Feasibility of aspirin and/or vitamin D3 for men with prostate cancer on active surveillance with Prolaris® testing.

Objectives: To test the feasibility of a randomised controlled trial (RCT) of aspirin and/or vitamin D3 in active surveillance (AS) low/favourable intermediate risk prostate cancer (PCa) patients with Prolaris® testing. Patients and Methods: Newly-diagnosed low/favourable intermediate risk PCa patients (PSA ≤ 15 ng/ml, International Society of Urological Pathology (ISUP) Grade Group ≤2, maximum biopsy core length <10 mm, clinical stage ≤cT2c) were recruited into a multi-centre randomised, double-blind, placebo-controlled study (ISRCTN91422391, NCT03103152). Participants were randomised to oral low dose (100 mg), standard dose (300 mg) aspirin or placebo and/or vitamin D3 (4000 IU) versus placebo in a 3 × 2 factorial RCT design with biopsy tissue Prolaris® testing. The primary endpoint was trial acceptance/entry rates. Secondary endpoints included feasibility of Prolaris® testing, 12-month disease re-assessment (imaging/biochemical/histological), and 12-month treatment adherence/safety. Disease progression was defined as any of the following (i) 50% increase in baseline PSA, (ii) new Prostate Imaging-Reporting and Data System (PI-RADS) 4/5 lesion(s) on multi-parametric MRI where no previous lesion, (iii) 33% volume increase in lesion size, or radiological upstaging to ≥T3, (iv) ISUP Grade Group upgrade or (v) 50% increase in maximum cancer core length. Results: Of 130 eligible patients, 104 (80%) accepted recruitment from seven sites over 12 months, of which 94 patients represented the per protocol population receiving treatment. Prolaris® testing was performed on 76/94 (81%) diagnostic biopsies. Twelve-month disease progression rate was 43.3%. Assessable 12-month treatment adherence in non-progressing patients to aspirin and vitamin D across all treatment arms was 91%. Two drug-attributable serious adverse events in 1 patient allocated to aspirin were identified. The study was not designed to determine differences between treatment arms. Conclusion: Recruitment of AS PCa patients into a multi-centre multi-arm placebo-controlled RCT of minimally-toxic adjunctive oral drug treatments with molecular biomarker profiling is acceptable and safe. A larger phase III study is needed to determine optimal agents, intervention efficacy, and outcome-associated biomarkers.

Real world use of oral treatments in interstitial cystitis/bladder pain syndrome in the UK: Outcome of a cross sectional study.

BACKGROUND: To describe the oral treatments people living with interstitial cystitis/bladder pain syndrome (IC/BPS) are using to treat their urologic condition in the UK. METHOD: A questionnaire hyperlink encompassing current and previous medications taken for IC/BPS with other sociodemographic and diagnostic indices was available to the Bladder Health UK website. Interested and fully consented individuals accessed and completed the survey. RESULTS: A total of 601 accessed the questionnaire of whom 173 participants responded (response rate: 28.7%) with a mean ± SD O'Leary/Sant scores of 20.12 ± 9.38. A sample size of 171 was estimated to be used in the survey. A fifth of the participants were not on any treatment at all. Amitriptyline was the most prevalent medication in use both alone and in combination. A shift in the use of unapproved (for IC/BPS) antidepressant, smooth muscle relaxant, opioids, gabapentenoids, and antibiotics was observed in the sample. There were no significant differences between the mean (SD) O'Leary/Sant scores of cohorts currently taking oral medications and those not taking it. More than two-thirds of the participants had been diagnosed with the disease more than 5 years. Just under a half (47.4%) of participants reported a history of allergy. CONCLUSION: Our study provides contemporary evidence that the treatments used for managing IC/BPS encompass a broad range of medications both recommended and not recommended by current guidelines. The latter suggests patients are willing to try novel treatments when more conventional ones are ineffective.

The relationship between illness perception and worsening of interstitial cystitis/painful bladder syndrome symptoms: A cross-sectional study.

OBJECTIVE: To evaluate disease perception in a cohort of patients with interstitial cystitis/painful bladder syndrome (IC/PBS) using the Brief Illness Perception-Questionnaire (BIP-Q) and to evaluate how this might relate to disease severity. MATERIALS AND METHODS: The study is a cross-sectional survey amongst members of Bladder Health UK who had previously received a clinical diagnosis of IC/PBS. A hyperlink containing the questionnaire was sent to the patient group's website and interested members accessed and completed the survey. Participants' inclusion was based on a prior clinical diagnosis of IC/PBS, current O'Leary Sant scores supportive of the diagnosis, and age between 18 and 80. A sample size of 171 was used in the study. The Brief Illness Perception Questionnaire (BIP-Q) and the O'Leary/Sant symptoms and problem indices questionnaire were used to collect data. A multivariable logistic regression analysis was used to test the relationship between items of BIP-Q and severity of IC/PBS. Content analysis was used for the causal domain and subsequently analysed as percentages. RESULTS: Six hundred and one members accessed the questionnaire of whom 159 returned completed questionnaires. One hundred and twenty-two of 159 (≥75%) respondents believe that their illness will continue indefinitely. The majority of the respondents indicated that IC/PBS had a negative impact on their daily lives, caused them worry and made them emotionally unstable. Of the 8 BIP-Q items, those most predictive of disease severity were (adjusted odd ratio and confidence intervals): consequence 0.094 (0.023-0.386); treatment control 2.702 (1.256-5.812); identity 0.141 (0.033-0.600); concern 9.363 (1.521-57.632). CONCLUSIONS: Our findings show that IC/PBS negatively impacts participant's quality of life and emotional wellbeing. Higher expectation for treatment benefit and increasing levels of patient concern are predictive for severity of IC/PBS.

Increased plasma CD14 levels 1 year postpartum in women with pre-eclampsia during pregnancy: a case-control plasma proteomics study.

Epidemiological data suggest that pre-eclampsia (PE) is associated with an increased risk of post-delivery metabolic dysregulation. The aim of the present case-control observational study was to examine the global plasma proteomic profile 1 year postpartum in women who developed PE during pregnancy (n = 5) compared to controls (n = 5), in order to identify a novel predictive marker linking PE with long-term metabolic imbalance. Key findings were verified with enzyme-linked immunosorbent assay (ELISA) in a separate cohort (n = 17 women with PE and n = 43 controls). One hundred and seventy-two proteins were differentially expressed in the PE vs. control groups. Gene ontology analysis showed that Inflammatory|Immune responses, Blood coagulation and Metabolism were significantly enriched terms. CD14, mapping to the inflammatory response protein network, was selected for verification based on bibliographic evidence. ELISA measurements showed CD14 to be significantly increased 1 year postpartum in women with PE during pregnancy compared to controls [PE group (median ± SD): 296.5 ± 113.6; control group (median ± SD): 128.9 ± 98.5; Mann-Whitney U test p = 0.0078]. Overall, the identified proteins could provide insight into the long-term disease risk among women with PE during pregnancy and highlight the need for their postpartum monitoring. CD14 could be examined in larger cohorts as a predictive marker of insulin resistance and type II diabetes mellitus among women with PE.

Distribution of mast cell subtypes in interstitial cystitis: implications for novel diagnostic and therapeutic strategies?

AIMS: To identify the presence and geographical distribution of mast cell (MC) subtypes: MCT (tryptase positive-chymase negative) and MCTC (tryptase positive-chymase positive) in bladder tissue. METHODS: Bladder tissue was obtained from patients with painful bladder syndrome/interstitial cystitis (n=14) and normal histology from University Hospital Southampton tissue bank. Sequential tissue slices were immunohistochemically stained for MC subtypes using anti-MC tryptase (for MCT and MCTC) and anti-MC chymase (for MCTC). Stained sections were photographed, and positively stained MCs were quantified using ImageJ. Data were analysed using descriptive statistics and individual paired t-tests. RESULTS: There was a significant difference in the density of MCs between each layer of the disease bladder, with the greatest accumulation within the detrusor (p<0.001). There was a significant increase in MCTC subtype in the lamina (p=0.009) in painful bladder syndrome/interstitial cystitis. CONCLUSIONS: Our results suggest that mastocytosis is present within all layers of disease bladder, especially the muscle layer. The varying increase in MC subtypes in the lamina and mucosa may explain the variability in painful bladder syndrome/interstitial cystitis symptoms. A high influx of MCTC in the mucosa of individuals who also had ulceration noted within their diagnostic notes may be of the Hunner's ulcer subclassification. These findings suggest a relationship between the pathogenesis of MC subtypes and the clinical presentation of painful bladder syndrome/interstitial cystitis. A cohort study would further elucidate the diagnostic and/or therapeutic potential of MCs in patients with painful bladder syndrome/interstitial cystitis.

Diluted honey inhibits biofilm formation: potential application in urinary catheter management?

AIMS: Biofilms are ubiquitous and when mature have a complex structure of microcolonies in an extracellular polysaccharide and extracellular DNA matrix. Indwelling medical devices harbour biofilms which have been shown to cause infections and act as reservoirs for pathogens. Urinary catheters are often in place for considerable periods of time and are susceptible to both encrustation and biofilm formation. Strategies for minimising biofilm occurrence underpin an active research area in biomedicine. Manuka honey has, inter alia, well-established antibacterial properties. This study aims to assess the influence of honey on early biofilm formation in an established in vitro model. METHODS: An established model of early biofilm formation using static bacterial cultures in vinyl 96-well plates was used to grow Escherichia coli, strain ATC 25922 and Proteus mirabilis, strain 7002. Planktonic cells were removed and the residual biofilm was stained with crystal violet, which were subsequently eluted and quantified spectrophotometrically. Manuka honey (Unique Manuka Factor 15+) was added either with the bacteria or up to 72 hours after. RESULTS: Biofilms in this model was developed over 3 days, after which growth stalled. Mixed (1:1) cultures of E. coli and P. mirabilis grew slower than monocultures. In mixed cultures, honey gave a dose-dependent reduction in biofilm formation (between 3.3 and 16.7%w/v). At 72 hours, all concentrations inhibited maximally (p<0.001). Application of honey to cultures after 24 and 48 hours also reduced the adherent bacterial biomass (p<0.05-p<0.01). CONCLUSION: Manuka honey at dilutions as low as 3.3% w/v in some protocols and at 10% or above in all protocols tested significantly inhibits bacterial attachment to a vinyl substrate and reduces further early biofilm development. No augmentation of growth over untreated controls was observed in any experiment.

Artemisinins as potential anticancer agents: uptake detection in erythrocytes using Fourier transform infrared spectroscopy and cytotoxicity against bladder cancer cells.

AIMS: Semisynthetic derivatives of the antimalarial drug artemisinin may also possess anticancer properties. The ability to detect artemisinin uptake and distribution in cells would facilitate live cell imaging without labelling. This study describes mid-range infrared absorption spectra for three artemisinin variants and attempts to detect their presence in a simple cell model (erythrocytes). Cytotoxicity assays assess potential anticancer properties against bladder cancer cells. METHODS: Mid-range Fourier transform infrared spectra were obtained from dry preparations of dihydroartemisinin (DHA), artesunate (ART) and artemether (ARTE). Erythrocytes were prepared from normal blood and incubated for 30 min at 37°C with the three artemisinin derivatives. Cytospin preparations were prepared on aluminium foil for spectroscopy. Potential for growth inhibition in the RT112 bladder carcinoma cell line was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide residual viable biomass method. RESULTS: Spectra were obtained from the three native compounds. Repeat scans after 8 weeks showed ART and ARTE to be stable, stored under manufacturer's recommendations. DHA exhibited marked changes over the same period. It was possible by subtraction to detect DHA in cytospins, but not ART or ARTE. The fit between the subtraction spectrum and that of the native compound was >80%. DHA and ART showed strong cytotoxic potential against RT112 cells. CONCLUSIONS: The artemisinin derivatives tested exhibit unique mid-range infrared absorption spectra which can be used to monitor degradation and, for DHA, can be detected by subtraction in loaded erythrocytes rendering future imaging studies feasible. Its cytotoxic efficacy against RT112 cells suggests bladder cancer as a possible target disease.

Tissue factor and tissue factor pathway inhibitor in women with a past history of preeclampsia: implication for a hypercoagulable state postpregnancy.

Preeclampsia (P-EC) is a multisystem disorder of pregnancy whose cause and pathogenesis remain poorly understood. However, abnormal haemostasis and endothelial dysfunction are thought to be implicated. Women with a past medical history of P-EC have a baseline hypercoagulable state postpregnancy. The aim of this study is to examine the relationship between tissue factor (TF) and TF pathway inhibitor (TFPI) in women who have had P-EC within the last 3 years (more than 6 months postpartum) and their normal counterparts. Blood specimens were collected from women known to have had P-EC within the last 3 years (n = 26) and aged-matched healthy women without past history of P-EC in previous pregnancy (n = 26). Plasma TF and TFPI levels were measured using ELISAs. Women who have had P-EC showed increased TF levels compared with their normal counterparts, whereas TFPI levels were reduced. Neither parameter differed significantly when the groups were tested against each other. Interestingly, the TF/TFPI ratio was significantly increased (P = 0.024) when the two groups were compared. In summary, there was a trend towards increased TF and reduced TFPI levels in the P-EC group. Such a tendency was not statistically significant. However, the TF/TFPI ratio was significantly increased when the groups were compared. Our findings suggest an imbalance between TF/TFPI levels in women with past history of P-EC postpregnancy. This may contribute to the development of maternal hypercoagulable states and may predispose women with a history of P-EC to cardiovascular risks later in life.

Fourier transform infrared spectroscopy imaging of live epithelial cancer cells under non-aqueous media.

AIMS: Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging. METHODS: The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope. RESULTS: Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells. CONCLUSIONS: The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.

Time-lapse live cell imaging and flow analysis of multidrug resistance reversal by verapamil in bladder cancer cell lines.

OBJECTIVES: To examine the effects of verapamil on the intracellular drug pharmacokinetics of epirubicin using alternative dosing schedules. The results might inform the choices for optimizing clinical chemotherapy. METHODS: Sensitive parental (MGH-U1) and multidrug resistant (MDR) (MGH-U1R and MGH-U1-MMC) bladder cancer cell lines were used. Fluorescence time-lapsed studies were performed on cells incubated with epirubicin alone or combined with verapamil. Flow cytometry was performed after the alternative dosing regimens. RESULTS: Verapamil reversed the epirubicin localization patterns in MDR cells. Time-lapse imaging showed that nuclear epirubicin accumulation in MDR cells with verapamil followed the parental curve. The maximal reversal took >60 minutes. Flow cytometry showed increased epirubicin uptake in MDR cells co-incubated with verapamil. Preincubation was not as effective as co-incubation. CONCLUSIONS: The results of our model indicate that longer exposure to MDR-class drugs, exemplified by epirubicin, increases uptake and the MDR reversing action of co-treatment with verapamil. The present results highlight the need for additional clinical trials of drug dosing and scheduling for combination intravesical chemotherapy regimens.

Antiprostasome antibodies are not an appropriate prognostic marker for prostate cancer.

OBJECTIVE: Antiprostasome antibodies (APAs) have been identified in serum of patients with prostate cancer and have been proposed as a new marker for metastatic disease. This study reassesses the role of APAs as a prognostic indicator for prostate cancer. MATERIAL AND METHODS: Serum samples from healthy controls (n=7) and patients with prostate cancer (n=22) were assayed for APAs using an enzyme-linked immunosorbent assay. RESULTS: APAs in varying amounts were present in healthy individuals as well as in men with prostate cancer. Higher levels were inversely and significantly associated with prostate-specific antigen (PSA). No significant relationships were noted between APA levels and other parameters such as age, time since diagnosis, metastatic status, Gleason histological score and hormonal treatment. CONCLUSIONS: The presence of serum APA is unlikely to be a strong prognostic indictor for prostate cancer on an individual basis as false positives will occur. However, such immune reactions which may be associated with PSA in cancer patients are in any case of interest in both the biology of prostate cancer and male fertility. The source of prostasomal antigen may be of critical importance to the outcome of the assay. However, immune reactions to prostasomes may be of considerable interest and warrant continued investigation.

The direct effect of nuclear pores on nuclear chemotherapeutic concentration in multidrug resistant bladder cancer: the nuclear sparing phenomenon.

PURPOSE: Multidrug resistance commonly limits effectiveness in treating malignancy with chemotherapy. Multidrug resistance has classically been described as a cell membrane phenomenon. Multidrug resistant cells are known to specifically exclude chemotherapy from the nucleus, resulting in lower nuclear concentrations than in the cytoplasm. This phenomenon is known as nuclear sparing and little is known of its etiology. We hypothesized that a component of the nuclear membrane, the nuclear pore, is responsible for this phenomenon. MATERIALS AND METHODS: In this in vitro study we used the drug sensitive urothelial cancer cell line Massachusetts General Hospital urothelial 1 sensitive and its multidrug resistant subline Massachusetts General Hospital urothelial 1 resistant. After quantitative assessment of nuclear pores resistant and sensitive cells were fused using polyethylene glycol and laser scanning confocal microscopy was used to identify if drug resistant and sensitive nuclei can coexist within the same cell. The effect of inhibiting nuclear pore function using the specific pore inhibitor, wheat germ agglutinin, was assessed in whole cells using confocal microscopy and cytotoxicity assay as well as in isolated nuclei. RESULTS: Nuclear pores appeared more numerous in multidrug resistance cells. Cell fusion experiments showed that multidrug resistance and sensitive nuclei could coexist with the same cell milieu. Wheat germ agglutinin reversed multidrug resistance in whole cells and isolated nuclei. CONCLUSIONS: Multidrug resistance is a complex phenomenon occurring at many cellular levels, of which all may be potential therapeutic targets. The nuclear pore is involved in this process, which is to our knowledge a previously undescribed phenomenon. These experiments suggest that it may act to export drug from the nucleus, which is a process inhibited by wheat germ agglutinin.

Current drug therapy for prostate cancer: an overview.

Prostate cancer is the most common cancer amongst men in the USA and the second most common malignant cause of male death worldwide after lung cancer. The life time risk of having microscopic evidence of prostate cancer for a 50 year old man is 42%. Prostate cancer is thus becoming an increasingly significant global health problem in terms of mortality, morbidity, as well as economically. This review, discusses current medical therapeutic options for prostate cancer including traditional treatments using luteinising hormone releasing analogues (LHRH), anti-androgens and estrogen treatments, and the use of novel drugs directed against molecular targets considered important in oncogenesis and metastasis. Prostate cancer chemoprevention using 5alpha-reductase inhibitors and the role of gene therapy are also considered.

Does human semen contain a functional haemostatic system? A possible role for Tissue Factor Pathway Inhibitor in fertility through semen liquefaction.

There is already evidence that a few components of the haemostatic system exist in semen. If these comprise a functional system, they may have a role in seminal clotting and liquefaction processes and ultimately may influence fertility. What might be expected in semen as collected from fertility clinics i.e., after having both coagulated and subsequently liquefied is uncertain. It does however still contain significant amounts of Tissue Factor (TF) although its effect on semen quality remains poorly understood. The present study analyses semen for Tissue Factor Pathway Inhibitor (TFPI). Measurements were made in seminal plasma, swim-up sperm and prostasomes and its relationship with conventional fertility parameters assessed. TFPI antigen levels in seminal plasma were measured in a total of 176 subjects using an Enzyme-linked immunosorbent assay (ELISA). These include sub-fertile (n=37), normally fertile (n=40), fertile sperm donor (n=34), vasectomized subjects (n=65) and in a further group defined by normality in several parameters derived from the World Health Organization (WHO) fertility criteria and termed "pooled normal semen parameters" (PNSP). For characterization studies, both TFPI activity and antigen were measured on whole semen, swim-up sperm and prostasome-rich fraction (n=5). TFPI levels were significantly higher in normal men as compared to sub-fertile (P<0.01) or vasectomized subjects (P<0.001). TFPI levels were even higher in the donor quality semen and the PNSP group. TFPI levels also correlated with semen liquefaction time, normal semen viscosity, sperm progression, percentage of motile sperm and sperm counts (density). In conclusion, the present finding substantiates the concept of an active clotting system in human semen. TFPI could regulate the activity of abundant TF, as it does elsewhere. Given a functional set of coagulation factors in semen, the TF/TFPI balance might impinge on its liquefaction and hence on global fertility.

The nuclear membrane in multidrug resistance: microinjection of epirubicin into bladder cancer cell lines.

OBJECTIVE: To assess whether microinjecting epirubicin into cells showing multidrug resistance (MDR, common to many cancers, including bladder cancer, with resistance to, e.g. anthracyclines and mitomycin C) spares the nucleus, as when these drugs accumulate, distribution in MDR cells characteristically spares the nucleus, suggesting that the nuclear membrane is responsible for excluding cytotoxic drugs from MDR nuclei. MATERIALS AND METHODS: Nuclear exclusion of drugs is an important feature of resistance in MDR cells, as many MDR-susceptible drugs have cytotoxic actions within the nucleus. Drug accumulation in 'classical' P-glycoprotein-mediated MDR cells is greatly reduced by efflux. Microinjection of epirubicin into the cytoplasm of MDR cells bypasses the P-glycoprotein efflux pump on the plasma membrane. Nuclear sparing would directly implicate the nuclear membrane in this phenomenon. Because of their fluorescence properties, which allow study by confocal microscopy and flow cytometry, anthracyclines have also been used extensively to investigate MDR. Thus sensitive (MGH-U1 and RT112) and MDR (MGH-U1R and MGH-U1-MMC) bladder cancer cell lines were used. Adherent cells from each cell line were individually microinjected with epirubicin (0.5 mg/mL) and a 77 kDa fluorescein isothiocyanate (FITC)-dextran (0.5 mg/mL). The pattern of nuclear epirubicin uptake in injected cells was then evaluated by confocal microscopy. The 77 kDa FITC-dextran allowed easier identification of injected cells and was also excluded from their nuclei. RESULTS: Sensitive bladder cancer cell lines all showed a nuclear accumulation pattern of epirubicin, consistent with their normal uptake after exposure to epirubicin. The MDR cell lines showed the characteristic nuclear-sparing pattern of epirubicin uptake, similar to the normal uptake pattern after epirubicin exposure. The 77 kDa FITC-dextran showed clearly which cells had been microinjected, and was excluded from the nuclei of all injected cells. Cell viability was confirmed by acridine-orange staining after initial visualization of injected cells. CONCLUSION: The nuclear membrane is responsible for the nuclear exclusion of epirubicin in MDR cells. Further work is necessary to determine the mechanisms involved.

Quantitation of seminal factor IX and factor IXa in fertile, nonfertile, and vasectomy subjects: a step closer toward identifying a functional clotting system in human semen.

Coagulation factor (F) IX is a zymogen of the plasma serine proteases, one that plays an essential role in the regulation of normal blood coagulation. Congenital defects of FIX synthesis or function cause hemophilia B (originally called hemophilia C). Factor IX is activated by Tissue Factor (TF):FVII/FVIIa complex and FXIa. Subsequent to its activation, FIXa combines with FVIIIa on the platelet surface and activates FX to FXa. Human semen forms a semi-solid gelatinous coagulum, which then liquefies within 5-20 minutes in vitro. In spite of evidence demonstrating the importance of the seminal coagulation and liquefaction process in terms of global fertility and despite the fact that the seminal coagulum is composed of fibrin-like material, it has always been addressed from the perspective of High Molecular Weight Seminal Vesicle (HMW-SV) proteins (Semenogelin I and II) and their cleavage by prostate-specific antigen rather than the conventional hemostatic factors. In this study and as part of our continuing investigation of human seminal clotting factors, we report here on seminal FIX and FIXa in normal, subfertile, and vasectomized subjects. Factors IX and FIXa were studied in a total of 119 semen specimens obtained from subfertile (n=18), normally fertile (n=34), and fertile sperm donors (n=27) and vasectomy subjects (n=40). Seminal FIX and FIXa levels were also measured in a group defined by normality in several parameters derived from the World Health Organization fertility criteria and termed "pooled normal semen parameters." Both FIX and FIXa were quantifiable in human semen. There was a wide individual variation in FIX and FIXa levels within groups. Despite the group size, statistically significant associations with fertility-related parameters were infrequent. There is a positive correlation between FIX and its activation product, FIXa (n=36; r=0.51; P <.05). Factor IXa elevation in the high sperm-clump group was significant (P <.05), and days of abstention correlated with FIXa levels (n=63; r=0.3; P <.05). The key finding of the present study is that both FIX and FIXa are present in concentrations that are not dissimilar to plasma levels and that are apparently functional, as the activated form is also present. This fact, taken with other reports of coagulation factors in semen, raises the likelihood that a functional set of hemostatic coagulation proteins exists in semen, potentially to interact with the HMW-SV proteins and the prostate-specific antigen system.

Epirubicin and meglumine gamma-linolenic acid: a logical choice of combination therapy for patients with superficial bladder carcinoma.

BACKGROUND: Anthracyclines have been established as first-line drugs for intravesical use in the treatment of patients with superficial bladder carcinoma, although they result only in a modest reduction in tumor recurrence rates. The essential fatty acid gamma-linolenic acid (GLA) also is an effective cytotoxic agent against superficial bladder carcinoma when it is applied topically. The objective of this study was to assess the efficacy of combined epirubicin and GLA with the purpose of developing a suitable model for modification of existing intravesical regimens. METHODS: The human urothelial carcinoma cell lines MGH-U1 and RT112 were used in standard cytotoxicity assays and were exposed to meglumine GLA (MeGLA) and epirubicin in two-dimensional concentration matrices. A thiozolyl blue (methyl-thiazoldiphenyl tetrazolium) assay was used to determine residual cell biomass. Drug interaction was quantified by median-effect analysis software (CalcuSyn), and the evaluation of drug uptake utilized fluorescence confocal microscopy (FCM) and flow cytometry. RESULTS: MeGLA caused a significant enhancement of anthracycline uptake, viewed by FCM, from 92 fluorescence units to 222 fluorescence units (P < 0.001). Flow cytometry confirmed the increased drug uptake and showed that the mean epirubicin content per cell increased from 23 to 57 units and from 8 to 24 units for MGH-U1 and RT112 cells, respectively (99% confidence interval < 0.3). This resulted in improved cytotoxicity, and it was shown that the drugs acted synergistically with all but the highest MeGLA concentrations. CONCLUSIONS: The efficacy of epirubicin was enhanced significantly when it was used in combination with most concentrations of MeGLA (< 300 microg/mL), and the two agents acted synergistically. There was a corresponding increase in epirubicin uptake by cells under these conditions. At high MeGLA concentrations, however, anthracycline solubility was compromised, and drug synergy was lost.