Construction of micelles and hollow spheres via the self-assembly behavior of poly(styrene-alt-pHPMI) copolymers with poly(4-vinylpyridine) derivatives mediated by hydrogen bonding interactions.

This study describes the preparation of hydrogen bonding connected micelles, consisting of a poly(styrene-alt-(para-hydroxyphenylmaleimide)) [poly(S-alt-pHPMI)] core and a poly(4-vinylpyridine) (P4VP) derivative shell in a selective solvent. The aim was to modify hydrogen bonding interaction sites at the core/shell interface by synthesizing P4VP derivatives in three different sequences, namely, P4VP homopolymers, PS-co-P4VP random copolymers, and block copolymers. TEM images showed the successful self-assembly of poly(S-alt-pHPMI)/PS-co-P4VP inter-polymer complexes into spherical structures. To dissolve the core structures, 1,4-dibromobutane was used as a cross-linking agent to tighten the PS-co-P4VP shell. The morphologies, particle sizes, hydrogen bonding, cross-linking reaction, and core dissolution were confirmed by TEM, DLS, FTIR, and AFM analyses. Poly(S-alt-pHPMI)/PS41-r-P4VP59 hydrogen bonding connected micelles, cross-linked micelles, and hollow spheres were larger and more irregular than poly(S-alt-pHPMI)/P4VP inter-polymer complexes due to the random copolymer architecture and the decrease in intermolecular hydrogen bonds. However, poly(S-alt-pHPMI)/PS68-b-P4VP32 resulted in rod- or worm-like structures after core dissolution.

Intra- and Intermolecular Hydrogen Bonding in Miscible Blends of CO2/Epoxy Cyclohexene Copolymer with Poly(Vinyl Phenol).

In this study, we synthesized a poly(cyclohexene carbonate) (PCHC) through alternative ring-opening copolymerization of CO2 with cyclohexene oxide (CHO) mediated by a binary LZn2OAc2 catalyst at a mild temperature. A two-dimensional Fourier transform infrared (2D FTIR) spectroscopy indicated that strong intramolecular [C-H···O=C] hydrogen bonding (H-bonding) occurred in the PCHC copolymer, thereby weakening its intermolecular interactions and making it difficult to form miscible blends with other polymers. Nevertheless, blends of PCHC with poly(vinyl phenol) (PVPh), a strong hydrogen bond donor, were miscible because intermolecular H-bonding formed between the PCHC C=O units and the PVPh OH units, as evidenced through solid state NMR and one-dimensional and 2D FTIR spectroscopic analyses. Because the intermolecular H-bonding in the PCHC/PVPh binary blends were relatively weak, a negative deviation from linearity occurred in the glass transition temperatures (Tg). We measured a single proton spin-lattice relaxation time from solid state NMR spectra recorded in the rotating frame [T1ρ(H)], indicating full miscibility on the order of 2-3 nm; nevertheless, the relaxation time exhibited a positive deviation from linearity, indicating that the hydrogen bonding interactions were weak, and that the flexibility of the main chain was possibly responsible for the negative deviation in the values of Tg.

Meso/Microporous Carbons from Conjugated Hyper-Crosslinked Polymers Based on Tetraphenylethene for High-Performance CO2 Capture and Supercapacitor.

In this study, we successfully synthesized two types of meso/microporous carbon materials through the carbonization and potassium hydroxide (KOH) activation for two different kinds of hyper-crosslinked polymers of TPE-CPOP1 and TPE-CPOP2, which were synthesized by using Friedel-Crafts reaction of tetraphenylethene (TPE) monomer with or without cyanuric chloride in the presence of AlCl3 as a catalyst. The resultant porous carbon materials exhibited the high specific area (up to 1100 m2 g-1), total pore volume, good thermal stability, and amorphous character based on thermogravimetric (TGA), N2 adsoprtion/desorption, and powder X-ray diffraction (PXRD) analyses. The as-prepared TPE-CPOP1 after thermal treatment at 800 °C (TPE-CPOP1-800) displayed excellent CO2 uptake performance (1.74 mmol g-1 at 298 K and 3.19 mmol g-1 at 273 K). Furthermore, this material possesses a high specific capacitance of 453 F g-1 at 5 mV s-1 comparable to others porous carbon materials with excellent columbic efficiencies for 10,000 cycle at 20 A g-1.

Development of Quantitative Methylation-Specific Droplet Digital PCR (ddMSP) for Assessment of Natural Tregs.

Regulatory T cells (Tregs) suppress immune responses in vivo in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs are classified as CD4+CD25highCD127low FOXP3+ cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/µL to less than 1 copy/µL (R 2 = 0.99) even in the presence of non-selective gDNAs. CD4+CD25highCD127lowFOXP3+ human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion in vitro. These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.

Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles.

BACKGROUND: Human pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS: We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS: IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α. CONCLUSIONS: The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

Factors affecting Salmonella-based combination immunotherapy for prevention of type 1 diabetes in non-obese diabetic mice.

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFß, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFß or PPI+TGFß+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFß+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.

Synthesis of Sodium Complexes Supported with NNO-Tridentate Schiff Base Ligands and Their Applications in the Ring-Opening Polymerization of L-Lactide.

A series of sodium complexes bearing NNO-tridentate Schiff base ligands with an N-pendant arm were synthesized and used as catalysts for the ring-opening polymerization of L-lactide (L-LA). Electronic effects of ancillary ligands coordinated by sodium complexes substantially influence the catalysis, and ligands with electron-donating groups increase the catalytic activity of the sodium complexes for catalyzing L-LA polymerization. In particular, a sodium complex bearing a 4-methoxy group has the highest activity with conversion up to 95% within 30 s at 0 °C and a low polydispersity index of 1.13, whereas the 4-bromo group showed the poorest performance with regard to the catalytic rate of L-LA polymerization in the presence of benzyl alcohol (BnOH). (1)H NMR pulsed-gradient spin-echo diffusion experiments and single-crystal X-ray analyses showed that sodium complexes [L(H)Na(THF)]2 and [L(4-Cl)Na(THF)]2 were dinuclear species in both solution and the solid state. The kinetic results indicated a first-order dependence on each of [[L(4-Cl)Na]2], [l-LA], and [BnOH].

Reduced MIR130A is involved in primary immune thrombocytopenia via targeting TGFB1 and IL18.

MicroRNAs (miRNAs) play a vital role in the regulation of immunological functions and prevention of autoimmune disease. The abnormal expressions of several miRNAs in patients with the acquired autoimmune disease, immune thrombocytopenia (ITP), have been reported. However, the exact mechanism of miRNAs in the pathogenesis of ITP is currently not well understood. This study examined the miRNA expression profile of peripheral blood mononuclear cells (PBMCs) in ITP patients by miRNA array and TaqMan real-time polymerase chain reaction. MIR130A expression was found to be significantly decreased in PBMCs from patients with active chronic ITP compared with that of normal controls. Subsequently, dual-luciferase reporter gene analysis was used to validate that MIR130A targeted the transforming growth factor-beta1 (TGFB1) and interleukin 18 (IL18) genes. In addition, we also monitored the dynamic expression of MIR130A and its targeted genes pre- and post-treatment of ITP patients and determined that the expression of MIR130A and TGFB1 was up-regulated, whereas IL18 expression was down-regulated after effective treatment. In conclusion, this study suggests that reduced MIR130A is involved in ITP via targeting of TGFB1 and IL18 expression.

Foxp3+ Treg expanded from patients with established diabetes reduce Helios expression while retaining normal function compared to healthy individuals.

Foxp3(+) regulatory T cells (Treg) play a crucial role in regulating immune tolerance. The use of Treg to restore immune tolerance is considered an attractive novel approach to inhibit autoimmune disease, including type 1 diabetes (T1D), and to prevent rejection of organ transplants. In view of the goal of developing autologous Treg-based cell therapy for patients with long-term (>15 years) T1D, it will be necessary to expand a sufficient amount of functional Treg in vitro in order to study and compare Treg from T1D patients and healthy subjects. Our results have demonstrated that there is a comparable frequency of Treg in the peripheral blood lymphocytes (PBLs) of patients with long-term T1D relative to those in healthy subjects; however, Th1 cells, but not Th17 cells, were increased in the T1D patients. Further, more Treg in PBLs from T1D patients than from healthy subjects expressed the CD45RO(+) memory cell phenotype, suggesting they were antigen-experienced cells. After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity. Although there was no difference in Helios expression on Treg in PBLs, in vitro expansion led to fewer Helios-expressing Treg from T1D patients than healthy subjects. While more Th1-like Treg expressing IFN-γ or TNF-α were found in the PBLs of T1D patients than healthy controls, there was no such difference in the expanded Treg. Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8(+) effector T cells equally well. Our findings demonstrate that a large number of ex vivo expanded functional Treg can be obtained from long-term T1D patients, although fewer expanded Treg expressed a high level of Helios. Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.

PTPN22 -1123G > C polymorphism is associated with susceptibility to primary immune thrombocytopenia in Chinese population.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by autoantibody-mediated platelet destruction. Multiple factors have been implicated in ITP pathogenesis, including T-lymphocyte dysfunctions. The protein tyrosine phosphatase, non-receptor type 22 (PTPN22) gene encodes lymphoid-specific phosphatase (LYP), a critical negative regulator of T cell activation. Single nucleotide polymorphisms (SNPs) of PTPN22 have been broadly associated with susceptibilities to various autoimmune disorders. Here we conducted a case-control study investigating whether the PTPN22 -1123G > C SNP contributes to the risk of ITP in Chinese population. The study included 191 ITP cases and 216 ethnically matched normal controls. Genotyping of -1123G > C SNP was performed using a single-base extension (SBE) and mass spectrometry method. Allelic and genotypic frequencies were compared between the case-control groups by the chi-square test. We observed significant overrepresentation of -1123G allele (p = 0.034, odds ratio (OR) = 1.374, 95% confidence interval (CI) [1.024-1.843]) and GG genotype (P = 0.038, OR = 1.951, 95% CI [1.031-3.694]) in the patients compared with the controls. Stratified analysis by gender and age of disease onset revealed comparable observations in both male and adult ITP cohorts. These data suggest a moderate association of PTPN22 -1123G > C SNP with susceptibility to ITP. Together with previous reports, our finding provides further evidence for PTPN22 being a general autoimmunity gene.

Inhibition of S-phase kinase-associated protein 2 (Skp2) reprograms and converts diabetogenic T cells to Foxp3+ regulatory T cells.

Autoreactive pathogenic T cells (Tpaths) and regulatory T cells (Tregs) express a distinct gene profiles; however, the genes and associated genetic/signaling pathways responsible for the functional determination of Tpaths vs. Tregs remain unknown. Here we show that Skp2, an E3 ubiquitin ligase that affects cell cycle control and death, plays a critical role in the function of diabetogenic Tpaths and Tregs. Down-regulation of Skp2 in diabetogenic Tpaths converts them into Foxp3-expressing Tregs. The suppressive function of the Tpath-converted Tregs is dependent on increased production of TGF-ß/IL-10, and these Tregs are able to inhibit spontaneous diabetes in NOD mice. Like naturally arising Foxp3(+) nTregs, the converted Tregs are anergic cells with decreased proliferation and activation-induced cell death. Skp2 down-regulation leads to Tpath-Treg conversion due at least in part to up-regulation of several genes involved in cell cycle control and genes in the Foxo family. Down-regulation of the cyclin-dependent kinase inhibitor p27 alone significantly attenuates the effect of Skp2 on Tpaths and reduces the suppressive function of converted Tregs; its effect is further improved with concomitant down-regulation of p21, Foxo1, and Foxo3. In comparison, Skp2 overexpression does not change Tpath function, but significantly decreases Foxp3 expression and abrogates the suppressive function of nTregs. These findings support the critical role of Skp2 in functional specification of Tpaths and Tregs, and demonstrate an important molecular mechanism mediating Skp2 function in balancing immune tolerance during autoimmune disease development.

Decreased expression of MBD2 and MBD4 gene and genomic-wide hypomethylation in patients with primary immune thrombocytopenia.

Genome-wide hypomethylation has been confirmed in patients with primary immune thrombocytopenia (ITP). Proteins containing methylcytosine-binding domain (MBD) are involved in promoter methylation as transcriptional repressors and promote the gene-silencing effect of DNA methylation. The purpose of this study was to investigate the methylation pattern of T cells and the relationship between genomic methylation and the expression of MBD2 and MBD4 in ITP patients. DNA deoxymethylcytosine content of CD4(+) cells from peripheral blood mononuclear cells was measured by enzyme-linked immunoassay. Real-time polymerase chain reaction was performed to quantify the transcription levels of MBD2 and MBD4 in peripheral blood mononuclear cells and CD4(+) cells. DNA dmC content in CD4(+) cells of ITP patients was significantly lower than in the controls (p = 0.001). The mRNA level of MBD2 and MBD4 in CD4(+) cells of ITP patients was statistically lower than those of the controls (p < 0.001). Positive correlations between methylation indexes and expression of each enzyme were observed in the control group (r(2) = 0.718, p = 0.004 for MBD2; r(2) = 0.608, p = 0.015 for MBD4). However, inverse correlations were found in ITP patients (r(2) = 0.604, p = 0.008 for MBD2; r(2) = 0.498, p = 0.027 for MBD4). Our results indicate that decreased expression of MBD2 and MBD4 might involve in the pathogenesis of ITP.

Human umbilical cord mesenchymal stem cells ameliorate mice trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Mesenchymal stem cells (MSCs), which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. In this study we investigated whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could ameliorate colitis in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model. TNBS-treated colitic mice were infused with hUC-MSCs or vehicle control. The mice were sacrificed on day 1, 3, and 5 after infusion, and their clinical and pathological conditions were evaluated by body weight, colon length, and histological analysis. The expression levels of proinflammatory cytokine proteins in colon were examined by ELISA. The homing of hUC-MSCs was studied by live in vivo imaging and immunofluorescent microscopy. hUC-MSCs were found to migrate to the inflamed colon and effectively treated the colitic mice with improved clinical and pathological signs. The levels of IL-17 and IL-23 as well as IFN-γ and IL-6 were significantly lower in the colon tissues of the hUC-MSC-treated mice in comparison with the vehicle-treated mice. Coculture experiments showed that hUC-MSCs not only could inhibit IFN-γ expression but also significantly inhibit IL-17 production by lamina propria mononuclear cells (LPMCs) or splenocytes of the colitic mice or by those isolated from normal animals and stimulated with IL-23. Systemically infused hUC-MSCs could home to the inflamed colon and effectively ameliorate colitis. In addition to the known suppressive effects on Th1-type immune responses, hUC-MSC-mediated modulation of IL-23/IL-17 regulated inflammatory reactions also plays an important role in the amelioration of colitis.

Killer cell Ig-like receptor (KIR) 3DL1 down-regulation enhances inhibition of type 1 diabetes by autoantigen-specific regulatory T cells.

Both Foxp3(+) regulatory T cells (Tregs) and antigen-expanded Foxp3(-) Tregs play an important role in regulating immune responses as well as in preventing autoimmune diseases and graft rejection. Molecular mechanisms modulating Treg function remain largely unclear, however. We report here on the expression and function of an inhibitory killer cell Ig-like receptor, KIR3DL1, in a nonobese diabetic (NOD) mouse-derived autoantigen-specific Treg (2D2), which protects from type 1 diabetes (T1D) in adoptive transfer experiments. This gene is not expressed in T1D pathogenic T cells (Tpaths) or non-Tpath T cells. KIR genes are known to play an important role in regulating natural killer (NK) cell function, but their role in Tregs and T1D is unknown. To examine whether KIR3DL1 expression may modulate Treg function, we used shRNA to down-regulate KIR3DL1 expression (2D2-shKIR). We find that KIR3DL1 down-regulation enhances in vitro function, as measured by improved suppression of target cell proliferation. Antibody blockade of IL-10 but not IL-4 partially abrogated suppressive function. In vivo function is also improved. Adoptive transfer of 2D2-shKIR into 10-wk-old NOD mice prevented spontaneous insulitis and T1D, and the inhibitory effect was further improved if the cells were transferred earlier into 6-wk-old NOD mice. These studies indicate that KIR3DL1 expression may negatively regulate Treg function and suggest a previously undescribed target for improving immune tolerance for potential treatment of autoimmune diseases like T1D.

Association of cytotoxic T-lymphocyte antigen 4 gene polymorphisms with idiopathic thrombocytopenic purpura in a Chinese population.

Idiopathic thrombocytopenic purpura (ITP) is an acquired organ-specific autoimmune hemorrhagic disease with many immune dysfunctions. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T-lymphocyte surface molecule that can down modulate and terminate immune responses. Recently, several studies have confirmed that some polymorphisms of this gene can influence its expression level, therefore speculating that they might be associated with autoimmune diseases. In order to investigate the role of the CTLA-4 gene in ITP, we investigated -318 and CT60 polymorphisms of the CTLA-4 gene in 186 ITP patients and 162 healthy controls through polymerase chain reaction (PCR)-restriction fragment length polymorphism. No significant differences were revealed in genotypes and allele distributions between the patients with ITP and the controls in both sites. Similar results were observed between the two groups when stratified by first onset age and disease course including acute childhood, chronic childhood, acute adult, and chronic adult. In the conclusion, these two single-nucleotide polymorphisms in CTLA-4 are not associated with susceptibility to ITP in a Chinese population.

[Human coagulation factor IX gene therapy in murine hemophilia B by hydrodynamic delivery and site-specific genomic integration].

OBJECTIVE: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.

[Construction and identification of a novel adeno-integrase hybrid system for hemophilia B].

This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.

Factor VIII gene mutations profile in 148 Chinese hemophilia A subjects.

BACKGROUND: Hemophilia A (HA) is a common X-linked recessive bleeding disease caused by mutations in FVIII gene. The identification of mutation in HA subjects can lead to more accurate diagnosis and contribute to the genetic counseling/prenatal diagnosis. OBJECTIVES: Our objective is to identify the FVIII defects in 148 unrelated Chinese HA subjects and to analyze the potential consequence of novel mutations. METHODS: FVIII: C was assayed using one-stage method, and FVIII inhibitor was tested using Bethesda method. Intron 22 and 1 inversions were identified by PCR technique. Non-inversion mutations of FVIII gene were identified by direct sequencing. Novel mutations were further analyzed based on a B-domain deleted FVIII crystallographic structure and bioinformatics tools. RESULTS: The intron 22 and 1 inversions affected 57 and three severe subjects, respectively. Sixty-seven different mutations were identified in non-inversion subjects including 35 novel mutations that were not reported previously. Novel mutations include five nonsense mutations, 15 missense mutations, three insertions, eight small deletions, two splice site mutations and two partial gene deletions. The potential deleterious effects of these novel missense mutations include disruption of the protein core, impairment of inter-domain interaction and FVIII binding with other proteins. CONCLUSION: Similar to other races, intron 22 and one inversions are also recurrent mutation in severe HA subjects monitored in our centre. Sixty-seven mutations (52% novel reported) among 88 non-inversion subjects represent the high degree of heterogeneity of FVIII gene mutations causing HA. Characteristic of HA FVIII gene mutations extend our insight into structure-function relationship of the FVIII molecule.

[Combined deficiency of factors V and VIII caused by a novel compound heterozygous mutation of gene Lman1].

Combined deficiency of factor V and VIII (F5F8D) is a rare, autosomal recessive disorder caused by mutations of either lman1 or mcfd2. To identify mutations of these two genes in a Chinese F5F8D family, the samples of peripheral blood were collected from the proband and her parents. Coagulation tests were carried out, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg) and coagulate activity of FV, FVIII (FV:C, FVIII:C). The genomic DNA was extracted, then all the exons and intron/exon boundaries of these two genes were amplified by polymerase chain reaction (PCR). The products were finally analyzed by direct sequencing. The results showed that the proband's APTT, PT, TT, Fg, FV:C and FVIII:C were 82.2 sec, 19.6 sec, 18.6 sec, 2.9 g/L, 7.1% and 18.7% respectively, while those parameters of the parents were all within the normal range. Two pathogenic mutations were identified in lman1 gene of the proband: one was the heterozygous c.912_913insA in exon 8 resulting in a frameshift of p.Glu305fsX20; the other was the heterozygous c.1366C > T in exon 11 resulting in p.Arg456X. The proband's father and mother were heterozygous for c.1366C > T and c.912_913insA respectively. It is concluded that F5F8D of the proband is caused by a novel compound heterozygous mutation of the lman1 gene, which has never been reported.

Th1 (CXCL10) and Th2 (CCL2) chemokine expression in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired organ-specific autoimmune disease with a polarization of T(h)1. Both the T(h)1 chemokine CXCL10 and T(h)2 chemokine CCL2 have been studied in several autoimmune diseases, but the status of these chemokines in ITP is still unknown. The aims of this study were to determine the expression of CXCL10 and CCL2 and their receptors, CXCR3 and CCR2, in ITP patients, and to conduct a preliminary study of the pathogenic roles of these factors in ITP. Plasma samples from 49 patients with ITP and 24 normal healthy subjects were assayed for CXCL10 and CCL2 plasma concentration by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction was performed to determine the mRNA expression of these chemokines and their receptors in the PBMNC of 24 normal controls and 28 active ITP patients as well as splenocytes of nine ITP patients. The CXCL10 levels in the plasma samples from patients with active ITP were significantly higher than those from healthy controls (p = 0.007) and decreased to normal levels in patients with remission ITP. In contrast, CCL2 levels were similar in patients with active disease, patients in remission, and control subjects. PBMNC of patients with active disease expressed more CXCL10 mRNA (p = 0.031) but less CCR2 mRNA (p = 0.005). Lower peripheral platelet count correlated with higher CXCL10 levels and CXCL10/CCL2 ratios. Our study demonstrated that plasma levels of CXCL10 and CXC10/CCL2 ratio were higher in patients with active ITP than in healthy donors, and had an association with platelet counts of the patients. CXCL10 might be a pathogenic factor of this disorder.