Quantification of Bevacizumab Activity Following Treatment of Patients With Ovarian Cancer or Glioblastoma.

Highly sensitive reporter-gene assays have been developed that allow both the direct vascular endothelial growth factor (VEGF) neutralizing activity of bevacizumab and the ability of bevacizumab to activate antibody dependent cellular cytotoxicity (ADCC) to be quantified rapidly and in a highly specific manner. The use of these assays has shown that in 46 patients with ovarian cancer following four cycle of bevacizumab treatment, and in longitudinal samples from the two patients that respond to bevacizumab therapy from a small cohort of patients with glioblastoma, that there is a reasonably good correlation between bevacizumab drug levels determined by ELISA and bevacizumab activity, determined using either the VEGF-responsive reporter gene, or the ADCC assays. One of the two primary non-responders with glioblastoma exhibited high levels of ADCC activity suggesting reduced bevacizumab Fc engagement in vivo in contrast to the other primary non-responder, and the two secondary non-responders with a decreasing bevacizumab PK profile, determined by ELISA that exhibited low to undetectable ADCC activity. Drug levels were consistently higher than bevacizumab activity determined using the reporter gene assay in serial samples from one of the secondary non-responders and lower in some samples from the other secondary non-responder and ADCC activity was markedly lower in all samples from these patients suggesting that bevacizumab activity may be partially neutralized by anti-drug neutralizing antibodies (NAbs). These results suggest that ADCC activity may be correlated with the ability of some patients to respond to treatment with bevacizumab while the use of the VEGF-responsive reporter-gene assay may allow the appearance of anti-bevacizumab NAbs to be used as a surrogate maker of treatment failure prior to the clinical signs of disease progression.

A Novel System for the Quantification of the ADCC Activity of Therapeutic Antibodies.

Novel ADCC effector cells expressing the V-variant or F-variant of FcγRIIIa (CD16a) and firefly luciferase under the control of a chimeric promoter incorporating recognition sequences for the principal transcription factors involved in FcγRIIIa signal transduction, together with novel target cells overexpressing a constant high level of the specific antigen recognized by rituximab, trastuzumab, cetuximab, infliximab, adalimumab, or etanercept, confer improved sensitivity, specificity, and dynamic range in an ADCC assay relative to effector cells expressing a NFAT-regulated reporter gene and wild-type target cells. The effector cells also contain a normalization gene rendering ADCC assays independent of cell number or serum matrix effects. The novel effector and target cells in a frozen thaw-and-use format exhibit low vial-to-vial and lot-to-lot variation in their performance characteristics reflected by CVs of 10% or less. Homologous control target cells in which the specific target gene has been invalidated by genome editing providing an ideal control and a means of correcting for nonspecific effects were observed with certain samples of human serum. The novel effector cells and target cells expressing noncleavable membrane-bound TNFα have been used to quantify ADCC activity in serum from patients with Crohn's disease treated with infliximab and to relate ADCC activity to drug levels.

Improved analytical methods for the detection and quantification of neutralizing antibodies to biopharmaceuticals.

Biopharmaceuticals are used extensively for the treatment of a number of chronic debilitating and fatal diseases such as cancer and inflammatory or autoimmune diseases. Although biopharmaceuticals are in general well tolerated, the development of anti-drug antibodies can impair their safety and efficacy. Assessment of immunogenicity is essential for a more effective and rational use of biopharmaceuticals, and is dependent upon the establishment of efficient standardized assays that allow direct comparison of immunogenicity data with clinical outcome. Although regulatory authorities recommend the use of cell-based assays that reflect the mechanism of action of the drug for the detection of neutralizing anti-drug antibodies, conventional cell-based assays are difficult to standardize and often give variable results. A number of strategies have been adopted to improve the performance of cell-based assays, including quantification of drug-induced proteins using either real-time RT-PCR or branched DNA to detect mRNA, or ELISAs to detect protein, bridging assays using immobilized cells and the use of reporter gene assays. The relative merits and limitations of each of these methods is reviewed herein.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists.

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

Immunogenicity and other problems associated with the use of biopharmaceuticals.

Biopharmaceuticals are used widely for the treatment of cancer, chronic viral hepatitis, inflammatory, and autoimmune diseases. Biopharmaceuticals such as interferons are well tolerated for the most part with the most common adverse events observed being 'flu-like' symptoms that resolve rapidly after initial treatment. Prolonged treatment is associated, however, with more serious adverse events including leucopenia, thrombocytopenia, and neuropsychiatric effects, which may necessitate dose reduction or even cessation of treatment in some patients. Recombinant growth factors, such as erythropoietin (EPO), granulocyte colony-stimulating factor, or granulocyte macrophage colony-stimulating factor, are for the most part well tolerated, although severe complications have been reported in patients with cancer or chronic kidney disease treated with EPO. Similarly, treatment of patients with cancer with high doses of interleukin-2 is associated with significant toxicity. Treatment of chronic inflammatory diseases, such as rheumatoid arthritis, psoriasis, and Crohn's disease, with antitumor necrosis factor-alpha monoclonal antibodies is associated with an increased risk of granulomatous infections and, in particular, tuberculosis. The monoclonal antibody, natalizumab, that targets alpha4 integrins is effective in the treatment of multiple sclerosis but is associated with the activation of JC virus and development of progressive multifocal leukoencephalopathy. Repeated administration of recombinant proteins can cause a break in immune tolerance in some patients resulting in the production of a polyclonal antibody response that can adversely affect pharmacokinetics and clinical response. In addition, neutralizing antibodies that cross react with nonredundant essential proteins such as EPO can cause severe autoimmune reactions.

Type I interferons mediate the innate cytokine response to recombinant fowlpox virus but not the induction of plasmacytoid dendritic cell-dependent adaptive immunity.

Type I interferons (IFNs) are considered to be important mediators of innate immunity due to their inherent antiviral activity, ability to drive the transcription of a number of genes involved in viral clearance, and their role in the initiation of innate and adaptive immune responses. Due to the central role of type I IFNs, we sought to determine their importance in the generation of immunity to a recombinant vaccine vector fowlpox virus (FPV). In analyzing the role of type I IFNs in immunity to FPV, we show that they are critical to the secretion of a number of innate and proinflammatory cytokines, including type I IFNs themselves as well as interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-1beta, and that deficiency leads to enhanced virus-mediated antigen expression. Interestingly, however, type I IFNs were not required for adaptive immune responses to recombinant FPV even though plasmacytoid dendritic cells (pDCs), the primary producers of type I IFNs, have been shown to be requisite for this to occur. Furthermore, we provide evidence that the importance of pDCs may lie in their ability to capture and present virally derived antigen to T cells rather than in their capacity as professional type I IFN-producing cells.

One-step assay for quantification of neutralizing antibodies to biopharmaceuticals.

Assessment of immunogenicity is an important part of biopharmaceutical drug safety evaluation and a prerequisite for the development of less immunogenic and safer biopharmaceuticals since anti-drug antibodies can impair the activity and compromise the safety of biopharmaceuticals. Although regulatory authorities recommend cell-based assays for detection of neutralizing antibodies (NAbs), such assays are difficult to standardize, and ill adapted to high-throughput analysis. These limitations have been overcome by the development of a unique one-step cell-based assay that allows both drug activity and drug NAbs to be quantified rapidly and with a high degree of precision simply be adding reporter cells to a sample. The reporter cells have been engineered to express firefly luciferase (FL) under the control of a drug-responsive promoter, and to express the drug of interest, the production of which is normalized relative to the expression of Renilla luciferase (RL) transcribed from a common doxycycline-inducible promoter. Residual drug levels present in a sample are first quantified by determination of FL expression, autocrine drug synthesis is then induced, and NAb activity is quantified from the difference in the ratio of FL/RL expression in the presence or absence of the sample. Since assay results are normalized relative to the expression of an internal standard, results are independent of cell number or differences in cell viability thus affording a high degree of assay precision and reducing serum matrix effects to a minimum. This unique assay platform is ideally suited for high-throughput analysis, is applicable to most biopharmaceuticals, and will facilitate standardization and comparison of immunogenicity data. The performance of the one-step assay is illustrated for interferon alpha2 (IFNalpha2) used widely to treated chronic hepatitis C (HCV) infection and neoplastic disease.

Safety, Tolerability, and Immunogenicity of Interferons.

Interferons (IFNs) are class II cytokines that are key components of the innate immune response to virus infection. Three IFN sub-families, type I, II, and III IFNs have been identified in man, Recombinant analogues of type I IFNs, in particular IFNα2 and IFNß1, have found wide application for the treatment of chronic viral hepatitis and remitting relapsing multiple sclerosis respectively. Type II IFN, or IFN gamma, is used principally for the treatment of chronic granulomatous disease, while the recently discovered type III IFNs, also known as IFN lambda or IL-28/29, are currently being evaluated for the treatment of chronic viral hepatitis. IFNs are in general well tolerated and the most common adverse events observed with IFNα or IFNß therapy are "flu-like" symptoms such as fever, headache, chills, and myalgia. Prolonged treatment is associated with more serious adverse events including leucopenia, thrombocytopenia, increased hepatic transaminases, and neuropsychiatric effects. Type I IFNs bind to high-affinity cell surface receptors, composed of two transmembrane polypeptides IFNAR1 and IFNAR2, resulting in activation of the Janus kinases Jak1 and Tyk2, phosphorylation and activation of the latent cytoplasmic signal transducers and activators of transcription (STAT1) and STAT2, formation of a transcription complex together with IRF9, and activation of a specific set of genes that encode the effector molecules responsible for mediating the biological activities of type I IFNs. Systemic administration of type I IFN results in activation of IFN receptors present on essentially all types of nucleated cells, including neurons and hematopoietic stem cells, in addition to target cells. This may well explain the wide spectrum of IFN associated toxicities. Recent reports suggest that certain polymorphisms in type I IFN signaling molecules are associated with IFN-induced neutropenia and thrombocytopenia in patients with chronic hepatitis C. IFNγ binds to a cell-surface receptor composed of two transmembrane polypeptides IFGR1 and IFGR2 resulting in activation of the Janus kinases Jak1 and Jak2, phosphorylation of STAT1, formation of STAT1 homodimers, and activation of a specific set of genes that encode the effector molecules responsible for mediating its biological activity. In common with type I IFNs, IFNγ receptors are ubiquitous and a number of the genes activated by IFNγ are also activated by type I IFNs that may well account for a spectrum of toxicities similar to that associated with type I IFNs including "flu-like" symptoms, neutropenia, thrombocytopenia, and increased hepatic transaminases. Although type III IFNs share the major components of the signal transduction pathway and activate a similar set of IFN-stimulated genes (ISGs) as type I IFNs, distribution of the IFNλ receptor is restricted to certain cell types suggesting that IFNλ therapy may be associated with a reduced spectrum of toxicities relative to type I or type II IFNs. Repeated administration of recombinant IFNs can cause in a break in immune tolerance to self-antigens in some patients resulting in the production of neutralizing antibodies (NABs) to the recombinant protein homologue. Appearance of NABs is associated with reduced pharmacokinetics, pharmacodynamics, and a reduced clinical response. The lack of cross-neutralization of IFNß by anti-IFNα NABs and vice versa, undoubtedly accounts for the apparent lack of toxicity associated with the presence of anti-IFN NABs with the exception of relatively mild infusion/injection reactions.

Locally administered TLR7 agonists drive systemic antitumor immune responses that are enhanced by anti-CD40 immunotherapy.

Topical application of tumors with the TLR7 agonist imiquimod is an effective adjunct treatment for a range of primary dermatological cancers. However, for therapy to be effective against a broad range of solid tumor types, it must promote a strong systemic antitumor response that targets metastases in addition to primary tumor. We therefore investigated the potential of locally delivered imiquimod to stimulate an effective systemic antitumor response in a murine model of malignant mesothelioma (AB1-HA) with primary and distal tumors (dual tumor). Persistent delivery of imiquimod into primary tumor significantly retarded tumor growth in all treated mice compared with vehicle control. This local antitumor immune response required both CD8 T cells and NK cells, but not CD4 T cells, and was reliant on type I IFN induction. In vivo CTL studies and Ly6A/E staining of lymphocytes suggested that local imiquimod treatment had indeed induced a systemic, Ag-specific CD8 response. However, notably this response was not sufficient to retard the growth of an untreated distal tumor. Because local imiquimod treatment did not induce significant CD4 T cell responses, we investigated the efficacy of combining imiquimod with agonistic CD40 Ab (as a surrogate for CD4 T cell help). Combination of locally delivered imiquimod with systemic anti-CD40 immunotherapy not only significantly enhanced the local antitumor response, with 30% complete resolution, but it was also effective at significantly retarding growth of distal tumor. These results demonstrate that antitumor responses induced by locally delivered TLR7 agonists can be harnessed systemically for treating distal tumor.

Targeting poly(I:C) to the TLR3-independent pathway boosts effector CD8 T cell differentiation through IFN-alpha/beta.

Poly(I:C) is an adjuvant used for antitumor treatment and vaccines because of its prominent effects on CD8 T cells and NK cells. Poly(I:C) binds TLR3 and this interaction is thought to be central for driving cell-mediated immune responses. We investigated the importance of TLR3 in poly(I:C)-mediated endogenous CD8 T cell responses using the pathogenic T cell stimulant Staphylococcus aureus enterotoxin A. While the responsive CD8 T cells expanded comparably in both wild-type and TLR3(-/-) mice, differentiation of effector CD8 T cells was enhanced by poly(I:C) in the TLR3(-/-) mice. A higher percentage of Ag-specific CD8 T cells became IFN-gamma and TNF-alpha producers in the absence of TLR3 signaling. Consistent with this boosted response was the observation that TLR3-deficient cells synthesized less IL-10 compared with TLR3-sufficient cells in response to poly(I:C). Ultimately, however, the fundamental mechanism of CD8 effector T cell differentiation through the TLR3-independent pathway was shown to be completely IFN-alpha/beta-dependent. Administration of IFN-alpha/beta-neutralizing Abs abolished the poly(I:C) effects in TLR3(-/-) mice. These findings reveal specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly(I:C) is authenticated as a therapeutic adjuvant used in vaccines.

Adjuvant activity of type I interferons.

Type I interferons (IFNs) produced primarily by plasmacytoid dendritic cells (pDCs) as part of the innate immune response to infectious agents induce the maturation of myeloid DCs and enhance antigen presentation. Type I IFNs also enhance apoptosis of virus-infected cells, stimulate cross priming and enhanced presentation of viral peptides. Type I IFNs are powerful polyclonal B-cell activators that induce a strong primary humoral immune response characterized by isotype switching and protection against virus challenge. Type I IFNs stimulate an IgG2a antibody response characteristic of Th1 immunity when ad-mixed with influenza virus vaccine and injected intramuscurarly (i.m.) or administered intranasally. The adjuvant activity of type I IFNs has been shown to involve direct effects of IFN on B-cells, effects on T-cells, as well as effects on antigen presentation. Oromucosal administration of type I IFNs concomitantly with i.m. injection of vaccine alone can also enhance the antibody response to influenza vaccination by enhancing trafficking of antigen-presenting cells towards the site of vaccination. Recombinant IFNs are potent adjuvants that may find application in both parenterally and mucosally administered vaccines.

Effect of sublingual administration of interferon-alpha on the immune response to influenza vaccination in institutionalized elderly individuals.

A randomized double-blind placebo-controlled study was conducted to determine the effect of sublingual administration of IFNalpha on the immune response to influenza vaccination in elderly institutionalized individuals. Sublingual administration of 10 million IU of IFNalpha immediately prior to vaccination, reduced the geometric mean haemagglutination inhibitory (HAI) and IgG2 circulating antibody titers, and the secretory IgA (sIgA) response in saliva, to the New York strain of influenza A virus, 21 days post-vaccination, without detectable drug-related local or systemic toxicity. IFN treatment did not inhibit the immune response to the other components of the vaccine; the New Caledonia strain of influenza A virus, or the Jiangsu strain of influenza B virus. At the dose tested sublingual administration of IFNalpha reduces the immune response to influenza vaccination in elderly institutionalized individuals.

Targeting the effector site with IFN-alphabeta-inducing TLR ligands reactivates tumor-resident CD8 T cell responses to eradicate established solid tumors.

Effective antitumor CD8 T cell responses may be activated by directly targeting the innate immune system within tumors. We investigated this response by injecting a range of TLR agonists into established tumors using a mouse model of malignant mesothelioma stably transduced with the hemagglutinin (HA) gene as a marker Ag (AB1-HA). Persistent delivery of the dsRNA mimetic poly(I:C) into established AB1-HA tumors resulted in complete tumor resolution in 40% of mice, with the remaining mice also showing a significant delay in tumor progression. Experiments in athymic nude mice along with CD8 depletion and IFN-alphabeta blocking studies revealed that tumor resolution required both CD8 T cells and type I IFN induction, and was associated with local changes in MHC class I expression. Surprisingly, however, tumor resolution was not associated with systemic dissemination or tumor infiltration of effector CD8 T cells. Instead, the antitumor response was critically dependent on the reactivation of tumor-resident CD8 T cell responses. These studies suggest that, once reactivated, pre-existing local CD8 T cell responses are sufficient to resolve established tumors and that in situ type I IFN is a determining factor.

Involvement of the Gab2 scaffolding adapter in type I interferon signalling.

Interferons (IFNs) are pleiotropic cytokines involved in the regulation of physiological and pathological processes. Upon interaction with their specific receptors, IFNs activate the Jak/STAT signalling pathway. Numerous studies suggest, however, that the classical Jak/STAT pathway cannot alone account for the wide range of IFN's biological effects. To better understand the role of alternative signalling pathways in the type I IFNs response, we analyzed novel tyrosine-phosphorylated proteins following IFN-alpha2 stimulation. We showed for the first time that the Grb2-associated binder 2 (Gab2) protein is differentially phosphorylated upon the IFN subtype employed and the cells stimulated. We demonstrated that IFNAR1 physically interacts with Gab2. Moreover, the cellular content of Gab2 varies as a function of IFN receptor chain expression levels, and in particular of the ratio of IFNAR1 to IFNAR2, suggesting that Gab2 and IFNAR2 compete for interaction with IFNAR1. Analysis of Gab2 deletion mutants indicates that IFNAR1 might interact with a Gab2 region containing p85-PI3'kinase binding sites. Our results shed new light on recent data involving both Gab2 and type I IFNs in osteoclastogenesis and oncogenesis.

SCRG1, a potential marker of autophagy in transmissible spongiform encephalopathies.

The Scrg1 gene was initially discovered as one of the genes upregulated in transmissible spongiform encephalopathies (TSE). Scrg1 encodes a highly conserved, cysteine-rich protein expressed principally in the central nervous system. The protein is targeted to the Golgi apparatus and large dense-core vesicles/secretory granules in neurons. We have recently shown that the Scrg1 protein is widely induced in neurons of scrapie-infected mice, suggesting that Scrg1 is involved in the host response to stress and/or the death of neurons. At the ultrastructural level, Scrg1 is associated with dictyosomes of the Golgi apparatus and autophagic vacuoles of degenerative neurons. It is well known that apoptosis plays a major role in the events leading to neuronal cell death in TSE. However, autophagy was identified in experimentally induced scrapie a long time ago and was recently reevaluated as a possible cell death program in prion diseases. The consistent association of Scrg1 with autophagic structures typical of scrapie is in agreement with the recruitment of Golgi-specific proteins in this degradation process and we suggest that Scrg1 might be used as a specific probe to identify neuronal autophagy in TSE.

Interferons alpha and gamma induce p53-dependent and p53-independent apoptosis, respectively.

Type I interferon (IFN) enhances the transcription of the tumor suppressor gene p53. To elucidate the molecular mechanism mediating IFN-induced apoptosis, we analysed programmed cell death in response to type I (IFNalpha) or type II (IFNgamma) treatment in relation to p53 status. In two cell lines (MCF-7, SKNSH), IFNalpha, but not IFNgamma, enhanced apoptosis in a p53-dependent manner. Furthermore, only IFNalpha upregulated p53 as well as p53 target genes (Noxa, Mdm2 and CD95). The apoptotic response to IFNalpha decreased in the presence of ZB4, an anti-CD95 antibody, suggesting that CD95 is involved in this process. When p53 was inactivated by the E6 viral protein or the expression of a p53 mutant, IFNalpha-induced apoptosis and p53 target genes upregulation were abrogated. Altogether these results demonstrate that p53 plays a pivotal role in the IFNalpha-induced apoptotic response. IFNalpha-induced PML was unable to recruit p53 into nuclear bodies and its downregulation by siRNA did not alter CD95 expression. In contrast, IFNgamma-induced apoptosis is p53-independent. CD95 and IFN-regulatory factor 1 (IRF1) are directly upregulated by this cytokine. Apoptotic response to IFNgamma is decreased in the presence of ZB4 and strongly diminished by IRF1 siRNA, implicating both CD95 and IRF1 in IFNgamma-induced apoptotic response. Taken together, these results show that in two different cell lines, IFNalpha and IFNgamma, induce p53-dependent -independent apoptosis, respectively.

An essential role for IFN-alpha in the overexpression of Fas ligand on MRL/lpr lymphocytes and on their spontaneous Fas-mediated cytotoxic potential.

Lymphocytes from aged autoimmune MRL/lpr mice overexpress Fas ligand (FasL), and are cytotoxic against Fas+ target cells. This cytotoxic potential is only partly due to FasL, as wild-type MRL+/+ lymphocytes are not able to kill Fas+ targets after induction of FasL. In addition, serum levels of interferon-alpha (IFN-alpha) increase in parallel with the Fas-dependent cytotoxic potential of lymphocytes from MRL/lpr mice as they age. To understand the mechanisms underlying these observations, combined suppression subtractive hybridization (SSH) and RT-PCR were used to study differential gene expression in splenocytes from MRL/lpr mice compared with splenocytes from MRL+/+ mice. Twenty-two genes were upregulated transcriptionally in MRL/lpr splenocytes compared with their MRL+/+ counterparts. Furthermore, 9 of these genes were also upregulated after treatment of MRL/lpr splenocytes with IFN-alpha, and 4 were strongly downregulated. MRL/lpr lymphocytes were also found to be hyperresponsive to IFN-alpha. Thus, MRL/lpr lymphocytes overexpressed mRNA for the IFN-alpha receptor (IFNAR-1 and IFNAR-2) chains of the IFN-alpha/beta receptor and exhibited high endogenous levels of both Stat1 and phosphorylated Stat1 proteins. Lymphocytes from young MRL/lpr mice, with low Fas-dependent cytotoxic activity, were found to become highly cytotoxic against Fas+ targets after treatment with IFN-alpha. These data suggest that IFN-alpha plays an important role in the physiopathology of the systemic lupus erythematosus (SLE)-like syndrome that occurs in MRL/lpr mice.

Scrg1, a novel protein of the CNS is targeted to the large dense-core vesicles in neuronal cells.

Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.

Oromucosal cytokine therapy: mechanism(s) of action.

Oromucosal cytokine therapy allows large amounts of cytokines to be administered with improved outcome and without dose limiting toxicity. Orally administered cytokines exert their effects by a novel two pronged mechanism of action. Firstly, specific populations of immuno-competent effector cells are activated in the oral cavity and migrate to the site of virus replication. Secondly, chemokines produced in the lymphoid tissue of the oral cavity enter the peripheral circulation and redirect activated lymphocytes to eliminate virus infected cells. Oromucosal IFN therapy constitutes an alternative and improved means of therapy for diseases such as chronic viral hepatitis which are currently treated parenterally with IFN alpha. The oral route also has obvious advantages for ease of administration and improved patient compliance. Furthermore, the availability of a well tolerated form of IFN therapy will also allow Type I IFNs to be used for the treatment of diseases such as upper respiratory tract virus infections, for which parenteral IFN therapy is currently precluded due to unacceptable toxicity.