Characteristics of HIV-1 molecular transmission networks and drug resistance among men who have sex with men in Tianjin, China (2014-2018).

BACKGROUND: In Tianjin, China, there is a relatively high prevalence of HIV in men who have sex with men (MSM). The number of HIV cases in Tianjin is also increasing. We investigated the HIV molecular transmission network, genetic tropisms, and drug resistance mutations in Tianjin. METHODS: Blood samples were collected from 510 newly diagnosed antiretroviral therapy (ART)-naïve HIV-1-infected subjects among MSM in Tianjin. Partial pol and env genes were sequenced and used for phylogenetic, genetic tropism, and genotypic drug resistance analyses. Molecular clusters were identified with 1.5% genetic distance and 90% bootstrap support. RESULTS: Among the 436 HIV-1 pol sequences obtained from the study participants, various genotypes were identified, including CRF01_AE (56.9%), CRF07_BC (27.8%), B (7.3%), CRF55_01B (4.1%), unique recombinant forms (URFs) (3.7%), and CRF59_01B (0.2%). A higher prevalence of X4 viruses was observed in individuals infected with CRF55_01B (56.3%) and CRF01_AE (46.2%) than with other subtypes. Of all 110 sequences in the 36 clusters, 62 (56.4%) were observed in 23 CRF01_AE clusters and 18 (16.4%) in four CRF07_BC clusters. Eight sequences clustered with at least one other shared the same drug resistance mutation (DRM). In different cluster sizes, the distributions of individuals by age, presence of sexually transmitted disease, and presence of DRMs, were significantly different. CONCLUSION: We revealed the characteristics of HIV molecular transmission, tropism, and DRMs of ART-naïve HIV-infected individuals among the MSM population in Tianjin. Identifying infected persons at risk of transmission is necessary for proposing counseling and treating these patients to reduce the risk of HIV transmission.

Retrospective analysis of clinical features in 134 coronavirus disease 2019 cases.

We aimed to describe the clinical features in coronavirus disease 2019 (COVID-19) cases. We studied 134 critically ill COVID-19 cases from 30 December 2019 to 20 February 2020 in an intensive care unit (ICU) at Wuhan Jinyintan Hospital. Demographics, underlying diseases, therapy strategies and test results were collected and analysed from patients on admission, admission to the ICU and 48 h before death. The non-survivors were older (65.46 (s.d. 9.74) vs. 46.45 (s.d. 11.09)) and were more likely to have underlying diseases. The blood group distribution of the COVID-19 cases differed from that of the Han population in Wuhan, with type A being 43.85%; type B, 26.92%; type AB, 10% and type O, 19.23%. Non-survivors tend to develop more severe lymphopaenia, with higher C-reactive protein, interleukin-6, procalcitonin, D-dimer levels and gradually increased with time. The clinical manifestations were non-specific. Compared with survivors, non-survivors more likely to have organ function injury, and to receive mechanical ventilation, either invasively or noninvasively. Multiple organ failure and secondary bacterial infection in the later period is worthy of attention.

Cardiac injuries in patients with coronavirus disease 2019: Not to be ignored.

OBJECTIVE: To describe the clinical features of coronavirus disease 2019 (COVID-19). METHODS: We recruited 73 patients with COVID-19 [49 men and 24 women; average age: 58.36 years (SD: 14.31)] admitted to the intensive care unit of Wuhan Jinyintan Hospital from December 30, 2019 to February 16, 2020. Demographics, underlying diseases, and laboratory test results on admission were collected and analyzed. Data were compared between survivors and non-survivors. RESULTS: The non-survivors were older (65.46 [SD 9.74]vs 46.23 [12.01]) and were more likely to have chronic medical illnesses. Non-survivors tend to develop more severe lymphopenia, with higher C-reactive protein, interleukin-6, D-dimer, and hs-Troponin I(hs-TnI) levels. Patients with elevated hs-TnI levels on admission had shorter duration from symptom onset to death. Increased hs-TnI level was related to dismal prognosis. Death risk increased by 20.8% when the hs-TnI level increased by one unit. After adjusting for inflammatory or coagulation index, the independent predictive relationship between hs-TnI and death disappeared. CONCLUSIONS: Cardiac injury may occur at the early stage of COVID-19, which is associated with high mortality. Inflammatory factor cascade and coagulation abnormality may be the potential mechanisms of COVID-19 combined with cardiac injury.

Large-scale high-density culture of hepatocytes in a liver microsystem with mimicked sinusoid blood flow.

In vitro engineering of liver tissue is a rapidly developing field for various biomedical applications. However, liver tissue culture is currently performed on only a small scale with a low density of hepatocytes. In this study, a simple design was introduced in a liver microsystem to enhance the transport of nutrients (e.g., oxygen and glucose) for the three-dimensional large-scale, high-density culture of hepatocytes. In this design, convection across the cell culture zone was generated to mimic sinusoid blood flow (SBF) based on the pressure difference between two fluids flowing in a countercurrent manner on either side of the cell culture zone. First, the distributions of living and dead cells in different culture subzones under various perfusion flow rates were observed, analysed, and compared. Then, the enhanced transport of nutrients was experimentally validated in relation to the viability of cells and theoretically explained by comparing the fluid velocity and oxygen concentration distribution in the cell culture zone in counterflow and coflow modes. Finally, the functions of the SBF-mimicked liver microsystem were assessed on the basis of specific metabolites, synthesized proteins, and bilirubin detoxification of hepatocytes, with collagen and alginate as extracellular matrices. Under this design, the density of hepatocytes cultured at the 3-mm-thickness scale reached ~7 × 107  cells/ml on Day 7, and the metabolism and detoxification functions of the cells worked well. In addition, a liver rope-like structure and sphere-like clusters of cells were observed. This work provides insight for the design of a bionic liver microsystem.

Peptide-modified vemurafenib-loaded liposomes for targeted inhibition of melanoma via the skin.

Vemurafenib is a chemotherapeutic drug recently approved by the FDA to treat melanoma. Because the drug is usually delivered orally, the route of administration readily causes damage to major organs with limited antitumor efficacy and bioavailability. In this study, we developed a peptide-modified vemurafenib-loaded liposome for the targeted inhibition of subcutaneous melanoma via the skin. First, the peptide-modified vemurafenib-loaded liposomes (Vem-TD-Lip) were prepared and characterized with respect to the size, shape and charge; the loading efficiency of vemurafenib; and the stability. Then, the intracellular uptake of these liposomes, their limited cytotoxicity, the selective inhibition of melanoma cells harboring BRAF mutations, and the liposome permeation ability were confirmed through in vitro experiments. Finally, the safety and antitumor activity of Vem-TD-Lip were evaluated in vivo. The results showed that transdermal delivery of Vem-TD-Lip effectively targeted and inhibited subcutaneous melanoma in male mice, the administration of Vem-TD-Lip through skin was better than that through oral administration and intravenous injection in terms of reducing damage to major organs and enhancing antitumor efficacy, and the peptide TD significantly enhanced the delivery of Vem-TD-Lip across the skin. This work provides a new strategy for delivering vemurafenib to target and inhibit subcutaneous melanoma.

Evaluation of serum D-dimer, fibrinogen, and CA19-9 for postoperative monitoring and survival prediction in resectable pancreatic carcinoma.

BACKGROUND: We sought to investigate the efficacy of serum D-dimer, fibrinogen, and CA19-9 for postoperative monitoring and prediction of survival in patients with resectable pancreatic carcinoma (PC). METHODS: One hundred and nineteen patients with resectable PC were enrolled. Serum D-dimer, fibrinogen, and CA19-9 values were analyzed before surgery and at the stages of relapse-free and progression disease. RESULTS: D-dimer, fibrinogen, and CA19-9 were significantly higher at the active stage of PC than those at the relapse-free stage [1059.2 (1690.1) ng/ml vs 485.18 (289.84) ng/ml, (3.71 ± 0.83) g/l vs (2.75 ± 0.52) g/l, 207.2 (681.8) U/ml vs 24.5 (30) U/ml, respectively, p < 0.01]. Patients with elevated preoperative D-dimer had significantly shorter overall survival (18.9 ± 1.9 months vs 29.2 ± 2.6 months, p < 0.01) and progression-free survival (10.6 ± 1.2 months vs 20.4 ± 2.4 months, p < 0.01) than did those with low D-dimer. The correlation between CA19-9 values and survival depended on the threshold value of CA19-9: when the threshold value was 37 U/ml, there was no correlation between CA19-9 and survival; when the threshold value was 253.8 U/ml (median CA19-9 for the enrolled patients), patients with elevated preoperative CA19-9 had significantly shorter overall survival (19.9 ± 2. 1 months vs 29.0 ± 2. 7 months) and progression-free survival (11.5 ± 1.5 months vs 21.0 ± 2. 6 months) than did the patients with low CA19-9 (p < 0.01); when the threshold value was 1000 U/ml, the overall survival was 15.5 ± 2.3 months vs 28.0 ± 2.0 months and the progression-free survival 8.9 ± 1.9 months vs 19.1 ± 1.9 months (p < 0.01). There was no correlation between fibrinogen and overall survival (25.8 ± 2.1 months vs 21.2 ± 2.9 months; p = 0.096) and progression-free survival (17.8 ± 2.1 months vs 12.7 ± 1.7 months; p = 0.168). CONCLUSIONS: For postoperative monitoring of patients with resectable PC, D-dimer, fibrinogen, and CA19-9 may be used as markers for monitoring disease relapse, but only preoperative D-dimer could predict survival.

Luminex xMAP combined with Western blot improves HIV diagnostic sensitivity.

Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (p<0.05). Western blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (p<0.1). Luminex xMAP combined with Western blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis.

[Epidemic analysis of HIV-1 incidence using BED-CEIA assay in Tianjin, China, 2010].

OBJECTIVE: To explore the epidemic characteristics of HIV-1 recent infection among high-risk populations in Tianjin and to provide reasonable evidence for intervention strategy. METHOD: Blood samples were detected using BED-CEIA to identify recent HIV-1 infection, and HIV/AIDS cases in Tianjin 2010 were collected to analyze epidemic data. RESULTS: 349 of reported HIV/AIDS cases were confirmed from total 777 207 people. ALL of 87 HIV-1 recent infections were males and mainly detected from men who have sex with men (MSM), voluntary counseling and testing (VCT) and STD outpatients, the HIV-1 incidence of each group was 1.48%, 0.38%, 0.17% respectively. There were not significant difference in recent HIV-1 infection rate between other areas and locals. The homosexual transmission was 80.46% among recent HIV-1 infections. CONCLUSION: The results of this study showed that the homosexual contact was primary route of HIV transmission in Tianjin. The infection of HIV was serious among MSM, effective interventions should be taken for HIV/AIDS control and prevention urgently.

[Survey on recent infection of human immunodeficiency virus among men who have sex with men in Tianjin during 2008 - 2009].

OBJECTIVE: To study the situation of HIV infections among men who have sex with men (MSM) in Tianjin during 2008 - 2009 and to provide reasonable evidence for intervention strategy. METHODS: Transect investigations in MSM were conducted three times during 2008 - 2009. Blood samples were collected and detected to identify the recent HIV infection with IgG-capture BED-enzyme immunoassay (BED-CEIA) before HIV incidence was estimated. RESULTS: 1799 specimens were tested and the HIV prevalence rates of each study were 6.7%, 8.6% and 6.2%, while the incidence rates were 2.7%, 2.5% and 2.8%, respectively. The estimated incidence rates among these testees were 5.36% and 5.52% per year in 2008 and 2009. RESULTS: of this study showed that the HIV incidence stabilized at high level among MSM in Tianjin, calling for the effective interventions be taken for HIV/AIDS control and prevention.

A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads.

AIM: To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. METHODS: The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. RESULTS: The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. CONCLUSION: The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction.

AIM: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening. METHODS: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3' end. IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening. RESULTS: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn(2+). The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay. CONCLUSION: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

[Experimental study of adoptive immunotherapy using CD3AK cells].

AIM: To analyze the immunological properties and biological activity of a monoclonal antibody (mAb) against CD3 molecule(yCD3), and to observe the tumor-suppressive activity of CD3AK cells in vitro and in vivo. METHODS: FCM was used to test the specificity of yCD3 and the immunological phenotype and cytokine production of CD3AK. 3H-TdR assay was used to measure the transformation of lymphocytes activated by yCD3. LDH assay was used to analyze the cytotoxic activity of CD3AK in vitro. Mice bearing tumors were used to observe the anti-tumor effect of CD3AK cells. RESULTS: yCD3 could bind specifically to T cells. 5 microg yCD3 could competitively inhibit 70% of standard anti-CD3 antibody to bind with CD3 molecules on cell membrane. 8 microg/L of yCD3 stimulated the proliferation of peripheral blood lymphocytes, which could be further boosted by IL-2 or anti-CD28 antibodies. Among activated CD3AK cells, CD3+, CD8+, and CD25+ cells increased. IL-2 and IFN-gamma producing CD3+ cells were also increased, to 3.29- and 2.47- fold, respectively, under the co-stimulation of anti-CD28 antibody. When the ratio of effective cells and target cells was 80:1, 57.54% target cells were killed. As compared with control, the percent of tumor inhibition in CD3AK cells treated tumor-bearing mice was 33.17%, and the inhibition rate of lung metastasis was 39.70%. The CD3AK cells treatment was more effective when combined with LAK cells. CONCLUSION: yCD3 could activate T cells and significantly induce the tumor-suppressive activity of CD3AK cells in vitro and in vivo, which lays a foundation for adoptive immunotherapy against tumors in clinical medicine.

[The application of SCGE-KIAS in monitoring of DNA damage in lymphocytes of tumor patients treated with cyclophosphamide].

Single cell gel electrophoresis assay (SCGE), also named as alkaline comet assay, was a simple, rapid and sensitive method to evaluate DNA damage. In this study SCGE technique was used to monitor DNA damage difference in tumor patients caused by chemotherapy, DNA damage distribution frequency and DNA damage characters were analyzed by komet image analysis system (KIAS). The results showed that cyclophosphamide greatly caused DNA damage in lymphocytes of tumor patients. There was significant difference of peripheral blood lymphocyte DNA damage between tumor patients and healthy controls. Tail length of lymphocytes were 33.69 +/- 7.56 micro m, and tail DNA% we re 31.51 +/- 5.4 6% in 10 cancer patients treated with cyclophosphamide, while Tail length were 1 6.2 +/- 1.5 micro m and tail DNA% were 7.46 +/- 1.15% in healthy controls. there was great significant difference on tail length and tail DNA% values between cancer patients and healthy controls (P < 0.01). In conclusion, the successful measurement of DNA damage caused by Cyclophosphamide treatment means that the alkaline comet assay as a valuable tool can be very useful in cancer epideminology study, and also be valuable to evaluate DNA damage status of patients in clinic.

[Construction of GPI-CTLA4Ig chimeric molecule and its expression on CHO-dhfr(-) cells].

AIM: To develop a novel immunosuppressant GPI-CTLAIg modified by glycosyl-phosphatidylinositol(GPI). METHODS: GPI-modified CTLAIg was produced by linking-up of CTLA4Ig with GPI-modification signal sequences from decay-accelerating factor (DAF). Chimeric molecule GPI-CTLA4Ig gene was cloned into eukaryotic expression vector pCI-dhfr. Using lipofectine-mediated gene transfer technique, pCI-GPI-CTLA4Ig was transfected into CHO-dhfr(-) cells, and the transfectants were screened by methotrexate (MTX). Expression of the recombinant protein was assessed by RT-PCR, ELISA, cell immunofluorescence staining and Western blot, and the purification of expressed protein was performed by protein A affinity chromatography. RESULTS: The chimeric molecule GPI-CTLA4Ig has been constructed and stablely expressed on CHO-dhfr(-) cells. CONCLUSION: GPI-modified CTLAIg will may be used as novel immunosuppressant for suppressing reaction in graft rejection.