Host Peripheral Blood Mononuclear Cells Research Articles

Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

Distinct transcriptomic signatures define febrile malaria depending on initial infective states, asymptomatic or uninfected

BackgroundCumulative malaria parasite exposure in endemic regions often results in the acquisition of partial immunity and asymptomatic infections. There is limited information on how host-parasite interactions mediate the maintenance of chronic symptomless infections that sustain malaria transmission.MethodsHere, we determined the gene expression profiles of the parasite population and the corresponding host peripheral blood mononuclear cells (PBMCs) from 21 children (< 15 years). We compared children who were defined as uninfected, asymptomatic and those with febrile malaria.ResultsChildren with asymptomatic infections had a parasite transcriptional profile characterized by a bias toward trophozoite stage (~ 12 h-post invasion) parasites and low parasite levels, while early ring stage parasites were characteristic of febrile malaria. The host response of asymptomatic children was characterized by downregulated transcription of genes associated with inflammatory responses, compared with children with febrile malaria,. Interestingly, the host responses during febrile infections that followed an asymptomatic infection featured stronger inflammatory responses, whereas the febrile host responses from previously uninfected children featured increased humoral immune responses.ConclusionsThe priming effect of prior asymptomatic infection may explain the blunted acquisition of antibody responses seen to malaria antigens following natural exposure or vaccination in malaria endemic areas.

Immunogenicity analysis of BPV-1 positive equine sarcoid-derived cultured fibroblasts

Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)− 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/− 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies.

Immunomodulatory effects of two recombinant arginine kinases in Sarcoptes Scabiei on host peripheral blood mononuclear cells.

As an important zoonotic parasitic disease with global distribution, scabies causes serious public health and economic problems. Arginine kinase (AK) is involved in cell signal transduction, inflammation, and apoptosis. Two AKs were identified in Sarcoptes scabiei, but their functions in the host immune response remain unclear. rSsAK-1 and rSsAK-2 were expressed, purified, and immunolocalized. The effects of rSsAK-1 and rSsAK-2 on rabbit PBMC proliferation, apoptosis, and migration; Bcl-2, Bcl-xl, Fas, Bax, and NF-κB transcription levels; and IL-2, IFN-γ, IL-4, IL-10, TGF-β1, and IL-17 secretion were detected. rSsAK-1 and rSsAK-2 were cloned and expressed successfully. Both enzymes were ~57 kDa and contained 17-kDa tagged proteins, and had good catalytic activity and immunoreactivity. The proteins were located in the S. scabiei exoskeleton, chewing mouthparts, legs, stomach, and intestine. SsAK-1 and SsAK-2 were secreted in the pool and epidermis of the skin lesions, which may be involved in S. scabiei-host interaction. rSsAK-1 and rSsAK-2 significantly promoted cell proliferation, induced cell migration, inhibited apoptosis, and increased Bcl-2, Bcl-xl and NF-κB (p65) transcription levels concentration-dependently, and inhibited IL-2, IFN-γ, and IL-10 secretion and promoted IL-4 and IL-17 secretion. rSsAK-1 and rSsAK-2 might increase Bcl-2 and Bcl-xl expression by activating the NF-κB signaling pathway to promote cell proliferation and inhibit apoptosis, which induced PBMC survival. By inducing PBMC migration to the infection site, rSsAK-1 and rSsAK-2 shifted the Th1/Th2 balance toward Th2 and changed the Th17/Treg balance, which indicated their immune role in S. scabiei allergic inflammation.

Impact of Plasmodium falciparum small-sized extracellular vesicles on host peripheral blood mononuclear cells

Background: Exagerated immune activation has a key role in the pathogenesis of malaria. During blood-stage infection, Plasmodium falciparum can interact directly with host immune cells through infected red blood cells (PfiRBCs), or indirectly by the release of extracellular vesicles (EVs). Here, we compared the impact of PfiRBCs and P. falciparum small-sized EVs (PfsEVs, also known as exosomes) from a Kenyan clinical isolate (PfKE12) adapted to short-term laboratory culture conditions on host peripheral blood mononuclear cells (PBMC). Methods: PfsEVs were isolated from cell-free culture-conditioned media by ultracentrifugation while mature trophozoite PfiRBCs were purified by magnetic column separation. The PfsEVs and the PfiRBCs were co-cultured for 18 hours with PBMC. Cellular responses were quantified by cell surface expression of activation markers (CD25, CD69) and cytokine/chemokine levels in the supernatant. Results: Relative to negative control conditions, PfsEVs induced CD25 expression on CD4+, CD19+ and CD14+ cells, while PfiRBCs induced on CD19+ and CD14+ cells. Both PfsEVs and PfiRBCs induced CD69 on CD4+, CD8+ and CD19+ cells. In addition, PfiRBCs induced higher expression of CD69 on CD14+ cells. CD69 induced by PfiRBCs on CD4+ and CD19+ cells was significantly higher than that induced by PfsEVs. Secretion of MIP1α, MIP1β, GM-CSF, IL-6, IL-8, and TNFα were significantly induced by both PfsEVs and PfiRBCs whereas MCP-1, IL-10, IL-17α were preferentially induced by PfsEVs and IP-10 and IFN-γ by PfiRBCs. Prior exposure to malaria (judged by antibodies to schizont extract) was associated with lower monocyte responses to PfsEVs. Conclusions: PfsEVs and PfiRBCs showed differential abilities to induce secretion of IL-17α and IFN-γ, suggesting that the former are better at inducing Th17, whilst the latter induce Th1 immune responses respectively. Prior exposure to malaria significantly reduces the ability of PfsEVs to activate monocytes, suggesting immune tolerance to PfsEVs may play a role in naturally acquired anti-disease immunity.

Unveiling the Immunomodulatory Characteristics of Haemonchus contortus Ephrin Domain Containing Protein in the Parasite-Host Interactions.

Simple SummaryHaemonchus contortus excretory/secretory products (HcESPs) contain many proteins that can perform various functions including modulating the host immune response. Recent studies indicate that IL-9 can be secreted by a specialized population of T cells called Th9 cells, which mediate anti-parasite immunity. Furthermore, HcESPs could enhance goat peripheral blood mononuclear cells (PBMCs) derived Th9 cells production. Ephrin domain containing protein (EPH) was identified as one of the HcESPs that can be isolated from different stages of this helminth. Nonetheless, the understanding of immunomodulatory roles of EPH on Th9 and other immune cells remains limited. In this study, the correlation between recombinant H. contortus Ephrin domain containing protein(rHcEPH)and goat PBMCs significantly enhanced Th9 cells differentiation, IL-9 expression, cell apoptosis efficiency, and cell migration, whereas cell proliferation was suppressed significantly depending on the concentration. Our findings illustrated that rHcEPH protein is linked to modulate the host immune cells and could enhance protective immunity by inducing Th9 cells secreted IL-9 in vitro.Ephrin domain containing protein (EPH), a significant excreted and secreted product (ESPs) of Haemonchus contortus, has been identified to have antigenic functions. Over the past years, a new subset of CD4 + T named as T helper 9 cells that secrete interleukin-9 (IL-9) as a signature cytokine is associated with tumor immunity and allergy. Nonetheless, the understanding of immunomodulatory roles of EPH on goat Th9 and other immune cells remains limited. Herein, EPH from H. contortus (HcEPH) was cloned and expressed in pET-28a. Immunofluorescence assay (IFA) was carried-out to localize rHcEPH within H. contortus adult worms and to bind with goat peripheral blood mononuclear cells (PBMCs). Besides, the impact of rHcEPH on signature cytokine IL-9 expression in goat PBMCs was evaluated. Flow cytometry was employed to examine Th9 cells production and cell apoptosis. The results revealed success in the expression and localization of rHcEPH in surface of adult H. contortus gut sections. According to IFA analysis, the rHcEPH protein was capable to react precisely with anti-H. contortus antibodies. Further functional analysis showed that correlation between rHcEPH and host PBMCs significantly enhanced Th9 cell differentiation, IL-9 expression, cell apoptosis efficiency, and cell migration, whereas cell proliferation was suppressed significantly depending on the concentration. Our observations indicated that rHcEPH protein is linked to modulate the host immune cells and could enhance protective immunity by inducing Th9 responses.

Impact of Plasmodium falciparum small-sized extracellular vesicles on host peripheral blood mononuclear cells

Background: Exagerated immune activation has a key role in the pathogenesis of malaria. During blood-stage infection, Plasmodium falciparum can interact directly with host immune cells through infected red blood cells (PfiRBCs), or indirectly by the release of extracellular vesicles (EVs). Here, we compared the impact of PfiRBCs and P. falciparum small-sized EVs (PfsEVs, also known as exosomes) from a Kenyan clinical isolate (PfKE12) adapted to short-term laboratory culture conditions on host peripheral blood mononuclear cells (PBMC). Methods: PfsEVs were isolated from cell-free culture-conditioned media by ultracentrifugation while mature trophozoite PfiRBCs were purified by magnetic column separation. The PfsEVs and the PfiRBCs were co-cultured for 18 hours with PBMC. Cellular responses were quantified by cell surface expression of activation markers (CD25, CD69) and cytokine/chemokine levels in the supernatant. Results: Relative to negative control conditions, PfsEVs induced CD25 expression on CD4+, CD19+ and CD14+ cells, while PfiRBCs induced on CD19+ and CD14+ cells. Both PfsEVs and PfiRBCs induced CD69 on CD4+, CD8+ and CD19+ cells. In addition, PfiRBCs induced higher expression of CD69 on CD14+ cells. CD69 induced by PfiRBCs on CD4+ and CD19+ cells was significantly higher than that induced by PfsEVs. Secretion of MIP1α, MIP1β, GM-CSF, IL-6, IL-8, and TNFα were significantly induced by both PfsEVs and PfiRBCs whereas MCP-1, IL-10, IL-17α were preferentially induced by PfsEVs and IP-10 and IFN-γ by PfiRBCs. Prior exposure to malaria (judged by antibodies to schizont extract) was associated with lower monocyte responses to PfsEVs. Conclusions: PfsEVs and PfiRBCs showed differential abilities to induce secretion of IL-17α and IFN-γ, suggesting that the former are better at inducing Th17, whilst the latter induce Th1 immune responses respectively. Prior exposure to malaria significantly reduces the ability of PfsEVs to activate monocytes, suggesting immune tolerance to PfsEVs may play a role in naturally acquired anti-disease immunity.

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro.

During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-γ cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-β1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.

Defining the complement C3 binding site and the antigenic region of Haemonchus contortus GAPDH.

Haemonchus contortus is an economically important parasite that survives the host defense system by modulating the immune response. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is secreted by the parasite and the host responds by producing anti-enzyme antibodies. The enzyme inhibits complement cascade, an arm of the innate immunity, by binding to complement C3. In this study, the C3 binding site and the antigenic region of the enzyme were identified by generating short recombinant fragments and deleting a defined region of the enzyme. Using these proteins in ligand overlay and plate binding assay, the C3 binding region of GAPDH was localized within the 38 residues represented by 77-114 amino acids whereas one of the antigenic regions was identified in between 77 and 171 amino acids. In addition, deletion of amino acids 77 to 171 from GAPDH (fragment AB) also showed weak immunogenicity but lacked C3 binding activity. Fragment D comprising 95 residues (77-171), had both the C3 binding activity as well as immunogenicity like the parent enzyme, also stimulated host peripheral blood mononuclear cells in vitro. This truncated GAPDH moiety was stable at refrigerated temperature for at least 12weeks and appears as a promising new therapeutic tool considering its longer shelf life as compared to the parent protein.

The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.

Recombinant Brugia malayi pepsin inhibitor (rBm33) exploits host signaling events to regulate inflammatory responses associated with lymphatic filarial infections

Prolonged existence of filarial parasites and their molecules within the host modulate the host immune system to instigate their survival and induce inflammatory responses that contribute to disease progression. Recombinant Brugia malayi pepsin inhibitor (rBm33) modulates the host immune responses by skewing towards Th1 responses characterized by secretion of inflammatory molecules such as TNF-α, IL-6, nitric oxide (NO). Here we also specified the molecular signaling events triggered by rBm33 in peripheral blood mononuclear cells (PBMCs) of filarial endemic normals (EN). rBm33 predominantly enhanced the levels of nitric oxide in cultured PBMCs but did not result in oxidative stress to the host cells. Further, rBm33 treatment of human PBMCs resulted in higher GSH/GSSG levels. MYD88 dependent activation was found to be associated with rBm33 specific inflammatory cytokine production. rBm33 triggered intracellular signaling events also involved JNK activation in host PBMCs. In addition, c-Fos and not NF-κB was identified as the transcription factor regulating the expression of inflammatory cytokines in rBm33 stimulated PBMCs. rBm33 marked its role in filarial pathology by altered levels of growth factors but did not have a significant impact on matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) activity of host PBMCs. Thus, the study outlines the signaling network of rBm33 induced inflammatory responses within the host immune cells.

The N- and C-terminal carbohydrate recognition domains of Haemonchus contortus galectin bind to distinct receptors of goat PBMC and contribute differently to its immunomodulatory functions in host-parasite interactions

BackgroundHco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus. A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells (PBMC) to facilitate the immune evasion. Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains (CRD) of Hco-gal-m had different sugar binding abilities. However, whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated.ResultsWe found that the N-terminal CRD of Hco-gal-m (MNh) and the C-terminal CRD (MCh) could bind to goat peripheral blood mononuclear cells by distinct receptors: transmembrane protein 63A (TMEM63A) was a binding receptor of MNh, while transmembrane protein 147 (TMEM147) was a binding receptor of MCh. In addition, MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis, while MNh was much more effective in inhibiting NO production. Moreover, MNh could suppress the transcription of interferon-γ (IFN-γ), but MCh not.ConclusionsOur data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response. These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

Transmembrane protein 63A is a partner protein of Haemonchus contortus galectin in the regulation of goat peripheral blood mononuclear cells.

BackgroundHco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells.MethodsA yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes.ResultsWe found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered.ConclusionsOur results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0816-3) contains supplementary material, which is available to authorized users.

Transcriptional and proteomic analysis reveal recombinant galectins of Haemonchus contortus down-regulated functions of goat PBMC and modulation of several signaling cascades in vitro

In this study, a combined proteomic and transcriptomic analysis was performed to understand the mechanisms underlying the immunomodulation induced by recombinant galectins of Haemonchus contortus (rHco-gal-m/f) on goat peripheral blood mononuclear cells (PBMC). We demonstrated that rHco-gal-m/f could be distinguished by antisera from goats experimentally infected with H. contortus and bound to the surface of goat PBMC. Following rHco-gal-m/f exposure, 16 differentially expressed proteins were identified, which function in biological processes such as stimulus response, biological regulation and localization. According to Gene Ontology Annotation, 15 proteins (93.8%) had binding activity and 9 proteins (56.3%) had catalytic activity. A series of transcriptomic analyses were performed subsequently to assess the expression change of certain pathway members. The integrated results of proteomic and transcriptomic analysis suggested that the activation of VEGF pathway, free radical producing pathway, NFκB pathway and ubiquitin-proteasome pathway was inhibited following exposure to rHco-gal-m/f, while the TLR pathway and CASPASE pathway were activated. Cytokine production and T cell differentiation were also influenced. Cell migration assays and ELISA were performed and the results were in accordance with the change of the proteins and genes. The protein and gene profiles determined here identified several mechanisms underlying the rHco-gal-m/f-induced immunomodulation of goat PBMC. This research provided insight into the interactive relationship between parasitic nematode galectins and host PBMC. It also shed new lights on the understanding of molecular mechanisms of helminthic immune evasion.

Closing two doors of viral entry: Intramolecular combination of a coreceptor- and fusion inhibitor of HIV-1

We describe a novel strategy in which two inhibitors of HIV viral entry were incorporated into a single molecule. This bifunctional fusion inhibitor consists of an antibody blocking the binding of HIV to its co-receptor CCR5, and a covalently linked peptide which blocks envelope mediated virus-cell fusion. This novel bifunctional molecule is highly active on CCR5- and X4-tropic viruses in a single cycle assay and a reporter cell line with IC50 values of 0.03–0.05 nM. We demonstrated that both inhibitors contribute to the antiviral activity. In the natural host peripheral blood mononuclear cells (PBMC) the inhibition of CXCR4-tropic viruses is dependant on the co-expression of CCR5 and CXCR4 receptors. This bifunctional inhibitor may offer potential for improved pharmacokinetic parameters for a fusion inhibitor in humans and the combination of two active antiviral agents in one molecule may provide better durability in controlling the emergence of resistant viruses.

Selective depletion of donor alloreactive T cells without loss of antiviral or antileukemic responses

AbstractPoor immune reconstitution after haploidentical stem cell transplantation results in a high mortality from viral infections and relapse. One approach to overcome this problem is to selectively deplete the graft of alloreactive cells using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient peripheral blood mononuclear cells (PBMCs), and this can result in graft versus host disease (GVHD). We have refined this approach using recipient Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines (LCLs) as stimulators to activate donor alloreactive T cells. Our studies demonstrate that allodepletion with an anti-CD25 immunotoxin following stimulation with HLA-mismatched host LCLs more consistently depleted in vitro alloreactivity than stimulation with host PBMCs, as assessed in primary mixed lymphocyte reactions (MLRs). Allodepletion using this approach specifically abrogates cytotoxic T-cell responses against host LCLs. In interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assays, antiviral responses to adenovirus and cytomegalovirus (CMV) were preserved following allodepletion. Likewise, using HLA-A2–pp65 tetramers, we have shown that the frequency of CMV-specific T cells is unaffected by allodepletion. Moreover, the donor anti-EBV response is partially retained by recognition of EBV antigens through the nonshared haplotype. Finally, we studied whether allodepletion affects the response to candidate tumor antigens in myeloid malignancies. Using HLA-A2–PR1 tetramer analysis, we found that the frequency of T cells recognizing the PR1 epitope of proteinase 3 was not significantly different in allodepleted and unmanipulated PBMCs from patients with chronic myeloid leukemia (CML) undergoing transplantation. Based on these data, we have embarked on a phase 1 clinical trial of addback of allo-LCL–depleted donor T cells in the haplo-identical setting.

Significance of HTLV-1 proviral load quantification by real-time PCR as a surrogate marker for HTLV-1-infected cell count

We developed a real-time (RT) PCR quantitative assay to measure the level of the integrated viral genome of HTLV-1 in host peripheral blood-mononuclear cells (PB-MNC) from healthy carriers and patients with adult T-cell leukemia (ATL). All of the clinical specimens were serologically and molecularly characterized by enzyme-linked immunosorbent assay (ELISA) and Southern blot hybridization (SBH) analyses. The assay system for quantifying the proviral copy level was sensitive, accurate, and reproducible over a wide range of density from 100 to 0.1% with a coefficient of variation (%) of 4.5 to 9.6. The proviral load of the healthy carriers and patients with ATL was 301 +/- 339 copies per 10(4) MNC (3 +/- 3.4%) on average and varied depending on the ATL cell number and the SBH band-status of single or multiple bands. In ATL cases with multiple bands detected by SBH analysis, their ATL cells were shown to harbor multiple copies within one ATL cell, so that the corrected copy number interpolated by the band number in SBH was closely equivalent to the expected ATL cell number in PB, corresponding to the virus-infected cell burden. The proviral load in healthy carriers ranged from 0.1 to 15% of PB-MNC, and, in combination with the fraction (%) of ATL-like flower cells defined by PB smear morphology, enabled carriers to be subgrouped into three categories. This result indicates that the detection of proviral load by(RT) PCR is sufficient and relevant to monitor the infected cell number in the PB and to evaluate the HTLV-1 pathologic status.

CD4-independent, CCR5-dependent simian immunodeficiency virus infection and chemotaxis of human cells.

Most simian immunodeficiency virus (SIV), human immunodeficiency virus type 2 (HIV-2), and HIV-1 infection of host peripheral blood mononuclear cells (PBMCs) is CD4 dependent. In some cases, X4 HIV-1 chemotaxis is CD4 independent, and cross-species transmission might be facilitated by CD4-independent entry, which has been demonstrated for some SIV strains in CD4(-) non-T cells. As expected for CCR5-dependent virus, SIV required CD4 on rhesus and pigtail macaque PBMCs for infection and chemotaxis. However, SIV induced the chemotaxis of human PBMCs in a CD4-independent manner. Furthermore, in contrast to the results of studies using transfected human cell lines, SIV did not require CD4 binding to productively infect primary human PBMCs. CD4-independent lymphocyte and macrophage infection may facilitate cross-species transmission, while reacquisition of CD4 dependence may confer a selective advantage for the virus within new host species.

Use of a Probe to Repeat Sequence of the Y Chromosome for Detection of Host Cells in Peripheral Blood of Bone Marrow Transplant Recipients

In situ hybridization for the Y chromosome (Y-ISH) was used to identify residual host cells in the peripheral blood of 51 recipients of sex-mismatched allogeneic marrow not depleted of T cells following conditioning with high-dose cyclophosphamide and total body irradiation (TBI). One patient who rejected the graft showed rapid reappearance of host cells after transient donor marrow engraftment. Host cells were detected at low levels in 49 of the remaining 50 patients. Host peripheral blood mononuclear cells (PBMC) decreased with time and reached a plateau at 1.0 +/- 0.2% within four weeks after transplantation, while the percentage of host granulocytes (GRAN) reached a plateau at background level. The mean absolute numbers of host PBMC or GRAN were less than 0.015 x 10(9)/L and did not vary significantly over the period studied. Neither the percentages nor the absolute numbers of host PBMC or GRAN were significantly affected by HLA-matching, TBI dose-intensity, pretransplant remission status, subsequent development of acute or chronic graft-versus-host disease or relapse after transplantation. The authors conclude that it is common to find a few residual host cells in the peripheral blood of allogeneic marrow transplant recipients, and the presence of these cells has no clinical significance.