Alterations in mtDNA: a qualitative and quantitative study associated with cervical cancer development.

OBJECTIVE: High-risk human papillomaviruses are the causative agent of cervical carcinogenesis. Additionally, a number of other unknown factors are also instrumental in the development of cancer. The aim of this present study was the analysis of the mutations in the D-loop region of mitochondrial DNA, and 4.997 bp deletion during cervical cancer development. Our research also extended to the relationship between mtDNA copy number, ROS (reactive oxygen species) production and the MnSOD (manganese superoxide dismutase) expression level. METHODS: The study group consisted of postoperative tissues from patients diagnosed with L-SIL, H-SIL and squamous cell cervical carcinomas. A quantitative real-time polymerase chain reaction was used to determine the copy number of the mitochondrial DNA, and MnSOD mRNA expression levels. A PCR amplification and a sequencing of DNA were used for the identification of HPV DNA and mtDNA mutations. RESULTS: A total of 62 point mutations in the D-loop region of mtDNA were found in study patients. The mitochondrial DNA copy number increased during cervical cancer development when compared to the corresponding tissues in the control samples. About 70% of the mtDNA copy number have a 4.997 bp deletion in L-SIL. We also observed an increase in ROS generation during cervical cancer development. CONCLUSION: Alterations in mtDNA both qualitatively (by mutations) and quantitatively (by mtDNA copy number) are associated with cervical cancer developments. High levels of mtDNA copy with a 4.997 bp deletion in L-SIL cells can be associated with the susceptibility of cells to HPV persistent infection and cervical cancer development.

Association of the IGF-I promoter P1 polymorphism with risk of cervical cancer.

Human papillomaviruses (HPV16, HPV18, HPV31, HPV33) are etiological agents in the development of cervical cancer. HPVs infect epithelial cells and depend on epithelial differentiation for the completion of their life cycle. Insulin-like growth factor I (IGF-I) is a potent mitogen involved in the regulation of cell proliferation and apoptosis of many cell types including normal and transformed epithelial cells. Deregulation of IGF-I expression and action is linked to diverse pathologies including cancer. A polymorphism in the P1 promoter region of the IGF-I gene may directly influence its expression. Using the PCR-SSCP method and sequencing of DNA, we identified a single nucleotide polymorphism (SNP) at -383(C>T) position of promoter P1 of the IGF-I in 16% of the study HPV-positive women with precancerous and cancerous lesions. In vitro, we observed that the SNP at-383(C>T) site significantly increased the reporter gene expresion in the HepG2 cell line, but not in the HeLa cell line relative to the wild type promoter. It suggests that the studied SNP can change expression of the IGF-I gene in distinct ways in different types of tissues. Deregulation of expression of the IGF-I gene can affect normal epithelium development and in case of HPV infection can potentially disrupt the virus life cycle and stimulate its passage into the oncogenic life cycle or persistent viral infections. Therefore, we propose that SNP C>T at the -383 position of P1 promoter may be one of the helpful prognostic markers in the diagnosis of cervical cancer development of women with persistent infection in the ectocervical epithelium. We have not found any association between the polymorphism CA repeats in the promoter P1 region of the IGF-I gene and suceptibility to HPV infection and cervical cancer development. The (CA)19 allele was the most common in the study of this group of women.

The prevalence of leptotrichia amnionii in cervical swabs of HPV positive and negative women.

UNLABELLED: The aim of the present study was to determine the prevalence of Leptotrichia amnionii in cervical swabs of women and its possible correlation with HPV infection and the stage of cervical cancer. MATERIAL AND METHODS: A total of 139 cervical swabs from healthy women with normal cytology, with dysplastic changes and with cervical cancer were tested for the presence of L.amnionii and high-risk HPV DNA by PCR methods. RESULTS: L. amnionii was found in normal vaginal flora and in women with bacterial vaginosis (BV), which suggests that it may be oportunistic pathogen. L. amnionii infection was diagnosed in 13.7% (19/139). Statistical analysis showed that there was positive association (p < 0.01) between the presence of L.amnionii in women with cervical cancer (38.5%) and its presence in women without cancer (11.1%). On the other hand, there was no statistically significant association between L.amnionii and HPV infections. CONCLUSION: The data presented in this study show for the first time the prevalence of L. amnionii infection in cervical specimens collected from 2004-2006 in Poznan and Lublin, Poland, and its association with HPV infection and the stage of carcinogenesis of the cervix.

Prevalence of Chlamydia trachomatis and herpes simplex virus 2 in cervical carcinoma associated with human papillomavirus detected in paraffin-sectioned samples.

PURPOSE: The aim of the study was to evaluate the frequency of occurrence of HPV and co-infection: Chlamydia (C.) trachomatis and HSV-2 in cervical cancer. MATERIAL AND METHODS: The study group consisted of 570 paraffin-sectioned samples of patients with cervical cancer. In order to identify viral and bacterial DNA in DNA isolated from archival, postoperative material, PCR analysis was performed using starters complementary to various types of HPV, HSV-2 and C. trachomatis. RESULTS: In patients with squamous cell cervical cancer the presence of 33 types of HPV was found in 90% (468/520). HPV 16 infections occurred in 69.4% (325/468), while HPV 18 infections were present in 30.5% (143/468) of cases. In the control group C. trachomatis and HSV-2 were observed in four cases (4/50), which constitute 8.0%. In the tissue sections from patients with squamous cell cervical carcinoma, C. trachomatis was identified in 26% (135/520) and HSV-2 in 28% (145/520). In the group of patients with adenocarcinoma C. trachomatis infections were found in 24% (12/50) and herpes virus was identified in 30% (15/50). Statistically significantly higher frequency of occurrence of HSV-2 and C. trachomatis was observed in paraffin-sectioned samples for patients with invasive cervical cancer compared to the control group, without neoplastic lesions (p < 0.05). No correlation was found between frequency of occurrence of HPV and C. trachomatis and of HPV and HSV-2 detected in paraffin-sectioned samples for cervical carcinoma.

Remote Estimation of Video-Based Vital Signs in Emotion Invocation Studies.

The goal of this study is to examine the influence of various imitated and video invoked emotions on the vital signs (respiratory and pulse rates). We also perform an analysis of the possibility to extract signals from sequences acquired with cost-effective cameras. The preliminary results show that the respiratory rate allows for better separation of some emotions than the pulse rate, yet this relation highly depends on a subject. The invoked positive emotion resulted in a respiratory rate difference >1.8 bpm, comparing to the average respiration rate of all neutral results (in 89% of observations). Visual facial expression in many cases was insufficient for emotion recognition (in video based experiment only 11.4% of visual responses were classified as an expected emotion).

Pose-Invariant Face Detection by Replacing Deep Neurons with Capsules for Thermal Imagery in Telemedicine.

The aim of this work was to examine the potential of thermal imaging as a cost-effective tool for convenient, nonintrusive remote monitoring of elderly people in different possible head orientations, without imposing specific behavior on users, e.g., looking toward the camera. Illumination and pose invariant head tracking is important for many medical applications as it can provide information, e.g., about vital signs, sensory experiences, injuries, wellbeing. In the performed experiments, we investigated the influence of different modifications of images (rotation, displacement of facial features, and displacement of facial quarters) on the prediction accuracy. Specifically, two models were tested on the set of collected low-resolution thermal images: Inception V3 Convolutional Neural Network (CNN) and Hinton's Capsule Network. The preliminary results confirm that the prediction ability of the model based on capsules can deal with different head orientations much better than CNN (for the 45° head rotation Capsule Network achieved ~% accuracy while CNN only 9.5%).

Long Distance Vital Signs Monitoring with Person Identification for Smart Home Solutions.

Imaging photoplethysmography has already been proved to be successful in short distance (below 1m). However, most of the real-life use cases of measuring vital signs require the system to work at longer distances, to be both more reliable and convenient for the user. The possible scenarios that system designers must have in mind include monitoring of the vital signs of residents in nursing homes, disabled people, who can't move, constant support for people regardless of the performed activity (e.g., during sleeping), infants, etc. In this work we verified the possibility of remote pulse estimation at a distance above 5m. Additionally, we integrated the deep learning algorithm for person tracking and identification, even when facial features are not visible. In this way, we enabled the collection of user specific measurements to create personalized vital signs patterns and we provided the support for monitoring of multiple people using one video stream. The preliminary results showed that it is possible to accurately (RMSE $< 2.8$ beats per minute) extract pulse from visible light sequences acquired with a webcam at a distance of 6m after applying a proper image pre-processing algorithm.

The motion influence on respiration rate estimation from low-resolution thermal sequences during attention focusing tasks.

Global aging has led to a growing expectancy for creating home-based platforms for indoor monitoring of elderly people. A motivation is to provide a non-intrusive technique, which does not require special activities of a patient but allows for remote monitoring of elderly people while assisting them with their daily activities. The goal of our study was to evaluate motion performed by a person focused on a specific task and check if this motion disrupts estimation of respiration rate. The preliminary results show that it is possible to reliable estimate respiration rate by focusing attention of a patient on a certain activity. The respiratory rate analyzed for silent reading task was estimated with mean error 0.27 breaths per minute (bpm), while for reading aloud task with 1.18 bpm. The observed head motion during the reading aloud task was 1.5 higher that for silent reading and about two times smaller for a case in which subjects were not focused on any task.

Amniotic band syndrome in dichorionic diamniotic twin pregnancy.

BACKGROUND: Amniotic band syndrome (ABS) is a rare disorder which leads to a number of deformities of the fetus body. The treatment depends on the severity of the defect and the extent of the deformity. CASE REPORT: A 36-year-old primigravida with a dichorionic diamniotic (DCDA) twin pregnancy was diagnosed during the first-trimester ultrasonography with fetal lower part edema of one twin caused by amniotic bands. A selective termination of the affected fetus was performed. The remainder part of the pregnancy was normal. A healthy newborn was delivered at term. After delivering the placenta, the presence of fetus papyraceus was detected. The amniotic bands were unidentifiable in the pathologist's examination. A reliable ultrasonographic diagnosis enables the detecting ABS in early pregnancy. CONCLUSION: In the case of ABS in DCDA twin pregnancy, conducting a selective termination of the affected fetus creates the opportunity for the proper development of the healthy fetus as well as reaching its full maturity. HIPPOKRATIA 2017, 21(1): 46-48.

Chlamydia trachomatis and herpes simplex virus 2 infection in vulvar intraepithelial neoplasia associated with human papillomavirus.

BACKGROUND: The role of viral and bacterial co-infection is stressed in VIN. A view that VIN is a sexually transmitted disease made the area of research larger and stimulated scientists to seek other sexually transmitted factors, among which Chlamydia trachomatis and Herpes simplex are frequently examined. PURPOSE: The aim of the study was to evaluate the frequency of occurrence of HPV DNA and the frequency of co-infection with Herpes virus type 2 and Chlamydia trachomatis in VIN. MATERIAL AND METHODS: We identified archival diagnostic phase tissue specimens from 41 cases of vulvar intraepithelial neoplasia III. From the same paraffin blocks containing material from the margins of surgical sections during vulvectomy, normal epithelial tissue fragments were collected. They constituted the control group. Lesion characteristics were examined in comparison with the presence of HPV DNA, HSV-2 and Chlamydia trachomatsis. Identification was performed using PCR. RESULTS: In the study group HPV infection was found in 75.6% of cases. In 73% of cases it was HPV 16. In the control group we found HPV 16 DNA in only one case (2.43%). In the HPV positive study group HPV 16 was found in 30 (30/31) cases. In only one case (1/31) it was HPV 18 type. In the study group of 41 cases with VIN, HSV-2 infection was found in six cases (14.63%). In comparison with the control group (9.75%) the difference was not statistically significant. The frequency of occurrence of Chlamydia trachomatis in the analyzed study material was 14.63% (6/41) and in the control group it was 9.75% (4/41). The difference was not statistically significant. Statistical analyses of correlations between the occurrence of DNA HPV and HSV-2 as well as of HPV and Chlamydia trachomatis showed no correlation in either case. CONCLUSION: No correlation was found between the frequency of occurrence of HPV and HSV-2 and HPV and Chlamydia trachomatis in either group.

Apoptosis of HeLa cell lines incubated with retinol.

PURPOSE: The aim of the study was to evaluate a soluble form of APO-1/Fas antigen in supernatants from HeLa cell line culture after 24 and 72 h of incubation with selected retinoic acid concentrations. MATERIALS AND METHODS: HPV18-positive cell lines were cultivated with All-trans-retinoic acid (ATRA) at concentrations of 1 x 10(-6) and 1 x 10(-9) M. The cultures were incubated for 24 and 72 h. A control culture with 3 microl of DMSO was incubated under identical conditions. The concentrations of soluble APO-1/Fas antigen in cell culture supernatants were estimated using an ELISA method. RESULTS: The culture with 72-h incubation with retinoic acid proved to be toxic to cells and was excluded from the analysis. The results obtained showed a significantly lower concentrations of soluble APO-1/Fas antigen in supernatants from cell lines incubated with retinol for 24 h than in the controls. CONCLUSIONS: The higher concentrations of soluble APO-1/Fas antigen in supernatants from the HeLa cell line without retinol may constitute a protective mechanism of the cells infected with the virus before undergoing Fas/FasL-dependent apoptosis. Lower concentrations of sAPO-1/Fas antigen in the supernatant from HeLa cell culture incubated with retinol may suggest that mechanisms protecting infected cells against Fas/FasL-mediated apoptosis become defective under the influence of retinol. Our studies confirm that Vitamin A and its analogues inhibit the proliferation of cells associated with HPV infection and suggest promising effects of retinoid therapy in inhibiting the progression of early cervical lesions to cancer.

Content of beta-carotene in blood serum of human papillomavirus infected women with cervical dysplasias.

Studies were carried out in 528 women hospitalized in the Department of Obstetrics and Gynecology Medical Academy in Lublin. Besides the control group, patients were classified according to the observed histopathological changes in the cervix (CIN) and found infections with human papillomavirus (HPV). In all cases beta-carotene content in blood serum was examined. HPV infection was probably a cause of decrease of beta-carotene content. It was found that with increased advancement of cervical dysplasia the level of beta-carotene in serum decreased.

Frequency of HPV infection and GSH levels in plasma of women with cervical dysplasia.

Human papillomavirus (HPV) can be a cause of cervical intraepithelial neoplasia (CIN) and cervical cancer. The level of antioxidant compounds in plasma (retinol, alfa-tocoferol, vitamins C and E, glutathione) can modulate the progression of latent HPV infection to subclinical lesions and CIN. Our studies show that in the cases of HPV infection glutathione (GSH) content decreased, but glutathione disulfide (GSSG) levels increased. The quantitative GSH/GSSG ratio reflects the above changes of both peptides which, the more distinct, the greater the pathological changes in the uterine cervix.

Folate deficiency and cervical intraepithelial neoplasia.

The presence of HPV, using the Digene Hybrid Capture System, was identified in a group of 324 women with CIN and in 228 women with normal cytological smears. Risk of occurrence of CIN was 40 times higher for high risk HPV types. The serum folic acid level and the level of antioxidant compounds in plasma (retinol, alpha-tocopherol, vitamins C and E) in women of the studied and control group was determined by HPLC (high-performance liquid chromatography-reversed phase). Statistically lower levels of folic acid were found in the women with CIN-HPV (+) (OR: 7.5: 95% CI: 1.2-9.7). Studies have shown that lower levels of antioxidants coexisting with low levels of folic acid increases the risk of CIN development.

Evaluation of DNA mismatch repair system in cervical dysplasias and invasive carcinomas related to HPV infection.

UNLABELLED: The aim of this study was to answer the question whether the products of hMSH2 and hMLH1 genes take part in the mutation track of cervical carcinoma. METHODS: IgG1 monoclonal antibodies (Pharmingen) detecting epitopes characteristic of hMLH1 and hMSH2 were used in the present study. The value of the half-quantitative H-score coefficient was calculated. Its threshold value was 0.4. Identification of 16 and 18 HPV types was performed by PCR. RESULTS: An intensified hMLH1 protein expression was observed both in the squamous epithelial carcinomas and cervical adenocarcinomas (H-score of 1.44 and 0.98, respectively) as compared to the control (H-score of 0.9). However, a decreased expression of hMSH2 protein was observed in the analysed cases of carcinoma (0.9 and 0.7) as compared to the control group (1.2). An intensified expression in G3 for hMLH1 and higher hMLH1 in comparison to hMSH2 was observed. CONCLUSIONS: 1. A considerable expression of hMLH1 and hMLH1 proteins was observed in the tissues with invasive cervical carcinoma not only within epithelial but also in stromal cells. 2. More intense expression of hMLH1 and hMSH2 was observed in invasive carcinomas and CIN than in the non-neoplastic cervical tissue lesions (erosion). 3. A stronger expression was observed for the hMLH1 than for the hMSH2 proteins--contrary to the cases of carcinomas of the uterine corpus and endometrial carcinoma.

DNA adducts in squamous cell cervical carcinomas associated with HPV infection.

UNLABELLED: The aim of the study was qualitative and quantitative evaluation of DNA adducts in squamous cell cervical carcinomas associated with oncogenic HPV infection. MATERIAL: The study material consisted of oncogenic tissue collected during the surgeries of seven women aged 37 to 52 who were undergoing surgical treatment due to squamous cell cervical carcinoma. The control group consisted of 3 tissue fragments from morphologically normal cervix collected from patients undergoing surgery due to uterine myomas. METHODS: DNA from the tissues was isolated using Genomic Prep Plus kit from A&A Biotechnology, Austria. Amplification reactions detecting HPV DNA presence in the tissue fragments were performed using specific starters allowing for amplification of conservative genome fragments within L1, E6 and E7 Papilloma viruses. After extraction, the DNA specimens underwent enzymatic digestion to nucleotides and marked on the 5' end using gamma32P-post labeling technique. Division and quantitative evaluation of DNA adducts was performed using thin-layer chromatography (TLC) on PEI-cellulose plates. Qualitative radioactivity measurements were performed using Bio-Imaging analyzer in quantitative mode. RESULTS: In all fragments, including the control, HPV 16 and/or 18 DNA was found. Mean adduct content in cervical carcinoma tissues was 289 adducts per 10(9) nucleotides and was higher than mean adduct content in control tissues (57 adducts per 10(9) nucleotides). CONCLUSIONS: The study results suggest that the content of DNA adducts in squamous cell cervical cancer associated with HPV infection may serve as a molecular marker of oncogenesis in this organ.

Relationship between HPV infection of the cervix and blood serum levels of steroid hormones among pre- and postmenopausal women.

Human papillomaviruses (HPVs) are frequently present in anogenital lesions but little is known about their role in carcinogenesis. There are steroid hormone response elements in virus genomes that influence expression patterns of viral genes. Activity of the elements may contribute to development of neoplasia in case of hormone level anomalies. Our study was to determine whether the presence of HPV DNA in cervical smears correlates with abnormal levels, of steroid hormones in blood serum. One hundred women aged 40-62 participated in the tests and were divided into two groups: premenopausal and postmenopausal (45 and 55 individuals, respectively). Presence of HPV DNA in cervical smears was detected by PCR and Southern blot hybridisation. Progesterone and estradiol levels in blood serum were measured by radioimmunoassay. Our study showed a higher prevalence of HPV DNA in women with higher levels of progesterone in blood serum. A relationship between hormone level and HPV DNA prevalence should alert clinicians about using hormone contraceptives and hormone replacement therapy.

Content of folic acid and free homocysteine in blood serum of human papillomavirus-infected women with cervical dysplasia.

The authors estimated the concentrations of folic acid and free homocysteine in the blood serum of women with CIN III (cervical intraepithelial neoplasia-Burghard's classification) infected with DNA HPV (human papillomaviruses) of type 16 and/or 18. The control group consisted of 49 patients with normal cytological smears without HPV infection. Types 16 and/or 18 DNA HPV were found in 50 patients. This women qualified for the studied group. The sequence of DNA HPV type 16 and/or 18 was identified with the PCR method (polymerase chain reaction). The high-performance liquid chromatography (HPLC) method was employed to evaluate the levels of folic acid and free homocysteine in the blood serum of the examined patients. Significantly lower levels of folic acid and higher levels of free homocysteine were observed in the blood serum of HPV-positive patients with CIN III. The correlation was found between serum concentrations of folic acid and free homocysteine in both groups.

The diagnostic value of cytomorphological traits in low and high risk type HPV infections.

The purpose of the study was to establish the diagnostic value of cytomorphological traits present in cervical smears in determining HPV infection. We examined 528 women, most of them between 18 and 34 years of age and sexually active, for the prevalence of HPV infection and compared the results of HPV DNA detection with the presence of cytomorphological traits of HPV infection in their cervical smears. The Digene Hybride Capture System technique was employed to detect HPV DNA in cytological material; cervical smears were examined by an experienced pathologist, who did not have prior knowledge of the HPV DNA test results. None of the cytomorphological traits examined proved to be pathognomonic for HPV infection. The diagnosis of HPV infection cannot be determined with certainty by a routine cytological examination. Only 15% of cervical smears from women infected with HPV showed classic cytomorphological traits of HPV infection - koilocytosis and/or dyskeratosis. We conclude that if more than three non-classic traits of HPV infection are found in a cervical smear, HPV infection can be suspected.

Clinical and morphological analysis of endometriosis in women of the Lublin Macroregion between 1987 and 1992.

Histopathological examinations performed between 1987 and 1992 at the Department of Pathology of the Lublin Medical Academy were analysed statistically. Endometriosis was found in 885 cases, i.e. 13.3% of all gynecological cases. An increased frequency as well as a shift in the peak age of incidence to the third decade, were found.