The Fungal Endophyte Epicoccum dendrobii as a Potential Biocontrol Agent Against Colletotrichum gloeosporioides.

Anthracnose caused by Colletotrichum gloeosporioides is one of most serious fungal diseases on Chinese fir (Cunninghamia lanceolata). Eight fungal endophytes were isolated from a young heathy branch of Chinese fir and screened against the pathogen in vitro. One isolate, designated as SMEL1 and subsequently identified as Epicoccum dendrobii based on morphological and phylogenetic analyses, suppressed mycelial growth of Colletotrichum gloeosporioides on dual-culture plates. Additionally, E. dendrobii metabolites significantly decreased the biomass of Colletotrichum gloeosporioides. E. dendrobii was able to enter the internal tissues of the host plant via stomatal cells. Metabolites of E. dendrobii significantly inhibited conidial germination and appressorium formation, which at least partly explained why the endophyte significantly inhibited lesion development caused by Colletotrichum gloeosporioides on various host plants. We further confirmed that some components with antifungal activity could be extracted from E. dendrobii using ethyl acetate as an organic solvent. To our knowledge, this is the first report of E. dendrobii as a potential biocontrol agent against a fungal phytopathogen.

A High-Quality Draft Genome Sequence of Colletotrichum gloeosporioides sensu stricto SMCG1#C, a Causal Agent of Anthracnose on Cunninghamia lanceolata in China.

Colletotrichum has a broad host range and causes major yield losses of crops. The fungus Colletotrichum gloeosporioides is associated with anthracnose on Chinese fir. In this study, we present a high-quality draft genome sequence of C. gloeosporioides sensu stricto SMCG1#C, providing a reference genomic data for further research on anthracnose of Chinese fir and other hosts.

Shoot Blight on Chinese Fir (Cunninghamia lanceolata) is Caused by Bipolaris oryzae.

Chinese fir (Cunninghamia lanceolata) is a significant timber species that has been broadly cultivated in southern China. A shoot blight disease on Chinese fir seedlings was discovered in Fujian, China and a fungus was then consistently associated with the symptoms. This fungus was determined to be causing this disease, among others by fulfilling Koch's postulates. Based on morphological characteristics and multilocus phylogenetic analyses with the sequences of the internal transcribed spacer, partial glyceraldehyde-3-phosphate dehydrogenase gene, partial translation elongation factor 1-α gene, and partial 28S large subunit ribosomal RNA gene, the fungus was identified as Bipolaris oryzae. These characteristics and phylogenetic analyses clearly support that this pathogen is different from B. sacchari, which was, until now, considered to be the causal agent of a similar blight on Chinese fir in Guangdong, China. The fungus was also shown to be strongly pathogenic to rice, one of the most susceptible hosts to B. oryzae. Crop rotation involving rice is often carried out with Chinese fir in southern China, a practice that most likely increases the risk of shoot blight on C. lanceolata. To our knowledge, shoot blight caused by B. oryzae is reported for the first time in a gymnosperm species.

[Real-time quantitative detection of E2A-PBX1 fusion gene in children with acute lymphoblastic leukemia and its clinical application in minimal residual disease monitoring].

OBJECTIVE: To establish a real-time reverse transcription-polymerase chain reaction (RT-PCR) for quantitative detection of E2A-PBX1 fusion gene mRNA in acute lymphoblastic leukemia (ALL) children and to explore its clinical significance in minimal residual disease monitoring and prognosis evaluation. METHODS: Real-time RT-PCR was used to quantitatively detect the mRNA expression of E2A-PBX1 gene in 11 newly diagnosed ALL patients at diagnosis (11 cases), complete remission (11 cases) and periods of relapse (3 cases). Ten children with normal bone marrow cell morphology and without hematopathy or tumor diseases were used as the control group. RESULTS: The median expression levels of E2A-PBX1 fusion gene in the ALL group at diagnosis and the relapse group were significantly higher than in the control and complete remission groups (P<0.01). Compared with E2A-PBX1 negative patients on day 33 during induction of remission, the recurrence rate increased and disease free survival rate at 3 year decreased significantly in E2A-PBX1 positive patients decreased (P<0.05). CONCLUSIONS: Measurement of E2A-PBX1 levels by real-time RT-PCR is useful for monitoing minimal residual disease, prediction of relapse and individual treatment. The expression level of E2A-PBX1 gene on day 33 during induction of remission can be used for prognosis evaluation.

Correction: Deng et al. Distinct Roles of Ena ATP Family Proteins in Sodium Accumulation, Invasive Growth, and Full Virulence in Colletotrichum gloeosporioides. J. Fungi 2023, 9, 566.

In the original publication [...].

Distinct Roles of Ena ATP Family Proteins in Sodium Accumulation, Invasive Growth, and Full Virulence in Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides, a significant fungal pathogen of crops and trees, causes large economic losses worldwide. However, its pathogenic mechanism remains totally unclear. In this study, four Ena ATPases (Exitus natru-type adenosine triphosphatases), homology of yeast Ena proteins, were identified in C. gloeosporioides. Gene deletion mutants of ΔCgena1, ΔCgena2, ΔCgena3, and ΔCgena4 were obtained through the method of gene replacement. First, a subcellular localization pattern indicated that CgEna1 and CgEna4 were localized in the plasma membrane, while the CgEna2 and CgEna3 were distributed in the endoparasitic reticulum. Next, it was found that CgEna1 and CgEna4 were required for sodium accumulation in C. gloeosporioides. CgEna3 was required for extracellular ion stress of sodium and potassium. CgEna1 and CgEna3 were involved in conidial germination, appressorium formation, invasive hyphal development, and full virulence. The mutant of ΔCgena4 was more sensitive to the conditions of high concentrations of ion and the alkaline. Together, these results indicated that CgEna ATPase proteins have distinct roles in sodium accumulation, stress resistance, and full virulence in C. gloeosporioides.

[Analysis of A Pedigree with Hereditary Coagulation Factor â « Deficiency Caused by Compound Heterozygous Mutations].

OBJECTIVE: To analysis clinical phenotype and potential genetic cause of a family affected with hereditary coagulation factor Ⅻ deficiency. METHODS: The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-Dimer (D-D), coagulation factor Ⅻ activity (FⅫ:C) and coagulation factor Ⅻ antigen (FⅫ:Ag) were determined for phenotype diagnosis of the proband and his family members(3 generations and 5 people). Targeted capture and whole exome sequencing were performed in peripheral blood sample of the proband. Possible disease-causing mutations of F12 gene were obtained and further confirmed by Sanger sequencing. The corresponding mutation sites of the family members were analyzed afterwards. The online bioinformatics software AutoPVS1 and Mutation Taster was used to predict the effects of mutation sites on protein function. RESULTS: The APTT of the proband was significantly prolonged, reaching 180.9s. FⅫ:C and FⅫ:Ag of the proband was significantly reduced to 0.8% and 4.17%, respectively. The results of whole exome sequencing displayed that there were compound heterozygous mutations in F12 gene of the proband, including the c.1261G＞T heterozygous nonsense mutation in exon 11 (causing p.Glu421*) and the c.251dupG heterozygous frameshift mutation in exon 4 (causing p.Trp85Metfs*53). Both mutations are loss of function mutations with very strong pathogenicity, leading to premature termination of the protein. AutoPVS1 and Mutation Taster software predicted both mutations as pathogenic mutations. The results of Sanger sequencing revealed that c.1261G＞T heterozygous mutation of the proband was inherited from his mother, for which his brother and his daughter were c.1261G＞T heterozygous carriers. Genotype-phenotype cosegregation was observed in this family. CONCLUSION: The c.1261G＞T heterozygous nonsense mutation in exon 11 and the c.251dupG heterozygous frameshift mutation in exon 4 of the F12 gene probably account for coagulation factor Ⅻ deficiency in this family. This study reports two novel pathogenic F12 mutations for the first time worldwide.

Involvement of Protein Kinase CgSat4 in Potassium Uptake, Cation Tolerance, and Full Virulence in Colletotrichum gloeosporioides.

The ascomycete Colletotrichum gloeosporioides is a causal agent of anthracnose on crops and trees and causes enormous economic losses in the world. Protein kinases have been implicated in the regulation of growth and development, and responses to extracellular stimuli. However, the mechanism of the protein kinases regulating phytopathogenic fungal-specific processes is largely unclear. In the study, a serine/threonine CgSat4 was identified in C. gloeosporioides. The CgSat4 was localized in the cytoplasm. Targeted gene deletion showed that CgSat4 was essential for vegetative growth, sporulation, and full virulence. CgSat4 is involved in K+ uptake by regulating the localization and expression of the potassium transporter CgTrk1. CgSat4 is required for the cation stress resistance by altering the phosphorylation of CgHog1. Our study provides insights into potassium acquisition and the pathogenesis of C. gloeosporioides.

Biotinidase deficiency characterized by skin and hair findings.

Biotinidase deficiency is a rare hereditary metabolic disease. Only a few cases have been reported in China, almost all of which have been in the pediatric population. We report a case of a girl with characteristic skin and hair findings with a negative family history, although her grandparents were consanguineous. The metabolites in the proband's blood and urine increased prominently, and the percentage of biotinase was 1.168%, much lower than normal. Genotyping identified two heterozygous mutations, which were C.1457T>A (p.L486Q) and C.1491dupT (p.L498Ffs*13) in the BTD gene. The diagnosis of biotinidase deficiency was established. No relevant reports about the missense mutation at the mutation site C.1457T>A (p.L486Q) of the BTD gene have been retrieved. Biotin replacement therapy was administered in the dose of 20 mg/d. The dermatitis subsided after 1 month, and the hair color was almost normal after 3 months. This reminds dermatologists to include biotinidase deficiency in their clinical differential when faced with children's intractable dermatitis, yellow hair, and alopecia.

Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction.

BACKGROUND: Patients carrying the HongKongαα (HKαα) allele and -α/ααα could be misdiagnosed as -α/αα by the current conventional thalassemia detection methods, leading to inaccurate genetic counseling and an incorrect prenatal diagnosis. This study was aimed to accurately analyze the genotypes of HKαα carriers and -α/ααα. METHODS: Samples were collected in our hospital from July 2017 to October 2019. Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB). Anti-4.2 multiplex-PCR was used to confirm carriers of the ααα duplication with -α deletion. Two-round nested PCR and multiplex ligation-dependent probe amplification (MLPA) were applied to accurately identify and confirm their genotypes. For data analysis, we used descriptive statistics and Fisher's exact tests. RESULTS: Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples. The results showed that α, ß, and αß compound thalassemia were identified in 1190 (46.78%), 1286 (50.55%), and 68 (2.67%) cases, respectively. A total of 227 samples from thalassemia patients were identified as -α/αα by Gap-PCR, and the genotypes of two samples were uncertain. There was a difference between Gap-PCR and combined groups (Gap-PCR combined with nested PCR and MLPA) in detecting HKαα (P < 0.05). Among the 229 patients, 20 patients were identified as HKαα carriers and one was identified as -α/ααα by two-round nested PCR and MLPA, including 15 patients with HKαα/αα, three with HKαα/αα and ß-thalassemia coinheritance, one with HKαα/--, one with HKαα/-α and ß-thalassemia coinheritance, and one with -α/ααα and ß-thalassemia coinheritance. CONCLUSIONS: ααα and HKαα genotypes of patients carrying -α need to be detected to reduce the misdiagnosis rate of patients carrying HKαα and -α3.7/ααα alleles. More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.

[Mutation analysis of the PAX6 gene in a family with congenital aniridia and cataract].

OBJECTIVE: To identify the mutation in the PAX6 gene in a family with congenital aniridia and cataract. METHODS: Total genomic DNA was extracted from peripheral blood leukocytes of 12 family members including three living affected members and 96 unrelated healthy controls. The coding exons 4-13 of the PAX6 gene with intronic flanking sequences were amplified by polymerase chain reaction (PCR). By comparing sequences of the affected members with that of normal individuals, the disease-causing mutation was detected by direct DNA sequencing. RESULTS: A PAX6 mutation was identified in the 3 patients, which did not exist in the unaffected members and unrelated healthy individuals. The nonsense mutation of C to T was detected at the nucleotide 1143, which converted the Arg codon (CGA) to a stop codon(TGA) (R261X) in exon 10. CONCLUSION: The mutation (R261X) detected in the present study is considered to result in the occurrence of congenital aniridia and cataract in the Chinese family.

[Polyclonal antibody preparation and characterization of mouse Rnf141].

OBJECTIVE: To obtain the Rnf141 polyclonal antibody and to study its function. METHODS: Rnf141 cDNA was obtained by PCR amplification and inserted into the expression vector pGEX-5X-3, which encoding the GST protein to generate a recombinant plasmid. The recombinant was transformed into E. coli BL21 (DE3). After inducing with 1 mmol/L IPTG at 25 degrees C, the GST-Rnf141 fusion protein was expressed at a high level as a soluble form. Purified fusion protein with Glutathione Sephorase 4B chromatography was applied to immune rabbits to prepare the polyclonal antibody. Western blot and immunohistochemistry were applied to confirm the specificity of the resulting antibody. RESULTS: pGEX-Rnf141 recombinant plasmid was constructed successfully and the resulting. GST-Rnf141 fusion protein could be expressed in E. coli BL21(DE3) at a relatively high level, standing for 46% of total bacteria protein. The titer of the antiserum was 1:128000 and the polyclonal antibody could recognize GST-Rnf141 fusion protein and Rnf141 protein in different tissues of mouse. The high expression of Rnf141 protein was observed in spleen and brain of mouse. CONCLUSION: Rnf141 polyclonal antibody was prepared successfully and this will provide material for further studies of Rnf141 expression and function.

Pleiotropic Roles of ChSat4 in Asexual Development, Cell Wall Integrity Maintenance, and Pathogenicity in Colletotrichum higginsianum.

Potassium has an important role to play in multiple cellular processes. In Saccharomyces cerevisiae, the serine/threonine (S/T) kinase Sat4/Hal4 is required for potassium accumulation, and thus, regulates the resistance to sodium salts and helps in the stabilization of other plasma membrane transporters. However, the functions of Sat4 in filamentous phytopathogenic fungi are largely unknown. In this study, ChSat4, the yeast Sat4p homolog in Colletotrichum higginsianum, has been identified. Target deletion of ChSAT4 resulted in defects in mycelial growth and sporulation. Intracellular K+ accumulation was significantly decreased in the ChSAT4 deletion mutant. Additionally, the ΔChsat4 mutant showed defects in cell wall integrity, hyperoxide stress response, and pathogenicity. Localization pattern analysis indicated ChSat4 was localized in the cytoplasm. Furthermore, ChSat4 showed high functional conservation with the homolog FgSat4 in Fusarium graminearum. Taken together, our data indicated that ChSat4 was important for intracellular K+ accumulation and infection morphogenesis in C. higginsianum.

[Relationship between podocyte molecule's distribution and expression, foot process morphology and proteinuria].

OBJECTIVE: To investigate the association between nephrin, podocin and alpha-actinin of the glomerular podocyte molecules, the morphometric change of podocyte foot process and the development of proteinuria. METHODS: Puromycin aminonucleoside (PAN) nephrosis was established. Immunofluorescence staining, image analysis and real time quantitative PCR were employed to study the distribution and quantitation of glomerular expression of nephrin, podocin and alpha-actinin. Morphometric methods were applied to evaluate the morphology change of podocyte foot processes under electron microscopy. RESULTS: (1) Before the onset of proteinuria, 2 days after PAN injection, the podocyte foot process became swollen nephrin and podocin staining were changed into discontinuous pattern accompanied by the decrease of podocin staining intensity. The foot process became more swollen on day 5,and podocin intensity continued to decrease. Meanwhile, nephrin decreased significantly both in protein intensity and at mRNA level. (2) When heavy proteinuria [(130.8+/-30.7) mg/d, P=0.02] occurred, complete effacement of podocyte foot processes was revealed; both podocin and nephrin staining intensity decreased dramatically(P<0.01), and no linear staining could be observed; nephrin and podocin mRNA gained back. (3) During the recovery of proteinuria, the foot process morphology recovered stepwise; both the intensity of nephrin and podocin increased. When proteinuria disappeared, the podocyte foot process reappeared; podocin immunofluorescence intensity recovered whereas nephrin did not; the intensity of alpha-actinin increased significantly and the distribution changed too. (4) Podocyte foot process volume density correlated negatively with nephrin(r(nephrin)=-0.78, P=0.000 1) and podocin immunofluorescence intensity(r(podocin)=-0.76, P=0.000 1), respectively. CONCLUSION: Before the onset of proteinuria, the first response of podocyte is the swollen foot process, the distribution change of nephrin and podocin and the decreased podocin intensity. There was close relationship between nephrin, podocin protein level and distribution pattern with the development of proteinuria and podocyte foot process effacement, whereas no major role was found for mRNA of nephrin and podocin. Our results suggest that proteinuria might occur after series of events of nephrin and podocin distribution change, their protein and mRNA expression level decrease, and morphology change of podocyte foot process.

[Retrospective clinical features and renal pathological analysis of 15 children with anti-neutrophil cytoplasmic antibody-associated vasculitis].

OBJECTIVE: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a disorder with poor prognosis. This study aimed to improve the diagnosis and treatment of ANCA associated vasculitis of children, to analyze the clinical features, pathological characteristics and the prognosis of children with ANCA-associated vasculitis. METHOD: Fifteen children with ANCA associated vasculitis who were hospitalized from 2003 to 2012 in our hospital were included. Their data of pre-diagnosis status, clinical manifestations, renal pathology, treatment and prognosis were reviewed retrospectively. RESULT: Of the 15 children, 11 were girls and 4 boys with a mean age of 10.7 years. Fourteen children were categorized as microscopic polyangitis. The time to diagnosis varied from 0.5 month to 40 months. Hematuria and proteinuria were revealed by urine analysis in all of them, only 6 children complained with gross hematuria or edema of oliguria. Decreased glomerular filtration rate was revealed in 13 children, 8 of whom had a creatinine clearance rate of less than 15 ml/(min·1.73 m(2)). Twelve children underwent renal biopsy, crescent formation was found in 11 children. Most of the crescents were cellular fibrous crescents or fibrous crescents. Six children were diagnosed as crescentic nephritis; the process of rapidly progressive nephritis was only observed in 2 children. Segmental glomerulosclerosis or global glomerulosclerosis were found in 10 children, 3 of them were diagnosed as sclerotic glomerulonephritis. Anemia and pulmonary injury were the most common extra renal manifestations. Other extra renal manifestations included rash, pain joint, gastrointestinal symptoms, abnormal findings of cardiac ultrasonography and headache. Eight children were treated with steroid combined with cyclophosphamide, 4 were treated with steroid and mycophenolate mofetil, 2 were treated with steroid, cyclophosphamide and mycophenolate mofetil, 3 children were treated with plasma exchange. Fourteen children were followed up for 0.5 month to 4 years. The renal function did not recover in children with creatinine clearance rate of less than 30 ml/(min·1.73 m(2)), who showed crescentic glomerulonephritis or sclerotic glomerulonephritis. The children who had creatinine clearance rate of more than 30 ml/(min·1.73 m(2))had better prognosis. CONCLUSION: More attention should be paid to ANCA-associated vasculitis among school age girls with anemia or pulmonary diseases. The renal damage was serious in children; however, the clinical manifestations were not obvious. Children with a creatinine clearance rate of less than 30 ml/(min·1.73 m(2)) had poor prognosis. Early accurate diagnosis is very important.

[Characteristics of repeated renal biopsy-proven primary focal segmental glomerulosclerosis in children].

OBJECTIVE: To analyze the characteristics of repeated renal biopsy-proven primary focal segmental glomerulosclerosis (PFSGS) in 8 children, and to reveal the relationship between clinical features and pathology, between the two times of renal biopsy pathology, and the indications for repeated renal biopsy. METHOD: The records of cases who ever experienced renal biopsy in this hospital were reviewed, of whom 8 cases of repeated renal biopsy-proven PFSGS were enrolled. The clinical manifestations, the reason why they had renal biopsy again, the difference in renal pathological findings, between the two biopsies and their therapeutic response. The classification of focal segmental glomerulosclerosis (FSGS) was based on the new criteria suggested by D'Agati in 2004. RESULT: Of the 8 cases, age of onset ranged from 1 to 12 years, all were diagnosed as nephrotic syndrome (NS), the age of first biopsy ranged from 1.1 to 15.0 years, and the follow-up period was 10 months to 14 years. The reason for repeated biopsy was poor therapeutic response, continuous heavy proteinuria, or the progressive renal dysfunction. Four cases had the both biopsies in this hospital, and the first renal pathology showed minimal change disease (MCD), mesangial proliferation, FSGS CELL type and FSGS GTL type. After the second biopsy, they were additionally treated with immunosuppressive agents or switched to another one, 2 cases with FSGS COLL type presented renal dysfunction or end stage renal disease (ESRD), 1 case who developed the disease at 1.4 years of age, presented renal dysfunction at 10 months follow-up. The remaining 5 cases acquired complete remission. CONCLUSION: FSGS is a clinicopathological syndrome, NS predominates clinically. It often indicates pathologic transformation when the patients show poor therapeutic response or continuous heavy proteinuria without remission. Mesangial proliferation can convert into FSGS, and the subtype of FSGS can shift. FSGS COLL type and onset at young age may suggest poor prognosis.

Calcium imaging in gentamicin ototoxicity: increased intracellular calcium relates to oxidative stress and late apoptosis.

OBJECTIVES: To estimate intracellular calcium changes in gentamicin (GM) ototoxicity using calcium imaging. To investigate GM-induced physiologic changes in auditory cells including cell viability, apoptosis, and oxidative stress. METHODS: Varying concentrations of GM were applied to the HEI-OC1 cochlear cell line. Calcium imaging tracked changes in intracellular calcium concentration during GM cytotoxicity. Cell viability and intracellular reactive oxygen species (ROS) levels also were measured. RESULTS: Little change in calcium levels occurred in HEI-OC1 cells exposed to less than 35 mM GM. However, calcium rose continuously in cells exposed to more than 60 mM GM. With administration of intermediate concentrations of 40 or 50 mM GM, calcium increased variably in different cells, returning to baseline in some cases, or rising continuously in others. Upon increase of GM concentration, intracellular calcium concentration and ROS were increased, and cell viability was decreased due to late apoptosis. CONCLUSION: This study shows that GM increased intracellular calcium, ROS, and late apoptosis of HEI-OC1 cells derived from cochlear tissue. Increase of intracellular calcium is related to GM-induced apoptosis and oxidative stress. Calcium imaging can be used to determine change of intracellular calcium concentrations and apoptosis in GM ototoxicity.

[Detection of WT1 gene in acute myeloid leukemia children by real-time fluorescent quantitative RT-PCR and its clinical significance].

Objective of this study was to establish a SYBR Green Ireal-time reverse transcription-polymerase chain reaction (RT-PCR) for quantitative detection of WT1 gene mRNA in children with acute myeloid leukemia (AML) and investigate its clinical significance. SYBR Green Ireal-time RT-PCR was used to quantitatively detect the mRNA expression of WT1 gene in 30 newly diagnosed AML patients, 12 cases of remission (30), 18 relapsed patients and 30 cases of normal bone marrow cell morphology, and dynamically to detect the expression of WT1 gene in 20 newly diagnosed AML children. ABL served as internal reference gene, and the 2(-ΔΔct) method was used to calculate the relative expression. The results showed that (1) the expression of WT1 gene in newly diagnosed AML children was higher than that of the normal controls and the patients with remission (p < 0.001); there were no significant difference of WT1 gene expression between AML patients with remission and normal controls (p > 0.05), which were same as in relapsed patients and newly diagnosed patients (p > 0.05); (2) WT1 gene in 20 newly diagnosed AML children highly expressed before the children were initially treated, decreased when they were complete remission, then expression increased again when their AML relapsed. The WT1 gene expression level began to rise in 5 cases before clinical relapse at 5 - 7 months; (3) the complete remission rate (CR) and 3 year overall survival (OS) did not show significant difference between the WT1-positive group and negative group when dynamically monitoring WT1 gene expression of 20 newly diagnosed children with AML. 3-year OS of WT1-positive group at the 22 - 30 days after initial treatment was significantly lower than that of the negative group (p < 0.05). It is concluded that SYBR Green Ireal-time RT-PCR is a rapid, efficient, sensitive and specific method. WT1 gene in AML childhood plays a role of cancer-promoting. The change of WT1 gene expression level contributes to evaluate the therapeutic efficacy, detect the minimal residual diseases and analyze the prognosis.

[Clinicopathological study of 212 children with primary focal segmental glomerular sclerosis].

OBJECTIVE: To evaluate the correlation between clinico-pathological features and outcome of children with primary focal segmental glomerular sclerosis (FSGS). METHOD: A total of 212 pediatric patients with D'Agati (2004) primary FSGS were included in this study between 1997 and 2008. According to FSGS histologic classification criteria, 5 pathologic variants were recognized: collapsing (COLL), cellular (CELL), glomerular tip lesion (GTL), perihilar, and not otherwise specified (NOS). Retrospective analysis of the therapeutic response, the relationship between the clinical efficacy and pathology and the outcome of the patients was made. RESULTS: Of the 212 patients, 178 (83.9%) had nephritic syndrome (NS), 97 (45.8%) had simple NS, 81 (38.2%) had nephritis-type NS, GTL variants were mostly appeared to be nephritic syndrome (n = 28) and COLL variants were the fewest (n = 11). The difference between the two variants had statistical significance (P < 0.05). Fourteen cases (6.6%) had nephrotic proteinuria, 20 cases (9.4%) had proteinuria with micro-hematuria. According to histologic classification, NOS (n = 86, 40.6%) was the most common type; perihilar type was seen in 25 cases (11.8%); CELL was seen in 58 cases (27.4%), COLL in 12 cases (5.6%), GTL in 31 cases (14.6%). Chronic tubular injury was present in most cases. CEL variants were mostly found in the early infancy. GTL and NOS variants initially appeared to be responsive to steroids, but subsequently became resistant or frequently recurrent; CELL and COLL appeared to be primarily steroid resistant, GTL and COLL variants had statistically significant differences (P < 0.05). The patients were followed-up for 5 months to 10 years. A response to therapy was observed in 50%, COLL FSGS had the highest rate of ESRD; 2 years renal survival rates were 67%, 3 years were 41%. CONCLUSIONS: FSGS is defined as a clinicopathologic syndrome manifesting proteinuria and focal and segmental glomerular sclerosis with foot process effacement. The location of the sclerosis within the glomeruli proved to have prognostic significance. Collapsing glomerulopathy is the most aggressive variant of FSGS. Compared with other variants, GTL variant may be the best type. Different histologic variants of FSGS have substantial differences in clinical features at the time of biopsy diagnosis and substantial differences in renal outcomes. Prolonged treatment of FSGS-NS with corticosteroids and immune suppressive agents may have some effects in achieving sustained remission and improve prognosis in children.

[Clinical and pathological characteristics of children with dense deposit disease].

OBJECTIVE: To analysis the clinical and pathological characteristics of children with dense deposit disease (DDD). METHODS: 12 Children diagnosed as DDD by electron microscope were enrolled in this study. The clinical and pathological data were analyzed. RESULTS: Of the 12 cases, 7 were males and 5 females, mean age 9.1 +/- 3.9 (5-13) years at onset, the duration from onset to renal biopsy was 1 month to 5 years and the follow-up period was 1-9 years. All cases had heavy proteinuria >50 mg/(kg x d), and persistent microscopic hematuria with recurrent gross hematuria during the course. Seven cases had hypertension (> or = 140/100 mm Hg, 1 mm Hg =0. 133 kPa), 5 cases had transient or recurrent abnormal renal function, and mild to severe anemia were observed in 8 cases respectively. All the cases had lower serum C3 (0.15-0.55 g/L). Clinically, 10 cases were diagnosed as nephritic syndrome (one case had partial lipodystrophy at the same time), and 2 cases were diagnosed as acute nephritic syndrome. Immunofluorescence study showed intense deposition of C3 along GBM, TBM and the wall of Bowman's capsule in a ribbon-like pattern and in the mesangial regions as coarse granules in all the cases. Under light microscopy, 9 cases showed the feature of membrane proliferative glomerulonephritis (MPGN), 1 case with focal segmental glomerulosclerosis (FSGS), 1 case with endocapillary proliferative glomerulonephritis (EnPGN) and 1 case with proliferative sclerosis (PSGN). Crescents were seen in 3 cases. Under electron microscopy, ribbon-like or linear electron-dense intramembranous deposits were identified in the lamina dense of GBM, and often along TBM and the wall of Bowman's capsule. All patients showed steroid resistance. After methylprednisone treatment, some patients showed transient remission. During the follow- up stage of 1-9 years, 3 cases showed normal urinalysis, 5 cases showed partial remission, 2 cases progressed to end stage renal disease (ESRD) and 2 cases were lost. CONCLUSION: DDD is an in dependently rare disease with pathological-clinical varieties. Children with DDD presented with persistently lower C3, heavy proteinuria, recurrent gross hematuria and anemia. The characteristic immunopathologic finding is intense deposition of C3 along the GBM. Under electron microscopy, ribbon-like or linear electron-dense deposits in the lamina dense of the GBM, TBM and the wall of Bowman's capsule. Electron microscopic examination to demonstrate the intramembranous dense deposits is definitive diagnosis, regardless of the finding of light microscopy. All of them showed steroid resistant. Patients with steroid and CTX treatment showed some clinical improvement of their urinalysis.