[A clinical study of haploidentical hematopoietic stem cell transplantation in the treatment of pediatric patients with acquired severe aplastic anemia: single center experience].

Objective: To investigate the efficacy of haplotype hematopoietic stem cell transplantation in the treatment of acquired severe aplastic anemia (SAA) in children. Methods: The clinical characteristics of 59 pediatric patients with SAA, including 26 cases VSAA, 37males and 22 females, 47 cases typeⅠ and 12 cases typeⅡ, undrerwent haplo-HSCT in our hospital between December 1st, 2011 and December 1st, 2017 were retrospectively analyzed. Among 59 patients, 56 patients with a median age of 4.5 (1.2-14.8) years and median weight of 43 (12-80) kg underwent their first HSCT and 3 patients underwent their second HSCT. All patients received the following conditioning regimen: busulfan, cyclophosphamide, and rabbit ATG or Bu (-, CTX) , fludarabineand rabbit ATG. The prophylaxis of acute graft versus host disease (aGVHD) was cyclosporine (CsA) , MMF and methotrexate. All patients received bone marrow transfusion on day 01 and peripheral stem cell transfusion on day 02 from haploid donor. The median dose of donor mononuclear cell counts was 15.60 (7.74-21.04) ×108/kg of recipient weight and CD34+ cell counts was 4.86 (3.74-7.14) ×106/kg of recipient weight. Results: Neutrophils and platelets of all 59 children were implanted. The median implantation time of granulocytes and platelets were 13 (10-19) d, 19 (9-62) d, respectively. The incidence of grade Ⅰ-Ⅱ aGVHD was 45.76% (27 cases) and grade Ⅲ/Ⅳ 13.56% (8 cases) , The incidence of chronic GVHD was 8.47% (5 cases) , The incidences of CMV and EBV viremia were 59.32% (35 cases) and 28.81% (17 cases) , respectively. The median follow-up was 30 (8-80) months, 57 patients survived with disease free, 2 patients died of GVHD. Both of the estimated 5-year OS and DFS rates were (96.4±2.5) %. Conclusion: Haplo-HSCT could improve the outcomes of SAA children.

Dynamic Virus-Bacterium Interactions in a Porcine Precision-Cut Lung Slice Coinfection Model: Swine Influenza Virus Paves the Way for Streptococcus suis Infection in a Two-Step Process.

Swine influenza virus (SIV) and Streptococcus suis are common pathogens of the respiratory tract in pigs, with both being associated with pneumonia. The interactions of both pathogens and their contribution to copathogenesis are only poorly understood. In the present study, we established a porcine precision-cut lung slice (PCLS) coinfection model and analyzed the effects of a primary SIV infection on secondary infection by S. suis at different time points. We found that SIV promoted adherence, colonization, and invasion of S. suis in a two-step process. First, in the initial stages, these effects were dependent on bacterial encapsulation, as shown by selective adherence of encapsulated, but not unencapsulated, S. suis to SIV-infected cells. Second, at a later stage of infection, SIV promoted S. suis adherence and invasion of deeper tissues by damaging ciliated epithelial cells. This effect was seen with a highly virulent SIV subtype H3N2 strain but not with a low-virulence subtype H1N1 strain, and it was independent of the bacterial capsule, since an unencapsulated S. suis mutant behaved in a way similar to that of the encapsulated wild-type strain. In conclusion, the PCLS coinfection model established here revealed novel insights into the dynamic interactions between SIV and S. suis during infection of the respiratory tract. It showed that at least two different mechanisms contribute to the beneficial effects of SIV for S. suis, including capsule-mediated bacterial attachment to SIV-infected cells and capsule-independent effects involving virus-mediated damage of ciliated epithelial cells.

[Role of STAT1 on the regulation the human hsp90 alpha gene expression].

OBJECTIVE: To investigate the role of STAT1 on the regulation of human hsp90 alpha gene expression. METHODS: We first transfected Jurkat cells with the STAT1 expression construct and analyzed the expression of hsp90 alpha gene expression via quantitative RT-PCR system. Then we co-transfected the STAT1 expression construct and the CAT reporter gene driven by different length of 5' flanking sequence of hsp90 alpha gene. Western blot was carried out to detect the level of tyrosine phosphorylation in Jurkat cells with and without heat shock treatment (42 degrees C 1 h). By electrophoretic mobility shift assays (EMSA), we evaluated the DNA binding activity of a STAT1 responsible element located in the regulatory region of hsp90 alpha gene in Jurkat cell nuclear extracts. RESULTS: The mRNA level of hsp90 alpha gene in Jurkat cells was decreased when transfected by STAT1 expression construct, over-expression of STAT1 down-regulates the expression of CAT reporter gene with the present of a distal fragment from -1756 to -1463 within the 5' flanking regulatory sequences of hsp90 alpha gene. The tyrosine phosphorylation of STAT1 was detectable in Jurkat cells and increased when subjected to heat shock. Electrophoretic mobility shift assays (EMSA) results showed that STAT1 could bind to its responsible element in the regulatory region of hsp90 alpha gene. CONCLUSION: STAT1 could negatively regulate the human hsp90 alpha gene expression.

[Gene expression changes after heat shock by cDNA array analysis].

OBJECTIVE: To study gene expression changes of cells in response to heat stress, we isolated total RNA from Jurkat cells before and after heat shock treatment. METHODS: cDNA was labeled with alpha-32P-dATP during reverse transcription of RNA and then used as probe to cDNA expression array. Autoradiogram images were analyzed by ESTblot software. RESULTS: After heat shock, the expression level of some forty genes increased while that of sixteen genes decreased. In addition to the elevated expression of heat shock genes, expression of c-Jun and CLK-1 increased most remarkably. The genes with notably decreased expression were integrin alpha-4 and transforming growth factor beta. The elevated expression of c-Jun and hsp90 alpha was further confirmed by Northern blot analysis. CONCLUSIONS: The expression of some genes in Jurkat cells changes after heat shock treatment. Heat shock induces elevated expression of hsp, c-Jun, CLK-1 gene while decreases expression of integrin alpha-4 and transforming growth factor beta.

Hepatitis B genotypes and the response to interferon therapy.

BACKGROUND/AIMS: Possible pathogenic differences among hepatitis B virus (HBV) genotypes have been observed; however, the response to interferon therapy among HBV genotypes remains unknown. We therefore analyzed the efficacy of interferon alfa in the treatment of chronic hepatitis B patients with different HBV genotypes. METHODS: Fifty-eight genotype B or C infected chronic hepatitis B patients who had been treated with interferon alfa-2b were retrospectively studied. The response to interferon was defined as normalization of serum aminotransferase level, loss of hepatitis B e antigen and HBV DNA 48 weeks post-treatment. RESULTS: Baseline data of both groups of patients were comparable; however, genotype C patients had a higher serum aminotransferase level and a higher frequency of core promoter mutation. The response rate was 41% and 15% in genotype B and C patients, respectively (p=0.045). In those with higher serum aminotransferase levels, the response rate was 50% and 17%, respectively (p=0.025). Additionally, younger age and genotype B infection may predict a better response to interferon alfa. CONCLUSIONS: HBV genotype C, compared to genotype B, is associated with a higher frequency of core promoter mutation, and a lower response rate to interferon alfa therapy.

Regulation of human hsp90alpha gene expression.

Mammalian HSP90alpha and HSP90beta are encoded by two individual genes. On the basis of the upstream sequences of the human hsp90alpha gene, GenBank accession number U25822, we have constructed CAT reporter plasmids driven by individual fragments of the hsp90alpha gene. We found that (1) the proximal heat shock element complex located at -96/-60 enhances hsp90alpha promoter expression; (2) heat shock induction depends upon the coexistence of distal heat shock element at -1031/-1022 and the proximal heat shock element complex of the hsp90alpha gene; (3) unlike hsp90beta, downstream sequences of the transcription start site inhibit hsp90alpha expression. We conclude that the regulatory mechanisms for the expression of hsp90alpha and hsp90beta genes are different.

A different experience of hepatic adenoma in Taiwan with emphasis on clinical manifestations and ultrasonographic features.

BACKGROUND/AIMS: Hepatic adenoma is a rare benign hepatic tumor and it is difficult to differentiate it from other focal hepatic tumors. Ultrasonography has become the choice of methods to detect focal hepatic lesions. The study aims at analyzing ultrasonographic features of hepatic adenoma. METHODOLOGY: A total of 8 patients with pathologically proven hepatic adenoma were studied retrospectively during an 8-year period. The ultrasound scanners used were Toshiba SSA-100A, Toshiba SSA-240 and Aloka 630. The ultrasonographic features and clinical data were analyzed. RESULTS: There were 7 males and 1 female. The mean age was 50 years. Of the 8 cases, 2 symptomatic cases had a tumor larger than 10 cm. The remaining 6 cases were asymptomatic and had tumors smaller than 5 cm. The echogenicity was variable in these tumors. An irregular sonolucent was only noted in a 15 cm tumor and was histologically proven to be internal bleeding. All the tumors were well-defined, however, a hypoechoic rim was obvious only in the isoechoic and hyperechoic tumors. CONCLUSIONS: Ultrasonographic features of some hepatic adenomas are different from those of hepatocellular carcinomas and hemangiomas, although the differential diagnosis cannot be made in small hypoechoic tumors. When ultrasonography is used more widely, more asymptomatic patients with small-sized hepatic adenoma will be detected, even in male subjects. The concept about the pathogenesis of hepatic adenomas may be changed.

RAPD analysis of natural populations of Acanthopanax brachypus.

Random Amplified Polymorphic DNA (RAPD) analysis is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population. The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan An, Shanxi Province. Through more than 2,000 PCRs, deep-going RAPD analysis was carried out on DNA samples from 49 individuals. The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2%, 18.6% and 5.4%; the average genetic distances within population, 0.055, 0.036 and 0.008; the average genetic distances between populations (I-II), (I-III) and (II-III), 0.105, 0.096 and 0.060. The genetic diversity of A. brachypus within and between populations was found, for the first time, to be rather poor, thus revealing innate factors as the cause contributing to its endangered status. In addition, our work also provides basic materials for elucidating the underlying cause of its endangerment and for its protection biology.

Factors associated with orthostatic hypotension in the Chinese population in Taiwan.

Factors associated with orthostatic hypotension are heterogeneous, and some of the factors are interrelated and interdependent, which may confound their relationships to orthostatic hypotension. To investigate the factors that were most likely related to orthostatic hypotension, a study of community-dwelling persons (419 men and 309 women) was conducted. Blood pressures and heart rates were measured after the subjects had been recumbent for 5 min and upright for 1 min. A total of 119 persons (16.3%) experienced orthostatic hypotension. Univariate analysis showed that orthostatic hypotension was associated with the following variables: hypertension, diabetes mellitus, cerebrovascular disease, proteinuria, abnormal renal function, or medications use. Those patients with orthostatic hypotension were older in age and had a higher body mass index, seated blood pressure, plasma creatinine, hemoglobin A1c, fasting and 2-h postload glucose levels than those without orthostatic hypotension. Multivariate analysis revealed that diabetes mellitus, hypertension, and age were independently associated factors for orthostatic hypotension. The higher the level of plasma hemoglobin A1c (%) elevation, the higher the likelihood of orthostatic hypotension manifestation. Clinically, elderly persons or patients with hypertension or diabetes mellitus should receive regular monitoring of supine and upright blood pressure in order to detect orthostatic hypotension and prevent its complications.

[A novel conservative structure found in the carp mitochondrial tRNA(phe) gene].

A nucleotide sequence for the tRNA(phe) gene of Carp mitochondria was determined. Sequence data comparisons made among the whale, human, Xenopus laevis, bovine, mouse, chicken and carp, showed that a novel conservative structure was found in the D. stem (dihydrouridine stem), which was known had variant nucleotides in any other vertebrate mitochondrial tRNA and cytoplasmic tRNA genes. This conservative structures contains 13 bp. When we compared the front 7 bp of the conservative structure with the eukaryotic RNA Pol III recognitive A domain, we found these two kinds of different species had partly homologue. As the mitochondrial tRNA(phe) gene is located between the displacement loop and mitochondrial rRNA gene, we inferred that the novel conservative structure might have some extra interesting functions.

Detection of minimal residual disease in childhood acute lymphoblastic leukemia after termination of therapy.

Using IgH and TCR gamma gene rearrangements as gene markers, we detected minimal residual disease (MRD) by means of the polymerase chain reaction (PCR) and restriction analysis. Of 18 children with acute lymphoblastic leukemia (ALL), MRDs were detected in 9 patients after termination of therapy. All 18 patients had been followed for 1.5 to 102 months after detection. Three of the nine MRD-positive patients relapsed within 3 to 6 months; none of the nine MRD-negative patients relapsed. We suggest that MRD negativity at the end of therapy might be an important factor for long-term disease-free survival, because the negative cases had a very low risk of relapse. Because the outcome for MRD-positive cases is more difficult to evaluate, patients with MRD after termination of therapy should be monitored.

[Structure analysis of the mitochondrial tRNA(Cys) gene and the original region of light-strand replication from carp].

In this study, we have determined the nucleotide sequences of the cysteine tRNA gene and the original region of light-strand replication from carp (Cyprinus carpio L.) mitochondrion, which were further represented respectively in cloverleaf secondary structure and stem loop structure. By comparing the nucleotide sequences of 5 vertebrate tRNA(Cys) genes, it is found that carp mitochondrial tRNA(Cys) gene have many unusual structural features differing from those of cytoplasmic tRNA(Cys) gene. The carp mitochondrial original region of light-strand replication consists of 36 bases which include 11 base pairs in its stem and 14 bases in its loop. By making a comparison between the nucleotide sequences of the carp original region of light-strand replication and those of other 10 vertebrates, it is further found that stem sequence of carp is very conservative while loop sequence is very variable. This result indicates that stem structure may play an important role in the replication of light strand.

Structure of the mitochondrial URFA6L gene and tRNA(Lys) gene from CARP.

The carp mitochondrial URFA6L gene consists of 165 base pairs. The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene. Their nucleotide sequences exhibit 68% homology. The carp URFA6L gene encodes a protein of 54 amino acids. The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids (proline, tryptophan, leucine, isoleucine and tyrosine). A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase 8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function as ATPase8. The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in clover-leaf secondary structure. Similar to amphibian and mammalian mitochondrial tRNA(Lys) genes, the carp mitochondrial tRNA(Lys) gene also has some unusual structural features as compared with its cytoplasmic counterpart. A comparison between the nucleotide sequences of the tRNA(Lys) gene from 7 vertebrates showed that the most conservative portions are the anticodon loop, nucleotides 8 and 9, the variable loop, the anticodon stem and the aminoacyl stem. The least conservative portions are the D-loop and the T-loop. These structural features may show that the mitochondrial tRNA(Lys) has a different interaction with mitochondrial ribosome.

A method for simultaneous detection of the transcripts of different cellular genes.

In this paper, a method for preparing radioactive-labelled first-strand cDNA has been described and the major procedures and reaction conditions have been studied. Some advantages provided by this method for simultaneously detecting the expression of multiple cellular genes are discussed.

Construction and screening of Plasmodium falciparum cDNA library.

The HAINAN isolate of Plasmodium falciparum FCC1/HN was cultured in vitro in large quantities. The total parasite mRNA was purified and reverse transcribed into cDNA. The cDNA fragments were inserted into lambda gt11 to construct a P. falciparum FCC1/HN erythrocytic stage cDNA library. Inhibitory monoclonal antibodies (McAbs) M26-32, F6-C2, and F6-D3 were used to screen the cDNA library expressed in E. coli. A total of 27 positive clones were found to react with M26-32 alone and 34 clones with both M26-32 and F6-C2. These expressed proteins may be candidates for use in malaria vaccine.

Amino acid sequence of rhizopuspepsin isozyme pI 5.

The complete amino acid sequence of an aspartic protease from Rhizopus chinensis, rhizopuspepsin isozyme pI 5, has been determined. Partial sequences were first obtained from the isolated isozyme by a combination of chemical and proteolytic enzyme cleavages, peptide purifications, and Edman degradations. About one-half of the sequence was revealed by this approach. To complete the amino acid sequence, a cDNA library of R. chinensis in pBR322 was constructed. An oligonucleotide probe was synthesized based on the sequence Trp-Trp-Gly-Ile-Thr, and about 40 positive clones were identified by colony hybridization. A clone, 33E2, which had an insert size of about 1.1 kilobase pairs, was found to contain the entire coding region of rhizopuspepsin isozyme pI 5. The sequence of rhizopuspepsin contains 325 amino acid residues. The alignment of the rhizopuspepsin sequence against other aspartic proteases revealed expected homology, with the closest similarity to penicillopepsin which shares 39% identical residues. Porcine pepsin shares about 36% identical residues with rhizopuspepsin.