Significant Proliferative Response Research Articles

A Novel Dehydrated Human Umbilical Cord Particulate Medical Device: Matrix Characterization, Performance, and Biocompatibility for the Management of Acute and Chronic Wounds.

Chronic wounds present a significant socioeconomic burden forecasted to increase in prevalence and cost. Minimally manipulated human placental tissues have been increasingly employed and proven to be advantageous in the treatment of chronic wounds, showing improved clinical outcomes and cost-effectiveness. However, technological advances have been constrained by minimal manipulation and homologous use criteria. This study focuses on the characterization of a novel dehydrated human umbilical cord particulate (dHUCP) medical device, which offers a unique allogeneic technological advancement and the first human birth tissue device for wound management. Characterization analyses illustrated a complex extracellular matrix composition conserved in the dHUCP device compared to native umbilical cord, with abundant collagens and glycosaminoglycans imbibing an intricate porous scaffold. Dermal fibroblasts readily attached to the intact scaffold of the dHUCP device. Furthermore, the dHUCP device elicited a significant paracrine proliferative response in dermal fibroblasts, in contrast to fibrillar collagen, a prevalent wound device. Biocompatibility testing in a porcine full-thickness wound model showed resorption of the dHUCP device and normal granulation tissue maturation during healing. The dHUCP device is a promising advancement in wound management biomaterials, offering a unique combination of structural complexity adept for challenging wound topographies and a microenvironment supportive of tissue regeneration.

Recombinant antigen 5 from Polybia paulista wasp venom (Hymenoptera, Vespidae): Antigen-specific antibody production and functional profile of CD4+ T cells in the immune response

Polybia paulista is a neotropical social wasp related to severe accidents and allergic reactions cases, including anaphylaxis, in southeastern Brazil. Antigen 5 (Poly p 5) is a major allergenic protein from its venom with potential use for component-resolved diagnostic. Therefore, the previous characterization of the immune response profile triggered by Poly p 5 should be evaluated. Recombinant Poly p 5 (rPoly p 5) was used to sensitize BALB/c mice with six weekly intradermal doses, and the specific antibody production and the functional profile of CD4+ T cells were assessed. rPoly p 5 induced the production of specific immunoglobulins (sIg) sIgE, sIgG1 and sIgG2a, which could recognize natural Poly p 5 presented in the venom of four different wasp species. rPoly p 5 stimulated in vitro the CD4+ T cells from immunized mice, which showed a significant proliferative response. These antigen-specific CD4+T cells produced IFN-γ and IL-17A cytokines and increased ROR-γT transcription factor expression. No differences between the control group and sensitized mice were found in IL-4 production and GATA-3 and T-bet expression. Interestingly, increased CD25+FoxP3+ regulatory T cells (Tregs) frequency was observed in the splenocyte cell cultures from rPoly p 5 immunized mice after the in vitro stimulation with both P. paulista venom extract and rPoly p 5. Here we showed that rPoly p 5 induces antigen-specific antibodies capable of recognizing Antigen 5 in the venom of four wasp species and modulates antigen-specific CD4+ T cells to IFN-γ production response associated with a Th17 profile in sensitized mice. These findings emphasize the potential use of rPoly p 5 as an essential source of a major wasp allergen with significant immunological properties.

The effect of immunosuppression or thymectomy on the response to tetanus revaccination in myasthenia gravis

ObjectiveTo determine the effect of tetanus toxoid (TT) revaccination on circulating B-, T- and NK-cell compartments in myasthenia gravis (MG) patients. MethodsLymphocyte (sub)populations and differentiation stages were assessed by flow cytometry in 50 TT revaccinated MG patients. TT-specific proliferative responses were explored in PBMC cultures. ResultsIn patients treated with azathioprine B- and NK cell numbers were strongly decreased. Lymphocyte (sub)populations remained unaffected upon TT revaccination. t All patients showed a significant TT-induced proliferative response. ConclusionTT revaccination is effective in MG patients with stable disease irrespective of their thymectomy status and medication and does not alter the composition of the lymphocyte compartment.

755 Rat engineered heart tissue is a novel in vitro model to evaluate cardiomyocyte proliferation and fibroblast activation after injury

Abstract Aims Adult mammals, including humans, fail to regenerate the majority of the lost cardiomyocytes (CMs) that are replaced with scar tissue after injury. This lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs 7 days after birth. An in vitro model that recapitulates these changes has not been developed yet. Using rat engineered heart tissues (rEHTs) we have developed a custom-made cryoinjury system to test the hypothesis that maturation of CMs in EHTs regulates the proliferative response of CMs after injury. Methods rEHT were generated using neonatal rat heart cells. A discrete lesion was produced on the mid-section of mature (Day 18) and immature (Day 6) EHTs using a custom-made system based on liquid nitrogen and a 23G needle and medium was supplemented with EdU for 48 h. Results Cryoinjury in mature EHTs produces a localized injury, preserving their residual contractile activity that does not recover over time. We observed a significant increase of EdU+CMs post injury (6.3 ± 1.9% vs. 10.1 ± 1.6%) without significant changes in Ki67+ and pH3+ CMs suggesting that cryoinjury in mature rEHTs induces DNA synthesis but not CM proliferation. Injury in mature EHTs induced also significant proliferation and activation of fibroblasts with collagen deposition. Interestingly, cryoinjury performed in immature EHTs stimulated a significant proliferative response in CMs Conclusions Similar to adult rodents, we show that cryoinjury induces DNA synthesis in CMs without proliferative response and contractile recovery. On the other hand, cryoinjury in immature EHTs leads to CMs proliferation. Moreover, mature EHT fibroblast response to injury retraces the activation progression of cardiac fibroblast after infarction characterized by proliferation, increase of activation markers, increase of collagen deposition suggesting EHTs as a novel model to investigate the biology of cardiac regeneration upon injury.

Abstract 11992: Rat Engineered Heart Tissue is a Novel in vitro Model to Evaluate Cardiomyocyte Proliferation and Fibroblast Activation After Injury

Introduction: Adult mammals, including humans, fail to regenerate the majority of the lost cardiomyocytes (CMs) that are replaced with scar tissue after injury. This lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs 7 days after birth. An in vitro model that recapitulates these changes has not been developed yet. Hypothesis: Using rat engineered heart tissues (rEHT) we have developed a custom-made cryoinjury system to test the hypothesis that maturation of CMs in EHTs regulates the proliferative response of CMs after injury. Methods: rEHT were generated using neonatal rat heart cells. A discrete lesion was produced on the mid-section of mature (day 18) and immature (day 6) EHTs using a custom-made system based on liquid nitrogen and a 23G needle and medium was supplemented with EdU for 48h. Results: Cryoinjury in mature EHTs produces a localized injury, preserving their residual contractile activity that does not recover over time. We observed a significant increase of EdU + CMs post injury (6.3 ± 1.9% vs. 10.1 ± 1.6 %) without significant changes in Ki67 + and pH3 + CMs suggesting that cryoinjury in mature rEHTs induces DNA synthesis but not CM proliferation. Injury in mature EHTs induced also significant proliferation and activation of fibroblasts with collagen deposition. Interestingly, cryoinjury performed in immature EHTs stimulated a significant proliferative response in CMs. Discussion: Similar to adult rodents, we show that cryoinjury induces DNA synthesis in CMs without proliferative response and contractile recovery. On the other hand, cryoinjury in immature EHTs leads to CMs proliferation. Moreover, mature EHT fibroblast response to injury retraces the activation progression of cardiac fibroblast after infarction characterized by proliferation, increase of activation markers, increase of collagen deposition suggesting EHTs as a novel model to investigate the biology of cardiac regeneration upon injury.

Zebrafish Blunt-Force TBI Induces Heterogenous Injury Pathologies That Mimic Human TBI and Responds with Sonic Hedgehog-Dependent Cell Proliferation across the Neuroaxis.

Blunt-force traumatic brain injury (TBI) affects an increasing number of people worldwide as the range of injury severity and heterogeneity of injury pathologies have been recognized. Most current damage models utilize non-regenerative organisms, less common TBI mechanisms (penetrating, chemical, blast), and are limited in scalability of injury severity. We describe a scalable blunt-force TBI model that exhibits a wide range of human clinical pathologies and allows for the study of both injury pathology/progression and mechanisms of regenerative recovery. We modified the Marmarou weight drop model for adult zebrafish, which delivers a scalable injury spanning mild, moderate, and severe phenotypes. Following injury, zebrafish display a wide range of severity-dependent, injury-induced pathologies, including seizures, blood–brain barrier disruption, neuroinflammation, edema, vascular injury, decreased recovery rate, neuronal cell death, sensorimotor difficulties, and cognitive deficits. Injury-induced pathologies rapidly dissipate 4–7 days post-injury as robust cell proliferation is observed across the neuroaxis. In the cerebellum, proliferating nestin:GFP-positive cells originated from the cerebellar crest by 60 h post-injury, which then infiltrated into the granule cell layer and differentiated into neurons. Shh pathway genes increased in expression shortly following injury. Injection of the Shh agonist purmorphamine in undamaged fish induced a significant proliferative response, while the proliferative response was inhibited in injured fish treated with cyclopamine, a Shh antagonist. Collectively, these data demonstrate that a scalable blunt-force TBI to adult zebrafish results in many pathologies similar to human TBI, followed by recovery, and neuronal regeneration in a Shh-dependent manner.

Human Desmocollin 3‒Specific IgG Antibodies Are Pathogenic in a Humanized HLA Class II Transgenic Mouse Model of Pemphigus

Pemphigus is a potentially lethal autoimmune bullous skin disorder, which is associated with IgG autoantibodies against desmoglein (DSG) 3 and DSG1. Notably, a subset of patients with pemphigus presents with a similar clinical phenotype in the absence of anti-DSG IgG, suggesting the presence of serum IgG reactive with desmosomal components other than DSG1 or DSG3. We and others have previously shown that such patients have serum IgG autoantibodies against desmocollin 3 (DSC3), a component of desmosomes, which induce loss of keratinocyte adhesion exvivo. Moreover, DSC3 hypomorphic mice show a severe blistering phenotype of the mucous membrane, which is highly characteristic of pemphigus. These findings prompted us to study the induction and regulation of anti-human DSC3 IgG in humanized mice transgenic for HLA-DRB1∗04:02, which is a highly prevalent haplotype in pemphigus. We show that IgG from sera of immunized mice induces acantholysis in a dispase-based keratinocyte dissociation assay through the activation of p38 MAPKs and EGFR. Passive IgG transfer from mice immunized with recombinant human DSC3 into neonates did not induce intraepidermal loss of adhesion presumably owing to the lack of homology between human and mouse DSC3. Exvivo stimulation of splenocytes from DSC3-immunized mice with human DSC3 leads to a significant proliferative IFN-γ and IL-4 T-cell response, which is restricted by HLA-DR/HLA-DQ. These findings suggest that the induction of pathogenic anti-DSC3 IgG is associated with DSC3-specific T cells that recognize DSC3 in association with HLA-DRB1∗04:02.

Antigen-specific response of CD4+ T cells and hepatic lymph node cells to Fasciola hepatica-derived molecules at the early and late stage of the infection in sheep

The immunomodulatory capacity of F. hepatica antigens is probably one of the main reasons for the development of a driven non-protective Th2 immune response. In this study, we analysed the cellular response of hepatic lymph node cells and CD4+ T cells in terms of proliferative response, efficiency of antigen presentation and cytokine production, to F. hepatica-derived molecules, at early and late stages of the infection. Thirty-one sheep were allocated into five groups and were slaughtered at 16 dpi and 23 wpi. In order to analyse antigen-specific response, the following F. hepatica recombinant molecules were used: rFhCL1, rFhCL2, rFhCL3, rFhCB1, rFhCB2, rFhCB3, rFhStf-1, rFhStf-2, rFhStf-3 and rFhKT1. A cell proliferation assay using hepatic lymph node cells and an antigen presentation cell assay using CD4+ T cells were performed. At 16 dpi, all molecules but rFhStf-2 and rFhKT1 elicited a significant cell proliferative response on hepatic lymph node cells of infected animals. At both early and late stage of the infection, antigen presentation of rFhCB3 and rFhCL2 resulted in higher stimulation index of CD4+ T cells which was IL-2 mediated, although no statistically significant when compared to uninfected animals. Significant cytokine production (IL-4, IL-10 and IFN-γ) was conditioned by the antigen-specific cell stimulation. No CD4+ T cell exhaustion was detected in infected sheep at the chronic stage of the infection. This study addressed antigen-specific response to F. hepatica-derived molecules that are involved in key aspects of the parasite survival within the host.

Abstract 1787: YH29407, a novel IDO1 inhibitor, enhances the anti-tumor effects through increased tumor-reactive T cell functions in solid tumor

Abstract Background: The indoleamine 2,3-dioxygenase (IDO), is an enzyme responsible for catabolizing tryptophan (Trp) to kynurenine (Kyn). The depletion of tryptophan and the increase in kynurenine exert important immunosuppressive functions by activating T regulatory cells and myeloid-derived suppressor cells. Therefore, overexpression of IDO is associated with poor clinical outcome. In this study, we evaluated pharmacokinetic and pharmacodynamic profiles and anti-cancer immune responses of YH29407, a novel IDO1 inhibitor. Methods: MC38 murine tumor models were established for pharmacokinetic and pharmacodynamic profiling. The kynurenine levels in plasma and tumor of YH29407, epacadostat and BMS-986205 following 3 day treatment were detected. The antitumor effects of YH29407 100 mg/kg QD/BID, epacadostat 100 mg/kg BID and BMS-986205 100 mg/kg QD for 10 day administration were evaluated in MC38 models. Various immunologic profiles were evaluated after 5 day treatment of individual inhibitors. Results: The AUC of kynurenine in plasma was decreased by 81.4% with YH29407 100 mg/kg BID, 62.7% with epacadostat 100 mg/kg BID, and 19.8% with BMS-986205 100 mg/kg QD treatment, respectively, compared to control group. The intratumoral concentration of kynurenine was also reduced by 99.4%, 89.9% and 1.1% with 3 day treatment of YH29407 100 mg/kg BID, epacadostat 100 mg/kg BID, and BMS-986205 100 mg/kg QD treatment, respectively, compared to control group. The intratumoral concentration of YH29407 BID, epacadostat BID and BMS-986205 QD at 24 hours after 3 day treatment was 9.697 µg/g, 5.478 µg/g and 0.025 µg/g, respectively. These results indicate favorable pharmacokinetic property of YH29407. Compared to epacadostat and BMS-986205, YH29407 100 mg/kg BID treatment exhibited significant proliferative response of splenocytes (P <0.05). IFN-γ secretion levels were also significantly increased by YH29407 treatment, compared to epacadostat and BMS-986205 treatment (P <0.05). Profiling of serum cytokine levels revealed significant increase of Granzyme B, IFN-γ, IL-12, and MMP2 and decrease of FASL, compared to the vehicle group. Upregulation of IFN-γ in CD3+ tumor-infiltrating lymphocytes was significantly induced in the YH29407 treated group compared to epacadostat and BMS-986205 treated groups (P <0.05). Treatment with YH29407 100 mg/kg BID induced significant tumor growth inhibition, compared to epacadostat 100 mg/kg BID and BMS-986205 100 mg/kg QD. Conclusion: The novel IDO1 inhibitor, YH29407, with improved pharmacokinetic and pharmacodynamic profiles, showed antitumor inflammatory reprogramming leading to enhanced antitumor efficacy. Citation Format: Kyoung-Ho Pyo, Ho-Woong Kang, Sun Min Lim, Do Hee Kim, Dong Kwon Kim, Gyu-Jin Lee, Jong-Suk Park, Se-Woong Oh, Byoung Chul Cho. YH29407, a novel IDO1 inhibitor, enhances the anti-tumor effects through increased tumor-reactive T cell functions in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1787.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

A longitudinal analysis of TCRβ repertoire diversity in immune tolerance of pediatric patients post-UCBT: T cell clonal deletion over clonal anergy

Background & Aim The mechanisms of long-term immune tolerance after HLA mismatched unrelated cord blood transplant (UCBT) is not understood. Both central (clonal deletion) and peripheral mechanisms (anergy, Treg, or Tr1 mediated suppression) are suspected. In this study, we set up serial experiments to test for each of these mechanisms in the GvH direction. Next-generation TCR sequencing was applied for tracking and quantitating alloreactive T cell clones within the TCRVβ repertoire. Methods, Results & Conclusion Methods: 12 patients (median age 1y/o) with non-malignant diseases were enrolled on a reduced-intensity conditioning trial (NCT01852370) with Alemtuzumab 2 weeks pre-UCBT. Patients were tolerant if completed immunocompetent, off immunosuppression for months without GvHD or rejection. T cell responses to host antigen-presenting cells (APC) were measured utilizing EBV transformed B cells in seronegative children by mixed lymphocyte reaction (MLR) and cytotoxic T lymphocyte (CTL) reaction, along with cytokine profiling and ImmunoSEQ® monitoring the fate of host-reactive T cell clones. To identify the key mechanisms of tolerance, we depleted Tregs with Denileukin diftitox prior to MLR and CTL. The involvement of Tr1 or anergy was examined either by blocking IL10R or by adding low dose IL2, respectively. Result and discussion In tolerant patients, there was no significant proliferative or CTL response towards host APC by donor-derived T cells while they responded vigorously to 3rd party APC (Fig1A). Similarly, cytokine measurements by Bioplex revealed host-specific non-responsiveness (Fig1B) similar to anti-self-responses of healthy volunteers. TCRβ clonotype tracking with ImmunoSEQ® revealed the disappearance of host-specific clones identifiable from an aliquot of the infused CB graft (pre-UCBT) (Fig1C). These alloreactive T cell clones became undetectable even in the presence of exogenous IL2 (Fig1D). There was no indication of Treg or Tr1 involvement in sustaining tolerance since neither deletion of Tregs, nor IL-10R blockade could reverse T cell hypo-reactivity in MLR, CTL, and Bioplex assays. In summary, the rapid acquisition of immune tolerance in the GvH direction post-UCBT is characterized by hypo-reactivity towards host APC in proliferation, cytotoxicity, cytokine secretion. While a small degree of anergy may also contribute, ImmunoSEQ® data confirmed that clonal deletion is a dominant mechanism towards long term immune tolerance.

Expression of Breast Cancer-Related Epitopes Targeting the IGF-1 Receptor in Chimeric Human Parvovirus B19 Virus-Like Particles.

Breast cancer is a worldwide health problem, and the complexity of the disease, as well as the lack of treatment specificity, generates an urgent need for developing prophylactic and therapeutic measures. Searching for novel epitope-based approaches able to induce tumour immunity, we designed virus-like particles (VLPs) derived from Human parvovirus B19 assembled of chimeric VP2 proteins displaying two epitopes from the insulin-like growth factor-1 receptor (IGF-1R). Here, we present the generation of two chimeric VP2s that retain the stability, solubility and conditions of purification and assembly of the native VP2. We generated versatile chimeric multiepitope anti-cancer vaccine candidates, which prevented and delayed tumour growth when used in a prophylactic scheme of 4 weekly immunizations prior to 4T1 cell inoculation in female BALB/c mice. The presence of specific antibodies against the displayed epitopes suggests their participation in the protective effect; in contrast, no significant proliferative T-cell responses were recorded following stimulation by specific epitopes. The results comprise an approach whereby fusing desired epitopes from cancer to the N-terminus of B19 VP2 protein can generate a library of chimeric VP2-desired epitopes for further assembly in a designed and personalized epitope delivery system.

Reconstitution of T-Cell Subsets Following Thymoglobulin-Induced Depletion in High Immunologic Risk and Donation After Cardiac Death Renal Transplant Recipients

IntroductionDepletion therapy in high immunologic risk (HR) patients by antithymocyte globulin (rATG) induces lymphopenia and subsequent compartmental repopulation of T-cell subsets. rATG is also given to patients receiving kidneys from donations after cardiac death (DCDs) to mitigate innate immune activation associated with the DCD process. MethodsWe compared the T-cell response with rATG in both HR and DCD kidney recipients. We examined the reconstitution of T-cell subsets after rATG treatment in HR and DCD recipients (n = 19 per group) by multicolor flow cytometry. ResultsFollowing treatment, there was a rapid drop in the frequency of T cells in both groups, which persisted over 28 days. HR patients had an early surge in the frequency of CD4+ naïve, effector-memory, and regulatory T cells. Although we found a significant proliferation of the T cells in both groups, the DCD cohort had a blunted response as well as reduced CD4+ T-cell immune-reactivity compare with the HR group. ConclusionsOur data suggest that there is a lack of significant homeostatic proliferative response in DCD recipients following rATG, and CD4+ T cells may be less reactive in the DCD group than previously thought, indicating that rATG treatment may not have to be considered a first-line induction therapy in DCD recipients.

Adjuvanticity of aqueous extracts of Artemisia rupestris L. for inactivated foot-and-mouth disease vaccine in mice

Several methods have been developed to improve the efficacy of foot-and-mouth disease virus (FMDV) vaccine. The study aims to determine whether aqueous extracts of Artemisia rupestris L. (AEAR) as an immunoactivator in combination with inactivated FMDV vaccine can promote immune responses in mice. Intramuscular co-immunization in ICR mice with different doses of AEAR plus FMDV vaccine could substantially improve the FMDV-specific antibody production (IgG, IgG1, and IgG2a) and lead to significant lymphocyte proliferative responses. Th1-type immune responses were also observed, including proliferative responses of CD8+, CD4+, CD4+CD44+, and CD8+CD44+ T cells and the killing efficacy of cytotoxic T lymphocyte (CTL) responses. AEAR also elicited the higher levels of IL-4 and IFN-γ in CD4+ T cells as well as the higher level of IFN-γ in CD8+ T cells. The medium dose of AEAR induced the significant adjuvant activity. Further tests in mice indicated that AEAR could activate DCs maturation by increasing the expression levels of co-stimulatory molecules (CD40, CD86, CD80, and MHC-II) on dendritic cells (DCs) from splenocytes and reduce the activity of regulatory T cells (Treg). Abnormal behaviors, side effects or death were not observed in immunized mice. AEAR could boost humoral and cell-mediated immunity elicited by FMDV vaccine, especially Th1-type immune responses.

Human Remyelination Promoting Antibody Stimulates Astrocytes Proliferation Through Modulation of the Sphingolipid Rheostat in Primary Rat Mixed Glial Cultures.

Remyelination promoting human IgMs effectively increase the number of myelinated axons in animal models of multiple sclerosis. Hence, they ultimately stimulate myelin production by oligodendrocytes (OLs); however, their exact mechanism of action remains to be elucidated, and in particular, it remains unclear whether they are directly targeting OLs, or their action is mediated by effects on other cell types. We assessed the effect of remyelination promoting antibody rHIgM22 on the proliferative response and on the ceramide/sphingosine 1-phosphate rheostat in mixed glial cell cultures (MGCs). rHIgM22 treatment caused a time-dependent increase in PDGFαR protein in MGCs. Forty-eight hours of treatment with rHIgM22 induced a dose-dependent proliferative response (evaluated as total cell number and as EdU(+) cell number) in MGCs. When the proliferation response of MGCs to rHIgM22 was analyzed as a function of the cell types, the most significant proliferative response was associated with GLAST(+) cells, i.e., astrocytes. In many cell types, the balance between different sphingolipid mediators (the "sphingolipid rheostat"), in particular ceramide and sphingosine 1-phosphate, is critical in determining the cell fate. rHIgM22 treatment in MGCs induced a moderate but significant inhibition of total acidic sphingomyelinase activity (measured in vitro on cell lysates), the main enzyme responsible for the stimulus-mediated production of ceramide, when treatment was performed in serum containing medium, but no significant differences were observed when antibody treatment was performed in the absence of serum. Moreover, rHIgM22 treatment, either in the presence or in absence of serum, had no effects on ceramide levels. On the other hand, rHIgM22 treatment for 24h induced increased production and release of sphingosine 1-phosphate in the extracellular milieu of MGC. Release of sphingosine 1-phosphate upon rHIgM22 treatment was strongly reduced by a selective inhibitor of PDGFαR. Increased sphingosine 1-phosphate production does not seem to be mediated by regulation of the biosynthetic enzymes, sphingosine kinase 1 and 2, since protein levels of these enzymes and phosphorylation of sphingosine kinase 1 were unchanged upon rHIgM22 treatment. Instead, we observed a significant reduction in the levels of sphingosine 1-phosphate lyase 1, one of the key catabolic enzymes. Remarkably, rHIgM22 treatment under the same experimental conditions did not induce changes in the production and/or release of sphingosine 1-phosphate in pure astrocyte cultures. Taken together, these data suggest that rHIgM22 indirectly influences the proliferation of astrocytes in MGCs, by affecting the ceramide/sphingosine 1-phosphate balance. The specific cell population directly targeted by rHIgM22 remains to be identified, however our study unveils another aspect of the complexity of rHIgM22-induced remyelinating effect.

Immunomodulatory effect of anise (<i>Pimpinella anisum</i>) in BALB/c mice

Purpose: The aim of this work was to investigate the effect of anise decoction consumption on lymphocytes activity, complete blood count (CBC) and nitric oxide (NO) production in BALB/c mice.Methods: BALB/c mice were given anise tea instead of drinking water and the effect on selected immune parameters was analyzed after 1 and 2 weeks of treatment.Results: Cell activity of anise treated mice was significantly higher than control group at week 2 as revealed by mixed lymphocyte reaction (MLR2. The spleen cells of anise treated mice showed a significant higher lymphocyte proliferative response to in vitro challenge with phytohaemagglutinin (PHA).compared to controls at week two of treatment. The increase in mouse foot thickness as indicator of delayed type of hypersensitivity (DTH) was less in anise treated mice compared to control group. Nitric oxide production by peritoneal macrophages in response to activation with lipopolysaccharides (LPS) was reduced by anise treatment after 1 and 2 weeks of treatment andno significant changes in CD4 and CD8 were noticed either at week 1 or 2 of treatment.Conclusion: This study provides preclinical evidence that anise possesses immunomodulatory activity when administered orally in mice and selectively activates cell-mediated immune mechanismsKeywords: Immunomodulatory, Anise, Traditional medicine

Construction of pLLO vector encoding truncated form of Listeriolysin O as molecular adjuvant for DNA vaccine studies

Background: The major problem of DNA vaccine is less immunogenicity of them verses other killed or live whole organism vaccines therefore adjuvants for use in this kind vaccines is very necessary. Genetic adjuvants with bacterial sources are an appropriate approach to modulate immune responses to DNA vaccines. Listeria Monocytogenes proteins such as Listeriolysin O (LLO) with CD4 and CD8 epitopes can be as an adjuvant to initiate both innate and adaptive immune responses if the protein cytotoxicity can be eliminated. Herein we constructed a truncated LLO plasmid as genetic adjuvant and tested it in combination with a DNA construct as a model vaccine.Materials and Methods: About 1340bp of the 5' end of whole LLO gene was amplified by PCR on DNA purified from Listeria Monocytogenes. Sequential sub cloning of truncated LLO into the Xho I/EcoRV sites of pcDNA3.1 plasmid, downstream of CMV promoter was done. pLLO plasmid was transfected to HEK293T cell line by lipofection method. LLO protein expression from transiently transfected 293T cell lysates was confirmed by western blotting. Then the adjuvant activity of LLO in BALB/c mice model was analyzed using proliferation test.Results: Double digestion of pLLO plasmid with the enzymes that were applied for cloning led to the isolation of two fragments with expected sizes. The final plasmid was also confirmed following sequencing reactions. Moreover, expression of LLO was evidenced in transfected 293T cells, compared to non-transfected controls. In vivo study was shown, high significant proliferative responses in LLO co-immunization pattern.Conclusion: In the DNA vaccine study, LLO co-administration plasmid could be a suitable genetic adjuvant to enhance cellular immune response of vaccine.

Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides.

Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine.

Local induction of IgT responses to pathogens and microbiota in the gill of rainbow trout

Gas exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin (Ig) responses against pathogens in specialized respiratory organs have only been described in tetrapods. We have previously shown that IgT is an Ig specialized in gut and skin mucosal immunity. Thus, we tested the hypothesis that IgT might play a pivotal role in mucosal immunity of teleost gills. In this study, we provide the first structural and functional characterization of all teleost Igs, including secreted IgD, IgM and IgT at a mucosal surface of a teleost fish. Here we show that IgT B cells represent the major B cell subset in the gill filaments. In contrast to reported results by others, we found that all gill B cells expressing surface IgM, also expressed surface IgD and that the percentage of B cells solely expressing either IgD or IgM was negligible. We found that the majority of bacterial microbiota in the gill mucosa is coated with IgT and, to a much lesser degree with IgM and IgD. More crucially, significant specific-IgT immune responses against Ichthyophthirius multifiliis (Ich) and Flavobacterium columnare were measured in the gill mucus, while pathogen-specific IgM responses were almost exclusively detected in the serum. Pathogen-specific IgD titers were absent both in gill mucus and serum. Importantly, we found significant IgT B-cell proliferative responses in the gill but not in the spleen or head kidney of fish that survived Ich infection. Moreover we also found that Ichand F. columnare-specific IgT titers were locally produced by gill explants of survivor fish while they were absent in the spleen or head kidney explants of the same animals. In addition to showing that IgT is the main Ig player in gill mucosal immunity, the observed generation of local IgT B cell proliferative and pathogen-specific IgT responses in the gills provides the first demonstration of locally induced B cell and secretory Ig responses in the mucosa of a teleost. Moreover, this represents the first study in which a bacterial pathogen is shown to induce dominant IgT responses in a fish mucosal site, thus strongly suggesting that IgT is induced by a variety of pathogens in addition to parasites. Our findings also have special relevance from an applied perspective as they may lead to the development of fish vaccines and immunostimulants that have the capacity to induce gill IgT mucosal immune responses.

Simultaneous kinetic analysis of resting and proliferative B and T lymphocytes following pathogenic challenge in rainbow trout (Oncorhynchus mykiss)

Very little is known with regards to the mechanisms involved in the induction of adaptive Band Tcell responses in teleost systemic and mucosal lymphoid organs. This knowledge in fish species has been held back for decades due to the absence of reliable monoclonal antibodies recognizing different fish leukocytes subpopulations. In the past we have produced mAbs to rainbow trout IgT and, more recently to rainbow trout CD4, which have enabled the characterization of IgT B cell and CD4 T cell subsets respectively. The potential of these antibodies is shown here by a newly developed multicolor flow cytometry strategy that allows the concomitant detection of IgT B cells, IgM B cells, CD41/CD4-2 T cells, CD4-2 T cells and CD4 monocytes/macrophages in both systemic and mucosal lymphoid organs. This multicolor assay was thereafter used as a fast and efficient method to analyze the kinetics of resting and proliferative B and T cells over a two week period, upon challenge of fish with a sublethal dose of Yersinia ruckeri. Overall, our results identified the spleen as the major site for CD4 T cell proliferation with significant proliferative responses occurring already 3 days post infection. Lower CD4 T cell proliferative responses were detected in head kidney, gill and gut. The B lymphocytes (IgT and IgM) proliferated in spleen, head kidney and gills, thus pointing to the gills as a main inductor site in mucosal responses to Y. ruckeri. To further explore the initial stages of lymphocyte mobilization and how this correlates with the development of adaptive immune responses in mucosal lymphoid tissues, we have applied this powerful approach to analyzing B and T cell response kinetics to Ichthyophthirius multifiliis, a parasite with a tropism for the skin and gills of trout. Additionally, to gain deeper insights into the spatial organization and interaction of B/T lymphocytes within the systemic and mucosal lymphoid organs, we have complemented the flow cytometry analyses with multicolor fluorescence microscopy imaging. This combined approach offers unprecedented opportunities for understanding the geography and the mechanisms by which adaptive immune responses in systemic and mucosal lymphoid organs are induced in teleost fish. In turn, such analysis will not only shed light into the pathways used by teleosts to develop adaptive immune responses, but will also provide the knowledge to rationally design better fish vaccines.