Evaluation of a two-test strategy for HIV screening in a low-prevalence setting and the indications for optimizing clinical management.

Objectives: To evaluate a two-test strategy for HIV screening in the low-prevalence population and to assess the feasibility of utilizing the optimal signal-to-cutoff (S/CO) threshold on the chemiluminescence immunoassay(CMIA) and an additional rapid test on the gold immune-chromatography assay (GICA) for screening positive patients and optimization of clinical management. Methods: We conducted a retrospective study of samples analyzed by the fourth-generation Architect HIV Ag/Ab combo assay (CMIA) in a large medical center between June 2017 and August 2020. Reactive samples underwent a second screening test using the rapid test GICA, followed by Western blot (WB) as the confirmatory test. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal S/CO. We calculated sensitivity, specificity, and predictive value based on our population. The performance of the single-test strategy (CMIA) was compared with that of the two-test strategy (CMIA and GICA). Logistic regression was used to analyze the factors of clinical characteristics leading to false positive results. Results: A total of 220558 samples were screened by CMIA, and 429 patients met the inclusion criteria. Of these, CMIA produced 199 false-positive results with a median S/CO of 1.93(IQR1.45-3.68) and 230 positive results with a median S/CO of 455.1 (IQR169.3-709.7). The optimal S/CO of the single-test strategy was 8.82, which achieved a sensitivity of 100% and a positive predictive value (PPV) of 90.9%. The two-test strategy (CMIA and GICA) provided a sensitivity of 100% and a PPV of 98.7%, which best correlated with the confirmatory test WB. The combination of S/CO 8.82 on the CMIA assay and additional test results of GICA can be defined as four types used to interpret HIV serostatus. The false positive rate (FPR) was high in the female, the age≤18 group, the pre-operative patients, and the patients from the clinical departments of Pediatrics, Gynecology and Obstetrics, and Oncology, etc. Conclusions: The false positive rate is high in the low-prevalence setting by using CMIA. The two-test strategy (CMIA and GICA) is recommended for HIV screening in hospitals. Hopefully, the clinicians will be able to interpret HIV serostatus and facilitate clinical decision-making while waiting for the confirmatory results.

Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling.

OBJECTIVES: Autophagy is divided into three phases: autophagosome engulfment of intracellular organelles and proteins, autophagosome fusion with lysosomes, and autolysosome degradation. The farnesyl pyrophosphate synthase inhibitor ibandronate (IBAN) has in vivo cardioprotective properties, potentially via anti-oxidant effects. Whether autophagy is involved in the cardioprotective effect of IBAN remains unexplored. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated in vitro with IBAN to assess autophagy induction. Lysosomal activation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling were assessed using a LysoTracker assay, acridine orange staining and western blotting. An MTS assay was used to assess cellular proliferation. Autophagy was inhibited using chloroquine or RNA silencing of autophagy-related 7 (Atg7) expression. RESULTS: IBAN induced autophagy in HUVECs. Moreover, IBAN activated lysosomal function, which is pivotal to autophagy induction. PI3K/AKT/mTOR activity was inhibited in IBAN-treated HUVECs, indicating the involvement of this pathway in IBAN-induced autophagy. Inhibition of autophagy using either chloroquine or Atg7 siRNA potentiated inhibition of HUVEC growth by IBAN, suggesting the involvement of non-autophagy pathways in the antiproliferative effects of IBAN. CONCLUSIONS: These findings provide insights into the role of autophagy in the cardioprotective effects of IBAN and the molecular mechanisms underlying autophagy induction by IBAN.

Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis.

Mechanistic target of rapamycin (mTOR) controls cell growth and metabolism in response to environmental and metabolic signals. Rapamycin robustly extends the lifespan in mammals and has clinical relevance in organ transplantation and cancer therapy but side effects include male infertility. Here, we report that chronic rapamycin treatment causes spermatogenic arrest in adult male mice due to defects in sex body formation and meiotic sex chromosome inactivation (MSCI). Many sex chromosome-linked genes were up-regulated in isolated pachytene spermatocytes from rapamycin-treated mice. RNA-Seq analysis also identified mRNAs encoding the core piRNA pathway components were decreased. Furthermore, rapamycin treatment was associated with a drastic reduction in pachytene piRNA populations. The inhibitory effects of rapamycin on spermatogenesis were partially reversible, with restoration of testis mass and sperm motility within 2 months of treatment cessation. Collectively, we have defined an essential role of mTOR in MSCI and identified a novel function as a regulator of small RNA homeostasis in male germ cells.

Exosomal MALAT1 derived from oxidized low-density lipoprotein-treated endothelial cells promotes M2 macrophage polarization.

Oxidized low-density lipoprotein (oxLDL)-induced injury and apoptosis of endothelial cells are important initial events in numerous cardiovascular diseases. Following activation by oxLDL, monocytes adhere to endothelial cells, migrate into the subendothelial spaces and then undergo differentiation into macrophages, which subsequently induces the formation of atherosclerotic lesions. However, the mechanisms underlying the activation of macrophage differentiation by oxLDL-treated endothelial cells remain unclear. In the present study, it was demonstrated that exosomal metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was increased in oxLDL-treated human umbilical vein endothelial cells. When co-cultured with monocytes, exosomes extracted from oxLDL-treated HUVECs were endocytosed. Furthermore, exosomes derived from oxLDL-treated endothelial cells were revealed to promote M2 macrophage polarization, as reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA analyses demonstrated increases in the expression of M2 macrophage markers, including macrophage mannose receptor 1 (also termed CD206), arginase-1 and interleukin (IL)-10, and decreases in the expression of the M1 macrophage marker, IL-12. Furthermore, the suppression of MALAT1 expression in monocytes was demonstrated to reverse exosome-mediated M2 macrophage polarization. In conclusion, the results of the present study revealed a novel mechanism underlying the onset of atherogenesis associated with endothelial cells and macrophages: Exosomal MALAT1 derived from oxLDL-treated endothelial cells promoted M2 macrophage polarization. This result may provide a novel scientific basis for the understanding of atherosclerosis progression.

Exosomes derived from oxidized LDL-stimulated macrophages attenuate the growth and tube formation of endothelial cells.

Oxidized low-density lipoprotein (oxLDL) has a critical role in the development of atherosclerosis. The participation of oxLDL­stimulated macrophages has been well­established in atherosclerosis, however the underlying mechanisms are unclear. Macrophage­derived exosomes are actively released and are involved in numerous physiological and pathological processes. However, the function of exosomes secreted by oxLDL­stimulated macrophages in atherosclerosis remains unknown. Exosomes from oxLDL­treated macrophages and controls were co­cultured with endothelial cells and the exosomes were taken up by endocytosis. Cell Counting Kit­8 and tube formation assay results revealed that exosomes derived from oxLDL­stimulated macrophages reduced the growth and tube formation ability of endothelial cells. Suppression of exosomal secretion by oxLDL­stimulated macrophages rescued the growth and tube formation ability of endothelial cells. Therefore, the results of the present study indicate that oxLDL­stimulated macrophages may attenuate the growth and tube formation of endothelial cells, at least in part through exosomal transfer. This may provide novel targets for the development of atherosclerosis therapeutics.

Concentrations and analysis of health risks of ambient air metallic elements at Longjing site in central Taiwan.

The concentrations of particulates and metallic elements that were bound to total suspended particulates in ambient air at Long Cyuan Elementary School (LCYES), Lung Ching Elementary School (LCHES) and Long Shan Primary School (LSPS) sampling sites in the Longjing area were measured. Significant difference tests were conducted at LSPS, LCYES and LCHES sites. Finally, carcinogenic and non-carcinogenic risk values for LSPS, LCYES and LCHES sites in the Longjing district were evaluated. The results show that the most average particulate and metallic element concentrations were highest in October, November, January, February, March, April, August, and September The average particulate and metallic element concentrations at LCHES were higher than at the other sampling sites. The Concentration Scatter Diagrams reveal the absence of significant variation among the LSPS, LCYES and LCHES sampling sites in the Longjing district. Therefore, these sampling sites are inferred to have similar emission sources. The children and adults inhalation carcinogenic risks which referenced US EPA method were all within acceptable ranges. Non-carcinogenic risks revealed that all metallic elements considered herein were harmless to human health.

Individualized dual antiplatelet therapy based on platelet function testing in patients undergoing percutaneous coronary intervention: a meta-analysis of randomized controlled trials.

BACKGROUND: High on-treatment platelet reactivity (HPR) represents a strong risk factor for thrombotic events after PCI. We aim to evaluate the efficacy and safety of individualizing intensified dual antiplatelet therapy (DAPT) in PCI-treated patients with HPR based on platelet function testing (PFT). METHODS: Electronic databases were searched for randomized control trials that reported the clinical outcomes of using an intensified antiplatelet protocol with P2Y12 receptor inhibitor comparing with standard maintenance dose of clopidogrel on the basis of platelet function testing. Clinical endpoints were assessed. RESULTS: From 2005 to 2016, thirteen clinical studies comprising 7290 patients were included for analysis. Compared with standard antiplatelet therapy with clopidogrel, the intensified protocol based on platelet function testing was associated with a significant reduction in major adverse cardiovascular events (RR:0.55, 95% CI: 0.36-0.84, p = 0.005), cardiovascular death (RR:0.60, 95% CI: 0.38-0.96, p = 0.03), stent thrombosis (RR:0.58, 95% CI: 0.36-0.93, p = 0.02) and target vessel revascularization (RR:0.33, 95% CI: 0.14-0.76, p = 0.009). No significant difference was found in the rate of bleeding events between intensified and standard protocol. CONCLUSIONS: Compared with standard clopidogrel therapy, individualized intensified antiplatelet therapy on the basis of platelet reactivity testing reduces the incidence of cardiovascular events in patient undergoing PCI, without increasing the risk of bleeding.

Continuous microfluidic assortment of interactive ligands (CMAIL).

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 10(5) CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 10(9) individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%-347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

Association of BAFF and IL-17A with subphenotypes of primary Sjögren's syndrome.

AIM: Primary Sjögren's syndrome (pSS) is an autoimmune disease affecting exocrine glands. Both autoreactive T cells and B cells are involved in the development of pSS, but their exact contribution to the pathogenesis is not clear. Here, we aimed to investigate the association of B-cell activating factor (BAFF) and interleukin (IL)-17A with subphenotypes of pSS. METHODS: Peripheral blood samples were collected from 31 pSS patients and 28 healthy controls. The serum levels of BAFF and IL-17A were quantified by sandwich ELISA. RESULTS: The increased circulating BAFF levels are associated with higher immunoglobulin G (IgG) levels (P = 0.0167) and anti-Ro/SS antigen A autoantibody (P = 0.032), while the elevated circulating levels of IL-17A are associated with lower C3 levels (P = 0.0213) and higher focus score of salivary gland tissue (P = 0.002). CONCLUSION: Our results show that BAFF and IL-17A are associated with different subphenotypes of pSS, suggesting both humoral and cellular immune response are involved in the pathogenesis of pSS.

Primary amyloidosis mimicking Crohn's disease: a case report.

Amyloidosis is an uncommon disease that results from the extracellular deposition of abnormal fibrillary protein. This paper reports a case of primary amyloidosis with predominant involvement of the gastrointestinal tract and heart as a mimicker of Crohn's disease in a sixty-seven years old man admitted with repeated diarrhea and fatigue. This patient poorly responded to 5-aminosalicylic acid and quickly developed dyspnea and hypotension. The further laboratory test revealed a monoclonal protein detected by serum protein electrophoresis. Biopsy of abdominal wall fat pad revealed amyloid substance deposited and positive Congo red staining, which was diagnosed as primary amyloidosis.

Complete protection of mice against lethal murine cytomegalovirus challenge by immunization with DNA vaccines encoding envelope glycoprotein complex III antigens gH, gL and gO.

Human cytomegalovirus infects the majority of humanity which may lead to severe morbidity and mortality in newborns and immunocompromised adults. Humoral and cellular immunity are critical for controlling CMV infection. HCMV envelope glycoprotein complexes (gC I, II, III) represent major antigenic targets of antiviral immune responses. The gCIII complex is comprised of three glycoproteins, gH, gL, and gO. In the present study, DNA vaccines expressing the murine cytomegalovirus homologs of the gH, gL, and gO proteins were evaluated for protection against lethal MCMV infection in the mouse model. The results demonstrated that gH, gL, or gO single gene immunization could not yet offer good protection, whereas co-vaccination strategy apparently showed effects superior to separate immunization. Twice immunization with gH/gL/gO pDNAs could provide mice complete protection against lethal salivary gland-derived MCMV (SG-MCMV) challenge, while thrice immunization with pgH/pgL, pgH/pgO or pgL/pgO could not provide full protection. Co-vaccination with gH, gL and gO pDNAs elicited robust neutralizing antibody and cellular immune responses. Moreover, full protection was also achieved by simply passive immunization with anti-gH/gL/gO sera. These data demonstrated that gCIII complex antigens had fine immunogenicity and might be a promising candidate for the development of HCMV vaccines.

A single dose of DNA vaccine based on conserved H5N1 subtype proteins provides protection against lethal H5N1 challenge in mice pre-exposed to H1N1 influenza virus.

BACKGROUND: Highly pathogenic avian influenza virus subtype H5N1 infects humans with a high fatality rate and has pandemic potential. Vaccination is the preferred approach for prevention of H5N1 infection. Seasonal influenza virus infection has been reported to provide heterosubtypic immunity against influenza A virus infection to some extend. In this study, we used a mouse model pre-exposed to an H1N1 influenza virus and evaluated the protective ability provided by a single dose of DNA vaccines encoding conserved H5N1 proteins. RESULTS: SPF BALB/c mice were intranasally infected with A/PR8 (H1N1) virus beforehand. Six weeks later, the mice were immunized with plasmid DNA expressing H5N1 virus NP or M1, or with combination of the two plasmids. Both serum specific Ab titers and IFN-gamma secretion by spleen cells in vitro were determined. Six weeks after the vaccination, the mice were challenged with a lethal dose of H5N1 influenza virus. The protective efficacy was judged by survival rate, body weight loss and residue virus titer in lungs after the challenge. The results showed that pre-exposure to H1N1 virus could offer mice partial protection against lethal H5N1 challenge and that single-dose injection with NP DNA or NP + M1 DNAs provided significantly improved protection against lethal H5N1 challenge in mice pre-exposed to H1N1 virus, as compared with those in unexposed mice. CONCLUSIONS: Pre-existing immunity against seasonal influenza viruses is useful in offering protection against H5N1 infection. DNA vaccination may be a quick and effective strategy for persons innaive to influenza A virus during H5N1 pandemic.