HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency.

Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways.

Cited2 in hematopoietic stem cell function.

PURPOSE OF REVIEW: Transcription co-regulator Cited2 is essential for mouse development. Recent work has shown that Cited2 plays important roles in normal hematopoiesis in fetal liver and adult bone marrow. This review focuses on the function of Cited2 in the maintenance of hematopoietic stem cells (HSCs) and its potential role in the metabolic regulation of HSCs. RECENT FINDINGS: Fetal liver cells from Cited2 null embryos give rise to reduced numbers of hematopoietic colonies and display significantly impaired hematopoietic reconstitution capacity. In adult mice, conditional deletion of Cited2 markedly reduces the number of HSCs and compromises hematopoietic reconstitution in mice receiving a transplant of Cited2 deficient bone marrow cells. Additional deletion of Ink4a/Arf or p53 in a Cited2-deficient background restores HSC functionality. Meanwhile, Cited2 deficient HSCs display loss of quiescence, which can be partially rescued by additional deletion of hypoxia inducible factor-1α. SUMMARY: Cited2 is an essential regulator in fetal liver and adult hematopoiesis. Further studies into the function of Cited2 and the underlying mechanism in the metabolic regulation of HSCs will provide a better understanding of the connection between energy metabolism and HSC quiescence and self-renewal. Investigations of the pathologic role of Cited2 in leukemogenesis may yield useful information in developing effective therapeutic strategies.

A new testing platform using fingerstick blood for quantitative antibody response evaluation after SARS-CoV-2 vaccination.

Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 µg/mL and was found to be linear in the range of 0.51-1000 µg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses.

A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics.

The pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0-7 days, 8-14 days, and 2-8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.

A high-throughput Anti-SARS-CoV-2 IgG testing platform for COVID-19.

BACKGROUND: Serology tests for detecting the antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can identify previous infection and help to confirm the presence of current infection. OBJECTIVE: The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgG antibody detection. RESULTS: Clinical agreement studies were performed in 107 COVID-19 patient serum samples and 226 negative donor serum/plasma samples. Positive percent agreement (PPA) was 46.15 % (95 % CI: 19.22 % ∼74.87 %), 61.54 % (95 % CI: 31.58 % ∼86.14 %), and 97.53 % (95 % CI: 91.36 % ∼99.70 %) for samples collected on 0-7 days, 8-14 days, and ≥15 days from symptom onset, respectively. Negative Percent Agreement (NPA) was 98.23 % (95 % CI: 95.53 % ∼99.52 %). No cross-reactivity was observed to patient samples positive for IgG antibodies against the following pathogens: HIV, HAV, HBV, RSV, CMV, EBV, Rubella, Influenza A, and Influenza B. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. CONCLUSION: An anti-SARS-CoV-2 IgG antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up anti-SARS-CoV-2 IgG testing.

A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics. METHODS: Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance. RESULTS: The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6-99.7%) and 92% sensitivity (95% CI, 86.1-95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC of 0.79 for precancerous lesions cfDNA. CONCLUSIONS: The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

Kinetics of plasma cfDNA predicts clinical response in non-small cell lung cancer patients.

Tyrosine kinase inhibitors (TKIs), VEGF/VEGF receptor inhibitors (VEGFIs) and immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers including non-small-cell lung cancer (NSCLC). This study aims to evaluate the utility of plasma cell-free DNA (cfDNA) as a prognostic biomarker and efficacy predictor of chemotherapy (CT) with or without these precision therapies in NSCLC patients. Peripheral cfDNA levels in 154 NSCLC patients were quantified before and after the first target cycle of chemotherapy. The correlations of cfDNA with tumor burden, clinical characteristics, progression-free survival (PFS)/disease-free survival (DFS), objective response ratio (ORR), and therapy regimens were analyzed respectively. Baseline cfDNA, but not post-chemotherapeutic cfDNA, positively correlates with tumor burden. Notably, cfDNA kinetics (cfDNA Ratio, the ratio of post-chemotherapeutic cfDNA to baseline cfDNA) well distinguished responsive individuals (CR/PR) from the non-responsive (PD/SD). Additionally, cfDNA Ratio was found negatively correlated with PFS in lung adenocarcinoma (LUAD), but not lung squamous-cell carcinoma (LUSC) which may be due to a limited number of LUSC patients in this cohort. LUAD patients with low cfDNA Ratio have prolonged PFS and improved ORR, compared to those with high cfDNA Ratio. When stratified by therapy regimen, the predictive value of cfDNA Ratio is significant in patients with chemotherapy plus VEGFIs, while more patients need be included to validate the value of cfDNA Ratio in other regimens. Thus, the kinetics of plasma cfDNA during chemotherapy may function as a prognostic biomarker and efficacy predictor for NSCLC patients.

Evaluation of a novel liquid biopsy-based ColoScape assay for mutational analysis of colorectal neoplasia and triage of FIT+ patients: a pilot study.

Circulating cell free tumour derived nucleic acids are becoming recognised as clinically significant and extremely useful biomarkers for detection of cancer and for monitoring the progression of targeted drug therapy and immunotherapy. Screening programmes for colorectal cancer in Europe use the Fetal Immunochemical Test (FIT) test as a primary screener. FIT+ patients are referred to immediate colonoscopy and the positive predictive value (PPV) is usually 25%. In this article, we report a study employing the ColoScape assay panel to detect mutations in the APC, KRAS, BRAF and CTNNB1 genes, in order to collect preliminary performance indicators and plan a future, larger population study. The assay was evaluated on 52 prospectively collected whole-blood samples obtained from FIT+ patients enrolled in the CRC screening programme of ASL NAPOLI 3 SUD, using colonoscopy as confirmation. The assay's sensitivity for advanced adenomas was 53.8% and the specificity was 92.3%. The PPV was 70.0% and negative predicitive value (NPV) was 85.7%. Workflow optimisation is essential to maximise sensitivity. Of note, four of the six positive cases missed by ColoScape had a less than suboptimal DNA input (data not shown). Had they been ruled out as inadequate, sensitivity would have increased from 53.8% to 69%. However, as stated previously, this is not a clinical trial, but rather an initial, preliminary technical evaluation. In conclusion this study shows that ColoScape is a promising tool and further studies are warranted in order to validate its use for the triage of FIT+ patients.

TCR spectratyping revealed T lymphocytes associated with graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Clonal expansion of T cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been observed, but their characteristics remain to be fully elucidated. We report here that CD8(+) T cells were the dominant T lymphocytes seen and T-cell repertoire diversity decreased dramatically during the first 3 months after allo-HSCT. Patients with GVHD grade II - IV had significantly lower T-cell repertoire diversity compared with non-GVHD patients. TCR beta variable gene (TCRBV) subfamily 8, 5.1, 5.2, 4, and 13 were the five most frequently expanded subfamilies among these patients. Among the 49 over-expanded clones identified, clonotype "TCR3-5" and "TCR18-5" were isolated from four patients with HLA-A2 allele and skin GVHD. Their frequencies correlated well with skin symptoms (i.e. rash). Moreover, they were detected in donors but not detected in recipients before transplantation. Lastly, three common TCRBV CDR3 motifs shared by T cells related with GVHD were discovered: TGDS, GLAG, and GGG. These findings suggest that TCR spectratyping is helpful for revealing GVHD-related T cells and may have utility in early diagnosis.

[Real-time PCR array for simultaneous detection of 37 kinds of fusion genes in leukemia].

OBJECTIVE: To develop a real-time PCR array for simultaneous quantitative detection of translocations/chromosomal aberrations in patients with leukemia, and to investigate the feasibility and utility thereof. METHODS: By construction and optimization a set of specific primes (totally 82 primers), an array containing 66 parallel PCR reactions was developed. That array was used on the specimens of bone marrow or peripheral blood from 31 patients with leukemia to detect simultaneously 37 fusion genes and 4 proto-oncogene activations often occurring in patients with leukemia. Eva Green fluorescent dye method was chosen in the protocol. Relative quantification was performed by Ct analysis and the result was expressed as the ratio of the target gene versus the internal control gene (ABL). Six patients with chronic myelocytic leukemia (CML) among the 31 cases underwent prior to and after treatment so as to study the expression changes of fusion genes and/or proto-oncogene. RESULTS: The established PCR array showed high efficiency of amplification and good sensibility (232 copies/microl) in the fusion gene detected. The standard curve had a satisfying linear range (10(2) approximately 10(8) copies/microl), showing a good reproducibility. Fourteen fusion genes, including PML/RARalpha, PLZF/RARalpha, BCR/ABL, MLL/AF1, MLL/AF6, MLL/AF10, AML/Eto, CBFbeta/MYH11, TLS/ERG, TEL/AML1, MOZ/CBP, MLL/hCDCrel, LAF4/MLLT2, and FIP1L1/PDGFRalpha, and activation of all 4 proto-oncogenes were found in the 31 samples. In one patient, 5 fusion genes and activation of 2 proto-oncogenes were observed. Such results were compared with those of RT-nested PCR in 28 samples. The comparison showed that this array was a bit less sensitive than RT-nested PCR, however, without significant difference between them (P = 0.009). The expression of BCR/ABL fusion gene, WT1 gene, and EVI1 gene decreased after treatment in the 6 CML patients, which was in accordance with the clinical features. CONCLUSION: The PCR array newly-established successfully detects various leukemia related fusion genes and proto-oncogene activation. It is useful in molecule diagnosis and monitoring minimal residual disease in leukemia, and therapeutic effect monitoring.

[Role of molecular screening for common fusion genes in the diagnosis and classification of leukemia].

OBJECTIVE: To assess the value of common fusion genes analysis in the diagnosis and classification of leukemia by multiplex RT-PCR. METHODS: The multiplex RT-PCR, including 8 parallel PCR reactions, could screen 86 mRNA breakpoints or splice variants at the same time, which was important for the diagnosis and prognosis of leukemia. Bone marrow samples from 161 cases of leukemia and 8 cases of myelodysplastic syndrome (MDS) were involved in the study. The distribution of common fusion genes in leukemia was analyzed by the method mentioned above in combination with clinical and morphological features. RESULTS: Ten fusion genes were detected in 115 cases of leukemia, including AML1/ETO, PML/RAR alpha, PLZF/RAR alpha, dupMLL, MLL/AF6, MLL/AF10, CBFbeta/MYH11, BCR/ABL, Hox11, and EVI1 BCR/ABL was positive in all the 52 cases of chronic myeloid leukemia; PML/RAR alpha was found in 21 of 25 acute promyelocytic leukemia (APL), and PLZF/RAR alpha was detected in one case of APL. Sixteen cases of 17 AML1/ETO-positive acute leukemia (AL) belonged to FAB-M2 subtype, and one case was mixed leukemia. Three of 4 AL cases carrying CBFbeta/MYH11 were M4 subtype, and one was M5 subtype. MLL aberrations were found in 16 AL, in which all MLL/AF6 translocation existed in M5 subtype with classic monoblastic characters. Furthermore, BCR/ABL was detected in 5 acute lymphoblastic leukemia (ALL) cases. Fusion genes were also found in 2 MDS cases, of which AML1/ETO positive-MDS-RAEB progressed to AML rapidly. CONCLUSION: Screening of common fusion genes by multiplex RT-PCR is an important tool which could provide useful and reliable molecular genetic information for the diagnosis and treatment of leukemia.

[The characteristics of anti-B-cell malignance cytotoxic T lymphocytic clones induced by lymphoma relative peptides].

OBJECTIVE: To analyze if the cytotoxic T-cell (CTLs) are peptide-special and HLA restrict, and what is the sequence characteristic of TCRbeta genes. METHODS: Using an antigen-specific T-cell expansion system in vitro, the peripheral blood mononuclear cells (PBMCs) from healthy HLA-A0201 positive donor were stimulated by PBMCs and T2 cells loading the IgHV1-QLVQSGAEV nonapeptide or IgHV3-QLVQSGAEV, B-lymphoma-related nonapeptides, as antigen presenting cells (APCs) once a week for four weeks so as to obtain peptide-specific CTLs. PBMCs from non-HLA-A * 0201 positive donors were used as controls. The immunophenotypes of the CTLs (CD3, CD4, or CD8) were identified by flow cytometry. The proportions of CD8 and peptide/tetramer double positive cells were assayed by using peptide/ HLA tetramer method, The IFN-gamma-releasing capacity of the CTLs incubated together with different target cells was assayed by ELISA. The changes of lymphocyte clones were analyzed TCR beta genes were identified by RT-PCR and spectral type method and then sequenced. RESULTS: After four times stimulation, the CD4/CD8 ratio of the cultured cell decreased obviously from 1.43 to 0.10 (P < 0.05), showing a proliferation of CD8(+) CTLs. The frequency of CD8 and IgHV1-QLVQSGAEV/ HLA-A * 0201 tetramer double positive CTLs was 49.83%, significantly higher than that before stimulation (0.04%). The IFN-gamma secretion detected by ELISA indicated that these CTLs were capable of recognizing the target cells in a peptide-specific and MHC-restricted way. Spectral type method showed that the TCRbeta repertoires were skewed in only a few TCR families. CONCLUSION: The peptides derived from IgHV succeeds in generation of peptide-specific CTLs in vitro in a clonality manner. These CTLs are capable of recognizing the target cells in a peptide-specific and MHC-restricted way, Understanding the function of these CTLs and the molecular structures of these TCR identification-related antigen peptides help discover the interaction between the B-cell and T-cell clones.

[Comparison of therapeutic effects of low-dose versus high-dose interferon alpha-2b treatment on chronic myelocytic leukemia: a prospective randomized study].

OBJECTIVE: To compare the therapeutic effects of low-dose and high-dose interferon alpha-2b (IFN) treatment on chronic myelocytic leukemia (CML). METHODS: A real-time quantitative reverse transcriptase PCR (RQ-PCR) method was established to detect the fusion gene bcr-abl expression, thereby studying the reduction of leukemic cells. Thirty newly diagnosed CML patients, 21 males and 9 females, aged 14 - 69, were treated with hydroxyurea to keep the white blood cell count less than 20 x 10(9)/L, and then randomized into 2 groups: high-dose IFN group receiving IFN alpha-2b 5MIU 6 times per week for 3 - 6 months and low-dose IFN group receiving IFN alpha-2b 3MIU every other day for 3 - 6 months. Bone marrow was collected every month to Real-time PCR was used to detect the expression of bcr-abl mRNA. Mononuclear cells were isolated and RNA was extracted to detect the expression of fusion gene bcr-abl and a control gene GAPDH. The results were reported as the number of bcr-abl copies/GAPDH copy. RESULTS: The established real-time quantitative PCR method could detect the bcr-abl molecules as low as 50 copies. The intra-assay coefficient of variation (CV) was less than 5% and the inter-assay CV was 5.13%. The median bcr-abl fusion gene expression level of 30 CML patients before IFN therapy was 0.098 (range: 0.010 - 5.799). The bcr-abl expression level decreased by 19.37% and 24.86% in the low-dose and high-dose IFN groups respectively after 3 months' therapy. No significant difference was observed between the two groups (P = 0.398). Relatively more side effects were observed in the high-dose IFN group than in low-dose group. CONCLUSION: RQ-PCR is a reliable method to monitor CML therapy by analyzing fusion gene bcr-abl expression. There is a difference in bcr-abl fusion gene expression levels among the newly diagnosed patients, and low-dose IFN is as effective as high-dose IFN in reducing bcr-abl expression but with less side effects.

Cited2 is required for the maintenance of glycolytic metabolism in adult hematopoietic stem cells.

Mammalian adult hematopoietic stem cells (HSCs) reside in the hypoxic bone marrow microenvironment and display a distinct metabolic phenotype compared with their progenitors. It has been proposed that HSCs generate energy mainly through anaerobic glycolysis in a pyruvate dehydrogenase kinase (Pdk)-dependent manner. Cited2 is an essential regulator for HSC quiescence, apoptosis, and function. Herein, we show that conditional deletion of Cited2 in murine HSCs results in elevated levels of reactive oxygen species, decreased cellular glutathione content, increased mitochondrial activity, and decreased glycolysis. At the molecular level, Cited2 deficiency significantly reduced the expression of genes involved in metabolism, such as Pdk2, Pdk4, and lactate dehydrogenases B and D (LDHB and LDHD). Cited2-deficient HSCs also exhibited increased Akt signaling, concomitant with elevated mTORC1 activity and phosphorylation of FoxOs. Further, inhibition of PI3/Akt, but not mTORC1, partially rescued the repression of Pdk4 caused by deletion of Cited2. Altogether, our results suggest that Cited2 is required for the maintenance of adult HSC glycolytic metabolism likely through regulating Pdk2, Pdk4, LDHB, LDHD, and Akt activity.

HLA-DRB1*09 is associated with increased incidence of cytomegalovirus infection and disease after allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, we have little information on the clinical association of various human leukocyte antigen (HLA) alleles with CMV infection. We reviewed medical records of 60 patients who underwent allo-HSCT. The effect of the 7 most frequent HLA alleles on the incidence of CMV infection and disease was analyzed, including HLA-A*02, A*11, A*24, B*13, B*40(60), DRB1*15, and DRB1*09. All the patients were monitored for CMV infection at least once weekly within 3 months. CMV infection was found in 38 (63.3%) patients on a median of day 36 (range: 16-89). Diagnosis of CMV disease was established in 6 (10.0%) patients, comprising pneumonia (n = 2), enterocolitis (n = 2), and hemorrhagic cystitis (n = 2). CMV disease was successfully treated using ganciclovir or foscarnet combined with immune globulins in 4 patients. The other 2 patients died without improvement of CMV disease. In multivariate analysis, grade II-IV acute graft-versus-host disease (aGVHD), CMV seronegative donors, and HLA-DRB1*09 were associated with increased incidence of CMV infection and disease after allo-HSCT. We suggest that more cautions should be taken to prevent CMV infection in patients with HLA-DRB1*09 after allo-HSCT.

Identification of disease-associated proteins by proteomic approach in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic systemic inflammatory disorder of the axial skeleton and shows significant inherited susceptibility. Auto-immune responses have been traditionally considered as a putative pathogenetic event in AS. However, no consistent self-antigen has been identified to responsible for the disorders in AS to this day. In this study, serum protein profiles of AS patients and healthy controls from a large Chinese AS family were investigated by two dimensional electrophoresis analysis. A group of four highly expressed protein spots was observed in all AS patients' profiles and subsequently identified as isoforms of haptoglobin precursor (pre-Hp) by ESI-Q-TOF MS/MS. Increased expression of haptoglobin precursor were also observed in sera of sporadic AS patients. Moreover, bioinformatics analysis revealed epitopes derived from haptoglobin precursor with high affinity binding to HLA-B( *)2705, a primary subtype associated with AS. These results indicate that pre-Hp may be involved in the pathogenesis of AS.

[Expression of PRAME gene in acute leukemia and its clinical significance].

This study was aimed to detect the expression levels of preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia (AL) and to evaluate the clinical significance of PRAME gene. The quantitative detection method was established by SYBR Green I real-time quantitative RT-PCR, then PRAME mRNA was measured by this method in 55 cases of acute leukemia, out of which 43 cases were acute myeloid leukemia (AML), 9 cases were acute lymphocytic leukemia (ALL) and other types leukemia were 3 cases. In addition, expression of PRAME gene was also analyzed in 7 cases of non-malignant hematological diseases and 8 healthy volunteers. K562 cell line was used as a positive control. The results showed that the expression of PRAME gene was found in 35 cases of acute leukemia, the positive percentage was 64%. No expression could be detected in any of the non-malignant hematological diseases and healthy volunteers. In 35 PRAME positive cases, 28 cases were AML, which mainly belonged to M3, M4 and M2 subtypes, and 5 cases was ALL. In 31 fusion gene positive cases, 23 cases were PRAME positive, and in 24 fusion gene negative cases 12 cases were PRAME positive. No significant relationship was found between PRAME expression level and clinical characteristics (age, sex, WBC count, blast cells in BM). The expression of PRAME gene decreased or disappeared in 6 patients achieving complete remission (CR). It is concluded that the PRAME gene expresses in 64% AML patients, which mainly belonged to M3, M4 and M2 subtypes, no expression could be detected in any of the non-malignant hematological diseases and healthy volunteers. There is remarkable difference in the level of PRAME transcript of the 35 cases and the expression of PRAME gene decreases or disappears when the patients achieved complete remission. These results suggest that PRAME expression in acute leukemia may be a useful marker to detect the minimal resi-dual disease (MRD) and to determine the response to therapy in AL patients.

[Characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigens].

To understand the characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigen, the characteristics of T cells were analyzed using T cell receptor beta variable region (TCRbetaV) gene spectrotyping in a case of antiphospholipid syndrome (APS). The results indicated that in the case of APS there were 2 dominant T cell clones. The TCRbetaVs sequences of the 2 T cell clones showed the TCRbetaVs belonged to 8 and 23 gene families respectively. The peptides of third complementarity-determining regions (CDR3) in the TCRbetaVs were CASSLLVAGGPRAYNEQFFGPG and CASSLAGFGQPQHFGDG. Comparing the motifs in CDR3 with another autoimmune disease, the motif YNEQFFGPG in TCRbetaV8 and motif QHFGDG in TCRbetaV23 were identical with that of idiopathic thrombocytopenic purpura and systemic lupus erythematosus reported before. In conclusion, some T cell clones proliferating in these autoimmune diseases may recognize the same antigens.