Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3+CD56+ Subset through the Co-Culturing Dendritic Cells

Cytokine-induced killer (CIK) cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs). We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6) in DCs at the expense of Th2 inducing phenotype (IL-13) and regulatory phenotype (PD-L1, IDO). Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet) and the downregulation of the Th2 signature (GATA-3) and the Th17 marker (RORC) on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.     

Tropism of Avian Influenza A (H5N1) Virus to Mesenchymal Stem Cells and CD34+ Hematopoietic Stem Cells

The presence of abnormal hematologic findings such as lymphopenia, thrombocytopenia, and pancytopenia were diagnosed in severe cases of avian influenza A H5N1. Whether direct viral dissemination to bone marrow (BM) cells causes this phenomenon remains elusive. We explore the susceptibility of the two stem cell types; hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) isolated from human BM cells or cord blood, to infection with avian H5N1 viruses. For the first time, we demonstrated that the H5N1 virus could productively infect and induce cell death in both human stem cell types. In contrast, these activities were not observed upon human influenza virus infection. We also determined whether infection affects the immunomodulatory function of MSCs. We noted a consequent dysregulation of MSC-mediated immune modulation as observed by high cytokine and chemokine production in H5N1 infected MSCs and monocytes cocultures. These findings provide a better understanding of H5N1 pathogenesis in terms of broad tissue tropism and systemic spread.     

Induction of specific immune responses against the Plasmodium vivax liver-stage via in vitro activation by dendritic cells

Due to chronic morbidity, the risk of increasing drug resistance and the existence of the hypnozoite stage in Plasmodium vivax malaria, there is a need to find out how hosts develop immunity to compromise the malaria parasites. Here we focused on an in vitro model for immunotherapy and vaccine development. Immunosuppressive mechanisms in malaria include inhibition of T cell response and suppression of dendritic cell function. Using in vitro activation of lymphocytes by malaria antigen-pulsed dendritic cells could overcome the limitation of antigen presentation during acute infections. Here we showed that the sporozoite-pulsed dendritic cell could elicit cytotoxicity against liver stage of P. vivax. Analysis using immunophenotypic markers showed maturation of the dendritic cells and stimulation of cytotoxic T cells. Functional assay of the in vitro-activated cytotoxic T cells showed enhancement of specific killing of the P. vivax exoerythrocytic stages within infected hepatocytes. This model may be useful for vaccine development against human malaria.     

Syringe-push membrane absorption as a simple rapid method of urine preparation for clinical proteomics

Background

The analysis of urinary proteome might reveal biomarkers of clinical value. However, current methods of urine preparation for down-stream proteomic analysis are complicated, time-consuming, and/or expensive. This study aims to develop a robust, simple, inexpensive and readily accessible urine preparation method to facilitate clinical proteomic workflow.

Result

Syringe-push membrane absorption (SPMA) was successfully developed by a combination of 5-ml medical syringe and protein-absorbable membrane. Comparing three membranes i.e., nitrocellulose, polyvinylidene difluoride (PVDF) and Whatman no.1, nitrocellulose combined with SPMA (nitrocellulose-SPMA) provided the greatest quality of proteome profile as demonstrated by 2-DE. The quality of the proteome profile and the performance of nitrocellulose-SPMA were systematically compared with three current methods of urine preparation (i.e., ultrafiltration, dialysis/lyophilization and precipitation). While different methods of urine preparation provided comparable proteome quality, nitrocellulose-SPMA had better working performance due to acceptable recovery yield, less workload, short working time, high accessibility and low unit cost. In addition, protein absorbed on nitrocellulose harvested from the SPMA procedure could be stored as a dried membrane at room temperature for at least 1-month without protein degradation or modification.

Conclusions

SPMA is a simple rapid method of preparing urine for downstream proteomic analysis. Because of it is highly accessible and has long storage duration, this technique holds potential benefit for large-scale multi-center research and future development of clinical investigation based upon urinary proteomic analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9087-4) contains supplementary material, which is available to authorized users.     

A suspected new species of Leishmania, the causative agent of visceral leishmaniasis in a Thai patient

A suspected new species of Leishmania is described as the causative agent of the third reported case of autochthonous visceral leishmaniasis in a Thai man living in Southern Thailand. The results of PCR-restriction fragment length polymorphism and sequence analysis of the internal transcribed spacer 1 of ssrRNA and the mini-exon genes were different from those of previously reported Leishmania species. A direct agglutination test (DAT) revealed that antibody against Leishmania infection was detected in nine domestic cats. No potential vectors could be identified. A large-scale epidemiological survey of leishmaniasis should be urgently conducted since visceral leishmaniasis is considered an emerging disease of public health concern in Thailand.     

Antitumor efficacy and intratumoral distribution of SN-38 from polymeric depots in brain tumor model

We investigate antitumor efficacy and 2D and 3D intratumoral distribution of 7-ethyl-10-hydroxycamptothecin (SN-38) from polymeric depots inside U-87MG xenograft tumor model in nude mice. Results showed that polymeric depots could be used to administer and controlled release of a large amount of SN-38 directly to the brain tumor model. SN-38 released from depots suppressed tumor growth, where the extent of suppression greatly depended on doses and the number of depot injections. Tumor suppression of SN-38 from depots was three-fold higher in animals which received double injections of depots at high dose (9.7 mg of SN-38) compared to single injection (2.2 mg). H&E staining of tumor sections showed that the area of tumor cell death/survival of the former group was two-fold higher than those of the latter group. Fluorescence imaging based on self-fluorescent property of SN-38 was used to evaluate the intratumoral distribution of this drug compared to histological results. The linear correlation between fluorescence intensity and the amount of SN-38 allowed quantitative determination of SN-38 in tumor tissues. Results clearly showed direct correlation between the amount of SN-38 in tumor sections and cancer cell death. Moreover, 3D reconstruction representing the distribution of SN-38 in tumors was obtained. Results from this study suggest the rationale for intratumoral drug administration and release of drugs inside tumor, which is necessary to design drug delivery systems with efficient antitumor activity.     

Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects.     

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection

Leishmania martiniquensis, a zoonotic hemoflagellate, is a causative agent of cutaneous (CL) and visceral leishmaniasis (VL) among humans and animals. This organism, first reported in Martinique Island, now has become an emerging infectious agent in Thailand. Symptomatic cases of L. martiniquensis infection among humans have continuously increased. In the meantime, asymptomatic infection of this novel species has seriously created national public health awareness and concern to prevent and control disease transmission. The unsuccessful serological test using the commercial rK39 dipstick based on antigen from Leishmania donovani to detect the antibodies against VL among infected Thai patients has encouraged us to further explore a new sensitive and specific antigenic epitope. In this study, we determined the sequences and expressed recombinant proteins of kinesin 39 (k39), heat shock protein 70 (hsp70), heat shock protein 83 (hsp83), and glycoprotein 63 (gp63) of L. martiniquensis to evaluate the diagnostic efficiency to detect antibodies against L. martiniquensis in patient sera. The preliminary results from western blot analysis have suggested that K39 is the most sensitive recombinant protein to detect L. martiniquensis. Moreover, this recombinant protein reacts with antibodies against L. donovani and Leishmania infantum, making it a promising antigen for further development of a universal rapid diagnostic tool for VL.     

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

Background

Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.

Results

A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val₃₂₇ - Asn₃₅₆) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp₂₉₈-Val₃₁₃ peptide was shortened to Asp₂₉₈-Arg₃₁₀ in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.

Conclusions

The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.     

Prevalence and associated risk factors of Leishmania infection among immunocompetent hosts,a community-based study in Chiang Rai,Thailand

BackgroundLeishmaniasis is an emerging infectious disease reported in the north and south of Thailand of which patients with HIV/AIDS are a high risk group for acquiring the infection. A lack of information regarding prevalence, and the risk association of Leishmania infection among asymptomatic immunocompetent hosts needs further investigation. Information on potential vectors and animal reservoirs in the affected areas is also important to control disease transmission.MethodsAn outbreak investigation and a cross-sectional study were conducted following one index case of cutaneous leishmaniasis (CL) caused by L. martiniquensis in an immunocompetent male patient reported in August 2015, Chiang Rai Province, Thailand. From September to November 2015, a total of 392 participants at two study areas who were related to the index case, 130 students at a semi-boarding vocational school and 262 hill tribe villagers in the patient’s hometown, were recruited in this study. The nested internal transcribed spacer 1-PCR (ITS1-PCR) was performed to detect Leishmania DNA in buffy coat, and nucleotide sequencing was used to identify species. Antibody screening in plasma was performed using the Direct Agglutination Test (DAT), and associated risk factors were analyzed using a standardized questionnaire. Captured sandflies within the study areas were identified and detected for Leishmania DNA using nested ITS1-PCR. Moreover, the animal reservoirs in the study areas were also explored for Leishmania infection.ResultsOf 392 participants, 28 (7.1%) were positive for Leishmania infection of which 1 (4.8%) was L. martiniquensis, 12 (57.1%) were L. orientalis and 8 (38.1%) were Leishmania spp. Of 28, 15 (53.6%) were DAT positive. None showed any symptoms of CL or visceral leishmaniasis. Risk factors were associated with being female (adjusted odds ratio, AOR 2.52, 95%CI 1.01–6.26), increasing age (AOR 1.05, 95%CI 1.02–1.08), having an animal enclosure in a housing area (AOR 3.04, 95%CI 1.13–8.22), being exposed to termite mounds (AOR 3.74, 95%CI 1.11–12.58) and having domestic animals in a housing area (AOR 7.11, 95%CI 2.08–24.37). At the semi-boarding vocational school, six Sergentomyia gemmea samples were PCR positive for DNA of L. orientalis and one S. gemmea was PCR positive for DNA of L. donovani/L. infantum. Additionally, one Phlebotomus stantoni was PCR positive for DNA of L. martiniquensis, and one black rat (Rattus rattus) was PCR positive for DNA of L. martiniquensis.ConclusionThis information could be useful for monitoring Leishmania infection among immunocompetent hosts in affected areas and also setting up strategies for prevention and control. A follow-up study of asymptomatic individuals with seropositive results as well as those with positive PCR results is recommended.