Róbert Póka

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Variable response of Hermansky-Pudlak syndrome to prophylactic administration of 1-desamino 8D-arginine in subsequent pregnancies

Hermansky-Pudlak syndrome is characterised by oculocutaneous albinism and haemorrhagic diathesis. The bleeding tendency that is associated with this autosomal recessive disease is caused by storage-pool deficiency and has been reported to be controllable by prophylactic administration of 1-desamino 8D-arginine (desmopressin, DDAVP). The DDAVP prophylaxis at the first delivery of our patient did not prevent the severe haemorrhagic sequeal requiring transfusion of packed red cells and platelets, but the same preventive measure was successful at her second childbirth. Response to prophylactic DDAVP administration varies between as well as within patients with Hermansky-Pudlak syndrome.

Randomised trial comparing combinations of cyclophosphamide and cisplatin without or with doxorubicin or 4′-epi-doxorubicin in the treatment of advanced ovarian cancer

Forty‐eight patients with FIGO stage III and IV epithelial carcinomas of the ovary were entered in this randomised trial. Radical surgery was performed and no residual tumor with a diameter greater than 2 cm was left behind. Of these patients 62.5% (10/16) had a complete or partial response on cyclophosphamide + cisplatin (CP) 87.5% (14/16) on cyclophosphamide + doxorubicin + cisplatin (CAP) and cyclophosphamide + 4′‐epi‐doxorubicin + cisplatin (CEP). The median time to progression was 3.5 months on CP, 12.5 months on CAP and 11.0 months on CEP. Patients treated with CAP combination chemotherapy had generally longer progression‐free survival (log rank χ2 = 5.4; P = 0.04). No significant difference was found, however, between patients on CAP and CEP. The median survival times were 12.5 months on CP, 26.5 months on CAP and 14.0 months on CEP. Patients treated with CAP combination chemotherapy had generally longer survival (logrank χ2 = 9.08; P = 0.0099). No significant difference was found, however, between patients on CAP and CEP in terms of survival. Asymptomatic mild‐to‐moderate laboratory test toxicity occurred in 6–12% of patients on CP, 6–12% on CAP and no toxicity of this type and grade on CEP. Nausea and vomiting were also less severe and less frequent in the CEP group. Cardiotoxicity was seen in 12.5% (2/16) only in the CAP group.

The prognostic significance of HPV‐16 genome status of the lymph nodes, the integration status and p53 genotype in HPV‐16 positive cervical cancer: a long term follow up

Objective Prognostic evaluation of HPV‐16 genome status of the pelvic lymph nodes, the integration status of HPV‐16 and p53 codon 72 polymorphism in cervical cancer.

Female child sexual abuse within the family in a Hungarian county.

Background: The aim of the study was to analyze the characteristics of intrafamiliar female child sexual abuse and to explore common features that may be utilized as targets for possible methods of prevention. We also described the medical and legal approaches to handling child neglect. Methods: This was a descriptive, cross-sectional study on 52 sexually abused girls under the age of 18 at the Department of Obstetrics and Gynecology, Medical and Health Science Center of Debrecen. We prospectively recorded the data of all cases. Intrafamiliar events were defined if the victim and perpetrator belonged to the same family. Legal outcomes were also recorded. Results: During the 16-year period, 209 cases of sexual abuse were seen in our clinic, 52 of them had been involved in child sexual abuse within the family. This accounts for 25% of adolescent cases. Eighty-six percent of the victims were pupils, 50% of them were between 11 and 14 years of age. The perpetrator was the victim’s father in 44%, and the stepfather in 40%. There was a slight difference between the type of abuse among the pre- and postpubertal group of victims, but statistically it was not significant. The abuse occurred on multiple occasions in 52%. The occurrence rate of assault was the highest in the summer season (58%), mostly in the afternoon (42%) and it took place almost exclusively at home (98%). The mother accompanied the victim in 38% of the cases and the police in 40%. Vaginal penetration was the type of abuse in 75%, and sexual perversion in 25%. Six victims were physically injured, the presence of sperm could be confirmed on vulvovaginal smears in 2 cases. One pregnancy conceived. Nine cases were reported to the police and as a result of legal proceedings, 5 perpetrators have been sentenced. Conclusion: The majority of crimes take place within the family and are disclosed after multiple episodes. The small proportion of reported sexual assaults is the consequence of the lack of harmony between the Hungarian conditions of emergency care and the criminal law. Prevention calls for attention at all levels of child education, observation at off-school times, early involvement of health professionals, applying standardized medical guidelines and the modification of jurisdiction.

Detection of human papillomavirus deoxyribonucleic acid in the female genital tract

A total of 336 biopsies, scrapes and exfoliated cells from the cervix and from the lower genital tract were screened for human papilloma (HP) viral sequences of types 6, 11, 16 and 18 by Southern blot, dot blot and filter in situ (FISH) hybridizations with cloned 32P-radiolabeled HPV DNA probes. The specimens included cervical intraepithelial neoplasias (CIN I–III), carcinoma in situ and invasive carcinoma of the cervix and vagina, adenocarcinomas, vulvar and vaginal condylomata acuminata and healthy epithelial samples. The oncogenic HPV 16 was found in 46% of the cervical carcinomas. Most of the type 16 occurences (75%) represented the third stage of inooperable cases. Similarly, HPV 18 was also most frequently present in this stage as well as in carcinoma in situ and in CIN III (25%, 18%). At the same time, in condylomata acuminata, types 6 and 11 were detectable in 88.7% of cares. In all, 13.5% of the normal samples harboured HPV DNA.

Stage-related superoxide anion production of granulocytes of gynecologic cancer patients

OBJECTIVE To measure superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 58 gynecologic cancer patients and compare to that of healthy controls. METHODS PMNLs were separated from peripheral blood samples by Ficoll and subsequent Percoll gradient sedimentation. Baseline and phorbol-dibutyrate (100 nmol/l) stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome c (50 micromol/l) absorbance. Differences between the mean superoxide anion production of different patient groups and the control group were assessed by Students t-test. RESULTS The mean superoxide anion production of PMNLs of healthy controls was 1.855 nM/min/3 x 10(5) cells (SD=0.211 nM/min/3 x 10(5) cells). Superoxide anion production of gynecologic cancer patients and healthy controls varied in a wide range. PMNLs of patients had lower baseline and stimulated activity than those of healthy volunteers. The frequency of a mean superoxide production at least 2 x SD below the control showed a parallel increase with advancing stage. CONCLUSION Granulocytes of gynecologic cancer patients have reduced capacity and inducibility of superoxide anion production already at an early stage of disease.

The Enhanced Inhibitory Effect of Different Antitumor Agents in Self-Microemulsifying Drug Delivery Systems on Human Cervical Cancer HeLa Cells

The aim of this study was to develop topical self-microemulsifying drug delivery systems (SMEDDS) containing antitumor agents (bleomycin, cisplatin and ifosfamide) and to investigate their inhibitory potential in SMEDDS on human cervical cancer HeLa cells. The physicochemical properties of cytostatic drug loaded SMEDDS were characterized. The cytotoxicity of main components of SMEDDS was also investigated. Their IC50 values were determined. HeLa cells were treated by different concentrations of cisplatin, bleomycin and ifosfamide alone and in various SMEDDS. The inhibitory effect on cell growth was analyzed by MTT cell viability assay. Inflammation is a driving force that accelerates cancer development. The inhibitory effect of these antitumor agents has also been tested on HeLa cells in the presence of inflammatory mediators (IL-1-β, TNF-α) as an in vitro model of inflamed human cervix. Significant differences in the cytotoxicity of cytostatic drugs alone and in SMEDDS have been found in a concentration-dependent manner. The self-micro emulsifying system may potentiate the effectiveness of bleomycin, cisplatin and ifosfamide topically. The effect of SMEDDS containing antitumor agents was decreased significantly in the presence of inflammatory mediators. According to our experiments, the optimal SMEDDS formulation is 1:1:2:6:2 ratios of Isopropyl myristate, Capryol 90, Kolliphor RH 40, Cremophor RH40, Transcutol HP and Labrasol. It can be concluded that SMEDDS may increase the inhibitory effect of bleomycin, ifosfamide and cisplatin on human cervical cancer HeLa cells. Inflammation on HeLa cells hinders the effectiveness of SMEDDS containing antitumor agents. Our results might ensure useful data for development of optimal antitumor formulations.

HPV- and node status in cervical cancer long-term results

OBJECTIVE To evaluate prognostic significance of HPV-status in cervical cancer and to compare that with the prognostic significance of lymph-node status. METHODS Cervical cancer biopsy specimens from primaries and, in surgical cases, from pelvic lymph-nodes too were analysed for the presence of human papillomavirus type 16 DNA-sequences using PCR technique. The management of surgical cases with two exceptions included Wertheims hysterectomy predominantly with preoperative local radiotherapy and also with postoperative local and external beam radiotherapy depending on the histology. Non-surgical cases were treated with combined local and external radiotherapy to pelvic fields. RESULTS Patients have been followed up for an average of 37 months after treatment ranging between 0 and 102 months. The mean progression-free survival time of surgical and non-surgical cases were 43 and 28 months, respectively. Patients with HPV-16 positive biopsies from the cervical primary had an average progression-free survival of 37 months, the same as those with HPV-16 negative cervical biopsies. Those patients who were found to carry HPV-16 DNA in their surgically removed pelvic lymph-nodes had an average of 27 months progression-free survival. The mean progression-free survival among histologically node-positive and node-negative surgical cases were 23 and 42 months, respectively. The mean progression-free survival time of node-positive cases with HPV-16 positive cervical primary was 7.5 months while that of patients with HPV-16 negative cervical biopsy was 38 months. Among histologically node-negative patients, HPV-16 positive and negative cases had an average progression-free survival time of 38 and 46 months, respectively. CONCLUSIONS Among those under investigation the most important factors to predict progression-free survival were surgically amenable disease, histologically negative pelvic lymph-nodes and HPV-16 negative cervical biopsies, though this latter one proved significant only among surgical cases.

Phagocytic index of neutrophil granulocytes and monocytes in healthy and preeclamptic pregnancy

Neutrophil granulocytes and monocytes have been intensively studied, but there is no scientific data on one of their most important functions, namely the phagocyte function in pregnancy and preeclampsia. The aim of this study was to examine this function. Twenty-five healthy pregnant, 25 preeclamptic pregnant, and 20 healthy, non-pregnant women were enrolled into our study. Cells were isolated from peripheral blood samples, marked and evaluated for the phagocytic index with an immunofluorescent microscope after phagocytosing the zymosan molecules. The phagocytic function of monocytes and neutrophil granulocytes decreased significantly in healthy pregnancy compared with non-pregnant women and in preeclampsia, and it decreased significantly compared with healthy pregnancy. Decreased phagocytic function in healthy pregnancy can be a part of the maternal immunosuppression, which is essential for the protection of the hemiallograft fetus. Further reduction of phagocytic function may be one of the immunoregulatory abnormalities found in preeclampsia.