Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Erythropoiesis changes with increasing age in the elderly Chinese.

INTRODUCTION: Erythropoiesis slowly decreases with increasing age, which may be reflected in red blood cell (RBC) parameters. This multicentre collaborative study aimed to investigate the changes in erythropoiesis with increasing age in a healthy Chinese population. METHODS: A total of 14,591 healthy individuals (6,713 aged at least 60 y and 7,878 aged below 60 y) from seven cities across China were enrolled. K2-EDTA anticoagulant blood samples were analysed. The results are presented as median and 2.5-97.5th percentile. RESULTS: RBC parameters showed some differences between the two groups divided by the age of 60 in the Chinese population. The median, 2.5th and 97.5th percentile values of RBC, haemoglobin (HGB) and haematocrit (HCT) in patients aged ≥ 60 y were significantly lower than in those ˂ 60 y. The values of mean cell volume (MCV), mean cell haemoglobin (MCH) and red cell distribution width (RDW) were higher in the group aged ≥ 60 y. Men had significantly higher RBC, HGB, HCT, MCV, MCH and RDW indices than women. The prevalence of anaemia gradually increased with age in men and was higher than that in women after 50. The median haemoglobin and MCV in Nanning and Guangzhou were lower than those in other regions. CONCLUSION: RBC parameters varied with increasing age and differed between males and females, indicating that erythropoiesis decreases in the elderly Chinese population. Subsequent studies should be conducted for age- and sex-specific reference intervals in healthy elderly Chinese populations.

Protein analysis of extracellular vesicles to monitor and predict therapeutic response in metastatic breast cancer.

Molecular profiling of circulating extracellular vesicles (EVs) provides a promising noninvasive means to diagnose, monitor, and predict the course of metastatic breast cancer (MBC). However, the analysis of EV protein markers has been confounded by the presence of soluble protein counterparts in peripheral blood. Here we use a rapid, sensitive, and low-cost thermophoretic aptasensor (TAS) to profile cancer-associated protein profiles of plasma EVs without the interference of soluble proteins. We show that the EV signature (a weighted sum of eight EV protein markers) has a high accuracy (91.1 %) for discrimination of MBC, non-metastatic breast cancer (NMBC), and healthy donors (HD). For MBC patients undergoing therapies, the EV signature can accurately monitor the treatment response across the training, validation, and prospective cohorts, and serve as an independent prognostic factor for progression free survival in MBC patients. Together, this work highlights the potential clinical utility of EVs in management of MBC.

Two dimensional nanosheets as immunoregulator improve HIV vaccine efficacy.

Two-dimensional (2D) nanosheets as carriers have shown promising potential for surface-displaying or loading various drugs. Nevertheless, developing sheet-like materials themselves into an immunoregulator has never been realized so far. In this study, we take advantage of the immunoregulatory effects of rare earth elements themselves and develop water-soluble erbium-dysprosium 2D nanosheets (2D NSs). Such 2D NSs can target lymph nodes and activate macrophages to improve vaccine efficacy in mice significantly. Transcriptome analysis further reveals that six critical molecules (Msr1, Ccr2, Serpinb9, Klrk1, Klrd1, Klrc1) closely correlate with 2D NS-mediated immunoregulation in vivo. For the first time, the present work realizes a proof-of-concept for designing immunoregulatory 2D NSs and shows a promising potential of 2D NSs for improving the immunoprophylaxis/immunotherapy of vaccines.

Surface-modified mesoporous nanofibers for microfluidic immunosensor with an ultra-sensitivity and high signal-to-noise ratio.

How to balance the sensitivity and signal-to-noise ratio of immunosensor remains many challenges during various diseases diagnosis. Here we develop a new microfluidic immunosensor based on surface-modified mesoporous nanofibers, and simultaneously realize an ultra-sensitivity and high signal-to-noise ratio for the detection of multiple biomarkers. In the current study, we fabricated titanium dioxide (TiO2)-based mesoporous electrospinning nanofibers, and modified nanofiber surface with both octadecylphosphonic acid (OPA) and poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (PEO-PPO-PEO). Such nanofibers as solid substrate are covered on microfluidic channels. The porosity of our nanofibers dramatically increased the adsorption capability of antibodies, realizing an ultra sensitivity of biomarker detection. PEO-PPO-PEO modification can significantly block non-specific absorptions, obtaining a satisfied signal-to-noise ratio. For the detection of HIV p24 and interleukin 5 (IL-5), our immunosensor increased 6.41 and 6.93 fold in sensitivity and improved 504.66% and 512.80% in signal-to-noise ratio, in compared with gold standard immunoassay (ELISA) used in the clinic. Our immunosensor also broaden the linear range for the detection of HIV p24 (0.86-800 pg/ml) and IL-5 (0.70-800 pg/ml), in compared with ELISA which is 5.54-500 pg/ml for HIV p24 and 4.84-500 pg/ml for IL-5. Our work provided a guideline for the construction of advanced point-of-care immunosensor with an ultra-sensitivity and high signal-to-noise ratio for disease diagnosis.

Increasing the Assembly Efficacy of Peptidic ß-Sheets for a Highly-Sensitive HIV Detection.

Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.

Low-cost thermophoretic profiling of extracellular-vesicle surface proteins for the early detection and classification of cancers.

Non-invasive assays for early cancer screening are hampered by challenges in the isolation and profiling of circulating biomarkers. Here, we show that surface proteins from serum extracellular vesicles labelled with a panel of seven fluorescent aptamers can be profiled, via thermophoretic enrichment and linear discriminant analysis, for cancer detection and classification. In a cohort of 102 patients, including 6 cancer types at stages I-IV, the assay detected stage I cancers with 95% sensitivity (95% confidence interval (CI): 74-100%) and 100% specificity (95% CI: 80-100%), and classified the cancer type with an overall accuracy of 68% (95% CI: 59-77%). For patients who underwent prostate biopsies, the assay was superior to the analysis of prostate-specific antigen levels (area under the curve: 0.94 versus 0.68; 33 patients) for the discrimination of prostate cancer and benign prostate enlargement, and also in the assessment of biochemical cancer recurrence after radical prostatectomy. The assay is inexpensive, fast, and requires small serum volumes (<1 µl), and if validated in larger cohorts may facilitate cancer screening, classification and monitoring.

Application of fluorescence in situ hybridization in the detection of bladder transitional-cell carcinoma: A multi-center clinical study based on Chinese population.

OBJECTIVE: To evaluate the diagnostic value of fluorescence in situ hybridization (FISH) in bladder cancer. METHODS: We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008. Receiver operating characteristic (ROC) curve analysis was performed and the area under curve (AUC) values were calculated for both the FISH and urine cytology tests. RESULTS: A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population. A total of 4807 patients with hematuria were prospectively, randomly enrolled for the simultaneous analysis of urine cytology, FISH testing, and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen. Overall, the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%, while that of cytology was 33.4% (p < 0.001). The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7% and 89.6%, respectively (p = 0.004). The sensitivity values of FISH for low and high grade bladder cancer were 82.6% and 90.1%, respectively (p = 0.002). CONCLUSION: FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages. Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.

Molecular Design of ß-Sheet Peptide for the Multi-Modal Analysis of Disease.

Intermolecular forces constrain peptide conformation. However, the role of each intermolecular force in constraining peptide conformation remains poorly understood. In this work, we show that aromatic-aromatic interactions drive peptides into ß-sheets, and the hydrophobic effect determines the assembly speed of peptides. By using intermolecular forces to artificially control the assembly of ß-sheets, a multi-modal analytical system was developed that allows five readouts and dual qualitative-quantitative analysis, and satisfies both point-of-care testing (POCT) and laboratory-based testing. For Mycoplasma Pneumoniae diagnosis, this system eradicates misdiagnosis (from 30 % to 0 %) and broadens the linear range by three-fold, both of which are critical for guiding therapy. This work not only illustrates exact roles of intermolecular forces in driving the formation of ß-sheets, but also provides a guideline for the construction of a multi-modal analytical system for disease diagnosis.

Microfluidic co-flow of Newtonian and viscoelastic fluids for high-resolution separation of microparticles.

The microfluidic passive control of microparticles largely relies on the hydrodynamic effects of the carrier media such as Newtonian fluids and viscoelastic fluids. Yet the viscoelastic/Newtonian interfacial effect has been scarcely investigated, especially for high-resolution particle separation. Here we report a microfluidic co-flow of Newtonian (water or PBS) and viscoelastic fluids (PEO) for the size-dependent separation of microparticles. The co-flow condition generates a stable viscoelastic/Newtonian interface, giving rise to the wall-directed elastic lift forces that compete with the center-directed lift forces, and efficiently hinders the migration of microparticles from the Newtonian to the viscoelastic fluid in a size-dependent manner. An almost complete separation of a binary mixture of 1 µm and 2 µm polystyrene particles is achieved by the co-flow of water and a very dilute PEO solution (100 ppm), whereas the sole use of water or PEO could not lead to an efficient separation. This co-flow microfluidic system is also applied for the separation of Staphylococcus aureus (1 µm) from platelets (2-3 µm) with >90% efficiencies and purities.

Peptidic ß-sheet binding with Congo Red allows both reduction of error variance and signal amplification for immunoassays.

Although conventional enzyme-linked immunosorbent assays (ELISA) and related assays have been widely applied for the diagnosis of diseases, many of them suffer from large error variance for monitoring the concentration of targets over time, and insufficient limit of detection (LOD) for assaying dilute targets. We herein report a readout mode of ELISA based on the binding between peptidic ß-sheet structure and Congo Red. The formation of peptidic ß-sheet structure is triggered by alkaline phosphatase (ALP). For the detection of P-Selectin which is a crucial indicator for evaluating thrombus diseases in clinic, the 'ß-sheet and Congo Red' mode significantly decreases both the error variance and the LOD (from 9.7ng/ml to 1.1 ng/ml) of detection, compared with commercial ELISA (an existing gold-standard method for detecting P-Selectin in clinic). Considering the wide range of ALP-based antibodies for immunoassays, such novel method could be applicable to the analysis of many types of targets.

Activity of Colistin in Combination with Meropenem, Tigecycline, Fosfomycin, Fusidic Acid, Rifampin or Sulbactam against Extensively Drug-Resistant Acinetobacter baumannii in a Murine Thigh-Infection Model.

Few effective therapeutic options are available for treating severe infections caused by extensively drug-resistant Acinetobacter baumannii (XDR-AB). Using a murine thigh-infection model, we examined the in vivo efficacy of colistin in combination with meropenem, tigecycline, fosfomycin, fusidic acid, rifampin, or sulbactam against 12 XDR-AB strains. Colistin, tigecycline, rifampin, and sulbactam monotherapy significantly decreased bacterial counts in murine thigh infections compared with those observed in control mice receiving no treatment. Colistin was the most effective agent tested, displaying bactericidal activity against 91.7% of strains at 48 h post-treatment. With strains showing a relatively low minimum inhibitory concentration (MIC) for meropenem (MIC ≤ 32 mg/L), combination therapy with colistin plus meropenem caused synergistic inhibition at both 24 h and 48 h post-treatment. However, when the meropenem MIC was ≥64 mg/L, meropenem did not significantly alter the efficacy of colistin. The addition of rifampin and fusidic acid significantly improved the efficacy of colistin, showing a synergistic effect in 100% and 58.3% of strains after 24 h of treatment, respectively, while the addition of tigecycline, fosfomycin, or sulbactam did not show obvious synergistic activity. No clear differences in activities were observed between colistin-rifampin and colistin-fusidic acid combination therapy with most strains. Overall, our in vivo study showed that administering colistin in combination with rifampin or fusidic acid is more efficacious in treating XDR-AB infections than other combinations. The colistin-meropenem combination may be another appropriate option if the MIC is ≤32 mg/L. Further clinical studies are urgently needed to confirm the relevance of these findings.

[Evaluation of PFA P2Y assay in monitoring platelet function in elderly patients with cardiovascular disease receiving clopidogrel treatment].

OBJECTIVE: To evaluate the value of a new platelet function test PFA P2Y (PFA-200) in monitoring clopidogrel treatment for cardiovascular disease in elderly patients. METHODS: Fifty-six elderly patients receiving clopidogrel therapy in the Department of Cardiology of General Hospital of PLA from March to August in 2016 and 85 healthy volunteers were recruited for analysis. All the subjects underwent PFA P2Y, LTA (light transmittance aggregometry) and TEG (Thromboelastograph) tests, and Spearman correlation coefficients were used to test the associations between test results. The agreement among the 3 platelet function test methods was assessed using Cohen's kappa coefficient. RESULTS: Correlation coefficient (r) was -0.701 (P<0.001) between PFA P2Y and LTA, and 0.475 (P<0.001) between PFA P2Y and TEG. The agreement was 75% between PFA P2Y and LTA and 67.9% between PFA P2Y and TEG. The κ value was 0.434 (P=0.001) between PFA P2Y and LTA and 0.242 (P=0.046) between PFA P2Y and TEG. With ADP-induced maximum platelet aggregation rate of LTA >50% as the laboratory clopidogrel resistance, the cut-off value of PFA P2Y was 119 s (AUC=0.733) with a sensitivity of 75.6% and a specificity of 73.3%. CONCLUSION: PFA P2Y has a moderate correlation and agreement with LTA, but has a poor correlation and agreement with TEG. PFA P2Y can be useful for assessing the effects of clopidogrel therapy and the association of the cut-off value (119 s) with the long-term clinical ischemic events needs be confirmed in further study.

[Clinical significance of new urine red blood cell parameter in different kinds of glomerulonephritis].

OBJECTIVE: To investigate the clinical value of new kinds of urinary erythrocyte morphology parameter in discriminating different pathology types of glomerulonephritis. METHODS: All of the 52 urine samples were from glomerulonephritis patients who had been diagnosed by renal biopsy results. The change of the percentage of acanthocytes, the size of RBC, the shape of RBC between the primary glomerulonephritis (39 cases) and secondary glomerulonephritis (13 cases) urine were detected by AVE-764 fully automatic urine cell analyzer. RESULTS: Acanthocytes could be found in both primary glomerulonephritis and secondary glomerulonephritis. Of the patients whose acanthocytes percentages above 10%, 94.1% had primary glomerulonephritis and 5.9% had secondary glomerulonephritis. The picture of size-shape phase were classified as strip-type, inverted triangle-type and hanging tail-type. 95.2% Strip-type cases were from primary glomerulonephritis patients. Triangle-typenormally cases were all from primary glomerulonephritis patients. Hanging tail-type cases were all from secondary glomerulonephritis. CONCLUSION: High acanthocytes percentage is most common in primary glomerulonephritis, going with the size and shape of RBC can be useful in the differential diagnosis of different pathology types of glomerulonephritis.

Comparison between a new platelet count drop method PL-11, light transmission aggregometry, VerifyNow aspirin system and thromboelastography for monitoring short-term aspirin effects in healthy individuals.

Platelet function has been described by many laboratory assays, and PL-11 is a new point-of-care platelet function analyzer based on platelet count drop method, which counts platelet before and after the addition of agonists in the citrated whole blood samples. The present study sought to compare PL-11 with other three major more established assays, light transmission aggregometry (LTA), VerifyNow™ aspirin system and thromboelastography (TEG), for monitoring the short-term aspirin responses in healthy individuals. Ten healthy young men took 100 mg/d aspirin for 3-day treatment. Platelet function was measured via PL-11, LTA, VerifyNow and TEG, respectively. The blood samples were collected at baseline, 2 hour, 1 day during the aspirin treatment and 1 day, 5 ± 1 days, 8 ± 1 days after the aspirin withdrawal. Moreover, 90 additional healthy subjects were recruited to establish a reference range for PL-11. Platelet function of healthy subjects decreased significantly 2 hours after 100 mg/d aspirin intake and began to recover during 4-6 days after the aspirin withdrawal. Correlations between methods were PL-11 vs. LTA (r = 0.614, p < 0.01); PL-11 vs. VerifyNow (r = 0.829, p < 0.01); PL-11 vs. TEG (r = 0.697, p < 0.001). There was no significant bias between PL-11 and LTA at baseline (bias = 1.94%, p = 0.804) using Bland-Altman analysis, while the data of PL-11 were significantly higher than LTA (bias = 24.02%, p < 0.001) during the aspirin therapy. The reference range for PL-11 in healthy young individuals was from 66.8 to 90.5% (95%CI). When aspirin low-responsiveness was defined as LTA > 20%, the cut-off values for each method were, respectively: PL-11 > 50%, VerifyNow > 533 ARU, TEG > 60.2%. The results of different platelet function assays were uninterchangeable for monitoring aspirin response and correlations among them were also varied. Correlations among PL-11 and other three major assays suggested the ability of PL-11 to assess the treatment effects of aspirin. But a large cohort study is needed to confirm the cut-off value of aspirin response detected by PL-11.

[Clinical and cytomorphological features of bone marrow metastasis of non-hematological malignant carcinoma].

OBJECTIVE: To analyze the clinical manifestations and hematologic parameters and observe the cytomorphological features of metastatic tumors in the bone marrow originating from different primary sites. METHODS: The clinical data of 77 patients with bone marrow metasta tumors admitted between 2009 and 2014 between 2009 and 2014 in General Hospital of PLA were studied retrospectively to analyze the indications of laboratory examinations (hematological laboratory tests, tumor markers, peripheral blood films, and bone marrow aspirates). RESULTS: Of the 77 patients analyzed, 64.9% were over 50 years of age. The most common clinical characteristics were bone pain (65%), anemia with thrombocytopenia (63.6%) and leukoerythroblastic reaction (61%). The hematological abnormalities included elevation of ESR, ALP, LDH, tumor markers, and hypoproteinemia. Cytological examination of bone marrow aspiration samples revealed different morphological characteristics of the metastatic cells from different primary sites; in most of the cases, scattered or clustered metastatic cells and degenerative tumor cells were found on the edge of the bone marrow smears. CONCLUSION: Detection of the primary tumor site is difficult by cytological examination of bone marrow aspiration samples, but the cytological findings can be of value in the diagnosis of neuroblastoma, small cell lung cancer and gastric cancer (signet ring cell carcinoma). A definite diagnosis of bone marrow metastatic tumor relies on a combined evaluation of the disease history, clinical symptoms and laboratory findings.

Induction of adhesion molecule expression in co-culture of human bronchial epithelial cells and neutrophils suppressed by puerarin via down-regulating p38 mitogen-activated protein kinase and nuclear factor κB pathways.

OBJECTIVE: In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions. METHODS: Neutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot. RESULTS: In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05). CONCLUSIONS: Coculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Application and development of review criteria on Sysmex XT-2100 in Chinese at a large third-grade class-A hospital.

BACKGROUND: Hematology analysis is an essential component of patient assessment and used in screening, diagnosis, and the planning of care. The objective of the study was to find out the suitable hematology review criteria for large scale general hospitals in China via the analysis of experimental data from Sysmex XE-2100 hematology analyzer. METHODS: A total 1486 blood samples were detected with the Sysmes XE-2100. Based on hematology review criteria suggested by international consensus group and a positive smear finding new optimal review rules were determined. RESULTS: With the International Article 41 Review Rules, the true positive ratio (TP), the false positive ratio (FP), the true negative ratio (TN), and the false negative ratio (FN) was 14.0% (208/1486), 31.49% (468/1486) 52.42% (779/1486), and 2.09% (31/1486), respectively. With the help of Laboman 4.2 software (the Sysmex Corporation), 19 rules for review of automated CBC and WBC differential were set up. With our review rules, the TP, FP, TN, and FN was 13.86% (206/1486), 25.17% (374/1486), 58.75% (873/1486) and 2.22% (33/1486), respectively. The review rules were validated, the FN was 0.96%, blasts and immature cells were not omitted. CONCLUSIONS: The review criteria can be developed in light of the rules of the International Consensus Group for Hematology Review, but should be improved depending on different laboratory's requirements.

The effects of HIV Tat DNA on regulating the immune response of HIV DNA vaccine in mice.

BACKGROUND: HIV trans-activator protein (Tat) is the crucial factor to control HIV transcription, and is usually considered as an important immunogen for the design of HIV vaccine. Recent studies reported some special bio-activities of Tat protein on immunoregulation. However, to date, few studies have focused on exploring the effects of Tat expression plasmid (pTat) on regulating the immune responses induced by HIV DNA vaccines. In this study, our main objective is to investigate the immunoregulation mediated by pTat in mice. METHODS: Four gene-coding plasmids (pTat, pGag, pEnv and pPol) were constructed, and the gene expression was detected by western blot method. The effects of pTat on regulating the immune responses to antigens Gag, Env, Pol were assessed by enzyme-linked immunospot and enzyme-linked immunosorbent assay. The data was analysed by one-way analysis of variance. RESULTS: After two immunizations, mice vaccinated with antigen expressing plasmid (pGag, pEnv or pPol) plus pTat exhibited significantly stronger IFN-gamma response than that vaccinated with the corresponding antigen alone. Moreover, mice receiving two injections of antigen plus pTat exhibited the same strong IFN-gamma response as those receiving three injections of antigen alone did. Furthermore, addition of pTat not only induced a more balanced Th1 and Th2 response, but also broadened IgG subclass responses to antigens Gag and Pol. CONCLUSION: pTat exhibited the appreciable effects on modulating immune responses to HIV antigens Gag, Env and Pol, providing us interesting clues on how to optimize HIV DNA vaccine.