Effect of the Lactococcus Lactis 11/19-B1 Strain on Atopic Dermatitis in a Clinical Test and Mouse Model.

Some lactic acid bacteria (LAB) are known to improve atopic dermatitis (AD) through the regulation and stimulation of the host immune system. In this study, we found that ingestion of yogurt containing Lactococcus lactis 11/19-B1 strain (L. lactis 11/19-B1) daily for 8 weeks significantly improved the severity scoring of atopic dermatitis (SCORAD) system score from 38.8 ± 14.4 to 24.2 ± 12.0 in children suffering from AD. We tried to identify which LAB species among the five species contained in the test yogurt contributed to the improvement in AD pathology using an AD mouse model induced by repeated application of 1-fluoro-2, 4-dinitrobenzene (DNFB). AD-like skin lesions on the dorsal skin and ear were most improved by L. lactis 11/19-B1 intake among the five LAB species. In addition, analysis of CD4+ T cell subsets in Peyer's patches (PPs) and cervical lymph nodes (CLNs) indicated that the intake of L. lactis 11/19-B1 generally suppressed all subsets related to inflammation, i.e., Th1, Th2 and Th17, instead of activating the suppressive system, Treg, in the AD mouse model. Histological observations showed ingestion of L. lactis 11/19-B1 significantly suppressed severe inflammatory findings, such as inflammatory cell filtration, epidermal erosion and eosinophil infiltration. These results suggest that the immunomodulatory effects of L. lactis 11/19-B1 contribute to improvements in AD pathology.

Two children with obesity-related glomerulopathy identified in a school urinary screening program.

The incidence of obesity-related glomerulopathy (ORG) has increased over the last decade, but there have been few reports on ORG in Japanese children. Reported herein are two children with ORG identified on school urinary screening (SUS). Patient 1 was a 12-year-old boy in whom proteinuria was first detected on SUS. His body mass index (BMI) was 33.8 kg/m(2) and he had hypertension and hyperuricemia. Patient 2, a 10-year-old boy, also had proteinuria identified on SUS. His BMI was 34.8 kg/m(2) , and he had fatty liver, hyperuricemia, and hypercholesterolemia. Both were diagnosed with ORG based on obesity, proteinuria, and renal pathological findings. After treatment, including candesartan, food restriction and physical exercise, urinary protein excretion was decreased in both cases. We believe that such school urinary screening programs may be effective for the early identification and treatment of children with ORG.

The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis.

Agmatine, an endogenous metabolite of arginine, selectively suppresses growth in cells with high proliferative kinetics, such as transformed cells, through depletion of intracellular polyamine levels. In the present study, we depleted intracellular polyamine content with agmatine to determine if attrition by cell death contributes to the growth-suppressive effects. We did not observe an increase in necrosis, DNA fragmentation, or chromatin condensation in Ha-Ras-transformed NIH-3T3 cells administered agmatine. In response to Ca(2+)-induced oxidative stress in kidney mitochondrial preparations, agmatine demonstrated attributes of a free radical scavenger by protecting against the oxidation of sulfhydryl groups and decreasing hydrogen peroxide content. The functional outcome was a protective effect against Ca(2+)-induced mitochondrial swelling and mitochondrial membrane potential collapse. We also observed decreased expression of proapoptotic Bcl-2 family members and of execution caspase-3, implying antiapoptotic potential. Indeed, we found that apoptosis induced by camptothecin or 5-fluorourocil was attenuated in cells administered agmatine. Agmatine may offer an alternative to the ornithine decarboxylase inhibitor difluoromethyl ornithine for depletion of intracellular polyamine content while avoiding the complications of increasing polyamine import and reducing the intracellular free radical scavenger capacity of polyamines. Depletion of intracellular polyamine content with agmatine suppressed cell growth, yet its antioxidant capacity afforded protection from mitochondrial insult and resistance to cellular apoptosis. These results could explain the beneficial outcomes observed with agmatine in models of injury and disease.

IgA nephropathy in a patient with dominant dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a rare and severe hereditary dermatosis. On the other hand, IgA nephropathy is the most common form of glomerulonephritis in childhood and adults, and clinically characterized by microhematuria and proteinuria and histologically by deposition of immunoglobulin A in mesangial lesions. Several renal complications of recessive DEB including IgA nephropathy and amyloidosis have been reported. However, there have been no reports on dominant DEB associated with IgA nephropathy. We report here for the first time a 17-year-old girl with dominant DEB associated with IgA nephropathy. The patient has suffered from episodes of urinary, upper airway, and skin infections. At 17 years of age, proteinuria and hematuria were detected, with a high value of serum IgA. Renal biopsy was performed, and immunofluorescence microscopic examination revealed segmental deposits of IgA in mesangial lesions, with many glomeruli exhibiting diffuse segmental mesangial-proliferative glomerulonephritis. We diagnosed dominant DEB associated with IgA nephropathy on the basis of proteinuria, hematuria, and deposits of IgA in mesangial lesions on immunofluorescence microscopic examination, and diffuse segmental mesangial-proliferative glomerulonephritis. These findings suggest that repeated skin infections might have contributed to the pathogenesis of IgA nephropathy in this patient.

The antiproliferative effects of agmatine correlate with the rate of cellular proliferation.

Polyamines are small cationic molecules required for cellular proliferation. Agmatine is a biogenic amine unique in its capacity to arrest proliferation in cell lines by depleting intracellular polyamine levels. We previously demonstrated that agmatine enters mammalian cells via the polyamine transport system. As polyamine transport is positively correlated with the rate of cellular proliferation, the current study examines the antiproliferative effects of agmatine on cells with varying proliferative kinetics. Herein, we evaluate agmatine transport, intracellular accumulation, and its effects on antizyme expression and cellular proliferation in nontransformed cell lines and their transformed variants. H-ras- and Src-transformed murine NIH/3T3 cells (Ras/3T3 and Src/3T3, respectively) that were exposed to exogenous agmatine exhibit increased uptake and intracellular accumulation relative to the parental NIH/3T3 cell line. Similar increases were obtained for human primary foreskin fibroblasts relative to a human fibrosarcoma cell line, HT1080. Agmatine increases expression of antizyme, a protein that inhibits polyamine biosynthesis and transport. Ras/3T3 and Src/3T3 cells demonstrated augmented increases in antizyme protein expression relative to NIH/3T3 in response to agmatine. All transformed cell lines were significantly more sensitive to the antiproliferative effects of agmatine than nontransformed lines. These effects were attenuated in the presence of exogenous polyamines or inhibitors of polyamine transport. In conclusion, the antiproliferative effects of agmatine preferentially target transformed cell lines due to the increased agmatine uptake exhibited by cells with short cycling times.

Development of glomerular endothelial cells, podocytes and mesangial cells in the human fetus and infant.

The process of glomerular development consists of four developmental stages: vesicle (V) stage, S-shaped body (S) stage, capillary loop (C) stage and maturation (M) stage. However, the development of glomerular endothelial, mesangial and epithelial cells in fetal and infant kidneys remains unclear. In order to determine the characteristics of human glomerular development, we investigated the process of glomerular development by staining fetal and infant kidneys for CD31, CD34 and FB21, markers for endothelial cells, alpha-smooth muscle actin (alpha-SMA), a marker for mesangial cells, and nephrin, a marker for podocytes. These series of studies were carried out on kidneys obtained at autopsy from 27 fetuses and 5 infants. The fetuses were divided into the following 5 groups according to gestational age; 13-19, 20-24, 25-29, 30-34 and 35-39 weeks. In each group, glomerular development was classified according to the developmental stage and the staining patterns for CD31, CD34, FB21, alpha-SMA and nephrin. The proportion of V-stage development in 100 glomeruli examined was highest at 13-19 weeks. After 20 weeks, the V-stage proportion decreased gradually, and the proportion of S stage became highest at 20-24 weeks. The C-stage proportion was highest at 25-29 weeks, while the M-stage proportion was highest in infants aged 1-6 months. The staining patterns for CD31, CD34 and FB21 were similar in endothelial cells after 25 weeks of gestation. Staining of alpha-SMA and nephrin was first observed in the S stage. In conclusion, maturation of endothelial cells starts at 25 weeks and is completed by 35 weeks of gestation. Epithelial cells and mesangial cells first appear during the S stage.

Efficacy of single dose of oral mizoribine pulse therapy two times per week for frequently relapsing nephrotic syndrome.

We assessed the efficacy of a single dose of oral mizoribine (MZB) pulse therapy two times per week for children with frequently relapsing nephrotic syndrome (FRNS). Eleven children with FRNS in remission were treated with oral MZB pulse therapy (daily dose 6 mg/kg; maximum total dose 300 mg). We compared their clinical manifestations before and after oral MZB pulse therapy and examined the changes in serum MZB concentration in each patient on the days when MZB was administered. Eight patients had no subsequent relapses (responders), and prednisolone could be discontinued. Although 2 of the other 3 patients (nonresponders) had one relapse and the remaining patient had two relapses, both the dosages of prednisolone and frequency of relapse after oral MZB pulse therapy were significantly lower than before oral MZB pulse therapy. The peak blood concentration and AUC0-4 of MZB in the responders were higher than in the nonresponders. None of patients had severe adverse effects, such as uricacidemia, leukopenia, liver dysfunction or alopecia. Oral mizoribine pulse therapy consisting of a single dose two days a week may be effective and safe in some FRNS patients.

Agmatine suppresses mesangial cell proliferation by modulating polyamine metabolism.

Polyamines play an essential role in the growth and differentiation of mammalian cells. The depletion of intracellular polyamines results in the suppression of growth. Proliferation of glomerular mesangial cells (MC) is the most common pathologic change in many forms of glomerulonephritis. Agmatine is a metabolite of arginine via arginine decarboxylase (ADC), highly expressed in the kidney, and unique in its capacity to suppress intracellular polyamine levels required for proliferation. As agmatine enters mammalian cells via the polyamine transport system, its antiproliferative effects may preferentially target cells with increased proliferative kinetics. In the present study, we evaluated the antiproliferative effects of agmatine on human MC in vitro. MC proliferation was stimulated with 20% fetal bovine serum (FBS) or platelet-derived growth factor (PDGF-BB, 20 ng/ml). Cell proliferation was measured using the (4.3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) proliferation assay. Intracellular polyamine levels were assayed by high performance liquid chromatography, and cell death was assessed by cellular DNA fragmentation enzyme-linked immunosorbent assay. The MTT proliferation assay showed that agmatine significantly suppressed proliferation of human MC treated with 20% FBS or 5% FBS + PDGF as compared to human MC treated with 5% FBS. Polyamine levels were markedly lower in cells treated with agmatine, and proliferation was rescued by administration of putrescine. The fragmented DNA was hardly detected in agmatine-treated human MC. In summary, human MC stimulated to increase their proliferative kinetics are significantly more sensitive to the antiproliferative effects of agmatine than normally cultured cells. Suppressed proliferation of the agmatine-treated human MC is not due to increased cell death. These results suggest that agmatine is a promising drug candidate for the treatment of human mesangial proliferative glomerulonephritis.

Renal effects of Coxsackie B4 virus in hyper-IgA mice.

For clarification of the pathogenetic role of viral infection in chronic glomerulonephritis, the renal effects of Coxsackie B4 virus (CB4) were examined in hyper-IgA (HIGA) mice. In experiment 1, HIGA mice (n = 75) were inoculated intravenously with live CB4 and inactivated CB4 once a month from 1 to 12 mo of age. In experiment 2, HIGA mice (n = 45) were inoculated intravenously with live CB4 and inactivated CB4 once at 6 wk of age. In experiment 3, 60 mice were inoculated intravenously with carbon and live or inactivated CB4 once at 6 wk of age. Mice in the control group were inoculated with vehicle. The kidneys were extirpated from five mice of each group killed with time after inoculation for histologic evaluation. The scores for mesangial IgA deposition, PCNA-positive cells, and matrix at 20 wk were higher in mice with live CB4 than in mice with inactivated CB4 or without CB4. On electron microscopic examination, swelling and detachment of endothelial cells from 24 h after inoculation and increase of serum IFN-gamma concentration were found in mice with live CB4. Many carbon particles were present in peripheral and central zones of the mesangium from 5 to 10 d in mice with carbon and live CB4. These results suggest that CB4 provokes exacerbation of renal pathologic findings in HIGA mice via endothelial injury, IFN-gamma production, and dysfunction of the mesangial pathway.

Efficacy of tonsillectomy pulse therapy versus multiple-drug therapy for IgA nephropathy.

We evaluated the efficacy of tonsillectomy plus pulse prednisolone, warfarin, and dipyridamole including methylprednisolone pulse (tonsillectomy plus pulse therapy), versus prednisolone, warfarin, and dipyridamole including mizoribine (PWDM) for the treatment diffuse IgA nephropathy (IgAN) in children. The patients were randomly assigned to be treated by tonsillectomy plus pulse therapy for 2 years (Group A, n=16) or PWDM for 2 years (Group B, n=16). The clinical features and pathological findings in both groups were analyzed prospectively. The mean urinary protein excretion after 6 months of treatment in both groups had decreased significantly compared with pre-therapy. The activity index (AI) in both groups was lower at the time of the second biopsy than at the time of the first biopsy. The chronicity index (CI) in Groups A and B did not differ between the first and second biopsy. At the latest follow-up examination none (0%) of the patients in either group had renal insufficiency. None of the patients in Group A, but six patients in Group B experienced an acute exacerbation of IgAN as a result of tonsillitis (P<0.05). In conclusion, although there was no untreated control group in this study, the results suggested that tonsillectomy plus pulse therapy is as effective as PWDM in ameliorating proteinuria and histological severity in IgAN patients and in preventing acute exacerbation of IgAN by tonsillitis.

Glomerulonephritis associated with chronic infection from long-term central venous catheterization.

There have been few reports on immune complex-mediated glomerulonephritis associated with chronic infection from long-term central venous catheterization in adulthood. We report here on a 13-year-old boy with nephritis who exhibited glomerulonephritis that had been induced by the long-term use of central venous catheters, and its resolution after extraction of the central venous catheter. A diagnosis of glomerulonephritis associated with chronic infection caused by long-term central venous catheterization was made, based on the absence of clinical findings after removal of the catheter, hypocomplementemia, pathology findings resembling membranoproliferative glomerulonephritis, and detection of Staphylococcus epidermidis from culture of the removed catheter culture. For clinicians using long-term central venous access for parenteral feeding, rapid catheter exchange is necessary for patients with fever of unknown origin.

The leukotriene B4 receptor antagonist ONO-4057 inhibits mesangioproliferative changes in anti-Thy-1 nephritis.

OBJECTIVE: ONO-4057 is a specific leukotriene B4 (LTB4) receptor antagonist which inhibits human neutrophil aggregation, chemotaxis and degranulation induced by LTB4. This study was conducted to evaluate the role of LTB4 in glomerulonephritis, and to examine whether ONO-4057 moderated anti-Thy-1 nephritis. METHODS: Experiment 1: Sixty Wistar rats were divided into three groups. Rats of Group A (n = 20) underwent intraperitoneal administration of placebo as a control group, rats of Group B (n = 20) first underwent intraperitoneal administration of 100 mg/kg ONO-4057 and rats of Group C (n = 20) first underwent intraperitoneal administration of 300 mg/kg ONO-4057 daily from day 3 before anti-Thy-1 antibody (OX7) injection to day 14 after OX7 injection, respectively. Experiment 2: Forty rats were divided into two groups. ONO-group (n = 20) was treated with 300 mg/kg BW of ONO-4057 and placebo-group (n = 20) with placebo daily from days 1 to 13 after OX7 injection. Urine and blood samples were collected and the kidneys were extirpated from five rats of each group sacrificed at 3 h, 24 h, day 7 or day 14 after the injection of OX7 in both experiments. Urinary protein excretion, renal function and pathological findings were analysed in each group of both experiments. RESULTS: (1) Glomerular infiltration by polymorphonuclear leucocytes (PMNs) and macrophages at 3 h was less in Groups B and C than in Group A, and matrix scores at day 7 were lower in Groups B and C than in Group A. Injury scores did not differ among the groups. (2) Urinary protein excretion at day 7 was less in Group C than in Group A. (3) Neither pathological findings nor urinary protein excretion differed between ONO-group and placebo-group. CONCLUSION: These results suggest that LTB4 is associated not with the pathogenesis of complement-dependent mesangial cell lysis but with that of mesangial proliferative change in anti-Thy-1 nephritis.

Detection of enteroviruses in renal biopsies from patients with immunoglobulin A nephropathy.

Viruses have been suspected to be one of the causes of IgA nephropathy (IgAN). Recent studies have detected viruses in renal tissues of patients with IgAN. Enteroviruses have been reported as pathogenic agents in some renal diseases. We previously reported that group B coxsackieviruses cause pathological changes in experimentally infected mouse kidney. The aim of the present study was to examine the participation of enteroviruses in the pathogenesis of renal diseases including IgAN. Renal biopsies of ten patients with IgAN (group 1) and of 19 patients with non-IgAN renal disease (group 2) were analyzed by polymerase chain reaction (PCR) for the presence of enteroviral RNA. Positive PCR results were obtained for three patients (30%) of group 1. We confirmed by sequencing that the positive PCR products were derived from strains of enteroviruses. One of these three patients also had a positive result for lymphocytes from peripheral blood. In contrast, enteroviral RNA was detected in none of the 19 patients of group 2. The incidence of enteroviral RNA detection in patients of group 1 was higher than that in group 2 (P<0.05). Our findings suggest that enteroviral infection may have the possibility of becoming one of the factors involved in the mechanism of onset or evolution of IgAN.

Oral mizoribine pulse therapy for patients with steroid-resistant and frequently relapsing steroid-dependent nephrotic syndrome.

BACKGROUND: We investigated the efficacy of oral mizoribine pulse therapy (mizoribine-pulse) for cyclosporin (CyA)-dependent, steroid-resistant nephrotic syndrome (SRNS) and frequently relapsing, steroid-dependent nephrotic syndrome (FR-SDNS). METHODS: One child with CyA-dependent SRNS and eight children with CyA-dependent FR-SDNS were treated with mizoribine-pulse (daily dose: 10 mg/kg; maximum total dose 500 mg). We compared clinical manifestations before and after mizoribine-pulse, and studied the changes in serum mizoribine concentration in each patient on days when mizoribine was administered. RESULTS: Four patients had no subsequent relapses (responders). Two of the four responders discontinued prednisolone and CyA, the other two discontinued CyA. Although each of the five other patients (non-responders) experienced single subsequent relapses, the dosages of prednisolone and CyA after mizoribine-pulse were decreased significantly compared with before mizoribine-pulse. The peak blood concentration of mizoribine in the responders was higher than in the non-responders (3.6+/-0.9 vs 1.8+/-0.4 microg/ml). CONCLUSIONS: Mizoribine-pulse may be effective for some patients with CyA-dependent SRNS and FR-SDNS.

Myeloid-related protein 8 expression on macrophages is a useful prognostic marker for renal dysfunction in children with MPGN type 1.

BACKGROUND: To clarify the role of subclasses of macrophages in chronic glomerulonephritis, we evaluated the relationship between myeloid-related protein 8 (MRP8) and MRP14 expression on macrophages and the progression of membranoproliferative glomerulonephritis (MPGN). METHODS: We enrolled 35 patients with MPGN type 1 who had a normal creatinine clearance at the time of their first renal biopsy and divided them into 2 groups based on clinical status at the time of their most recent examination: 12 patients with normal urine test results and 12 patients with minor urinary abnormalities at the latest observation (group 1) and 7 patients with persistent nephropathy and 4 patients with renal insufficiency (group 2). The first and second renal biopsy findings and MRP8 and MRP14 expression on macrophages were investigated in both groups. RESULTS: Mean scores for positive glomerular and interstitial MRP8 and CD68 staining at the time of the first and second biopsies were significantly higher in group 2 than group 1. At the time of the second biopsy, mean scores for interstitial CD68-positive (CD68 +) staining were higher in group 2 than group 1. Mean scores for glomerular and interstitial MRP8 + and CD68 + staining at the time of the first biopsy correlated with the chronicity index at the time of second biopsy in both groups. CONCLUSION: Results suggest that MRP8 expression on macrophages in glomeruli and interstitial lesions at the first biopsy can be a useful prognostic marker for renal dysfunction in children with MPGN type 1.

IgA deficiency and membranous glomerulonephritis presenting as nephrotic syndrome.

Selective IgA deficiency associated with glomerulonephritis is rare and no previous reports in childhood have been made of the association of IgA deficiency and membranous glomerulonephritis (MGN). We report a 5-year-old boy with selective IgA deficiency and MGN. He presented with nephrotic syndrome. Percutaneous renal needle biopsy showed diffuse global thickening on light microscopy and heavy IgG and moderate C3 deposits were found on immunofluorescence. Electron microscopy detected extensive global subepithelial deposition of electron-dense material with frequent intramembranous extension and spike formation. The pathological diagnosis was diffuse MGN stage 1. Oral prednisolone (1 mg kg(-1) day(-1)), angiotensin-converting enzyme inhibitors (ACEI), and angiotensin II receptor blocker (ARB) were given resulting in reduction of proteinuria. The prednisolone dose was gradually tapered and discontinued after 2 months. At present the patient has been in complete remission for 10 months despite the discontinuance of prednisolone. In conclusion, our treatment with corticosteroid, ACEI and ARB reduced proteinuria and was effective for our case with selective IgA deficiency and MGN.

Uninephrectomy induces progressive glomerulosclerosis and apoptosis in anti-Thy1 glomerulonephritis.

Administration of the anti-Thy1 antibody in rats induces reversible glomerulonephritis resembling human mesangiolytic and mesangioproliferative diseases. The purpose of the present study was to design a model of irreversible glomerulosclerosis, using the anti-Thy1 antibody injection after uninephrectomy, and examine it, focusing on apoptosis in the process of progressive sclerotic changes. Wistar rats were divided into three groups: one-kidney groups (group I and III) and a two-kidney group (group II). All groups were injected with the anti-Thy1 antibody (OX-7) at day 0, and group I and III were uninephrectomized at day -6. Only group III rats were given a half dose of OX-7 as compared with group I and II. Rats were killed for histological examinations at days 7, 14 and 30. In group I, progressive glomerular lesions, such as glomerular adhesion to Bowman's capsule, crescent formation, and collapse of capillary tufts were observed at days 14 and 30. No significant differences were observed in the pathological findings between group I and III. There was a significantly higher number of glomerular terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in group I as compared to group II at days 7 and 14. Moreover, the glomerular expression of transforming growth factor-beta, heparan sulfate proteoglycan and chondroitin sulfate proteoglycan significantly increased in group I as compared to group II at days 7 and 14. Progressive glomerulosclerosis can be induced in the rat by a single injection of the anti-Thy1 antibody after unilateral nephrectomy. It is suggested that apoptosis and extracellular matrix accumulation play an important role in the development of glomerulosclerosis.

Mizoribine oral pulse therapy for steroid-dependent nephrotic syndrome.

There have been reports of the use of mizoribine (MZB) oral pulse therapy for the treatment of systemic lupus erythematosus. We report its efficacy in a 9-year-old girl with steroid- and cyclosporine-dependent nephrotic syndrome (NS). The patient experienced relapses of NS when prednisolone was tapered to 20 mg/day after discontinuing cyclosporine due to biopsy proven toxicity. When methylprednisolone pulse therapy combined with prednisolone therapy (40 mg/day) failed to result in a complete remission after 3 weeks, oral MZB pulse therapy (total dose of 500 mg, 10 mg/kg per day in three divided daily doses twice a week) was given. This therapy was continued for 9 months and resulted in complete remission of the NS for 6 months despite the discontinuation of prednisolone. The serum concentration of MZB was above 2.5 microg/ml for about 10 h (from 3 h after the first dose of MZB to 2 h after the final dose). Thus, our results suggest that this regimen may be effective for patients with steroid-dependent NS.