In vitro modeling of tumor interclonal interactions using breast cancer cell lines.

AIM: To study the peculiarities of ecological relationships of breast cancer (BC) cell lines MCF-7, BT-474 and MDA-MD-231 under co-culturing conditions. MATERIALS AND METHODS: Three BC cell lines: luminal A - MCF-7, luminal B - BT-474 and triple-negative - MDA-MD-231 were co-cultured pairwise. Immunocytochemistry was used to differentiate the cell lines in the wells. The effect of the cell-free culture medium on the growth rate of the alternate cell line in the pair was also evaluated. RESULTS: It was shown that when BT-474 cells were co-cultured with MCF-7 and BT-474 cells were co-cultured with MDA-MD-231, two types of ecological interactions could be observed: commensalism and amensalism, respectively. While the cells do not interact with each other in contact, the supernatants of single cultures of MCF-7 and MDA-MD-231 exert the same effect on BT-474 as co-cultivation of BT-474 with these cells. CONCLUSIONS: The paracrine mechanism of intercellular interaction between different human BC cell lines has been demonstrated. The models used in population ecology can be applicable to identify the types of interaction between cell lines.

Breast tumour cell subpopulations with expression of the MYC and OCT4 proteins.

The MYC and OCT4 genes are known factors associated with maintaining pluripotency and are linked with a more aggressive course, progression, and resistance to therapy in cancer. Determining the subpopulations of tumour cells expressing the Myc and Oct4 proteins will provide an opportunity to understand which tumour cell subpopulations expressing MYC and OCT4 are associated with metastasis and resistance and which subpopulations can be targeted by anti-MYC and anti-OCT4 therapy. The study included paraffin-embedded tissue from tumours from 27 patients with luminal B breast cancer obtained after neoadjuvant chemotherapy (NACT). Immunofluorescence staining was used to identify subpopulations of tumour cells expressing Myc, Oct4 and Snai2 (Opal™ 7-Color Kit (PerkinElmer, Hopkinton, MA). The following tumour cell subpopulations were identified with the Myc and Oct4 proteins and the Snai2 EMT marker: stem/progenitor tumour cells with/without Myc, Oct4 or Snai2 expression; differentiated tumour cells with/without Myc, Oct4 or Snai2 expression; and other nontumour cells (CK7-EpCAM-CD44+/-Myc+/-(Oct4, Snai2)+/-) within the inflammatory infiltrate in the tumour parenchyma and stroma. The circulating tumour cell subpopulations with Oct4 protein expression in the bloodstream were studied by flow cytometry. It was found that in patients with partial regression (PR) in response to NACT, the frequency of tumour stem cells was 3.6-fold increased (p = 0.038) in the non-EMT state (CK7+EpCam+CD44+Snai2-). In patients with metastases, there was a statistically significant 2.5-fold increase in the frequency of differentiated tumour cells with Myc expression (CK7+EpCam+CD44-Myc+) and a 2.7-fold increase in the frequency of cells with Oct4 expression (CK7+EpCam+CD44-OCT4+). In the next stage, the frequencies of subpopulations with expression of the Oct4 protein and signs of EMT among circulating tumour cells (CTCs) were determined. In patients with metastases, the frequency of tumour stem cells in the EMT state (CD326+CD44+CD24-CD325+) (p = 0.015) was more than fourfold increased, and the frequency of progenitor tumour cells with expression of the Oct4 stem protein (CD326+CD44+CD24+Oct4+) (p = 0.016) was almost sixfold higher than that in patients without metastases. Nonstem (differentiated) tumour cells with expression of the stemness proteins Myc and Oct4 were present in the breast tumour. Their content was significantly higher in residual tumours after NACT in patients who subsequently developed metastases compared with that in patients without metastases. Such cells are a new in situ marker of metastasis.

[The chemical toxicological investigation of gamma-hydroxybutyric acid in biological objects and the interpretation of the results of the analysis]. / Khimiko-toksikologicheskoe issledovanie GOMK v bioob''ektakh i interpretatsiia rezul'tatov analiza.

The authors propose a method for the quantitative determination of gamma-hydroxybutyric acid (GHBA) in biological objects including biological fluids (blood, urine) and tissues with the use of gas chromatography with mass-selective detector. The samples for the analysis were prepared by liquid-liquid extraction by butyl acetate with subsequent derivatization using a N,O-Bis(trimethylsilyl)trifluoroacetamide/Trimethylchlorosilane mixture (BSTFA + 1%TMCS). The graded graphs were linear in the range of GHBA concentrations from 26.4 to 1321.6 mg/l. Coefficients of correlation for all the graphs were higher than 0.999. The threshold of detectability for GHBA in blood, urine, and internal organs was 10 mg/l, the quantification limit concentration 26 mg/l.

[Organization of quality control of the forensic medical investigations]. / Organizatsiia kontrolia kachestva sudebno-khimicheskikh issledovanii.

The article describes the system of quality control at all stages of forensic chemical investigations (including pre-analytical, analytical, and post-analytical ones) carried out by the Saint-Petersburg Bureau of Forensic Medical Expertise It is shown that standardization and control taken together make up a solid foundation for obtaining the reliable and reproducible results of forensic chemical investigations. Responsibility for the quality control at all stages of forensic chemical investigations lies on the heads of the respective departments. Special staff members are appointed to supervise performing each concrete form of control and keeping the relevant documentation.

Comprehensive meta-analytical summary on human papillomavirus association with head and neck cancer.

An etiological role of high risk human papillomavirus (HPV) in the development of cervical cancer has been well established. Hence, attention of researchers has been focused on the role of HPV in pathogenesis of other malignancies, such as head and neck cancers. An analysis of epidemiological data on the prevalence of HPV infection among healthy people and patients with precancerous lesions and/or cancer is an important step in understanding the role of HPV in head and neck carcinogenesis. More and more data de-monstrate the impact of HPV infection on disease outcome. HPV-positive patients have been shown to have better responses to radiotherapy and better overall and disease-free survival than HPV-negative patients. This review presents data of the meta-analysis based on a large number of original studies on HPV prevalence in patients with precancerous lesions and in patients with oral, oropharyngeal and laryngeal cancers as well as findings on the impact of HPV infection on survival of these patients.

Integrative and episomal forms of genotype 16 of human papillomavirus in patients with cervical intraepithelial neoplasia and cervical cancer.

The study involved 500 patients with LSIL (low grade squamous intraepithelial lesion), HSIL (high grade squamous intraepithelial lesion), stage I-IV cervical cancer, infected with human papillomavirus (HPV), as well as 235 women without pathological changes in cervical mucosa. The comprehensive survey included colposcopy, cytological and histological analysis, detection and genotyping of high-risk human papillomavirus. Viral load and physical status of HPV16 DNA was evaluated in cases of mono-infection (n = 148). The prevalence of virus-positive cases among the patients with LSIL/NSIL, cervical cancer patients and healthy women was 69.2%, 76.7% and 51.9%, respectively. An association between the severity of disease and high viral load was revealed. The frequency of integrated DNA was strongly increased in patients with a high viral load. The frequency of episomal forms was either reduced or not detecteable in patients with high viral load as compared to patients with low viral load. It is reasonable to suggest that a high HPV16 viral load may cause an increase in the frequency of integration of virus DNA into the cellular/host genome. This suggests that a high HPV16 viral load may be considered as a risk factor for prognosis of cervical intraepithelial neoplasia and cervical cancer.

Genetic aspects of HPV infection detection in tumor and adjacent tissues of patients with laryngeal squamous cell carcinoma.

The article describes methods for the human papillomavirus (HPV) detection in tumor and adjacent (morphologically intact) tissues of patients with laryngeal squamous cell carcinoma (LSSC) in terms of viral pathogenesis. Comparative evaluation of the principles and techniques for HPV detection was performed. Advantages and disadvantages of the HPV detection methods are described. Approaches for DNA and HPV oncoproteins E6-E7 identification are substantiated. The results of our research into the qualitative and quantitative detection of HPV in the tumor and adjacent tissues of patients with Lssc are described. The research was conducted using commercial test systems Amplisens HPV HR screen-titre-FL and Amplisens HPV HR genotype-FL. Based on these results we developed the algorithm of HPV detection in samples of tumor tissue of patients with Lssc. The need for typing HPV-positive tissue samples with low concentration of HPV DNA was discussed.

[Morphological changes of testicular tissues in rats of different age groups, depending on the time of being in the abdominal cavity].

Pathological changes in the testes of animals of different age groups with the experimentally simulated cryptorchidism were evaluated. Animals were divided into 3 groups according to the time of fixation of testes into the abdominal cavity for a term of 2, 3 and 4 weeks. The changes were recorded at the time of bringing down the testes into the scrotum and at 14th and 28th day after bringing down. An assessment of pathological changes and the dynamics of the reparative capacity of testicular tissue in animals of different age groups at different stages of the study was performed. Experimental cryptorchidism leads to marked degenerative changes in the testes, up to the destruction of the elements of the testicular tissue in both groups. Comparative analysis of reparative capabilities revealed that potential of recovery of testicular tissue in young group of animals is much higher than that of mature animals.

[The role of nitric oxide synthesis induction and inhibition in regulation of blood neutrophil cell death during oxidative disbalance].

Modeling oxidative stress in vitro with 5mM H2O2 has demonstrated a protective role of nitric oxide on realization of constitutional blood neutrophil cell death. The NO-synthase inductor L-arginine and the inhibitor of nitric oxide synthesis, L-NAME, influenced on the amount of annexin-positive cells, the content of Bax protein, reactive oxygen species, cyclic nucleotides, and calcium homeostasis in neutrophils under conditions realizing programmed death during oxidative stress in vitro and under acute inflammation. During oxidative stress L-arginine normalized the increased intracellular Ca2+ level and the cAMP/cGMP ratio due to increase of cGMP level, stabilized metabolism and prolonged neutrophil life. During acute inflammation NO induction was insufficient for limitation of Ca2+ release into cytosol and for onset of the apoptotic effect; blockade of NO synthesis deteriorated this situation by activating neutrophil apoptosis due to the sharp increase in Ca2+ content and reduction of cyclic nucleotides in cytosol. The protective effect of NO on neutrophil cell death during oxidative dysbalance is not associated with regulation of apoptotic protein Bax.

Regulatory role of nitric oxide in neutrophil apoptosis.

Apoptosis of blood neutrophils from healthy donors was studied under conditions of cell culturing with different concentrations of H(2)O(2), selective NO synthase inhibitor, and inductor of NO synthesis (L-arginine). In vitro incubation of neutrophilic leukocytes with 5 mM H(2)O(2) led to activation of the apoptotic program in neutrophils, which was seen from increased content of Bax protein in the cells and increased number of apoptotic cells in the culture. Increased content of annexin-positive cells after incubation of neutrophil culture with NO synthase inhibitor suggests involvement of NO in the regulation of neutrophil apoptosis under conditions of oxidative stress, while L-arginine prevented H(2)O(2)-induced programmed cell death.

[Suppurative complications of chickenpox in children]. / Gnoinye oslozhneniia vetrianoi ospy u detei.

In 1979-1990 213 children with local (n = 155) and generalized (n = 58) purulent complications of chickenpox, developing on days 2-6 after the eruptions, underwent treatment. They occurred mostly in children aged from 12 months to 3 years. Treatment of local forms caused no difficulties and was successful in all cases. A very severe course was encountered in 25 children with the development of ligneous phlegmon and toxico-infectious shock; 6 of them died. The extent of the lesion in these cases included more than 10% of the skin surface. The treatment consisted in multiple incisions, the prescription of antibiotics in maximal doses, and immunostimulating and infusion therapy.

[Gas-chromatographic laparocentesis in the diagnosis of closed injuries of hollow abdominal organs]. / Gazokhromatograficheskii laparotsentez v diagnostike zakrytykh povrezhdenii polykh organov zhivota.

A principally new method of diagnostics of closed injuries of hollow organs of the abdomen was used in clinical practice in 36 patients. It consists in performing laparocentesis in combination with gas chromatography. The technique, indications and contraindications to gas chromatographic laparocentesis as a method of diagnostics of such injuries are described.

[Use of the indirect hemagglutination reaction for detecting antibodies to Pseudomonas pyocyanea in acute suppurative destructive pneumonia]. / Primenenie reaktsii nepriamoi gemaggliutinatsii dlia vyiavleniia antitel k sinegnoinoi palochke u bol'nykh ostroi gnoinoi destruktivnoi pnevmoniei.

No less than 4-fold increase in the antibody titres to Pseudomonas aeruginosa during the infectious process served as laboratory confirmation of its participation in the infectious process. In 49 of 91 patients hemagglutining titre exceeded the diagnostic one (1:640). In 9 patients (chiefly in infants) hemagglutinin titre remained low, but there was a rise of antibody level to Pseudomonas aeruginosa during the disease. High hemagglutinin titres were noted in 12 patients on admission to the clinic, with reduction of the antibody titres at the late periods of the disease. Antibody titres remained unchanged during the disease in 15 patients. In 3 cases the indirect hemagglutination test was assesed as negative. In the rest of the patients hemagglutinin titres varied within the range of the diagnostic titre. Thus, the indirect hemagglutination test with erythrocytic diagnostic agent permitting to determine antibodies to Pseudomonas aeruginosa could be used at the clinic in combination with bacteriological and other investigations for establishing etiology of the destructive process.

[Characteristics of the agents of acute suppurative destruction of the lungs in children]. / Kharakteristika vozbuditelei ostroi gnoinoi destruktsii legkikh u detei.

The authors carried out bacteriological studies of the blood, the pleural cavity puncture material, the discharge from the bronchi in bronchoscopy, and also of smears from the nasopharynx of 40 children with acute purulent destruction of the lungs. Staphylococci, often in association with various Gram-negative bacilli, chiefly of the Pseudomonas genus, and Enterobacteriaceae family, prevailed in the cultures of the pathological material. Pathogenic staphylococcus was found in the nasopharynx of patients in 65% of cases. Along with staphylococci in over half of the patients there were revealed Gram-negative bacteria, and yeast-like Candida fungi. In the majority of patients the microbes isolated from the pharynx and the lungs were identical. In these cases bacteriological examination of pharyngeal smears can serve as an auxiliary method of laboratory diagnosis. The isolated straphylococcus strains possessed the majority of the pathogenicity signs; in the great majority of cases they were resistant to the antibiotics the most widely used in the therapeutic practice; among them strains of phage types 80, 54, 75, 77, and 81 prevailed. Thus, at different periods of the disease various types of the microbes prevailed in the materials obtained from the patients. In acute purulent destruction of the lungs bacteriological examination should be carried in dynamics.