MOLECULAR MARKERS OF THE PROGRESSION OF CONJUNCTIVAL NEOPLASTIC EPITHELIAL LESIONS.

Conjunctival epithelial lesions vary from benign to borderline malignancy to malignant, therefore it is extremely important to detect the molecular markers of the malignant progression of conjunctival intraepithelial lesions. The aim of our study was to analyse the molecular markers of the progression of conjunctival intraepithelial lesions. We have analysed Ki67, PHH3, Bcl2, P53, P63 and CK7 using standard immunohistochemistry. In addition, we have calculated the squamous-glandular index based on the evaluation of H&E stained specimens and as the ratio of P63/CK7. The results of our study indicated that the presence of squamous epithelium is significantly increased during the progression of conjunctival intraepithelial lesions, and therefore the squamous-glandular index is also increased. In addition, it is possible to divide conjunctival intraepithelial lesions as low grade and high grade lesions based on the distribution of proliferation and apoptosis markers.

MICROENVIRONMENT ALTERATIONS IN CONJUNCTIVAL NEOPLASTIC LEOSIONS WITH DIFFERENT PROLIFERATION-APOPTOTIC CHARACTERISTICS.

Different studies indicate that tumor infiltrating lymphocytes (TILs) and tumor associated neutrophils (TANs) play an important role during the progression of malignant tumors. We have analysed the distribution of tumor associated neutrophils (TANs) and tumor infiltrating lymphocytes (TILs) in different conjunctival lesions, with different proliferation and apoptotic characteristics. The distribution of TILs and TANs were evaluated in standard haematoxylin and eosin (H&E) stained sections using the digital pathology software QuPathin normal conjunctiva, actinic keratosis, pterigea, conjunctival intraepithelial neoplasias (CoIN1-3) and conjunctival squamous cell carcinoma (CSCC). In addition, the expression of following markers were investigated using standard immunohistochemistry: Ki67, Bcl2, p53, CD3, CD8 and Foxp3. The study results indicated that the number of TILs and TANs are significantly increased during the progression of conjunctival intraepithelial lesions. Also, the number of TILs and TANs significantly correlate with higher proliferation index, lower apoptotic index and the p53 mutation status.

Evaluation of state opioid substitution treatment program in Georgia.

AIMS: Evaluation of State Opioid Substitution Treatment OST (methadone and buprenorphine/naloxone- Addnok-N) program in Georgia and optimization of the routine measurement instrument. Patients were recruited from 4 Tbilisi and 5 regional State Programs in May-October 2013. 2 structured self-questionnaires (one - anonymous for sensitive questions) were developed for patients to assess demographics, retention in treatment, mean drug dose, HIV and Hepatitis C and B status, illicit drug and alcohol use, social activities, crime involvement, health status, HIV risk behavior, treatment compliance and satisfaction. 608 patients (7 females) were surveyed (512 - on Methadone, 96 - on buprenorphine/naloxone). 337 (1 female) patients completed an anonymous questionnaire. Mean age - 39.43±8.7 (21-65 years). 10 (1.64%) respondents were HIV positive; 448 (73.68%) - HCV+ and 24 (3.95%) - HBV+; average methadone dose - 39.27±22.2mg; buprenorphine/naloxone - 7.4±3.6 mg; 64 (40%) of employed began working while in program; 365 (60%) have been in treatment for less than 1 year, and 146 (24%) - for 1-3 years vs. 258 (51%) out of 506 patients surveyed in 2011. 494 (81.2%) reported improvement of social status and 508 (83.5%) - of health status. 305 (90.5%) out of 337 reported no- and 30 (8.9%) - reduction of criminal activity. 467 (76.81%) patients attended individual and 200 (32.9%)-group psychotherapy sessions with various frequencies. The common adverse events: sleep disturbances - 48.84%; weakness - 50.82%; mood disturbances - 42.44%, and heaviness - 36.35%. 257 (46%) reported using of alcohol; 16 - opioids; 29 - sedative/hypnotics; 8 - marijuana and 1 - ATS past 30 days; 55 - drug injection and 11 - sharing of any injection equipment past 6 months. State OST program is effective in Georgia in terms of reduction of illegal drug use, injection risk behavior and criminal activity, and on the other hand - improving of social activity and general health. Treatment retention is less as compared with 2011 survey.

Needs assessment and treatment compliance at state opioid substitution treatment programes in Georgia.

AIMS: conduct needs assessments and treatment compliance evaluations in MMT and Suboxone Substitution State Programs in Georgia (Republic of). 506 patients (2 females) were surveyed (92% on Methadone, 8% on Suboxone) from 6 Tbilisi and 4 regional State Programs in 2011 November. Mean age - 40±8,56 (22-65) year; 254 (51.4%) were in treatment for 1-3 year. Evaluation was carried out on the base of structured self-questionnaire that covers demographics, drug use history, general drug use trends, psychotherapeutic sessions' acceptance and open label question regarding treatment challenges and satisfaction. 305 (60.3%) attended individual and 57 (11.3%) group psychotherapy sessions with 50.79% attending once/month or rare. The main reason given for therapy non-attendance - no needs for it (29.48%); the main drugs before admission - heroin (80.04%), buprenorphine (53.49%); Main drugs used in Georgia nowadays - desomorphine ("crocodile"), alcohol and marihuana. Commonly used drugs by program patients (136 positive answers) - alcohol-13.62%, marihuana-10.39%, pregabalin - 8.17%, opioids- 6.62% (mostly-"crocodile"), home-made stimulants-6.23%, sedatives -5.45%. 55.4% are extremely satisfied with treatment, 82.4% - with program staff. Patients' main wishes- free of charge programs (46.4%) and provide take-home doses (22.07%). Methadone and Suboxone ST are being well accepted in Georgia and appear to be reducing illegal opioid use. However, the psychotherapeutic sessions' attendance is very low.

Evaluation of the quality of life (Whoqol-Bref) among methadone and suboxone substitution state program patients and healthy volunteers in Georgia.

AIMS: to evaluate QOL patients being treated with methadone and Suboxone in State-sponsored programs in Georgia. The WHOQOL-BREF (26 questions) version was administered to patients in State substitution program and healthy volunteers to assess their overall function and life satisfaction in physical, mental, social health, and environment domains. Domain scores were calculated and converted to 4-20 and 0-100 scales, identical to the WHOQOL-100. 485 patients (309 from 6 Tbilisi and 176 from 5 regional centers) and 50 healthy volunteers (13 male, 37 female) were surveyed. Significant differences were observed between new admitted patients (0-3 month) and healthy controls by mean physical (47.5 vs. 51.94; CI 95%); psychological (55.0 vs. 60.50; CI 95%) and environmental (46.2 vs. 52.2; CI 95%) domains, but not by social relationships or between Tbilisi and regions. The Social domain scores were raised in accordance with time spent in treatment reaching a maximum improvement within 1-3 years (social- 72.8 vs. 67.7; CI 95%), further with few descending tends round the healthy people's scores. These pilot data show decrements in QOL among patients entering maintenance treatment with improvements in the course of maintenance treatment. It is possible that the increased indicators in social domain up to especially high level within the first 3 years is the result of subjective factors, with the subsequent return to healthy community level.

Integrity of human YACs during propagation in recombination-deficient yeast strains.

Several isogenic strains with defects in recombination/repair genes (RAD1, RAD50, RAD51, RAD52, RAD54, and RAD55) were examined for their ability to propagate accurately a variety of linear and circular yeast artificial chromosomes (YACs) containing human DNA inserts. To assess YAC stability, the human DNA inserts were internally marked by an ADE2-pBR-URA3 cassette. Following selection for Ura- clones on 5-fluoroorotic acid containing medium, the following types of YAC deletions were identified: (i) those caused by homologous recombination with a telomeric pBR sequence; (ii) internal deletions, presumed to occur by recombination between commonly occurring DNA repeats such as Alu and LINE sequences; and (iii) deletions leading to loss of part of a YAC arm. rad52 host strains, but not other recombination-deficient strains, decreased the rate of all types of YAC deletions 25- to 400-fold. We have also developed and tested kar1 strains with a conditional RAD52 gene that allow transfer of a YAC from any host into a recombination-deficient background. These strains provide an efficient tool for stabilization of YACs and are useful for allowing additional recombinational modification of YACs.

Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast (YACs).

Mammalian DNAs cloned as artificial chromosomes in yeast (YACs) frequently are chimeras formed between noncontiguous DNAs. Using pairs of human and mouse YACs we examined the contribution of recombination during transformation or subsequent mitotic growth to chimeric YAC formation. The DNA from pairs of yeast strains containing homologous or heterologous YACs was transformed into a third strain under conditions typical for the development of YAC libraries. One YAC was selected and the presence of the second was then determined. Co-penetration of large molecules, as deduced from co-transformation of markers identifying the different YACs, was > 50%. In approximately half the cells receiving two homologous YACs, the YACs had undergone recombination. Co-transformation depends on recombination since it was reduced nearly 10-fold when the YACs were heterologous. While mitotic recombination between homologous YACs is nearly 100-fold higher than for yeast chromosomes, the level is still much lower than observed during transformation. To investigate the role of commonly occurring Alu repeats in chimera formation, spheroplasts were transformed with various human YACs and an unselected DNA fragment containing an Alu at one end and a telomere at the other. When unbroken YACs were used, between 1 and 6% of the selected YACs could incorporate the fragment as compared to 49% when the YACs were broken. We propose that Alu's or other commonly occurring repeats could be an important source of chimeric YACs. Since the frequency of chimeras formed between YACs or a YAC and an Alu-containing fragment was reduced when a rad52 mutant was the recipient and since intra-YAC deletions are reduced, rad52 and possibly other recombination-deficient mutants are expected to be useful for YAC library development.

[The causes of instability of artificial mini-chromosomes in yeasts mutant for chl genes]. / Izuchenie prichin nestabil'nosti iskusstvennykh mini-khromosom u mutantov drozhzhei po genam chl.

In mutants chl2, chl3, chl5, and chl6, which control mitotic chromosome transmission, the behaviour of the centromeric plasmids with various genes was analyzed. The main cause of chromosome instability in chl2, chl5, and chl6 is chromosome loss during cell division (1:0 segregation). The main cause of chromosome instability in chl3. is nondisjunction (2:0 segregation). According to this, the chl3 mutant, but not other chl's, cannot maintain the mini-chromosome with SUP11 gene. This gene causes cell death in high copy number. Chromosome nondisjunction in chl3 is also confirmed by the data on the mini-chromosome carrying CUP1 gene responsible for copper-resistance in yeast. The copper resistancy level in chl3 transformants is much higher than in chl5 or wild type transformants. Elevated copper resistance of chl3 transformants is caused by the transit accumulation of CUP1-carrying mini-chromosome in part of the cell population as a result of segregation mistakes upon cell divisions. Thus, the CHL3 gene is a new gene that controls the process of mitotic chromosome disjunction in yeast.

[Behavior in a cell of artificial mini-chromosomes during induced transcription of centromeric DNA]. / Povedenie v kletke iskusstvennykh mini-khromosom pri indutsirovannoi transkriptsii tsentromernoi DNK.

Hybrid yeast plasmids were constructed, containing the centromere loci CEN3 under the control of two inducible yeast promoters--GAL10 and PHO5. It was shown, that during the induction of transcription from the GAL10 promoter the decrease in mitotic stability of minichromosome is affected both by partial disruption of centromere function by transcription and by influence of galactose on the number of residual cell divisions. In two strains the activity of GAL10 promoter was considerably higher than that of the PHO5 promoter. It is proposed to use the effect of minichromosome destabilization for evaluation of the relative promoter strength.

[Chromosome stability in saccharomycete yeasts]. / Izuchenie stabil'nosti khromosom u drozhzhei sakharomitsetov.

Mutants with high instability of chromosome III designated Chl+ (chromosome loss) were obtained after irradiation with UV the Z4221-3c1 haploid disomic for chromosome III. The Chl+ mutants can be divided into two classes: 1) CL2, CL3, CL7, CL9, CL11, CL12, CL13 with elevated level of spontaneous inter- and intragenic recombination; 2) CL4, CL8 which unstable maintenance of chromosome III not accompanied with elevation of mitotic recombination frequency. The CL4 and CL8 mutants also reveal, in contrast to other mutants, unstable maintenance of artificial mini-chromosomes with chromosomal replicator ARS1 and centromeric loci CEN3, CEN4, CEN5, CEN6, CEN11. Substitution of ARS1 for other yeast replicators (ARS2, ARS of 2 micron plasmid) leads to no stabilization of mini-chromosomes in mutants. The noncentromeric plasmids containing homologous replicator (or replicators) from Candida maltosa are maintained with the same frequency both in wild type and in mutants. So, the stability of mini-chromosomes in CL4 and CL8 is not connected with uneffective replication of these chromosomes. Instability of chromosome III and mini-chromosomes in CL4 and CL8 is controlled by two nonallelic genes designated chl14 and chl18. We suppose that these genes control the process of centromere interaction with mitotic spindle microtubules.

The stability of chromosomes in yeast.

CL mutants with high instability of chromosome III were UV-induced in haploid strain disomic for chromosome III. The obtained CL mutants can be divided into two groups: (1) CL2, CL3, CL7, CL11-CL13 with elevated level of spontaneous inter- and intragenic recombination and (2) CL4, CL8 in which instability of chromosome III is not accompanied by elevation of mitotic recombination frequency. CL4 and CL8 mutants also show unstable maintenance of artificial minichromosomes with different chromosomal replicators and centromeric loci. The instability of chromosome III and minichromosomes in CL4 and CL8 is determined by two nonallelic genes designated ch14 and ch18. The role of ch14 and ch18 genes in mitotic chromosome transmission is discussed.