Pulmonary hypertension- a novel phenotypic hypothesis of Kabuki syndrome: a case report and literature review.

BACKGROUND: Pediatric pulmonary hypertension (PH) is a serious and rare disease that is often derived from genetic mutations. Kabuki syndrome (KS) is a chromosomal abnormality disease that has its origin in the mutation of lysine methyltransferase 2D(KMT2D). Recent evidence has shown that KMT2D mutations are associated with pediatric pulmonary disorders. However, the relationship between the clinical courses of PH and the KMT2D mutation is reported in extremely few cases. Therefore, in this paper, a case was presented and previous literature was reviewed for better understanding of the correlation between pediatric PH and KMT2D mutations. CASE PRESENTATION: A 3-year-old girl was transferred to our center for severe cough, shortness of breath, fatigue and fever. Physical examination revealed facial deformities and growth retardation. Echocardiography showed a small atrial septal defect (ASD), and right heart catheterization indicated a significant increase in pulmonary vascular pressure and resistance. The genetic test suggested that she had a KMT2D gene mutation. The patient was finally diagnosed with KS. She was given targeted drugs to reduce pulmonary vascular pressure, but the effect was unsatisfactory. CONCLUSIONS: KS can be complicated with multiple organ malformations and dysfunction. With the progress of next generation sequencing, an increasing number of new phenotypes related to KMT2D mutations have been reported. A bold hypothesis is proposed in this article, that is, PH may be a new phenotype associated with KMT2D mutations. It is suggested that KS and PH should be differentiated from each other to avoid delayed diagnosis and treatment in clinical practice. There is no specific drug for KS treatment. The prognosis of children with inherited PH is usually poor, and lung transplantation may increase their survival rates.

SjHSP70, a recombinant Schistosoma japonicum heat shock protein 70, is immunostimulatory and induces protective immunity against cercarial challenge in mice.

High levels of protective immunity can be induced in different animals immunized with radiation-attenuated (RA) Schistosoma cercariae or schistosomula. However, the schistosome-derived molecules responsible for the strong protective effect elicited by RA schistosome larvae have not been identified or characterized. The 70-kDa heat shock proteins of schistosomes are considered major immunogens, and may play an important role in stimulating high levels of innate and adaptive immune responses in an RA schistosome vaccine model. Here, we demonstrate the immunobiological functions of Schistosoma japonicum heat shock protein 70 (SjHSP70) by investigating its expression profile in RA-schistosomula-derived cells, evaluating the protection induced by recombinant SjHSP70 (rSjHSP70) against cercarial challenge, and assaying the humoral and cellular immune responses to rSjHSP70 in BALB/c and C57BL/6 mice. The expression of SjHSP70 on the surfaces of cells from RA or normal schistosomula was determined with flow cytometry. Its expression was significantly higher on early RA schistosomula cells than on the cells from normal parasites. The protection afforded both BALB/c and C57BL/6 mice vaccinated with rSjHSP70 alone, rSj22.6 (a membrane-anchoring protein of S. japonicum) alone, or a combination of rSj22.6 and rSjHSP70 without adjuvant was evaluated. rSjHSP70 alone induced the highest protective effect against S. japonicum cercarial challenge, followed by the rSj22.6 plus rSjHSP70 combination and then rSj22.6 alone, in both mouse strains. Like ISA206 adjuvant, rSjHSP70 enhanced the protective efficacy induced by rSj22.6 in the C57BL/6 mouse strain. Antigen-specific IgG1 and IgG2a responses were detected with enzyme-linked immunosorbent assays in mice immunized with rSjHSP70 alone, rSj22.6 alone, or the rSj22.6 plus rSjHSP70 combination. Immunization with rSjHSP70 or the rSj22.6 plus rSjHSP70 combination induced mixed Th1/Th2-type antibody responses in BALB/c mice and a Th2-type antibody response in C57BL/6 mice. The profiles of cytokine production by splenic lymphocytes in both strains of mice immunized with the antigens described above were detected in vitro using a Cytometric Bead Array. The profiles of the proinflammatory cytokines interferon γ, tumor necrosis factor α, interleukin 6 (IL-6), and IL-17A and the regulatory cytokine IL-10 induced by the rSj22.6 plus rSjHSP70 combination were similar to those induced by rSj22.6 emulsified with the ISA206 adjuvant control. Like the ISA206 adjuvant, rSjHSP70 protein enhanced the proinflammatory and Th2-type or regulatory cytokine production induced by the rSj22.6 antigen. These results indicate that SjHSP70 is exposed on the surfaces of cells from RA schistosomula, and that rSjHSP70 protein is a promising protective antigen with a potential adjuvant function. Thus, SjHSP70 protein might play a key role in the protective immunity elicited by the RA schistosome vaccine.

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin.

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

Assessment of fibrosis during the development of fatty liver in rabbits using real-time shear-wave elastography.

Nonalcoholic and alcoholic rabbit models of fatty liver were established by feeding on high-fat diet and alcohol, respectively, and fatty liver stiffness at different pathological stages was assessed with real-time shear-wave elastography (SWE), so as to investigate the fibrosis process during the development of fatty liver. The fatty liver stiffness of rabbit in nonalcoholic and alcoholic groups was higher than that in the control group, and that in alcohol group was higher than that in the nonalcoholic group (P<0.01). The elasticity modulus of liver in fatty liver rabbits of nonalcoholic and alcoholic groups showed a positive correlation with progression of liver fibrosis (P<0.01). Real-time SWE, as a noninvasive diagnostic method, can objectively reflect the liver stiffness change and progression of liver fibrosis during the development of fatty liver.

Xanthatin, a novel potent inhibitor of VEGFR2 signaling, inhibits angiogenesis and tumor growth in breast cancer cells.

Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer treatment. In this study, we described a novel VEGFR2 inhibitor, xanthatin, which inhibits tumor angiogenesis and growth. The biochemical profiles of xanthatin were investigated using kinase assay, migration assay, tube formation, Matrigel plug assay, western blot, immunofluorescence and human tumor xenograft model. Xanthatin significantly inhibited growth, migration and tube formation of human umbilical vascular endothelial cell as well as inhibited vascular endothelial growth factor (VEGF)-stimulated angiogenesis. In addition, it inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream signaling regulator. Moreover, xanthatin directly inhibit proliferation of breast cancer cells MDA-MB-231. Oral administration of xanthatin could markedly inhibit human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that xanthatin inhibits angiogenesis and may be a promising anticancer drug candidate.

Interactions Between Targeted Ultrasound Contrast Agents With Anti-Human Interleukin 8 Monoclonal Antibody and Activated Endothelial Cells.

The study aimed to explore the role of interleukin 8 (IL-8) in atherosclerotic plaques and develop a new method for the evaluation of endothelial function by assessing the interactions between the injured endothelial cells and the targeted ultrasound agent that carried anti-human IL-8 monoclonal antibody. Anti-human IL-8 monoclonal antibodies were associated to the shells of SonoVue microbubbles by covalent conjugation technology. The specific interaction between the microbubbles and the normal or injured endothelial cells was observed using an inverted microscope. The microbubble adherence was quantified by calculating the ratio of the adherent microbubbles to endothelial cells. The results were compared with the control microbubbles. There were rare adherences of control microbubbles to the normal or injured endothelial cells, whereas the targeted microbubbles could adhere to endothelial cells well. Importantly, compared with the normal endothelial cells, a significantly higher number of targeted microbubbles bound to the injured endothelial cells. The ultrasound agents with anti-human IL-8 monoclonal antibody can specifically bind to the injured endothelial cell, which provides a new insight to the noninvasive detection of endothelial dysfunction using ultrasound imaging techniques.

[Expression and purification of recombinant N-terminal protein of SARS S1 subunit expressed in E. Coli].

OBJECTIVE: To express and purify the recombinant N-terminal protein of SARS virus S1 subunit and to study its role in SARS immune response. METHODS: The gene encoding N-terminal 334 amino acid residuals of SARS virus S1 subunit was cloned and expressed in E. Coli. After purification, the recombinant protein was identified by anti-SARS positive sera from recovered SARS patients. The sera from health donors, which were collected before the out-break of SARS, were used as negative control in the study. RESULTS: Sequencing analysis confirmed that the desired DNA sequence in recombinant plasmid was correct and had the same sequence of natural N-terminal of SARS virus S1 subunit. The molecular weight of recombinant fusion protein is about 64 000. The recombinant S1 protein could react with three antibody positive samples from recovered SARS patients, which showed specific bands at 64 000, but not with the control samples according to results of western blot. CONCLUSION: The recombinant N-terminal protein of SARS virus S1 subunit displays specific reaction with SARS antibody and may provide a good tool for further research of immune response to SARS virus.

[Preparation of the polypeptide antibody against human transcriptional factors TFDP1 and TFDP3].

AIM: To prepare the polyclonal antibody against human transcriptional factors DP3(TFDP3) and DP1(TFDP1) and identify their reactivity and specificity. METHODS: Two totally different peptides between TFDP1 and TFDP3 were chosen as antigens to inoculate rabbits. The proteins from HEK293 cells were transiently transfcted with eukaryotic expression vectors of TFDP1 and TFDP3. The reactivity and specificity of antibodies were detected by Western blot. RESULTS: The amino acid residues of TFDP3 17-26 and TFDP1 17-32 were selected and synthesized to produce polypeptide antibodies. Western blot analysis demonstrated that anti-TFDP3 antibody could specifically recognize TFDP3 protein, while anti-TFDP1 antibody could both recognize TFDP1 and bind TFDP3 protein. CONCLUSION: The polypeptide antibodies against TFDP3 and TFDP1 have been prepared. The anti-TFDP3 antibody has no cross-reaction with TFDP1.

Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F.

The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFDP3 is apparently distinct from TFDP1 in function. Although TFDP3 retained the capacity to bind to E2F proteins, the resulting heterodimers failed to interact with the E2F consensus sequence. In contrast to the stimulatory effect of TFDP1, TFDP3 inhibited E2F-mediated transcriptional activation. Consistent with this observation, we found that ectopic expression of TFDP3 impaired cell cycle progression from G(1) to S phase instead of facilitating such a transition as TFDP1 does. Sequence substitution analysis indicated that the DNA binding domain of TFDP3 was primarily responsible for the lack of DNA binding ability of E2F-TFDP3 heterodimers and the inhibition of E2F-mediated transcriptional activation. Fine mapping further revealed four amino acids in this region, which were critical for the functional conversion from activation by TFDP1 to suppression by TFDP3. In conclusion, these studies identify a new DP protein and a novel mechanism whereby E2F function is regulated.

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.