Immune-competent Hosts Research Articles

Can miRNA712_3p be a promising biomarker for early diagnosis of toxoplasmosis?

Objective: To assess the role of miRNA712_3p as a specific biomarker in early detection of toxoplasmosis in plasma of mice acutely infected with Toxoplasma gondii. Methods: Real-time PCR was used to measure the level of miRNA712_3p in plasma of infected mice. Immune-competent and immune-suppressed mice were examined, three and five days post-infection. Results: Results revealed significant up-regulation of plasma miRNA712_3p in both immune- competent and immune-compromised groups in comparison to the control non-infected group. Additionally, an increase in the level of miRNA712_3p was noticed correspondently in the parasite density detected in liver impression smears. Conclusions: miRNA712_3p can be used as a novel biomarker for the detection of Toxoplasma gondii infection in both immune-competent and immune-compromised host.

Cancer immune resistance: can theories converge?

Immune oncology (IO) is challenged to expand its usefulness to a broader range of cancers. A second generation of IO agents acting beyond the realm of Checkpoint Inhibitor Therapy (CIT) is sought with the intent of turning immune-resistant cancers into appealing IO targets. The published literature proposes a profusion of models to explain cancer immune resistance to CIT that largely outnumber the immune landscapes and corresponding resistance mechanisms. In spite of the complex and contradicting models suggested to explain refractoriness to CIT, the identification of prevailing mechanisms and their targeting may not be as daunting as it at first appears. Here, we suggest that cancer cells go through a conserved evolutionary bottleneck facing a Two-Option Choice to evade recognition by the immune competent host: they can either adopt a clean oncogenic process devoid of immunogenic stimuli (immune-silent tumors) or display an entropic biology prone to immune recognition (immune-active tumors) but resilient to rejection thanks to the recruitment of compensatory immune suppressive processes. Strategies aimed at enhancing the effectiveness of CIT will be different according to the immune landscape targeted.

Cerebral Toxoplasmosis in a Patient with Systemic Lupus Erythematosus

Toxoplasma gondii is a globally distributed protozoan parasite that causes toxoplasmosis and is frequently associated with infections in the immune compromised and in neonates. On initial infection, the parasite enters the host through the intestinal epithelium, usually after oocysts are ingested. Additionally, vertical transmission from mother to fetus can occur as well as infection from organ transplantation. In immune competent hosts, presentation is commonly flu-like with fever, headache and lymphadenopathy. The host’s immune response typically contains the infection, limiting it to tissue cysts comprised of slowly replicating bradyzoites. In immune deficient hosts, when tissue cysts containing bradyzoites rupture, the compromised immune system is unable to halt progression of the parasite. Bradyzoites differentiate into rapidly replicating tachyzoites and as the parasite proliferates, host cell death occurs and necrotic foci develop. Most commonly, immune deficient hosts present with encephalitis, pneumonitis, and chorioretinitis [1]. We report a case of cerebral toxoplasmosis in a female patient with systemic lupus erythematosus (SLE) being treated with hydroxy chloroquine, mycophenolate mofetil and prednisone.

Prevalence and risk of hepatitis E virus infection in the HIV population of Nepal

BackgroundInfection with the hepatitis E virus (HEV) can cause acute hepatitis in endemic areas in immune-competent hosts, as well as chronic infection in immune-compromised subjects in non-endemic areas. Most studies assessing HEV infection in HIV-infected populations have been performed in developed countries that are usually affected by HEV genotype 3. The objective of this study is to measure the prevalence and risk of acquiring HEV among HIV-infected individuals in Nepal.MethodsWe prospectively evaluated 459 Human Immunodeficiency Virus (HIV)-positive individuals from Nepal, an endemic country for HEV, for seroprevalence of HEV and assessed risk factors associated with HEV infection. All individuals were on antiretroviral therapy and healthy blood donors were used as controls.ResultsWe found a high prevalence of HEV IgG (39.4%) and HEV IgM (15.3%) in HIV-positive subjects when compared to healthy HIV-negative controls: 9.5% and 4.4%, respectively (OR: 6.17, 95% CI 4.42–8.61, p < 0.001 and OR: 3.7, 95% CI 2.35–5.92, p < 0.001, respectively). Individuals residing in the Kathmandu area showed a significantly higher HEV IgG seroprevalance compared to individuals residing outside of Kathmandu (76.8% vs 11.1%, OR: 30.33, 95% CI 18.02–51.04, p = 0.001). Mean CD4 counts, HIV viral load and presence of hepatitis B surface antigen correlated with higher HEV IgM rate, while presence of hepatitis C antibody correlated with higher rate of HEV IgG in serum. Overall, individuals with HEV IgM positivity had higher levels of alanine aminotransferase (ALT) than IgM negative subjects, suggesting active acute infection. However, no specific symptoms for hepatitis were identified.ConclusionsHIV-positive subjects living in Kathmandu are at higher risk of acquiring HEV infection as compared to the general population and to HIV-positive subjects living outside Kathmandu.

It Ain't Called Crypto for Nothing

SESSION TITLE: Fungal Infections 2 SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: In the age of Chest CTs, pulmonary nodules are a common finding and determining the etiology and need for treatment can be challenging. The differential for pulmonary nodules includes infection, rheumatologic manifestations, malignancy, and inflammatory lung diseases. In the work up of these findings, patient characteristics, risk factors, and clinical history help to narrow the potential causes. However, in medicine, things are often not as simple, and a simplistic approach to pulmonary nodules may lead to missed diagnoses. In this case series, we present two cases of biopsy-proven Cryptococcal pneumonia in immune competent pts. CASE PRESENTATION: In both cases, our patients were M in their 60s, without any known immune dysfunction, malignancy, HIV, or history of chronic steroid use. The first patient (pt A) presented to the ED, with c/o SOB, chest pain, subacute dry cough, and fatigue. CTA of the chest was performed which revealed multiple large pulmonary nodules and adenopathy. The second patient (pt B) presented for a second opinion given progressive SOB, fatigue, and weight loss with pulmonary nodules refractory to antibiotic therapy. In both cases, an infectious work-up was completed and included fungal testing and rheumatologic screening which were all negative. PET CTs were obtained and in both cases the lesions were highly PET avid. Biospies were subsequently performed (Pt A CT guided biopsy, Pt B VATs wedge biopsy) and revealed Crytoccoccus neoformans. Pts LPs' were negative for CNS involvement. Both pts were started on Fluconazole with clinical and radiographic improvement. DISCUSSION: Cryptococcal pneumonia is a relatively rare diagnosis, even more rare in immune competent patients. Findings on chest imaging often include nodular opacities with or without cavitation, adenopathy, and pleural effusions. These findings however, are non-specific and are associated with multiple disease states. Despite a low pre-test probability, biopsy-proven Cryptococcal pneumonia was identified and subsequently treated in these immune competent patients. CONCLUSIONS: Although a rare entity in immune competent hosts, Cryptococcal pneumonia should remain on the differential in the work-up of pulmonary nodules in the immune competent host. Reference #1: Cryptococcal pneumonia: the great mimicker, Lasya Thambidurai MD, et al. British Journal of Radiology. Published Online: January 05, 2017 Reference #2: Lindell RM, Hartman TE, Nadrous HF, Ryu JH. Pulmonary cryptococcosis: CT findings in immunocompetent patients. Radiology 2005; 236: 326-31 DISCLOSURE: The following authors have nothing to disclose: Amit Bharara, Rebecca Potfay, George Kallingal No Product/Research Disclosure Information

Cutaneous Squamous Cell Carcinoma Leading to a Broken Heart

SESSION TITLE: Student/Resident Lung Cancer SESSION TYPE: Student/Resident Case Report Slide PRESENTED ON: Sunday, October 29, 2017 at 04:30 PM - 05:30 PM INTRODUCTION: Cardiac metastases have been found in 6-20 % of autopsies of patients with metastatic malignancies (1). Secondary cardiac neoplasms, particularly lymphoma, esophageal, lung, breast cancers and melanoma are the most common (2). We report the case of an immuno-competent 58 year old man with widespread metastatic infiltration of the heart and lungs from primary cutaneous squamous cell carcinoma (cSCC). CASE PRESENTATION: 58 year old male, non-smoker, with history of resected cSCC of the back presented a year later with a new left axillary mass. Biopsy was positive for metastatic SCC. PET-CT demonstrated left metastatic axillary lymph nodes and bilateral lung nodules for which he began cisplatin and cetuximab. He became dyspneic and oxygen dependent, developing small pleural effusions that were negative for malignancy. Echocardiogram showed a hyperdynamic left ventricle without pericardial effusion. CT angiogram of chest was negative for pulmonary embolus however demonstrated progression of bilateral pulmonary nodules and lymphadenopathy. Bronchoscopy was notable for nonobstructive endobronchial lesions in the left mainstem with biopsy revealing SCC. Persistent tachycardia preceded cardiopulmonary arrest without successful resuscitation. Autopsy revealed widely metastatic squamous cell carcinoma involving heart and bilateral lungs. The heart surprisingly revealed numerous foci of metastatic moderately to poorly differentiated squamous cell carcinoma involving the septum, anterior right ventricular wall, posterior left ventricular wall, aortic arch and foci of pericardium. DISCUSSION: Cardiac metastases are clinically more difficult to diagnose pre-mortem due to nonspecific signs and symptoms. The first approach is often echocardiography, but CT and Cardiac MRI may highlight some important imaging findings (3). The prognosis of patients with cardiac metastasis remains dismal with a 5 years survival of 7% reported in the literature (2). CONCLUSIONS: Dermatologic SCC has been documented to have a potentially aggressive and fatal nature in organ transplant recipients due to long-term immunosuppression however our case illustrates this in an immune-competent host. Cardiac metastasis should be considered in patients presenting with cardiac symptoms especially when there is a history of advanced cancer. When cardiac metastases are discovered the prognosis is poor (2). Reference #1: Cheruvu B, Cheruvu P, Boyars M. An unusual case of metastasis to the left side of the heart: a case report. J Med Case Reports. 2011;5:23. doi: 10.1186/1752-1947-5-23 Reference #2: Hunter JM, Reid AL, Stride PJ, Dettrick A. Right ventricular inflow tract obstruction secondary to metastatic cutaneous squamous cell carcinoma. BMJ Case Reports. 2012;2012:bcr2012006556. doi:10.1136/bcr-2012-006556 Reference #3: Villa A, Eshja E, Dallavalle S, Bassi EM, Turco A. Cardiac metastases of melanoma as first manifestation of the disease. Journal of Radiology Case Reports. 2014;8(4):8-15. doi:10.3941/jrcr.v8i4.1700 DISCLOSURE: The following authors have nothing to disclose: Tulip Jhaveri, Kamil Rechache, Christopher Tretter, Carla Lamb No Product/Research Disclosure Information

An unusual case of community acquired pneumonia in an immune competent host due to Pseudomonas Aeruginosa: an example of clinical management without hospitalization

An unusual case of community acquired pneumonia in an immune competent host due to Pseudomonas Aeruginosa: an example of clinical management without hospitalization

IFN-Lambda: The Key to Norovirus’s Secret Hideaway

The cellular tropism of noroviruses in immune-competent hosts remains enigmatic. In recent studies in Cell Host & Microbe and Immunity, Lee etal. (2017) and Tomov etal. (2017) determine that norovirus innate immune evasion enables infection of intestinal epithelial cells, escape from adaptive immunity, virus persistence, and shedding.

Systemic cryptococcosis in an immune-competent child.

Crytococcus neoformans is an encapsulated yeast that frequently affects immune-compromised patients, although increasingly being detected in the immune-competent host as well. We report a case of disseminated cryptococcosis in a young child in whom no immune deficiency was yet identified. A 4-year-old child presented with high-grade fever, intermittent abdominal pain and generalized skin eruptions for the past two months. He had pallor, firm lymphadenopathy, skin lesions with scarring and firm hepatosplenomegaly. Magnetic resonance imaging of brain and bone-marrow aspiration were normal. Fine-needle-aspiration-cytology of cervical lymph nodes demonstrated Cryptococcus. Serum latex-agglutination test showed a positive titer (1:256). Cryptococcus culture was sterile. The patient received intravenous liposomal amphotericin-B and oral flucytosine for 8 weeks followed by oral fluconazole. Disseminated cryptococcosis with involvement of reticuloendothelial and dermatological systems is rare. Early diagnosis and timely management of associated complications would be life saving.

Abstract 3968: Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth

Abstract Glioblastoma (GBM) development and therapeutic resistance has been accompanying with the tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). TAMs are heterogeneous cell populations of immune regulatory myeloid-derived suppressor cells (MDSCs) and polarization of anti-tumor macrophages (M1) into pro-tumor macrophages (M2). We investigated the role of myeloid cell NF-κB signaling in orthotopic GBM model using immune deficient and immune competent hosts. Interestingly, conditional deletion of canonical NF-κB signaling (p65) with Lysm-Cre (p65 KO) in myeloid cells, significantly inhibited syngeneic GL261 tumor growth in immune-competent mice compared to control mice. We studied the TAMs recruitment to the tumor and their polarization under the influence of TME. P65 KO mice displayed decreasing trend of immune cell infiltration (CD45), which phenotyped as decreased F4/80+, CD68+, CD206+ (M2) and Gr1+CD11b+ (MDSCs) macrophages, compared to control mice. This was associated with the increased CD80+ (M1) macrophages, increasing trend of CD4+ and CD8+ cytotoxic T cells, and decreased CD44+ mesenchymal cancer stem cells (CSCs) populations in the TME. Cytokine array data indicated that loss of canonical NF-κB signaling within the TAMs was implicated in increased production of IFNγ, IGF1, MCP1, MIP1α, and TNFα cytokines. Co-culture of T cells with p65 KO or control MDSCs identified increased proliferation of T cells with p65 KO MDSCs compared to control MDSCs. Conversely, GBM patient-derived xenografts and U251 GBM cell line-derived tumors showed increasing trend of growth in immune-deficient mice, following the transplantation of p65 KO bone marrow (BM) compared to control BM. Pro-tumor macrophages and CSCs were increased and T cell populations were decreased in human tumors grown in immune deficient mice transplanted with p65 KO BM, compared with control BM. In addition, analysis of human data set revealed higher expression of p65 subunit of NF-κB complex in brain tumor stroma compared to the tumor cells. The study suggests that canonical NF-κB signaling in TAMs is required for the tumor-promoting macrophage polarization and GBM growth in immunocompetent host compared to immune deficient host. Therefore, targeting myeloid-specific NFκB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity. Citation Format: Bhagelu R. Achyut, Jennifer Bradford, Kartik Angara, Mohammad Rashid, Meenu Jain, Thaiz Borin, ASM Iskander, Roxan Ara, Ali Arbab. Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3968. doi:10.1158/1538-7445.AM2017-3968

Abstract 3965: Epithelial membrane protein-2 is a novel regulator of immune editing in breast cancer

Abstract Cancer immuno-editing is a process that describes the interaction between immune cells and tumor cells. This interaction can result in elimination of a developing tumor, tumor dormancy, or tumor cells that are capable of surviving in an immune-competent host. We have recently uncovered a novel mechanism of immunoediting by the tetraspan protein Epithelial membrane protein or EMP2. We propose that EMP2 serves as a bridge between innate and adaptive immunity via tumor mediated type I interferon expression. Most tumor cells evade the immune system through suppression or ignorance. EMP2 levels promote type I interferon expression which was found to correlate with PDL1 expression in a number of cell lines. Concordantly, in vivo, in multiple breast cancer cell lines, EMP2 promoted tumor growth. In contrast, reduction in EMP2 levels correlated with a reduction in tumor size and a corresponding increase in tumor associated macrophages. Given that tumors with lower levels of EMP2 grow poorly, we hypothesized that targeting EMP2 may provide a therapeutic benefit. Recently, we have developed a panel of novel IgG1 monoclonal antibodies to EMP2 and tested their ability to treat both xenograft and syngeneic mouse models. Our results show that anti-EMP2 IgG1 significantly reduces tumor load in both models in part through the recruitment of M1 macrophages. To explore this observation more fully, experiments using murine syngeneic models were employed. Anti-EMP2 therapy elicits a robust ADCC response, producing an immune infiltrate rich in macrophages, NK cells, and lymphocytes. In addition, we show that CD8 cells show reduced exhaustion in the presence of anti-EMP2 antibodies. Conclusions: EMP2 is a protein upregulated in a number of cancers in women including breast, ovarian, and endometrial tumors. Our work collectively show that EMP2 is a novel immune regulator where its expression creates an immunosuppressive tumor microenvironment. We thus predict that targeting EMP2 may improve therapeutic outcomes for cancers in women. Citation Format: Jessica Tsui, Negin Ashki, Yuling Chang, Deven Patel, Jasmine Sjarif, Madhuri Wadehra. Epithelial membrane protein-2 is a novel regulator of immune editing in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3965. doi:10.1158/1538-7445.AM2017-3965

Abstract SY25-02: Antigenic strength vs. tumor microenvironment in determining cancer immunosurveillance

Abstract We evaluated immune surveillance in pancreatic ductal adenocarcinoma (PDA), which exhibits far fewer missense mutations and potential neoepitopes than classically described carcinogen-induced, highly mutated tumors. In contrast to these immunogenic tumor models from which the theory of elimination-equilibrium-escape was determined, we found no evidence for immunoediting in a mutant Kras-driven murine model of PDA. These tumors progress the same with or without a functional adaptive immune system, similar to other mouse tumors that have also been shown to grow independently of T cells. Tumors arising in T cell–depleted mice grew unchecked in immune-competent hosts. We further show that KPC pancreatic tumors have few if any predicted neoepitopes derived from somatic tumor missense mutations and that an immunoediting phenotype can be rescued by ectopic expression of a neoantigen (OVA) in the same tumor model. Thus, the manifestations of cancer immune surveillance in the KPC model depend on tumor antigenic strength; the lack of neoepitopes is not a consequence of immunoediting in this model. These findings suggest an alternative biologic mechanism in which tumor outgrowth reflects immune quiescence, which is linked to, and regulated by, poor tumor antigenicity—a conclusion with potential relevance to human cancers with a low burden of mutations and minimal neoepitopes. Citation Format: Robert H. Vonderheide. Antigenic strength vs. tumor microenvironment in determining cancer immunosurveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY25-02. doi:10.1158/1538-7445.AM2017-SY25-02

Abstract 66: Anthracycline-based antibody drug conjugates with potent immune-stimulatory functions

Abstract Antibody drug conjugates (ADCs) are highly potent and selective anti-tumor drugs, combining the specific targeting of monoclonal antibodies with the potency of small molecule toxic payloads. Here, we employed enzymatic, site-specific conjugation to generate homogenous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a non-cleavable peptide linker, using the anti-HER-2 antibody trastuzumab (part of trastuzumab emtansine) and the anti-CD30 antibody cAC10 (part of brentuximab vedotin). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as trastuzumab emtansine and brentuximab vedotin. Anti-tumor activity in an immune-competent host involved activation of the immune system, as shown by evaluation of a trastuzumab-PNU ADC in a Kadcyla-resistant HER2-positive orthotopic breast cancer model. Depletion of CD8 T cells severely reduced the anti-tumor activity of the ADC, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was entirely inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. In summary, we present a novel ADC format endowed not only with highly potent cytotoxicity, but also effective immune-stimulatory functions. Citation Format: Roger R. Beerli. Anthracycline-based antibody drug conjugates with potent immune-stimulatory functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2017-66

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells

Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer.

Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 Klebsiella pneumoniae Clonal Lineage Producing KPC-Type Carbapenemases.

ST258-K. pneumoniae (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.

Association of Cytokines and Chemokines in Pathogenesis of Breast Cancer.

Breast cancer touches women's life worldwide. Expected outcome is not achieved due to molecular heterogeneity and complex biology despite substantial advancement in diagnosis, prevention and treatment of breast cancer. Patients with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2) positive tumors receive hormone ablation and Her2 directed therapy. While patients diagnosed with triple-negative breast cancer receive chemotherapy in both the early and advanced stages. However, chemotherapeutic efficacies are not the same in every patient, which has fostered a major effort to identify new targets to treat breast cancer. Positive therapeutic outcome after immune checkpoint inhibitors emphasizes the significance of the host immune system in breast cancer. Cancer develops in immune competent host wherein cytokines, while shaping the immune system, also serve as growth signals for cancer cells. The dynamics of cross talk between immune system and cancer cells mediated by cytokines and chemokines changes during cancer initiation, progression, and therapeutic interventions. Hence, better understanding of molecular footprint of cancer cells, as well as crosstalk between cancer cells and host immune system is needed to develop patient specific treatment and management of breast cancer.

Pragmatic Combination of Available Diagnostic Tools for Optimal Detection of Intestinal Microsporidia.

Diarrhea is a debilitating condition in HIV infected individuals and with the finding that almost 1/4 cases of diarrhea in HIV are due to microsporidia, there is a dire need to institute measures for its detection on a regular basis. Keeping this in mind the study aims to determine the burden of intestinal microsporidiosis in HIV seropositive patients presenting with and without diarrhea and to compare the ability of microscopy and PCR in its detection.The study group consisted of 120 patients divided into four groups HIV seropositive with/without diarrhea, and HIV seronegative with/without diarrhea. Performance of four staining techniques including Modified Trichrome, Calcofluor White, Gram Chromotrope and Quick hot Gram Chromotrope stains were evaluated against PCR in diagnosing enteric microsporidiosis from stool samples.Overall prevalence of intestinal microsporidiosis was 10.83%. The same for HIV seropositive patients with diarrhea was 23.33%, HIV seropositive patients without diarrhea and in immune-competent hosts with diarrhea was 10% each. Enterocytozoon bieneusi was found to predominate. Calcofluor white stain detected maximum microsporidia in stool samples (76.92%), followed by Modified Trichrome stain (61.5%), PCR (46.15%) and Gram Chromotrope and Quick hot Gram Chromotrope stains (38.4% each). PCR exhibited the best performance with a sensitivity and specificity of 100%. Our data suggests screening of stool samples with either Modified Trichrome or Calcofluor white stain followed by PCR confirmation thus leading to maximum detection along with speciation for complete cure.

EBV-Directed T Cell Therapeutics for EBV-Associated Lymphomas.

Epstein Barr virus (EBV) is a human gamma herpes virus that establishes latency in B cells after primary infection. EBV generally only causes a mild, self-limiting viral illness but is also associated with several malignancies including posttransplantation lymphoproliferative disorder in the immunosuppressed host as well as Hodgkin and non-Hodgkin lymphoma in the immune competent host. The expression of EBV antigens by lymphoma has important applications as targets for adoptive T cell therapy. However, as many lymphomas only express subdominant EBV antigens that are less immunogenic, novel strategies are needed to manufacture EBV-specific T cell products specific for Latent Membrane Protein 1 (LMP1) and LMP2, which are expressed in lymphomas with type II and III latency. While several techniques for manufacturing EBV-CTLs are described in the literature, this chapter focuses on one method for generating Good Manufacturing Practice (GMP)-compliant EBV-specific T cell products that are enriched with LMP1 and LMP2.