Helen K. W. Law

Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents

Mesenchymal stem cells (MSCs) are an important cellular component of the bone marrow microenvironment for supporting haemopoiesis. However, their response to high‐dose chemotherapy remains unknown. We assessed the acute direct effects of individual chemotherapeutic agents on human MSCs (hMSCs). Using an in vitro culture system, the chemosensitivity of hMSCs was determined by XTT (2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulphophenyl)‐5‐[(phenylamino) carbonyl]‐2H‐tetrazolium hydroxide) assay in comparison with that of NB‐4 cells, a leukaemic cell line, and normal peripheral blood mononuclear cells. The recovery of cell numbers following exposure to chemotherapeutic agents and chemotherapy‐induced apoptosis of hMSCs were evaluated. Human MSCs were resistant to chemotherapeutic agents commonly used in bone marrow transplantation (BMT) (i.e. busulphan, cyclophosphamide and methotrexate). However, they were relatively sensitive to a panel of cytotoxic agents, such as paclitaxel, vincristine, etoposide and cytarabine. Furthermore, different recovery patterns were noted. There was sustained suppression in hMSCs following 3u2003d exposure to paclitaxel, cytarabine and etoposide. In contrast, significant recovery was seen in hMSCs treated with dexamethasone and vincristine respectively. Human MSCs have different patterns of response to a panel of chemotherapeutic agents commonly used in BMT or cancer therapy. Understanding this variation is important in optimizing conditioning regimens for BMT.

Mannose-Binding Lectin in Severe Acute Respiratory Syndrome Coronavirus Infection

Abstract Little is known about the innate immune response to severe acute respiratory syndrome (SARS) coronavirus (CoV) infection. Mannose-binding lectin (MBL), a key molecule in innate immunity, functions as an ante-antibody before the specific antibody response. Here, we describe a case-control study that included 569 patients with SARS and 1188 control subjects and used in vitro assays to investigate the role that MBL plays in SARS-CoV infection. The distribution of MBL gene polymorphisms was significantly different between patients with SARS and control subjects, with a higher frequency of haplotypes associated with low or deficient serum levels of MBL in patients with SARS than in control subjects. Serum levels of MBL were also significantly lower in patients with SARS than in control subjects. There was, however, no association between MBL genotypes, which are associated with low or deficient serum levels of MBL, and mortality related to SARS. MBL could bind SARS-CoV in a dose- and calcium-dependent and mannan-inhibitable fashion in vitro, suggesting that binding is through the carbohydrate recognition domains of MBL. Furthermore, deposition of complement C4 on SARS-CoV was enhanced by MBL. Inhibition of the infectivity of SARS-CoV by MBL in fetal rhesus kidney cells (FRhK-4) was also observed. These results suggest that MBL contributes to the first-line host defense against SARS-CoV and that MBL deficiency is a susceptibility factor for acquisition of SARS

Decreased yield, phenotypic expression and function of immature monocyte‐derived dendritic cells in cord blood

Dendritic cells are critical for the induction of both primary immune responses and immunological tolerance, as well as for the regulation of T‐helper 1 (Th1) and 2 (Th2) immune responses. As neonates are notably deficient in Th1 response and cord blood transplantation is noted to result in less graft‐versus‐host disease (GvHD), we compared the phenotypic and functional characteristics of monocyte‐derived dendritic cells (DCs) that favour Th1 development from cord blood and adult peripheral blood to understand the underlying mechanisms of these observations. Our results showed that: (1) after culture for 7u2003d with interleukin (IL)‐4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), cord blood monocytes generated less CD1a+ cells than adult peripheral blood monocytes, and the CD1a+ cell percentage decreased thereafter; (2) compared with adult blood DCs, cord blood DCs had reduced intensity of expression of CD1a and MHC class II molecules, but the expression levels of CD11c and CD86 were similar; (3) the endocytotic ability of cord blood DCs was reduced compared with adult blood DCs, and this function was related to reduced mannose receptor (MR)‐positive cells; (4) furthermore, the ability of cord blood DCs to stimulate CD3+ T cells in an allogeneic mixed lymphocyte reaction was significantly lower than that of adult blood DCs. These results suggested that the dysfunction of cord blood monocytes in differentiating into professional DCs will affect the activation of naive T cells, especially Th1 development, and may be related to the susceptibility to different infections in the neonates, as well as the lower incidence of GvHD in cord blood transplantation.

Enumeration of human antigen-specific naive CD8 T cells reveals conserved precursor frequencies

The number of antigen-specific naive CD8(+) T cells is believed to be important in the shaping of adaptive immune responses, and is predictive for the magnitude of priming responses in mouse models. Because of extremely low precursor frequencies, knowledge about these cells comes from indirect techniques and estimations. Here, we present a strategy based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric cytometry, which permitted direct detection and analysis of CD8(+) T cells reactive for 6 different naive epitopes (MART-1(26-35), HIV-1 Gag p17(77-85), hepatitis C virus [HCV] NS3(1406-1415), HCV Core(132-140), NY-ESO-1(157-165), and cytomegalovirus [CMV] pp65(495-503)). Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6 x 10(-6) to 1.3 x 10(-4)), but conserved frequencies among humans. Development of a procedure for direct assessment of T-cell precursor frequency in humans has important implications, with particular relevance to vaccine development and monitoring of tumor and self-reactive T cells.

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

ABSTRACT Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the viruss success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.

MHC expression kinetics and immunogenicity of mesenchymal stromal cells after short-term IFN-γ challenge

OBJECTIVEnUnder the influence of interferon-gamma (IFN-gamma), mesenchymal stromal cells (MSCs) are conditional antigen-presenting cells, which have immunosuppressive potential. Apart from IFN-gamma upregulation of major histocompatibility complexes class I and II (MHC-I and MHC-II) expression, the underlying kinetics and mechanisms have not been described previously. This information is helpful to delineate how human MSCs can be modulated by IFN-gamma in different clinical scenarios.nnnMATERIALS AND METHODSnHere, we demonstrated that IFN-gamma-treated MSCs underwent classical signal transduction pathway via phosphorylation of signal transducers and activators of transcription-1, activation of interferon regulatory factor-1, and class II transactivator comparable to that of primary human blood macrophages.nnnRESULTSnIFN-gamma markedly induced expression of MHC-I instantly, while its effects on MHC-II were less dramatic and delayed up to 4 days. This is due to a slower intracellular transport of the MHC-II antigen to the membrane surface. More important is that MSCs showed a reduction in their proliferation by 50% without evidence of cell death after prolonged IFN-gamma treatment for 8 days. High-dose IFN-gamma-treated MSCs (500 U/mL) could initiate T-cell activation as indicated by expression of CD25 and proliferation of allogeneic T cells.nnnCONCLUSIONSnThe summative IFN-gamma effects will adversely affect the immunoprivilege status and lifespan of MSCs.

Functional Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Production by Avian Influenza Virus–Infected Macrophages

Abstract Severe human disease associated with influenza A H5N1 virus was first detected in Hong Kong in 1997. Its recent reemergence in Asia and high associated mortality highlight the need to understand its pathogenesis. We investigated the roles of death receptor ligands (DRLs) in H5N1 infection. Significant up-regulation of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TNF-α, but not Fas ligand (FasL) mRNA, was detected in human monocyte–derived macrophages (MDMs) infected with avian influenza viruses A/Hong Kong/483/97 (H5N1/97) or its precursor, A/Quail/Hong Kong/G1/97. H5N1/97-infected MDMs exhibited the strongest induction of apoptosis in Jurkat T cells, and it could be reduced by TRAIL–receptor 2 blocking antibody. Furthermore, influenza virus infection enhanced the sensitivity of Jurkat T cells to apoptosis induced by TNF-α, TRAIL, and FasL. Our data suggested that functional TRAIL produced by influenza virus–infected MDMs was related to their cytotoxicity and that the enhanced sensitization to DRL-induced apoptosis detected in avian influenza may contribute to disease pathogenesis

Differential Expression of Chemokines and Their Receptors in Adult and Neonatal Macrophages Infected with Human or Avian Influenza Viruses

Abstract In 1997, avian influenza virus H5N1 was transmitted directly from chicken to human and resulted in a severe disease that had a higher mortality rate in adults than in children. The characteristic mononuclear leukocyte infiltration in the lung and the high inflammatory response in H5N1 infection prompted us to compare the chemokine responses between influenza virus–infected adult and neonatal monocyte-derived macrophages (MDMs). The effects of avian influenza virus A/Hong Kong/483/97 (H5N1) (H5N1/97), its precursor A/Quail/Hong Kong/G1/97 (H9N2) (H9N2/G1), and human influenza virus A/Hong Kong/54/98 (H1N1) (H1N1/98) were compared. Significantly higher expression of CCL2, CCL3, CCL5, and CXCL10 was induced by avian influenza viruses than by human influenza virus. Moreover, the increase in CCL3 expression in H5N1/97-infected adult MDMs was significantly higher than that in neonatal MDMs. Enhanced expression of CCR1 and CCR5 was found in avian virus–infected adult MDMs. The strong induction of chemokines and their receptors by avian influenza viruses, particularly in adult MDMs, may account for the severity of H5N1 disease

Ganoderma lucidum polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function.

BackgroundPrevious studies demonstrated Ganoderma lucidum polysaccharides (GL-PS), a form of bioactive β-glucan can stimulate the maturation of monocyte-derived dendritic cells (DC). The question of how leukemic cells especially in monocytic lineage respond to GL-PS stimuli remains unclear.ResultsIn this study, we used in vitro culture model with leukemic monocytic cell-lines THP-1 and U937 as monocytic effectors cells for proliferation responses and DCs induction. We treated the THP-1 and U937 cells with purified GL-PS (100 μg/mL) or GL-PS with GM-CSF/IL-4. GL-PS alone induced proliferative response on both THP-1 and U937 cells but only THP-1 transformed into typical DC morphology when stimulated with GL-PS plus GM-CSF/IL-4. The transformed THP-1 DCs had significant increase expression of HLA-DR, CD40, CD80 and CD86 though not as high as the extent of normal monocyte-derived DCs. They had similar antigen-uptake ability as the normal monocyte-derived DCs positive control. However, their potency in inducing allogeneic T cell proliferation was also less than that of normal monocyte-derived DCs.ConclusionOur findings suggested that GL-PS could induce selected monocytic leukemic cell differentiation into DCs with immuno-stimulatory function. The possible clinical impact of using this commonly used medicinal mushroom in patients with monocytic leukemia (AML-M4 and M5) deserved further investigation.

Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors

BackgroundHuman mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.ResultsWe selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria.ConclusionIn conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.