Urinary exosome as a potential biomarker for urinary tract infection.

Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.

Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.

BACKGROUND: Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients. METHODS: Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4-2 and bone metastatic C4-2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody. RESULTS: Among proteins upregulated in C4-2 and C4-2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4-2 and C4-2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.

TMPRSS2 Met160Val polymorphism: significant association with sporadic prostate cancer, but not with latent prostate cancer in Japanese men.

OBJECTIVES: To investigate the association between the TMPRSS2 Met160Val polymorphism and the risk of prostate cancer in Japanese men. METHODS: Genomic DNA samples from 518 Japanese sporadic prostate cancer patients, 433 controls and 154 Japanese men who were diagnosed as having latent prostate cancer based on autopsy results were genotyped for the TMPRSS2 Met160Val polymorphism using a TaqMan assay. Logistic regression analyses were carried out to estimate the odds ratios and 95% confidence intervals. The relationship between the presence of the polymorphism, and clinicopathology and survival was also examined. RESULTS: The T allele frequency of the control group was 0.372, of the sporadic prostate cancer group was 0.435 and of the latent prostate cancer group was 0.370. The CT and TT genotypes were significantly associated with risk for sporadic prostate cancer; age-adjusted odds ratios (95% confidence intervals) were 1.418 (1.027-1.960) for CT, 1.907 (1.224-2.990) for TT and 1.524 (1.123-2.072) for CT/TT genotypes. There was no significant association observed between the TMPRSS2 Met160Val polymorphism and the risk for latent prostate cancer. The TMPRSS2 Met160Val polymorphism was not significantly associated with any clinicopathological features or prognosis. CONCLUSIONS: The TMPRSS2 Met160Val polymorphism is a genetic risk factor for sporadic prostate cancer in a Japanese population.

[Relevant factors concerning phenomena related to the process of initiating dialysis in elderly patients with chronic renal failure: predicting the outcomes of subsequent phenomena at the stage of the initial phenomenon].

AIM: We previously investigated the relevant factors concerning each individual phenomenon related to the process of initiating dialysis in elderly patients with chronic renal failure. Background factors that were identified as relevant factors were significant in terms of enabling us to predict the outcome of each phenomenon in new patients. However, the significance of these factors in predicting the outcomes of subsequent phenomena at the stage of the initial phenomenon was unclear. This was attributed to the fact that the subjects with phenomena decreased in number and because of changes in characteristics (hereafter, changes in subjects) with the progression of the process of initiating dialysis. In the present study, we aimed to identify relevant factors for predicting the outcomes of subsequent phenomena at the stage of the initial phenomenon. For this purpose, we assumed that "progression of the process of initiating dialysis does not result in significant reductions in the number of cases and causes only minor changes in characteristics". METHODS: We studied a total of 152 patients with advanced chronic renal failure aged >or=60 years. Background factors were investigated in all patients. The following phenomena were analyzed: acceptance of dialysis, urgency of initiating dialysis, alleviation of disease, and returning home. In order to identify new relevant factors, we focused on the order and condition of the process by which each background factor was narrowed down during logistic regression analysis for each phenomenon. We determined which background factor to focus on for each phenomenon based on changes in background factors. RESULTS: Age and cognitive function were related to the urgency of initiating dialysis and alleviation of disease. Age, walking ability, and cognitive function were related to returning home. Age was eliminated at the final stage of logistic regression analysis for alleviation of disease and at the penultimate stage of logistic regression analysis for returning home. CONCLUSION: We simulated the restoration of cases lost during the process of initiating dialysis in order to determine the relationship of age to alleviation of disease as well as returning home. Although age significantly affected alleviation of disease, its relationship to returning home was unclear. Age was thus identified as a new relevant factor for alleviation of disease. In addition, all relevant factors identified from investigation of each phenomenon enabled prediction of outcomes of subsequent phenomena at the stage of the initial phenomenon.

Effects of a Gentle, Self-Administered Stimulation of Perineal Skin for Nocturia in Elderly Women: A Randomized, Placebo-Controlled, Double-Blind Crossover Trial.

BACKGROUND: Somatic afferent nerve stimuli are used for treating an overactive bladder (OAB), a major cause of nocturia in the elderly. Clinical evidence for this treatment is insufficient because of the lack of appropriate control stimuli. Recent studies on anesthetized animals show that gentle stimuli applied to perineal skin with a roller could inhibit micturition contractions depending on the roller's surface material. We examined the efficacy of gentle skin stimuli for treating nocturia. METHODS: The study was a cross-over, placebo-controlled, double-blind randomized clinical study using two rollers with different effects on micturition contractions. Participants were elderly women (79-89 years) with nocturia. Active (soft elastomer roller) or placebo (hard polystyrene roller) stimuli were applied to perineal skin by participants for 1 min at bedtime. A 3-day baseline assessment period was followed by 3-day stimulation and 4-day resting periods, after which the participants were subjected to other stimuli for another 3 days. The primary outcome was change in the frequency of nighttime urination, for which charts were maintained during each 3-day period. RESULTS: Twenty-four participants were randomized, of which 22 completed all study protocols. One participant discontinued treatment because of an adverse event (abdominal discomfort). In participants with OAB (n = 9), change from baseline in the mean frequency of urination per night during the active stimuli period (mean ± standard deviation, -0.74 ± 0.7 times) was significantly greater than that during placebo stimuli periods (-0.15 ± 0.8 times [p < 0.05]). In contrast, this difference was not observed in participants without OAB (n = 13). CONCLUSIONS: These results suggest that gentle perineal stimulation with an elastomer roller is effective for treating OAB-associated nocturia in elderly women. Here the limitation was a study period too short to assess changes in the quality of sleep and life. TRIAL REGISTRATION: UMIN Clinical Trial Registry (CTR) UMIN000015809.

Effect of phorbol 12,13-dibutyrate on smooth muscle tone in rat stomach fundus.

We investigated the effects of phorbol 12,13-dibutyrate (PDBu), a typical protein kinase C (PKC) activator, on smooth muscle tone in the rat stomach fundus. In 5-hydroxytriptamine (5-HT)-precontracted stomach fundus strips, PDBu induced dose-dependent relaxation, but 4alpha-phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not induce relaxation. A PDBu-induced dose-dependent relaxation was also observed in strips precontracted with platelet-activating factor (PAF), carbachol, or 60 mM K+. In stomach fundus strips pretreated with PDBu, the contractile responses to 5-HT and PAF were completely blocked, but those induced by carbachol and endothelin-1 (ET-1) were only partially inhibited. In stomach fundus strips preincubated with carbachol in Ca2+-free medium, the Ca2+-induced contraction was decreased by preincubation with PDBu. In strips preincubated with 5-HT, PAF, or ET-1 in Ca2+-free medium, Ca2+-induced contractions were greatly inhibited by pretreatment with PDBu. These results suggest that in rat stomach fundus strips, PDBu-induced relaxation is mediated by activation of PKC. We speculate that a major factor mediating the relaxant action of PDBu in rat stomach fundus smooth muscle is represented by a reduction in Ca2+ influx via an inhibition of Ca2+ channels.

Diabetes-related changes in contractile responses of stomach fundus to endothelin-1 in streptozotocin-induced diabetic rats.

The contractile response of the stomach fundus to endothelin-1 (ET-1) was examined in streptozotocin (STZ)-induced diabetic rats. In STZ-diabetic rats (versus age-matched control rats) (a) ET-1 caused a longer-lasting contraction of stomach fundus strips, and (b) in the dose-response curve, the ET-1-induced contraction was significantly greater for a given concentration (3 x 10(-7) to 10(-7) M). Although repeated application of ET-1 led to desensitization, the desensitization was less pronounced in STZ-diabetic rats than in the controls. The density of the binding sites for [(125)I]-ET-1 was increased in the diabetic stomach fundus (versus the controls), but Kd values were similar between the two groups. The ET(B) receptor mRNA expression level was significantly increased in the diabetic stomach fundus. These results suggest that the diabetes-related enhancement of the ET-1-induced contraction of the stomach fundus may be due to an increase in the ET(B) receptor population.

[Refusal of initiation of dialysis by elderly patients with chronic renal failure].

AIM: Refusal of dialysis is not uncommon in elderly patients with chronic renal failure. In this study, we retrospectively inspected our dealings with patients who refused our offer to initiate dialysis. In addition, we discussed how to grasp the meaning of this phenomenon. METHODS: We treated 152 patients with advanced chronic renal failure aged 60 years and over at Tokyo Metropolitan Geriatric Hospital. The patients fulfilling the following two criteria were considered to be refusal cases. The first criterion was that an acceptance of the initiation of dialysis could not be obtained in spite of repeated counseling. The second criterion was that a definite outcome was precipitated by the development of severe uremic symptoms. In every refusal case, clinical characteristics and household members were surveyed. Verbal expressions of the reasons for refusal were retrieved from medical charts. The outcome was also studied. RESULTS: The two criteria were fulfilled in 7 cases. The male/female ratio was 5:2. The age was 78 +/- 7 years (mean +/- standard deviation). All but one cases were ambulatory, and all cases had normal cognitive function. Four cases were married, and the other cases had lost their partners. The number of household members was 3.9 +/- 1.8. We speculated that every case could maintain a good quality of life even after the initiation of dialysis. Representative expressions of the reasons for refusal were "I have already lived fully" and "I would prefer to accept death rather than dialysis". The outcome was urgent initiation of dialysis (five cases) and death (two cases). The time between initial counseling and the outcome was 115 +/- 37 days. CONCLUSION: Accepting or refusing dialysis therapy is a selection related to life or death. We must make an effort to obtain consent to initiating dialysis if patients are assessed as suitable for dialysis.

[Acceptance of dialysis therapy in elderly patients with chronic renal failure].

The aim of the present study is to clarify relevant factors concerning acceptance of dialysis therapy in elderly patients with chronic renal failure. Patients with advanced renal failure aged 60 years and over (152 cases) were investigated. The male/female ratio was 85:67. The age was 76 +/- 7 years (mean +/- standard deviation). The proportion of patients with acceptance of dialysis to patients with non-acceptance was 121:31. In all patients, the cause of renal failure (non-diabetes/diabetes), serum albumin level, comorbid conditions, ambulation, cognitive function, marital status, and presence of younger cohabitants were surveyed. The patients were divided into two groups for each category. Patients were categorized as the young-old (aged 60 to 74 years) and the old-old (aged 75 years and over). Serum albumin level was categorized as either low (less than 3.5 g/dl) or normal (3.5 g/dl and over). The number of patients who accepted dialysis therapy was evaluated for each group. Intergroup comparisons were carried out by the chi 2 test. Statistically significant factors were age (p < 0.0001), serum albumin level (p = 0.016), ambulation (p = 0.011), cognitive function (p < 0.0001), and marital status (p = 0.009). Multivariant logistic regression analysis was also performed using background factors as explanatory variables and acceptance or non-acceptance of dialysis therapy as a dependent variable. The factors presented by the nominal scale were converted to dummy variables. Statistically significant factors were age (p < 0.0001) and cognitive function (p < 0.0001). Serum albumin level, ambulation, and marital status were significant only in the chi 2 test. This could be explained by the close correlations of these factors with age and cognitive function. The old-old category and poor cognitive function were dominant factors with regard to non-acceptance of dialysis therapy.

[Analyses of factors concerning the short-term outcome of elderly patients beginning dialysis].

The aim of the present study is to clarify relevant factors concerning the short-term outcome of elderly patients beginning dialysis. One hundred nineteen patients aged 60 years and over who had newly started dialysis at our hospital were studied. The male/female ratio was 70:49. The age was 74 +/- 7 years (mean +/- standard deviation). In all patients, the timing of referral to a nephrologist (early/late), the urgency of the initiation of dialysis (non-urgent/urgent), the cause of renal failure (nondiabetes/diabetes), serum albumin concentration, comorbid conditions (cerebrovascular disease, ischemic heart disease, etc.), ambulation, cognitive function, and the outcome (relief/death) were surveyed. Twelve patients did not obtain relief and finally died. The influence of the timing of referral on the urgency of the initiation of dialysis was studied. Furthermore the influence of the urgency of the initiation of dialysis on the outcome was studied. The chi 2 test was used for statistical comparisons. The need for urgent dialysis was less among early referral cases as compared with late referral cases (p < 0.0001). The incidence of death was more frequent in urgent dialysis than in non-urgent dialysis (p = 0.016). Multivariate logistic regression analysis was performed using background factors as explanatory variables and the outcome as a dependent variable. Statistically significant factors were the urgency of the initiation (p = 0.040), serum albumin concentration (p = 0.022), and cerebrovascular disease (p = 0.002). The most common cause of death was severe infectious diseases (pneumonia, sepsis). It was speculated that background factors associated with the outcome could contribute to the onset and the progression of infectious diseases.

[A pitfall in analyses of relevant factors concerning phenomena in the process of initiating dialysis: alterations in the characteristics of subjects diminish the significance of age].

In our previous studies, relevant factors concerning the main phenomena related to the process of initiating dialysis were examined in elderly patients with chronic renal failure. Examined phenomena were as follows: (1) the acceptance of dialysis; (2) the urgency of initiating dialysis; (3) short-term outcome; (4) returning home. Multivariate logistic regression analysis was used to determine relevant factors. Although we speculated that age should be a relevant factor for each phenomenon, the phenomenon on which age had some impact was only the first. We suspected the existence of a pitfall, through which the relation of age was lost in the second, the third, or the fourth phenomenon. The fact that every phenomenon had its own relevant factors was thought to be an important clue to the discovery of pitfalls. Relevant factors were derived from both the number of dropout-patients and their demographic and clinical status. From the viewpoint of nondropout-patients, the progression of the process of initiating dialysis might alter the characteristics of subjects for successive phenomena In this study, we set out to investigate whether alterations in the characteristics of subjects were pitfalls. Alterations were regarded as a fall of the mean age, an increment of the rate of the patients with ability to walk, and an increment of the rate of the patients with normal cognitive function. In addition, the old-old patients tended to have limited numbers of those who had the ability to walk and normal cognitive function. In other words, aging changes in ambulatory and cognitive function were not brought to subjects. These alterations may cause the loss of the relation of age to each phenomenon. Thus, we presumed these alterations to be pitfalls. We must clarify whether aging changes are brought to subjects beforehand in analyses that include the old-old patients as subjects.

[Differences in features between young-old and old-old patients beginning dialysis].

We investigated differences in features between young-old and old-old patients beginning dialysis. Patients aged over 60 years who had newly started dialysis (121 cases) were studied. These were 71 men and 50 women. The age was 74 +/- 7 years (mean +/- standard deviation). The patients were divided into the young-old group (aged 60 to 74 years) and the old-old group (aged over 75 years), 64 patients were young-old and 57 were old-old. In every patient, the cause of renal failure (non-diabetes/diabetes), body indexes, comorbid conditions, laboratory data of nutritional status, ambulation, cognitive function, and psychosocial status (acceptance of dialysis therapy, marital status, younger cohabitants, and engagement in work) were surveyed. Data were assessed in each group. Intergroup comparisons were made using Student's t-test, the chi 2 test, and the Mann-Whitney's U-test. Diabetes was frequent in the young-old group. The results concerning body indexes, comorbid conditions, and laboratory data reflected age-related phenomena or skew distribution of diabetic patients. Though differences were not statistically significant, both the percentage of patients with inability to walk and the percentage of patients with impaired cognitive function were lower in the old-old. These results could not be interpreted as age-related phenomena or skew distribution of diabetic patients. From the data of psychosocial indexes, it would speculate that the young-old had some advantage over the old-old for initiation of dialysis. We look for the explanation to psychosocial aspects of dialysis therapy.

A gentle mechanical skin stimulation technique for inhibition of micturition contractions of the urinary bladder.

Effects of gentle skin stimulation of various segmental areas on the micturition contractions of the urinary bladder were examined in anesthetized male rats. The bladder was expanded by infusing saline via urethral cannula until the bladder produced rhythmic micturition contractions as a consequence of rhythmic burst discharges of vesical pelvic efferent nerves. Gentle stimulation was applied for 1 min by slowly rolling on top of skin with an elastomer "roller". Rolling on the perineal area inhibited both micturition contractions and pelvic efferent discharges during and after stimulation. Stimulation of the hindlimb, abdomen and forelimb inhibited micturition contractions after stimulation ended, in this order of effectiveness. During stimulation of the perineal skin, the reflex increase in pelvic efferent discharges in response to bladder distension to a constant pressure was also inhibited up to 45% of its control response. The inhibition of the micturition contractions induced by perineal stimulation was abolished, to a large extent by the opioid receptor antagonist naloxone and completely by severing cutaneous nerves innervating the perineal skin. We recorded unitary afferent activity from cutaneous branches of the pudendal nerve and found that the fibers excited by stimulation were low-threshold mechanoreceptive Aß, Aδ and C fibers. Discharge rates of afferent C fibers (7.9 Hz) were significantly higher than those of Aß (2.2 Hz) and Aδ (2.9 Hz) afferents. The results suggest that low frequency excitation of low threshold cutaneous mechanoreceptive myelinated and unmyelinated fibers inhibits a vesico-pelvic parasympathetic reflex, mainly via release of opioids, leading to inhibition of micturition contraction.

Association of rs6983561 polymorphism at 8q24 with prostate cancer mortality in a Japanese population.

UNLABELLED: We conducted present study to address whether the rs6983561 polymorphism, an established genetic marker for prostate cancer susceptibility, was a prognostic indicator. We genotyped 518 Japanese patients with prostate cancer and analysed their survival retrospectively. As a result, patients with the CA/CC genotype of rs6983561 survived significantly longer than those with the AA genotype (P = .033). BACKGROUND: Genome-wide association studies have revealed several genetic variants at 8q24 that are associated with prostate cancer susceptibility. Rs6983561 (A/C) is a single-nucleotide polymorphism located at 8q24 that has been established as a genetic risk marker for prostate cancer susceptibility. The present study investigated the association between the rs6983561 polymorphism and prostate cancer mortality in a Japanese population. PATIENTS AND METHODS: The study examined 518 native Japanese male patients with sporadic prostate cancer. Germline DNA samples were obtained from all participants and genotyping of rs6983561 was performed using a TaqMan assay. Observation periods were from the date of diagnosis of prostate cancer to May 21, 2010. The Cox proportional hazards model was used to estimate the cause-specific survival (CSS) and the overall survival (OS). RESULTS: Patients with the CA/CC genotype of rs6983561 survived significantly longer than those with the AA genotype. In a multivariate model, the hazard ratios (HRs) and 95% confidence intervals (CIs) of the CSS and the OS for the rs6983561 polymorphism were 2.438 (1.262 - 5.046, P = .007) and 1.957 (1.142 - 3.485, P = .014), respectively. When the analysis was restricted to subjects with metastatic disease, the HRs of the CSS and the OS were 3.353 (95% CI, 1.689 - 7.446; P = 3.76 x 10(-4)) and 3.361 (95% CI, 1.741 - 7.136; P = 1.70 x 10(-4)), respectively. CONCLUSION: In the Japanese population examined in this study, the rs6983561 polymorphism at 8q24 was significantly associated with prostate cancer mortality, especially among patients with metastatic disease.

Primary aldosteronism due to unilateral adrenal microadenoma in an elderly patient: efficacy of selective adrenal venous sampling.

We encountered a case of drug-resistant hypertension and hypokalemia. Laboratory data suggested primary aldosteronism (PA). Computed tomography imaging appeared normal for a long duration with a left-sided nodule appearing far later; adrenal scintigraphy was first normal, and the second test showed right-sided uptake. However, a repeat selective adrenal venous sampling (SAVS) indicated a left-sided lateralization of the hypersecretion of aldosterone. Left adrenectomy was performed, and his clinical symptoms improved. The histopathological findings demonstrated the aldosterone-producing microadenoma with secondary micronodules. In conclusion, SAVS should be performed to determine the laterality of PA with obscure CT imaging.

Elucidation with the protease alpha-chymotrypsin of the inhibitory modulating action of endogenous neuropeptide Y over sympathetic neurotransmission in rat vas deferens.

The physiological role of endogenous neuropeptide Y (NPY) in sympathetic neurotransmission was examined in rat and guinea pig vas deferens (VD), using alpha-chymotrypsin (alpha-CT). NPY-like immunoreactivity was detected in the longitudinal muscle layer of VD densely in rats but sparsely in guinea pigs, and it disappeared following surgical denervation. Under blockade of the prejunctional alpha(2)-adrenergic autoinhibition, alpha-CT potentiated the phasic contraction in rat, but not guinea pig, VD induced by trains of transmural nerve stimulation (TNS) in a frequency-dependent manner, which was reproducible during repeated applications and not affected by pretreatment with capsaicin. In contrast, alpha-CT did not potentiate the twitch response or contractions induced respectively by a single pulse TNS or by direct electrical stimulation to the smooth muscle. Exogenously applied NPY suppressed the twitch response, which was cancelled by alpha-CT, and excitatory junction potentials, although it affected neither spontaneous junction potentials nor the direct electrical stimulation-induced contraction. These observations provided further evidence to support that NPY is released endogenously by TNS at high frequency, acting prejunctionally to suppress sympathetic neurotransmission. Thus, the protease alpha-CT proved itself to be a useful tool to reveal a functional role of endogenously released peptides.

Prominent induction of cyclin B1 in G2/M renal cancer cells with butyrolactone 1.

BACKGROUND: Butyrolactone 1 (BL) is a cyclin dependent kinase (CDK) inhibitor derived from Aspergillus terreus. None of the present drugs are effective for the treatment of renal cell carcinoma. The use of BL is expected to promote a new type therapy of renal cancer. METHODS: We investigated three human renal cancer cell lines: ACHN, OS-RC-2 and RCC10RGB, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and two-color flow cytometry. Simultaneous measurements of DNA content and cyclin expression allowed us to perform cell cycle specific analysis. Western blot analysis was performed using ACHN to represent cell lines. RESULTS: BL inhibited cell proliferation and caused cell accumulation at G2/M phase associated with the emergence of the third peak. Moreover, BL induced cyclin B1 over-expression in G2/M cells. These changes were quite definite, whereas cyclins D1, E and A showed no changes at all. Cyclin B1 accumulation was confirmed by western blot analysis. The chronological observation revealed that the emergence of the third peak preceded the regression of the increased cyclin B1 positive G2/M cells. These results suggested that BL accelerated cyclin B1 accumulation in G2/M cells, which then shifted to G1 phase without cell division. New G1 cells started DNA synthesis most likely as endoreduplication to form the third peak and the mechanism of cyclin B1 accumulation converted into down-regulation. CONCLUSION: BL induced significant cell kinetic interference in the tested human renal carcinoma cell lines. This might indicate the possibility of a new medical treatment modality for renal cancer.

Interleukin-2 blocks the antitumour activity caused by depletion of CD25 cells in a murine renal adenocarcinoma model.

OBJECTIVE: To test the effectiveness of antimouse CD25 monoclonal antibody (mAb) against murine renal adenocarcinoma (RENCA) cells, as immunoregulatory/suppressor cells are known to be involved in tumour development in vivo, but the functions of these cells are not yet clear, and eliminating naive CD25 (interleukin-2 receptor alpha)-positive T cells elicits potent immune responses to syngeneic tumours in vivo. MATERIALS AND METHODS: Aliquots of 1 x 10(4) or 1 x 10(5) RENCA cells were implanted into the subcapsule of the left kidney of syngeneic male Balb/c mice. Mice were injected with 125 micro g of antimouse CD25 mAb to deplete CD25(+) cells before RENCA implantation. Then 10(4) units of recombinant human interleukin-2 (rhIL-2) were subcutaneously injected twice daily for 7 days. Fourteen or 25 days later the tumour size was determined by laparotomy, and cells sorted using two-colour flow cytometry. RESULTS: Depletion of naive CD25(+) cells with anti-CD25 mAb and rhIL-2 administration effectively induced anti-RENCA tumour activity in Balb/c hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth. RENCA implantation reduced the proportion of CD4(+) cells among splenocytes, whereas anti-CD25 mAb treatment increased it. The proportion of CD25(+)CD8(+) cells among splenocytes and that of CD25(+) cells among CD8(+) cells were markedly reduced by co-administration of anti-CD25 mAb and rhIL-2 with RENCA implantation. Both CD4(+) and CD8(+) cells were stained around the remnant microscopic RENCA tumour after anti-CD25 mAb treatment. CONCLUSION: Either depletion of naive CD25(+) cells or rhIL-2 administration suppressed RENCA tumour growth in murine hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth.