Targeted high-throughput sequencing technique for the molecular diagnosis of primary immunodeficiency disorders.

The aim of this study was to investigate the usefulness of targeted high-throughput sequencing (HTS) for the molecular diagnosis of primary immunodeficiency diseases (PID).A total of 56 clinically diagnosed or suspected PID patients were divided into 4 groups according to the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015 and their chief clinical presentations. Patients and their biological family members were examined by targeted HTS, which sequenced the exons and ±10 bp flanking introns of 171 PID-related genes panel. All significant variants were confirmed by PCR-Sanger sequencing. Pathogenicity of the variants was evaluated by using bioinformatics.A total of 117 variants in 73 genes were found in 56 patients. Accurate molecular diagnosis of PID was made in 13 (23.2%) patients, and 12 novel mutations were detected in these patients. Twenty-seven patients carried heterozygous variants that are probably pathogenic in ≥2 genes; 16 patients had only 1 missense variant, or had several variants but not >1 variant was deleterious as evaluated by bioinformatics. The meaning of the targeted HTS results of these patients remains to be studied.Targeted HTS can make a precise molecular diagnosis of PID and detect more novel pathogenic mutations. More and more variations with ambiguous significance are discovered and explanation of these variations is a challenge to the clinicians.

IP3R-Grp75-VDAC1-MCU calcium regulation axis antagonists protect podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropathy rat model.

BACKGROUND: The mechanism of podocyte apoptosis is not fully understood. In addition, the role of the inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (Grp75)/voltage-dependent anion channel 1 (VDAC1)/mitochondrial calcium uniporter (MCU) calcium regulation axis, which is located at sites of endoplasmic reticulum (ER) mitochondria coupling, in the mechanism of podocyte apoptosis is unclear. This study aimed to understand the roles of this axis in podocyte apoptosis and explore potential targets for podocyte protection. METHODS: The expression of IP3R, Grp75, VDAC1, and MCU and mitochondrial Ca2+ were analyzed during Adriamycin- or angiotensin II-induced apoptosis in cultured mouse podocytes. The interaction between IP3R, Grp75, and VDAC1 was investigated using co-immunoprecipitation experiments. The effects of IP3R, Grp75, and MCU agonists and antagonists on mitochondrial Ca2+ and apoptosis were investigated in cultured podocytes. The podocyte-protective effects of an MCU inhibitor were further investigated in rats with Adriamycin-induced nephropathy. RESULTS: Increased expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction among the IP3R-Grp75-VDAC1 complex, mitochondrial Ca2+ overload, and increased active caspase-3 levels were confirmed during Adriamycin- or angiotensin II-induced mouse podocyte apoptosis. Agonists of this axis facilitated mitochondrial Ca2+ overload and podocyte apoptosis, whereas specific antagonists against IP3R, Grp75, or MCU prevented mitochondrial Ca2+ overload and podocyte apoptosis. A specific MCU inhibitor prevented Adriamycin-induced proteinuria and podocyte foot process effacement in rats. CONCLUSIONS: This study identified a novel pathway in which the IP3R-Grp75-VDAC1-MCU calcium regulation axis mediated podocyte apoptosis by facilitating mitochondrial Ca2+ overload. Antagonists that inhibit Ca2+ transfer from ER to mitochondria protected mouse podocytes from apoptosis. An MCU inhibitor protected podocytes and decreased proteinuria in rats with Adriamycin-induced nephropathy. Therefore, antagonists to this pathway have promise as novel podocyte-protective drugs.

Decrease of galectin-3 in keratinocytes: A potential diagnostic marker and a critical contributor to the pathogenesis of psoriasis.

Psoriasis-specific proteins dysregulated in keratinocytes and involved in the pathophysiological process of psoriasis remains elusive. We report here that epidermal galectin-3 expression is significantly downregulated in lesional skin, but not in non-lesional skin in psoriasis patients, nor in a group of diseases known as psoriasiform dermatitis clinically and histologically similar to psoriasis. The deficiency of epidermal galectin-3 is sufficient to promote development of psoriatic lesions, as evidenced by more severe skin inflammation in galectin-3 knockout (gal3-/-) mice, compared to wild-type mice, after imiquimod treatment, and in skin from gal3-/- mice grafted onto wildtype mice. The development of psoriatic-like lesions is attributable to 1) the spontaneously tuning up of psoriasis signatures in keratinocytes through JNK pathway; and 2) neutrophil accumulation caused by the enhanced leukocyte-recruiting capacity associated with overexpression of S100A7-9 and CXCL-1, 8 in keratinocytes with impaired galectin-3 expression. Psoriasis-like skin inflammation is significantly improved in gal-3-/- mice both by inhibition of neutrophils accumulation with a selective CXCR2 antagonist of SB225002, and by intracutaneous injection of recombinant galectin-3. Overall, these findings offer promising galectin-3-related diagnostic and therapeutic resolutions of psoriasis.

[Clinical Features and Prognosis of 227 cases of Acute Myeloid Leukemia with Cross-lineage Antigen Expression].

OBJECTIVE: To analyse the clinical features and prognostic significance of cross-lineage antigen expression in patients with acute myeloid leukemia(AML) in order to establish individualized treatment for a better outcome and prognosis. METHODS: A total of 227 cases (exduding M3) were detected by flow cytometry for immune phenotype. The CD7(-)CD56(-)CD19(-) AML served as control. The clinical features, treatment response and prognosis of CD7(+) group, CD56(+) group and CD19(+) group were compared. RESULTS: The detection rate of CD56(+),CD7(+) and CD19(+) in AML was 15.9%, 25.1% and 11.0%, respectively. There were no differences between CD56(+) AML, CD7(+) AML, CD19(+) AML, and CD56(-)CD7(-)CD19(-) AML in the proportion of blast cells, white blood cell count, hemoglobin level, platelet count and MDS transformed AML rate. The CR after the first course chemotherapy and cumulative CR in CD56(+) AML patients were lower than those in the control group (20.0% vs 58.1%, P=0.0099; 73.3% vs 87.5%, P=0.04). The median time of CR in CD56(+) AML was longer than that in the control group (118 days vs 46 days, P=0.04). The PFS time and OS time of CD56(+) AML were shorter than those in the control group (245 days vs 580 days, P=0.037; 494 days vs 809 days, P=0.04). The CR after the first course chemotherapy and cumulative CR in CD19(+) AML patients were higher than those in the control group(75.0% vs 58.1%, P=0.46; 100% vs 87.5%, P=0.02). The median time of CR in CD19(+) AML was shorter than that in the control group (28 days vs 46 days, P=0.02). The PFS time and OS time of CD19(+) AML tended to be longer than those in the control group (P=0.13, P=0.07, respectively). The median PFS and OS were not reached at the time of last follow-up. The CR after the first course chemotherapy, cumulative CR and median time to CR in CD7(+) AML patients were not different from those in the control group (53.1% vs 58.1%, P=0.67; 87.1% vs 87.5%, P=0.44; 50 days vs 46 days, P=0.44). No differences of PFS and OS were observed between CD7(+) AML and the control. CONCLUSION: CD56(+) AML patients respond poorly to treatment, frequently relapse after complete remission and have a low survival rate. These patients need more intensive chemotherapy or in combination with other treatments. The interval of MRD detection should be shortened to find out relapse earlier. CD19(+) AML patients have a good treatment outcome and often accompanies with AML1/ETO fusion gene, which is known to be a good prognostic marker. Aberrant expression of CD7 on AML cells is not a poor prognostic factor in this study.

[Expression of CD56 and CD19 in Patients with Newly Diagnosed Multiple Myeloma and Their Relationship with Karyotypes and Prognosis].

OBJECTIVE: To study the relationship between surface markers of CD56 and CD19 and karyotypes and prognosis in multiple myeloma. METHODS: A total of 126 cases of newly diagnosed multiple myeloma in the first hospital of Peking university from 2011 to 2015 were enrolled in this study. Cytogenetic abnormalities and immunophenotypes were detected by using fluorescence in situ hybridization and flow cytometry respectively before chemotherapy. Bone marrow smear was used for detection of abnormal plasma cell infiltration. By combining with their basic data, the relationship between immunophenotypes, cytogenetics and prognosis of MM was analyzed. RESULTS: (1) The median of myeloma cells in the 126 patients was 0.24（0.01-0.97）; the median of myeloma cells in 116 patients who have immunophenotype datas was 0.25（0.01-0.97）； the median of myeloma cells in CD19 positive patients was 0.11（0.01-0.53）; the median of myeloma cells in CD19 negative patients was 0.26（0.01-0.97）. The median of myeloma cells in CD19 positive patients was much lower than that in CD19 negative patients（P=0.036）. (2)In 116 patients detected by the immunophenotype, the myeloma cells expressed CD19,CD20,CD56 and CD117. Compared with CD56 negative patients(45/116,38.79%),CD56 positive patients(71/116,61.21%) had a clearly favorable disease outcome（OS was 53.0 month vs 31.0 month,P=0.016; PFS was 37.5 months vs 18.4 months, P=0.036）. (3)CD19 positive patients was 16.38%（19/116）,CD19 negative patients was 83.62%（97/116）； CD19 positive MM and CD19 negative MM had no difference in OS and PFS. (4)CD117 positive rate in CD19 positive patients was 42.11%(8/19), the CD117 positive rate in CD19 negative patients was 18.57%(18/97), the CD19 expression positively correlated with CD117 expression. (5)FISH detection was done for 67 newly diagnosed MM patients, 8 patients showed normal karyotypes(11.94%), 59 patients had abnormal karyotypes(88.06%). The most common abnormal karyotypes were IgH rearragement which occurred in 47 patients(70.15%). Other abnormal karyotypes included 1q21+, del(13q14),del(13q14.3),del(17p13) . These abnormal karyotypes occurred in 37 patients（55.22%）,31 patients（46.27%）,33 patients（49.25%） and 13 patients（19.40%） respectively. In comparison with CD19 negative MM patients, the incidence rate of 1q21+ and del(13q14.3) was significantly lower in CD19 positive patients(1q21+:33.33% vs 61.54%,P=0.016; del(13q14.3): 33.33% vs 53.85%,P=0.043）. CONCLUSION: The prognosis of CD56 positive MM patients is better than that of CD56 negative MM patients, CD19 negative MM has more abnormal karyotypes and bone marrow infiltration,but they have no statistical prognostic differences.

Reduced AKT phosphorylation contributes to endoplasmic reticulum stress-mediated hippocampal neuronal apoptosis in rat recurrent febrile seizure.

AIMS: Febrile seizure (FS) is one of the most common types of seizures in childhood. Recurrent FS can result in hippocampus injury and thus impair learning capacity and memory, while the underlying molecular mechanisms are still elusive. Studies indicated that endoplasmic reticulum stress (ERS), involved in many diseases including some neurodegenerative diseases, can increase the expression of tribbles-related protein 3 (TRIB3), which thus inhibits the activity of AKT. The current study assessed whether ERS, TRIB3 and AKT signalling is involved in the hippocampus injury following recurrent FS. MAIN METHODS: Recurrent FS was induced in Sprague-Dawley (SD) rats by using a heated water-bath. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to assess hippocampus apoptosis, and electron microscopy was used to examine ultrastructural changes. Protein expression and localization of TRIB3, glucose-regulated protein 78(GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) as well as AKT were examined by using western blot and double immunofluorescence staining. Knockdown of TRIB3 was studied in primary cultured neurons treated with hyperthermia. KEY FINDINGS: As compared with control, apoptosis of hippocampus was significantly induced in FS group. Abundance of TRIB3, GRP78 and CHOP was remarkably elevated, while phosphor-AKT decreased significantly in hippocampus of rats with recurrent FS. Double immunofluorescence indicated that phosphor-AKT was not detected in cells with induction of TRIB3 in FS rats. Hyperthermia-treated cells showed up-regulates TRIB3 expression and that TRIB3 reduces AKT phosphorylation. SIGNIFICANCE: These results show that recurrent FS may induce injury of hippocampal cell by interfering with AKT activation through ERS-mediated up-regulation of TRIB3.

Comparison of Multiple Methods for Determination of FCGR3A/B Genomic Copy Numbers in HapMap Asian Populations with Two Public Databases.

Low FCGR3 copy numbers (CNs) has been associated with susceptibility to several systemic autoimmune diseases. However, inconsistent associations were reported and errors caused by shaky methods were suggested to be the major causes. In large scale case control association studies, robust copy number determination method is thus warranted, which was the main focus of the current study. In the present study, FCGR3 CNs of 90 HapMap Asians were firstly checked using four assays including paralog ratio test combined with restriction enzyme digest variant ratio (PRT-REDVR), real-time quantitative (qPCR) using TaqMan assay, real-time qPCR using SYBR Green dye and short tenden repeat (STR). To improve the comparison precision reproductively, the results were compared with those from recently released sequencing data from 1000 genomes project as well as whole-genome tiling BAC array data. The tendencies of inconsistent samples by these methods were also characterized. Refined in-home TaqMan qPCR assay showed the highest correlation with array-CGH results (r = 0.726, p < 0.001) and the highest concordant rate with 1000 genome sequencing data (FCGR3A 91.76%, FCGR3B 85.88%, and FCGR3 81.18%). For samples with copy number variations, comprehensive analysis of multiple methods was required in order to improve detection accuracy. All these method were prone to detect copy number to be higher than that from direct sequencing. All the four PCR based CN determination methods (qPCR using TaqMan probes or SYBR Green, PRT, STR) were prone to higher estimation errors and thus may lead to artificial associations in large-scale case-control association studies. But different to previous reports, we observed that properly refined TaqMan qPCR assay was not inferior to or even more accurate than PRT when using sequencing data as the reference.

[Mutational analysis of the methyl-CpG-binding protein 2 (MECP2) gene in male autism patients].

OBJECTIVE: To investigate mutations in the methyl-CpG-binding protein 2 (MECP2) gene in male autism patients by PCR, denaturing high-performance liquid chromatography (DHPLC) and sequencing to explore the role of mutations in MECP2 in autism patients. METHODS: We recruited DNA samples from 44 male autism patients who matched the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DMS-IV) standards. DHPLC was used to screen the mutations in MECP2 gene, and DNA sequencing was performed for the samples with positive DHPLC results. The family members were further investigated in the patients with missense mutations in MECP2 gene. RESULTS: Four cases were found to have mutations in MECP2 gene, including missense mutations of c.590C>T(T197M)in one case and c.602C>T(A201V)in one case, and synonymous mutations of c.1053C>G in one case and c.897C>T in one case. In addition, we found C>T variation in intron 3 at the +74 bp before exon 4, a SNP (rs2071569) usually detected in Chinese population. In the case with c.602C>T(A201V)mutation, his mother and maternal grandfather had the same mutation. His mother had normal phenotype, but his maternal grandfather had depressive disease. CONCLUSION: Mutations in MECP2 are present in male autism patients with relatively higher prevalence, suggesting that these mutations may play roles in the pathogenesis of autism.

[A1381T and -1793G/C polymorphisms of vWF gene impact the plasma vWF levels in Yugur, Tibetan and Han nationalities of China].

The aim of this study was to investigate the similarities and differences of A1381T (rs216311) and -1793G/C (rs7966230) single nucleotide polymorphisms (SNP) in Chinese Yugur, Tibetan, and Han nationalities and their influence on plasma vWF concentration in order to explore the sensitivity of these 3 nationalities to vWF-related diseases. Peripheral venous blood was obtained from 322 Yugur, 399 Tibetan, and 120 Han healthy people. The DNA were then extracted. vWF gene A1381T and -1793G/C polymorphisms were analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequenced when it was necessary. The vWF:Ag level in plasma was determined by ELISA. The results showed that the genotype distribution of vWF gene at both A1381T and -1793G/C loci in Yugur, Tibetan and Han nationalities was different with statistically significance (P < 0.05). GG genotype of A1381T locus accounted for 69.9% in Yugur nationality, which was much higher than 56.6% and 53.3% in Tibetan and Han nationalities respectively(P < 0.01); AA genotype of A1381T locus expressed a low level of vWF in plasma. For the -1793G/C locus, the proportion of CG genotype in Yugur was much higher than that in Han, CC genotype expressed a high level of vWF in plasma. The plasma vWF levels with different nationalities and the polymorphism of vWF gene were significantly different. It is concluded that the polymorphisms of vWF gene at both A1381T and -1793G/C loci in Yugur, Tibetan and Han are significantly different; the polymorphism of vWF gene influences the plasma vWF level; the plasma vWF levels in Yugur and Tibetan are significantly higher than that in Han, which may be associated with the living environment and habits.

[Effects of ICAM-1 gene K469E, K56M polymorphisms on plasma sICAM-1 expression levels in Chinese Yugur, Tibetan and Han nationalities].

This study was purposed to investigate the intercellular cell adhesion molecule-1 (ICAM-1) gene K469E (A/G) (rs5498) and K56M (A/T) (rs5491) single nucleotide polymorphisms (SNP) and soluble ICAM-1 (sICAM-1) levels in plasma in three Chinese populations of Yugur, Tibetan and Han nationalities, to analyze comparatively the genotypes and allele frequencies distribution in different ethnic groups, and to explore the effects of ICAM-1 K469E and K56M polymorphism and sICAM-1 levels in plasma. EDTA-anticoagulant venous blood from Yugur(327 cases), Tibetan (400 cases) and Han (126 cases) people was collected, the DNA was extracted by using whole blood genomic DNA extraction kit, DNA SNP were analyzed by PCR-RFLP, genotype was judged by gel scan imaging system after agarose gel electrophoresis, the gene sequence was determined and the distribution of ICAM-1 genotypes and allele frequencies were compared among different ethnic groups, besides, the group representativeness was tested via the Hardy-Weinberg genetic equilibrium. Finally, the human sICAM-1 plasma levels were detected by using human ICAM-1 ELISA kit. The results showed that DNA sequencing result was consistent with PCR-RFLP analysis. In Yugur, Tibetan and Han nationalities, the KK, KE and EE three genotypes at ICAM-1 K469E gene locus were detected, the genotype distribution was not statistically significantly different, while the K, E allele frequency distribution was statistically significantly different (P < 0.05). Both of genotype and allele frequency distribution between Yugur, Tibetan and Han nationalities were statistically significantly different (P < 0.05). In K56M site only KK, KM two genotypes were detected, but the MM genotype was not detected in the three ethnic groups; the difference of two genotypes and K, M allele frequencies between Yugur and Han population was statistically significantly different (P < 0.05). Among three ethnic groups, the sex ratio and age distribution of K469E, K56M genotypes and allele frequencies of ICAM-1 gene were not significantly different, and distribution was in accordance with Hardy-Weinberg genetic equilibrium (P > 0.05). The plasma sICAM-1 level at ICAM-1 K469E allele locus in K individuals [(253 ± 122), (185 ± 97) µg/L] was higher than that at non-K allele [(145 ± 110) µg/L, P < 0.01]; the plasma sICAM-1 level of ICAM-1 K56M sites with KK genotype [(253 ± 122) µg/L] was higher than that of the KM genotypes [(168 ± 103) µg/L, P < 0.01]. In Yugur and Tibetan groups, the plasma sICAM-1 levels [(224 ± 80), (214 ± 111) µg/L] were higher than that in the Han group [(175 ± 125)µg/L, P < 0.05]. Pairwise comparison indicated that the plasma sICAM-1 levels between Yugur and Han group were statistically significantly different (P < 0.01), that was significantly different between Tibetan and Han group (P < 0.05). It is concluded that in Yugur, Tibetan and Han population, the genotypes and gene frequencies of two amino acid sites K469E and K56M in ICAM-1 were KK/KE-type, KK-type and K allele, moreover, the ratio of them in Yugur and Tibetan group was higher than that in Han, while there is not significant difference in sex ratio and age distribution, therefore, ICAM-1 genotype and allele frequency distribution in this study had ethnic representativeness. ICAM-1 gene K469E and K56M polymorphisms were likely to affect the plasma sICAM-1 expression level. K469E gene K allele may be a genetic risk factor, while K56M gene M allele a may be genetic protective factor for some diseases.

[Screening of mitochondrial deoxyribonucleic acid 3271T > C, 8356T > C, 9176T > C/G and 13513G > A mutations in mitochondrial encephalomyopathies].

OBJECTIVE: To investigate the spectrum of mitochondrial DNA (deoxyribonucleic acid) 3271T > C, 8356T > C, 9176T > C/G and 13513G > A mutations in Chinese patients with mitochondrial encephalomyopathies. METHODS: Peripheral blood samples were collected from 500 mitochondrial encephalomyopathies patients clinically diagnosed as mitochondrial encephalomyopathy lactic acidosis & stroke-like episodes (MELAS), myoclonus epilepsy & ragged-red fibers (MERRF) or Leigh's syndrome from October 2005 to October 2009. The methods of PCR- polymerase chain reaction-restriction fragment length polymorphism (RFLP) and PCR-sequencing were performed to identify the mutations. RESULTS: No patients with the 3271T > C, 8356T > C, 9176T > C/G or 13513G > A mutations were identified. CONCLUSION: The mutations of 3271T > C, 8356T > C, 9176T > C/G and 13513G > A are rare causes of mitochondrial encephalomyopathies in Chinese patients.

[Role of cytokines and gene expression characteristics in cultured lymphocytes ex vivo for adoptive immunotherapy].

Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy. This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stimulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines. The experiment was divided into 4 groups. The levels of total T lymphocytes (CD3(+)), T helper lymphocytes (CD3(+)CD4(+)), cytotoxic T-lymphocyte (CD3(+)CD8(+)), memory T cells (CD3(+)CD8(+)CD45RO(+)), naive T cells (CD3(+)CD8(+)CD45RA(+)), Th2 cells (CD3(+)CD30(+)), B cells (CD19(+)), NK cells (CD56(+)), naive T regulatory cells (CD4(+)CD25(+)), precise T regulatory cells (CD4(+)CD25(+)FOXP3(+)) were detected by flow cytometry. The expression levels of house-keeping gene (mad1, pten), T helper cells transcriptional regulatory gene t-bet (Th1), gata3 (Th2), cytokine IFN-γ(Th1), IL-4(Th2) were detected by using RT-PCR. The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects. Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation. The expression of IFN-γ gene was significantly increased; the T helper differentiation-related gene t-bet, gata3 also increased evidently, while expression change of house-keeping gene mad1 and pten were not evident. Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation. It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines, and the different cytokines play different roles in cell differentiation.

Copy number variation of FCGR3A rather than FCGR3B and FCGR2B is associated with susceptibility to anti-GBM disease.

Anti-glomerular basement membrane antibody disease (anti-GBM disease) is a rare disorder characteristic of universally poor outcome. Fcgamma receptors (FcgammaRs) play important roles in anti-GBM disease based on evidence from animal models. Copy number variation (CNV) influences disease susceptibility. The FcgammaRs genes show CNV, and CNV of the FCGR3B gene is associated with glomerulonephritis in systemic lupus erythematosus and anti-neutrophil cytoplasmic antibody-associated small vasculitis. Here, we investigated CNV of three FCGR genes, including two (FCGR3A and FCGR3B) for activating FcgammaRs and one (FCGR2B) for inhibitory FcgammaR by duplex quantitative real-time PCR. Copy numbers were analyzed by Applied Biosystems CopyCaller Software v1.0. We first demonstrated the distribution of CNV of FCGR3A, FCGR3B and no CNV of FCGR2B in Chinese population (including 47 anti-GBM patients and 146 healthy controls). The frequency of CNV of FCGR3A was observed to be significantly higher than matched healthy controls (27.7 versus 12.3%, P = 0.013, odds ratio 1.21-6.10). Considering previous report about gene knock-out animal models and CNV effect of FCGR3A, we thus propose that CNV in members of FCGR family should have different roles in the pathogenesis of human anti-GBM disease.

[Effects of in vivo transfer of human chemokine-like factor 1 gene on cardiac function after acute myocardial infarction in rats].

OBJECTIVE: To study the effects of chemokine-like factor 1(CKLF1)-plasmid transfer on cardiac function in a rat acute myocardial infarction (AMI) model. METHODS: Thirty male SD rats were randomly devided into 3 groups. One hundred micrograms of CKLF1-plasmid, empty plasmid or saline were injected intramuscularly with in vivo electroporation, respectively. Rats were subjected to left coronary artery ligation on the 6th day after gene transfer. Ultrasonic cardiography and hemodynamics were conducted and evaluated on the 22nd day after gene transfer. Then, the animals were sacrificed for determination of percentage of myocardial infarcion. RESULTS: The left ventricular ejection fraction in CKLF1 group (67.02% +/- 12.24%) was significantly higher than that in the saline group (43.64% +/- 7.82%) and empty plasmid group (47.56% +/- 4.10%), P<0.05. Fractional shortening of left ventricle in CKLF1 group (33.83% +/- 10.15%) was higher than that in saline group (18.49% +/- 3.96%) and empty plasmid group (20.85% +/- 2.24%), P<0.05. The maximal velocity of left ventricular pressure ascensus was higher in CKLF1 group [(5 720.01 +/- 826.32) mmHg/s, 1 mmHg=0.133 kPa] than in saline group [(3 955.69 +/- 685.91) mmHg/s] and in empty plasmid group [(4 412.03 +/- 500.74) mmHg/s)], P<0.05. And the maximal velosity of left ventricular pressure descensus was higher in CKLF1 group [(4 636.23 +/- 407.17) mmHg/s] than in saline group [(2 984.82 +/- 615.24) mmHg/s] and in empty plasmid group [(2 963.87 +/- 419.36) mmHg/s], P<0.05. While the percentage of myocardial infarction in CKLF1 group (29.63% +/- 3.93%) was smaller than that in saline group (38.01% +/- 5.48%) and in empty plasmid group (37.50% +/- 6.33%), P<0.05. CONCLUSION: CKLF1 gene transfer can limit the mass of myocardial infarction and improve post-infarction cardiac function.

Role of autoantibodies against the linker subdomains of envoplakin and periplakin in the pathogenesis of paraneoplastic pemphigus.

BACKGROUND: The presence of autoantibodies against multiple epidermal proteins is an important feature in paraneoplastic pemphigus (PNP). Circulating anti-desmoglein 3 autoantibody, the major pathogenic autoantibody in pemphigus vulgaris (PV), has been proved pathogenic in PNP. Because of many clinical differences between PNP and PV, we speculate about the involvement of other autoantibodies in the pathogenesis of PNP. Envoplakin (EPL) and periplakin (PPL) are recognized by most PNP sera. Their linker subdomains are highly homologous and necessary for the association of intermediate filaments. METHODS: We characterized the autoantibodies against the linker subdomains of EPL and PPL in PNP patients' sera and their associated tumors by enzyme-linked immunosorbent assay (ELISA) and immunofluorence. We also applied the purified autoantibodies against EPL and PPL from PNP sera to cultured human epidermal keratinocytes (HEK), to evaluate the changes of cell-cell adhesion. RESULTS: Autoantibodies against EPL and PPL were detected in most PNP patients by ELISA, and the decrease of these autoantibodies after removal of the tumors was roughly comparable to the improvement of clinical symptoms. Cultured tumor cells from PNP patients secreted these autoantibodies. Specific immunoglobulin receptors for EPL and PPL were found on B lymphocytes in tumors from PNP. Furthermore, purified anti-EPL and anti-PPL autoantibodies from PNP sera were capable of dissociating cultured human epidermal keratinocytes. CONCLUSION: Autoantibodies against EPL and PPL may also be pathogenic in PNP.

[Two novel mutations of GJB1 gene associated with typical X-linked Charcot-Marie-Tooth disease].

OBJECTIVE: To analyze the relationship between phenotype and genotype and the role of immune cells in the pathogenesis of X-linked Charcot-Marie-Tooth disease (CMT1X). METHODS: The probands of the two families with X-linked dominant inherited peripheral neuropathy were evaluated clinically, electrophysiologically, pathologically and genetically. The available family members were genetic analyzed and the novel mutations were compared with other known ones. RESULTS: (1) In both families, affected members presented progressive weakness and wasting of distal extremities and it seems that males suffered more severely than affected females with onset in the first decade of their life. Proband of family 1 showed moderately elevated CSF protein and marked increase of IgG-syn in CSF.(2) Nerve conduction velocity (NCV) of the peripheral nerves was intermediately slow in both motor and sensory nerves exhibiting the features of demyelination. Brain-stem auditory evoked potentials (BAEPs) was abnormal in the proband of family 1: delayed I-III interpeak intervals were recorded but with normal III-V interpeak intervals. (3) Sural nerve biopsy in the probands of the two families showed a prominent distinguished loss of myelinated fibers and a few clusters of regenerating axons without conspicuous onion-bulb formations. Thinly myelinated fibers was prominent in family 2 but not in family 1. Immunohistochemical staining showed that there were positive CD68 cells in the endoneurial space and lamellar sheath. (4) By genetic testing, we identified two novel missense mutations of GJB1 gene, which resulted in Ile127Phe amino acid substitution in family 1(located in the intracellular loop of connexin 32) and Asp178Gly amino acid substitution in family 2 (located in the 2(nd) extracellular loop of CX32), respectively. Both mutations were highly conserved in low species and were predicted to be possibly damaging through Polyphen prediction tool. CONCLUSION: The two novel GJB1 gene mutations cause a spectrum of clinical manifestations of CMT1X in both families. However, the mutations site of CX32 alone cannot predict these phenotypic variations in CMT1X fully. The immune system may be involved in the pathogenesis of the disease.

[In vivo interleukin-10 gene transfer down-regulates myocardial matrix metalloproteinase and myocardial collagen expressions in rats with acute myocardial infarction].

OBJECTIVE: We investigated the in vivo effects of recombinant adenovirus-associated virus type-2 (AAV-2) mediated interleukin-10 (IL-10) gene transfer on the expression of matrix metalloproteinase (MMP)-2, 9, tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I and type III in a rat acute myocardial infarction model. METHOD: Male Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 6): sham operation group, MI/AAV2 group, and MI/AAV2-IL-10 group (10(10) vg/ml x 0.1 ml injection at peri-infarct regions immediately post MI). Five days later, the expressions of MMP-2 and MMP-9 were measured by RT-PCR, Western blot and zymography. The expression of TIMP-1 was measured by RT-PCR and Western blot. Collagen type I and type III were assessed by RT-PCR and immunohistochemical stain. RESULTS: The myocardial expressions of MMP-2, MMP-9 and collagen contents in MI/AAV2 group were significantly increased than those in sham operation group. Myocardial expressions of MMP-2, MMP-9 were significantly decreased and the expression of TIMP-1 significantly increased in the MI/AAV2-IL-10 group than those in MI/AAV2 group. Moreover, the expressions of collagen type I, collagen type III and the ratio of I/III collagen in border zones of infarcted myocardium were decreased by 47.6% (P < 0.01), 23.6% (P < 0.05), and 17.9% (P < 0.05) respectively, while the expression of TIMP-1 increased by 73.1%(P < 0.05) in MI/AAV2-IL-10 group compared to MI/AAV2 group. CONCLUSION: In vivo myocardial IL-10 transfer reduced myocardial MMP and collagen expression and increasing the TIMP expression.

[Expression of major histocompatibility complex class II antigens on lungs in rat with bleomycin-induced pulmonary fibrosis].

OBJECTIVE: To explore the expression levels of MHC II molecules and its regulator genes CIITA on bleomycin-induced pulmonary fibrosis in rats, and to investigate the underlying immunologic mechanisms of pulmonary fibrosis. METHODS: The rats were treated with either a single intratracheal bleomycin injection (fibrosis group) or a normal saline injection (control group). The pathologic changes of lung tissues stained with HE and Masson were observed, and the contents of hydroxyproline were detected on the 7th and 28th day respectively after bleomycin administration. The expression of MHC II molecules in the lung tissues was evaluated with immunohistochemistry techniques, and the percentage of MHC II+ cells was measured. The amounts of total CIITA and type I, III and IV CIITA mRNA of lung tissues were measured by real-time PCR using Taqman probe. RESULTS: (1)The percentage of MHC II+ cells in lung tissues increased significantly in fibrosis group compared with that of control group on the 7th day and the 28th day [(0.10 +/-0.03) vs (0.06+/-0.02), P < 0.05; (0.15+/-0.03) vs (0.06+/-0.01),P < 0.01, respectively]; In fibrosis group, the percentage on the 28th day was higher than that on the 7th day [(0.15+/-0.03) vs (0.10+/-0.03), P < 0.05]; (2) Compared with control group on the 7th day, total CIIA mRNA increased 170.4% [(2.89+/-1.07) vs (1.07+/-0.46), P < 0.05], type I CIIA increased 258.8% [(0.77+/-0.38) vs (0.21+/-0.09), P < 0.05], while type IV CIITA decreased 87.2% [(0.39+/-0.15) vs (3.01+/-0.79), P < 0.01]; On the 28th day, total CIITA mRNA increased 98.6% [(4.14+/-1.15) vs (2.08+/-0.76), P < 0.05], type I CIIA increased 137.1% [(0.79+/-0.34) vs (0.33+/-0.23), P < 0.05], type IV CIITA mRNA still decreased, but there was no significant difference [(2.98 +/-0.92) vs (3.95+/-0.93), P > 0.05]; In fibrosis group, type IV CIIA mRNA was 667.3% [(2.98+/-0.92) vs (0.39+/-0.15), P < 0.01] higher on the 28th day than that on the 7th day; Type III CIIA mRNA levels of both groups had no significant difference. CONCLUSION: MHC II/CIITA system of lung tissues was probably involved in the development of rat pulmonary fibrosis.

Endogenously generated sulfur dioxide and its vasorelaxant effect in rats.

AIM: The present study was designed to explore the endogenous production and localization of the sulfur dioxide (SO2)/aspartate aminotransferase pathway in vascular tissues of rats and to examine its vasorelaxant effect on isolated aortic rings,as well as the possible mechanisms. METHODS: The content of SO2 in the samples was determined by using high performance liquid chromatography with fluorescence detection. Aspartate aminotransferase activity and its gene expression were measured by an enzymatic method and quantitative RT-PCR, respectively. Aspartate aminotransferase mRNA location in aorta was detected by in situ hybridization. The vasorelaxant effect of SO2 on isolated aortic rings of the rats was investigated in vitro. L-type calcium channel blocker, nicardipine, and L-type calcium channel agonist, Bay K8644, were used to explore the mechanisms by which SO2 relaxed the aortic rings. RESULTS: Aorta had the highest SO2 content among the vascular tissues tested (P<0.01). The aortic aspartate aminotransferase mRNA located in endothelia and vascular smooth muscle cells beneath the endothelial layer.Furthermore, a physiological dose of the SO2 derivatives (Na2SO3/NaHSO3) relaxed isolated artery rings slightly, whereas higher doses (1-12 mmol/L) relaxed rings in a concentration-dependent manner. Pretreatment with nicardipine eliminated the vasorelaxant response of the norepinephrine-contracted rings to SO2 completely. Incubation with nicardipine or SO2 derivatives successfully prevented vasoconstriction induced by Bay K8644. CONCLUSION: Endogenous SO2 and its derivatives have a vasorelaxant function, the mechanisms of which might involve the inhibition of the L-type calcium channel.

[Identification of an ideal noninvasive method to detect A3243G gene mutation in MELAS syndrome].

OBJECTIVE: To identify a better non-invasive method to detect the carrier of mitochondrial A3243G mutation, a cause of mitochondrial encephalopathy-lactic acidosis-stroke like episode (MELAS) syndrome. METHODS: DNA was extracted from the peripheral blood, urine, hair follicle, and saliva of 25 MELAS syndrome patients carrying A3243G mutation and their mothers and other maternal relatives, 33 persons in number, and the muscle tissues from 5 patients obtained by biopsy. A3243G mutation was detected by PCR-RFLP method, and the A3243G mutation ratio was identified by measuring the density of each band and calculation with the software AlphaEase 5.0. RESULTS: A3243G mutations were detected in all tissues of the 25 MELAS patients. The A3243G mutation ratio in urine was 62% +/- 9%, significantly higher than that in the blood [(36% +/- 10%), t = -11.13, P < 0.01]. A3243G mutations were detected in at least one tissue of the 28 maternal relatives. The A3243G mutation rates in their urine samples was 33.0% (5.0% - 70.4%), significantly higher than that in their blood samples [8.0% (0 - 33.3%), z = -4.197, P < 0.01]. There was no significant difference in A3243G mutation ratio among the samples of hair follicle, saliva, and blood. CONCLUSION: The A3243G mutation ratio in urine is significantly higher than those in blood samples of the patients and their maternal relatives. A noninvasive method, A3243G mutation ratio analysis of urine is superior to that in blood.