High susceptibility of human dendritic cells to avian influenza H5N1 virus infection and protection by IFN-alpha and TLR ligands

There is worldwide concern that the avian influenza H5N1 virus, with a mortality rate of >50%, might cause the next influenza pandemic. Unlike most other influenza infections, H5N1 infection causes a systemic disease. The underlying mechanisms for this effect are still unclear. In this study, we investigate the interplay between avian influenza H5N1 and human dendritic cells (DC). We showed that H5N1 virus can infect and replicate in monocyte-derived and blood myeloid DC, leading to cell death. These results suggest that H5N1 escapes viral-specific immunity, and could disseminate via DC. In contrast, blood pDC were resistant to infection and produced high amounts of IFN-alpha. Addition of this cytokine to monocyte-derived DC or pretreatment with TLR ligands protected against infection and the cytopathic effects of H5N1 virus.     

A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity

At least two distinct human dendritic cell (DC) subsets are produced in the bone marrow and circulate in the peripheral blood-precursor myeloid DCs (pre-mDCs) and plasmacytoid DCs (PDCs). Both lineages of DCs are instrumental in antiviral innate immunity and shaping Th1 adaptive immune responses. PDCs are the most potent IFN-alpha-producing cells to viral pathogens. Dengue, an acute flavivirus disease, provides a model to study DC responses to a self-limited human viral infection. We analyzed circulating DC subsets in a prospective study of children with dengue across a broad range of illness severities: healthy controls; mild, nondengue, presumed viral infections; moderately ill dengue fever; and, the most severe form of illness, dengue hemorrhagic fever. We also examined PDC responses in monkeys with asymptomatic dengue viremia and to dengue virus exposure in vitro. The absolute number and frequency of circulating pre-mDCs early in acute viral illness decreased as illness severity increased. Depressed pre-mDC blood levels appeared to be part of the typical innate immune response to acute viral infection. The frequency of circulating PDCs trended upward and the absolute number of circulating PDCs remained stable early in moderately ill children with dengue fever, mild other, nondengue, febrile illness, and monkeys with asymptomatic dengue viremia. However, there was an early decrease in circulating PDC levels in children who subsequently developed dengue hemorrhagic fever. A blunted blood PDC response to dengue virus infection was associated with higher viremia levels, and was part of an altered innate immune response and pathogenetic cascade leading to severe disease.     

Multimerization of Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) and Familial Chylomicronemia from a Serine-to-Cysteine Substitution in GPIHBP1 Ly6 Domain

GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia.     

Protective Efficacy of Baculovirus Dual Expression System Vaccine Expressing Plasmodium falciparum Circumsporozoite Protein

We have previously developed a new malaria vaccine delivery system based on the baculovirus dual expression system (BDES). In this system, expression of malaria antigens is driven by a dual promoter consisting of the baculovirus-derived polyhedrin and mammal-derived cytomegalovirus promoters. To test this system for its potential as a vaccine against human malaria parasites, we investigated immune responses against the newly developed BDES-based Plasmodium falciparum circumsporozoite protein vaccines (BDES-PfCSP) in mice and Rhesus monkeys. Immunization of mice with BDES-PfCSP induced Th1/Th2-mixed type immune responses with high PfCSP-specific antibody (Ab) titers, and provided significant protection against challenge from the bites of mosquitoes infected with a transgenic P. berghei line expressing PfCSP. Next, we evaluated the immunogenicity of the BDES-PfCSP vaccine in a rhesus monkey model. Immunization of BDES-PfCSP elicited high levels of anti-PfCSP Ab responses in individual monkeys. Moreover, the sera from the immunized monkeys remarkably blocked sporozoite invasion of HepG2 cells. Taken together with two animal models, our results indicate that this novel vaccine platform (BDES) has potential clinical application as a vaccine against malaria.     

Evaluation of In Vitro Cross-Reactivity to Avian H5N1 and Pandemic H1N1 2009 Influenza Following Prime Boost Regimens of Seasonal Influenza Vaccination in Healthy Human Subjects: A Randomised Trial

Introduction

Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses.

Methods

In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose.

Results

Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated.

Conclusion

Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01044095","term_id":"NCT01044095"}}NCT01044095     

Intra-arterial administration of TNF-α followed by arterial ablation is an effective therapy for a regionally confined TNF-resistant rat mammary adenocarcinoma

Tumor necrosis factor-alpha (TNF-alpha) is an immunomodulatory cytokine that has exhibited anti-tumor activity in a variety of experimental systems. However, the toxicities associated with systemic administration of TNF-alpha have limited its clinical utility and have led to the investigation of targeted delivery techniques with the ability to present the TNF-alpha dose directly to the vascular bed of the tumor. The intra-arterial (IA) administration of TNF-alpha to patients with liver metastases represents one such approach, and recent work suggests that subsequent ablation of the tumor's arterial supply via embolization may enhance the efficacy of intra-arterial treatments (hepatic chemoembolization). The present study was undertaken to test the hypothesis that IA administration of TNF-alpha is superior to the intravenous (IV) route for inhibition of tumor growth in a regionally confined rat mammary adenocarcinoma model that provides for ablation of the arterial supply to the tumor following cytokine therapy. Rats bearing hind limb mammary adenocarcinomas received single IA or IV infusions of 8 x 10(5), 1 x 10(6), and 1.5 x 10(6) units of TNF-alpha via the common femoral artery (CFA) followed 1 h later by ligation of the artery. Control animals received either no treatment or IA infusion of 2% normal rat serum (NRS) followed by ipsilateral CFA ligation. Tumor size was measured every other day after treatment. Tumor growth inhibition occurred in the first 5 to 10 days after treatment. IV administration of TNF-alpha did not result in visual tumor necrosis or significant reduction in the rate of tumor growth. IA administration of TNF-alpha resulted in statistically significant diminution of tumor size as compared to untreated controls and animals receiving IA 2% normal rat serum (NRS; P<0.05 at days 6, 8 and 10), regardless of the dose employed. The maximum growth inhibition with IA TNF-alpha was a 91% reduction in tumor volume that was achieved with a dose of 1 x 10(6) U TNF-alpha. These results demonstrate improved anti-tumor activity with the IA administration of TNF-alpha over the IV route in a regionally confined mammary adenocarcinoma. IA administration of biologic response modifiers like TNF-alpha may therefore be a useful approach for the hepatic chemoembolization of breast adenocarcinomas metastatic to the liver.     

Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis

The ability of dendritic cells (DCs) to shape the adaptive immune response to viral infection is mediated largely by their maturation and activation state as determined by the surface expression of HLA molecules, costimulatory molecules, and cytokine production. Dengue is an emerging arboviral disease where the severity of illness is influenced by the adaptive immune response to the virus. In this report, we have demonstrated that dengue virus infects and replicates in immature human myeloid DCs. Exposure to live dengue virus led to maturation and activation of both the infected and surrounding, uninfected DCs and stimulated production of tumor necrosis factor alpha (TNF-alpha) and alpha interferon (IFN-alpha). Activation of the dengue virus-infected DCs was blunted compared to the surrounding, uninfected DCs, and dengue virus infection induced low-level release of interleukin-12 p70 (IL-12 p70), a key cytokine in the development of cell-mediated immunity (CMI). Upon the addition of IFN-gamma, there was enhanced activation of dengue virus-infected DCs and enhanced dengue virus-induced IL-12 p70 release. The data suggest a model whereby DCs are the early, primary target of dengue virus in natural infection and the vigor of CMI is modulated by the relative presence or absence of IFN-gamma in the microenvironment surrounding the virus-infected DCs. These findings are relevant to understanding the pathogenesis of dengue hemorrhagic fever and the design of new vaccination and therapeutic strategies.     

Potentiation of cytotoxic effect of lymphotoxin by anti-cancer drugs and elevated temperatures

The cytotoxic lymphokine, lymphotoxin (LT), has been shown to possess antitumor effect in vitro and in vivo. We examined the effect of the combination of partially purified LT with anti-cancer drugs and elevated temperatures on mouse transformed fibroblast cell line, L-929, and two human carcinoma of the cervix cell lines, HeLa and ME180. The cells were treated for 7 hr with Adriamycin, cisplatin, or bleomycin. These cells were then incubated for 24 hr in the presence of LT. At the end of the incubation period, cytotoxicity was measured by the neutral red dye uptake assay. There was 10- to 47-fold potentiation of cytotoxicity of LT on L-929 cells. The potentiation of cytotoxicity on human carcinoma of cervix cell lines ranged from 3- to 23-fold. L-929 cells and ME180 cells were incubated for 7 hr at 40 or 42 degrees C followed by 24 hr of incubation in the presence of LT. The elevated temperature treatment also enhanced (5- to 9-fold) the cytotoxic effect of LT. DNA, RNA, and protein syntheses of the ME180 cells was measured following incubation at 42 degrees C. It was observed that all three parameters were suppressed by incubation at this temperature. It was, therefore, possible that the repair of LT damaged cells was hampered by the elevated temperature treatment. It is suggested that LT may have a potential as an anti-tumor agent in combination with selected therapeutic drugs and hyperthermia.     

Human cytokines, tumor necrosis factor, and interferons: gene cloning, animal studies, and clinical trials

Presented is a comprehensive program designed to isolate human cytokine genes and investigate their relative induction, and to analyze cytokine activities in cell culture, animal tumor models, and human clinical trials. Human cytokine cDNAs have been isolated from a cDNA library made from normal human peripheral blood leukocytes (PBLs) treated with Sendai virus and the relative induction of tumor necrosis factor (TNF), alpha and gamma interferons (IFN-alpha, IFN-gamma), and interleukin-1 beta IL-1 beta) genes has been analyzed. In the Sendai virus-induced PBL system, IL-1 beta mRNA was shown to be approximately twofold higher than TNF or IFN-alpha mRNA whereas IFN-gamma mRNA was 50-100-fold lower than TNF or IFN-alpha mRNA. The cytotoxic activity of TNF was analyzed on several cell lines and IFN-alpha and IFN-gamma were shown to potentiate TNF cytotoxicity about 2-200-fold depending on cell lines. The LD50 for recombinant TNF in BALB/c mice was determined to be 6 X 10(7) U/kg and the therapeutic dose of recombinant TNF in sarcoma 180 bearing BALB/c mice was 3 X 10(5) U/kg, indicating a wide therapetic index. Phase I clinical trials of recombinant TNF given I.V. indicated a tolerated dose of 150,000 U/kg with biphasic half-life (T-1/2) of 2 and 31 min following TNF injection. Phase II trials of TNF and trials of TNF combined with IFN-alpha are in progress. These studies indicate that cytokines such as TNF and IFN-alpha are subject to similar induction systems, potentiate each other's activities, and can be tolerated at specific doses for potential therapeutic use.     

IL-8 and IDO expression by human gingival fibroblasts via TLRs

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-alpha enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-gamma enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-gamma, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.