Development of a targeted gene panel for the diagnosis of Gorlin syndrome.

Gorlin syndrome is a rare autosomal dominant disease caused by mutations in the PTCH1, PTCH2, and SUFU genes. Each symptom of the disease has a different time point of onset, which makes early diagnosis based solely on symptoms challenging. In this study, a gene panel was developed to overcome the challenges in the diagnosis of Gorlin syndrome and allow diagnosis using a single test. A custom panel was generated for four genes associated with Gorlin syndrome: PTCH1, PTCH2, SMO, and SUFU. Twenty-seven samples from 12 patients with Gorlin syndrome and three asymptomatic blood relatives of the patients were examined. This panel was highly reliable with a high Q30 quality score, on-target ratio, and coverage. The panel was time- and cost-efficient and enabled the detection of more mutations than whole-exome sequencing for the same patient. Pathogenic mutations in both PTCH1 and PTCH2 were detected in five of the 12 patients with Gorlin syndrome who were diagnosed based on clinical symptoms. Using this panel, the same mutation was identified in the patients and their blood relatives. In summary, this panel facilitated the highly reliable genetic diagnosis of Gorlin syndrome at a low cost, using only blood samples.

Analysis of radiotherapy in 1054 patients with primary central nervous system lymphoma treated from 1985 to 2009.

AIMS: Data on primary central nervous system lymphoma that had been collected through surveys for four consecutive periods between 1985 and 2009 were analysed to evaluate outcomes according to treatment. MATERIALS AND METHODS: All had histologically proven disease and had received radiotherapy. No patients had AIDS. Among 1054 patients, 696 died and 358 were alive or lost to follow-up. The median follow-up period for surviving patients was 37 months. RESULTS: For all patients, the median survival time was 24 months; the 5 year survival rate was 25.8%. Patients treated with methotrexate-based chemotherapy and radiation had a higher 5 year survival rate (43%) than those treated with radiation alone (14%) and those treated with non-methotrexate chemotherapy plus radiation (20%), but differences in relapse-free survival were smaller among the three groups. The 5 year survival rate was 25% for patients treated with whole-brain irradiation and 29% for patients treated with partial-brain irradiation (P = 0.80). Patients receiving a total dose of 40-49.9 Gy had a higher 5 year survival rate (32%) than those receiving other doses (21-25%, P = 0.0004) and patients receiving a whole-brain dose of 30-39.9 Gy had a higher 5 year survival rate (32%) than those receiving ≥40 Gy (13-22%, P < 0.0005). Patients receiving methotrexate-based chemotherapy and partial-brain radiotherapy (≥30 Gy) had a 5 year survival rate of 49%. CONCLUSIONS: The optimal total and whole-brain doses may be in the range of 40-49.9 and <40 Gy, respectively, especially in combination with chemotherapy. Patients receiving partial-brain irradiation had a prognosis similar to that of those receiving whole-brain irradiation. With methotrexate-based chemotherapy, partial-brain radiotherapy may be worth considering for non-elderly patients with a single tumour.

DNA vaccination targeting macrophage migration inhibitory factor prevents murine experimental colitis.

Previous studies have shown that neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibody reduces intestinal inflammation in mice. In this study we tested whether or not anti-MIF autoantibody induced by DNA vaccine targeting MIF protects mice against experimental colitis. Mice were administered a MIF-deoxyribonucleic acid (DNA) vaccine by introducing oligonucleotides encoding helper T epitope into the cDNA sequence of murine MIF by in vivo electroporation. Preventive effects of this method against dextran sulphate sodium-induced (DSS) colitis were evaluated. Mice administered with MIF-DNA vaccine raised values of autoantibody significantly. The clinical and histological findings of colitis induced by 3·0% DSS solution were ameliorated significantly in mice treated with MIF-DNA vaccine compared with saline or pCAGGS-treated mice given DSS. Myeloperoxidase activity, infiltration of F4/80-positive staining cells and the levels of proinflammatory cytokines were suppressed in the colon of MIF-DNA vaccine treated mice compared with saline or pCAGGS-treated mice exposed to DSS. Our results suggest that immunization with helper T epitope DNA-vaccine targeting MIF may be a useful approach for the treatment of colitis including inflammatory bowel diseases.

A novel DNA vaccine-targeting macrophage migration inhibitory factor improves the survival of mice with sepsis.

Sepsis is a common and frequently fatal condition and there is an urgent need for new therapies that will further reduce sepsis-induced mortality. Macrophage migration inhibitory (MIF) factor is important in the regulation of innate and adaptive immunity and is believed to play a key regulatory role in sepsis and autoimmune disease. As MIF deficiency or immunoneutralization protects mice or rats from fatal endotoxic shock or other inflammatory diseases, we examined whether DNA vaccination against this molecule would also be protective. DNA vaccines can stimulate both humoral and cellular immunity simultaneously and have been shown to be effective against a variety of pathogens or cytokine-driven pathologies. Mice were immunized with a MIF/tetanus toxin (TTX) DNA vaccine and sepsis was then induced by lipopolysaccharide or cecal ligation and puncture. The MIF/TTX DNA-vaccinated mice were protected from the lethal effect of sepsis compared with control-vaccinated mice in both models. Compared with the control-vaccinated mice, the MIF/TTX DNA-vaccinated mice also showed significantly lower serum tumor necrosis factor (TNF)-alpha protein levels and reduced mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, macrophage inflammatory protein-2 and Toll-like receptor-4 in the lungs. Thus, the MIF/TTX DNA vaccine may be useful for the prophylaxis of septic shock.

Activation of the SIRT1 pathway and modulation of the cell cycle were involved in silymarin's protection against UV-induced A375-S2 cell apoptosis.

Silymarin, derived from the milk thistle plant, Silybum marianum, has been traditionally used in the treatment of liver disease. Our previous study demonstrated that silymarin has an anti-apoptotic effect against UV irradiation. In this study, SIRT1, a human deacetylase that was reported to promote cell survival, was activated by silymarin (5 x 10(- 4) mol/L) in UV-irradiated human malignant melanoma, A375-S2 cells, followed by down-regulated expression of Bax and decreased release of cytochrome c. Cleavage of procaspase-3 and digestion of its substrates, the inhibitor of caspase-activated DNase (ICAD) and poly(ADP-ribose) polymerase (PARP), were also reduced. Consistent with its protective effect on UV-induced apoptosis, silymarin (5 x 10(- 4) mol/L) also increased G(2)/M phase arrest, possibly providing a prolonged time for efficient DNA repair. Consequently, that silymarin protected A375-S2 cell against UV-induced apoptosis was partially through SIRT1 pathway and modulation of the cell cycle distribution.

Dracorhodin perchlorate induces apoptosis in HL-60 cells.

Dracorhodin perchlorate, an anthocyanin red pigment, induces human premyelocytic leukemia HL-60 cell death through apoptotic pathway. Caspase -1, -3, -8, -9, and -10 inhibitors partially reversed the cell death induced by dracorhodin perchlorate. Caspase-3 and -8 were activated followed to the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Dracorhodin perchlorate up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-XL. The cell death was accompanied with phosphorylation of ERK, JNK and p38 MAPK and partially reduced by MEK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB 203580). Taken together, dracorhodin perchlorate-induced apoptosis in HL-60 cells via up-regulation of Bax, activation of caspases and ERK/p38/JNK MAPKs.

Fluctuations in carotid arterial distensibility during the menstrual cycle do not influence cardiovagal baroreflex sensitivity.

AIM: Fluctuations in autonomic nervous functions throughout the menstrual cycle and the underlying mechanism concerning them are not well known. This study was designed to test the hypothesis that fluctuations in cardiovagal baroreflex sensitivity (BRS) throughout the menstrual cycles of young women are due to fluctuations in carotid arterial distensibility. METHODS: In eight eumenorrhoeic healthy young women (18-24 years), we determined the variations in the carotid arterial distensibility coefficient (DC; via simultaneous ultrasonography and applanation tonometry), cardiovagal BRS (phase IV of the Valsalva manoeuvre and the sequence method; up- or down-sequence spontaneous BRS), and serum oestradiol and progesterone concentrations at five points in the menstrual cycle (menstrual = M, follicular = F, ovulatory = O, early luteal = EL, and late luteal = LL). RESULTS: Serum oestradiol and progesterone levels were consistent with the predicted cycle phases. Carotid arterial DC fluctuated cyclically, increasing significantly from the M (52.4 +/- 4.9 x 10(-3) kPa(-1), mean +/- SE) and F (52.7 +/- 4.4) phases to the O (57.6 +/- 4.4) phase and declining sharply in the EL (46.0 +/- 4.0) and LL (45.1 +/- 3.0) phases (F = 6.37, P < 0.05). Contrary to our prediction, however, cardiovagal BRS by the Valsalva manoeuvre (P = 0.73) or sequence method (up-sequence spontaneous BRS; P = 0.84: down-sequence spontaneous BRS; P = 0.67) did not change significantly during the menstrual cycle. CONCLUSION: The results suggest that, although carotid arterial distensibility fluctuates with the changes in ovarian hormone levels that occur during the menstrual cycle, the fluctuations in carotid arterial distensibility do not influence cardiovagal BRS.

Effect of aging on carotid artery stiffness and baroreflex sensitivity during head-out water immersion in man

To examine the possible age-related blood pressure (BP) deregulation in response to central hypervolemia, we measured spontaneous baroreflex sensitivity (SBRS), carotid arterial compliance (CC), and R-R interval coefficient of variation (RRICV) during basal and thermoneutral resting head-out-of-water immersion (HOWI) in 7 young (YG = 24.0 ± 0.8 years) and 6 middle-aged/older (OL = 59.3 ± 1.3 years) healthy men. Compared with basal conditions (YG = 19.6 ± 4.0 vs OL = 6.1 ± 1.5 ms/mmHg, P < 0.05), SBRS remained higher in YG than OL during rest HOWI (YG = 23.6 ± 6.6 vs OL = 9.3 ± 2.1 ms/mmHg, P < 0.05). The RRICV was significantly different between groups (YG = 6.5 ± 1.4 vs OL = 2.8 ± 0.4 percent, P < 0.05) under HOWI. The OL group had no increase in CC, but a significant increase in systolic BP (basal = 115.3 ± 4.4 vs water = 129.3 ± 5.3 mmHg, P < 0.05) under HOWI. In contrast, the YG group had a significant increase in CC (basal = 0.16 ± 0.01 vs water = 0.17 ± 0.02 mm²/mmHg, P < 0.05) with no changes in systolic BP. SBRS was positively related to CC (r = 0.58, P < 0.05 for basal vs r = 0.62, P < 0.05 for water). Our data suggest that age-related vagal dysfunction and reduced CC may be associated with SBRS differences between YG and OL groups, and with BP elevation during HOWI in healthy older men.

[A case of renal cell carcinoma diagnosed by MAG3 scintigraphy because of moderate renal dysfunction].

An 84-year-old man presented at our hospital with complaints of severe gross hematuria and lower right abdominal pain. A right renal mass was detected by ultrasound sonography and plain computerized tomography (CT) scan, but an exact diagnosis was not obtained. Because the patient presented with moderate renal dysfunction and severe gross hematuria, we were unable to perform imaging studies using contrast material or ureteroscopic instruments. Finally, mercaptoacetylglycyl-glycylglycine (MAG3) scintigraphy and magnetic resonance imaging (MRI) demonstrated renal cell carcinoma, and we performed transarterial embolization (TAE) therapy using ethanol and gel foam. Based on their efficacy and noninvasiveness, we conclude that MAG3 scintigraphy and MRI are the optimal modalities for imaging in patients with renal dysfunction.

[Simultaneously detected double malignancies on a duplicated kidney associated with atrophied counterpart: a case report].

A case of simultaneous double malignant tumor in the same kidney, associating renal cell carcinoma with renal pelvic transitional cell carcinoma, in a 70 year-old-male was reported. On January 6, 2000 he presented with macroscopic hematuria. There were no remarkable findings on cystoscopic examination. Drip infusion pyelography and multidetector-row computed tomography demonstrated a tumor mass on the upper pole of the left kidney and atrophic right kidney. Systemic chemotherapy with CDDP, MTX and ADR was performed preoperatively. Then, hemi-left nephrectomy underwent with the diagnosis of renal pelvic tumor and renal tumor. The surgical specimen was pathologically diagnosed as transitional cell carcinoma of the renal pelvis and renal cell carcinoma of its upper pole. This is the 32nd case of simultaneous occurrence of renal cell carcinoma and transitional cell carcinoma in the same kidney in the Japanese literature.

[A case of patent urachus in an adult male].

A 31-year-old man was referred to our hospital for evaluation of urachal rest. The history of his present illness dated back to birth, when the umbilicus was projected and urinary discharge was noted. At that time, the symptom of discharge had spontaneously subsided. At the age of 22, however, the patient again experienced discharge from the umbilicus. Although he did not seek treatment, after six years this symptom disappeared. Around this time, however, pyuria was revealed during medical examination, and abdominal ultrasonography (US) suggested the presence of urachal rest. At the time of hospitalization, physical examination revealed that the patient's right testis was not palpable. He was diagnosed with patent urachus with hemilateral aplasia and monorchism by US, computed tomography, magnetic resonance imaging and cystoscopy. The patient subsequently underwent radical operation. Patent urachus in adults is very rare, and only a few cases have been reported. To our knowledge, only one previously reported case involved a recurrence after spontaneous healing. Further, this is the first report of a patient with patent urachus with hemilateral aplasia and monorchism. Radical operation is generally recommended, based on the fact that very few cases heal conservatively.

Blood glucose and insulin concentrations are reduced in humans administered sucrose with inosine or adenosine.

Recently we found that some nucleosides such as inosine or adenosine inhibited alpha-glucosidase from rat intestine. The aim of this study was to determine whether these nucleosides are sucrase inhibitors in humans as well as rats. Blood glucose and insulin responses were examined in 23 healthy volunteers (18 males and 5 females) administered sucrose with inosine and 8 (males) administered sucrose with adenosine. The initial increase in plasma glucose and serum insulin concentrations at 30 min after loading sucrose (50 g) alone were significantly reduced by co-administration of inosine (2.5 and 1.0 g) or adenosine (2.5 g). The total increases in the areas under the plasma glucose and serum insulin concentration curves for 3 h after administration of the same amount of sucrose with inosine were also significantly less than those when the volunteers were administered sucrose alone. These results in humans agree with the findings obtained in our previous studies in rats. These nucleosides may be used as one of the components of artificial sweeteners when mixed with sucrose and may be useful as food additives to suppress increases in blood glucose and insulin.

[Clinical statistics on patients and operations during a 20-year period (1978-1997) at Department of Urology, Jikei University School of Medicine].

A statistical analysis was made of diseases, patients, and operations at our department in the four affiliated hospitals of Jikei University School of Medicine from 1978 through 1997. Our findings were as follows. Newly diagnosed urogenital malignancies, except testicular tumors, have been increasing in the last two decades among both outpatients and inpatients. Urogenital infections, including tuberculosis, have gradually been decreasing in the last two decades. The treatment of urinary stone diseases was markedly altered by the induction of extracorponeal shock wave lithotripsy.

Transient expression of megakaryocyte-derived protein immunoreactive with an antiserum to cartilage oligomeric matrix protein in developing rat liver.

We analyzed a megakaryocyte-derived protein immunoreactive with an antiserum to cartilage oligomeric matrix protein (COMP) in the developing rat liver. Staining with the anti-COMP antiserum in the developing rat liver increased during embryogenesis, and was strongest in the livers of 17-day-old embryos. However, staining in the liver was not detected at eight days after birth or thereafter. The stained cells were found to be megakaryocytes. We partially purified the protein showing cross-reaction with the antiserum to COMP from a megakaryocyte-rich cells fraction in 17-day-old embrionic rat livers. The molecular weight of this protein (approximately 95 kDa) was close to the molecular weight of COMP (105 kDa). Amplification of an RT-PCR fragment (225 bp) corresponding to part of COMP mRNA was detected in cartilage, but not in megakaryocytes of fetal liver or bone marrow. Based on these results, the fetal rat liver megakaryocyte-derived protein that reacted with the antiserum against COMP was thought to contain a common epitope with COMP from cartilage, but to be a different protein from COMP.

Controlled study of circadian rhythm of blood pressure in patients with aldosterone-producing adenoma compared with those with essential hypertension.

OBJECTIVE: The circadian rhythm of blood pressure in patients with aldosterone-producing adenoma (APA) is not well understood. We evaluated the circadian blood pressure rhythm in such patients by comparison with that in patients with essential hypertension (EHT). The latter are characterized by a nocturnal blood pressure decline, the so called 'dipping' blood pressure pattern. DESIGN AND METHODS: A total of 12 patients with APA and 36 patients with EHT who were matched by age and sex, and who had no severe organic disorders, were hospitalized to control their diet (low sodium) and activities. Ambulatory blood pressure monitoring was conducted for 24 h. The 24-h blood pressure was divided into waking blood pressure (0600-2130 h) and sleeping blood pressure (2200-0530 h). RESULTS: The two groups showed no significant differences in age, sex, serum creatinine, plasma glucose, daily urinary sodium excretion, and left ventricular mass index. Although the 24-h mean blood pressure was higher in APA (112 +/- 8 mmHg) than EHT (102 +/- 12 mmHg), the dipping mean blood pressure ratio (%), which was calculated from the sleeping and waking blood pressures, did not differ significantly between the two groups (93.2 +/- 5.4 versus 92.8 +/- 5.9). CONCLUSION: The dipping ratio of blood pressure in patients with APA resembled that of patients with EHT. Variables that would influence the circadian rhythm of blood pressure were controlled during study. The results suggest that a circadian blood pressure in patients with APA is of the dipping type, characterized by a nocturnal blood pressure decline, when a low sodium diet is ingested.

Serum glucose and insulin response in rats administered with sucrose or starch containing adenosine, inosine or cytosine.

Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine. The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625-0.125 g/kg of body weight). The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides. The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides.

Macrophage migration inhibitory factor up-regulates expression of matrix metalloproteinases in synovial fibroblasts of rheumatoid arthritis.

Neutral matrix metalloproteinases (MMPs) are responsible for the pathological features of rheumatoid arthritis (RA) such as degradation of cartilage. We herein show the up-regulation of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin) mRNAs of cultured synovial fibroblasts retrieved from rheumatoid arthritis (RA) patients in response to macrophage migration inhibitory factor (MIF). The elevation of MMP-1 and MMP-3 mRNA was dose-dependent and started at 6 h post-stimulation by MIF, reached the maximum level at 24 h, and was sustained at least up to 36 h. Interleukin (IL)-1beta mRNA was also up-regulated by MIF. These events were preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1, a common inhibitor of these proteases, was slightly up-regulated by MIF. Similarly, mRNA up-regulation of MMP-1 and MMP-3 was observed in the synovial fibroblasts of patients with osteoarthritis. However, their expression levels were much lower than those of RA synovial fibroblasts. The mRNA up-regulation by MIF was inhibited by the tyrosine kinase inhibitors genestein and herbimycin A, as well as the protein kinase C inhibitors staurosporine and H-7. On the other hand, the inhibition was not seen after the addition of the cyclic AMP-dependent kinase inhibitor, H-8. The mRNA up-regulation of MMPs was also inhibited by curcumin, an inhibitor of transcription factor AP-1, whereas interleukin-1 receptor antagonist, an IL-1 receptor antagonist, failed to inhibit the mRNA up-regulation. Considering these results, it is suggested that 1) MIF plays an important role in the tissue destruction of rheumatoid joints via induction of the proteinases, and 2) MIF up-regulates MMP-1 and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1- dependent pathways, bypassing IL-1beta signal transduction.

[A case of primary renal carcinoid tumor].

The patient was a 44-year-old woman in whom a mass of the right kidney was found when she underwent abdominal ultrasonography in a physical checkup. The computed tomography of abdomen and the magnetic resonance imaging of abdomen revealed a 3 x 3 cm solid mass at the right kidney. Excretory urograms showed a deformity of the right collecting system due to a mass lesion. The specimen of the resected tumor showed a cellular arrangement specific to carcinoid tumor and positive reaction of Grimelius stain. Electron microscopy confirmed the presence of dense-core granules in the tumor cells. Gastrointestinal investigation after the operation revealed no particular findings suggesting a carcinoid syndrome. No evidence of metastasis or recurrence was noted for 5 months after the operation. This was a very rare case of primary renal carcinoid tumor, representing the 12th case in the literature in Japan.

[Clinical studies on inverted papilloma of the urinary tract].

BACKGROUND: Inverted papilloma of the urinary tract is believed to be a benign neoplasm based on its histologic morphology and clinical behavior. In recent years, however, several investigators have warned against too optimistic an approach, emphasizing the possibility of malignant cellular transformation within the lesion, or its eventual association with other urothelial tumors such as transitional cell carcinoma or carcinoma in situ. We here report on 35 clinicopathologically diagnosed cases of inverted papilloma, and present the clinical significance attributed to these lesions in view of the current literature. PATIENTS AND METHODS: From 1976 to 1997, 35 cases of inverted papilloma of the urinary tract were treated at our hospital. This report presents the clinical features of these cases, the results of prognosis research, an investigation of the cases in which inverted papilloma and transitional cell carcinoma were found to co-exist, and a discussion of the recurrent cases of inverted papilloma found in previous literature. RESULTS: The patients ranged from 24 to 77 years of age, with a mean of 54 years, and included 4 women and 31 men. The most frequently occurring symptom was grosshematuria, and more than 90% of the 35 cases occurred in the bladder. In 2 of the 35 cases, transitional cell carcinoma coexisted with the inverted papilloma, at a different location in the bladder in one case and within the same neoplasm in the ureter in the other case. Clinical courses after treatment were followed in 29 of the 35 cases, with a follow-up period of from 8 months to 19 years (mean follow-up, 5 years and 4 months). Of these 29 cases, 2 showed recurrence, one at 16 and one at 30 months after the initial resection. Many previous reports show that the association of inverted papilloma and transitional cell carcinoma is stronger in the upper urinary tract and recurrence of inverted papilloma almost always happens with 2 years. CONCLUSION: This study suggests that some cases of urinary inverted papilloma show recurrence or malignant potential. Our results indicate that all cases of urinary inverted papilloma should be treated and followed as cases of low-grade transitional cell carcinomas. Consequently, all cases must be followed for two years or more after the initial operation.

[A case of primary transitional cell carcinoma of the prostate responsive to combination chemotherapy with methotrexate, epirubicin and cisplatin].

A 72-year-old man with the chief complaint of macrohematuria was referred to our hospital in February 1996. On transrectal examination, the prostate was stony hard without nodules. Cystoscopic findings and biopsies did not confirm a transitional cell carcinoma or carcinoma in situ in the bladder. However transurethral and transrectal biopsy specimens of prostate showed transitional cell carcinoma invading the prostate stroma. Pelvic CT scan revealed a lymph node metastasis in the left iliac artery region. Under the diagnosis of primary transitional cell carcinoma of the prostate, radical cystoprostatectomy and lymph node dissection were performed after two courses of neoadjuvant systemic chemotherapy. Pathologically no viable transitional cell carcinoma could be found in any of the specimens. Thus neo-adjuvant systemic chemotherapy was very effective in this case.