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Objective To explore the features and risk factors of bacterial skin infections (BSIs) in hospitalized patients with bullous pemphigoid (BP). Methods Records were retrospectively reviewed for 110 hospitalized patients with BP admitted to Peking University First Hospital between 2013 and 2019. Bacterial species and drug resistance were assessed, and then the underlying risk factors for BSIs were evaluated. Results Infections were present in 40% (44/110) of the patients. Staphylococcus aureus (72.7%, 32/44) was the most common bacterium, and it was highly resistant to penicillin (81.3%, 26/32), erythromycin (62.5%, 20/32), and clindamycin (56.3%, 18/32), but 100.0% sensitive to vancomycin and tigecycline. Coronary heart disease (P = .02; odds ratio [OR], 12.68), multisystem comorbidities (P = .02; OR, 3.67), hypoalbuminemia (P = .04; OR, 3.70), high levels of anti-BP180 antibodies (>112.4 U/mL; P = .003; OR, 6.43), and season (spring: reference; summer: P = .002; OR, 23.58; autumn: P = .02; OR, 12.19; winter: P = .02; OR, 13.19) were significantly associated with BSIs. Conclusions Hospitalized patients with BP had a high incidence of BSIs, and those patients with underlying risk factors require careful management to prevent and control BSIs.

Objectives We aimed to describe the clinical and histopathologic features of Castleman disease (CD), particularly emphasizing its associations with paraneoplastic pemphigus (PNP) and prognosis. Methods We retrospectively enrolled 123 CD patients at our center. Clinical, pathologic, and laboratory data were reviewed. Results Fifty percent of the patients had PNP. Compared with those without PNP, patients with PNP-associated CD had more hyaline vascular (HV) variants (83.9% vs 57.4%), fewer mixed cellular variants (16.1% vs 24.6%), and no plasmacytic variants (0% vs 18.0%). Thirty-eight of 87 patients with the HV variant of CD (HV-CD) had stroma-rich (SR) features, and the incidence rate was higher in those with PNP-associated CD than in those without PNP (48.4% vs 13.1%, P < .001). The SR variant was associated with higher PNP-associated IgG titers than SR absence before surgery (median 1:160 vs 1:80, P = .019) or after surgery (median 1:160 vs 1:40, P = .013). The SR variant was also an unfavorable prognostic factor for CD survival in univariate analysis. The 3-year survival rates were 47.5% among those with PNP and 87.7% among those without PNP (P < .001). Conclusions PNP is associated with specific subtypes of CD and affects survival. The SR variant of HV-CD positively correlates with the incidence of PNP.

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

Solasonine, the main active ingredient of Solanum nigrum L., has been reported to exert extensive antitumor activity. However, the antitumor effects in acute monocytic leukemia and the exact mechanisms involved are unknown. In this study, we investigated the role of solasonine on inhibiting the progression of acute monocytic leukemia. Our findings showed that solasonine inhibited the proliferation of acute monocytic leukemic cell lines (THP-1 and MV4-11) in vitro. Solasonine promoted apoptosis and induced cell cycle arrest in the G2/M phase. Analysis of RNA-seq data suggested that solasonine correlated with increased expression of genes in the AMPK/FOXO3A pathway. Inhibition of AMPK with compound C followed by treatment with solasonine showed that solasonine reduced apoptosis, caused less cell cycle arrest, and inactivated the AMPK/FOXO3A axis in THP-1 and MV4-11 cells. Solasonine also inhibited tumor growth by the activation of the AMPK/FOXO3A axis. In conclusion, solasonine inhibited the progress of acute monocytic leukemia in vitro and in vivo and triggered the apoptosis and cell cycle arrest in the G2/M phase by upregulating the AMPK/FOXO3A pathway.

Paraneoplastic autoimmune multiorgan syndrome is a complex and deadly disease. We retrospectively reviewed the clinical features and risk factors for paraneoplastic autoimmune multiorgan syndrome in 145 Chinese patients. The most common neoplasm was Castleman disease (56%), and patients with Castleman disease tended to be younger (≤ 42 years old: 83% vs. 29%) and to have a greater proportions of lichen planus-like lesions (47% vs. 27%) and bronchiolitis obliterans (49% vs. 29%), compared to other neoplasm-associated patients. Among all 145 patients in the study, the survival rates were 84% at 1 year, 65% at 3 years, and 54% at 5 years. Kaplan-Meier curve analysis revealed that mortality was associated with older age (> 42 years), neoplasm type, labial lesions, and larger skin lesion area (> 17.5% of the body surface area). However, only older age and larger skin lesion area were independent factors associated with mortality in multivariate analysis. We suggest that patients with Castleman disease and paraneoplastic autoimmune multiorgan syndrome have many unique characteristics and the underlying risk factors for death require further exploration.

Autoimmune blistering diseases (AIBDs) are a group of fatal diseases with specific autoantibodies. BIOCHIP mosaic is a novel and all-in-one measure used for the rapid diagnosis of AIBDs. To evaluate the diagnostic accuracy based on BIOCHIP mosaic (FA1501-1005-60) in Chinese patients with AIBDs. Seventy-seven patients with AIBDs and 20 controls were enrolled. The BIOCHIP mosaic was performed using both serum and plasma samples. Based on BIOCHIP mosaic, the data from paired plasma and serum samples demonstrated a high degree of concordance (Cohen’s kappa = 0.896–1.000) for autoantibodies against Dsgl, Dsg3, BP180-NC16A-4X, BP230gC, prickle-cell desmosomes, and pemphigoid antigens. Moreover, BIOCHIP mosaic also demonstrated a high degree of consistency for the detection rate of anti-Dsgl, Dsg3, plakins, BP180-NC16A-4X and non-collagenous domain of type VII collagen autoantibodies for the diagnosis of pemphigus foliaceus (77.3%), pemphigus vulgaris (88.6%), paraneoplastic pemphigus (100.0%), bullous pemphigoid (92.8%) and epidermolysis bullosa acquisita (99.0%), respectively. Using BIOCHIP mosaic, serum and plasma samples may be used interchangeably at 1/10 dilution. Overall, the BIOCHIP mosaic was shown to be a useful and accurate tool for the diagnosis of AIBDs.

Background Infections were the primary cause of death (34.3–55.5%) in patients with pemphigus. Skin was usually the origin of infections. The study aimed to explore features and associated factors of bacterial skin infections (BSIs) in inpatients with pemphigus. Methods One hundred and seventy-seven inpatients with pemphigus hospitalizing from November 2014 to April 2019 were continuously recruited through Peking University First Hospital’s inpatient records inpatients with pemphigus hospitalizing from November 2014 to April 2019 were continuously recruited through Peking University First Hospital’s inpatient records. Then, we retrieved the clinical and laboratory data to explore the characteristics and associated factors of BSIs. Results Of patients enrolled, pemphigus vulgaris (PV, n = 142) and pemphigus foliaceus (PF, n = 9) were most common, followed by pemphigus erythematosus (PE, n = 25) and pemphigus vegetans (Pveg, n = 1). Eighty-seven of 177 (49.2%) inpatients developed BSIs, and they had a longer length of stay compared with inpatients without BSIs (median: 18.9 vs. 14.1 days, p = 0.008). Staphylococcus aureus was the most common bacteria (71.3%, 62/87) and highly resistant to penicillin (91.9%, 57/62). Higher levels of anti-Dsg1 autoantibodies (> 124.2 U/mL) (p 169.5 U/mL) (p = 0.03, OR = 2.074, 95% CI: 1.084–3.969) were underlying risk factors of BSIs when analyzed by binary regression analysis. As for Gram’s stain of bacteria, females had a lower rate of Gram-positive infections (p = 0.03). Patients using oral antibiotics (p = 0.05) had a higher rate of Gram-negative infections. Inpatients who were hospitalized in other hospitals within 2 weeks before the current admission had a higher rate of Gram-negative and co-infections (p = 0.03). Conclusions Inpatients with pemphigus had a high incidence of BSIs. Some factors were associated with the susceptibility of BSIs and bacterial species.

We describe a case of primary cutaneous aspergillosis caused by Aspergillus fumigatus, and elucidate the underlying genetic and immunological mechanisms. Routine clinical and laboratory investigations were performed. Whole-exome sequencing of the patient’s DNA suggested the presence of a CARD9 mutation, which was confirmed by Sanger sequencing. Innate and adaptive immunological responses of patient-derived CARD9-deficient cells were evaluated with ELISA and flow cytometry. Cutaneous and pulmonary aspergillosis models were established in Card9 knockout (KO) mice, which were compared with wild-type and immunosuppressed mice, to explore the pathogenesis and Aspergillus susceptibility. A 45-year-old man presented with a 37-year history of skin lesions on his face. A diagnosis of primary cutaneous aspergillosis was made through histopathology, immunohistochemistry, and tissue culture. Sanger sequencing of CARD9 showed a homozygous frame-shift mutation (c.819_820insG, p.D274fsX60), which led to the lack of CARD9 expression. Peripheral blood mononuclear cells from the patient showed selective impairment of proinflammatory cytokines, and Th1-, Th17-, and Th22-associated responses upon fungus-specific stimulation. The cutaneous aspergillosis model established in Card9 KO mice presented with persistent infection, with fungal germs and short hyphae in tissue, consistent with the patient’s lesions. Skin lesions in immunosuppressed mice were more severe, and led to death. Unlike our patient, Card9 KO mice were relatively susceptible to pulmonary aspergillosis, with reasons to be investigated. This is, to our knowledge, the first report that links cutaneous aspergillosis to CARD9 mutation. This work enriches both the phenotypic spectrum of CARD9 deficiencies and the genetic background of cutaneous aspergillosis.

Background/purpose Previous studies have shown that hemoporfin-mediated photodynamic therapy (PDT) was a treatment for port-wine stain (PWS). Our current study aimed to identify optimal hemoporfin dose. Methods A prospective, multicenter, double-blind, randomized clinical trial was conducted. Patients were assigned into low- or high-dose hemoporfin (2.5 mg/kg and 5 mg/kg intravenously, respectively), or control (placebo) group, at a rate of 2:2:1. Treatment efficacy was evaluated at week 8. Then, patients in control group were randomly assigned into either high- or low-dose hemoporfin group. Treatment reactions and adverse events were analyzed at week 16. Results A total of 100 patients (40, 40, 20 in low-, high-dose hemoporfin, and control group, respectively) were enrolled. Compared to low dose (40%) and control group (15%), a higher proportion of patients in high-dose group (75%) had achieved skin lesion improvements. Treatment satisfactions were graded highest in high-dose group. Compared to low-dose group (14.3%), high-dose group (46.0%) had more frequent skin hyperpigmentation, which disappeared 3-6 months after treatment. Other treatment reactions and adverse events were comparable between two groups. Conclusions Photodynamic therapy with 5 mg/kg hemoporfin could be an effective and safe treatment for PWS.

// Kun Chen 1 , Gang Wang 2 , Hongzhong Jin 3 , Jinhua Xu 4 , Xuejun Zhu 5 , Min Zheng 6 and Heng Gu 1 1 Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing, Jiangsu, China 2 Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shanxi, China 3 Peking Union Medical Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Peking, China 4 Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China 5 Department of Dermatology, Peking University First Hospital, Peking, China 6 Department of Dermatology, Second Affiliated Hospital, Zhejiang University, School of Medicine, Hangzhou, Zhejiang, China Correspondence to: Heng Gu, email: guhengy@yahoo.com.cn Min Zheng, email: minz@zju.edu.cn Keywords: psoriasis, epidemiology, phenotype, Chinese Han Received: November 17, 2016 Accepted: April 29, 2017 Published: June 12, 2017 ABSTRACT Psoriasis is a worldwide chronic inflammatory disease, involving both skin and joints. In order to characterize psoriasis in Han Chinese population, we conducted this nationwide prospective and hospital based survey, in which 56 hospitals with departments of dermatology participated, located in 33 cities across China. A total of 12,031 outpatients with psoriasis were registered during 2009 to 2010, which the data was collected by standard questionnaires. The main data acquisition included demographics, family history, disease status and other comorbidities. Physical and dermatological examination, including body surface area (BSA) and psoriasis area severity index (PASI) were applied to evaluate the disease severity. Descriptive statistics, 2 tailed t -test and chi-square test were used appropriately for the statistical analysis. From the study, we found that the male and female ratio of the patients was 1.49:1. Mean age of onset was 30.2 ± 14.5 years for males and 27.1 ± 15.6 years for females ( P < 0.05). Scalp was the most common onset site (52.8%), The mean PASI was 18.70 ± 10.01, indicating that most patients presenting at the hospitals had moderate-to-severe psoriasis and the majority was psoriasis vulgaris (96.5%). Among 12,031 patients, 23.1% had a family history of psoriasis,16.1% had comorbidities, and 29.9% had nail changes. The most important aggravation factor was season change (60.2%), followed by psychological stress (34.5%), and there significant differences between genders on trigger factors. In conclusion, this study characterizing psoriasis in Han Chinese population, could be used as basic data for future study.

Objectives MicroRNAs are powerful regulators in hepatocellular carcinoma (HCC) tumorigenesis. MicoRNA‐191 (miR‐191) has been reported to play an important role in HCC, However, the regulatory mechanism is still unclear. In this study, we investigated the role of miR‐191 in HCC and studied its underlying mechanisms of action. Materials and methods The expression of miR‐191 in HCC tissues was determined by quantitative real‐time PCR (qRT‐PCR). The role of miR‐191 in HCC cells was examined by using both in vitro and in vivo assays. Downstream targets of miR‐191 were determined by qRT‐PCR and Western blot analysis. Dual‐luciferase assays were performed to validate the interaction between miR‐191 and its targets. Results The expression of miR‐191 was significantly higher in HCC patients and a higher miR‐191 expression predicted poorer prognosis. Analysis of The Cancer Genome Atlas data sets suggested that miR‐191 positively correlated with cell cycle progression. Gain and loss of function assays showed that miR‐191 promoted cell cycle progression and proliferation. Luciferase reporter assay showed that miR‐191 directly targeted the 3'‐untranslated region of KLF6 mRNA. Furthermore, circular RNA has_circ_0000204 could sponge with miR‐191, resulting in inactivation of miR‐191. Conclusions Our study sheds light on the novel underlying mechanism of miR‐191 in HCC, which may accelerate the development of cancer therapy.

Various CD7-targeting immunotoxins have been tested for its potential in treating CD7+ malignant patients but none of those immunotoxins was approved clinically because of lacking enough efficacy and safety. Here we successfully constructed the monovalent and bivalent CD7 nanobody-based immunotoxins PG001 and PG002, both conjugated with a truncated derivative of Pseudomonas exotoxin A respectively. The prokaryotic system expressed immunotoxins not only maintained their binding specificity for CD7-positive cells with a Kd of 16.74 nM and 3.6 nM for PG001 and PG002 respectively, but also efficiently promoted antigen-restricted apoptosis of the CD7-positive leukemia cell lines Jurkat and CEM, and primary T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells with an in vitro cytotoxic activity (EC50) in the range of 23-30 pM for PG002. In NOD/SCID mice transplanted with CEM cells, PG001 and PG002 prevented engraftment of the cells and markedly prolonged mouse survival. Owing to the efficient antigen-restricted anti-leukemic activity of PG002, this CD7 nanobody-based immunotoxin exhibited a superior anti-CD7 positive malignancies activity than previously reported immunotoxins, and may represent a promising therapeutic strategy in treating CD7-positive leukemia and lymphoma, which still remain a significant clinical challenge.

BACKGROUND AND OBJECTIVES:Atopic dermatitis (AD) primarily affects infants and young children. Although topical corticosteroids (TCSs) are often prescribed, noncorticosteroid treatments are needed because compliance with TCSs is poor due to concerns about their side effects. In this longest and largest intervention study ever conducted in infants with mild-to-moderate AD, pimecrolimus 1% cream (PIM) was compared with TCSs.Methods:A total of 2418 infants were enrolled in this 5-year open-label study. Infants were randomized to PIM (n = 1205; with short-term TCSs for disease flares) or TCSs (n = 1213). The primary objective was to compare safety; the secondary objective was to document PIM’s long-term efficacy. Treatment success was defined as an Investigator’s Global Assessment score of 0 (clear) or 1 (almost clear).Results:Both PIM and TCSs had a rapid onset of action with >50% of patients achieving treatment success by week 3. After 5 years, >85% and 95% of patients in each group achieved overall and facial treatment success, respectively. The PIM group required substantially fewer steroid days than the TCS group (7 vs 178). The profile and frequency of adverse events was similar in the 2 groups; in both groups, there was no evidence for impairment of humoral or cellular immunity.Conclusions:Long-term management of mild-to-moderate AD in infants with PIM or TCSs was safe without any effect on the immune system. PIM was steroid-sparing. The data suggest PIM had similar efficacy to TCS and support the use of PIM as a first-line treatment of mild-to-moderate AD in infants and children.

Summary Background Paraneoplastic pemphigus (PNP) involves multiple organs, but little is known about its neurological involvement. Objectives To investigate the symptoms, prognosis and profiles of associated autoantibodies in myasthenia gravis (MG), and their correlations in patients with PNP. Methods Fifty-eight patients with PNP were assessed for myasthenic symptoms and laboratory evidence. Serum autoantibodies against acetylcholine receptor (AChR), acetylcholinesterase (AChE), titin, ryanodine receptor (RyR) and muscle-specific kinase (MuSK) were measured by enzyme-linked immunosorbent assay. Patients with pemphigus vulgaris (PV), pemphigus foliaceus (PF), connective tissue disease (CTD) and non-PNP MG (NP-MG), and healthy donors, served as controls. These autoantibodies in PNP were also compared in the presence or absence of dyspnoea or muscle weakness. Cox regression and log-rank tests were used for survival analysis. Results Overall 39% of patients with PNP experienced muscle weakness, and 35% were diagnosed with MG. Moreover, 35% had positive anti-AChR and 28% had anti-AChE antibodies, similarly to NP-MG (33% and 17%, respectively, P > 0·05). However, both were negative in all patients with PV, PF and CTD and healthy donors (P

Objectives Circular RNA, a type of RNA formed by a covalently closed loop, possesses sophisticated abilities of gene regulation in tumorigenesis and metastasis. However, the role of circRNAs on lung adenocarcinoma (LUAD) remains largely unknown. Materials and methods The role of cMras was examined both in vitro and in vivo. cMras expression in LUAD tissues was determined by quantitative real‐time PCR (qRT‐PCR). Downstream targets of cMras were predicted by bioinformatics tools and confirmed by RNA immunoprecipitation assay and luciferase assay. qRT‐PCR and western blot assay were used to detect the expression of specific targets. Results Thirty‐six paired LUAD and healthy tissues were collected and cMras resulted significantly downregulated in cancerous tissues. Its expression was negatively associated with tumour stages. cMras overexpression suppressed LUAD growth and metastasis, while endogenous cMras silencing resulted in the opposite effects. Bioinformatics analysis and experimental evidence confirmed that cMras was a sponge of miRNA‐567 and released its direct target, PTPRG. cMras overexpression decreased miR‐567 expression and subsequently increased PTPRG expression, while increased miRNA‐567 expression blocked the effects induced by cMras. Moreover, PTPRG was downregulated in LUAD and patients with low PTPRG expression exhibited significantly poor prognosis. These results suggested that cMras/miR‐567/PTPRG regulatory pathway might be associated to LUAD tumorigenesis and development. Conclusions A novel circular RNA cMras and its functions were identified, discovering a cMras/miR‐567/PTPRG regulatory pathway in LUAD tumorigenesis and development.

Background and Objectives Photodynamic therapy (PDT) has shown potentially beneficial results in treating port-wine stain, but its benefit–risk profile remains undefined. This study aimed to evaluate the efficacy and safety of PDT conducted with hemoporfin and a 532 nm continuous wave laser to treat port-wine stain clinically. Patients and Methods This randomized clinical trial was conducted in eight hospitals in China. Participants were adolescent and adult patients (age range: 14–65 years old) with port-wine stain. During stage 1 (day 1 to week 8) all patients were randomized at a 3:1 ratio to treatment (532 nm laser irradiation (96–120 J/cm2) with hemoporfin (5mg/kg; PDT-hemoporfin, n = 330)) or placebo groups (irradiation with placebo (PDT-placebo, n = 110)); during stage 2 (week 8 to 16) patients in both groups were offered treatment. Clinician-evaluators, who were blind to the study, classified each case on the following four-level scale according to assessment of before and after standardized pictures of the lesion area: no improvement

Autoantibodies against N-terminal domains and linker subdomains of envoplakin (EVPL) and periplakin (PPL) were frequently detected in sera of paraneoplastic pemphigus(PNP) patients.To further investigate finer epitopes of EVPL and PPL, and evaluate their associations with clinical aspects of PNP.We produced 12 overlapping truncated fragments of these regions in Escherichia coli, and measured their reactivities to sera of 65 PNP patients and 50 healthy volunteers by enzyme-linked immunosorbent assays (ELISA). Then appropriate statistics were performed to evaluate the correlation between antibodies against different fragments and clinical information of patients.EVPL-N1 (aa1-141) and EVPL-L3 (aa1684-1784) were recognized by PNP sera at the same sensitivity of 75.38% (49/65) with specificities of 98% and 92%, respectively. Although neoplasm types were not associated with any fragment, the ELISA of these fragments and indirect immunofluorescence on rat bladder complemented each other in detecting PNP. Meanwhile, levels of autoantibodies against EVPL-N3 were elevated with PNP accompanied with bronchiolitis obliterans or presented with lichen planus-like lesions (P0.05). No influence of autoantibodies against any fragments on prognosis of the patients was observed by Cox regression test, though antibodies against some fragments were higher in the dead patients.The study proved antigenic epitopes were mainly concentrated on EVPL-N1 and EVPL-L3 in PNP. Autoantibodies against EVPL-N3 might associate with those patients accompanied with bronchiolitis obliterans or presented with lichen planus-like lesions.

Olmsted syndrome (OS) is a rare congenital disorder characterized by palmoplantar and periorificial keratoderma, alopecia in most cases, and severe itching. The genetic basis for OS remained unidentified. Using whole-exome sequencing of case-parents trios, we have identified a de novo missense mutation in TRPV3 that produces p.Gly573Ser in an individual with OS. Nucleotide sequencing of five additional affected individuals also revealed missense mutations in TRPV3 (which produced p.Gly573Ser in three cases and p.Gly573Cys and p.Trp692Gly in one case each). Encoding a transient receptor potential vanilloid-3 cation channel, TRPV3 is primarily expressed in the skin, hair follicles, brain, and spinal cord. In transfected HEK293 cells expressing TRPV3 mutants, much larger inward currents were recorded, probably because of the constitutive opening of the mutants. These gain-of-function mutations might lead to elevated apoptosis of keratinocytes and consequent skin hyperkeratosis in the affected individuals. Our findings suggest that TRPV3 plays essential roles in skin keratinization, hair growth, and possibly itching sensation in humans and selectively targeting TRPV3 could provide therapeutic potential for keratinization or itching-related skin disorders.

Background/purpose: This phase IIa study aimed to study the efficacy and safety of hemoporfin in photodynamic therapy (PDT) with a 532 nm continuous laser for port-wine stain (PWS). Methods: In this 8-week open-labeled study in three centers, three different laser exposure times (532 nm continuous laser for 20, 30 and 40 min) were used in stage I, group A, stage II, group B and stage III, group C, respectively. Primary efficacy assessment was performed by an independent group of experts, who reviewed the standardized photos. Secondary efficacy assessment consisted of the subjective grading of the PWS fading by the investigators and the patients. Treatment reactions and adverse events (AE) were recorded separately. Results: Forty patients were initially enrolled in the study, but stage III had to be cancelled eventually for the safety of the patients. Patients in groups A and B showed similar satisfactory results in efficacy assessments, the total ‘response’ rate being 80.0% and 94.7% in groups A and B, respectively. The AE rates were also similar in the two groups. Self-limiting photosensitive dermatitis and hyperpigmentation were the most frequently observed AE. Conclusion: Hemoporfin-PDT is effective and safe for patients with PWS aged 16–50.

Burkitt lymphoma is a fast growing non-Hodgkin lymphoma that occurs primarily in young males. The causes of Burkitt lymphoma include chromosome rearrangement and virus infection, but accurate and complete reasons remain to be discovered. The available treatment for Burkitt lymphoma is chemotherapy and radiation therapy. It is a highly aggressive B-cell neoplasm with not all patients cured, in spite of current therapies. This study evaluated the effects of traditional Chinese medicine Marsdenia tenacssima (MTE) and its component compound Tenacigenoside A (TGTA) and 11α-O-benzoyl-12β-O-acetyltenacigenin B (TGTB) on human Burkitt lymphoma growth. It was observed that MTE, TGTA or TGTB inhibited cell growth and induced apoptosis of Burkitt lymphoma cells in culture. In lymphoma bearing NOD/SCID nude mice, both TGTA and TGTB inhibited tumor growth and improved animal survival. TGTA and TGTB significantly increased tumor cell apoptosis on lymphoma bearing mice, primarily through down-regulation of BCL2 and BCL-XL and up-regulation of BID.

Glucocorticoids are the first-line treatment for pemphigus vulgaris. Among 140 patients receiving systemic glucocorticoids, 124 patients achieved complete remission off or on a prednisone dose of ≤10 mg/day or less for 6 months or more. The mean average steroid controlling doses were 0.65, 0.62, 0.80, 1.08 and 1.38 mg/kg per day for the mucosal-dominant patients and the mild, moderate, severe and extensive cutaneous-involved patients, respectively (P < 0.001). The mean durations of the initial tapering after controlling doses started were 77.98, 48.78, 31.74 and 28.83 days when the disease was controlled with doses of 40 mg/day or less, 45-60 mg/day, 65-80 mg/day and more than 80 mg/day for the cutaneous-involved types, respectively (P < 0.005). Of the patients, 79.51% achieved complete remission within 3 years, 98.36% within 5 years and all within 6 years, which corresponded to a 50% yearly reduction of glucocorticoid dose. These successfully treated patients indicate that a severity-tailored initial dose of glucocorticoids, an initial tapering duration based on the initial dose and a subsequent 50% yearly tapering regimen may cure pemphigus vulgaris within 3-6 years.

Sera from paraneoplastic pemphigus (PNP) immunoprecipitate multiple antigens from human epidermal protein extract. In this study, we further characterized the autoantibodies in 12 PNP sera. Immunoblotting using recombinant linker subdomains of envoplakin, periplakin, desmoplakin, and bullous pemphigoid antigen I found that 11 of the 12 sera recognized linker subdomains of envoplakin and periplakin. We then synthesized 12 peptides covering the linker subdomain of envoplakin for ELISA. One of the peptides, peptide no. 8, was recognized by nine out of the 12 sera with a higher affinity. A method of ligand–receptor binding assay was designed and performed using this peptide labeled with fluorescence as the ligand. Peptide no. 8 bound to CD20+ cells in Castleman's tumors from the patients whose sera were positive to this peptide by ELISA. Our data suggest that the linker subdomain of plakin proteins may be one of the major areas recognized by PNP autoantibodies, and epitopes in the linker subdomain of envoplakin recognized by PNP autoantibodies with a high affinity are dispersed in several areas and are variable among PNP patients. We also demonstrate that B-lymphocyte clones specifically reacting to epidermal proteins exist in Castleman's tumors from PNP.

Summary Background Paraneoplastic pemphigus is an autoimmune mucocutaneous disease associated with Castleman's tumours, which when surgically removed often result in great improvement of mucocutaneous lesions. An IgG autoantibody against epidermal proteins is often used as a diagnostic marker for disease. Our aim was to ascertain the role of Castleman's tumours in production of the autoantibody and pathogenesis of paraneoplastic pemphigus. Methods We enrolled seven patients with paraneoplastic pemphigus associated with Castleman's disease and assessed the effect of removal of tumours on mucocutaneous lesions in six individuals and on autoantibody titre with indirect immunofluorescence in four patients. We cultured tumour cells from one patient and assayed the secreted autoantibody. Finally, we characterised the gene sequence and expression of the variable region of the immunoglobulin heavy chain (IgV H ) in tumour B cells from all patients by reverse transcription-PCR, DNA sequencing, and in-situ hybridisation. Findings Cutaneous lesions disappeared within 6–11 weeks after resection of tumours. Mucosal lesions also improved in this period, but lasted for 5–10 months overall. Autoantibody titre decreased and became undetectable within 5–9 weeks in three of four patients assessed. We identified secreted autoantibody, similar to that identified in patients' serum, in cultured tumour cells. The tumour B-cells of the seven patients shared and expressed two rearrangement patterns of complementarity determining region 3 (CDR3) of IgV H . Interpretation Secreted autoantibody from Castleman's tumours, which reacts against epidermal proteins, could be an essential factor in the pathogenesis of paraneoplastic pemphigus. We noted clonal rearrangement, resulting in similar variable regions of IgV H , in tumour B cells isolated from all seven patients. However, whether this pattern is associated with autoimmunity remains to be ascertained.

Epidermolytic hyperkeratosis (EHK) is a rare dominantly inherited skin disorder with erythroderma and hyperkeratosis. Mutations have been found in keratin 1 (K1) or keratin 10 (K10) gene. In the present study, we reported three sporadic and one familial Chinese EHK patients with their mutation findings. All the mutations turned out to be single heterozygous point substitutions. A novel mutation designated as E477K of K1 was identified in one patient, and previous reported mutations in codon 156 of K10, i.e. R156S, R156P, R156H were found in other patients. This is the first report of the keratin mutations in Chinese kindreds. The results showed that the possible correlation between the genotype and phenotype in these patients was complex, not only depended on the position of the mutation but also on the actual amino acid substitution. And palmoplantar keratoderma (PPKD) can be an accompanied symptom caused by either K1 or K10 mutation.

We describe a Chinese family with generalized atrophic benign epidermolysis bullosa (GABEB), a non-lethal variant of junctional epidermolysis bullosa. The proband was an offspring of consanguineous parents, had generalized blisters since birth and developed severe alopecia during early childhood. Ultrastructural examination of the proband's skin revealed fissures in the lamina lucida. Immunofluorescence assays using a monoclonal antibody recognizing the extracellular domain of the 180 kDa bullous pemphigoid antigen (BPAG2) showed loss of fluorescent signal in the basal membrane zone of the skin. DNA sequencing revealed a homozygous C-to-G transversion at nucleotide position 899 in exon 11 of the COL17A1 gene, which encodes BPAG2. This mutation results in serine to cysteine at position 265, which is located in a highly conserved region of the intracellular domain of BPAG2. We showed that the proband's father was heterozygous for this mutation. In addition, we found a novel polymorphic substitution of C-to-G at nucleotide position 798 in exon 10 of the COL17A1 gene, which results in an I233M change in BPAG2 and is a common polymorphic allele in a limited Chinese population.

A young woman with recurrent painful lesions on the dorsal aspects of her hands associated with arthralgia presented with pain and redness of both eyes. After extensive investigations, a diagnosis of erythema elevatum diutinum accompanied by peripheral ulcerative keratitis was made. The patient was treated with dapsone 50 mg, t.i.d., and responded well.

Lipoid proteinosis is caused by loss-of-function mutations in the glycoprotein extracellular matrix protein 1 (ECM1). We report here mutation analysis of the ECM1 gene in a Chinese family with lipoid proteinosis. A 10-year-old boy presented with a hoarse voice, acneiform scars and yellow skin nodules, as well as beaded eyelid papules and a thickened sublingual frenulum. Skin biopsy showed widespread deposition of hyaline material in the dermis and thickened basement membrane. His elder sister had the same clinical manifestations. The coding region of ECM1 was amplified and sequenced and both affected siblings were shown to have a novel homozygous single nucleotide substitution, c.658T>G, in exon 6, which converts cysteine to glycine, designated p.C220G. Both parents were heterozygous for this mutation which was not detected in 100 control chromosomes. Missense mutations in the ECM1 gene are an unusual finding in lipoid proteinosis, but this case adds to the spectrum of disease-associated mutations in this rare genodermatosis.

Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) remains challenge for the treatment of chronic myeloid leukemia (CML). IM resistance often results from unknown mechanisms with wild type BCR-ABL that have no effects on TKIs binding to ABL kinase domain. The basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed an initial bioinformatics screen on over represented CML genes, followed by a quantitative PCR screen of genes that were elevated in TKIs resistant CML samples. We identified a total of 33 candidate genes that were highly expressed in TKIs resistant patients. Among these genes, 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3) controlling the limiting step of glycolysis, was found to strongly associated with TKIs resistance. RNA interference of the expression of PFKFB3 and pharmacological inhibition of its kinase activity markedly increased the sensitivity of TKIs resistant CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 prevented CML cells growth and significantly improved the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment demonstrated that the Ets transcription factor PU.1 regulates the elevated expression of PFKFB3 in TKIs resistant CML cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent TKIs resistant CML. Disclosures No relevant conflicts of interest to declare.

Pure hair and nail ectodermal dysplasia (PHNED) is a congenital condition characterized by hypotrichosis and nail dystrophy. Autosomal-recessive PHNED has previously been mapped to chromosomal region 12q12-q14.1, which contains the type II hair keratin and HOXC clusters. Hoxc13-null mice are known to develop hair and nail defects very similar to those seen in human PHNED. We performed whole-exome sequencing in a consanguineous Chinese family affected by PHNED and identified a homozygous nonsense mutation (c.390CA [p.Tyr130(∗)]) in HOXC13 in all affected individuals. In an additional affected female from a consanguineous Afghan family, we found a 27.6 kb homozygous microdeletion involving the first exon of HOXC13. We examined HOXC13 expression in scalp specimen obtained from the index individual of the Chinese family and detected dramatically reduced mRNA levels in skin tissue and nearly absent protein staining in hair follicles, suggesting a mechanism of nonsense-mediated mRNA decay. We also observed markedly decreased expression of four HOXC13 target genes in the specimen. Taken together, our results demonstrate that loss-of-function mutations in HOXC13 cause autosomal-recessive PHNED and further highlight the importance of HOXC13 in hair and nail development.

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer (EMMPRIN), plays an essential role in tumor progression and metastasis, the expression of which on cell surface is a critical clinical testing index for cancer therapy. In this work, an electrochemical method to assay CD147/EMMPRIN expression on tumor cell surface is proposed. While the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by horseradish peroxidase (HRP) can be employed for electrochemical measurement, the signal enhancement amplified by gold nanoparticles (GNPs) can be also utilized in this study. Therefore, under optimized conditions, the fabricated biosensor responds linearly to the CD147/EMMPRIN concentration from 125 to 1000 pg/mL with a detection limit as low as 52 pg/mL. High sensitivity can also be achieved for the quantification of breast cancer cells in a linear range from 6.2 × 10(4) to 6.25 × 10(5) cells/mL. Moreover, the CD147/EMMPRIN expressed on a single breast cancer cell can be calculated as 2.57 × 10(4) molecules/cell. The proposed strategy in this study is considerably potential for monitoring the dynamic protein expression on cancer cells and for the effective cancer diagnosis and treatment in the future.

Background Hartnup disease is a rare autosomal-recessive abnormality of renal and gastrointestinal neutral amino acid transport associated with neurologic, psychiatric, and dermatologic symptoms. Mutations in the SLC6A19 gene have been proposed to be responsible for the underlying changes in this disorder. Aim To investigate a pedigree with Hartnup disorder and to search for the mutation in the SLC6A19 gene in this pedigree. Methods The encoding exons of the SLC6A19 gene were amplified and sequenced from genomic DNA samples. Amino acids were determined in urine samples from the proband and her family members. Results The proband and her brother had a homozygous mutation of c.850G > A in the SLC6A19 gene, causing G284R in the transmembrane domain of the SLC6A19 transporter, inherited from their parents who were heterozygous carriers. Their urine samples showed increased values of eight neutral amino acids. Conclusion We found a novel homozygous mutation of G284R in the transmembrane domain of the SLC6A19 transporter in the proband, with typical dermatologic and neurologic manifestations and increased levels of urinary neutral amino acids.

Background Castleman tumor, a rare lymphoproliferative disorder, is one of the associated tumors in paraneoplastic pemphigus. We analyzed the characteristics of a group of patients with Castleman tumor to clearly understand and to improve the prognosis of the disease. Observations Ten cases of paraneoplastic pemphigus associated with Castleman tumor treated in the Department of Dermatology, Peking University First Hospital, Beijing, China, from May 1, 1999, to March 31, 2004, were analyzed for clinical aspects, characteristics and histologic features of the tumors, and computed tomographic findings. Literature was reviewed and data were compared with our cases. Castleman tumor was a frequently reported neoplasm in association with paraneoplastic pemphigus in China. The disease was found to be caused by an autoimmune reaction originating from the B lymphocytes in the Castleman tumor. Conclusions Castleman tumor in association with paraneoplastic pemphigus is a commonly reported subtype of paraneoplastic pemphigus in China. Early detection and removal of the Castleman tumor are crucial for the treatment of this tumor-associated autoimmune disease.

Dyskeratosis congenita (DKC) is a rare and fatal congenital syndrome characterized by the triad of reticular skin pigmentation, nail dystrophy and mucosal leukoplakia, and the predisposition to bone marrow failure and malignancies. Mutations in DKC1 gene encoding dyskerin are responsible for the X-linked dyskeratosis congenita. Here we report mutation analysis of two Chinese pedigrees with dyskeratosis congenita. The 15 coding exons of DKC1 and their flanking regions were amplified from genomic DNA by PCR. DNA sequencing and restriction endonuclease digestion were used for mutation detection. Transition mutation of 1226C→T (P409L) found in the first pedigree is a novel mutation. In the second pedigree, the proband's mother phenotypically normal carried a de novo transition mutation of 1058C→T (A353 V) in one allele, and transmitted the mutant allele to her two sons who had typical manifestations of dyskeratosis congenita.

Ureaplasma urealyticum is a causative agent of non-gonococcal urethritis and is implicated in the pathogenesis of several other diseases. However, U. urealyticum is also frequently found in the normal genitourinary tract. To characterize the distribution pattern of biovars and genotypes in normal physical check-up women and in sex workers, cervical swabs taken from 261 physical check-up clients and 98 sex workers were cultured. Positive cultures were further biotyped and genotyped by PCR. The data indicate that a) U. urealyticum is more frequently isolated in sex workers than in physical check-up women (p < 0.001); b) infection with only one genotype (genotype 1, 3 or 6) of biovar 1 is frequently found in physical check-up women; c) biovar 2 infection and mixed infection caused by more than one genotype of biovar 1 are more prevalent in sex workers than in physical check-up women (p < 0.001 and p < 0.01, respectively); d) no difference in distribution of genotype 1, 3 and 6 of biovar 1 is found between sex workers and physical check-up women (p = 0.396); e) the PCR method described here is relatively simple, rapid and specific for the biotyping between biovar 1 and 2 and genotyping of genotypes 1, 3, 6, and 14 in biovar 1.

Dystrophic epidermolysis bullosa pruriginosa, a subtype of epidermolysis bullosa dystrophica and a heterogeneous inherited disease, is characterized by pruritus, excoriated nodular prurigo-like lesions, skin fragility, altered anchoring fibrils and loss of dermal-epidermal adhesion. Mutation in type VII collagen gene (COL7A1) is thought to be implicated in the underlying change for dystrophic epidermolysis bullosa pruriginosa. We report here a large family of dominant dystrophic epidermolysis bullosa pruriginosa. Mutation analysis using polymerase chain reaction and direct sequencing demonstrated a novel nucleotide substitution of 6899A-->G in exon 87 in one COL7A1 allele of the proband and 18 affected family members. This substitution was not found in 100 normal alleles. Polymerase chain reaction and sequencing of the cDNA, reverse transcribed from the proband's peripheral lymphocyte RNA, suggest that this mutation causes aberrant COL7A1 mRNA splicing of exon 87 skipping. Clinical features and pedigree analysis suggest that 6899A-->G substitution is a mutation with full penetrance and variable expressivity.

Ureaplasma urealyticum is a causative agent of nongonococcal urethritis and is implicated in the pathogenesis of several other diseases. The species is divided into 14 serovars and two biovars, of which biovar 1 is most commonly isolated from clinical specimens. Reported associations between individual serovars and diseases have been difficult to confirm because of practical difficulties with serotyping. The multiple-banded antigen (MBA) is the predominant U. urealyticum antigen recognized during infections in humans and probably has a significant role in virulence. The 5′ end of the MBA gene is relatively conserved but contains biovar, and possibly serovar, specificity. The 5′ ends of the MBA genes of standard strains of U. urealyticum biovar 1, consisting of serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and sequenced to identify serovar-specific differences. The 5′ end of the MBA gene sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14. Significant differences between the MBA gene sequences allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125–UMA269 and UMS-125–UMA269′. Serovars 1 and 6 were distinguished by restriction enzyme analysis of the amplicon and/or by PCR specific for serovar 6. These methods were used to identify and type U. urealyticum in 185 (46.3%) of 400 genital specimens from women. Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive specimens. Of 165 specimens containing U. urealyticum biovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and 25.5% contained serovars 1, 3/14, and 6, respectively. U. urealyticum was found in a significantly higher proportion of pregnant women than in sex workers and other women attending a sexually transmissible diseases clinic ( P < 0.01). The methods described are relatively rapid, practicable, and specific for serotyping isolates and for direct detection and identification of individual serovars in clinical specimens containing U. urealyticum biovar 1.

Background Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare and distinct type of T-cell lymphoma. It is frequently associated with hemophagocytic syndrome (HPS), whose etiologic factors always involve Epstein-Barr Virus(EBV) infection. Although EBV is absent in many cases of sporadic SPTCL, we found a case of SPTCL with full facial edema, HPS and EBV infection as the first symptom. Patient and treatment A 36-year-old female had presented with fever, full facial edema for 20 days before hospitalization. Remarkable abnormalities were found as hypersplenotrophy, pancytopenia, hypofibrinogenemia, high ferritin and soluble interleukin-2-receptor level, hemophagocytosis in bone marrow smear, which indicated HPS. Detection by PCR of EBV-DNA in peripheral blood was 9.73×104 copies. On the next day of her hospitalization, HLH-2004 protocol (VP-16+dexamethasone+cyclosporine A) was performed. Her temperature fell to normal on the 4th day. Full facial edema vanished gradually in the following days. After 8 weeks’ treatment, all the abnormalities in the laboratory examination returned to normal. Despite of cyclosporine A and prednisone were used as continual treatment, facial edema and fever relapsed accompanied by multiple skin lesions on her back, upper and lower limbs 4 months later. Reexamination of the laboratory index related above were abnormal once again. Copy number of EBV-DNA was 5.52×105. Histologic and immunohistochemical features of tissue biopsy from the skin lesion on her right arm revealed the evidence of SPTCL. Then, she was treated by asparaginase plus DICE regimen. The facial edema and skin lesion vanished gradually during the course of treatment. Now (one month after the first course of chemotherapy), the patient is in her second course of asparaginase plus DICE regimen and SPTCL remains remission. Conclusion This was not a typical case of SPTCL. The patient presented with overt skin lesion 4 months after the first symptom of full facial edema, which suggested SPTCL is a heterogenous disease with variable presentations. Awareness of this rare entity is critical to both ensure timely diagnosis and treatment. In addition, the pathogenesis of SPTCL remains unclear. Some malignant lymphomas are induced by chronic inflammation due to viral or bacterial infection. It is well known that EBV infection often causes malignant lymphomas of B-cell type, particularly in elderly peoples and immunocompromised individuals. Although EBV is absent in many cases of sporadic SPTCL, the virus appears to be implicated in the etiology of variant SPTCL occurring in the Asian population. Disclosures: No relevant conflicts of interest to declare.

Relapsed, refractory Hodgkin’s or non-Hodgkin’s lymphoma remains to be a challenge and lacks efficient treatment. A small number of tumor cells that escape from treatment become resistant or unresponsive to chemotherapeutic agents. These cells grow rapidly and soon regenerate into large tumors. Immune vaccination with dendritic cells (DCs) efficiently inhibits growth of small size tumor only. The progression of tumors induces accumulation of Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the lymphatic organs and vicinity of tumor cells. MDSCs enable tumor cells to escape from host or exogenous immune cells mediated surveillance and attack. Gemcitabine is the chemotherapeutic agent used for lymphoma treatment. Gemcitabine has been reported to selectively eliminate MDSCs in tumor bearing animals. Thus, gemcitabine not only eliminates the majority of tumor to leave fewer resistant cells, it also reduces MDSCs to improve the immune environment favorable for the subsequent tumor vaccination. In this presentation, we used A20 cells to establish a large size, murine B-cell lymphoma model. We confirmed the accumulation of MDSCs in the spleen of lymphoma-bearing mice. Gemcitabine induced increased apoptosis in isolated, cultured MDSCs. In vivo injection of gemcitabine eliminated the majority of MDSCs in the spleen of lymphoma-bearing mice. Gemcitabine treatment combined with intratumoral injection of DCs markedly reduced the size of tumor, and significantly improved the survival of the lymphoma-bearing mice. We demonstrated that intra-large-size-lymphoma injection with inactivated DCs in combination with gemcitabine chemotherapy enhanced the therapeutic efficacy of either DC-based vaccination or chemotherapeutic reagent alone. Together with other studies, our report supports the hypothesis that gemcitabine-mediated reduction in MDSCs significantly reduces the immune suppression in tumor-bearing animal environment. This study established novel experimental foundation for the combination of immune-chemotherapy to treat relapsed or refractory lymphoma in large size with enhanced efficacy. Disclosures: No relevant conflicts of interest to declare.

Abstract 965 Chronic Myelogenous Leukemia (CML) is driven by the fusion oncogene, BCR-ABL, which transforms normal hematopoietic stem cells (HSCs) to leukemic stem cells (LSCs). Tyrosine kinase inhibitors, such as imatinib mesylate, control the massive expansion of leukemic cells in most patients with CML, but cannot eradicate CML LSCs. Several genetic pathways have been shown to be critical for the growth and survival of CML LSCs, including signaling molecules, tumor suppressors, and metabolic regulators. However, the role of transcription factors in functional regulation of LSCs in CML has not been widely studied. GA Binding Protein (GABP) is an ets transcription factor that is required for entry of fibroblasts into the cell cycle, and expression of Gabpa (the DNA-binding component of the complex), alone, was sufficient to induce quiescent, serum-starved cells to enter the cell cycle. Thus, Gabp is both necessary and sufficient for cell cycle entry. Conditional deletion of Gabpa in mouse bone marrow decreased hematopoietic progenitor cells more than 100-fold, but hematopoietic stem cells (HSCs) were relatively preserved. Gabpα null HSCs exhibited significant cell cycle arrest. We sought to determine if the cell cycle arrest caused by Gabpa loss could impair development of CML cells in a mouse model. We used retroviral infection of bone marrow from 5-FU-treated mice (to enrich for stem and progenitor cells) to generate a rapidly fatal CML-like syndrome in mice. Bone marrow from mice with loxP-flanked (floxed) Gabpa and wild type control mice was infected with a retrovirus that co-expresses BCR-ABL, Cre recombinase, and green fluorescent protein (GFP). As expected, transplantation into recipient mice of control mouse bone marrow infected with BCR-ABL-Cre-GFP retrovirus caused a rapidly fatal myeloproliferative neoplasm, with a median survival of approximately three weeks; mice died with massive infiltration of GFP+ myeloid cells in peripheral blood cell, spleen, bone marrow, and other organs. In floxed Gabpa bone marrow, the retrovirus deleted floxed Gabpa in cells that express the BCR-ABL fusion oncogene, and these cells were identifiable based on GFP expression. Transplantation of floxed Gabpa bone marrow infected with BCR-ABL-Cre-GFP retrovirus failed to induce CML during six months of observation. Importantly, GFP+ peripheral blood granulocytes were observed for at least 6 months after transplantation; these CD11b+, Gr1+ cells continued to express BCR-ABL and were shown to be Gabpa null. These results indicate that the lack of Gabpa severely impaired the function of LSCs. In addition, secondary transplantation of bone marrow from these mice again demonstrated the presence of BCR-ABL-expressing peripheral blood myeloid cells. We conclude that Gabp transcription factor is required for the transformation of HSCs to LSCs by BCR-ABL. Furthermore, the persistence of BCR-ABL-expressing myeloid cells without the development of leukemia provides a unique model that permits analysis of the biological properties of BCR-ABL in vivo. The continued generation of BCR-ABL-expressing cells without CML development is unprecedented, and represents a unique model of leukemia tumor suppression. Disclosures: No relevant conflicts of interest to declare.

Assay of phosphotyrosine levels using flow cytometry has been used to identify patients with chronic myelogenous leukemia (CML) positive for the Bcr-Abl fusion gene. We hypothesized that clinical monitoring could identify treatment response through reductions in intra-granulocyte phosphotyrosine. Initially, we studied cell lines FDC-P1 and HL60 (Bcr-Abl–negative) and FDrv210 and K562 (Bcr-Abl–positive) with our technique. A fluorescein isothiocyanate-conjugated monoclonal antibody was used along with fluorescence-conjugated microspheres for reference (ratio of sample fluorescence: bead fluorescence=relative fluorescence unit [RFU]). Samples from 20 controls and 32 patients undergoing treatment were analyzed using the same method. Bcr-Abl status for each patient was confirmed using fluorescent in situ hybridization (FISH) or polymerase chain reaction gene amplification (PCR). Testing of cell lines consistently produced expected results. Patient values were found to be significantly higher than control values (P