Effect of two cleaning processes for bone allografts on gentamicin impregnation and in vitro antibiotic release.

Bone allografts are a useful and sometimes indispensable tool for the surgeon to repair bone defects. Microbial contamination is a major reason for discarding allografts from bone banks. To improve the number of safe allografts, we suggest chemical cleaning of the grafts followed by antibiotic impregnation. Comparison of two chemical cleaning processes for bone allografts aiming for antibiotic impregnation and consequently delivery rates in vitro. Bone chips of 5-10 mm were prepared from human femoral heads. Two cleaning methods (cleaning A and cleaning B) based on solutions containing hydrogen peroxide, paracetic acid, ethanol and biological detergent were carried out and compared. After the cleaning processes, the bone chips were impregnated with gentamicin. Bacillus subtilis bioassay was used to determine the gentamicin release after intervals of 1-7 days. Differences were compared with non-parametric Mann-Whitney U tests. The zones of inhibition obtained from the bone grafts cleaned with both cleaning processes were similar between the groups. The concentration of the released antibiotic was decreasing gradually over time, following a similar pattern for both groups. The cleaning procedure A as well as the cleaning procedure B for bone allografts allowed the impregnation with gentamicin powder in the same concentrations in both groups. The delivery of gentamicin was similar for both groups. Both cleaning procedures were easy to be carried out, making them suitable for routine use at the bone banks.

Improved limits on nu(e) emission from mu+ decay.

We investigated mu(+) decays at rest produced at the ISIS beam stop target. Lepton flavor (LF) conservation has been tested by searching for nu(e) via the detection reaction p(nu(e),e(+))n. No nu(e) signal from LF violating mu(+) decays was identified. We extract upper limits of the branching ratio (BR) for the LF violating decay mu(+)-->e(+)+nu(e)+nu(-) compared to the standard model (SM) mu(+)-->e(+)+nu(e)+nu(mu) decay: BR<0.9(1.7) x 10(-3) (90% C.L.) depending on the spectral distribution of nu(e) characterized by the Michel parameter rho=0.75(0.0). These results improve earlier limits by one order of magnitude and restrict extensions of the SM in which nu(e) emission from mu(+) decay is allowed with considerable strength. The decay mu(+)-->e(+)+nu(e)+nu(mu) often proposed as a potential source for the nu(e) signal observed in the LSND experiment can be excluded.

[Safety and acceptance of self application of cubic pessaries and urethral ring pessaries]. / [Sicherheit und Akzeptanz der Selbstanwendung von Würfel- und Urethraringpessaren]

OBJECTIVE: Flexible cubic and ring pessaries with a suburethral thickening are devices for the treatment of genital prolapse and urinary stress incontinence. Threads attached to the pessary enable overnight self-removal and self-application. Little is known about the safety and women's acceptance of these devices. METHODS: Eighteen women (age 36-85 years), 9 of them with failed surgery, tried this therapy (cubic pessary n = 6; ring pessary n = 12). The size of the pessary was chosen clinically. All patients were seen after a week and then monthly. RESULTS: All women showed up after 1 week. Twelve of 18 women felt comfortable with these devices at that time. During follow-up, 5/18 women wanted surgical therapy within the first 2 months, 3/18 refused the device later, 1/18 was lost to follow-up and 9/18 patients continued pessary therapy for a mean duration of 11 months. During 107 treatment months, only 2 mild complications were observed without the need to intervention (mild vaginal erosion, spotting). CONCLUSIONS: Pessary therapy is safe but has a dropout rate of approximately 50%. It can be an efficient alternative method in motivated women.

CD44 splice variant expression in normal and malignant uterine cervical epithelium.

CD44 expression was investigated immunohistochemically on paraffin sections obtained from 88 uterine cervical cancers and 31 normal cervices, using monoclonal antibodies against CD44 variant epitopes v4, v5, v6, v7/8, v9 and the standard form of the CD44 protein. Normal epithelium showed expression of all CD44 splice variants, at least in traces, and it was located predominantly in basal and parabasal cells. In cervical carcinomas CD44 expression was widely heterogeneous. CD44 variant v9 staining was moderate or strong in 86% of the tumors, and it was significantly correlated with CD44 v6 staining. Also a significant correlation between expression of CD44 v4 and v6 occurred. Highly significant correlations between CD44 expression of variants v4 and v6 and tumor stage as well as patients age were found. In addition, these variants were more frequently expressed in squamous cell carcinomas than in adenocarcinomas. However, in contrast to the recently reported data, we were not able to confirm the hypothesis that CD44 v6 represents a prognostic indicator in cervical cancer. In FIGO stages III and IV, patients with CD44 variant v4 positive tumors had a significantly longer disease-free and overall survival than patients with CD44 variant v4 negative tumors. In conclusion, our data indicate that CD44 v6 tissue expression cannot be considered as a prognostic factor in cervical cancer. Regarding the unexpected outcome of patients with CD44 v4 positive tumors, further investigations are needed to elucidate the exact clinical value of this variant isoform.

High serum levels of soluble CD44 variant isoform v5 are associated with favourable clinical outcome in ovarian cancer.

In 96 ovarian cancer patients, the present study investigates the clinical significance of pretreatment concentrations of soluble CD44 standard (CD44s) and its isoforms v5 and v6 determined in the serum and the ascitic fluid by means of recently developed enzyme-linked immunosorbent assays (ELISAs). Furthermore, CD44 serum concentrations in the ovarian cancer patients were compared with circulating CD44 levels in 50 healthy age-matched female blood donors. Whereas CD44s was found to be higher and CD44v5 to be lower in ovarian cancer patients than healthy control subjects, no statistical difference between the two cohorts was revealed for CD44 isoform v6. In the ascitic fluid samples, variant isoform v5 and v6 were demonstrated at lower concentrations than serum. Multivariate analysis of overall survival demonstrated that a high pretreatment serum level of soluble CD44 isoform v5 is independently associated with favourable clinical outcome in ovarian cancer. When circulating CD44 isoforms were compared with a panel of serum parameters known to be involved in the immunological network, an inverse correlation between serum CD44v5 levels and indicators of cellular immune system activation, such as soluble interleukin 2 receptor, immunostimulatory protein 90K and neopterin, became apparent.

Human peritoneal mesothelial cells are more potent than ovarian cancer cells in producing tumor marker CA-125.

The aim of the present study was to investigate the extent to which human peritoneal mesothelial cells (HPMCs) are able to participate in the release of tumor marker CA-125 in ovarian cancer and other conditions associated with an involvement of the peritoneum. For this purpose CA-125 shedding was measured in the supernatant culture medium of HPMCs obtained from various donors and seven well-established ovarian cancer cell lines (OVCAR-3, 2780, 2774, SKOV-6, SKOV-8, HOC-7, HTB-77). Furthermore, the influence of inflammatory cytokines [interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma)] on CA-125 release in normal and malignant cells was also studied. Constitutive CA-125 shedding was found to be about five times higher in HPMCs as compared with the investigated ovarian cancer cell lines. IL-1 beta and TNF-alpha treatment of HPMCs resulted in a significant reduction in CA-125 release; however, no consistent pattern in CA-125 secretion was found during incubation with either IL-1 beta or TNF-alpha in the various malignant cell lines. IFN-gamma, on the other hand, induced a highly significant increase in CA-125 secretion in ovarian cancer cells, but did not influence the shedding of CA-125 in HPMCs.

Interaction of retinoic acid and interferon-alpha in breast cancer cell lines.

Retinoids and Interferons have been demonstrated to synergistically amplify the inhibition of proliferation in cultured breast cancer cells. Recently we reported that interferon-gamma (IFN-gamma) modulates the action of retinoic acid (RA): IFN-gamma increased expression of retinoic acid receptor-gamma (RAR-gamma) and suppressed the increase of retinoic acid binding protein type II (CRABP-II) expression. To improve the understanding of mechanism mediating synergism we extended our studies to the type I interferon-alpha. Synergistic inhibition of proliferation could be detected also by IFN-alpha and RA in BT-20 and SKBR-3 breast cancer cell lines but not in MCF-7 cells. Neither IFN-alpha nor any retinoid tested alone were able to increase RAR-gamma message, only the combination of both had this ability. In MCF-7 breast cancer cell lines the combination of any retinoid with IFN-alpha increased CRABP II expression level compared with the retinoids alone. In contrast with SKBR-3 and BT-20 cells a combination of ATRA with IFN-alpha markedly reduced ATRA mediated CRABP II induction. These results suggest that two factors may be responsible for synergistic action of RA and IFN-alpha: the inhibition of the CRABP II expression and an IFN-alpha/RA mediated upregulation of RAR-gamma.

Prognostic value of CD44 splice variant expression in ovarian cancer.

In 44 ovarian cancers, CD44 variant (CD44v) expression was investigated immunohistochemically using a variant-specific polyclonal antibody. Patients with CD44v-positive carcinomas had a significantly shorter disease-free survival than patients with CD44v-negative tumors. Overall survival was also significantly reduced for stages III and IV of the International Federation of Gynecology and Obstetrics. Furthermore, a highly significant inverse correlation was observed between CD44v expression and preoperative platelet count. Urinary neopterin concentration, a marker of cell-mediated immunostimulation, did not differ between CD44v-positive and -negative ovarian cancer patients. Moreover, in seven ovarian carcinoma cell lines, modulation of CD44v expression was analyzed by living cell radioimmunoassay. Interferon-alpha, interferon-gamma, tumor necrosis factor, transforming growth factor-beta, all-trans retinoic acid and cisplatin did not affect CD44v expression.

Lupus vulgaris confirmed by polymerase-chain reaction.

We report a case of lupus vulgaris with typical clinical and histological findings. Mycobacterium tuberculosis was not only identified by a conventional culture technique, but also by a recently established system which has been designed to detect mycobacterial DNA in formalin-fixed, paraffin-embedded tissue by polymerase-chain reaction (PCR). Because results can be obtained within days, the PCR-based technique may markedly facilitate the diagnosis of skin tuberculosis.

Inhibition of ion conductances by osmotic shrinkage of Madin-Darby canine kidney cells.

Osmotic swelling of Madin-Darby canine kidney (MDCK) cells enhances the ion conductances of the cell membrane, which allows release of cellular ions and subsequent regulatory cell volume decrease. The present study has been performed to test whether cell shrinkage similarly affects the ion conductances of MDCK cell membranes. Increase of extracellular osmolarity by addition of 50 mM NaCl or 100 mM mannitol leads within 3 min to a hyperpolarization of the cell membrane, a marked increase of cell membrane resistance [by 223 +/- 38% (n = 8) and 228 +/- 21% (n = 5), respectively], as well as a moderate increase of the K+ selectivity of the cell membrane (by 37 +/- 13%, n = 9). Thus exposure to hypertonic extracellular fluid decreases the cell membrane conductances including the K+ conductance. Cell volume measurements reveal a regulatory cell volume increase, which is sensitive to both furosemide and dimethylamiloride. Extracellular ATP (10 microM), which activates calcium-sensitive K+ channels, hyperpolarizes the cell membrane close to the K+ equilibrium potential. The respective values are -69.9 +/- 3.1 mV (n = 9) in isotonic fluid, -79.4 +/- 1.8 mV (n = 9) within 3 min, and -76.4 +/- 1.8 mV (n = 7) within 16-h exposure to hypertonic extracellular fluid. This observation points to a sustained increase of intracellular K+ activity after exposure to hypertonic extracellular fluid.

Regulation of potassium conductance by prostaglandins in cultured renal epitheloid (Madin-Darby canine kidney) cells.

Madin-Darby canine kidney (MDCK) cells form arachidonic acid metabolites following stimulation of several hormones known to modify the ion conductances at the plasma membrane. The present study has been performed to elucidate the influence of arachidonic acid on the electrical properties of subconfluent MDCK cells. As a result, arachidonic acid (1 or 10 mumol/l) leads to a transient hyperpolarization of the cell membrane, followed by a transient depolarization and a second, sustained hyperpolarization. The effects are inhibited by cycloxygenase inhibitor indomethacin (1 mumol/l). The initial transient hyperpolarization is mimicked by prostaglandin E2 (PGE2, 0.1 mumol/l), the sustained hyperpolarization by both PGE2 (0.1 mumol/l) and PGF2 alpha (0.1 mumol/l). The transient hyperpolarization is paralleled by an increase of potassium selectivity and a decrease of cell membrane resistance and is thus the result of increased potassium conductance. The transient depolarization is paralleled by an increase of chloride selectivity, reflecting an increase of chloride conductance. The sustained hyperpolarization is paralleled by an increase of cell membrane resistance, and increase of potassium selectivity and a decrease of chloride selectivity, and is thus the result of decreasing chloride conductance. The observations reveal a role of prostaglandins in the regulation of ion conductances in MDCK cells, which could well participate in the transport regulation by hormones.

Progesterone inhibits K conductance in plasma membrane of cultured renal epitheloid MDCK cells.

Progesterone causes natriuresis, an effect largely attributed to displacement of aldosterone from its receptor. The present study, however, demonstrates that progesterone (0.1, 1, and 10 mumol/1, respectively) also causes a rapid, fully reversible depolarization of Madin-Darby canine kidney (MDCK) cells (by 1.3 +/- 0.5, 4.1 +/- 0.7 and 12.3 +/- 1.5 mV, respectively). 17 alpha-Hydroxyprogesterone and dihydroxytestosterone are, by two orders of magnitude, less effective, whereas cholesterol, aldosterone, hydrocortisone, and estradiol (each up to 10 mumol/l) did not significantly alter the potential difference across the cell membrane. The effect of progesterone is blunted by antiprogestogen RU 486 (5 mumol/l). The progesterone-induced depolarization is paralleled by a decrease of potassium selectivity and an increase of cell membrane resistance and is abolished in the presence of the potassium channel blocker barium (10 mmol/l), as well as in the presence of 40 mmol/l potassium in the extracellular fluid. Neither removal of extracellular chloride or bicarbonate nor amiloride, ouabain, or pretreatment with pertussis toxin abolish the depolarizing effect of 5 mumol/l progesterone. In conclusion, acute administration of progesterone depolarizes MDCK cells by decreasing the potassium conductance of the cell membrane.

Consequences of 6 weeks of strength training on red cell O2 transport and iron status.

Effects of endurance training on O2 transport and on iron status are well documented in the literature. Only a few data are available concerning the consequences of strenuous anaerobic muscular exercise on red cell function. This study was performed to test the influence of strength training alone on parameters of red cell O2 transport and iron status. Twelve healthy untrained males participated in a strength-training programme of 2-h sessions four times a week lasting 6 weeks. After 6 weeks a small but significant reduction of haemoglobin (Hb; -5.4 g.l-1) was found (p less than 0.05). Mean red cell volume did not change, but a pronounced decrease of mean cell Hb concentration (from 329.2 g.l-1, SE 2.5 to 309.8 g.l-1, SE 1.2; p less than 0.001) and mean corpuscular Hb (from 29.6 pg, SE 0.4 to 27.7 pg, SE 0.3; p less than 0.01) was observed. Serum ferritin decreased significantly by 35% (p less than 0.01); transferrin, serum iron and iron saturation of transferrin were unaltered. Serum haptoglobin concentration was diminished significantly by 30.5% (p less than 0.01). The reticulocyte count had already increased after 3 weeks of training (p less than 0.05) and remained elevated during the following weeks. Strength training had no significant influence on the O2 partial pressure at which Hb under standard conditions was 50% saturated, red cell 2,3-diphosphoglycerate and ATP concentration as well as on erythrocytic glutamate-oxalacetate transaminase activity. The data demonstrate that mechanical stress of red cells due to the activation of large muscle masses led to increased intravascular haemolysis, accompanied by a slightly elevated erythropoiesis, which had no detectable influence on Hb-O2 affinity. Training caused an initial depletion of body iron stores (prelatent iron deficiency). Although Hb had decreased by the end of the training phase a true "sports anaemia" could not be detected.

Ion channels in Madin-Darby canine kidney cells.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.