Familial focal segmental glomerulosclerosis with Alport-like glomerular basement changes caused by paired box protein 2 gene variant.

Paired box protein 2 (PAX2) gene variant causes renal coloboma syndrome (MIM#120330). Further, they are associated with focal segmental glomerulosclerosis and characterized by basement membrane changes similar to Alport syndrome.Herein, we report an 8-year-old boy who presented with proteinuria and decreased renal function. His paternal uncle has focal segmental glomerulosclerosis and renal failure, and his paternal grandmother has renal failure and is receiving peritoneal dialysis. Further, his father has stage 2 chronic kidney disease. At 3 years of age, his serum creatinine-estimated glomerular filtration rate was 40-50 mL/min/1.73 m2. At 8 years of age, his renal function further decreased and he had proteinuria (urinary protein/Cr 3.39 g/g Cr). Renal histopathology showed oligonephronia and focal segmental glomerulosclerosis. A partial basket-weave pattern, similar to Alport syndrome, was also observed on a transmission electron microscope, and low-vacuum scanning electron microscopy revealed coarse meshwork changes in the glomerular basement membrane. Genetic analysis revealed a PAX2 heterozygous variant (NM_003987.4:c.959C  >  G), a nonsense variant in which the serine at position 320 changes to a stop codon, in our patient and his father. PAX2 is a transcription factor that is important for the podocyte variant. However, podocytes with PAX2 gene variants may cause abnormal basement membrane production and repair, thereby resulting in Alport-like changes.

Ultrastructural and morphological analysis during progression of Bowen disease reveals a complex interplay between hyperkeratosis, cytokeratin expression, host immunity and amyloid deposition.

BACKGROUND: Bowen disease, one of the common skin cancers, is defined as squamous cell carcinoma in situ, characterized by atypical keratinocytes occupying the full thickness of the epidermis, and predominantly occurs on sun-protected skin. There is no existing data on the impact of tumour and immune cell interactions or cytokeratin expression on the pathology of Bowen disease. OBJECTIVES: We analysed dynamic changes in cytokeratin expression and immune cell composition during the development and progression of Bowen disease. MATERIALS & METHODS: Analysis was performed using immunohistochemistry and electron microscopy for samples from 140 patients with Bowen disease and 20 patients with invasive squamous cell carcinoma. We evaluated cytokeratin expression, the number of infiltrating immune cells and amyloid deposition by immunohistochemistry, and the ultrastructural relationship between tumour cells and immune cells by electron microscopy. RESULTS: The results showed that the expression of CK14 is associated with tumour progression, keratotic status and amyloid deposition and that the expression of CK10 is associated with accumulation of immune cells in Bowen disease. The findings of electron microscopy indicated repeated battles involving immune cells in response to tumour invasion. CONCLUSION: The expression of cytokeratins, hyperkeratosis, inflammatory infiltration and amyloid deposition are useful findings indicating the "stage" in Bowen disease.

Segment-Dependent Gene Expression Profiling of the Cartilaginous Fish Nephron Using Laser Microdissection for Functional Characterization of Nephron at Segment Levels.

For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.

Extracellular vesicles derived from Spirometra erinaceieuropaei plerocercoids inhibit activation of murine macrophage RAW264.7 cells.

Parasitic helminths modify host immune reactions to promote long-term parasitism. We previously purified a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF), from the excretory/secretory products of Spirometra erinaceieuropaei plerocercoids and reported its cDNA and genomic DNA sequences. In this study, we isolated extracellular vesicles (EVs) from the excretory/secretory products of S. erinaceieuropaei plerocercoids and found that they suppressed the production of nitric oxide and the gene expression of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 in lipopolysaccharide-stimulated macrophages. EVs are membrane-bound vesicles 50-250 nm in diameter and are localized in the whole bodies of plerocercoids. EVs from plerocercoids encapsulate a variety of unidentified proteins and microRNAs (miRNAs), which are non-coding RNAs that play essential roles in post-transcriptional gene regulation. The miRNAs of the EVs were analyzed, and 334,137 sequencing reads were mapped to the genomes of other organisms. A total of 26 different miRNA families were identified, including miR-71, miR-10-5p, miR-223, and let-7-5p, which have been reported to have immunosuppressive effects. We confirmed that P-ISF was present in the supernatant but not in the EVs by western blotting with an anti-P-ISF antibody. These results suggest that S. erinaceieuropaei plerocercoids suppress host immunity by releasing P-ISF and EVs.

Dual role of basophils in the pathogenesis of bullous pemphigoid elucidated by pathological and ultrastructural studies

Background: Bullous pemphigoid (BP), one of the most common autoimmune blistering disorders, is characterized by early erythematous and bullous lesions. Histopathologically, eosinophilia in the dermal tissue is a common finding in BP. In addition, basophils infiltrate the BP skin lesion. Although basophils are involved in the induction of type 2 immunity along with eosinophils, their role in both the erythema and blister, as well as the chronology of their involvement, have not been investigated. Objectives: To elucidate the role of basophils in BP development and resolution by performing early- and late-phase histopathological analysis of BP. Materials & Methods: A total of 25 patients with BP who underwent biopsy for both erythema and bullous lesions and were not taking oral steroids at the time of biopsy, were selected. Biopsy specimens of the erythematous (inflammatory) and bullous (resolution) phases were compared by histopathology, immunohistochemistry and electron microscopy. Results: During the early phase of BP, the number of basophils positively correlated with the number of eosinophils compared with other immune cells. In the late phase (bullous phase) of BP, the number of basophils significantly increased and more cell-cell contact between the basophils and M2 macrophages was noted, compared to the early phase Conclusions: Basophils are involved in the development of BP and its resolution, in part, via cell-cell contact with eosinophils or M2 macrophages, as demonstrated by pathological analysis.

Outcome From a Randomized Controlled Clinical Trial　- Improvement of Peripheral Arterial Disease by Granulocyte Colony-Stimulating Factor-Mobilized Autologous Peripheral-Blood-Mononuclear Cell Transplantation (IMPACT).

BACKGROUND: The clinical usefulness of peripheral blood (PB) mononuclear cell (MNC) transplantation in patients with peripheral arterial disease (PAD), especially in those with mild-to-moderate severity, has not been fully clarified.Methods and Results:A randomized clinical trial was conducted to evaluate the efficacy and safety of granulocyte colony-stimulating factor (G-CSF)-mobilized PBMNC transplantation in patients with PAD (Fontaine stage II-IV and Rutherford category 1-5) caused by arteriosclerosis obliterans or Buerger's disease. The primary endpoint was progression-free survival (PFS). In total, 107 subjects were enrolled. At baseline, Fontaine stage was II/III in 82 patients and IV in 21, and 54 patients were on hemodialysis. A total of 50 patients had intramuscular transplantation of PBMNC combined with standard of care (SOC) (cell therapy group), and 53 received SOC only (control group). PFS tended to be improved in the cell therapy group than in the control group (P=0.07). PFS in Fontaine stage II/III subgroup was significantly better in the cell therapy group than in the control group. Cell therapy-related adverse events were transient and not serious. CONCLUSIONS: In this first randomized, large-scale clinical trial of G-CSF-mobilized PBMNC transplantation, the cell therapy was tolerated by a variety of PAD patients. The PBMNC therapy was significantly effective for inhibiting disease progression in mild-to-moderate PAD.

Study of prolonged administration of lanthanum carbonate in dialysis patients.

Data of 36 months were accumulated regarding the effects of lanthanum carbonate (LA) on serum phosphate concentrations in dialysis patients. Fifty-three patients (average age and dialysis history 58.4 years and 9.1 years) were included in this study who have been receiving outpatient treatment since March 2009, and who have been unable to maintain serum phosphate concentrations of ≤6.0 mg/dL via traditional therapeutic agents used for hyperphosphatemia. Patients were given dosage of LA in addition to, or instead of, co-hyperphosphatemia treatments already being received. Mean dosages of calcium carbonate (CC) and sevelamer hydrochloride (SH) before starting LA administration were 1301.9 mg and 2462.3 mg, respectively. Dosage of LA for all cases was 750 mg at initial dose; 1528.3 mg at 5 months; and 1416.7 mg at 30 months. Dosage of other phosphate binders were 905.7 mg of CC and 820.8 mg of SH at 5 months; and 687.5 mg of CC and 1031.3 mg of SH at 30 months. Serum phosphorus levels (P levels) were significantly decreased at 1 month of LA administration, and continued until 30 months of La treatment. These results suggest that LA successfully controlled serum P and Ca concentrations simultaneously within target ranges without affecting serum intact parathyroid hormone concentration, although further long-term prospective cohort study on LA would be required.

Novel morphological study of solar lentigines by immunohistochemical and electron microscopic evaluation.

Solar lentigines (SL) are hyperpigmented lesions generally seen in elderly people. Their pathogenesis has not been completely elucidated. We examined 75 cases of SL using routine histopathology and immunohistochemistry. In addition, seven cases were evaluated by electron microscopy. Histopathologically, we observed vacuolar changes in the dermoepidermal junction in 85% of the cases. Dermal melanophages were seen in 77% of the cases. The immunohistochemical expression rates in the epidermis for cytokeratin (CK)15, CK14, CK10, p63 and nestin were 76%, 100%, 100%, 100% and 17%, respectively. In 58 cases showing dermal melanophages, expression rates of CD163 and factor XIIIa on melanophages were 79% and 83%, respectively. Double positivity for both proteins was identified in 44 cases (75%). Ultrastructurally, vacuolar structures were seen in the cytoplasm of basal cells and upper dermis in all cases examined. We observed elimination processes of damaged basal keratinocytes, which were probably produced by ultraviolet (UV) irradiation, into the papillary dermis. The segregated damaged cell bodies containing melanin granules seemed to be phagocytosed by poorly immunostimulatory macrophages labeled immunohistochemically by CD163 and factor X IIIa, contributing to prolonged pigmentation of SL. In addition, repeated basal keratinocyte damages may be in association with altered CK and p63 expression patterns in the constituent cells of SL.

Long-term clinical outcomes for patients with lower limb ischemia implanted with G-CSF-mobilized autologous peripheral blood mononuclear cells.

BACKGROUND: Many studies have described the clinical effects of treating critical limb ischemia with granulocyte colony-stimulating factor-mobilized autologous peripheral blood mononuclear cells (M-PBMNC); however, there are no long-term data available on survival, limb salvage, or prognostic factors. METHODS: To investigate the long-term clinical outcomes of M-PBMNC implantation, we reviewed data for 162 consecutive patients with limb ischemia who were treated with M-PBMNC implantation at 6 hospitals between 2001 and 2006. A subset of 123 patients with homogenous clinical profiles was selected for prognostic factor analysis. RESULTS: Of the 162 patients, 50 died during the follow-up period. The median follow-up time for surviving patients was 26.4 months. The 2-year survival rate was 65% for the 140 patients with arteriosclerosis obliterans (ASO), and 100% for the 11, 4 and 7 patients with thromboangiitis obliterans (TAO), diabetic gangrene (DG) and connective tissue disease (CTD), respectively. The 1-year amputation-free rates for ASO, TAO, DG and CTD were 70%, 79%, 75% and 83%, respectively. Common serious adverse events included heart failure (15 cases), myocardial infarction (15 cases), serious infection (13 cases), stroke (10 cases), and malignant tumor (9 cases). Significant negative prognostic factors associated with overall survival were ischemic heart disease and collection of a small number of CD34-positive cells. Factors associated with time-to-amputation and amputation-free survival were a combination of Fontaine classification and lower limb gangrene, and history of dialysis. CONCLUSIONS: Collection of a small number of CD34-positive cells and ischemic heart disease were associated with a reduction in overall survival.

[Reversal of pulmonary hypertrophic osteoarthropathy in surgically treated lung cancer].

A 61-year-old man was admitted for evaluation of an abnormal chest abnormal with progressive swelling in both hands, clubbing of all fingers and toes, and polyarthroceles. He was given a diagnosis of pulmonary hypertrophic osteoarthropathy (PHO) associated with primary lung cancer, and underwent an upper left lobectomy. Histopathological analysis revealed stage IIB adenocarcinoma of the lung with K-ras mutation, but with no evidence of epidermal growth factor receptor (EGFR). Postoperatively, his symptoms rapidly improved, and the preoperatively observed high levels of serum vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) decreased to normal levels after just 1 month. VEGF and IL-6 caused by the genetic mutation of K-ras might play a role in the pathogenesis of PHO with lung cancer.

Therapeutic neovascularization by the implantation of autologous mononuclear cells in patients with connective tissue diseases.

Vasculopathy in patients with connective tissue diseases (CTDs), including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE), is a serious complication that mainly affects small arteries and capillaries, reduces the blood flow and causes progressive tissue ischemia. Recently, CTD patients have been reported to have abnormalities in circulating endothelial progenitor cells (EPCs); these abnormalities are believed to contribute to the pathophysiology of vasculopathy and to the premature and accelerated development of atherosclerosis in CTD patients. Furthermore, we are currently conducting a clinical pilot study to determine the efficacy of implanting autologous mononuclear cells obtained from the bone marrow and peripheral blood into the ischemic digits or limbs of CTD patients. In this review, we discuss the role of EPCs in the process of neovascularization and in the pathophysiology of CTDs, and we describe a clinical pilot study on the use of autologous cell therapy for treating ischemic digits in patients with CTDs.

[Prader-Willi syndrome associated with obesity hypoventilation syndrome].

Prader-Willi syndrome (PWS) is a genetic disorder, characterized by shorter height, severe obesity and muscular hypotonicity. In particular, sleep disordered breathing (SDB) is a well-known complication in PWS. We encountered one case of PWS, complicated by typical obesity hypoventilation syndrome. A 23-year-old woman had been given a diagnosis of PWS as age 1, therefore she was treated with growth hormone replacement therapy, and with uvulopalatopharyngoplasty (UPPP) for her narrow throat. Her weight increased greatly to 96kg, body mass index (BMI) 51 kg/m2, resulting in hypersomnolence, cyanosis, heavy snoring, and nocturnal awakening. Eventually, she was admitted because of urinary incontinuence and loss of consciousness. On admission, she had severe hypoxia plus substantial hypercapnia, and her chest X-ray film showed severe cardiomegaly with massive pleural and pericardial effusion. On polysomnography (PSG) one week later, her apnea hypopnea index (AHI) was 16 with a mean nocturnal arterial saturation of 74%, mean percutaneous PCO2 59 Torr, which rose to 73 Torr during REM sleep. Non-invasive positive pressure ventilation (NPPV) was initiated, and improved her condition greatly. She was discharged, but continued to recieve NPPV, and her condition has stayed improved.

[Overlap syndrome involving obstructive sleep apnea syndrome associated with chronic obstructive pulmonary disease].

We reported a case of overlap syndrome involving severe obstructive sleep apnea syndrome (OSAS) associated with chronic obstructive lung disease (COPD). This patient was a 52-year-old heavy smoking man, who had suffered from snoring and apnea for five years, and was admitted to our hospital because of worsening dyspnea. His BMI was 25 Kg/M2, His jaw was very small and he had a narrow upper airway. Chest X-ray showed hyperlucency throughout both lung fields with a markedly dilatation pulmonary arteries. His PaO2 was 62Torr, PaCO2 was 47Torr, FEV(1.0%) was 59%, mean pulmonary artery pressure was 27 mmHg, PSG showed that AHI was 70, were most pronounced during rapid eye movement sleep. He was given a diagnosis of overlap syndrome of OSAS associated with COPD. Generally, Overlap syndrome was believed that chronic bronchitis type (blue bloater) was more frequent than emphysema type. This case was a very rare case, with no obesity, moderate COPD, associated with pulmonary hypertension and hypercapnea, and then to be severe OSAS. However we should be more careful about the OSAS associated with overlap syndrome of the Japanese patients, because to be one factor of exacerbation of respiratory failure.

Clinical study of therapeutic angiogenesis by autologous peripheral blood stem cell (PBSC) transplantation in 92 patients with critically ischemic limbs.

Patients with critically ischemic limbs due to maintenance hemodialysis and diabetes are increasing in number markedly in Japan. The difficulty of treating critically ischemic limbs is well recognized. Despite active medication and surgical therapy, many critically ischemic limbs are amputated. Ninety-two patients with critically ischemic limbs were treated by transplantation of autologous peripheral blood stem cells (PBSCs). The stem cells were mobilized into the peripheral blood by administration of granulocyte colony stimulating factor (G-CSF). The mobilized mononuclear cells were separated by an apheresis technique using a centrifuge. The separated mononuclear cells contained approximately 4.0 x 10(7) CD34-positive cells. The collected cell suspension was divided into aliquots of 0.5-1.0 ml and transplanted into the muscle of ischemic limbs at 50-70 transplantation points. At 1.5 months after PBSC transplantation, a strong immunostaining of CD34-positive cells and factor VIII, as well as capillary formation, was observed in the muscles into which stems cells had been transplanted. In each patient tested, the serum vascular endothelial growth factor (VEGF) level increased after stem cell transplantation; the mean VEGF level increased by 176%. Of 11 diabetic patients (DM) who were not receiving hemodialysis (HD), there were no amputees regardless of their Fontaine classification. Of 19 patients in the HD(+)DM(-) category, there were no amputations in Fontaine stage I, II, and III patients, whereas three limbs and one toe were amputated in Fontaine stage IV patients. Of 13 patients in the HD(-)DM(+) category, none of the Fontaine stage I, II, or III patients underwent amputation, but six Fontaine stage IV patients underwent amputation. Of 49 patients in the HD(+)DM(+) category, 38 (78%) were classified as Fontaine stage IV, 71% (27/38) of whom had a toe or a limb amputated. In nine patients over 80 years of age, one toe and one limb were amputated. Nondiabetic, nondialyzed patients with ischemic limbs are strongly indicated for stem cell transplantation regardless of Fontaine classification. Therapeutic angiogenesis is effective for critically ischemic limbs resulting from hemodialysis and diabetes until Fontaine stage III, but is of limited effectiveness for stage IV cases.

Pioglitazone inhibits the growth of human leukemia cell lines and primary leukemia cells while sparing normal hematopoietic stem cells.

Peroxisome proliferator-activated receptors (PPARs) compose a subfamily of nuclear hormone receptors functioning as transcriptional regulators. Originally, the PPARgamma ligand known as thiazolidinedione (TZD) was used for the treatment of diabetic patients. However, recent studies have shown that TZD also has an antitumor effect that inhibits cell growth in several types of human malignant neoplasms, including leukemia cell lines. Since pioglitazone is the only TZD currently available in clinics in Japan and the role of TZD in normal human hematopoietic cells or primary leukemia cells has not been previously reported, we investigated the effect of pioglitazone on human normal hematopoietic progenitor cells, primary leukemia cells, and leukemia cell lines (HL60, K562, U937, HEL, CEM, Jurkat, and NALM1). Pioglitazone inhibited the proliferation of leukemia cells in a dose-dependent manner. The viable cell numbers of HL60, K562, and Jurkat leukemia cell lines were profoundly reduced when the cells were cocultured with pioglitazone. Colony formation in the leukemia cell lines as well as the primary leukemia cells was significantly inhibited to 20-71% and 1-25% of that in control cultures by the addition of 100 and 300 microM of pioglitazone, respectively. However, the CFU-E and CFU-GM colonies of cells obtained from healthy volunteers were not altered in the presence of 100 microM of pioglitazone. Pioglitazone (300 microM) induced slight decrease of CFU-E and CFU-GM. BFU-E was more sensitive to pioglitazone than CFU-E and CFU-GM. Pioglitazone-induced growth inhibition in HL60 cells was associated with cell cycle arrest at the G1 phase, as has been reported for another TZD, troglitazone. Similar levels of PPARgamma protein were observed in both leukemia and normal bone marrow cells by Western blotting, suggesting that the expression of PPARgamma protein was not associated with the inhibitory potency of pioglitazone. In conclusion, our results suggest that pioglitazone may offer a new therapeutic approach to aid in the treatment of leukemia.

Influence of lymphocytes in malignant pleural effusion on the therapeutic efficacy of intrapleural OK-432 in lung cancer patients.

OBJECTIVES: Malignant pleural effusion, a common complication seen in advanced lung cancer patients, is often treated with intrapleural administration of chemical agents. In Japan, OK-432, a biological response modifiers, which activates the cytotoxic activity of lymphocytes and boosts antitumor immunity, is among the most frequently used chemical agents. The purpose of this study was to determine, in a case-control study, whether or not the rate of lymphocytes in malignant pleural effusion (lymphocyte rate) influences the therapeutic efficacy of intrapleural OK-432. PATIENTS AND METHODS: We enrolled 20 lung cancer patients with malignant pleural effusion treated with intrapleural OK-432 who were admitted to our hospital between January 2000 and December 2004. Therapeutic efficacy was assessed from the response rate, duration of chest drainage after treatment with intrapleural OK-432, time to progression of malignant pleural effusion, and survival time. RESULTS: Response rate in patients who had a high lymphocyte rate (the High lymphocyte rate group) was significantly higher than in patients who had a low lymphocyte rate (the Low lymphocyte rate group). Lymphocyte rate did not correlate with duration of chest drainage after treatment with intrapleural OK-432, time to progression of malignant pleural effusion, or survival time. CONCLUSIONS: The lymphocyte rate in malignant pleural effusion influences the response rate to treatment by intrapleural OK-432. In the High lymphocyte rate group, intrapleural OK-432 for malignant pleural effusion was effective. We conclude that intrapleural OK-432 is useful for malignant pleural effusion patients with a high lymphocyte rate before treatment.

An inhibitory effect of A20 on NF-kappaB activation in airway epithelium upon influenza virus infection.

Influenza is a major disease in humans. The reemergence of avian influenza A viruses has indicated that hyperinflammatory responses are closely related to the severity of disease. Influenza virus infection induces nuclear transcription factor kappaB (NF-kappaB) activation. NF-kappaB and NF-kappaB-dependent gene products promote lung inflammation and injury. Therefore, it is important to investigate the means to attenuate NF-kappaB activation. A20 is a cytoplasmic zinc finger protein that inhibits NF-kappaB activity, However, little is known about the role of A20 in influenza virus infection. Here, we have examined the role of A20 in influenza virus infection-induced NF-kappaB promoter activation in human bronchial epithelial cells. The results showed that (1) A20 protein and mRNA are inducible and expressed in the lung from mice and human bronchial epithelial cells upon influenza virus infection; (2) NF-kappaB promoter activation was induced in bronchial epithelial cells upon influenza virus infection; and (3) overexpression by transient transfection of A20 attenuated NF-kappaB promoter activation in bronchial epithelial cells. These results indicate that A20 may function as a negative regulator of NF-kappaB-mediated lung inflammation and injury upon influenza virus infection, thereby protecting the host against inflammatory response to influenza virus infection.