Babita Agrawal

The biological role of mucins in cellular interactions and immune regulation: Prospects for cancer immunotherapy

Among the human mucins, MUC1 is unique in its cell-surface transmembrane expression and its apparent signal-transduction functions. The high expression of MUC1 on many human cancers makes it an attractive target for immunotherapy. Immunization of human cancer patients with MUC1 peptides has resulted in the generation of both anti-MUC1 antibody and cytotoxic T lymphocyte responses. Recently, a novel immunoregulatory role for MUC1 has been suggested by experiments demonstrating that soluble MUC1 induces T-cell unresponsiveness, and that T cells appear to express and secrete MUC1 following their activation. MUC1 is an apparent paradox, having both adhesive and antiadhesive functions, and immunostimulatory and immunosuppressive activities.

Mucin 1-specific immunotherapy in a mouse model of spontaneous breast cancer.

Human mucin 1 (MUC1) is an epithelial mucin glycoprotein that is overexpressed in 90% of all adenocarcinomas including breast, lung, pancreas, prostate, stomach, colon, and ovary. MUC1 is a target for immune intervention, because, in patients with solid adenocarcinomas, low-level cellular and humoral immune responses to MUC1 have been observed, which are not sufficiently strong to eradicate the growing tumor. The hypothesis for this study is that enhancing MUC1-specific immunity will result in antitumor immunity. To test this, the authors have developed a clinically relevant breast cancer model that demonstrates peripheral and central tolerance to MUC1 and develops spontaneous tumors of the mammary gland. In these mice, the authors tested a vaccine formulation comprised of liposomal-MUC1 lipopeptide and human recombinant interleukin-2. Results indicate that when compared with untreated mice, immunized mice develop T cells that express intracellular IFN-&ggr;, are reactive with MHC class I H-2Db/MUC1 tetramer, and are cytotoxic against MUC1-expressing tumor cells in vitro. The presence of MUC1-specific CTL did not translate into a clinical response as measured by time of tumor onset, tumor burden, and survival. The authors demonstrate that some of the immune-evasion mechanisms used by the tumor cells include downregulation of MHC-class I molecule, expression of TGF-&bgr;2, and decrease in IFN-&ggr; -expressing effector T cells as tumors progress. Finally, utilizing an injectable breast cancer model, the authors show that targeting a single tumor antigen may not be an effective antitumor treatment, but that immunization with dendritic cells fed with whole tumor lysate is effective in breaking tolerance and protecting mice from subsequent tumor challenge. A physiologically relevant spontaneous breast cancer model has been developed to test improved immunotherapeutic approaches.

Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

Background Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection. Methodology Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed. Principal Findings HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05). Conclusions HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection.

MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment

Pancreatic cancer is a highly aggressive, treatment refractory disease and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas over-express altered forms of a tumor-associated antigen, MUC1 (an epithelial mucin glycoprotein), which is a target for immunotherapy. Using a clinically relevant mouse model of pancreas cancer that demonstrates peripheral and central tolerance to human MUC1 and develops spontaneous tumors of the pancreas, we have previously reported the presence of functionally active, low affinity, MUC1-specific precursor cytotoxic T cells (pCTLs). Hypothesis for this study is that MUC1-based immunization may enhance the low level MUC1-specific immunity that may lead to an effective anti-tumor response. Data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-γ and are cytolytic against MUC1-expressing tumor cells in vitro. However, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. We demonstrate that the CTL tolerance could be reversed at least in vitro with the use of anti-CD40 co-stimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-ß that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+CD25+ T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy.

Induction of Primary Human T Cell Responses against Hepatitis C Virus-Derived Antigens NS3 or Core by Autologous Dendritic Cells Expressing Hepatitis C Virus Antigens: Potential for Vaccine and Immunotherapy

Hepatitis C virus (HCV)-specific T cell responses have been suggested to play significant role in viral clearance. Dendritic cells (DCs) are professional APCs that play a major role in priming, initiating, and sustaining strong T cell responses against pathogen-derived Ags. DCs also have inherent capabilities of priming naive T cells against given Ags. Recombinant adenoviral vectors containing HCV-derived Core and NS3 genes were used to endogenously express HCV Core and NS3 proteins in human DCs. These HCV Ags expressing DCs were used to prime and stimulate autologous T cells obtained from uninfected healthy donors. The DCs expressing HCV Core or NS3 Ags were able to stimulate T cells to produce various cytokines and proliferate in HCV Ag-dependent manner. Evidence of both CD4+ and CD8+ T cell responses against HCV Core and NS3 generated in vitro were obtained by flow cytometry and Ab blocking experiments. Further, in secondary assays, the T cells primed in vitro exhibited HCV Ag-specific proliferative responses against recombinant protein Ags and also against immunodominant permissive peptide epitopes from HCV Ags. In summary, we demonstrate that the dendritic cells expressing HCV Ags are able to prime the Ag-specific T cells from uninfected healthy individuals in vitro. These studies have implications in designing cellular vaccines, T cell adoptive transfer therapy or vaccine candidates for HCV infection in both prophylactic and therapeutic settings.

Immunomodulation by hepatitis C virus-derived proteins: targeting human dendritic cells by multiple mechanisms

Hepatitis C virus (HCV) has the ability to persist in the majority of infected people. Strong, multispecific and sustained T-cell response is correlated with viral clearance. The mechanisms of chronicity by HCV are unclear. HCV could restrain the immune system and establish chronic infection by modulating dendritic cell (DC) function, T-cell function or both. DC dysfunction has been postulated to be either due to direct HCV infection or by the presence of HCV proteins. In this report, for the first time, we have examined whether soluble HCV proteins can impair DC function or directly inhibit T-cell responses in the cells obtained from healthy uninfected people. Our studies revealed that different HCV proteins used distinct mechanisms to down-regulate DC functions. Individual HCV proteins, Core, NS3, NS4, NS5 as well as fused Polyprotein (Core-NS3-NS4) were found to impair functions of both immature DCs and mature DCs by regulating the expression of co-stimulatory and antigen presentation molecules, strikingly reducing IL-12 secretion, inducing the expression of FasL to mediate apoptosis, interfering with allo-stimulatory capacity, inhibiting toll-like receptor signaling and inhibiting nuclear translocation of NFkappaB in DCs. Interestingly, HCV proteins did not directly inhibit T-cell proliferation. Our findings clearly demonstrate that HCV proteins impair T-cell responses indirectly by inhibiting DCs that could result in a sub-optimal cellular immune response allowing for persistent HCV infections. These studies delineate important mechanisms by which initial DC dysfunction can establish contributing to chronicity. Our data are in agreement with earlier observations that DCs are impaired in HCV infected people.

Immunogenicity and protective efficacy of a DNA vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekin ducks

The potential of CD154 (CD40L) as a powerful immunological adjuvant has been shown in various strategies. In this study we examine the immunogenicity and protective efficacy of a CD40-targeting avian influenza hemagglutinin (HA) subunit DNA vaccine in ducks. DNA constructs encoded the ectodomain of the HA protein of LPAI A/mallard/BC/373/2005 (H5N2) with or without fusion to the ectodomain of duck CD154. CD40-targeting significantly accelerated and enhanced humoral responses to the vector-encoded HA protein. In viral challenge experiments with A/chicken/Vietnam/14/2005 (H5N1), DNA immunization conferred partial protection against the genetically distant HPAI. The observed improved kinetics and magnitude of immune induction suggest that CD40-targeting holds promise for influenza A vaccine development.

HCV-core and NS3 antigens play disparate role in inducing regulatory or effector T cells in vivo: Implications for viral persistence or clearance

A distinguishing feature of HCV is its ability to persist in majority of the infected people. We investigated the role of HCV-core and NS3 in inducing effector T cells to mediate antiviral immunity. Our studies revealed that immunization with recombinant adenoviral vector containing HCV-core or NS3 leads to differential development of regulatory vs. effector T cells in mice, resulting in distinct outcomes of virus infection. For the first time, our studies directly demonstrate that HCV-core enhances both CD4(+) and CD8(+) T(regs) which possibly contribute to persistent infection, whereas HCV NS3 induces both CD4(+) and CD8(+) effector T cells to allow viral clearance.

MUC1 mucin is expressed on human T-regulatory cells: function in both co-stimulation and co-inhibition.

MUC1 mucin, an important protein of epithelial cells and epithelial-derived carcinomas, is also expressed on activated T cells, showing both positive and negative regulatory functions. It is currently unknown whether MUC1 is a true regulatory protein of T cells and what conditions lead to MUC1 co-stimulation versus co-inhibition. We have found that MUC1 is expressed on the majority of T-regulatory cells (CD4(+)/CD25(+)/FoxP3(+)) in humans (>90%) and that CD3/MUC1 co-stimulation leads to an increased number of T-regulatory cells. We also discovered that the immunoregulatory function is dependent upon the number of accessory (CD3(-)) cells present, with co-inhibition occurring with <5-10% accessory cells while co-stimulation begins with a reconstitution of ~50% accessory cells. Co-inhibition was also found to not be the result of the apoptosis but a separate and unknown pathway. This data further characterizes MUC1 as an immunoregulatory protein of T cells capable of giving a positive or negative stimulus.

Chemotherapeutic Interventions Against Tuberculosis

Tuberculosis is the second leading cause of infectious deaths globally. Many effective conventional antimycobacterial drugs have been available, however, emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has overshadowed the effectiveness of the current first and second line drugs. Further, currently available agents are complicated by serious side effects, drug interactions and long-term administration. This has prompted urgent research efforts in the discovery and development of new anti-tuberculosis agent(s). Several families of compounds are currently being explored for the treatment of tuberculosis. This review article presents an account of the existing chemotherapeutics and highlights the therapeutic potential of emerging molecules that are at different stages of development for the management of tuberculosis disease.