Clinical laboratory investigation of a patient with an extremely high D-dimer level: A case report.

BACKGROUND: D-dimer, a soluble degradation product of cross-linked fibrin, is commonly used as an important marker for the diagnosis of disseminated intravascular coagulation and differential diagnosis of thrombosis. Herein, we present a geriatric case with an unusually elevated D-dimer level. CASE SUMMARY: An 82-year-old woman, admitted to the ward with a diagnosis of chronic heart failure, was noted to have a remarkably elevated D-dimer level, beyond the qualified range (> 100 mg/L), utilizing the Innovating D-dimer for Sysmex CS-5100 System™. However, no evidence, including clinical symptoms, radiographic evidence of thromboembolic disease, and parallel fibrinogen degradation product values, suggested that this patient was at high risk of thrombopenia. To confirm the discrepancy, a series of approaches including sample dilution, re-analysis via alternative methods, and sample treatment with blockage of specific heterophilic antibodies were performed. A remarkable disappearance of the elevated D-dimer values was observed in the samples after they were subjected to these approaches (4.49, 9.42, 9.06, and 12.58 mg/L, respectively). This confirmed the presence of heterophilic antibodies in this case. In addition, a reduction in cardiac output due to the presence of cardiac failure could also be responsible for the existence of a hypercoagulable state in this case. CONCLUSION: In conclusion, the presence of heterophilic antibodies should be considered when an elevated D-dimer value is not in conformity with the clinical evidence, and a viral infection should be considered when interference by a heterophilic antibody exists.

Immunization with truncated envelope protein of Zika virus induces protective immune response in mice.

The global spread of Zika virus (ZIKV) as well as its unexpected link to infant microcephaly have resulted in serious public health concerns. No antiviral drugs against ZIKV is currently available, and vaccine development is of high priority to prepare for potential ZIKV pandemic. In the present study, a truncated E protein with the N-terminal 90% region reserved (E90) from a contemporary ZIKV strain was cloned and expressed in Escherichia coli, purified by a Ni-NTA column, and characterized by Western blotting assays. Immunization with recombinant E90 induced robust ZIKV-specific humoral response in adult BALB/c mice. Passive transfer of the antisera from E90-immunized mice conferred full protection against lethal ZIKV challenge in a neonatal mice model. Our results indicate that recombinant ZIKV E90 described here represents as a promising ZIKV subunit vaccine that deserves further clinical development.

The bone-preserving effects of exendin-4 in ovariectomized rats.

Exendin-4 was found to be beneficial to the skeleton in diabetic rodents. In this study, we assessed the changes of bone mineral densities (BMDs) and quality in non-diabetic ovariectomized (OVX) rats after treatment with exendin-4. The regulatory role of exendin-4 on osteoblastogenesis and adipogenesis in rat bone marrow stromal cells (BMSCs) was also explored. Three months after sham surgery or OVX, 18 5-month-old female Wistar rats were divided into three groups and received the following treatment for 8 weeks: (1) Sham + vehicle; (2) OVX + vehicle; and (3) OVX + exendin-4 20 µg/kg/day. Micro-CT and three-point bending test were used to evaluate the BMDs, bone morphometric parameters, and biomechanical properties. Real-time PCR and Western blot were performed to measure gene and protein expression after exendin-4 treatment in adipogenesis and osteoblastogenesis of rat BMSCs. Exendin-4 could improve trabecular volume, thickness, and number, increase BMD, and reduce trabecular spacing in the lumbar spine and femur of OVX rats. Exendin-4 had little impact on the mechanical resistance of femurs to fracture. When rat BMSCs were treated with exendin-4, the mRNA expression levels of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and collagen α1 (Coll-1) were increased, while those of peroxisome proliferators activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBPα) decreased. Exendin-4 treatment also resulted in increased expression levels of p38, p42/44, and ß-catenin proteins. Exendin-4 was anabolic to bone in OVX rats possibly by facilitating osteoblastogenesis while repressing adipogenesis during BMSC lineage differentiation.

The Anti-inflammatory Effect of the CXCR4 Antagonist-N15P Peptide and Its Modulation on Inflammation-Associated Mediators in LPS-Induced PBMC.

Inflammation was the important pathological process of many disease developments, but current therapeutic means for inflammatory diseases are not satisfactory. Chemokines and their receptors represent valuable targets for anti-inflammatory drug discovery. The N15P polypeptide (sequence: LGASWHRPDKCCLGY) is independently developed by our research group, it is a new CXCR4 antagonist drug derived from viral macrophage inflammatory protein-II (vMIP-II). This study aims to clarify the anti-inflammatory potency of N15P polypeptide on the lipopolysaccharide (LPS)-induced inflammation in vitro. In this study, we evaluated the anti-inflammatory effects of N15P polypeptide by the LPS-induced peripheral blood mononuclear cell (PBMC) model and measured the level of inflammatory factors (tumor necrosis factor alpha (TNF-α), IL-6, IL-8, nuclear factor kappaB (NF-κB), cyclooxygenase-2 (COX-2), Toll-like receptor 4 (TLR4), MyD88, phosphoinositide 3-kinase (PI3K), and Akt). The messenger RNA (mRNA) expressions of inflammatory factors were analyzed by real-time PCR (RT-PCR) microarray analysis, and the production of inflammatory factors was measured further by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that the expression of inflammatory factors (TNF-α, IL-6, IL-8, NF-κB, COX-2, TLR4, MyD88, PI3K, and Akt) was downregulated by N15P peptide, suggesting that N15P peptide has a strong inhibitory effect on the inflammatory responses induced by LPS.

A biologically active vMIP-II-IgG3-TfN fusion protein, secreted from methylotrophic yeast Pichia pastoris.

The viral macrophage inflammatory protein II (vMIP-II) which showed a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus. To explore the feasibility of using TfN as a carrier moiety for delivery of therapeutic proteins, a genetically engineered vMIP-II-IgG3-TfN fusion gene was loaded into the yeast expression vector pPICZα. The linearized recombinant plasmid pPICZα-vMIP-II-IgG3-TfN was transformed into X33 competent cells. The recombinant protein was expressed in methylotrophic yeast Pichia pastoris and was confirmed to have expected molecular mass of 48 kDa by SDS-PAGE. Using methods combining ammonium sulfate precipitation, dialysis, ultrafiltration and affinity chromatography, the vMIP-II-IgG3-TfN fusion protein was successfully purified from the supernatant of the broth. Western-blotting analysis showed that 6× His antibody recognized the purified vMIP-II-IgG3-TfN. CD spectrum revealed a positive peak at 196.5 nm and a negative peak at 209 nm. MALDI-TOF MS analysis showed that the purified vMIP-II-IgG3-TfN was an intact and homogeneous protein. The pepsin digestibility assay showed that the vMIP-II-IgG3-TfN fusion protein could be digested into small fragments by pepsin after 2 min treatment. The vMIP-II-IgG3-TfN fusion protein was found to be stable in human plasma for up to 48 h. Furthermore, in vitro bioactivity assay indicated that the vMIP-II-IgG3-TfN fusion protein can block the chemotaxis of U937 cells induced by SDF1α. In total, this study illustrates the development of an active vMIP-II-IgG3-TfN fusion protein expressed in P. pastoris.

Anti-HCMV and KSHV effect of a trapping ligand antagonist for herpesvirus-encoded GPCR.

We have found and synthesized a trapping ligand peptide H22-LP (the conservative sequence is NAHCALL) from a random phage library according to the broad-spectrum trapping receptor H22, which derived from the residue 14-35 near the N-terminal region of receptor US28 on HCMV. In this study, we will evaluate its potential as an efficient antagonist of US28 and the anti-virus activity, acting as a broad spectrum chemokine receptors antagonist. Stable expression of US28 and ORF74 in NIH/3T3 cells were successfully constructed in vitro. Flow cytomety was used to determine the concentration of Ca2+ induced by H22-LP, and the binding of H22-LP and US28 was confirmed by enzyme-linked immunosorbent assay (ELISA). Antivirus activity of H22-LP on HCMV and KSHV was evaluated by anti-virus experiments. Our data suggest that H22-LP is an effectual antagonist of receptor US28 of HCMV and ORF74 of KSHV in the transfection assay, and it has potential to inhibit infection of HCMV and KSHV. These results provide support for the development of anti-virus strategies based on targeted inhibiting the infection of herpesvirus.

[The viral acceptor antagonist to entrapment ligand H9 in vitro activeness and the function to human acceptor CX3-CR1].

AIM: To clarify the activeness of H9 in vitro and internalization and modulation of the surface chemokine receptor CX3CR1 induced by H9, To discuss the influence of H9 on the chemokine receptor CX3CR1. METHODS: Inhibition by chemotactic peptide on the physiological detection of chemokine induced cell migration activity. Flowcytometry examined the effection of H9 on intracellular calcium. Laser scanning confocal microscopy and flow cytometry were used to determine the quality and quantity of CX3CR1 internalization. RESULTS: H9 was able to block the migration induced by chemokine receptor. In the chemoattraction test, H9 was unable to induce the chemotactic movement, and it does not affect the signal transduction and activeness of cells. It was found that H9 could induce internalization with a maximal rate of 70%, at the concentration of 200 ng/mL. The internalized CX3CR1 molecules could recycled to the cell surface. CONCLUSION: H9 makes human CX3CR1 internalize. After internalizing, the CX3CR1 receptor recirculates the cell surface. It does not affect CX3CR1 physiology function. H9 could be used as a specificity anti-virus peptide.

[Acute toxicity of CCR5 antagonist C25P polypeptide in mice and its carcinogenicity in vitro].

OBJECTIVE: To study the acute toxicity of C25P polypeptide, a CCR5 antagonist, in mice and its carcinogenic effect in vitro. METHODS: The acute toxicity of C25P polypeptide in mice was assessed by determining the maximum tolerated dose (MTD). The mice were given C25P at the dose of 3.64 g/kg by tail vein injection, and the control mice received saline (40 ml/kg) injection. The mice were continuously observed for 14 days after the administration and sacrificed on day 14 for routine blood test, examination of the blood biochemistry and pathological examination. The carcinogenicity of C25P polypeptide in vitro was evaluated in cultured cell lines by chromosome aberration test, cell transformation test and non-anchorage dependent growth test. RESULTS: No mice died following administration of the drug, but 3 mice showed mild adverse reactions. The rats in both groups showed an increase in the body weight at a comparable rate. GPT increased and ALP decreased significantly in C25P polypeptide group (P<0.05). Most of the organs of the rats treated with in C25P polypeptide remained normal, but 3 mice showed pathologies in the lung, spleen and liver. Chromosome aberration test, cell transformation test and non-anchorage-dependent growth test all yielded negative results for C25P polypeptide. CONCLUSION: C25P polypeptide is a low-toxicity drug that produces no apparent acute toxicity in mice or obvious carcinogenicity in vitro.

[Study on dengue virus infection of human dendritic cells].

To study Dengue virus (DV) infection of human dendritic cells (DC). Monocyte isolated from healthy human peripheral blood were incubated in medium with GM-CSF and IL-4 for more than 7 days. DCs were then collected and identified by transmission electron microscope, immunohistochemistry and lymphocytes stimulatory ability. Dengue virus type II (DV-2) were infected with human dendritic cells(DC) in vitro, culture supernatants and cells were collected by different time postinfection (6h, 12h, 24h, 48h, 72h). Viral titers were evaluated by microplaque forming assay on C6/36 monolayer cells; DV antigen in human dendritic cells were demonstrated by an indirect immunofluorescent assay (IFA). Localization of DV in DC was observed under a transmission electron microscope. The viruses were detected in the culture supernatants as early as 6h after infection; the highest viral titers were obtained at 48h, and then declined to very low titers at 96h. DV-2 antigen was detected in infected DC by IFA. After infection for 48h, DV particles were obvious in cystic vesicle, vacuoles . Human dendritic cells are targets of dengue virus infection. DV could efficiently infect DC and produce virus particles, DC possibly plays a role in the pathogenesis of DV infection.

[Inhibitory effect of viral macrophage inflammatory protein-II on metastasis of breast cancer cell line MCF-7 through antagonising CXCR4].

BACKGROUND & OBJECTIVE: CXCR4-stromal cell-derived factor-1 (CXCR4-SDF-1alpha) system has been proved to be involved in targeting metastasis of breast cancer. Some antagonists of CXCR4 have inhibitory effects on metastasis of breast cancer. This study was to investigate effect of viral macrophage inflammatory protein-II (vMIP-II), an antagonist of CXCR4, on metastasis of breast cancer cell line MCF-7. METHODS: Proliferation of MCF-7 cells stimulated by vMIP-II of different concentrations (10, 50, 100, 500, and 1 000 ng /ml) was detected by MTT assay, clone formation rate was assessed by agar clone assay. Adhesion and chemotaxis assays were also used to evaluate the effect of vMIP-II on MCF-7 cells in different steps of metastasis. RESULTS: MCF-7 cells treated with vMIP-II of a series of concentrations for 72 h showed no proliferation change (P >0.05). vMIP-II (50-1 000 ng /ml) suppressed colony formation of MCF-7 cells in a concentration-dependent manner. After MCF-7 cells treated with 300 ng/ml of vMIP-II for different time (0, 0.5, 2, and 6 h), inhibition peak of cell adherence to fibronectin (FN) and Matrigel was observed. The number of migration was low in MCF-7 cells in the presence of vMIP-II of 500 ng/ml (24+/-10) was lower than that of control MCF-7 cells (60+/-9) (P< 0.05). CONCLUSIONS: The clone formation rate of MCF-7 cells may negatively correlates with the concentration of vMIP-II. vMIP-II may inhibit MCF-7 cells adhesion to FN and Matrigel, and suppress chemotactic activity of MCF-7 cells toward extracts of human lung protein.