On normalizing of urinary KIM-1 level to urine creatinine in patients with renal cell cancer.

KIM-1 (kidney injury molecule 1), a marker of acute kidney injury, is produced by epithelial cells of renal proximal tubules. Elevated KIM-1 levels in urine and plasma are associated with renal cell carcinoma (RCC). The aim of this study was to compare the significance of non-normalized uKIM-1 values and those normalized to urine creatinine, as urinary biomarkers in RCC. The uKIM-1, urine creatinine and their ratio (uKIM-1/Cre) were studied in 118 RCC patients and 58 apparently healthy subjects. The median of uKIM-1 in the healthy group was 0.71 ng/ml (1st and 3rd quartiles were 0.35 and 1.23, respectively) and in RCC patients it was 2.36 (1.43; 5.93) ng/ml. The medians of uKIM-1/Cre were 0.77 (0.49; 1.18) and 2.42 (1.41; 4.61) ng/mgCre, respectively. Stage I RCC is statistically significantly different from stages II-III and stage IV using uKIM-1/Cre values (p = 0.0056 and p = 0.0012, respectively); using uKIM-1 values significant differences occur only when comparing stages I and IV (p = 0.015). In both healthy individuals and RCC patients, uKIM-1/Cre levels were slightly lower in subgroups younger than 50 years than in subgroups older than 50 years, whereas a similar trend was observed for uKIM-1 only in patients. In healthy men and male patients, uKIM-1 levels were higher than in the corresponding groups of women (the differences were not statistically significant), but the use of uKIM-1/Cre values eliminated the gender differences. A high correlation was found between the concentrations of uKIM-1 and urine creatinine in three healthy subjects followed up for 3 weeks (Spearman's correlation coefficients were 0.758, 0.825 and 0.933, respectively). The data obtained are clear evidence of the need for normalization uKIM-1 to urine creatinine in RCC patients.

[The test system to identify mucin MUC1 in human blood serum using the technique of immune-enzyme analysis based on monoclonal antibody ICO25].

On the basis of genuine mouse monoclonal antibody ICO25 the test system IEA ICO25 was developed and standardized to quantitative detect tumor-associated antigen, mucin1 in human blood serum in format of inhibitory immune-enzyme analysis. The analytic characteristics of test-system correspond to the standards applied to immune-enzyme diagnostic kits. The results of identification of MUC1 in blood serum of healthy donors and female patients with breast pathology using IEA ICO25 fully correlate with the data concerning the detection of antigen CA15-3 using certified commercial kits. The test system IEA ICO25 can be used to detect MUC1 in human blood serum for research purpose.

[Antibodies to synthetic peptides for the detection of survivin in tumor tissues].

Survivin, an endogenous protein, is a promising marker for the diagnosis of cancer. The aim of the present work was to obtain antibodies specific to survivin and capable of detecting this protein in tumor tissues. Four peptides corresponding to fragments (1-22), (54-74), (80-88)-(153-165), and (118-144) of the survivin-2B sequence were selected and synthesized. Rabbits were immunized with the synthetic peptides. It has been shown that all peptides in a free state, without conjugation with a high-molecular-weight carrier, stimulate the production of antibodies capable of binding with recombinant survivin. Antipeptide antibodies were isolated from sera and their performance in the immunohistochemical detection of survivin in human tumor tissues was studied. It was shown that only antibodies to the (80-88)-(153-165) peptide bind to the survivin present in breast and bladder tumors. The ability of antibodies to this peptide to detect survivin in tumor tissue lysates was demonstrated by immunoblotting. The part of the sequence targeted by the antibodies against the (80-88)-(153-165) peptide was localized using truncated peptide fragments.

[Effect of substituents on photochemical and biological properties of 13,15-N-cycloimide derivatives of chlorin p6].

The effect of electron-accepting substituents in position 3 of the chlorine p6 macrocycle in neutral and carboxyl-containing negatively charged cycloimide derivatives of chlorin p6 (CIC) on the photochemical and biological properties of these photosensitizers was studied. A relationship between the structure and properties of CICs was analyzed on the basis of information on their photoinduced cytotoxicity, efficiency of the generation of reactive oxygen species, photostability, intracellular localization, quantitative parameters of accumulation in cells, and cellular pharmacokinetics. It was shown that these compounds can be used for the development of photosensitizers with intense light absorption at 740 nm, controlled intracellular localization, and a high photodynamic activity toward tumor cells.

[Photobiological properties of 13,15-N-(carboxymethyl)- and 13,15-N-(2-carboxyethyl)cycloimide derivatives of chlorin p6]. / Fotobiologicheskie svoistva 13,15-N-(karboksimetil)- i 13,15-N-(2-karboksiétil)tsikloimidnykh proizvodnykh khlorina p6.

Lipophilic derivatives of chlorin p6, 13,15-N-(carboxymethyl)cycloimide methyl ester (CIC1) and 13,15-N-(2-carboxyethyl)cycloimide methyl ester (CIC2), were shown to absorb light in 710 nm region and to be efficient IR photosensitizers. They exhibit similar phototoxicities on the cells of A549 human lung adenocarcinoma, which are 40- and 100-fold higher than those of chlorin p6 and the clinically used Photogem, respectively, and are not toxic in the absence of light irradiation. The confocal spectral imaging technique allowed us to demonstrate that the high phototoxicity of CIC1 and CIC2 is due to their ability to readily penetrate to cells and to be bound to the cell membranes and lipid-containing structures in the monomeric photoactive form. Under the irradiation, the membrane-bound CIC1 and CIC2 are characterized by high quantum yields of singlet oxygen generation (0.6 and 0.65, respectively) and the inability to produce hydroxyl radicals. A 1.5-microM content of CIC1 and CIC2 in the incubation medium provides for their average cytoplasmic concentrations of 21 and 16.5 microM, respectively. The incubation times to achieve 50% level of maximum accumulation for CIC1 and CIC2 in A549 cells are 30 +/- 6 and 24 +/- 12 min, and the times for 50% release of the dyes from the cells are 17 +/- 4 and 50 +/- 10 min, respectively. A diffuse distribution with the predominant accumulation in the membranes of the Golgi apparatus and mitochondria is characteristic of both CIC2 and CIC1, whereas, in addition, CIC1 is considerably accumulated in lipid droplets (cellular organelles responsible for the storage and metabolism of neutral lipids and steryl esters). Our results demonstrate that changes in the structure of the imide substituent could affect the intracellular localization and the rate of release of chlorin p6 cycloimide derivatives from cells while preserving their high photodynamic activity.

Potential of block copolymer- and immuno-conjugates for tumor-targeted delivery of Bowman-Birk soybean proteinase inhibitor.

The present work reports the effect of conjugation of the anticarcinogenic and antitumor soybean Bowman-Birk protease inhibitor (BBI) with amphiphilic block copolymer of ethylene oxide and propylene oxide (PEO-PPO) as well as with monoclonal antibody via clinical dextran (D) on tumor-targeted delivery of BBI.

Immunoconjugates of soybean Bowman-Birk protease inhibitor as targeted antitumor polymeric agents.

To enhance the antitumor potential of soybean Bowman-Birk inhibitor (BBI), the conjugate of BBI with an antibody via a macromolecular carrier was prepared. Clinical dextran (D) was used as a biocompatible biodegradable carrier for co-immobilization of BBI and antibody. A model immunoglobulin isolated from sheep serum (sIgG), raised against human IgM was utilized to develop the procedure of immunoconjugate synthesis. The molar ratio of the ingredients in the conjugate was the following BBI:D:sIgG=9:1:1. Comparison of the dose response curves for the native sIgG and the BBI-D-sIgG conjugate indicated that sIgG completely retained its specific activity (>90%) after modification with dextran. The determination of the Ki values for chymotrypsin interaction with the native BBI and the BBI-D-sIgG conjugate indicated high anti-chymotrypsin activity. In the next step, the monoclonal antibody (ICO 25 MAb) against the mucin-like human epithelial membrane antigen was used for conjugation as it is the most universal vector for targeting different agents to human tumors of epithelial origin. The influence of conjugation on the specificity of the Mab reaction with its antigen was studied. The conjugated MAb reacted with tumor cells of different epithelial genesis (breast, lung, gastric, ovarian and uterus tumors), but did not react with tumor cells of non-epithelial origin. It was shown that BBI-D-ICO 25 MAb conjugate has almost the same immunohistochemical activity as non-conjugated MAb. These results demonstrated the feasibility of exploiting the activities of covalently bound BBI and ICO 25 MAb for anticarcinogenic agent targeting.

Confocal raman microspectroscopy and imaging study of theraphthal in living cancer cells.

Binary systems combining a transition metal complex and ascorbate have been proposed recently for catalytic therapy of malignant tumors. The killing effect on tumor cells is achieved by production of free radicals in the course of accelerated oxidation of ascorbate by dioxygen in the presence of transition metal complexes. Further progress in the development of binary catalytic systems (BCSs) requires a special method for their investigation in cells and tissues, because neither component of BCSs fluoresces. Here a resonance Raman confocal spectral imaging (RR CSI) technique was introduced as a unique approach to monitor quantitatively the transition metal complexes within living cells. Intracellular accumulation, localization, and retention of theraphthal (TP), a catalyst of the advanced TP/ascorbate BCS, were investigated in A549 cells with the RR CSI technique. The cellular analysis was complemented with the detailed study of molecular interactions of TP in solution and environmental factors affecting the RR spectrum of TP. TP does not penetrate into membranes, it binds very weakly to DNA and RNA, but it readily forms complexes with proteins. Binding with Ca(2+) cations and decreasing pH below 6 induce aggregation of TP. By analyzing RR spectra recorded from every point within a TP-treated cell, three states of the agent were discriminated, namely, monomeric TP in polar environment, TP bound to proteins, and aggregated TP. Their cytoplasmic and nuclear distributions were mapped at different stages of uptake and efflux. By introducing organelle-selective fluorescent probes into drug-treated cells and measuring intracellular localization of both the probe and the drug, compartmentation of TP was revealed. Cell growth suppression by the TP/ascorbate system was measured, and probable molecular and organelle targets of radical damage were characterized.

[Study of localization and molecular interactions of biologically active compounds in living cells and tissue slices based on confocal microspectroscopy and reconstruction of the spectral images]. / Issledovaniia lokalizatsii i molekuliarnykh vzaimodeistvii biologicheski aktivnykh soedinenii v zhivykh kletkakh i srezakh tkanei na osnove metoda konfokal'noi mikrospektroskopii i rekonstruktsii spektral'nykh izobrazhenii.

The confocal spectral imaging (CSI) technique is described, its basic principles are considered, and a brief review of its applications to the study of biologically active compounds (BAC) within living cells and in tissue slices is presented. This technique is based on measurements and analysis of fluorescence or resonance Raman spectra in each point of the specimen under microscope with a three-dimensional resolution of about cubic micrometer. This technique is applicable to the study of stained fluorescent and nonfluorescent compounds. Unlike the conventional approaches based on the optical microscopy, the CSI technique opens the opportunity for the identification of complexes and microenvironment of BAC in intact cells and thin tissue slices (slices or sections), as well as for the analysis of localization and distribution of compounds of interest and their complexes in cellular organelles and tissue structures. The use of CSI technique in combination with the conventional biochemical and cytological methods makes it possible to significantly expand the informativeness of investigation of modes of action of new BAC.

[Metaplastic breast cancer]. / Metaplasticheskii rak molochnoi zhelezy.

Three groups of metaplastic mammary carcinoma are distinguished: squamous cell carcinoma, invasive ductal carcinoma with partial squamous metaplasia and carcinoma with formation of heterologous elements. Squamous cell carcinoma is subdivided into 5 variants: solid and spindle cell variants, carcinoma in preexisting cyst and fibroadenoma; carcinoma resulting from total squamous metaplasia of invasive ductal carcinoma. Squamous cell mammary carcinoma has a relatively favourable prognosis. Partial squamous metaplasia has minimal influence on the course of the disease.

[Various forms of breast cancer]. / O nekotorykh formakh raka molochnoi zhelezy.

844 observations of mammary carcinoma special forms were analyzed. In 48% of cases there was a combined structure, primary multiple carcinoma was in 23.6%, bilateral in 2.8% cases. Morphological and immunohistochemical investigation with use of monoclonal antibodies to the membrane antigen of lipid globules were performed. The clinical features of different forms are described.

[Pharmacokinetics of human milk lactoferrin in intact mice and tumor-carrying animals]. / Izuchenie farmakokinetiki laktoferrina zhenskogo moloka u intaktnykh myshei i zhivotnykh-opukholenositelei.

Pharmacokinetics of iodine containing and native lactoferrin from human milk was studied in intact mice and the animals with Ehrlich ascites carcinoma. Both these antigens, native and I-lactoferrin, were similarly distributed in intact mice body. The same pharmacokinetics was found in tumor-bearing animals. Exogenous lactoferrin penetrated mainly into liver tissue, then simultaneously with bile--into small intestine and further into large intestine. Distinct accumulation of the glycoprotein was not found in tumor tissue.

[An immunohistochemical study using monoclonal antibodies of the antigen of fat globule membranes in human milk]. / Immunogistokhimicheskoe izuchenie antigena membran zhirovykh globul zhenskogo moloka s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies (Mab) IKO-21 and IKO-25 obtained to the membrane antigen of the woman milk lipid globules were tested immuno- and cytochemically on the samples of human normal definitive and embryonal tissues and tumours. Mab IKO-21 are shown to react, apart from epithelial tissues, with a vascular endothelium and blood cells. Mab IKO-25 are specific to the epithelial tissue and malignant epithelial tumours. Their intensive reaction with the cells of malignant tumours of the mammary gland, lung, ovary and the type of their distribution in the organs and tissues enables their use in the differential diagnosis of malignant epithelial and non-epithelial tumours as for revealing metastases both in vitro and in vivo.

[Study of the integral antigen of the membranes of fatty globules of human milk]. / Izuchenie integral'nogo antigena membran zhirovykh globul zhenskogo moloka.

The glycoprotein with the molecular weight of 82,500 D (Ag3) was extracted from the native membranes of fatty globules of human milk and was purified by isoelectrofocusing. Polyclonal antisera (PCA) as well as monoclonal antibodies (MoAb)-ICO-21, ICO-22, ICO-26, ICO-29 against this membrane antigen were produced. The localization of the antigen in the human normal and malignant tissues was tested by the immunochemical technique. Apical staining of the Ag3 was in the acinus and duct cells of the normal breast. Weak staining with the antibodies to Ag3 was revealed in the epithelium cells of ovaries, bronchi, kidney tubules and uterine tubes. The far more pronounced staining of the Ag3 with PCA and MCA was observed in the cell cytoplasma both in the primary and metastatic breast and ovarian tumours. The specificity of the MoAb to Ags suggests that they will be useful in the diagnosis of the malignant disease of the breast and ovary.

[alpha 1-proteinase inhibitor in breast cancer]. / alfa1-Ingibitor proteinaz pri rake molochnoi zhelezy.

Level of alpha 1-proteinase inhibitor (alpha 1-Pi) and antitryptic activity in blood serum were assessed in 167 patients with breast cancer and 30 cases of benign lesions. An increase in blood serum-alpha 1-Pi level was shown to be associated with tumor advancement but could not be regarded as a marker of cancer. Concentration of the inhibitor exceeded 8 mg/ml in 83.4% of cases with metastatic breast cancer but was as a rule, equal to or below that value in metastasis--free patients. Those two groups were prognostically different. Morphological analysis established a correlation between blood serum-alpha 1-Pi level, on the one hand, and grade of malignancy, degree of pathologic changes in tumor stroma and extent of lymph node involvement, on the other.