[Immuno-PCR Assay for Quantitation of Antibodies to Epstein-Barr Virus].

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein-Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV-IgG1-biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.

Cancer-associated antigens and antigen arrays in serological diagnostics of malignant tumors.

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer-associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.

[Contemporary molecular-biological methods of cancer diagnosis and monitoring based on the humoral immune response to tumor-associated antigens].

Humoral immune response to tumor-associated antigens in cancer patients can be used as a basis for disease diagnosis and monitoring. Moreover, identification of molecular targets of such response may be used to develop antigen-specific anticancer vaccines. Here, we review the main approaches to identification and study of tumor-associated antigens recognized by serum antibodies. We also focus on the challenges that must be met before serological antigens can be used in clinical cancer diagnostics.

New mutations in the human p53 gene--a regulator of the cell cycle and carcinogenesis.

Mutations in the tumor suppressor gene p53 often lead to disarrangement of the cell cycle and of genetic integrity control of cells that may contribute to tumor development. We studied p53 gene mutations in 26 primary tumors of colorectal cancer patients. Mutations in p53 were found in 17 tumors (65.4%). All point mutations affected the DNA binding domain of p53 and were localized in exons 4-8 of the gene. Mutant p53 isoforms with altered domain structure and/or with alternative C-terminus arising from frameshift mutations or abnormal splicing were found in six tumors. Mutations Leu111Gln and Ser127Phe were shown in colorectal cancer for the first time. Isoforms p53-305 with C(4) insertion in codons 300/301 and p53i9* including an additional 44 nucleotides of the 3 -end of intron 9 were discovered for the first time. Mutations of p53 were associated with lymph node metastases and III/IV stage of tumors that are signs of unfavorable prognosis in colorectal cancer.

[House-dust mites allergen for specific immunotherapy by oral administration: experimental study].

The oral medicinal form of house-dust allergen has been prepared with the use of new technology. To produce the preparation, the standard water-saline extract obtained from house-dust mites (Dermatophagoides farinae) allergen has been used. This preparation is chemically stable and does not lose its biological activity for 2 - 3 years of storage. The microbiological purity of the allergen meets the requirements for oral preparations. The allergen has proved to be safe in cases of prolonged administration and has no chronic toxicity. As shown with the use of immunochemical methods, the preparation has high immunogenicity and lower allergenicity. This preparation may be recommended for clinical trials on a limited circle of subjects.

[Evaluation of specific activity of preparations of allergens from synanthropic insects].

Physical, chemical and immunobiological characteristics of allergens from synanthropic insects were studied by tests for anaphylaxis, indirect degranulation of mast cells test and ELISA. Sera from 20 patients with severe and intermediate atopic asthma with sensiblization to common allergens have been studied. All extracts of allergens from synanthropic insects (german cockroach, oriental cockroach, american cockroach, speckled feeder cockroach, cricket, common house fly, brown house moth, confused flour beetle, rice weevil, grain weevil) have specific activity. Extracts of allergens from common house fly, brown house moth, german cockroach and oriental cockroach had the strongest allergenic activity as measured by ELISA. Obtained allergens can be used for insect allergy diagnostics.

[Mites allergy in children from Tula region].

Characteristics of allergy to mites in children living in Tula region have been revealed. It was shown that mites from Pyroglyphidae (Dermatophagoides pteronyssinus, Dermatophagoides farinae), Cheyletidae and Glycyphagidae (G. destructor, G. domesticus) families play important role in development of atopic allergy in children from this region. Efficacy of plant-origin acaricide "Milbiol" as part of prophylactic measures was evaluated. Its use in children with mild and intermediate asthma led to decrease of number of wheezing episodes, improvement of respiratory function, lessening of clinical signs of allergic rhinitis, decrease or discontinuation of usage of vasoconstrictive preparations.

[Study of allergens from vegetables, fruits and berries]. / Izuchenie allergenov iz ovoshchei, fruktov i iagod.

Different methods of obtaining allergens from vegetables, fruit and berries have been evaluated. Two technologies for obtaining food allergens have been considered; of these, Coca's method has proved to be more technological, economical and less labor-consuming. The newly developed technology has made it possible to obtain stable, safe and specifically active allergens which may be used for diagnosing food allergy.

[Serological study of a repertoire of human cancer antigens and autoantigens]. / Serologicheskie issledovaniia reperatura rakovykh antgenov i autoanigenov cheloveka.

The spectrum of human antigens allows a monitoring of various pathological processes such as autoimmune disorders and tumorigenesis. Serological analysis of cDNA expression libraries (SEREX) is now used to search for new cancer-associated antigens, which are potential diagnostic markers or targets for immunotherapy of cancer. The results obtained for several solid tumors are reviewed. Groups of antigens detectable by SEREX, causes of immunogenicity of autoantigens, and prospective implication of antigens in diagnostics and monitoring of cancer are discussed.

Thrombin-mediated events implicated in mast cell activation.

It has been suggested that mast cells contain receptors for thrombin because binding of thrombin to peritoneal mast cells (PMCs) results in heparin release. Peritoneal mast cell responsiveness to different thrombin forms was examined by measuring ion conductance, intracellular pH, the concentration of cyclic guanosine monophosphate (cGMP), and release of histamine. Several types of receptors for thrombin are suggested by the results, which demonstrate that: (1) PMCs responded to alpha-thrombin and diisopryopyl-phosphoryl-alpha-thrombin (DIP-alpha-thrombin), but not to gamma-thrombin, by activation of Na/H exchange in reactions involving protein kinase C and by a simultaneous elevation in cell conductance and capacitance; (2) the initial 1-nmol/L alpha-thrombin-induced acidification of PMC cytoplasm was absent in Ca-free medium, and higher doses of alpha-thrombin induced a biphasic reaction (acidification preceeded alkalinization); and (3) PMC stimulation by alpha-thrombin at low concentrations (< 1 nmol/L) resulted in increase of cGMP and simultaneous decrease of histamine release, whereas thrombin concentrations > 1 mumol/L induced the acceleration of histamine release.

[The effect of formaldehyde modification on the physicochemical properties and specific activity of an allergen from Dermatophagoides farinae mites]. / Vliianie modifikatsii formal'degidom na fiziko-khimicheskie svoistva i spetsificheskuiu aktivnost' allergena iz kleshchei Dermatophagoides farinae.

D. farinae allergen has been modified with formaldehyde and its properties have been studied. This study has demonstrated that the formolation of D. farinae ticks allergen causes changes in isoelectric points of proteins and a shift of protein fraction to acidic pH values: 3.0-4.85; an insignificant decrease in the volume of gel filtration output on a column packed with Sephacryl S-300; a decrease in the esterase activity from 10.5 to 1.8 microM tosylarginine methyl ester/min. per mg of protein. As revealed with the use of microdot enzyme immunoassay, the modified allergen loses its capacity to bind with human specific IgE antibodies in vitro.

[Allergens from Dermatophagoides dust mites: origin, antigenic and structural characteristics, and therapeutic agents]. / Allergeny iz kleshchei domashnei pyli roda Dermatophagoides: priroda,antigennaia i strukturnaia kharakteristika, lechebnye preparaty.

Micromites (genus Dermatophagoides) are the major source of allergens in house dust. Four homologous classes of major allergens have been isolated from extracts of D. pteronyssinus and D. farinae mites. According to current theories, all major mite allergens are proteins of gastrointestinal origin. Group I mite allergens, Der pI and Der fI, are thermolabile glycoproteins with M(r) of 25 kDa. A comparison of primary structure of these proteins reveals a 30% homology with cathepsins B and H, papain and actinidine. Analysis of enzymatic activities reveals that group I allergens are proteolytic enzymes related to the class of cysteine proteinases. With regard to antigenic composition, Der pI and Der fI have three common and two species-specific epitopes. The amino acid sequence of the major allergenic determinant for Der pI has been established. Group II mite allergens, Der pII and Der fII, are single-chain thermostable proteins with M(r) of 10-14 kDa and are said to bear many common features with the lysozyme. Group III mite allergens are analogous to trypsin. A 50% homology of amino acid sequences of Der pIII and Der fIII to those of vertebrate and invertebrate serine proteinases has been found. To the fourth group of major mite allergens one may relate mite amylase (M(r) = 56-60 kDa). A high degree of homology has been established between group IV allergens and mammalian alpha-amylase. Mite allergens of all groups induce the production of specific IgE antibodies in human organism. The use of purified allergens increases the efficiency of diagnosis and treatment of mite-induced allergoses. Modified forms of mite allergens (allergoids, allergens adsorbed on carriers, liposome preparations, etc.) are helpful tools in specific immunotherapy.

[The mite allergen and allergoid stimulation of histamine secretion by mast cells]. / Stimuliatsiia kleshchevym allergenom i allergoidom sekretsii gistamina tuchnymi kletkami.

Rat peritoneal mast cells were incubated with serum from highly mite-sensitive patients. It was demonstrated that exposure of passive sensitized mast cells to allergen from mites Dermatophagoides farinae induced the release of histamine. Exposure of mast cells to 10 micrograms/ml and 50 micrograms/ml mite allergen resulted in an increase of histamine secretion to 48% of the basal level. The allergoid (formaldehyde-modified mite allergen) had poor histamine-releasing activity compared to allergen. The allergoid (50 micrograms/ml) induced a 2.5-fold decrease in histamine release. The allergen at the same concentrations and the same release as allergen in dose 0.1 microgram/ml.

[The effect of a mite allergen on Na/H metabolic activity in peritoneal mast cells]. / Vliianie kleshchevogo allergena na aktivnost' Na/H-obmena v peritoneal'nykh tuchnykh kletkakh.

Mite allergen interacting with mast cells treated with sera from bronchial patient sensitized to home dust Dermatophagoides farinae causes changes in intracellular pH. Regulation of pHi peritoneal mast cells is participated by Na/H metabolism probably activated by protein kinase C.

[Thymosin alpha(1)--an endogenous modulator of alpha-thrombin recognition site]. / Timozin al'fa1--éndogennyi moduliator tsentra uznavaniia alfa-trombina.

Thymosin alpha 1-inhibited fibrinogen clotting activity of alpha-thrombin, but not amidolysis of H-D-Phe-Pip-Arg-pNA. Modulation of thrombin interaction with rat peritoneal mast cells (RPMC) by suppressors of additional recognition binding site (thymosin and heparin) was studied. Thrombin-induced pHi changes of RPMC were controlled with pH-sensitive fluorescent dye, BCECF. Thrombin caused a biphasic changes in pHi: rapid cell acidification (0.02) followed by slow alkalinization (0.06 above baseline for 18 min). Thymosin suppressed thrombin-induced pHi increase above resting level. Similar changes in pHi were observed after modification of additional recognition binding site by heparin. Beta/gamma-thrombin with disrupted additional binding site was shown to induce only a decrease of pHi. It is concluded that thymosin alpha 1 is endogenous modulator of alpha-thrombin activity.

[Binding of FITC-labelled alpha-thrombin by peritoneal mast cells]. / Sviazyvanie mechennogo FITTs alpha-trombina peritoneal'nymi tuchnymi kletkami.

The interaction of bovine alpha-thrombin with peritoneal mast cells was studied using FITC-labeled enzyme. Thrombin was modified with FITC in the presence of heparin and was separated from heparin and free FITC by gel-filtration at HPLC yielding FITC-labeled alpha-thrombin with intact additional recognition binding site for high molecular substrates and cell receptors. Equilibrium studies have shown that the binding of thrombin to peritoneal mast cells is active independent, rapid, specific, saturable and reversible. Equilibrium between bound and free thrombin is attained within I min and Scatchard analysis indicates a population of approximately 54 x 10(3) sites/cell with a dissociation constant of 1.3 x 10(-9) M. FITC-labeled alpha-thrombin binds to peritoneal mast cells in a temperature-dependent manner with optimum at 37 degrees C. These results indicate that FITC-labeled alpha-thrombin binds to peritoneal mast cells with high affinity.

[Effect of non-fractionated and low-molecular heparin on the functional state of mast cells]. / Vliianie nefraktsionirovannogo i nizkomolekuliarnogo geparina na funktsional'nyi status tuchnykh kletok.

The state of the mast-cell population of rats treated with unfractionated and low-molecular weight heparins under stress conditions has been comparatively studied by the morphometrical assay. The stress was produced by 60 min immobilization followed by intravenous injection of unfractionated (UF) or low-molecular weight (LMW) heparin. The stress-induced heparin release from mast cells resulted in a 3.3-fold decrease of the index of saturation with heparin and in a significant increase of granulolysis and degranulation. The mast cell secretory status reached the preinjection level within 20 min in rats with UF heparin injected (15 unit/200 g). At the same time mast cells of rats with LMW heparin have no such ability. The data obtained indicate that LMW heparin in contrast to UF heparin cannot be accumulated (or accumulated very slowly) by mast cells. This fact as well as low affinity of LMW heparin to endothelium and blood platelets promote its preservation in blood for a long time.

[The incorporation of 35S-heparin into mast cells in the rat and its release into the circulatory bed]. / Vkliuchenie 35S-geparina v tuchnye kletki krysy i vydelenie ego v krovenosnoe ruslo.

It was shown that the intensity of the clearance of 35S-heparin in the blood is extremely high in the first 30 minutes after the introduction of the label. Further on the intensity of the clearance was decreased and by the 60th minute only 11 per cent of the radioactivity was left and by the 240th minute--3.7%. It was established that during intravenous introduction of 35S-heparin the obese cells are capable of accumulating it from the blood flow.