Female Age Affects the Mesenchymal Stem Cell Characteristics of Aspirated Follicular Cells in the In Vitro Fertilization Programme.

Aspirated follicular cells (AFCs) from the in vitro fertilization program can express various stem cell markers and are even able to differentiate into different types of cells in vitro. The female reproductive potential decreases with increasing age due to lowered ovarian reserve and oocyte quality, but data on the effect of female age on stem cell characteristics of AFCs are scarce. Therefore, the aim of this study was to elucidate whether female age affects the mesenchymal stem cell (MSC) characteristics of AFCs. Follicular aspirates were collected from 12 patients included in the in vitro fertilization programme with a normal ovarian reserve. Patients were divided into four age groups: Group A ≤ 30 years, Group B 31-35 years, Group C 36-39 years and Group D ≥ 40 years. After removal of the oocytes, AFCs were collected from follicular aspirates using hypo-osmotic technique and cultured in vitro, and their stemness was compared according to female age. The cultured AFCs were analysed for gene expression using the Human Mesenchymal Stem Cell RT2 Profiler™ PCR Array, for their potential for differentiation into adipogenic and osteogenic lineage, and for their expression of MSC-related markers using immunocytochemistry. We found that female age can significantly influence their stemness: expression of pluripotency and MSC-related genes, and their differentiation potential. Despite the relatively high expression of MSC-related genes, the AFCs of the oldest patients had the lowest potential to differentiate into osteogenic and adipogenic lineages in vitro, which may be related to their age and the changed ovarian function.

Geographical distribution of Slovenian BRCA1/2 families according to family origin: implications for genetic screening.

Knowledge of the geographical distribution of highly recurrent mutations may be useful for efficient screening in cancer families. Since the cloning of the BRCA1/2 genes, it is known that the wide spectrum of deleterious mutations shows high ethnic and geographic heterogeneity. In this study, we have tested probands from 582 breast/ovarian cancer families and positioned all 156 BRCA1/2 families on the map according to the family origin. We observed that high-risk families with the same recurrent mutation present a typical geographical distribution and that different recurrent mutations may show different distribution patterns. We then evaluated the genetic screening implications of this heterogeneous prevalence of the most recurrent mutations found [300T>G(c.181T>G), 1806C>T(c.1687C>T), 969ins7(c.844_850dupTCATTAC), 5382insC(c.5266dupC), 235G>A(c.116G>A) in BRCA1 and IVS16-2A>G(c.7806-2A>G) in BRCA2]. On the basis of these results, specific testing procedures for new incident cases may be offered according to their family origins and, according to the information regarding clusters revealed in this study, the individuals (especially those at low risk), originating from regions with clusters, might be screened preferentially for cluster mutations and analysis may be simplified according to the family origin.

The hTERT mRNA in plasma samples of early breast cancer patients, non-cancer patients and healthy individuals.

One of the most important changes, which make cancer cells immortal, is reactivation of the telomerase enzyme. Human telomerase is composed of at least two subunits, hTERT and hTR. Many investigators have already detected telomerase mRNA in bodily fluids. The first aim of our study was to find out if there is a difference in the appearance frequency of detectable hTERT mRNA in plasma of early breast cancer patients, non-cancer patients and healthy individuals. The second aim was to determine whether surgical removal of the tumor affects the presence of hTERT mRNA in plasma of early breast cancer patients. 87 patients with early breast cancer, 22 non-cancer patients and 21 healthy individuals were included in the study. From early breast cancer patients, two blood samples were collected, the first prior and the second 24 hours after the surgical removal of the tumor. From other individuals one blood sample was collected. The presence or absence of hTERT mRNA was determined from all blood samples. 47% of early breast cancer patients, 32% of non-cancer patients and 5% of healthy individuals tested positive for the presence of hTERT mRNA in plasma. The difference between early breast cancer patients and healthy individuals was statistically significant (p<0,001). Among early breast cancer patients, 26% were positive for the presence of plasma hTERT mRNA before and after the surgical removal of the tumor, 21% were positive before and negative after, 36% were negative before and after and 17% were negative before and positive after the surgical removal of the tumor. In conclusion, we found statistically significant difference of hTERT mRNA presence in plasma of early breast cancer patients when compared to healthy individuals. Second, we found that hTERT mRNA in plasma of early breast cancer patients is affected by the surgical removal of the tumor.

Specific T cell responses to Helicobacter pylori predict successful eradication therapy.

OBJECTIVE: The aim of our prospective study was to test a specific T cell response to Helicobacter pylori before therapy and compare it to the success of H. pylori eradication 12 months later. METHODS: A total of 14 dyspeptic patients and 10 patients with previous H. pylori eradication failure were recruited into the study; before therapy their gastric samples for H. pylori cultivation and blood samples for dendritic cell cultivation were obtained. H. pylori antigens were produced to prime dendritic cells for stimulation of T lymphocyte response. RESULTS: The level of cytokine response by T cells was measured and results were compared with the success of H. pylori eradication one year later. There was a significantly increased response in expression of IFN-gamma and IL-4 molecules by DCs stimulated T cells in subjects that successfully eradicated H. pylori compared with those who failed to eradicate the infection. Our results support the hypothesis that successful H. pylori eradication requires established anti-H. pylori immune response besides antibiotic treatment. CONCLUSION: Effective IFN-gamma cytokine response to H. pylori antigens seems to be of particular importance. Immunisation could be therefore beneficial for H. pylori eradication, while immunodeficiency could cause the failure in H. pylori eradication.

Prognostic significance of tyrosinase mRNA detected by nested RT-PCR in patients with malignant melanoma.

The aim of this study was to evaluate the role of tyrosinase mRNA appearance in blood of malignant melanoma (MM) patients, especially with advanced stages, for predicting the disease progression, and consequently the survival. The tyrosinase mRNA was measured by nested RT-PCR in peripheral venous blood samples obtained from 86 patients (53 male and 33 female) with mainly stage III and IV MM. The data were analyzed using standard methods for survival analysis and logistic regression. Tyrosinase was negative in the MM patients with the disease stage I or II, positive tyrosinase was in 11/50 patients with stage III and in 5/22 patients with stage IV. Systemic metastases developed in 14/16 patients with positive tyrosinase and in 41/70 with negative tyrosinase. The 3-year survival was 8% and 28% among the patients with positive and the patients with negative tyrosinase, respectively. The log rank test showed statistically significant better survival of tyrosinase negative patients when compared to tyrosinase positive patients (p=0.039). Multivariate analysis using logistic regression indicated tyrosinase to be a statistically significant prognostic factor for the survival of MM patients after controlling for Breslow and ulceration values (p=0.006). Positive tyrosinase in peripheral venous blood is statistically significant, and more importantly independent negative predictor of survival.

The immunohistochemical and serological determination of p53 protein in patients with malignant lymphomas.

The product of mutated p53 gene is a protein with abnormal conformation, impaired DNA binding, and a prolonged half life, the latter of which results in immunohistochemically detectable levels within nuclei of malignant cells. The present study was aimed at the immunohistochemical determination of p53 overexpression in patients with various histological types of nonHodgkin's lymphomas (NHL), with a particular interest in gastric lymphomas. In these patients, as well as in controls, also serological determinations of p53 protein were performed using an ELISA method. Immunohistochemical overexpression of p53 protein was found in 21% of NHL patients, with the highest incidence of p53 immunoreactivity in cases of Burkitt's lymphoma, follicle center lymphoma grade III, and diffuse large B-cell lymphoma. In gastric lymphomas the overall incidence of p53 immunoreactivity was as high as 46%. Serological ELISA determinations of p53 protein in NHL patients and in controls remained below the lowest detection limit of the method in all 128 cases. Considering that p53 mutations are associated with poor response to therapy, and consequently with poor prognosis, it is of great importance to determine the subset of patients that are particularly at risk for an unfavorable outcome and should be treated more aggressively. Immunohistochemical determinations of p53 overexpression represent a rapid and simple, yet somewhat imperfect technique for an estimation of the frequency of mutational events. On the other hand, serological determinations of p53 protein are completely inadequate for the evaluation of p53 status.

Telomerase activity as a biological marker in some gynecological tumors: comparison with tissue and serum CA 125.

BACKGROUND: Since telomerase activity is detectable in cancer cells but not in some normal somatic cells, it has been considered as a potential diagnostic marker as well as a target for possible anticancer strategies. The purpose of this study was to assess the value of telomerase activity determination in some gynecological tumors and to compare it with the CA 125 tissue and serum profile. PATIENTS AND METHODS: The telomerase activity was determined in 11 gynecological tumors: 7 ovarian carcinomas, 2 carcinomas of the fallopian tube and 2 cervical carcinomas, and compared to the activity in the normal peritoneal tissue of the same patients. Additionally, the levels of CA 125 were measured in the tumor and normal peritoneum tissue samples as well as in the patients' sera. RESULTS: In ovarian tumors, the telomerase activity was detected in 71.4% (5 out of 7), while in the carcinomas of the fallopian tube and cervical carcinomas such activity was not observed. Negative for telomerase activity were also all samples of peritoneum. The range of CA 125 in the tumor tissue was 99 U/g-803667 U/g of tissue and in the normal peritoneum 71 U/g-4925 U/g of tissue. CONCLUSION: In conclusion, telomerase activity could be detected in some of the gynecological tumors, but for clinical use as a diagnostic marker it is inferior to CA 125.

The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain.

The present study investigated the effect of inhibiting the expression of Na(v)1.8 (PN3/SNS) sodium channels by an antisense oligodeoxynucleotide (ODN) on bladder nociceptive responses induced by intravesical acetic acid infusion in rats. Animals were injected intrathecally with either Na(v)1.8 antisense or mismatch ODN. Control cystometrograms under urethane anesthesia during intravesical saline infusion exhibited intercontraction intervals (ICIs) that were significantly longer in antisense-treated rats than in mismatch ODN-treated rats. Intravesical infusion of 0.1% acetic acid induced bladder hyperactivity as reflected by a 68% reduction in ICIs in mismatch ODN-treated rats but did not significantly reduce ICIs in antisense-treated rats. The number of Fos-positive cells after acetic acid administration were significantly reduced in the L6 spinal cord from antisense-treated animals, compared with mismatch ODN-treated animals. In addition, Na(v)1.8 immunoreactivity was reduced in L6 dorsal root ganglion neurons in the antisense-treated rat. In patch-clamp recordings, the conductance density of TTX-resistant sodium currents in dissociated bladder afferent neurons that were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall was also smaller in antisense-treated rats than in mismatch ODN-treated rats, whereas no changes were observed in TTX-sensitive currents. These results indicate that the Na(v)1.8 TTX-resistant sodium channels are involved in the activation of afferent nerves after chemical irritation of the bladder. These channels represent a new target for the treatment of inflammatory pain from visceral organs such as the urinary bladder.

Transition metal complexes with thiosemicarbazide-based ligands. XLII. Bis(S-methylisothiosemicarbazido-N1, N4)bis(pyrazole-N2)-nickel(II) diiodide.

In the title compound, [Ni(C(2)H(7)N(3)S)(2)(C(3)H(4)N(2))(2)]I(2), the Ni(II) ion assumes a centrosymmetric distorted octahedral geometry. The two molecules of S-methylisothiosemicarbazide are coordinated as bidentate ligands via the terminal N atoms, forming five-membered chelate rings. The I atoms are approximately in the equatorial plane of the chelate rings at a similar distance from both. The five-membered chelate rings are almost planar and exhibit flattened envelope conformations. There is a weak intermolecular interaction between the lone pair of electrons of the S atom and the center of the pyrazole ring.

Regulation of Na+ channel distribution in the nervous system.

An important aspect of Na+ channel regulation is their distribution on neuronal membranes within the nervous system. The complexity of this process is brought by the molecular diversity of Na+ channels and differential regulation of their distribution. In addition, Na+ channel localization is a highly dynamic process depending on the status of the cell in vitro, and (patho)physiological condition of the organism in vivo. Nonetheless, the pharmacological manipulation of Na+ channel distribution should be possible and will hopefully bring safer and more-potent medicines in the future.

Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.

BACKGROUND: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. MATERIALS AND METHODS: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). RESULTS: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. CONCLUSION: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vaccine.

Detection of tyrosinase mRNA by an optimised nested RT-PCR in the peripheral blood of patients with advanced malignant melanoma.

Malignant melanoma (MM) has a high metastatic potential even in small primary lesions, and an early distinction between localized and regionally/distally advanced disease is of major importance for the patients' treatment and, consequently, for their survival. Exploiting the fact that tyrosinase is a tissue specific enzyme which is only expressed in normal skin melanocytes and MM cells that invade the blood during metastasizing, the objective of our study was to optimise the nested RT-PCR assay for the detection of tyrosinase mRNA and, hence, to detect circulating melanoma cells (CMC) in whole venous blood of MM patients. Eighteen MM patients (stage III and IV, according to AJCC) and 8 healthy subjects were included in our study. Following optimisation of the procedure, the lowest detection limit of 10 MM cells per 1 ml of the blood was achieved. Tyrosinase mRNA was detected in 27.8% (5/18) of blood samples from MM patients and in none of the healthy volunteers. Preliminary results of this study suggest that the method is sensitive and specific to the CMC detection in the peripheral blood and may thus be helpful in determining the disease stage and, consequently, in planning treatment.

The circulating auto-antibodies to p53 protein in the follow-up of lymphoma patients.

Mutant p53 proteins may be targets of the host immune system - consequently a certain proportion of cancer patients (the percentage varies according to the type of cancer) with tumors that carry p53 missense mutations develop circulating p53 antibodies. The present study was aimed at defining the occurrence of circulating antibodies to p53 protein in patients with various types of non-Hodgkin's lymphomas (NHL). Altogether, the sera of 108 cases with various histological types of NHL and of 20 healthy controls were assessed for the presence of antibodies to p53 protein with an ELISA method. In 73 cases of NHL, also the immunohistochemical staining for p53 antigen was performed to make a rough estimation of the frequency of mutational events. The development of autoantibodies to p53 protein was observed in approximately 7% of NHL patients (predominantly in the more aggressive variants of the disease, but also in one case of small lymphocytic lymphoma). This proportion represents roughly one third of the number of patients assessed (immunohistochemically) to carry a missense p53 mutation in their tumors. The autoantibodies to p53 protein can be used as a tumor marker (early appearance, high specificity) in the follow-up of a subset of NHL patients, but, unfortunately, this subset comprises only approximately 7% of NHL patients.

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice.

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Soluble tumor necrosis factor receptor I (sTNFRI) as a prognostic factor in melanoma patients in Slovene population.

Tumor necrosis factor alpha (TNF-alpha) and its receptors (TNFRI and TNFRII) which exist in soluble form as a product of cleavage of the extracellular domain of membrane integrated receptors, still rise debate about their importance. It was reported that TNF-alpha has numerous actions in diseases such as inflammation, autoimmunity, infectious diseases, septic shock and many types of cancer [1, 2]. Several authors have reported the significance of sTNFRI level in serum of cancer patients [3, 4]. This study was performed in collaboration with the Institute of Oncology of Slovenia. At least two different mouse monoclonal antibodies (MAbs) against human sTNFRI have been prepared to obtain a sensitive and reliable sandwich ELISA. It was compared with commercially available R&D and Endogen ELISAs for the determination of sTNFRI. Groups of patients with different stages of melanoma and epithelial ovarian carcinoma were tested and their clinical records were reexamined. Levels of sTNFRI were measured and compared with the normal serum levels of sTNFRI.

Differential distribution of the tetrodotoxin-sensitive rPN4/NaCh6/Scn8a sodium channel in the nervous system.

Voltage-gated sodium channels underlie the generation of action potentials in excitable cells. Various sodium channel isoforms have been cloned, functionally expressed and distinguished on the basis of their biophysical properties or differential sensitivity to tetrodotoxin (TTX). In the present study, we have investigated the immunolocalization of the TTX-sensitive sodium channel, rPN4/NaCh6/Scn8a, in discrete areas of the rat nervous system. Thus, in naïve animals, PN4 was abundantly expressed in brain, spinal cord, dorsal root ganglia (DRG) and peripheral nerve. The presence of PN4 at the nodes of Ranvier in the sciatic nerve suggests the importance of this sodium channel in peripheral nerve conduction. In addition, the pattern of PN4 immunolabeling was determined in DRG, spinal cord and sciatic nerve in rats subjected to chronic constriction nerve injury (CCI).

Effectiveness of a simply designed tumor vaccine in prevention of malignant melanoma development.

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57Bl/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier-maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5 x 10(5) viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macrophage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.

A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain.

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective "knock-down" of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN/SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inflammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.

Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury.

P2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.