Safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of Exendin-4-IgG4-Fc in healthy subjects: A phase 1, single-centre, randomized, double-blind, dose escalation study.

AIM: Novel long-acting drugs for type 2 diabetes mellitus may optimize patient compliance and glycaemic control. Exendin-4-IgG4-Fc (E4F4) is a long-acting glucagon-like peptide-1 receptor agonist. This first-in-human study investigated the safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of a single subcutaneous injection of E4F4 in healthy subjects. METHODS: This single-centre, randomized, double-blind, placebo-controlled phase 1 clinical trial included 96 subjects in 10 sequential cohorts that were provided successively higher doses of E4F4 (0.45, 0.9, 1.8, 3.15, 4.5, 6.3, 8.1, 10.35, 12.6 and 14.85 mg) or placebo (ChinaDrugTrials.org.cn: ChiCTR2100049732). The primary endpoint was safety and tolerability of E4F4. Secondary endpoints were pharmacokinetic, pharmacodynamic and immunogenicity profiles of E4F4. Safety data to day 15 after the final subject in a cohort had been dosed were reviewed before commencing the next dose level. RESULTS: E4F4 was safe and well tolerated among healthy Chinese participants in this study. There was no obvious dose-dependent relationship between frequency, severity or causality of treatment-emergent adverse events. Cmax and area under the curve of E4F4 were dose proportional over the 0.45-14.85 mg dose range. Median Tmax and t1/2 ranged from 146 to 210 h and 199 to 252 h, respectively, across E4F4 doses, with no dose-dependent trends. For the intravenous glucose tolerance test, area under the curve of glucose in plasma from time 0 to 180 min showed a dose-response relationship in the 1.8-10.35 mg dose range, with an increased response at the higher doses. CONCLUSION: E4F4 exhibited an acceptable safety profile and linear pharmacokinetics in healthy subjects. The recommended phase 2 dose is 4.5-10.35 mg once every 2 weeks.

No Overlap among Serum GAD65, NMDAR and AQP4 Antibodies in Patients with Neuromyelitis Optica Spectrum Disorders.

OBJECTIVE: To evaluate whether serum glutamic acid decarboxylase (GAD), N-methyl-D-aspartate-receptor (NMDAR), and aquaporin-4 (AQP4) autoantibodies coexist in patients with neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD). METHODS: Serum samples were collected from 98 patients with NMO/NMOSD. Serum GAD65, NMDAR and AQP4 antibodies were measured using a cell-based assay. RESULTS: A total of 63 patients (64.3%) had myelitis and optic neuritis and satisfied the revised diagnostic criteria for NMO. Longitudinally extensive transverse myelitis was seen on spinal cord magnetic resonance imaging, showing continuous T2-weighted signal abnormalities in at least three vertebral segments in 26 patients (26.5%); 5 patients (5.1%) had recurrent optic neuritis, and 4 patients (4.1%) had brain syndromes with optic neuritis and myelitis. None of the 98 patients had diabetes, stiff-man syndrome, or epilepsy. All 98 patients tested positive for AQP4 antibody. No patients tested positive for GAD65 and NMDAR antibodies. CONCLUSIONS: In the present study, we found no simultaneous presence of serum GAD65, NMDAR and AQP4 antibodies in patients with NMO/NMOSD.

Safety and immunogenicity of primary vaccination with a SARS-CoV-2 mRNA vaccine (SYS6006) in Chinese participants aged 18 years or more: Two randomized, observer-blinded, placebo-controlled and dose-escalation phase 1 clinical trials.

Vaccination plays a key role in preventing morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We aimed to evaluate the safety and immunogenicity of a SARS-CoV-2 messenger ribonucleic acid (mRNA) vaccine SYS6006. In the two randomized, observer-blinded, placebo-controlled phase 1 trials, 40 adult participants aged 18-59 years and 40 elderly participants aged 60 years or more were randomized to receive two doses of SYS6006 or placebo (saline). Adverse events (AEs) were collected through 30 days post the second vaccination. Immunogenicity was assessed by live-virus neutralizing antibody (Nab), spike protein (S1) binding antibody (S1-IgG), and cellular immunity. The result showed that 7/15, 9/15 and 4/10 adult participants, and 9/15, 8/15 and 4/10 elderly participants reported at least one AE in the 20-µg, 30-µg and placebo groups, respectively. Most AEs were grade 1. Injection-site pain was the most common AE. Two adults and one elder reported fever. No vaccination-related serious AE was reported. SYS6006 elicited wild-type Nab response with a peak geometric mean titer of 232.1 and 130.6 (adults), and 48.7 and 66.7 (elders), in the 20-µg and 30-µg groups, respectively. SYS6006 induced moderate-to-robust Nab response against Delta, and slight Nab response against Omicron BA.2 and BA.5. Robust IgG response against wild type and BA.2 was observed. Cellular immune response was induced. In conclusion, two-dose primary vaccination with SYS6006 demonstrated good safety and immunogenicity during a follow-up period of 51 days in immunologically naive population aged 18 years or more. (Trial registry: Chictr.org.cn ChiCTR2200059103 and ChiCTR2200059104).

Phase I study of a non-S2P SARS-CoV-2 mRNA vaccine LVRNA009 in Chinese adults.

BACKGROUND: COVID-19 caused by SARS-CoV-2 is a great threat to public health. We present the safety and immunogenicity data from a phase I trial in China of an mRNA vaccine (LVRNA009). METHODS: In the single-centre, double-blind, placebo-controlled and dose-escalation study, 72 healthy unvaccinated adults aged 18-59 years were randomized (3:1) to receive LVRNA009 with one of three vaccine dosage (25, 50 and 100 µg) or placebo, to evaluate for the safety, tolerability and immunogenicity of LVRNA009. RESULTS: All these participants received two injections 28 days apart. No adverse events higher than grade 2 were reported during the study. A total of 30 participants (42 %) reported solicited adverse reactions during the first 14 days after vaccinations. Of the events reported, fever (n = 11, 15 %) was the most common systemic adverse reaction, and pain at the injection site (n = 17, 24 %) was the most frequent solicited local adverse reaction. Anti-S-protein IgG and neutralising antibodies were observed to have been induced 14 days after the first dose, significantly increased 7 days after the second dose, and remained at a high level 28 days after the second dose. Specific T-cell responses peaked 7 days and persisted 28 days after second vaccination. CONCLUSION: LVRNA009 has demonstrated promising results in safety and tolerability at all three dose levels among Chinese adults. LVRNA009 at three dose levels could rapidly induce strong humoral and cellular immune responses, including binding and neutralising antibody production and IFN- γ secretion, which showed good immunogenicity. CLINICAL TRIAL REGISTRATION NUMBER: Clinicaltrials.gov NCT05364047; Chictr.org.cn ChiCTR2100049349.

Safety and immunogenicity of the SARS-CoV-2 ARCoV mRNA vaccine in Chinese adults: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Safe and effective vaccines are urgently needed to end the COVID-19 pandemic caused by SARS-CoV-2 infection. We aimed to assess the preliminary safety, tolerability, and immunogenicity of an mRNA vaccine ARCoV, which encodes the SARS-CoV-2 spike protein receptor-binding domain (RBD). METHODS: This single centre, double-blind, randomised, placebo-controlled, dose-escalation, phase 1 trial of ARCoV was conducted at Shulan (Hangzhou) hospital in Hangzhou, Zhejiang province, China. Healthy adults aged 18-59 years negative for SARS-CoV-2 infection were enrolled and randomly assigned using block randomisation to receive an intramuscular injection of vaccine or placebo. Vaccine doses were 5 µg, 10 µg, 15 µg, 20 µg, and 25 µg. The first six participants in each block were sentinels and along with the remaining 18 participants, were randomly assigned to groups (5:1). In block 1 sentinels were given the lowest vaccine dose and after a 4-day observation with confirmed safety analyses, the remaining 18 participants in the same dose group proceeded and sentinels in block 2 were given their first administration on a two-dose schedule, 28 days apart. All participants, investigators, and staff doing laboratory analyses were masked to treatment allocation. Humoral responses were assessed by measuring anti-SARS-CoV-2 RBD IgG using a standardised ELISA and neutralising antibodies using pseudovirus-based and live SARS-CoV-2 neutralisation assays. SARS-CoV-2 RBD-specific T-cell responses, including IFN-γ and IL-2 production, were assessed using an enzyme-linked immunospot (ELISpot) assay. The primary outcome for safety was incidence of adverse events or adverse reactions within 60 min, and at days 7, 14, and 28 after each vaccine dose. The secondary safety outcome was abnormal changes detected by laboratory tests at days 1, 4, 7, and 28 after each vaccine dose. For immunogenicity, the secondary outcome was humoral immune responses: titres of neutralising antibodies to live SARS-CoV-2, neutralising antibodies to pseudovirus, and RBD-specific IgG at baseline and 28 days after first vaccination and at days 7, 15, and 28 after second vaccination. The exploratory outcome was SARS-CoV-2-specific T-cell responses at 7 days after the first vaccination and at days 7 and 15 after the second vaccination. This trial is registered with www.chictr.org.cn (ChiCTR2000039212). FINDINGS: Between Oct 30 and Dec 2, 2020, 230 individuals were screened and 120 eligible participants were randomly assigned to receive five-dose levels of ARCoV or a placebo (20 per group). All participants received the first vaccination and 118 received the second dose. No serious adverse events were reported within 56 days after vaccination and the majority of adverse events were mild or moderate. Fever was the most common systemic adverse reaction (one [5%] of 20 in the 5 µg group, 13 [65%] of 20 in the 10 µg group, 17 [85%] of 20 in the 15 µg group, 19 [95%] of 20 in the 20 µg group, 16 [100%] of 16 in the 25 µg group; p<0·0001). The incidence of grade 3 systemic adverse events were none (0%) of 20 in the 5 µg group, three (15%) of 20 in the 10 µg group, six (30%) of 20 in the 15 µg group, seven (35%) of 20 in the 20 µg group, five (31%) of 16 in the 25 µg group, and none (0%) of 20 in the placebo group (p=0·0013). As expected, the majority of fever resolved in the first 2 days after vaccination for all groups. The incidence of solicited systemic adverse events was similar after administration of ARCoV as a first or second vaccination. Humoral immune responses including anti-RBD IgG and neutralising antibodies increased significantly 7 days after the second dose and peaked between 14 and 28 days thereafter. Specific T-cell response peaked between 7 and 14 days after full vaccination. 15 µg induced the highest titre of neutralising antibodies, which was about twofold more than the antibody titre of convalescent patients with COVID-19. INTERPRETATION: ARCoV was safe and well tolerated at all five doses. The acceptable safety profile, together with the induction of strong humoral and cellular immune responses, support further clinical testing of ARCoV at a large scale. FUNDING: National Key Research and Development Project of China, Academy of Medical Sciences China, National Natural Science Foundation China, and Chinese Academy of Medical Sciences.

Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

Dynamic alterations in the gut microbiota and metabolome during the development of methionine-choline-deficient diet-induced nonalcoholic steatohepatitis.

AIM: To investigate changes in gut microbiota and metabolism during nonalcoholic steatohepatitis (NASH) development in mice fed a methionine-choline-deficient (MCD) diet. METHODS: Twenty-four male C57BL/6J mice were equally divided into four groups and fed a methionine-choline-sufficient diet for 2 wk (Control 2w group, n = 6) or 4 wk (Control 4w group, n = 6) or the MCD diet for 2 wk (MCD 2w group, n = 6) or 4 wk (MCD 4w group, n = 6). Liver injury, fibrosis, and intestinal barrier function were evaluated after 2 and 4 wk of feeding. The fecal microbiome and metabolome were studied using 16s rRNA deep sequencing and gas chromatography-mass spectrometry. RESULTS: The mice fed the MCD diet presented with simple hepatic steatosis and slight intestinal barrier deterioration after 2 wk. After 4 wk of feeding with the MCD diet, however, the mice developed prominent NASH with liver fibrosis, and the intestinal barrier was more impaired. Compared with the control diet, the MCD diet induced gradual gut microbiota dysbiosis, as evidenced by a marked decrease in the abundance of Alistipes and the (Eubacterium) coprostanoligenes group (P < 0.001 and P < 0.05, respectively) and a significant increase in Ruminococcaceae UCG 014 abundance (P < 0.05) after 2 wk. At 4 wk, the MCD diet significantly reduced the promising probiotic Bifidobacterium levels and markedly promoted Bacteroides abundance (P < 0.05, and P < 0.01, respectively). The fecal metabolomic profile was also substantially altered by the MCD diet: At 2 wk, arachidic acid, hexadecane, palmitic acid, and tetracosane were selected as potential biomarkers that were significantly different in the corresponding control group, and at 4 wk, cholic acid, cholesterol, arachidic acid, tetracosane, and stearic acid were selected. CONCLUSION: The MCD diet induced persistent alterations in the gut microbiota and metabolome.

[Clinical implications of combined measurement bone marrow T cells intracellular IFNgamma and HLA-DRB1*1501 measurement in predicting the response to immunosuppressive therapy for aplastic anemia].

OBJECTIVE: To evaluate the clinical implication of combined measurement of bone marrow (BM) T lymphocyte intracellular IFNgamma with HLA-DRB1*1501 in predicting the response to immunosuppressive therapy (IST) in patients with aplastic anemia (AA). METHODS: Enrolled into the present study were 51 idiopathic AA patients treated with cyclosporine A (CsA) based IST. BM CD(8)(+) T lymphocyte intracellular IFNgamma was determined with flow cytometry and HLA-DRB1*1501 detected with PCR-sequence specific primer before treatment. The relationship between laboratory indices and clinical response were investigated and the potential usefulness of parameters in predicting the response to IST for AA was evaluated. RESULTS: These HLA-DRB1*1501 shows sensitivity of 45.7% (16/35) and specificity of 87.5% (14/16) respectively. Intracellular IFNgamma has sensitivity of 94.3% (33/35) and specificity of 62.5% (10/16), respectively. With combination of intracellular IFNgamma with HLA-DRB1*1501, the parallel test increases the sensitivity of 97.1% (34/35) and the negative predictive value of 90.0% (9/10). On the other hand, the serial test improves the specificity and positive predictive value which both achieve 93.7% (15/16). It could be calculated through a logistic regression equation that the probabilities of prediction of four subgroups of patients whose results are both positive reaction, a positive intracellular IFNgamma plus negative HLA-DRB1*1501, a negative intracellular IFNgamma plus positive HLA-DRB1*1501 and both negative reaction are 89.0%, 77.4%, 34.5% and 18.2%, respectively. CONCLUSIONS: Combination of BM T cells intracellular IFNgamma stain and HLA-DRB1*1501 phenotype can be a useful predictor for AA patients in immunosuppressive therapy. The patients with both positive results of the two tests may have more possibilities to response to IST. It may have an important implication for the majority of AA patients whose intracellular IFNgamma stain has a positive reaction.

MR-NTD: Manifold Regularization Nonnegative Tucker Decomposition for Tensor Data Dimension Reduction and Representation.

With the advancement of data acquisition techniques, tensor (multidimensional data) objects are increasingly accumulated and generated, for example, multichannel electroencephalographies, multiview images, and videos. In these applications, the tensor objects are usually nonnegative, since the physical signals are recorded. As the dimensionality of tensor objects is often very high, a dimension reduction technique becomes an important research topic of tensor data. From the perspective of geometry, high-dimensional objects often reside in a low-dimensional submanifold of the ambient space. In this paper, we propose a new approach to perform the dimension reduction for nonnegative tensor objects. Our idea is to use nonnegative Tucker decomposition (NTD) to obtain a set of core tensors of smaller sizes by finding a common set of projection matrices for tensor objects. To preserve geometric information in tensor data, we employ a manifold regularization term for the core tensors constructed in the Tucker decomposition. An algorithm called manifold regularization NTD (MR-NTD) is developed to solve the common projection matrices and core tensors in an alternating least squares manner. The convergence of the proposed algorithm is shown, and the computational complexity of the proposed method scales linearly with respect to the number of tensor objects and the size of the tensor objects, respectively. These theoretical results show that the proposed algorithm can be efficient. Extensive experimental results have been provided to further demonstrate the effectiveness and efficiency of the proposed MR-NTD algorithm.

Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel.

AIM: To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoli-posomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G(2)/M cells and induced apoptosis, but significantly decreased the percentage of cells in G(2)/G(1) and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

[Effectiveness and safety of rhIL-11 in the treatment of chemotherapy-induced thrombocytopenia].

OBJECTIVE: To investigate the effectiveness, safety and possible mechanism of recombinate human interleukin 11 (rhIL-11) in the treatment of chemotherapy-induced thrombocytopenia. METHODS: Thirty-four patients (totally 76 cycles) with chemotherapy-induced thrombocytopenia received subcutaneous injection of rhIL-11 at the dose of 25 microg.kg(-1).d(-1) for 4 to 16 days. Serum IL-11 level was measured by ELISA, and IL-11 R alpha expression was detected by RT-PCR. RESULTS: The mean baseline platelet count before chemotherapy was (135.0 +/- 54.3) x 10(9)/L for the 1st cycle and (259.4 +/- 64.5) x 10(9)/L for the 2nd cycle. The time to administer rhIL-11 was 7 to 16 days (median 12 days) in the 1st cycle and 4 to 10 days (median 6 days) in the 2nd, respectively (P < 0.05). The duration of post-chemotherapy platelet count below 50 x 10(9)/L was 7 to 13 days (median 10 days) for the 1st cycle and 3 to 8 days (median 5 days) for the 2nd, respectively (P < 0.05). Platelet count reached 300 x 10(9)/L or above in 30 chemotherapy cycles. The maximum platelet count was found to appear at D10 to D 17 (median D14), and negatively correlated with the pre-chemotherapy serum IL-11 level after administration of rhIL-11. Major adverse reactions included edema, headache, muscle and joint pain. CONCLUSION: rhIL-11 is effective and safe for the treatment of chemotherapy-induced thrombocytopenia, with a relatively slow but sustained effect on the recovery of platelet count. Pre-chemotherpy serum IL-11 level might predict the efficacy of rhIL-11.

Syndrome of inappropriate antidiuretic hormone secretion in patients with aquaporin-4 antibody.

The objective of this study was to analyze the frequency of syndrome of inappropriate antidiuretic hormone secretion (SIADH) in patients with positive aquaporin-4 (AQP4) antibodies and evaluate the relationship between SIADH and hypothalamic lesions in patients with NMO and NMO spectrum disorder (NMOSD). AQP4 antibodies were tested by an indirect immunofluorescence assay employing HEK-293 cells transfected with recombinant human AQP4. Clinical data of patients were analyzed retrospectively. In total, 192 patients with AQP4 antibodies were certified, of which 41 patients (21.4 %) were included in the present study. Six patients (14.6 %, 6/41) met the criteria of SIADH, of which hyponatremia was mild in one patient, and severe in five. Five patients experienced confusion or decreased consciousness. Four patients were diagnosed with NMO and two were diagnosed with recurrent optic neuritis. Magnetic resonance imaging showed 11 of 41 patients (26.8 %) had hypothalamic lesions. All patients with SIADH had hypothalamic abnormalities. Hyponatremia resolved in all patients after intravenous methylprednisolone and intravenous immunoglobulin therapy. SIADH is not rare in patients with NMO/NMOSD, especially in patients with lesions close to the hypothalamus.

[A study on early activation of T lymphocytes and soluble tumor necrosis factor receptor in patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of T cell early activation marker (CD(69)) on CD(4)(+) and CD(8)(+) lymphocytes in peripheral blood and the levels of soluble tumor necrosis factor receptor 1 (sTNF-R1) and sTNF-R2 in serum and bone marrow in patients with myelodysplastic syndrome (MDS). METHODS: Whole blood cell culture procedure was applied to activate T lymphocytes with phytohemagglutinin (PHA) (20 mg/L) in vitro. The expression rates of CD(69) on CD(4)(+) and CD(8)(+) lymphocytes at 0 h and 4 h after culture were analyzed with two-color flow cytometry. The levels of sTNF-R1 and sTNF-R2 in serum and bone marrow were measured with ELISA. RESULTS: There was an increase in the expression rates of CD(69) on CD(4)(+) and CD(8)(+) cells in RA and RAS patients (8.32% and 9.88% respectively) and in the expression rate of CD(69) on CD(8)(+) cells in RAEB and RAEB-T patients (7.92%) before PHA stimulation. CD(69) expression on CD(4)(+) and CD(8)(+) cells were significantly elevated in MDS patients after PHA stimulation (53.46% and 51.63% in RA + RAS; 42.93% and 41.96% in RAEB and RAEB-T) and the expression rate on CD(4)(+) cells was similar to that on CD(8)(+) cells. The levels of the two sTNF-R in MDS patients were elevated. sTNF-R1 in RA and RAS (1.58 +/- 0.68) micro g/L (PB), (2.10 +/- 0.26) micro g/L (BM); sTNF-R2 in RA and RAS (1.41 +/- 0.50) micro g/L (PB), (1.95 +/- 0.64) micro g/L (BM); sTNF-R1 in RAEB and RAEB-T (2.62 +/- 2.55) micro g/L (PB), (3.12 +/- 0.67) micro g/L (BM); sTNF-R2 in RAEB and RAEB-T (1.96 +/- 0.56) micro g/L (PB), (3.09 +/- 0.62) micro g/L (BM). The levels of sTNF-R2 in serum positively correlated to the expression rate of CD(69) on CD(8)(+) cells before PHA stimulation. CONCLUSION: It is indicate that the increased early activation and activated potentials of T lymphocytes, along with abnormally elevated immunologically active molecules play an important role in immune pathogenesis of patients with MDS.

[Monitoring of plasma concentration of imatinib mesylate in patients with chronic myeloid leukemia].

OBJECTIVE: To analyze the clinical efficacy of imatinib mesylate (IM) for Ph-positive or BCR-ABL positive chronic myeloid leukemia (CML) to couple the trough plasma concentrations (C mins) of IM with clinical responses and adverse events (AEs). METHODS: One hundred and one CML patients received IM therapy, and Cmins of IM were determined in 30 patients. RESULTS: (1) Cumulative complete hematological response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR) and negative BCR/ABL fusion gene rates were 96.6%, 86.5%, 77.5% and 47.2%, respectively, in CML-CP patients. In accelerated and blastic phases (AP and BC) patients, CHR, MCyR, CCyR and negative BCR-ABL fusion gene rates were 58.3%, 25.0%, 25.0%, 8.3%, respectively. (2) Mean Cmins of IM was significantly higher in the CCyR at 1 year [(1472 +/- 482) microg/L] group than in the non-CCyR at 1 years group [(1067 +/- 373) microg/L] (P < 0.05), and higher in the MMR at 1 year group than in the non-MMR at 1 years group [(1624 +/- 468) microg/L vs (1137 +/- 404) microg/L, P < 0.05]. CONCLUSION: IM significantly improves cytogenetic and molecular response, event-free survival, and overall survival for patients with Ph-positive CML. The Cmins of IM exerts a significant impact on clinical response (CCyR and MMR at 1 year).

[In vitro activation of bone marrow natural killer T cells of aplastic anemia patients].

OBJECTIVE: To investigate the quantitative and qualitative changes of TCRVα24(+)Vß11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer). METHODS: NKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry. RESULTS: In AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\]. CONCLUSION: Compared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.

[Influence of tumor necrosis factor-alpha and interferon-gamma on erythropoietin production and erythropoiesis in cancer patients with anemia].

OBJECTIVE: To explore impaired erythropoiesis and relative inadequacy of erythropoietin production in the anemic cancer patients and the correlation of tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) with inadequate erythropoietin (EPO) response and impaired erythropoiesis in cancer patients with anemia. METHODS: Fifty adult anemic and 15 non-anemic tumor patients were studied. Serum EPO levels were measured by radioimmunoassay (RIA) and serum soluble transferrin receptor (sTfR). TNF-alpha and IFN-gamma levels by enzyme-linked immunosorbent assay (ELISA). Log transformed EPO and sTfR values were used in statistical analysis. The R correlation analyses were performed. RESULTS: The mean serum immunoreactive erythropoietin level in anemic cancer patients [(23.11 +/- 10.00) IU/L] was not significantly higher than in healthy people (P = 0.053), but significantly lower than in IDA patients with similar degree of anemia [(43.00 +/- 22.00) IU/L, P < 0.01]. Both O/P EPO [0.88 (0.54-1.10)] and O/P sTfR [0.89 (0.57-1.22)] were significantly lower in anemic cancer patients than in controls and in non-anemic cancer patients. There was no significant difference between the latter two groups. Furthermore, the expected inverse linear relation between serum EPO and hemoglobin levels was absent in the anemic cancer patients, and so did the relation between serum sTfR and hemoglobin levels. There was no correlation between O/P EPO and O/P sTfR. The serum levels of both TNF-alpha and IFN-gamma in anemic cancer patients [(25.75 +/- 26.71) ng/L, (50.49 +/- 42.12) ng/L, respectively] were significantly higher than those in healthy controls (both P < 0.01) or in nonanemic cancer patients (both 0.01 < P < 0.05), and so did between non-anemic cancer patients and controls. The serum levels of TNF-alpha were inversely correlated with hemoglobin levels (r = - 0.40, P = 0.004), O/P EPO (r = -0.32, P = 0.025) or O/P sTfR (r = -0.36, P = 0.01); while serum levels of IFN-gamma were inversely correlated with hemoglobin levels (r = -0.36, P = 0.01) or O/P sTfR (r = 0.39, P = 0.006), but not with O/P EPO. Conclusions Anemia of cancer is due to impaired erythropoiesis and relative inadequacy of EPO production. TNF-alpha might inhibit EPO production and erythropoiesis, while IFN-gamma maybe directly inhibit erythropoiesis and be independent of EPO response inadequacy.

[Preliminary study of multivariable model in predicting response to immunosuppressive therapy in patients with aplastic anemia].

OBJECTIVE: To evaluate the potential usefulness of a multivariable model in predicting the response to immunosuppressive therapy (IST) in patients with aplastic anemia (AA), and its application to the clinical practice. METHODS: PB T cells subpopulation and BM T cells intracellular IFN-gamma and IL-4 were serially analyzed by flow cytometry (FCM) before and during treatment. HLA-DRB1 * 1501 phenotype was analyzed by PCR-SSP. The predictive potentials of different parameter combinations for clinical responsiveness were statistically assessed. RESULTS: In all evaluated parameters, CD8+ cell intracellular IFN-gamma had the relatively best diagnostic value with sensitivity and specificity of 94.3% and 62.5%, and positive and negative predictive value of 84.6% and 83.3% respectively. Positive CD8+ cell intracellular IFN-gamma plus Tc1/Tc2 < 50 could increase the positive predictive value to 92.3%. A multivariable model consisting of absolute neutrophil count (ANC), BM T cell intracellular IFN-gamma, Tc1/Tc2 ratio and HLA-DRB * 1501 phenotype of the patients was finally established. CONCLUSION: The multivariable model is superior to each of the single parameters in terms of predictive power of IST therapeutic outcome, and its higher accuracy and the clinical application make it potentially useful in practice.

[Change of expression of cell adhesion molecules CD11a and CD49d in patients with chronic aplastic anemia before and after therapy].

To explore the cell adhesion molecules (CAMs) CD11a and CD49d in patients with chronic aplastic anemia (CAA) and its clinical implications, the expression of CD11a and CD49d in mononuclear cell (MNC) of bone marrow (BM) and peripheral blood (PB) were measured using APAAP techniques in 20 patients with CAA before and after SSL/C therapy. The results showed that the expression of CD11a and CD49d in MNC of BM and CD11a in MNC of PB increased significantly (P < 0.05) after SSL/C therapy, and there was no significant change of CD49d in MNC of PB in both groups. In conclusion, the decrease of CAMs of CD11a and CD49d participated in the pathogenesis of CAA. The expression of CAMs increases with effective treatment, so the restoration or improvement of altered CAMs of CAA might be beneficial to the proliferation and differentiation of hematopoietic stem cell, and improvement of hematopoiesis in CAA.

[Synergistic effect of Apo2L and chemotherapeutic agents on leukemia cells].

OBJECTIVE: To explore if the antileukemic drugs Vp16 or Ara-C are able to upregulate DR5 gene expression and enhance Apo2L-induced apoptosis of HL-60 cells. METHODS: Cell apoptosis was determined by flow cytometry after annexin V/PI staining, the effect of Apo2L on fresh leukemia cells by MTT reduction assay, the expression of DR5 gene in HL-60 cells by semi-quantitative RT-PCR. RESULTS: 1. Apo2L induced apoptosis of HL-60 cells in a dose-dependent manner. 2. Apo2L inhibited the proliferation of fresh leukemia cells, but there was difference among different subtypes. 3. Vp16 or Ara-C upregulated DR5 gene expression and augmented Apo2L-induced apoptosis in HL-60 cells. CONCLUSION: Apo2L could induce apoptosis of HL-60 cells and inhibit the proliferation of fresh leukemia cells. Ara-C or Vp16 upregulated DR5 gene expression and increased the sensitivity of HL-60 to Apo2L-induced cytotoxicity. Apo2L might be a promising antileukemic agent for the treatment of leukemia.

[Immunophenotyping of peripheral blood leukemic cells by multi-parameter cytometry].

AIM: To explore the clinical application and significance of CD45/SCC gating strategy of multi-parameter cytometry and tricolor fluorescence-labeled monoclonal antibodies in immunophenotyping of peripheral blood leukocytes in leukemia. METHODS: Peripheral blood leukocytes were stained with CD45 combined with a panel of monoclonal antibodies to different lineage markers. CD45/SSC dot plot was formed and leukemic cell colony, once displayed, was gated for immunophenotyping. RESULTS: 144 patients with acute leukemia and 51 with chronic leukemia were enrolled. Distinguishable leukemic cell colony was found in 137 out of 144 specimens. 66 patients were immunologically classified as ALL and 67 as AML. 4 patients were unable to be classified immunophenotypically. 27 AL patients coexpressed markers of another cell lineage (20%). Immunophenotypic features of different types of leukemia were described. It is a common phenomenon that AL of one lineage coexpressed markers of another lineage. CONCLUSION: An accurate immunophenotyping of leukemia can be realised when leukemic cell colony is displayed and analyzed by CD45/SCC pattern and gating strategy, which is important for clinical treatment and prognosis judgment.