated donor stimulators much more efficiently when the donor and recipient differ from each other, not only in the major histocompatibility complex (MHC) class I (H-2Kb) (anti-B10.MBR B10.AKM) but also in the MHC classes I + II, i.e., Kb + Ib (anti-C57BL/6 B10.D2 (R101)). The differentiation of original pCTL-2 is enhanced as the result of a synergistic action of the Kb alloantigen and rIL-2 in small doses. The anti-Kb pCTL-2, after their separation from helper T cells non-adherent to the macrophage donor monolayer and their subsequent elution from it, give rise to pronounced differentiation in simplified conditions over 3 days in monoculture in the absence of both stimulators and rIL-2. It is suggested that spontaneous and highly efficient specific differentiation of the eluted pCTL-2 is due to a dramatically enhanced secretion of the CTL differentiation factor by pCTL-2 themselves, and in addition, rIL-2 can also contribute to the secretion of this factor.

[Characteristics of the conditions for memory cell differentiation--the initial and enriched precursors of secondary cytotoxic T-lymphocytes (pCTL-2) specific for the histocompatibility class I molecule]. / Osobennosti uslovii differentsirovki kletok pamiati--iskhodnykh i obogashchennykh predshestvennikov vtorichnykh tsitotoksicheskikh T-limfotsitov (pTsTL-2), spetsifichnykh k molekule gistosovmestimosti klassa I.

The in vivo induced pCTL-2 with phenotype L3T4- Lyt2- specific to the H-2Kb molecule, turn into effector CTL during 4 days in the mixed lymphocyte culture (with heat-treated donor stimulators) much more efficiently when donor and recipient are different from one another not only in MHC class I (anti-BIO, MBR BIO.AKM) but in I + II (Kb + Ib) ahti-C57BL/6 BIAD2(RIOI). The initial pCTL-2 differentiation in enhanced as a result of synergistic effect between the Kb alloantigen and rIL2. The anti-Kb pCTL-2, being separated from helper T cells by means of absorption onto the macrophage donor monolayer and elution from it, give rise to pronounced differentiation in simplified conditions, irrespective of the stimulator presence and without external rIL2. It is supposed that these phenomena are raised to secretion of the CTL differentiation factor by the eluted pCTL-2 themselves, and besides, rIL2 may promote for secretion of this factor additionally.

Difference between antigen-binding receptor repertoires in effector cytotoxic T lymphocytes and their secondary precursors (memory cells) specific to H-2Kb.

The fine specificity of antigen-binding receptors was compared in pCTL-2 and secondary effector CTL (cytotoxic T lymphocytes) induced in vivo with the H-2Kb alloantigen in recombinant inbred mice. The lymphocyte preparations were enriched by elution from macrophage monolayers of various origins, including the donor (B6 strain), the H-2Kb mutant bm1, the H-2Kk allele B10.A(4R) and the recipient strain B10.D2(R101) as a control. Anti-Kb pCTL-2 eluted from third-party bm1 or B10.A(4R) monolayers gave rise to CTL progeny that lysed, equally well, both donor TC and those third-party TC from whose monolayer the pCTL-2 had been eluted, but which were unable to lyse irrelevant third-party TC. The lytic activities of secondary CTL whose precursors had been eluted from bm1 or B10.A(4R) monolayers were 6 and 12 times lower, respectively, than pCTL-2 eluted from the donor monolayer. Opposite results were shown for receptors of enriched secondary anti-Kb effector CTL. Irrespective of their elution source, whether donor, mutant or allele variant, the eluted effector CTL were able to lyse the donor TC to a similar degree and much more than the given third-party TC; moreover, they retained cross-reactivity in all cases. It is suggested that CTL receptors are homogeneous in specificity for a whole composite immunodominant epitope and differ from each other only in the affinity/lability of the combining site. In contrast, pCTL-2 can be separated into fractions: receptors of each fraction are strictly specific (with high affinity) to a particular portion of the same composite CTL epitope. It seems likely that the pCTL-2 receptor antigen-binding site is modified during pCTL-2 in vivo differentiation into effector CTL.

[Allogeneic lymphocyte induction in vivo of highly active T-killers specific for the molecule of the H-2 class-I complex and the detection of their receptors in vitro]. / Induktsiia allogennymi limfotsitami in vivo vysokoaktivnykh T-killerov, spetsificheskikh k molekule kompleksa H-2 klassa I, i vyiavlenie ikh retseptorov in vitro.

The optimal conditions are found for in vivo irradiated lymphocyte induction of high cytotoxic T lymphocytes (CTL) specific to the H-2Kb molecule with a subsequent monoculture differentiation. B10.D2(R101) CTL have a pronounced excess as compared to B10.A (4R) CTL, with respect to lysis intensity of the same target cells (TC), and requires a lower term of the monoculture incubation in spite of their specificity to the same H-2Kb molecule. As the susceptibility of TC for CTL lysis is higher (M phi as compared to EL-4 thymoma cells), CTL are much more inactivated with the monoclonal antibodies to Lyt-2 and Lyt-3 antigens without complement. Anti-H-2Kb CTL differentiated in the monoculture cross react to TC bearing third-party H-2 molecules (Kk, Dq, Dk). Unlike a stable CTL adherence to the donor M phi monolayer, nonspecific CTL adherence to the syngeneic M phi monolayer declines in the presence of EGTA, and as a result of repeated detachment of lymphocytes. The findings give rise to study receptor affinity expressed on the in vivo induced CTL surface, the CTL receptor monoclonal antibody and the CTL differentiation factor.

[The main difference between the repertoire of receptors of effector T-killers and their secondary precursors (memory cells)]. / Kardinal'noe otlichie repertuara retseptorov éffektornykhh T-killerov ot ikh vtorichnykh predshestvennikov (kletok pamiati).

Receptor repertoire analysis of in vivo induced secondary cytotoxic T lymphocyte precursors (pCTL-2) was performed, using the technique of their specific adherence to macrophage monolayers with subsequent elution of the adherent pCTL-2 and their activation by heat-killed donor stimulator cells. The capacity of anti-H-2Kb pCTL-2 receptors to contact H-2Kbm has been revealed only in a minor pCTL-2 component whose progenitors were able to lyse mutant bm1 target cells (TC). Unlike poor cross-reactivity of CTL descended from anti-Kb, pCTL-2 which were eluted from the donor monolayer, CTL-progenitors of anti-Kb pCTL-2 eluted from bm 1 or B10. A (4R) third-party monolayers lyse in equal quantities the donor TC and those third-party TC from which they have been eluted, but fail to lyse other TC. Enrichment of pCTL-2 eluted from bm 1 or B10. A (4R) monolayers is 6- or 12-fold lower, respectively, than after their elution from the donor monolayer. The findings indicate that anti-class I MHC pCTL-2 are separated into fractions, with their receptors being strongly specific (with high affinity) to the particular fragment of the same complex epitope without cross-reactivity to other fragments. These data differentiate pCTL-2 receptors from effector CTL ones which are homogenous in specificity to a whole (single) complex epitope with variable degree of complementarity. A cardinal distinction of receptor repertoire between CTL, pCTL-2 and suppressor T cells specific to the same class I MHC molecule and alteration of the active site during pCTL-2 differentiation have been suggested.

[Response of lymphoid and hematopoietic tissues to the growth of syngeneic transplantable hemangiopericytomas in mice]. / Reaktsii limfoidnoi i krovetvornoi tkanei na rost singennoi perevivaemoi gemangioperitsitomy u myshei.

The growth of syngeneic hemangiopericytoma in CBA X X C57BL/6j)F1 male mice was attended by the development of leukemoid reaction. The spleen showed an abrupt increase of the area taken by the red pulp with a remarkable activation of erythropoiesis in the pulp. The peripheral blood developed erythrocytosis 7 days after transplantation of pericytoma followed by anemia.