Zoárd Tibor Krasznai

Role of ion channels and membrane potential in the initiation of carp sperm motility

The exposure of freshly spawned, immotile carp sperm to hypoosmotic media triggers the initiation of calcium-dependent flagellar motility. Intracellular calcium concentration has been thought to be the critical component in motility initiation, possibly acting through a novel signalling pathway. The sensitivity of sperm cells to changes of osmolality of the environment raises the question whether a mechanoregulated osmosensitive calcium pathway is involved in the activation mechanism of carp sperm motility. The sperm cells are in a depolarized state in the seminal plasma (W = –2.6 ± 3 mV) and they hyperpolarize upon hypoosmosis-induced activation of motility (W = –29 ± 4 mV). The intracellular sodium [Na + ]i, potassium [K + ]i and calcium [Ca 2+ ]i ion concentrations were determined in quiescent cells, and at 20, 60 and 300 s after activation. The [Na + ]i and [K + ]i of the quiescent cells were similar to the [Na + ]e and [K + ]e of the seminal plasma. Following hypoosmotic shock-induced motility, both [Na + ]i and [K + ]i decreased to one-fourth of the initial concentration. The [Ca 2+ ]i doubled at initiation of the motility of the sperm cells and remained unchanged for 5 min. Bepridil (50–250 µM), a blocker of the Na + /Ca 2+ exchanger, blocked carp sperm motility reversibly. Gadolinium, a blocker of stretch-activated channels (10–20 µM), inhibited sperm motility in a dose-dependent manner and its effect was reversible. Hypoosmotic shock fluidized the membrane and gadolinium treatment made it more rigid in both quiescent cells and hypotonic shock treated but immotile sperm cells. Based on these observations, it is suggested that, besides the well-known function of potassium and calcium channels, stretch-induced conformational changes of membrane proteins are also involved in the sperm activation mechanism of common carp.

Elevated human epididymis protein 4 concentrations in chronic kidney disease

Background Human epididymis protein 4 (HE4) has recently become an available tumour biomarker for detecting ovarian cancer along with the standard cancer antigen 125 (CA125). However, it is unknown if the levels of HE4 and CA125 may be altered in subjects who have impaired renal function with no ovarian disorders. Methods In 113 female patients at different stages of chronic kidney disease (CKD) with no ovarian and lung cancer and 68 subjects with normal renal and ovarian function, HE4 and CA125 concentrations were analysed by using chemiluminescent microparticle immunoassay (Architect®, Abbott) and electrochemiluminescent immunoassay (Modular E170®, Roche), respectively. Renal function was evaluated by measuring serum creatinine and urea concentrations (Cobas Integra-800®, Roche). Estimated glomerular filtration rate (eGFR in mL/min/1.73 m2) was calculated by the 4v-MDRD formula. Results Significantly increased HE4 concentrations (P < 0.0001) were found in individuals with differently decreased eGFR values (<90 mL/min/1.73 m2) compared with clinical controls. CA125 serum concentration was higher than normal in subjects with CKD3 (eGFR = 30–59 mL/min/1.73 m2), but significant elevation (P = 0.006) in CA125 concentrations was seen only in those who had severe renal failure (CKD4-5; eGFR < 30 mL/min/1.73 m2). These tendencies were independent of age in our study cohort, and seemed to be more evident among women in premenopausal status. Conclusions HE4 concentrations may be elevated in CKD patients with no ovarian and lung cancer. Thus, HE4 results should be interpreted cautiously in women with renal disorders.

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.

Pgp inhibition by UIC2 antibody can be followed in vitro by using tumor-diagnostic radiotracers, 99mTc-MIBI and 18FDG.

P-glycoprotein (Pgp, ABCB1) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing the phenomenon of multidrug resistance. It has been shown earlier that the combined application of a class of Pgp modulators (e.g. cyclosporine A and SDZ PSC 833) used at low concentrations and UIC2 antibody is a novel, specific, and effective way of blocking Pgp function (Goda et al., 2007). In the present work we study the UIC2 antibody mediated Pgp inhibition in more detail measuring the accumulation of tumor diagnostic radiotracers, 2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) and [(99m)Tc]hexakis-2-methoxybutyl isonitrile ((99m)Tc-MIBI), into Pgp(+) (A2780AD) and Pgp(-) (A2780) human ovarian carcinoma cells. Co-incubation of cells with UIC2 and cyclosporine A (CSA, 2μM) increased the binding of UIC2 more than 3-fold and reverted the rhodamine 123 (R123), daunorubicin (DNR) and (99m)Tc-MIBI accumulation of the Pgp(+) 2780AD cells to approx. the same level as observed in Pgp(-) cells. Similarly, 50μM paclitaxel (Pacl) increased UIC2 binding, and consequently reinstated the uptake of R123, DNR and (99m)Tc-MIBI into the Pgp(+) cells. Blocking Pgp by combined treatments with CSA+UIC2 or Pacl+UIC2 also decreased the glucose metabolic rate of the A2780AD Pgp(+) cells measured in (18)FDG accumulation experiments suggesting that the maintenance of Pgp activity requires a considerable amount of energy. Similar treatments of the A2780 Pgp(-) cells did not result in significant change in the R123, DNR, (99m)Tc-MIBI and (18)FDG accumulation demonstrating that the above effects are Pgp-specific. Thus, combined treatment with the UIC2 antibody and Pgp modulators can completely block the function of Pgp in human ovarian carcinoma cells and this effect can be followed in vitro by using tumor-diagnostic radiotracers, (99m)Tc-MIBI and (18)FDG.

Functional implications of membrane modification with semenogelins for inhibition of sperm motility in humans.

Semenogelin I and II (Sgs) are the major component of human semen coagulum. The protein is rapidly cleaved after ejaculation by a prostate-specific antigen, resulting in liquefaction of the semen coagulum and the progressive release of motile spermatozoa. Sgs inhibit human sperm motility; however, there is currently no information on its effect on the sperm membrane. This study investigated the role of Sgs on human sperm motility through regulation of membrane potential and membrane permeability. Fresh semen samples were obtained from normozoospermic volunteers, and studies were conducted using motile cells selected using the swim-up method. Sgs changed the characteristics of sperm motion from circular to straightforward as evaluated by a computer-assisted motility analyzer, and all parameters were decreased more than 2.5 mg/mL. The results demonstrate that Sgs treatment immediately hyperpolarized the membrane potential of swim-up-selected sperm, changed the membrane structure, and time-dependently increased membrane permeability, as determined through flow cytometric analysis. The biphasic effects of Sgs were time- and dose-dependent and partially reversible. In addition, a monoclonal antibody against Sgs showed positive binding to cell membrane proteins in fixed cells, observed with confocal fluorescence microscopy. These results demonstrate that Sgs modifies the membrane structure, indirectly inhibiting motility, and provides suggestions for a therapy for male infertility through selection of a functional sperm population using Sgs.

Seminal Vesicle Secretion 2 Acts as a Protectant of Sperm Sterols and Prevents Ectopic Sperm Capacitation in Mice

ABSTRACT Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.

18FDG, [18F]FLT, [18F]FAZA, and 11C-Methionine Are Suitable Tracers for the Diagnosis and In Vivo Follow-Up of the Efficacy of Chemotherapy by miniPET in Both Multidrug Resistant and Sensitive Human Gynecologic Tumor Xenografts

Expression of multidrug pumps including P-glycoprotein (MDR1, ABCB1) in the plasma membrane of tumor cells often results in decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy. It has been shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) is an effective way of blocking Pgp function. In the present work we investigated the suitability of four PET tumor diagnostic radiotracers including 2-[18F]fluoro-2-deoxy-D-glucose (18FDG), 11C-methionine, 3′-deoxy-3′-[18F]fluorothymidine (18F-FLT), and [18F]fluoroazomycin-arabinofuranoside (18FAZA) for in vivo follow-up of the efficacy of chemotherapy in both Pgp positive (Pgp+) and negative (Pgp−) human tumor xenograft pairs raised in CB-17 SCID mice. Pgp+ and Pgp− A2780AD/A2780 human ovarian carcinoma and KB-V1/KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of the treatment with an anticancer drug doxorubicin combined with UIC2 and CSA. The combined treatment resulted in a significant decrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp+ tumors. Our results demonstrate that 18FDG, 18F-FLT, 18FAZA, and 11C-methionine are suitable PET tracers for the diagnosis and in vivo follow-up of the efficacy of tumor chemotherapy in both Pgp+ and Pgp− human tumor xenografts by miniPET.

18FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp.

The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).

Intention-to-treat analysis of radical trachelectomy for early-stage cervical cancer with special reference to oncologic failures: Single-institutional experience in Hungary

Objective The aim of our study was to evaluate clinical and pathological data in order to draw eligibility criteria for oncologically sufficient radical trachelectomy (RT) in early-stage cervical cancer. Reviewing all cases of attempted RT performed at our unit, we focused attention on prognostic indicators of the need for additional oncologic treatment following RT. The analysis was extended by extensive literature review to include previously published cases of oncologic failures. Methods The authors retrospectively analyzed data of patients who underwent RT at the Department of Obstetrics and Gynecology, University of Debrecen. Electronic records and case notes of RT cases were reviewed to determine the incidence of abdominal and vaginal route, distribution of clinicopathologic data, and follow-up results of individual cases. Individual procedures were categorized as oncologically insufficient if additional oncologic treatment was necessary following RT. Theoretical eligibility criteria for RT in early-stage cervical cancer were determined retrospectively by selecting prognostic features that were associated with oncologic insufficiency from clinicopathologic indicators of the complete series. Results Twenty-four cases of RT were performed by the authors, 15 vaginal RTs with laparoscopic pelvic lymphadenectomy and 9 abdominal RTs with open pelvic lymphadenectomy. Fifteen of 24 cases proved oncologically sufficient. Three cases required immediate conversion to radical hysterectomy because of positive sentinel nodes and/or positive isthmic disc on frozen section. In further 5 cases, final pathology results indicated additional oncologic treatment, that is, radical hysterectomy (n = 2), chemoradiotherapy (n = 2), or chemotherapy (n = 1). One patient among immediately converted cases and another 3 among those who required additional oncologic treatment died of their disease later. There were no other cases of recurrences over a median follow-up of 34 months (range, 12–188 months). Factors that may predict oncologic insufficiency of RT were stage IB1 or greater, tumor size of greater than 2 cm in 1 dimension or greater than 15 mm in 3 dimensions, G3, nonsquamous/adeno histological type, stromal invasion of greater than 9 mm, and lymphovascular space involvement in the primary tumor. Conclusions Most cases of oncologically insufficient RTs have significant risk features that can be identified preoperatively. There is a need for more clinicopathologic data on oncologic failure of RT cases in order to improve patient selection.