17ß-Estradiol induces proliferation of endometrial NK cells (CD56+) in postmenopausal women.

OBJECTIVE: The aim of this report was to evaluate the impact of hormone replacement therapy (HRT) on lymphocytic infiltration of the endometrium in postmenopausal women. METHOD: This study included 58 Japanese patients who had undergone hysterectomy at the University Hospital of Occupational and Environmental Health, Japan. Before surgery, nine patients had received 17ß-estradiol (E2), 0.72 mg transdermally for 2-8 weeks (E2 group); 16 patients had received an Estra-1,3,5(10)-triene-3,16α, 17ß-triol (E3) vaginal tablet 0.5 mg per month five times (E3 group); and 19 patients had received 17ß-estradiol, 0.62 mg, and norethindrone acetate (P), 2.70 mg for 3-16 weeks (E2 + P group). Fourteen patients received no HRT (control group). We examined uterine tissue specimens immunohistochemically for CD45+, CD3+, CD4+, CD8+, CD20+, CD56+, and Ki67 antigen-positive cells. RESULTS: The numbers of CD56 + cells were significantly increased in the E2 group compared with all other groups (E2 vs. E3: 7.0 vs. 0.75, p = 0.017; E2 vs. E2 + P: 7.0 vs. 0.58, p = 0.009; E2 vs. CONTROL: 7.0 vs. 0.43, p = 0.010). The numbers of CD3+ cells were significantly increased in the E2 group compared with the control group (149.3 vs. 42.6, p = 0.008). CONCLUSION: 17ß-Estradiol induced the proliferation of endometrial uterine natural killer cells (CD56+) in postmenopausal women.

Mutation of DNASE1 in people with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.

3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects.

Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1-18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H(2)O(2) scavenging effect to exogenous H(2)O(2), while lactate had no scavenging effect. 3BP induced H(2)O(2) production. Pyruvate protected against H(2)O(2)-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.

D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects.

Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

Neuroradiological and neurofunctional examinations for patients with 22q11.2 deletion.

Since the neuroradiological features of patients with 22q11.2 deletion syndrome are not well-understood, examinations using functional imaging were performed in this study. Brain magnetic resonance imaging (MRI) and 1H-magnetic resonance spectroscopy (MRS) were performed using a clinical 3-Tesla MR imager in 4 patients with 22q11.2 deletion syndrome (2 boys and 2 girls; aged 2-6 years.) and 20 age- and sex-matched healthy control subjects. Furthermore, interictal 123I-iomazenil (IMZ) single photon emission computed tomography (SPECT) was examined in 2 of the 4 patients. Among the 4 patients with 22q11.2 deletion syndrome, 2 patients showed polymicrogyria and 1 patient showed agyria. Those patients with brain malformations also showed abnormal brain artery patterns and decreased accumulation of IMZ in 123I-IMZ SPECT. Although all 4 patients showed epileptic discharges in their electroencephalograms (EEG), one patient with polymicrogyria had no seizure episodes. Decreases in γ-aminobutyric acid (GABA) corresponding to the areas of polymicrogyria and/or epileptic discharges in EEG were shown in all patients except for the patient with agyria. Although consistent evidence was not seen in patients with 22q11.2 deletion syndrome in this study, brain malformations and disturbances of the GABAergic nervous system would be underlying mechanisms of the neurodevelopmental abnormalities in this syndrome.

Incidence of and risk factors for incisional hernia after closure of temporary ileostomy for colorectal malignancy.

PURPOSE: Incisional hernia is a major complication after stoma closure and can cause uncomfortable symptoms. In this study, we evaluated the risk factors for hernia formation with the aim of reducing the incidence of incisional hernia. METHODS: A total of 134 oncology patients underwent closure of a temporary loop ileostomy between May 2004 and December 2013. The incidence of incisional hernia was determined by routine follow-up computed tomography scanning every 6 months. The relationships between patients' characteristics, including age, sex, obesity, diabetes mellitus, surgical site infection, chronic obstructive pulmonary disease, hypertension, hypoalbuminemia, smoking, and presence of a midline hernia and the occurrence of incisional hernia were retrospectively evaluated. RESULTS: The median follow-up time was 47 months (range 8-130). Hernias occurred in 23.9% of patients (32/134). The median time to detection of hernias was 8 months (range 2-39). The Chi-squared test revealed significant differences in obesity (P = 0.0003), hypertension (P = 0.0057), and incisional hernia history (P = 0.0000) between patients with and without incisional hernia. Multivariable analysis and univariate analysis revealed that hypertension and the presence of midline incisional hernia were risk factors for incisional hernia. CONCLUSIONS: Hypertension and the presence of a midline incisional hernia were the major risk factors for incisional hernia after loop ileostomy closure. These risk factors can be addressed before planning surgery.

Increase of integrin-linked kinase activity in cultured podocytes upon stimulation with plasma from patients with recurrent FSGS.

Recurrent focal segmental glomerulosclerosis (FSGS) is a major challenge in the field of transplantation. Integrin-linked kinase (ILK) has emerged as a key mediator of podocyte-glomerular basement membrane (GBM) interactions. To clarify the involvement of plasma factors in FSGS recurrence, we examined the effects of plasma from FSGS patients with or without posttransplant recurrence on cultured podocytes, focusing particularly on ILK activity. Podocytes from a conditionally immortalized mouse podocyte cell line were treated with plasma from 11 FSGS patients, and ILK activity was determined using an immune complex kinase assay. Treatment with plasma from three patients with recurrence induced an increase in ILK activity. In contrast, no increase in ILK activity was observed in cultured podocytes treated with plasma from the remaining three patients with recurrence and five patients without recurrence. Cultured podocytes treated with plasma that induced ILK activity showed alterations of focal contact and detachment from the laminin matrix. In conclusion, this preliminary study provides experimental evidence suggesting the possible presence of circulating toxic factors in the plasma of some patients with recurrent FSGS, which induce an increase in podocyte ILK activity that may lead to the detachment of podocytes from the GBM.

Osteoprotegerin affects the responsiveness of fibroblast growth factor-23 to high oral phosphate intake.

BACKGROUND: Both fibroblast growth factor-23 (FGF-23) and osteoprotegerin (OPG) are associated with phosphate metabolism, and are produced by bone tissue. METHODS: In order to clarify the influence of bone turnover on phosphate metabolism, we examined the response of FGF-23 to an oral phosphate load in 4 groups of mice (2 OPG knockout (KO) and 2 wild-type (WT) groups) given either a high-phosphate diet or a normal diet by performing serum and urinary biochemical assays. RESULTS: Although there was no significant difference in serum phosphate/ calcium levels between the groups, the decrease in tubular reabsorption rate of phosphate (%TRP) by oral phosphate load was smaller in the OPG KO mice than in the WT mice. FGF-23 level was significantly increased by a high-phosphate diet in WT mice, but not in OPG KO mice. However, there was no significant difference of intact PTH and calcitriol levels between the OPG KO and WT mice. CONCLUSION: Therefore, OPG may play a key role in mediating the response of FGF-23 to an oral phosphate load in bone cells.

Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.

Angiotensin II (Ang II) has been implicated in the development of progressive glomerulosclerosis, but the precise mechanism of this effect remains unclear. In an experimental model, we have shown previously that TGF-beta plays a key role in glomerulosclerosis by stimulating extracellular matrix protein synthesis, increasing matrix protein receptors, and altering protease/protease-inhibitor balance, thereby inhibiting matrix degradation. We hypothesized that Ang II contributes to glomerulosclerosis through induction of TGF-beta. Ang II treatment of rat mesangial cells in culture increased TGF-beta and matrix components biglycan, fibronectin, and collagen type I at both the mRNA and protein levels in a time- and dose-dependent manner. Saralasin, a competitive inhibitor of Ang II, prevented the stimulation. Ang II also promoted conversion of latent TGF-beta to the biologically active form. Coincubation of mesangial cells with Ang II and neutralizing antibody to TGF-beta blocked the Ang II-induced increases in matrix protein expression. Continuous in vivo administration of Ang II to normal rats for 7 d resulted in 70% increases in glomerular mRNA for both TGF-beta and collagen type I. These results indicate that Ang II induces mesangial cell synthesis of matrix proteins and show that these effects are mediated by Ang II induction of TGF-beta expression. This mechanism may well contribute to glomerulosclerosis in vivo.

Peripheral blood stem cell mobilization by granulocyte colony-stimulating factor alone and engraftment kinetics following autologous transplantation in children and adolescents with solid tumor.

In 56 pediatric and adolescent patients (median age 7 years, range 1-21) with various solid tumors, peripheral blood stem cells (PBSC) were mobilized with granulocyte colony-stimulating factor (G-CSF) alone, and the yields of PBSC and engraftment kinetics following autologous peripheral blood stem cell transplantation (PBSCT) were evaluated retrospectively. Granulocyte colony-stimulating factor (10 microg/kg) was injected subcutaneously for mobilization when patients showed no influence of previous chemotherapy, and administration was continued for 5 days. The peaks of CD34+ cells and colony-forming units-granulocyte/macrophage in the blood were observed on days 4 through 6 of G-CSF administration in all patients. Peripheral blood stem cell harvest was commenced on day 5 of G-CSF treatment. Compared to the results in patients mobilized by chemotherapy plus G-CSF (N=18), the progenitor cell yields were lower in patients mobilized with G-CSF alone. However, there were no significant differences in WBC and ANC engraftment compared to the chemotherapy plus G-CSF mobilization group. Platelet recovery following autologous PBSCT was delayed in patients mobilized with G-CSF alone. The median time taken for ANC and platelet counts to reach 0.5 x 10(9) and 20 x 10(9)/l was 12 days (range: 9-28) and 15 days (8-55), respectively, in all patients who received PBSC mobilized by G-CSF alone. In summary, mobilization with G-CSF alone can mobilize sufficient CD34+ cells for successful autografting and sustained hematological reconstitution in pediatric and adolescent patients with solid tumors, and even in heavily pre-treated patients.

Suppressing effects of dietary supplementation of the organoselenium 1,4-phenylenebis(methylene)selenocyanate and the Citrus antioxidant auraptene on lung metastasis of melanoma cells in mice.

The modifying effects of the organoselenium 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and the Citrus antioxidant auraptene as dietary supplements on experimental pulmonary metastasis of B16BL6 murine melanoma cells were investigated in an i.v. injection model in mice. Seven groups of male C57BL/6 mice were fed a basal diet (control group) or the basal diet supplemented with p-XSC (4, 8, or 15 mg/kg) or auraptene (250, 500, or 1000 mg/kg). All mice were fed their respective diet for 2 weeks before and after i.v. injection of 1 x 10(5) viable melanoma cells. At termination of the study, the incidence of lung metastatic tumors was determined. Cross-sectional areas and tumor volumes were analyzed morphometrically. In addition, apoptotic indices of lung metastatic tumors of all groups were counted. The incidences of lung metastasis in mice fed the diet mixed with 8 or 15 mg p-XSC/kg were significantly smaller than that in mice fed the basal diet. The mean numbers of metastatic lung tumors were significantly lower in mice fed p-XSC (4, 8, and 15 mg/kg) and auraptene (500 and 1000 mg/kg) than in controls. Cross-sectional areas and volumes of the tumors were also significantly decreased in mice given p-XSC (8 or 15 mg/kg) and auraptene (500 mg/kg). Apoptotic indices in mice fed the diets mixed with p-XSC (4, 8, or 15 mg/kg) and auraptene (500 and 1000 mg/kg) were significantly greater than those in the control group. These results indicate that in mice, diet supplementation with p-XSC and auraptene reduces pulmonary metastasis of B16BL6 melanoma cells and inhibits the growth of these metastatic tumors in lung, in part, by inducing apoptosis. We suggest that these agents, especially p-XSC, may be valuable in preventing metastatic diseases in future studies in the clinic.

Effect of endothelin-1 (1-31) on extracellular signal-regulated kinase and proliferation of human coronary artery smooth muscle cells.

1. We have previously found that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs (1-31), without any further degradation products. In this study, we investigated the effect of synthetic ET-1 (1-31) on the proliferation of cultured human coronary artery smooth muscle cells (HCASMCs). 2. ET-1 (1-31) increased [3H]-thymidine incorporation and cell numbers to a similar extent as ET-1 at 100 nM. This ET-1 (1-31)-induced [3H]-thymidine uptake was not affected by phosphoramidon, an inhibitor of ET-converting enzyme. It was, however, inhibited by BQ123, an endothelin ET(A) receptor antagonist, but not by BQ788, an endothelin ET(B) receptor antagonist. 3. By using an in-gel kinase assay, we demonstrated that ET-1 (1-31) activated extracellular signal-regulated kinase 1/2 (ERK1/2) in a concentration-dependent manner (100 pM to 1 microM) in HCASMCs. ET-1 (1-31)-induced ERK1/2 activation was inhibited by BQ123, but not by BQ788 and phosphoramidon. Inhibition of protein kinase C (PKC) and ERK kinase also caused a reduction of ET-1 (1-31)-induced ERK1/2 activation, whereas tyrosine kinase inhibition had little effect. 4. Gel-mobility shift analysis revealed that the ERK1/2 activation was followed by an increase in transcription factor activator protein-1 DNA binding activity in HCASMCs. 5. Our results strongly suggest that ET-1 (1-31) itself stimulates HCASMC proliferation probably through endothelin ET(A) or ET(A)-like receptors. The underlining mechanism of cell growth by ET-1 (1-31) may be explained in part by PKC-dependent ERK1/2 activation. Since human chymase has been proposed to play a role in atherosclerosis, ET-1 (1-31) may be one of the mediators.

Colitis-related rat colon carcinogenesis induced by 1-hydroxy-anthraquinone and methylazoxymethanol acetate (Review).

Patients with long-standing ulcerative colitis (UC) have an increased risk for developing colorectal cancer (CRC) compared to the general population. For investigation of the mechanisms and prevention of UC and UC-related CRC, establishment of a promising animal model for such disease is important. 1-hydroxyanthraquinone (1-HAQ) present in certain medicinal plants such as Rubia tinctorum L. is a genotoxic and rodent colon carcinogen. Long-term feeding of 1-HAQ induced hyper-cell proliferation in rat colonic crypts with ulcerative changes, crypt abscess, severe inflammation and erosion before the occurrence of tumors, which are similar to those found in human UC. In addition, 1-HAQ has a synergistic effect with methylazoxymethaol (MAM) acetate on colon carcinogenesis. The polymerase chain reaction-single strand conformation polymorphism analysis revealed no mutations in Ki-ras and p53 in colonic neoplasms induced by MAM acetate + 1-HAQ, MAM acetate alone or 1-HAQ alone. Also, no mutations of APC were found in these tumors. These findings are similar to those found in human ulcerative colitis-associated colon cancer in contrast with sporadic colon cancers. A previous study revealed that induced colonic tumors had beta-catenin mutation with high frequency, suggesting tumor development by activation of the beta-catenin-Tcf signaling pathway. Increased expression in TNF-alpha and IL-1alpha was found in these induced colonic neoplasms, and the expression was more remarkable in colonic mucosa of rats exposed to MAM acetate + 1-HAQ, MAM acetate or 1-HAQ when compared with that in untreated rats. Thus, these cytokines may act as growth factors in rat colon carcinogenesis by MAM acetate and 1-HAQ and the synergistic effect of 1-HAQ with MAM acetate might be related to the biological effects of the cytokines expressed in the inflammatory conditions induced by 1-HAQ.

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells.

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.

Acute tubulointerstitial nephritis associated with Yersinia pseudotuberculosis infection.

Two siblings infected with Yersinia pseudotuberculosis suffered from acute renal failure about 2 weeks after the onset of the disease. Renal histology in both siblings showed acute tubulointerstitial nephritis. Yersinia pseudotuberculosis type VB was isolated from feces of one of them, antibodies to Yersinia pseudotuberculosis type VB in their sera were elevated. The results of the present study suggest that acute renal failure complicating infections with Yersinia pseudotuberculosis is due to acute tubulointerstitial nephritis.

Ultrastructural alterations of glomerular anionic sites in IgA nephropathy.

The ultrastructural alterations of glomerular anionic sites were studied in biopsy specimens from 34 patients with IgA nephropathy using polyethyleneimine (PEI). Prominent common findings in the glomeruli of the patients were few PEI particles in electron dense deposits in the mesangial and subepithelial area and marked reduction in glomerular anionic sites covered with deposits. The anionic sites of the glomerular basement membrane (GBM) and epithelial cell surface coat (ESC) appeared unaltered in the patients with hematuria and/or mild proteinuria. But in patients with proteinuria in the nephrotic range, focally discrete loss of anionic sites in the lamina rara externa (LRE) was seen and the number of anionic sites of the ESC were decreased with retraction of the foot processes. The anionic sites of the lamina rara interna showed much less change in these patients. Subepithelial deposits were often seen concomitantly with focal loss of anionic sites in the LRE at the site of the deposits, but subendothelial deposits had little influence on the anionic sites of the neighboring GBM. The anionic sites of GBM that showed focal thinning with small GBM projections were appreciably decreased in number, but those in split GBM were not decreased. These results suggest that either loss of the negative charge on the glomerular capillary wall associated with subepithelial immune deposition or morphological changes of the GBM contribute to the progression of proteinuria in IgA nephropathy.

Streptokinase gene variable region classification in streptococci: lack of correlation with post-streptococcal glomerulonephritis.

To investigate a possible causal role of streptokinase (SKase) in acute post-streptococcal glomerulonephritis (APSGN), the major variable region of SKase genes of Streptococcus pyogenes strains isolated from patients with and without APSGN were analyzed using the polymerase chain reaction, restriction enzyme analysis and the direct sequencing of SKase genes. In the APSGN-associated strains, six of nine revealed mutant classes corresponding to the nephritogenic classes I and II proposed by Johnston et al. [1992], the remaining three belonged to non-nephritogenic classes. In twenty strains not associated with APSGN, seventeen belonged to classes I and II, while three were from other classes. The major variable region of the SKase gene shows no apparent relation with induction of APSGN in humans, suggesting that unique classes of streptococcal SKase do not play a role in the pathogenesis of APSGN.

Detection of hepatitis C virus core protein in the glomeruli of patients with membranous glomerulonephritis.

Two Japanese patients suffered from membranous glomerulonephritis associated with hepatitis C virus (HCV) infection. Renal histologic changes were characterized by granular deposits of IgG and C3 along the capillary wall and numerous subepithelial deposits in glomeruli. Hypocomplementemia was present in one patient, but both cryoglobulins and rheumatoid factors were absent. HCV RNA was detected in both their sera by RT-PCR, both free and in the form of circulating immune complexes. The HCV core protein was found in the glomeruli from both patients by indirect immunofluorescence. These results suggest that in some patients chronic HCV infection causes membranous glomerulonephritis through immune complex deposition involving HCV proteins.

Selective inhibition of the bacterial peptidoglycan biosynthesis by the new types of liposidomycins.

We examined the inhibitory activity against bacterial peptidoglycan biosynthesis, mammalian glycoprotein biosynthesis and growth of BALB/3T3 cells of four different types of liposidomycins which have the structure with or without sulfate and/or 3-methylglutaric acid moieties. Liposidomycins inhibited peptidoglycan biosynthesis about 30 to 500 times more effectively than tunicamycin, whereas liposidomycins inhibited mammalian glycoprotein biosynthesis about 30 to 300 times less effectively than tunicamycin. When the cytotoxic effect of liposidomycins and tunicamycin on the growth of mammalian cells were compared, liposidomycins did not show toxicity against BALB/3T3 cell at 25 microg/ml, though tunicamycin inhibited cell growth by 50% at 0.05 microg/ml. On the basis of these results, it is concluded that liposidomycins are selective antibiotics showing highly specific inhibition toward bacterial peptidoglycan biosynthesis.