Host Immune Mechanisms Research Articles

Interactions between host immune response and antigenic variation that control Borrelia burgdorferi population dynamics.

The population dynamics of pathogens within hosts result from interactions between host immune responses and mechanisms of the pathogen to evade or resist immune responses. Vertebrate hosts have evolved adaptive immune responses to eliminate the infection, while many pathogens evade immune clearance through altering surface antigens. Such interactions can result in a characteristic pattern of pathogen population dynamics within hosts consisting of population growth after infection, rapid population decline following specific immune responses, followed by persistence at low densities during a chronic infection stage. Despite the medical importance of chronic infections, little is known about the conditions of the interactions between variable antigens and the adaptive immune system that cause the characteristic pathogen population dynamics. Using the vls antigenic variation system of the Lyme disease pathogen, Borrelia burgdorferi, as a model system, we investigated conditions of the interaction between the antigenic variation system and the adaptive immune response that can explain the within-host population dynamics of B. burgdorferi using mathematical modelling. This characteristic population dynamic pattern can be explained by models that assume a variable immune removal rate of antibody-bound B. burgdorferi. However, models with a constant immune removal rate could reproduce the rapid population decline of B. burgdorferi populations but not their long-term persistence within hosts using parameter values determined by fitting empirical data. The model predictions, along with the assumptions about the interactions between B. burgdorferi and the immune response, can be tested experimentally to estimate the likelihood that each mechanism affects B. burgdorferi population dynamics in real infections.

CD8+ T cells provide immune protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection.

Scrub typhus, caused by a Gram-negative obligately intracellular coccobacillus, Orientia tsutsugamushi, is a long neglected but important tropical disease. Orientia tsutsugamushi causes illness in one million people each year, and 1 billion people are at risk. Without appropriate diagnosis and treatment, the disease can cause severe multiorgan failure with a case fatality rate of 7–15%. The current gaps in knowledge of immunity include the unknown mechanisms of host immunity to O. tsutsugamushi. Using an intravenous (i.v.) disseminated infection mouse model, we observed that more CD8+ T cells than CD4+ T cells were present in the spleen of infected mice at 12 dpi. We also determined that Treg cells and the proportion of T cells producing IL-10 were significantly increased from 6 dpi, which correlated with the onset of illness, body weight loss, and increased bacterial loads. We further studied CD8-/-, MHC I-/- and wild type control (WT) C57BL/6J mice to determine the importance of CD8+ T cells and MHC I molecules. After infection with an ordinarily sub-lethal dose of O. tsutsugamushi, all CD8-/- and MHC I-/- mice were moribund between 12 and 15 dpi, whereas all WT mice survived. Bacterial loads in the lung, kidney, liver and spleen of CD8-/- and MHC I-/- mice were significantly greater than those in WT mice. Interferon-γ (IFN-γ) and granzyme B mRNA levels in the liver of CD8-/- and MHC I-/- mice were significantly greater than in WT mice. In addition, more severe histopathologic lesions were observed in CD8-/- mice. Finally, adoptive transfer confirmed a major role of immune CD8+ T cells as well as a less effective contribution by immune CD8 T cell-depleted splenocytes in protection against O. tsutsugamushi infection. These studies demonstrated the critical importance of CD8+ T cells in the host immune response during O. tsutsugamushi infection.

Kunkel Lecture: Fundamental immunodeficiency and its correction.

"Fundamental immunodeficiency" is the inability of the encoded immune system to protect an otherwise healthy host from every infection that could threaten its life. In contrast to primary immunodeficiencies, fundamental immunodeficiency is not rare but nearly universal. It results not from variation in a given host gene but from the rate and extent of variation in the genes of other organisms. The remedy for fundamental immunodeficiency is "adopted immunity," not to be confused with adaptive or adoptive immunity. Adopted immunity arises from four critical societal contributions to the survival of the human species: sanitation, nutrition, vaccines, and antimicrobial agents. Immunologists have a great deal to contribute to the development of vaccines and antimicrobial agents, but they have focused chiefly on vaccines, and vaccinology is thriving. In contrast, the effect of antimicrobial agents in adopted immunity, although fundamental, is fragile and failing. Immunologists can aid the development of sorely needed antimicrobial agents, and the study of antimicrobial agents can help immunologists discover targets and mechanisms of host immunity.

Gut bacterial peptides with autoimmunity potential as environmental trigger for late onset complex diseases: In–silico study

Recent evidences suggest that human gut microbiota with major component as bacteria can induce immunity. It is also known that gut lining depletes with ageing and that there is increased risk of autoimmune and inflammatory disorders with ageing. It is therefore likely that both may be correlated as depletion of gut lining exposes the gut bacterial antigens to host immune mechanisms, which may induce immunity to certain bacterial proteins, but at the same time such immunity may also be auto-immunogenic to host. This autoimmunity may make a protein molecule nonfunctional and thereby may be involved in late onset metabolic, autoimmune and inflammatory disorders such as, Diabetes, Rheumatoid Arthritis, Hyperlipidemias and Cancer. In this in-silico study we found a large number of peptides identical between human and gut bacteria which were binding to HLA-II alleles, and hence, likely to be auto-immunogenic. Further we observed that such autoimmune candidates were enriched in bacterial species belonging to Firmicutes and Proteobacteria phyla, which lead us to conclude that these phyla may have higher disease impact in genetically predisposed individuals. Functional annotation of human proteins homologous to candidate gut-bacterial peptides showed significant enrichment in metabolic processes and pathways. Cognitive trait, Ageing, Alzheimer, Type 2 diabetes, Chronic Kidney Failure (CKF), Chronic Obstructive Pulmonary Disease (COPD) and various Cancers were the major diseases represented in the dataset. This dataset provides us with gut bacterial autoimmune candidates which can be studied for their clinical significance in late onset diseases.

A Critical Role of Zinc Importer AdcABC in Group A Streptococcus-Host Interactions During Infection and Its Implications for Vaccine Development

Bacterial pathogens must overcome host immune mechanisms to acquire micronutrients for successful replication and infection. Streptococcus pyogenes, also known as group A streptococcus (GAS), is a human pathogen that causes a variety of clinical manifestations, and disease prevention is hampered by lack of a human GAS vaccine. Herein, we report that the mammalian host recruits calprotectin (CP) to GAS infection sites and retards bacterial growth by zinc limitation. However, a GAS-encoded zinc importer and a nuanced zinc sensor aid bacterial defense against CP-mediated growth inhibition and contribute to GAS virulence. Immunization of mice with the extracellular component of the zinc importer confers protection against systemic GAS challenge. Together, we identified a key early stage host-GAS interaction and translated that knowledge into a novel vaccine strategy against GAS infection. Furthermore, we provided evidence that a similar struggle for zinc may occur during other streptococcal infections, which raises the possibility of a broad-spectrum prophylactic strategy against multiple streptococcal pathogens.

Suppressor Cell-Depleting Immunotherapy With Denileukin Diftitox is an Effective Host-Directed Therapy for Tuberculosis.

Host-directed therapies that augment host immune effector mechanisms may serve as important adjunctive therapies for tuberculosis treatment. We evaluated the activity of denileukin diftitox in an acute mouse model of tuberculosis (TB) infection and analyzed the cellular composition and bacterial burden in lungs and spleens. These in vivo studies show that denileukin diftitox potentiates standard TB treatment in the mouse model, an effect which may be due to depletion of T-regulatory and myeloid-derived suppressor cells during TB infection. Our results indicate that denileukin diftitox and other suppressor cell-depleting therapies may be useful adjunctive, host-directed therapies for TB.

Immune Recognition of the Epidemic Cystic Fibrosis Pathogen Burkholderia dolosa.

Burkholderia dolosa caused an outbreak in the cystic fibrosis (CF) clinic at Boston Children's Hospital from 1998 to 2005 and led to the infection of over 40 patients, many of whom died due to complications from infection by this organism. To assess whether B. dolosa significantly contributes to disease or is recognized by the host immune response, mice were infected with a sequenced outbreak B. dolosa strain, AU0158, and responses were compared to those to the well-studied CF pathogen Pseudomonas aeruginosa In parallel, mice were also infected with a polar flagellin mutant of B. dolosa to examine the role of flagella in B. dolosa lung colonization. The results showed a higher persistence in the host by B. dolosa strains, and yet, neutrophil recruitment and cytokine production were lower than those with P. aeruginosa The ability of host immune cells to recognize B. dolosa was then assessed, B. dolosa induced a robust cytokine response in cultured cells, and this effect was dependent on the flagella only when bacteria were dead. Together, these results suggest that B. dolosa can be recognized by host cells in vitro but may avoid or suppress the host immune response in vivo through unknown mechanisms. B. dolosa was then compared to other Burkholderia species and found to induce similar levels of cytokine production despite being internalized by macrophages more than Burkholderia cenocepacia strains. These data suggest that B. dolosa AU0158 may act differently with host cells and is recognized differently by immune systems than are other Burkholderia strains or species.

CD56bright NK IL-7Rα expression negatively associates with HCV level, and IL-7-induced NK function is impaired during HCV and HIV infections.

Several lines of evidence support the concept that NK cells play an important role in control of hepatitis C virus (HCV) infection via cytokine secretion and cytotoxicity. IL-7 is a homeostatic cytokine with a role in T cell development, activation, proliferation, and cytokine secretion. The IL-7Rα chain [cluster of differentiation (CD)127] is expressed on NK cells, with greatest abundance on the CD56brightCD16dim/- (CD56bright) subset. Here, we measured CD127 expression on CD56bright, CD56dimCD16+ (CD56dim), or CD56negCD16+ (CD56neg) NK cell subsets of 25 uninfected donors (UD); 34 chronic HCV-infected, treatment-naïve; 25 HIV-infected, virally suppressed on antiretroviral therapy (ART); and 42 HCV-HIV-coinfected subjects on ART. Interestingly, CD127 expression on CD56bright NK cells negatively correlated with HCV plasma levels in HCV monoinfection and HCV-HIV coinfection. IL-7 induced CD69 expression, as well as IFN-γ production, in CD56bright NK cells and also enhanced the IFN-α-induced CD69 expression on these cells. The latter was impaired in HIV infection. Furthermore, IL-7 induced B cell lymphoma 2 (BCL-2) expression and cell cycling of CD56bright NK cells, and this effect was impaired in HCV- and HIV-infected subjects. Whereas IL-7-stimulated CD56bright NK cell degranulation appeared intact in all cohorts, we observed impaired IL-7-activated NK cell cytolytic function in HCV- and HIV-infected subjects. Finally, IL-7-induced phosphorylation of STAT-5 (pSTAT-5) signaling was impaired in NK cells of subjects with chronic viral infection, and this was reversible upon 6 mo of viral suppression with IFN-free HCV therapy. These results implicate that IL-7-dependent NK cell activation and effector function may be other host immune surveillance mechanisms that are impaired in viral infections.

CCL2/Monocyte Chemoattractant Protein 1 and Parathyroid Hormone Action on Bone.

Chemokines are small molecules that play a crucial role as chemoattractants for several cell types, and their components are associated with host immune responses and repair mechanisms. Chemokines selectively recruit monocytes, neutrophils, and lymphocytes and induce chemotaxis through the activation of G protein-coupled receptors. Two well-described chemokine families (CXC and CC) are known to regulate the localization and trafficking of immune cells in cases of injury, infection, and tumors. Monocyte chemoattractant protein 1 (MCP-1/CCL2) is one of the important chemokines from the CC family that controls migration and infiltration of monocytes/macrophages during inflammation. CCL2 is profoundly expressed in osteoporotic bone and prostate cancer-induced bone resorption. CCL2 also regulates physiological bone remodeling in response to hormonal and mechanical stimuli. Parathyroid hormone (PTH) has multifaceted effects on bone, depending on the mode of administration. Intermittent PTH increases bone in vivo by increasing the number and activity of osteoblasts, whereas a continuous infusion of PTH decreases bone mass by stimulating a net increase in bone resorption. CCL2 is essential for both anabolic and catabolic effects of PTH. In this review, we will discuss the pharmacological role of PTH and involvement of CCL2 in the processes of PTH-mediated bone remodeling.

Host-Virus Interaction of ZIKA Virus in Modulating Disease Pathogenesis

The Zika virus (ZIKV) is a newly emerging pathogen that has resulted in a worldwide epidemic. It primarily spreads either through infected Aedes aegypti or Aedes albopictus mosquitos leading to severe neurological disorders such as microcephaly and Guillain-Barré syndrome in susceptible individuals. The mode of ZIKV entry into specific cell types such as: epidermal keratinocytes, fibroblasts, immature dendritic cells (iDCs), and stem-cell-derived human neural progenitors has been determined through its major surface envelope glycoprotein. It has been known that oligosaccharides that are covalently linked to viral envelope proteins are crucial in defining host-virus interactions. However, the role of sugars/glycans in exploiting host-immune mechanisms and aiding receptor-mediated virus entry is not well defined. Therefore, this review focuses on host-pathogen interactions to better understand ZIKV pathogenesis.

ASSESSMENT OF CYTOTOXIC AND IMMUNOMODULATORY PROPERTIES OF PIPER NIGRUM LINN. (WHITE PEPPER) SEED EXTRACT

Macrophages are tissue-based phagocytes that play the central role in initiating defence mechanism of host immunity. Macrophage expresses inducible nitric oxide synthase (iNOS) that inhibit pathogen replication by releasing a variety of effector molecules which includes nitric oxide (NO). In the present study ethanol extract of Piper nigrum Linn. (white pepper) seed was investigated for its cytotoxicity and in vitro immunomodulatory properties using cell proliferation and NO determination assay with J774a-1, macrophage cell line. The ethanol seed extract was found to exhibit toxicity at higher concentrations of 50µg/ml and 100µg/ml. The seed preparation was observed to enhance both, the proliferation of macrophage cell with higher percentage proliferation of 29.24 at 12.5µg/ml and the production of NO with significant stimulation level of 47.74% at 12.5µg/ml compared to the control. Since the results show the modulator effect of Piper nigrum Linn. seed on macrophage cells, it could be considered to possess immunomodulatory potential.

Human immunity against EBV-lessons from the clinic.

The mammalian immune system has evolved over many millennia to be best equipped to protect the host from pathogen infection. In many cases, host and pathogen have coevolved, each acquiring sophisticated ways of inducing or protecting from disease. Epstein-Barr virus (EBV) is a human herpes virus that infects >90% of individuals. Despite its ubiquity, infection by EBV is often subclinical; this invariably reflects the necessity of the virus to preserve its host, balanced with sophisticated host immune mechanisms that maintain viral latency. However, EBV infection can result in various, and often fatal, clinical sequelae, including fulminant infectious mononucleosis, hemophagocytic lymphohistiocytosis, lymphoproliferative disease, organomegaly, and/or malignancy. Such clinical outcomes are typically observed in immunosuppressed individuals, with the most extreme cases being Mendelian primary immunodeficiencies (PIDs). Although these conditions are rare, they have provided critical insight into the cellular, biochemical, and molecular requirements for robust and long-lasting immunity against EBV infection. Here, we review the virology of EBV, mechanisms underlying disease pathogenesis in PIDs, and developments in immune cell–mediated therapy to treat disorders associated with or induced by EBV infection.

Participation of TLR2 and TLR4 in Cytokines Production by Patients with Symptomatic and Asymptomatic Chronic Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a serious public health issue. Its evolution involves an acute stage, characterized by no specific symptoms, and the chronic stage during most individuals are asymptomatic, but about 30-40% of them become symptomatic presenting the cardiac or digestive disease. Host immune response mechanisms involved in symptomatic or asymptomatic chronic disease are not fully understood. The pro-inflammatory cytokines are crucial in host resistance. However, a fine control of this inflammatory process, by action of anti-inflammatory cytokines, is necessary to avoid tissue injury. This control was found to be responsible for no clinical manifestations in asymptomatic individuals. Toll-like receptors (TLRs) are extremely important in defining the cytokine profile released in response to a micro-organism. We found that patients with the cardiac form predominantly released the pro-inflammatory cytokines: IFN-γ, TNF-α and IL-17 with the involvement of both, TLR2 and TLR4. In contrast, patients with asymptomatic disease release predominantly the anti-inflammatory cytokines IL-10 and TGF-β, but also with TLR2 and TLR4 participation. The mechanisms by which stimulation of the same TLRs results in release of different pattern of cytokines, depending on the patients group that is being evaluated, are discussed.

Diverse types of dermatologic toxicities from immune checkpoint blockade therapy.

Immunomodulatory drugs that leverages host immune mechanisms to destroy tumor cells have been met with great promise in the treatment of cancer. Immunotherapy, targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death 1 (PD-1) receptor and its ligand (PD-L1) have shown tremendous improvements in the survival of patients with advanced solid tumors. However, the development of dermatologic toxicity (DT) is a consequence to immunotherapy. Review of published reports of the DT to immunotherapy revealed patients receiving anti-CTCLA-4 antibody or anti-PD-1/PD-L1 antibody often develop a DT of any type and grade. In this article, of the 3825 patients who were treated with anti-PD-1 and of 556 patients receiving anti-PD-L1, DT of any type and grade were reported in 1474 (∼39%) and 95 (∼17%) of patients, respectively. The emergence of specific types of DT to immunotherapy is beginning to be recognized can be categorized into four groups: (a) inflammatory, (b) immunobullous, (c) alteration of keratinocytes and (d) alteration of melanocytes. Lichenoid dermatitis and bullous pemphigoid appear to be DT more associated with anti-PD-1/PD-L1 antibody. The DT profile in patients receiving immunotherapy is diverse, and early recognition of specific types of DT that clinicians may encounter is critical for optimal patient care.

Expression Pattern of Immunosurveillance-Related Antigen Is a Critical Prognostic Factor of Adult T-Cell Leukemia/Lymphoma

Introduction: Human T-cell lymphotropic virus (HTLV-1) carriers are often found in coastal areas of the Kyushu-Okinawa region in Japan. Of the patients infected with the virus, only 2-5% will develop adult T-cell leukemia/lymphoma (ATLL). ATLL is classified into four clinical subtypes as follows: acute, lymphoma, chronic, and smoldering. Chronic-type ATLL has a better prognosis compared with acute and lymphoma types. However, in half of patients, chronic-type ATLL progresses to leukemia/lymphoma crisis. Abnormal expressions of CD58 and beta-2 microglobulin (b2M) were recently reported to be involved in the immune escape mechanism of tumor cells in diffuse large B-cell lymphoma (Pasqualucci et. al. Cancer Cell 2014). In our previous study, acute-type ATLL had significantly higher rates of CD58 gene deletion, which was associated with leukemia/lymphoma crisis in chronic-type ATLL (Yoshida et. al. Cancer Res 2014). However, the clinicopathological significance of HLA expression in ATLL patients remains controversial. In addition, we previously reported that the expression of programmed cell death ligand 1 (PD-L1), one of the regulatory factors of tumor immune responses, influenced disease prognosis in ATLL patients. Especially PD-L1 expression in stromal cells in tumor microenvironments is associated with better prognosis of ATLL (Miyoshi et al. Blood 2016). The present study evaluated the clinical relevance of HLA, b2M, and PD-L1 expressions, with particular emphasis on predicting overall survival of ATLL patients.Methods: We report the clinicopathological profiles of 123 patients diagnosed as having ATLL at Kurume University (Kurume, Japan) according to the 2008 World Health Organization classification system. Among these patients, 45 had the acute type and 52 had the lymphoma type, with a median age of 66 years (range, 13-90 years). We examined the immunohistological expressions of HLA and b2M proteins in all the 123 patients by using tissue microarray. The expression patterns and clinicopathological features were also examined statistically. All data were analyzed by using the STATA software version 12.0 (StataCorp, College Station, Texas).Results: In the immunohistochemical study, 91 (74%) of the 123 patients were positive for HLA, including 60 who were cell membrane positive (HLAm+), 31 who demonstrated cytoplasmic localization (HLAcy+), and 32 who were negative for HLA (HLA−). In addition, 89 (72%) of the 123 patients were positive for b2M, including 54 who were cell membrane positive (b2Mm+), 35 who demonstrated cytoplasmic localization (b2Mcy+), and 34 who were b2M negative (b2M−). The HLAm+b2Mm+ group, which showed membrane staining for both HLA and b2M, accounted for 48 cases (39%) in the present study (Table 1). In terms of clinical features, no significant differences were found between the HLAm+b2Mm+ group and the other groups. Immunophenotypical evaluation revealed significantly higher rates of CD30-positive lymphoma cells (P = 0.004) and PD-L1-positive stromal cells in microenvironments (PD-L1+stromal cells ≥50/HPF: miPD-L1high) in the HLAm+b2Mm+ patients (P = 0.008) than in the patients in other groups. Disease-specific survival (DSS) curves showed that the HLAm+b2Mm+ group had significantly better prognosis than the other groups (Figure 1-a). Especially the HLAm+b2Mm+ group with miPD-L1high had an excellent prognosis, with a 5-year survival rate of 77% (Figure 1-b). The multivariate analysis demonstrated that the membrane expressions of both HLA and b2M in lymphoma cells with increased number of PD-L1-positive stromal cells were an independent factor of good prognosis in ATLL (Table2).Conclusions: The results of our study demonstrated the following three points: 1) The expression patterns of HLA and b2M in the patients with ATLL varied. 2) The HLAm+b2Mm+ group showed significantly better prognosis than the other groups. Especially the HLAm+b2Mm+ group with miPD-L1high demonstrated the most favorable prognosis. These results suggest that the host immune surveillance mechanism for lymphoma cells in the HLAm+b2Mm+ group with mPD-L1high is still functioning. 3) The membrane expressions of HLA and b2M in ATLL cells are useful as a predictive marker of good prognosis and increase in the number of PD-L1-positive stromal cells in tumor microenvironments. [Display omitted] [Display omitted] [Display omitted] DisclosuresAsano:Chugai: Honoraria; Jannsen: Honoraria. Ohshima:Chugai: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau.

Rituximab-Induced CD20-Mediated Signals and Suppression of PI3K-AKT Pathway Cooperates to Inhibit B-Cell Lymphoma Growth By Down-Regulation of Myc.

Rituximab, anti-CD20 monoclonal antibody, shows anti-lymphoma effects by either activating host immune mechanism against lymphoma cells such as ADCC and CDC or suppressing signal(s) important for growth of B-cell lymphoma. Accumulated evidence shows de-regulated PI3K-AKT pathway is important therapeutic target in B-cell lymphoma. Although studies suggested suppression of PI3K-AKT pathway is possibly critical in rituximab-induced anti-lymphoma signals, details of molecular mechanisms covering CD20-mediated signals and PI3K-AKT pathway are still unknown.We analyzed signals in B-cell lymphoma cell line, SD07, which had bi-allelic deletions of both CD20(MS4A1) and PTEN gene. Briefly, SD07 was established from a patient with diffuse large B-cell lymphoma(DLBCL)who showed refractory CD20-negative relapse(Eur.J. Haematol. 2012).We transiently restored protein expression of either or both of CD20 and PTEN by electroporation of expression plasmid having cDNA of those genes in SD07. In this experiment, expression of both proteins were clearly detected by western blot until 72 hours after transfection. CD20 protein was also detectable on cell surface of SD07 cells by flow cytometric(FCM) analysis at comparable levels of those of Daudi cells after transfection on CD20 cDNA.Our previous study showed expression of both c-myc RNA and protein, as judged by FCM, was induced by transient transfection of CD20 cDNA in SD07 cells. Western blot analysis also showed significant increase of myc protein 24hrs after transfection (vector=13.7±0.2, CD20=14.5±0.4, P<0.05, arbitrary density unit) and the increase was not affected by the addition of rituximab(20µg/ml)in culture. In contrast transfection of PTEN cDNA produced significant decrease of myc protein (PTEN=11.2±0.1, P<0.01). Myc protein levels was comparable between cells transfected with CD20 plus PTEN and PTEN alone. However in the presence of rituximab, myc protein expression was markedly downregulated in SD07 cells at both 24 hours(vector=13.7±0.2,CD20+PTEN=8.3±0.1,P=0.0008)and 48hours(vector=12.4±0.3 vs CD20+PTEN=11.2±0.1,P=0.04).24 hours after transfection, de-phosphorylation and down-regulation of retinoblastoma(RB) protein expression was evident only in SD07 cells transfected with both CD20 and PTEN, but not with either alone.Rituximab also augmented down-regulation of RB protein in SD07 cells transfected with both CD20 and PTEN.After transfection, in vitro cell growth of SD07, as evaluated by MTT, was as follows;vector=4.6±0.3, CD20=4.8±0.3(P=NS), PTEN=4.1±0.2(P<0.05), CD20+PTEN=3.6±0.2(P<0.01), CD20+PTEN+rituximab=2.7±0.2(P<0.01),OD570nm, days3)72 hours after transfection, by FCM, SD07 transfected with both CD20 and PTEN showed significant increase of subG1 DNA content potion compared with cells transfected with either alone(vector=2.5±0.2%,CD20=5.3±0.2%,PTEN=8.8 ±0.5%,CD20+PTEN= 16.1±0.6, P<0.01). Furthermore, addition of rituximabsignificantly increase of cells in subG1 in SD07 with transfected with both CD20 and PTEN(18.6±0.5%,P<0.01), suggesting suppression of PI3K-AKT is important for mediateing rituximab-induced apoptotis.Accordance with proliferative effect of CD20 inSD07, transfection of CD20 alone produced significant increase of phosphorylation of AktSer473(vector=11.2±2.3, CD20=13.3±2.3,pAkt/total Akt,P<0.05). Co-transfection of PTEN with CD20 suppress the CD20-induced phosphorylation of Akt in SD07 cells. (CD20+PTEN=12.8±2.3,P=NS).In summary, although data is limited in a cell line, it clearly shows rituximab-induced CD20-mediated signals and suppression of PI3K-AKT pathway by tumor suppressor PTEN cooperatively inhibits growth and induce apoptosis of DLBCL cell line, SD07. Myc protein is critical molecules in this particular cooperative pathways. The present result partly agree studies which showed that inhibition of PI3K-AKT signaling and down-regulation of myc is important in PTEN-induced cytotoxicity in GCB(germinal center B cell)type DLBCL. In addition, we show here myc is not only for PTEN, but also involved in rituximab-induced anti-lymphoma effect. The present result provide a rationale for combination of rituximab and PI3K inhibitor in the treatment of DLBCL. In this context, myc is a candidate of the therapeutic target in rituximab-based immunochemotherapy for B cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Nitric oxide and cytokine production by glial cells exposed in vitro to neuropathogenic schistosome Trichobilharzia regenti.

BackgroundHelminth neuroinfections represent a serious health problem, but host immune mechanisms in the nervous tissue often remain undiscovered. This study aims at in vitro characterization of the response of murine astrocytes and microglia exposed to Trichobilharzia regenti which is a neuropathogenic schistosome migrating through the central nervous system of vertebrate hosts. Trichobilharzia regenti infects birds and mammals in which it may cause severe neuromotor impairment. This study was focused on astrocytes and microglia as these are immunocompetent cells of the nervous tissue and their activation was recently observed in T. regenti-infected mice.ResultsPrimary astrocytes and microglia were exposed to several stimulants of T. regenti origin. Living schistosomulum-like stages caused increased secretion of IL-6 in astrocyte cultures, but no changes in nitric oxide (NO) production were noticed. Nevertheless, elevated parasite mortality was observed in these cultures. Soluble fraction of the homogenate from schistosomulum-like stages stimulated NO production by both astrocytes and microglia, and IL-6 and TNF-α secretion in astrocyte cultures. Similarly, recombinant cathepsins B1.1 and B2 triggered IL-6 and TNF-α release in astrocyte and microglia cultures, and NO production in astrocyte cultures. Stimulants had no effect on production of anti-inflammatory cytokines IL-10 or TGF-β1.ConclusionsBoth astrocytes and microglia are capable of production of NO and proinflammatory cytokines IL-6 and TNF-α following in vitro exposure to various stimulants of T. regenti origin. Astrocytes might be involved in triggering the tissue inflammation in the early phase of T. regenti infection and are proposed to participate in destruction of migrating schistosomula. However, NO is not the major factor responsible for parasite damage. Both astrocytes and microglia can be responsible for the nervous tissue pathology and maintaining the ongoing inflammation since they are a source of NO and proinflammatory cytokines which are released after exposure to parasite antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1869-7) contains supplementary material, which is available to authorized users.

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions.

The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies.

Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model.

African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus, ASF virus (ASFV). There is no vaccine to prevent the disease and current control measures are limited to culling and restricting animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pretoriuskop/96/4 (Pret4) virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 days post infection (dpi) showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response (ASFV-specific antibody and interferon (IFN)-γ responses, or specific cytokine profiles) and protection against challenge. With the exception of ASFV-specific antibodies in survivors challenged at 21 and 28 dpi, no association between the parameters assessed and protection could be established. These results, encompassing data from 65 immunized swine, underscore the complexity of the system under study, suggesting that protection relies on the concurrence of different host immune mechanisms.

Molecular and functional characterization of caspase-8 from the big-belly seahorse (Hippocampus abdominalis)

Apoptosis is a physiological process that can also participate in host immune defense mechanisms, including tumor growth suppression along with homeostasis and maturation of immune cells. Caspases are known to be involved in cellular apoptotic signaling; among them, caspase-8 plays an important role in the initiation phase of the apoptotic death cascade. In the current study, we molecularly characterized a caspase-8 homolog (designated as HaCasp-8) from Hippocampus abdominalis. The HaCasp-8 gene harbors a 1476 bp open reading frame (ORF) that codes for a protein of 492 amino acids (aa) with a predicted molecular mass of 55 kDa. HaCasp-8 houses the typical domain architecture of known initiator caspases, including the death effector domain and the carboxyl-terminal catalytic domain. As expected, phylogenetic analysis reflected a closer evolutionary relationship of HaCasp-8 with its teleostean similitudes. The results of our qPCR assays confirmed the ubiquitous expression of HaCasp-8 in physiologically important tissues examined, with pronounced expression levels in ovary tissues, followed by blood cells. HaCasp-8 expression at the mRNA level was found to be significantly modulated by lipopolysaccharide, polyinosinic:polycytidylic acid, Streptococcus iniae, and Edwardsiella tarda injection. Overexpression of HaCasp-8 could trigger a significant level of cell death in HEK293T cells, suggesting its putative role in cell death. Taken together, our findings suggest that HaCasp-8 is an important component in the caspase cascade, and its expression can be significantly modulated under pathogen stress conditions in the big-belly seahorse.