[Inhibitory Effect of Cinobufotalin on Macrophage Inflammatory Factor Storm and Its Mechanism].

OBJECTIVE: To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism. METHODS: THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot. RESULTS: 1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1ß, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway. CONCLUSION: Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.

[Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice].

OBJECTIVE: The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. METHODS: Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. RESULTS: On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. CONCLUSION: Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

[Curative Efficacy and Survival Analysis of Low-Intensity Traditional Chemotherapy Regimen in the Elderly Patients with Acute Myeloid Leukemia (non-M3)].

OBJECTIVE: To investigate the short-term and long-term curative efficacy of low-intensity traditional chemotherapy regimen for elderly patients with acute myeloid leukemia (AML, non-M3) and related adverse reactions, in order to explore whether low-intensity traditional chemotherapy regimen still has application value in the treatment of elderly AML patients today. METHODS: The clinical characteristics, treatment response and prognosis of 67 elderly patients with AML (non-M3) admitted to our hospital from June 2008 to December 2018 were retrospectively analyzed. All patients received low-intensity conventional chemotherapy (i.e. lower standard dose, and without new drugs listed in China since the 21st century), including DA, HA, CAG, etc. The CR rate, median survival time and 5-year cumulative survival rate of patients were evaluated, and the related indexes were compared with the data reported in domestic and foreign literatures at the same time. RESULTS: The CR rate was 55.2% (37/67), the median survival time was 13.7 months, and the 5-year cumulative survival rate was (24.4±6.3)% in patients received low-intensity tradional chemotherapeutic regimens. The CR rates of high-risk group and non-high-risk group were 38.7% (12/31) and 69.4% (25/36), respectively; the median survival time of high-risk group and non-high-risk group was 8.9 months and 25.2 months respectively; the 5-year cumulative survival rate of high-risk group was (10.2±6.6)% and that of non-high-risk group was (36.0± 9.4)%. Compared with the data reported in the literature at the same time, the data obtained from the low-intensity traditional chemotherapy regimen for the elderly AML did not have an obvious disadvantage, morever had relatively short bone marrow suppression time, low induction early mortality rate and low incidence of severe infection. CONCLUSION: At present, the low-intensity traditional chemotherapy regimen still has good curative effect and survival advantages for elderly AML patients, especially for non-high-risk patients. The adverse reactions are controllable, and the physical and economic conditions of the vast majority of patients can bear the treatment regimen.

Tumor-released autophagosomes induces CD4+ T cell-mediated immunosuppression via a TLR2-IL-6 cascade.

BACKGROUND: CD4+ T cells are critical effectors of anti-tumor immunity, but how tumor cells influence CD4+ T cell effector function is not fully understood. Tumor cell-released autophagosomes (TRAPs) are being recognized as critical modulators of host anti-tumor immunity during tumor progression. Here, we explored the mechanistic aspects of TRAPs in the modulation of CD4+ T cells in the tumor microenvironment. METHODS: TRAPs isolated from tumor cell lines and pleural effusions or ascites of cancer patients were incubated with CD4+ T cells to examine the function and mechanism of TRAPs in CD4+ T cell differentiation and function. TRAPs-elicited CD4+ T cells were tested for their suppression of effector T cell function, induction of regulatory B cells, and promotion of tumorigenesis and metastasis in a mouse model. RESULTS: Heat shock protein 90α (HSP90α) on the surface of TRAPs from malignant effusions of cancer patients and tumor cell lines stimulated CD4+ T cell production of IL-6 via a TLR2-MyD88-NF-κB signal cascade. TRAPs-induced autocrine IL-6 further promoted CD4+ T cells secretion of IL-10 and IL-21 via STAT3. Notably, TRAPs-elicited CD4+ T cells inhibited CD4+ and CD8+ effector T cell function in an IL-6- and IL-10-dependent manner and induced IL-10-producing regulatory B cells (Bregs) via IL-6, IL-10 and IL-21, thereby promoting tumor growth and metastasis. Consistently, inhibition of tumor autophagosome formation or IL-6 secretion by CD4+ T cells markedly retarded tumor growth. Furthermore, B cell or CD4+ T cell depletion impeded tumor growth by increasing effector T cell function. CONCLUSIONS: HSP90α on the surface of TRAPs programs the immunosuppressive functions of CD4+ T cells to promote tumor growth and metastasis. TRAPs or their membrane-bound HSP90α represent important therapeutic targets to reverse cancer-associated immunosuppression and improve immunotherapy.

BF211, a derivative of bufalin, enhances the cytocidal effects in multiple myeloma cells by inhibiting the IL-6/JAK2/STAT3 pathway.

Despite remarkable advances in multiple myeloma (MM) therapy, this condition remains incurable. BF211 is an active compound derived from bufalin, which is isolated from the Traditional Chinese Medicine, Chansu. In this study, we explored the cytotoxicity of BF211 in 20 tumor cell lines and discovered that the MM cell lines, ARP-1 and CAG, exhibited greater sensitivity to BF211. Compared with bufalin, BF211 induced a greater apoptotic effect and lower acute toxicity at nanomolar concentration. The IL-6/JAK2/STAT3 signaling pathway is essential to the progression and development of MM. We showed that exogenous IL-6 promoted MM cell proliferation in a dose-dependent manner and this effect was blocked by BF211. Furthermore, BF211 suppressed the phosphorylation of JAK2 and STAT3 both in vivo and in vitro. In a mouse MM xenograft model, BF211 significantly inhibited tumor growth and did not affect body weight. In conclusion, the anti-MM activity of BF211 is mediated mainly by suppressing the IL-6/JAK2/STAT3 signaling pathway. Thus, we suggest that BF211 warrants further investigation in clinical trials in MM.

Tumor cell-released autophagosomes (TRAP) enhance apoptosis and immunosuppressive functions of neutrophils.

Our previous studies have confirmed that tumor cell-released autophagosomes (TRAP) could induce the differentiation of B cells into IL-10+ regulatory B cells (Bregs) with suppressive activities on T lymphocytes. However, the mechanism of TRAP-mediated immune suppression is still largely unclear. Herein, we sought to assess the immunomodulatory effect of TRAPs on human neutrophils, a major immune cell type that infiltrates human tumor tissues. We found that TRAPs enriched from malignant effusions or ascites of cancer patients and tumor cell lines were rapidly and effectively phagocytized by neutrophils through macropinocytosis and promoted neutrophil apoptosis via reactive oxygen species (ROS) generation and caspase-3 activation. Moreover, the apoptotic neutrophils that have phagocytized TRAPs inhibited the proliferation and activation of CD4+ T and CD8+ T cells in a cell contact- and ROS-dependent manner. These findings define a novel TRAP-mediated mechanism in neutrophils that potentially suppresses the anti-tumor T cell immunity and highlight TRAPs as an important target for future tumor immunotherapy.

Progression of Large Lymphoma Is Significantly Impeded with a Combination of Gemcitabine Chemotherapy and Dendritic Cells Intra-Tumor Vaccination.

Relapsed, refractory lymphoma remains to be a challenge and lacks efficient treatment. Some tumor cells escape from treatment, become resistant to chemotherapeutic agents, and rapidly regenerate into large tumors. Lymphoma cells induce accumulation of Gr-1(+-)CD11b(+) myeloid derived suppressor cells (MDSCs) in lymphatic organs and their vicinity. MDSCs enable tumor cells to escape from immune cells mediated surveillance and attack. Gemcitabine is a chemotherapeutic agent that eliminates both tumor cells and MDSCs, improving the immune environment favorable for subsequent treatment. We evaluated the effects of low dose gemcitabine combined with intra-tumorally delivered dendritic cells (DCs) for the treatment of A20 large-size lymphoma. We showed that MDSCs increased markedly in lymphoma-bearing mice, and that gemcitabine significantly increased the apoptosis of MDSCs. Treatment of lymphoma with either gemcitabine or intra-tumoral DCs alone could not inhibit tumor growth or rescue lymphoma-bearing mice. Treatment of lymphoma with small dose gemcitabine followed by intra-tumorally injected DCs significantly improved the efficacy of either individual treatment by reducing MDSCs, inducing onsite DCs maturation, eliminating tumor cells, inhibiting tumor growth and relapse, and extending the survival of the lymphoma-bearing mice, partly through the induction of the IFNγ secreting cells and the activation of cytotoxic lymphocytes. We showed that NK cells and CD8(+ )T cells were the major effectors to mediate the inhibition of tumor growth. Thus, the observation that gemcitabine synergizes DCs mediated immunotherapy to improve the efficacy of large size lymphoma treatment provides an experimental basis for the combination of chemotherapy and immunotherapy for the efficient treatment of relapsed or refractory lymphoma.

The contribution of TNF-α in the amygdala to anxiety in mice with persistent inflammatory pain.

Tumor necrosis factor alpha (TNF-α) is known for its role in inflammation and pain, which are strongly associated with mood disorders such as anxiety and depression. The amygdala is a forebrain structure that modulates anxiety. However, little is known about the role of TNF-α in the development of anxiety in animals with chronic pain. In the present study, we examined TNF-α expression in the basolateral amygdala (BLA) following injection of complete Freund's adjuvant (CFA) into the hind paw of mice to induce inflammation. We also determined the effects of TNF-α expression on the development of anxiety in these mice. TNF-α expression was increased in the BLA during the chronic phase of CFA-induced peripheral inflammation. The local infusion of TNF-α-neutralizing antibody infliximab in the BLA reversed anxiety-like behaviors in mice with persistent inflammatory pain. In vitro slice recordings revealed that TNF-α significantly enhanced AMPA-receptor-mediated glutamatergic excitatory synaptic transmission and inhibited GABAA-receptor-mediated inhibitory synaptic transmission in the BLA. Our findings, therefore, provide strong evidence that TNF-α contributes to the development of anxiety in mice with persistent inflammatory pain.

Infliximab monotherapy for Chinese patients with moderate to severe plaque psoriasis: a randomized, double-blind, placebo-controlled multicenter trial.

BACKGROUND: Tumor necrosis factor-α is a key mediator in the pathogenesis of psoriasis. Infliximab is a monoclonal antibody that specifically binds to tumor necrosis factor-α. The purpose of this study was to validate the efficacy and safety of 5 mg/kg infliximab therapy in Chinese patients with moderate to severe plaque psoriasis. METHODS: In this multicenter, double-blind, placebo-controlled trial, 129 patients with moderate-to-severe psoriasis were randomized to the induction therapy (weeks 0, 2 and 6) with infliximab 5 mg/kg (n = 84) or placebo (n = 45), followed with infliximab 5 mg/kg scheduled at week 14 and week 22 in the infliximab group, and infliximab 5 mg/kg scheduled at weeks 10, 12 and 16 in the placebo group. The primary end point was the proportion of patients who achieved at least 75% improvement in Psoriasis Area and Severity Index (PASI 75 response rate) from baseline at week 10. RESULTS: At week 10, 81.0% of patients treated with infliximab (5 mg/kg) achieved a 75% or greater improvement compared with 2.2% of patients treated with placebo (P < 0.001). A significant improvement in PASI, Physician's Global Assessment (PGA) and Dermatology Life Quality Index (DLQI), was seen from week 6 through week 14 in the infliximab group compared with the placebo group. Through week 22, PASI, PGA, DLQI were well maintained. The incidence of adverse events for the infliximab treatment group was slightly higher in comparison to the placebo treatment group during the first 10 weeks without statistical significance. However, there were 3 cases of tuberculosis that developed during the 26 weeks treatment with infliximal. CONCLUSIONS: Infliximab treatment was effective as induction and maintenance treatments for Chinese patients with moderate to severe plaque psoriasis. Most drug-induced adverse events were mild to moderate, and well tolerated. Screening for tuberculosis is essential and prophylactic treatment should be given if necessary.

PU.1 is essential for CD11c expression in CD8(+)/CD8(-) lymphoid and monocyte-derived dendritic cells during GM-CSF or FLT3L-induced differentiation.

Dendritic cells (DCs) regulate innate and acquired immunity through their roles as antigen-presenting cells. Specific subsets of mature DCs, including monocyte-derived and lymphoid-derived DCs, can be distinguished based on distinct immunophenotypes and functional properties. The leukocyte integrin, CD11c, is considered a specific marker for DCs and it is expressed by all DC subsets. We created a strain of mice in which DCs and their progenitors could be lineage traced based on activity of the CD11c proximal promoter. Surprisingly, we observed levels of CD11c promoter activity that were similar in DCs and in other mature leukocytes, including monocytes, granulocytes, and lymphocytes. We sought to identify DNA elements and transcription factors that regulate DC-associated expression of CD11c. The ets transcription factor, PU.1, is a key regulator of DC development, and expression of PU.1 varies in different DC subsets. GM-CSF increased monocyte-derived DCs in mice and from mouse bone marrow cultured in vitro, but it did not increase CD8(+) lymphoid-derived DCs or B220(+) plasmacytoid DCs. FLT3L increased both monocyte-derived DCs and lymphoid-derived DCs from mouse bone marrow cultured in vitro. GM-CSF increased the 5.3 Kb CD11c proximal promoter activity in monocyte-derived DCs and CD8(+) lymphoid-derived DCs, but not in B220(+) plasmacytoid DCs. In contrast, FLT3L increased the CD11c proximal promoter activity in both monocyte-derived DCs and B220(+) plasmacytoid DCs. We used shRNA gene knockdown and chromatin immunoprecipitation to demonstrate that PU.1 is required for the effects of GM-CSF or FLT3L on monocyte-derived DCs. We conclude that both GM-CSF and FLT3L act through PU.1 to activate the 5.3 Kb CD11c proximal promoter in DCs and to induce differentiation of monocyte-derived DCs. We also confirm that the CD11c proximal promoter is not sufficient to direct lineage specificity of CD11c expression, and that additional DNA elements are required for lineage-specific CD11c expression.

[Induction of prostate cancer-specific CTLs with dendritic cells pulsed by different types of tumor antigens].

OBJECTIVE: To study the effectiveness of freeze-thaw antigens and acid eluted peptide antigens extracted from tumor cell-pulsed dendritic cells (DC) in inducing prostate cancer-specific cytotoxic T lymphocytes (CTL) in vitro. METHODS: Tumor antigens were extracted from the prostate cancer cell line PC-3 with the repeated freeze-thaw and weak acid elution methods. Peripheral blood mononuclear cells were cultured with recombinant human GM-CSF and IL-4 for inducing DCs in vitro. Then the DCs were pulsed with the two kinds of prostate cancer tumor antigens respectively and cultured with T cells for inducing CTLs. The activity of the tumor-specific CTLs were detected by LDH release assay. RESULTS: The protein content in the tumor antigens obtained from PC-3 (2 x 10(7)) by citric acid-phosphate buffer elution and that by the repeated freeze-thaw method were (212.2 +/- 7.9) microg and (963.0 +/- 25.3) microg, respectively. The two kinds of prostate cancer antigens-pulsed DCs had a significant role in inducing the PC-3 cell-specific CTLs, and the CTLs induced by acid-eluted peptide antigen-pulsed DCs exhibited an even more significant tumor-specific cytotoxicity than those induced by repeated freeze-thaw ([60.4 +/- 5.52]% vs. [43.7 +/- 4.11]%, P < 0.01). CONCLUSION: Both the weak acid elution and repeated freeze-thaw methods for extracting prostate cancer antigens can be used for in vitro sensitization of DCs. The DCs pulsed by either of the two kinds of antigens can activate CTLs, and the antigens extracted by weak acid elution are even more effective.

A novel selenium and copper-containing peptide with both superoxide dismutase and glutathione peroxidase activities.

Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.

[Basic and clinical research on paraneoplastic pemphigus].

Paraneoplastic pemphigus (PNP) is an autoimmune blistering skin disease associated with neoplasms. Clinically, it is characterized by severe mucosal erosions and various cutaneous lesions. Suprabasal acantholysis and cleft with scattered necrotic keratinocytes are the unique histopathological features of PNP. The pathogenic autoantibodies existed in PNP sera, and their production was correlated with the associated tumor. Early detection and resection of the tumor are essential for the treatment of the disease.

Development of ELISA for the specific determination of autoantibodies against envoplakin and periplakin in paraneoplastic pemphigus.

INTRODUCTION: In paraneoplastic pemphigus (PNP), indirect immunofluorescence microscopy using rat bladder sections is widely used to search for circulating autoantibodies which are predominantly directed against envoplakin and periplakin. A sensitive and specific detection system for envoplakin/periplakin-specific autoantibodies is not yet available. METHODS: Overlapping fragments spanning envoplakin and periplakin, respectively, were analyzed for their ability to bind PNP-specific autoantibodies by immunoblotting and ELISA in sera from patients with PNP (n=31), pemphigus vulgaris (n=30), and bullous pemphigoid (n=50) as well as healthy volunteers (n=140). The results were compared with those obtained by immunoblotting of extract of cultured human keratinocytes. RESULTS: Immunoblot analysis revealed that most sera contained antibodies against the N-termini of both plakins as well as against the C-terminus of envoplakin. By ELISA, reactivities against envoplakin(1-481), envoplakin(1626-2033), and periplakin(1-324) were found in 25 (80.6%), 25 (80.6%), and 23 (74.2%) PNP sera, and in 1 (1.2%), 3 (3.7%), and 2 (2.5%) control sera, respectively. CONCLUSIONS: The new ELISA based on an N-terminal fragment of envoplakin shows a high diagnostic accuracy to detect circulating autoantibodies in PNP. It is easy to be setup and standardized and can help to differentiate PNP patients from those with pemphigus vulgaris.

[Dendritic cell-based immunotherapy for multiple myeloma -- review].

Patients with multiple myeloma (MM) have increased constantly in recent years, but treatment for patients with MM is currently unsatisfactory and it is necessary to develop new complementary therapies. Dendritic cells (DCs) are specialized antigen-presenting cells capable of initiating and regulating immune responses. Vaccination with tumor antigen-pulsed DCs has shown to be safe and possesses therapeutic effect against many tumors. In this review, the various types of MM-associated antigens and clinical trials on DC-based immunotherapy in MM are summarized, the development of DC immunotherapy for MM patients in future trials is discussed.

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.

[Surgical treatment and prognosis analysis of localized retroperitoneal Castleman disease: a study of 20 cases].

OBJECTIVE: To investigate the clinical characteristics, surgical treatment and prognosis analysis of localized retroperitoneal Castleman disease (CD), and to improve the level of diagnosis and treatment of retroperitoneal Castleman disease with paraneoplastic pemphigus (PNP). METHODS: The clinical data of retroperitoneal CD with PNP from January 1993 to May 2009 were compared with CD without PNP retrospectively, including clinical features, tumor site, lab examination, surgical treatment, pathologic subtype and results of surgery. RESULTS: (1) Retroperitoneal Castleman disease more likely originated in para-kidney and iliac fossa with middle age of 36 years old, especially in left retroperitoneum. Of the 20 cases, 18 tumors (90%) were hyaline vascular variants and 2 were mixed variants. There was no statistical difference in gender, age, tumor site and pathological subtype between two groups. (2) Retroperitoneal CD with PNP more likely complicated with bronchiolitis obliterans (BO) and high level of serum CEA/CA242. (3) Retroperitoneal Castleman tumors had clear margin, intact envelop and were easily resectable, however the biological behavior of CD with PNP might tend malignant changing, invade adjacent blood vessel or seed locally, and eventually relapse after operation. (4) The 5-year survival rate of retroperitoneal CD with PNP was 42.8%, significantly lower than those without PNP. The average survival time was 30 months. Bronchiolitis obliterans and radical resection were the key effect in prognosis of retroperitoneal CD. CONCLUSIONS: Retroperitoneal CD with PNP has distinctive clinical features. Early diagnosis, prompt removal of tumor and termination secretion of causative antibody are critical to the management of this disease.

Compound heterozygosity for new splice site mutations in the plakophilin 1 gene (PKP1) in a Chinese case of ectodermal dysplasia-skin fragility syndrome.

Ectodermal dysplasia-skin fragility syndrome is a rare autosomal recessive inherited disease characterized by skin fragility, nail dystrophy and hyperkeratosis of palms and soles. Skin biopsy shows the loss of cell adhesion and the decrease of desmosomes in number and size. Mutations in PKP1 have been found to be the underlying cause of the syndrome. We report here a Chinese case of ectodermal dysplasia-skin fragility syndrome. Mutation analysis revealed compound heterozygosity for mutations in PKP1 of the proband. A new splice site mutation (c.1053 T>A+c.1054+1 G>T) near the 3' end of exon 5 and at the donor end of intron 5 on one allele was transmitted from the proband's mother. Another new splice site mutation (c.1835-2 A>G) near the acceptor end of intron 10 originated from her father. The absence of the mutant mRNA and plakophilin 1 protein in the proband's skin may result from the mechanism of nonsense-mediated mRNA decay induced by premature stop codons in PKP1 transcripts due to the two splice site mutations.