IgE-Based Therapeutic Combination Enhances Antitumor Response in Preclinical Models of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents 3% of all cancer cases and 7% of all cancer deaths in the United States. Late diagnosis and inadequate response to standard chemotherapies contribute to an unfavorable prognosis and an overall 5-year survival rate of less than 10% in PDAC. Despite recent advances in tumor immunology, tumor-induced immunosuppression attenuates the immunotherapy response in PDAC. To date, studies have focused on IgG-based therapeutic strategies in PDAC. With the recent interest in IgE-based therapies in multiple solid tumors, we explored the MUC1-targeted IgE potential against pancreatic cancer. Our study demonstrates the notable expression of FceRI (receptor for IgE antibody) in tumors from PDAC patients. Our study showed that administration of MUC1 targeted-IgE (mouse/human chimeric anti-MUC1.IgE) antibody at intermittent levels in combination with checkpoint inhibitor (anti-PD-L1) and TLR3 agonist (PolyICLC) induces a robust antitumor response that is dependent on NK and CD8 T cells in pancreatic tumor-bearing mice. Subsequently, our study showed that the antigen specificity of the IgE antibody plays a vital role in executing the antitumor response as nonspecific IgE, induced by ovalbumin (OVA), failed to restrict tumor growth in pancreatic tumor-bearing mice. Utilizing the OVA-induced allergic asthma-PDAC model, we demonstrate that allergic phenotype induced by OVA cannot restrain pancreatic tumor growth in orthotopic tumor-bearing mice. Together, our data demonstrate the novel tumor protective benefits of tumor antigen-specific IgE-based therapeutics in a preclinical model of pancreatic cancer, which can open new avenues for future clinical interventions.

Importance of strain selection in the generation of heterologous immunity to Glaesserella (Haemophilus) parasuis.

Glaesserella (Haemophilus) parasuis is a part of the microbiota of healthy pigs and also causes the systemic condition called Glässer's disease. G. parasuis is categorized by it capsular polysaccharide into 15 serovars. Because of the serovar and strain specific immunity generated by whole cell vaccines and the rapid onset of disease, G. parasuis has been difficult to control in the swine industry. This report investigated the protection afforded by the use of two serovar 5 isolates (Nagasaki and HS069) as whole cell, killed bacterins against homologous challenge and heterologous challenge with the serovar 1 strain 12939 to better understand bacterin generated immunity. Both bacterins induced a high antibody titer to the vaccine strain and the heterologous challenge strain. Protection was seen with both bacterins against homologous challenge; however, after heterologous challenge, the HS069 bacterin provided complete protection and all Nagasaki bacterin vaccinated animals succumbed to disease. The difference in protection appears to be due to differences in antibody specificity and the capacity of induced antibody to fix complement and opsonize G. parasuis, as shown by Western blotting and functional assays. This report shows the importance of strain selection when developing bacterin vaccines, as some strains are better able to generate heterologous protection. The difference in protection seen here can also be utilized to detect proteins of interest for subunit vaccine development.

Local and systemic immunosuppression in pancreatic cancer: Targeting the stalwarts in tumor's arsenal.

Late detection, compromised immune system, and chemotherapy resistance underlie the poor patient prognosis for pancreatic ductal adenocarcinoma (PDAC) patients, making it the 3rd leading cause of cancer-related deaths in the United States. Cooperation between the tumor cells and the immune system leads to the immune escape and eventual establishment of the tumor. For more than 20 years, sincere efforts have been made to intercept the tumor-immune crosstalk and identify the probable therapeutic targets for breaking self-tolerance toward tumor antigens. However, the success of these studies depends on detailed examination and understanding of tumor-immune cell interactions, not only in the primary tumor but also at distant systemic niches. Innate and adaptive arms of the immune system sculpt tumor immunogenicity, where they not only aid in providing an amenable environment for their survival but also act as a driver for tumor relapse at primary or distant organ sites. This review article highlights the key events associated with tumor-immune communication and associated immunosuppression at both local and systemic microenvironments in PDAC. Furthermore, we discuss the approaches and benefits of targeting both local and systemic immunosuppression for PDAC patients. The present articles integrate data from clinical and genetic mouse model studies to provide a widespread consensus on the role of local and systemic immunosuppression in undermining the anti-tumor immune responses against PDAC.

Generation and Evaluation of a Glaesserella (Haemophilus) parasuis Capsular Mutant.

Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer's disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.

Administration of granulocyte-colony stimulating factor (G-CSF) to pigs results in a longer mean survival time after exposure to Streptococcus suis.

The use of immunomodulators is a promising alternative to the use of antibiotics for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease. Previously we demonstrated a replication-defective adenovirus vector that expresses porcine granulocyte colony-stimulating factor (G-CSF) elicited a sustained neutrophilia, lasting nearly 3 weeks, which may be beneficial to prevent bacterial diseases during times of peak incidence. In a pilot study using the vectored G-CSF with a Caesarian-derived, colostrum-deprived (CDCD) pig model of Streptococcus suis disease, only 1 of 4 pigs given G-CSF developed disease, while 3 of 4 non-treated pigs developed Streptococcal disease. In a subsequent study using a larger number of pigs, although there was no difference in overall survival, there was a longer mean survival time in G-CSF treated pigs. S. suis infection is more severe in CDCD pigs than conventionally raised pigs, consequently results in the field may be superior to the ones reported in this study. Although there were positive effects from the use of G-CSF in this study, further research is needed to determine if improved clinical outcomes could be achieved under field conditions and whether the use of G-CSF in pigs to induce a sustained increase in circulating neutrophil numbers may be useful as an adjunct to antibiotics to diminish the severity of Streptococcal disease, especially during times of stress and pathogen exposure such as post-weaning.

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus).

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 µg/mL ConA, while 1 µg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.

Methicillin-Resistant Staphylococcus aureus Sequence Type (ST) 5 Isolates from Health Care and Agricultural Sources Adhere Equivalently to Human Keratinocytes.

Staphylococcus aureus is part of the nasal microbiome of many humans and has become a significant public health burden due to infections with antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA) strains. Several lineages of S. aureus, including MRSA, are found in livestock species and can be acquired by humans through contact with animals. These livestock-associated MRSA (LA-MRSA) isolates raise public health concerns because of the potential for livestock to act as reservoirs for MRSA outside the hospital setting. In the United States, swine harbor a mixed population of LA-MRSA isolates, with the sequence type 398 (ST398), ST9, and ST5 lineages being detected. LA-MRSA ST5 isolates are particularly concerning to the public health community because, unlike the isolates in the ST398 and ST9 lineages, isolates in the ST5 lineage are a significant cause of human disease in both the hospital and community settings globally. The ability of swine-associated LA-MRSA ST5 isolates to adhere to human keratinocytes in vitro was investigated, and the adherence genes harbored by these isolates were evaluated and compared to those in clinical MRSA ST5 isolates from humans with no swine contact. The two subsets of isolates adhered equivalently to human keratinocytes in vitro and contained an indistinguishable complement of adherence genes that possessed a high degree of sequence identity. Collectively, our data indicate that, unlike LA-MRSA ST398 isolates, LA-MRSA ST5 isolates do not exhibit a reduced genotypic or phenotypic capacity to adhere to human keratinocytes.IMPORTANCE Our data indicate that swine-associated livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 isolates are as capable of adhering to human skin and have the same genetic potential to adhere as clinical MRSA ST5 isolates from humans. This suggests that humans in contact with livestock have the potential to become colonized with LA-MRSA ST5 isolates; however, the genes that contribute to the persistence of S. aureus on human skin were absent in LA-MRSA ST5 isolates. The data presented here are important evidence in evaluating the potential risks that LA-MRSA ST5 isolates pose to humans who come into contact with livestock.

Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR.

Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 µg/mL) and concanavalin A (1 µg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1ß, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.

Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine.

Type I interferons, such as interferon alpha (IFN-α), contribute to innate antiviral immunity by promoting production of antiviral mediators and are also involved in promoting an adaptive immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating and costly viruses to the swine industry world-wide and has been shown to induce a meager IFN-α response. Previously we administered porcine IFN-α using a replication-defective adenovirus vector (Ad5-IFN-α) at the time of challenge with virulent PRRSV and demonstrated an increase in the number of virus-specific IFNγ secreting cells, indicating that the presence of IFN-α at the time of infection can alter the adaptive immune responses to PRRSV. In the current experiment, we explored the use of IFN-α as an adjuvant administered with live-attenuated PRRSV vaccine as a method to enhance immune response to the vaccine. Unlike the previous studies with fully virulent virus, one injection of the Ad5-IFN-α abolished replication of the vaccine virus and as a result there was no detectible adaptive immune response. Although IFN-α did not have the desired adjuvant effect, the results further highlight the use of IFN-α as a treatment for PRRSV infection.

Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains.

Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1ß, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.

Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation.

UNLABELLED: Parainfluenza viruses are known to inhibit type I interferon (IFN) production; however, there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, IFN-λR1/interleukin-10R2 (IL-10R2), which is primarily expressed by epithelial cells. Parainfluenza virus 3 (PIV-3) infection is highly restricted to the airway epithelium. We therefore sought to examine type III IFN signaling pathways during PIV-3 infection of epithelial cells. We used three strains of PIV-3: human PIV-3 (HPIV-3), bovine PIV-3 (BPIV-3), and dolphin PIV-1 (Tursiops truncatus PIV-1, or TtPIV-1). Here, we show that message levels of IL-29 are significantly increased during PIV-3 infection, yet downstream antiviral signaling molecules are not upregulated to levels similar to those of the positive control. Furthermore, in Vero cells infected with PIV-3, stimulation with recombinant IL-29/-28A/-28B does not cause upregulation of downstream antiviral molecules, suggesting that PIV-3 interferes with the JAK/STAT pathway downstream of the IFN-λR1/IL-10R2 receptor. We used Western blotting to examine the phosphorylation of Stat1 and Stat2 in Vero cells and the bronchial epithelial cell line BEAS-2B. In Vero cells, we observed reduced phosphorylation of the serine 727 (S727) site on Stat1, while in BEAS-2B cells Stat1 phosphorylation was decreased at the tyrosine 701 (Y701) site during PIV-3 infection. PIV-3 therefore interferes with the phosphorylation of Stat1 downstream of the type III IFN receptor. These data provide new evidence regarding strategies employed by parainfluenza viruses to effectively circumvent respiratory epithelial cell-specific antiviral immunity. IMPORTANCE: Parainfluenza virus (PIV) in humans is associated with bronchiolitis and pneumonia and can be especially problematic in infants and the elderly. Also seen in cattle, bovine PIV-3 causes respiratory infections in young calves. In addition, PIV-3 is one of a number of pathogens that contribute to the bovine respiratory disease complex (BRDC). As their name suggests, interferons (IFNs) are produced by cells to interfere with viral replication. Paramyxoviruses have previously been shown to block production and downstream signaling of type I IFNs. For the first time, it is shown here that PIV-3 can induce protective type III IFNs in epithelial cells, the primary site of PIV-3 infection. However, we found that PIV-3 modulates signaling pathways downstream of the type III IFN receptor to block production of several specific molecules that aid in a productive antiviral response. Importantly, this work expands our understanding of how PIV-3 effectively evades host innate immunity.

Development and application of specific cytokine assays in tissue samples from a bottlenose dolphin with hyperinsulinemia.

Chronic inflammation has been associated with insulin resistance and type 2 diabetes (T2D) in humans. Postmortem hepatic and splenic tissue from a 46-year-old geriatric male bottlenose dolphin (Tursiops truncatus) with insulin resistance (chronic hyperinsulinemia with hyperglycemia), chronic inflammation (white blood cell count greater than 12,000 cells/µL), and mild fatty liver disease was evaluated for elevated pro-inflammatory mediators. Cytokine mRNA expression in postmortem hepatic and splenic tissue, as determined by real-time PCR, included an array of cytokines: TGF-ß, TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-12p40, IL-13, and IL-18. Values from this dolphin were compared to a younger reference dolphin with no known chronic metabolic perturbations or inflammation. Levels of TGF-ß, TNF-α, and IL-4 were higher in the case dolphin's liver compared to that of the reference dolphin. In the case dolphin's spleen, IL-10 and IFN-γ mRNA was upregulated while IL-4 was less than the reference dolphin. IL-18 and IL-13 were upregulated in both tissues. Fluorescent immunohistochemistry (IHC) utilized the following antibodies: anti-porcine IL-6, anti-bovine IFN-γ, IL-4, and IL-10, anti-human TGF-ß, anti-ovine IL-1ß, and anti-dolphin IL-8. Fluorescent IHC in spleen from the case dolphin revealed staining of IL-4, IL-6, IL-8, and TGF-ß throughout the tissue. IL-10 and IFN-γ were seen to predominate in areas surrounding the follicles of splenic tissue. This is the first characterization of cytokine levels in dolphin hepatic and splenic tissue. While there are limitations to a case study, this report of inflammatory biomarkers in tissues of a dolphin with insulin resistance and fatty liver disease are similar to those observed in human patients.