Epidemiological survey and clinical investigation of pediatric IgA nephropathy.

BACKGROUND: Since school urinalysis screening was introduced in 1974, the number of cases requiring initiation of dialysis due to glomerulonephritis has been steadily decreasing and school urinalysis screening has been praised for contributing to the early detection and treatment of glomerulonephritis. However, the lack of nationwide epidemiological surveys is also a problem. METHODS: We conducted an epidemiological survey focusing on the frequency of occurrence of pediatric IgA nephropathy in Nishinomiya City. Subjects comprised 374,846 children who underwent school urinalysis screening from 2003 to 2012. Renal biopsy findings and clinical findings of these pediatric IgA nephropathy cases were retrospectively investigated. RESULTS: There were 37 (mean 3.7/year) newly diagnosed cases of pediatric IgA nephropathy in Nishinomiya City. The IgA nephropathy onset rate per 100,000 children who underwent school urinalysis screening was 9.9 cases/year. Compared to the histologic low grade group, the histologic high grade group had significantly higher urinary P/C ratio (P < 0.001). In the histologic high grade group, the number of cases of proteinuria remission 3 years after starting treatment was significantly higher in the group treated with steroids (P = 0.045). CONCLUSIONS: Our study found that 9.9 cases of pediatric IgA nephropathy were diagnosed per 100,000 in the pediatric population, which is equivalent to or slightly more than past reports. IgA nephropathy, which poses a high histologic risk, presents with heavy proteinuria; but the proteinuria remission rate following steroid therapy is high 3 years after treatment, which suggests that administration of steroids results in an improved clinical outcome.

[Clinical usefulness of CD68 staining in children with various glomerular diseases].

PURPOSE: Glomerular macrophage accumulation is a common feature of proliferative forms of human glomerulonephritis and kidney injury. Our present study was designed to investigate the role of macrophages in pediatric kidney diseases by using CD68 staining. MATERIAL AND METHODS: Seventy-four patients (39 boys and 35 girls) with pediatric kidney disease yielding 81 specimens were investigated. A monoclonal anti-human CD68 mouse antibody (KP1) was used as a macrophage marker in this study. Paraffin-embedded renal biopsy specimens were stained for immunohistochemical analysis. The average number of macrophages per glomerulus in each patient was calculated as the total number of CD68 (+) cells within all glomeruli divided by the total number of glomeruli in a single section and the average number of observed interstitial macrophages was calculated in 3-5 high power fields. RESULTS: Glomerular macrophage accumulations were increased with crescentic proliferative glomerulonephritis, mesangial proliferative glomerulonephritis, and focal segmental glomerulosclerosis. Glomerular and interstitial macrophage accumulations were correlated with hematuria, proteinuria and renal function (eGFR). In particular, activity and chronicity index, as well as the severity of glomerular IgA, C3, and fibrinogen deposition were correlated with glomerular macrophage accumulation. CONCLUSIONS: Macrophage accumulation observed by CD68 staining was a useful marker in providing a deeper understanding of the clinicopathologic state of children with chronic kidney diseases, and was effective in the selection of treatment.

Small cell osteosarcoma successfully treated by high-dose ifosfamide and methotrexate, combined with carboplatin and pirarubicin.

Small cell osteosarcoma (SCO) is the most rare subtype of osteosarcoma and has a poor prognosis. An 11-year-old boy presented with 2-month history of painful tumefaction in the lower leg. Imaging analysis demonstrated a mixture of osteolytic and osteosclerotic lesions in the proximal tibia and extraskeletal area. Histology of the open biopsy showed small round cells producing mucous matrix. Based on these findings, SCO was suspected. The patient received three cycles of neoadjuvant chemotherapy using high-dose ifosfamide, high-dose methotrexate, pirarubicin and carboplatin. Wide-margin resection was performed followed by tibial lengthening using the Ilizarov method and two cycles of adjuvant chemotherapy with the same drugs as for neoadjuvant chemotherapy. Histology of the resected specimen showed that almost all tumor cells were necrotized. Neither recurrence nor metastasis was found after 4 years. Our experience suggests that neoadjuvant chemotherapy, such as the one used here, would be exceedingly effective for SCO without serious non-hematological toxicities.

Germline PTCH1 mutations in Japanese basal cell nevus syndrome patients.

Basal cell nevus syndrome (BCNS or Gorlin syndrome, OMIM: 109400) is a rare autosomal dominant disorder with high penetrance. It is characterized by developmental anomalies and predisposition to tumors (for example, basal cell carcinoma (BCC) and medulloblastoma). PTCH1, the human homolog of the Drosophila patched gene, was identified as a gene responsible for BCNS. The PTCH1 protein is a Hedgehog (Hh) protein receptor and is pivotal for early development, stem cell maintenance and/or differentiation. We analyzed the six Japanese families with BCNS and identified six germline mutations in the PTCH1 gene. One family had a nonsense mutation (c.1196G>A), one had a 1-bp deletion (c.2029delA), two had 2-bp deletions (c.239_240delGA and c.1670_1671delCA) and one had a 58-bp duplication (c.1138_1195dup). They caused premature termination, resulting in the truncation of the PTCH1 protein. Analysis of a high-density single nucleotide polymorphism (SNP) mapping array showed a large approximately 1.2-Mb deletion, including the PTCH1 gene in one allele, in a family in which PTCH1 mutations were not identified at the sequence level. These data indicated that all the six families who were diagnosed with BCNS had mutations in the PTCH1 gene and that a single copy of a PTCH1 mutation causes BCNS.

Extramedullary relapse of acute lymphoblastic leukemia in childhood to the prostate.

A 6-year-old boy presented with the chief complaints of miction pain and pollakisuria. He had a past history of acute lymphoblastic leukemia (ALL), which subsided in response to chemotherapy at 3 years of age. Ultrasonography revealed urinary retention associated with bilateral hydronephrosis secondary to the prostate enlargement. Computed tomography and magnetic resonance imaging showed no other abnormal finding. Transrectal needle biopsy showed infiltration of leukemic cells in the prostate. Bone marrow puncture and cerebrospinal fluid aspiration revealed no leukemic cells, resulting in a diagnosis of extramedullary relapse of ALL in the prostate. Although he was successfully treated by chemotherapy, irradiation and his voiding function was improved, ALL relapsed in the left testis 1 year later. In spite of left orchiectomy, irradiation and additional chemotherapy, he died of bone marrow relapse and multiple organ failure. Extramedullary relapse of ALL in the prostate is very rare. To our knowledge, our case is the first well-documented report in the published work.

Disseminated necrotizing leukoencephalopathy following chemoradiation therapy for acute lymphoblastic leukemia.

Disseminated necrotizing leukoencephalopathy (DNL) is a potentially fatal complication of treatment involving intrathecal administration of chemotherapeutic agents such as methotrexate (MTX) alone or in combination with cranial radiotherapy (RT). We describe a case of acute lymphoblastic leukemia (ALL) treated with high-dose intravenous and intrathecal methotrexate combined with craniospinal RT resulting in DNL. Typical MR imaging features of progressive deep white matter lesions showing a characteristic pattern of enhancement after contrast was seen in this case. Deep white matter lesions with ring-like enhancement and calcifications were seen on CT; it showed a mass effect at one stage, which is not typical for DNL. Long-term clinical and imaging follow-up were helpful for the diagnosis in this case.

Clinical characteristics, etiologies and pathophysiology of patients with severe short stature with severe GH deficiency: questionnaire study on the data registered with the foundation for growth science, Japan.

In this study, we sent questionnaires to doctors treating severe short stature with severe GH deficiency (GHD) (height SDS (HtSDS) below -4 and all peak GH to provocative stimuli below 2 micro/L) (abbreviated as Severe Case), and obtained effective replies of 51 cases. The clinical characteristics, etiologies, and pathophysiology of these patients were examined. Among the 51 Severe Cases no consanguinity was observed, 44 were IGHD (24 males and 20 females), 3 were GH-1 gene deletion, 2 were Pit-1 gene mutation, and 2 were achondroplasia. HtSDS in these Severe Cases was already remarkably low at 12 (-3.0) and 24 months old (-3.9), while their birth weight and birth length were within normal ranges. Among 44 patients with IGHD, 12 were isolated GHD, and the remaining 32 were combined pituitary hormone deficiency (CPHD). Pituitary MRI was undergone in 25 idiopathic GHD, and abnormal findings (pituitary atrophy, interruption of stalk, and ectopic posterior lobe) were observed in 21 patients with CPHD. More than half of these patients had the history of breech delivery. Three patients with GH-1 gene mutation showed normal pituitary MRI, whereas one of two patients with Pit-1 mutation showed pituitary atrophy and narrowing of pituitary stalk. In conclusion, Severe Cases tended to have CPHD, and the incidence of Severe Case was only 0.6% of total IGHD. Although GHD due to genetic disorders is considered to be extremely rare (0.06% of total IGHD), the incidence reaches high levels (9.8%) among Severe Cases. Growth disorders in these Severe Cases seem to occur soon after delivery. Much earlier diagnosis and hGH treatment are desirable to attain better final height in the Severe Cases. GH-1 and Pit-1 gene analyses are crucial, when genetic abnormalities other than achondroplasia are suspected.

RAS-blocking bisphosphonate zoledronic acid inhibits the abnormal proliferation and differentiation of juvenile myelomonocytic leukemia cells in vitro.

Juvenile myelomonocytic leukemia (JMML) is a clonal myeloproliferative/myelodysplastic disorder of early childhood with a poor prognosis. JMML cells are characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) caused by a continuously activated GM-CSF receptor-retrovirus-associated sequence (RAS) signal transduction pathway through various molecular mechanisms, resulting in spontaneous GM colony formation in vitro. Bisphosphonate zoledronic acid (ZOL), a RAS-blocking compound, suppressed colony formation from bone marrow (BM) cells of 8 patients with JMML and 5 healthy control subjects without and with GM-CSF (10 ng/mL), respectively, in a dose-dependent manner in clonal culture. At 10 microM ZOL, however, spontaneous GM colony formation from JMML BM cells decreased to 3%, but the formation of G colonies containing granulocytes, but no macrophages, was enhanced, whereas 40% of GM colonies were retained and G colony formation was not affected in culture of normal BM cells with GM-CSF. In suspension culture, cytochemical and flow cytometric analyses showed that 10 microM ZOL also inhibited spontaneous proliferation and differentiation along monocyte/macrophage lineage of JMML BM cells but not the development of normal BM cells by GM-CSF. The inhibitory effect of ZOL on JMML cells was confirmed at a single-clone level and observed even at 3 microM. The current result offers a novel approach to therapy in JMML.

Relationship between Thy-1 expression and cell-cycle distribution in human bone marrow hematopoietic progenitors.

Analysis of the relationship between Thy-1 expression and cell-cycle distribution of hematopoietic stem cells (HSCs) showed that freshly isolated Thy-1+ and Thy-1- subsets of the CD34highCD38-flt-3-Lin- population were predominantly in G0/G1 phase and remained essentially quiescent, whereas after 6 days of cytokine stimulation, the Thy-1+ subset of the population entered the cycling state while the Thy-1- subset again remained quiescent. Expression of Thy-1 antigen resulted in a drastic increase in the percentage of cycling cells in CD34highCD38-flt-3-Lin-Thy-1+- as well as CD34highCD38-flt-3-Lin- Thy-1(-)-cell-initiated cultures. The Thy-1+ subset of the CD34highCD38-flt-3-Lin- population exists in the freshly isolated CD34highCD38-flt-3-Lin- Thy-1+ fraction, loses Thy-1 expression during 6 days, and re-expresses Thy-1 for an additional 2 days. Cell-cycle analysis demonstrated that this unique subset contains abundant S/G2M cells. Thus, Thy-1 expression appears to be an indicator of cell-cycle phase in targeting HSC, which might serve in the cell subset best suited for gene transfer.

Protection against bleomycin-induced lung injury by IL-18 in mice.

The role of interleukin (IL)-18 in the protection from interstitial pneumonia and pulmonary fibrosis induced by bleomycin (BLM) was investigated by comparing the severity of BLM-induced lung injuries between wild-type and C57BL/6 mice with a targeted knockout mutation of the IL-18 gene (IL-18-/- mice). IL-18-/- mice showed much worse lung injuries than wild-type mice, as assessed by the survival rate, histological images, and leukocyte infiltration in the bronchoalveolar lavage fluid and myeloperoxidase activity. In wild-type mice, administration of IL-18 before BLM instillation resulted in suppression of lung injuries, increases in the hydroxyproline content, and decreases in the granulocyte-macrophage colony-stimulating factor content in the lung. Preadministration of IL-18 also resulted in prevention of the reduction of the lung IL-10 content caused by BLM-induced damage of alveolar epithelial. BLM instillation suppressed superoxide dismutase (SOD) activity in IL-18-/- mice to a greater extent than in wild-type mice. Pretreatment of IL-18 augmented Mn-containing superoxide dismutase (Mn-SOD) messenger RNA expression and SOD activity in the lung and prevented the reduction of SOD activity caused by BLM in both wild-type and IL-18-/- mice. These results suggest that IL-18 plays a protective role against BLM-induced lung injuries by upregulating a defensive molecule, Mn-SOD.

Correlation of c-kit expression and cell cycle regulation by transforming growth factor-beta in CD34+ CD38- human bone marrow cells.

To investigate the relationship between c-kit expression and cell cycle regulation by endogenous transforming growth factor-beta (TGF-beta) in human bone marrow hematopoietic progenitor cells, CD34+ CD38- c-kit(low/-) and CD34+ CD38- c-kit(high) populations were cultured in stem cell factor, thrombopoietin, interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and anti-TGF-beta, and analyzed for cell cycle status. Arrest in G0/G1 was most prominent in the precultured CD34+ CD38- c-kit(low/-) subset (95.62 +/- 4.15%). While postcultured CD34+ CD38- c-kit(high) cells initiated from CD34+ CD38- c-kit(high) cells entered cell cycle within 36 hr, postcultured CD34+ CD38- c-kit(low/-) cells initiated from CD34+ CD38- c-kit(low/-) cells remained dormant until 36 hr and entered cell cycle within 90 hr. Anti-TGF-beta increased the percentage of S/G2M phase postcultured CD34+ CD38- c-kit(high) cells (from 19.08 +/- 11.95 to 47.04 +/- 2.93%), but no significant change was observed in postcultured CD34+ CD38- c-kit(low/-) cells. These results suggest that endogenous TGF-beta plays an important role in the cell cycle arrest of c-kit(high) but not c-kit(low/-) cells in CD34+ CD38- cells, which proliferate without undergoing differentiation. The different regulatory mechanism of cell cycle entry of the CD34+ CD38- c-kit(high) and CD34+ CD38- c-kit(low/-) subsets might be the result of differences in their sensitivity to endogenous TGF-beta.

Hepatocyte growth factor gene therapy retards the progression of chronic obstructive nephropathy.

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive tubular atrophy and interstitial fibrosis. Rupture of the balance between cell proliferation and apoptosis plays a critical role in renal atrophy. Hepatocyte growth factor (HGF) is a cytokine function on cell survival and tissue regeneration. We studied the effects and possible mechanisms of HGF gene therapy on tubular cell survival and anti-fibrosis in chronic obstructed nephropathy. METHODS: An in vivo transfection procedure of repeatedly transducing skeletal muscles with the HGF gene using liposomes containing the hemagglutinating virus of Japan (HVJ liposome) was tested on UUO rats. Expression of HGF and c-Met were examined by in situ hybridization, ELISA, or immunohistochemical staining. Interstitial fibrosis and macrophage infiltration were evaluated by Masson's Trichrome staining, alpha-smooth muscle actin and ED-1 immunostaining. Cell survival indices including proliferating cell nuclear antigen (PCNA), Bcl-2, Bcl-xL and Bax were measured by immunohistochemistry and Western blots. Apoptosis was determined by the TUNEL method. RESULTS: After HVJ-HGF gene transfer, endogenous HGF and c-Met were up-regulated in UUO kidneys. Renal fibrosis, macrophage infiltration and tubular atrophy were suppressed both at day 14 and 28 after UUO (P < 0.05 or 0.01). Tubular cell proliferation was activated while apoptosis was inhibited, especially at the late stage of UUO. Bcl-2 was enhanced in the HGF-transfected UUO rats, while no changes of Bcl-xL and Bax were found. CONCLUSIONS: In vivo HGF gene transfection retards the progression of chronic obstructed nephropathy and protects tubular cell survival in the long-term UUO model. Bcl-2 rather than Bcl-xL or Bax may contribute to the anti-apoptotic function of HGF.

Doppler echocardiographic differentiation of functional from anatomical pulmonary atresia: analysis using quantitative parameters.

Functional pulmonary atresia must be distinguished from anatomical atresia, which has an intact ventricular septum, to avoid inappropriate treatment, but there is a paucity of data regarding the echocardiographic features that differentiate these conditions. Echocardiographic findings in 5 neonates with functional atresia were compared to those in 5 with anatomical atresia. The left and right ventricular end-diastolic dimensions (LVDd, RVDd), percent of normal predicted LVDd, RVDd/LVDd, tricuspid valve ring diameter (TVD), percent of normal predicted TVD, grade of tricuspid regurgitation (TR), peak TR velocity, pulmonary valve ring diameter (PVD), percent of normal predicted PVD, the minimum diameter of the ductus and the peak velocity through it (PDA velocity) were measured. In addition, systolic pulmonary (PAp) and right ventricular pressure (RVp) from either PDA velocity or TR velocity, and calculated PAp/RVp were also estimated. There were significant differences in RVDd/LVDd, %TVD, and peak TR velocity between the 2 groups. All functional patients showed RVDd/LVDd >0.6, %TVD >100%, estimated RVp <50mmHg, PAp/RVp >0.85, and peak TR velocity <4m/s, whereas the findings in anatomical atresia patients were completely the opposite. In conclusion, a large RVDd/LVDd, TVD, PAp/RVp, low RVp and small TR velocity all suggest functional rather than anatomical pulmonary atresia, although there may be some exceptions such as severe Ebstein anomaly.