Purified Peripheral Blood Mononuclear Cells Research Articles

Optimized and Scalable Precoating-Free Reprogramming of Human Peripheral Blood Mononuclear Cells into iPSCs.

Human disease modeling has been profoundly transformed by the introduction of human induced pluripotent stem cells (iPSCs), marking the onset of a new era. This ground-breaking development offers a tailored framework for generating pluripotent cells from any individual, effectively enabling the development of cellular models for the study of human physiology and diseases on an unprecedented scale. Although technologies for iPSCs generation have advanced rapidly over the past two decades, protocols for reprogramming patient-derived somatic cells into stem cells still pose a major challenge for the development of automated pipelines capable of generating iPSCs at scales that are cost-effective, reproducible, and easy to implement. Most methods commonly rely on extracellular matrix protein mixtures or synthetic substrates to promote efficient proliferation of iPSCs. Nonetheless, employing these substances entails a laborious and time-consuming process, as the culture surface requires coating treatments before cell seeding. Here we describe a method for reprogramming blood-derived mononucleated cells that eliminates the need to precoat culture surfaces for the entire experimental flow. This procedure is suitable for fresh or frozen purified peripheral blood mononuclear cells (PBMCs) and allows seeding of reprogrammed cells in a culture medium containing a fragment of laminin-511, regardless of the method of reprogramming employed. Our protocol incorporates a streamlined workflow that optimizes key factors, including cell density, culture medium composition, and iPSC culture propagation techniques. Using a precoating-free approach, we eliminate the time-consuming steps, while our optimized subcloning method improves the scalability of the protocol, making it suitable for large-scale applications. Additionally, the automation-friendly nature of our protocol allows for high-throughput processing, reducing the labor and costs associated with manual handling. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Miniaturized and time efficient precoating-free reprogramming of fresh or frozen PBMCs Alternate Protocol: Erythroid progenitor cells (EPCs) enrichment and reprogramming into iPSCs using Sendai viral vectors Basic Protocol 2: Picking and precoating-free optimized expansion of iPSC clones.

Abstract P4-07-18: FerOX, an anthracyclines nanoformulation to lead toward immuno-mediated cancer remission, preserving T-cell immune competence

Abstract Background Chemotherapy with anthracyclines, such as doxorubicin (DOX), still remains a mainstay in cancer treatment. DOX kills tumor cells by direct interference with DNA replication, thus it is very effective against highly proliferating cells. Since DOX is not cell-specific drug, it displays severe side effects that limit its clinical use. Besides its direct cytotoxicity, it enhances also tumor immunogenicity, since DOX-induced immunogenic cancer cell death facilitates tumor antigens presentation. However, the generation of an adaptive immune response is one of the most highly- proliferative processes, thus it may be affected by DOX cytotoxicity, paradoxically preventing the establishment of a proper adaptive antitumor immune response. Therefore, the effect of DOX on dividing T-cells urges investigation. Methods We explored the phenotypic and functional effects of DOX on lymphocytes from healthy donors and from breast cancer (BC) patients. Purified peripheral blood mononuclear cells (PBMC) were subjected to an uptake assay and to a CFSE-based proliferation assay in vitro. Characterization of lymphocytes was made by flow cytometry (FC) and confocal microscopy. Innovative DOX nanoformulation with proved efficacy in cancer cell line, BC Patient-derived Organoids (PDO) and murine model of BC, has been assessed in parallel to liposomal DOX, evaluating both DOX uptake and their capability to preserve proliferation potential in different PBMC subpopulation. Results PBMC incubated in vitro with DOX displayed a dose-dependent DOX uptake that results in a dramatic proliferative impairment. The same behavior has been observed in BC patient-derived PBMC, isolated immediately after the end of first cycle of chemotherapy, which show a prompt DOX internalization coupled with a dramatic impairment of proliferative potential in comparison to the match-paired PMBC isolated before the treatment. Interestingly, FC characterization showed that DOX internalization is higher in CD8+ than CD4+ T-cells and mainly affects central memory, effector memory and terminal differentiated effector. Since pivotal to every adaptive immune response is the activation and massive proliferation of T cells from their resting state, we have exploited a DOX formulation in ferritin nanocages (FerOX), that displays a TfR1-mediated tumor specific homing in order to circumvent drug internalization in those immune cells. FerOX efficacy has been proved in BC-PDO, evidencing an inverse correlation between TfR1 expression and HFn-DOX IC50. HFn nanoformulation protects immune cells from DOX toxic effects, reducing DOX uptake and proliferative impairment, mainly preserving Naïve T-cells. The capability of FerOX to preserve T-cells competence to generate an immune response has been corroborated by an in vitro experiment aimed to mimic what could happens to patient’s PBMCs if they could be treated with FerOX. Conclusions As a whole, our data indicate that preventing T- cells chemotherapy-related toxicity through FerOX could allow the host adaptive immune system to fully benefit from the DOX-induced immunogenic cell death to vaccinate against cancer. This strategy could be promising for complete tumor eradication and might generate a long- term immunosurveillance of recurrences and metastases. Citation Format: Serena Mazzucchelli, Francesco Mainini, Francesco Andreata, Marta Truffi, Arianna Bonizzi, Francesca Piccotti, Marta Sevieri, Fabio Corsi. FerOX, an anthracyclines nanoformulation to lead toward immuno-mediated cancer remission, preserving T-cell immune competence [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-18.

Combined assessment of S- and N-specific IL-2 and IL-13 secretion and CD69 neo-expression for discrimination of post-infection and post-vaccination cellular SARS-CoV-2-specific immune response.

BackgroundAntibody‐based tests are available for measuring SARS‐CoV‐2‐specific immune responses but fast T‐cell assays remain scarce. Robust T cell‐based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination.MethodsOne hundred seventeen individuals (COVID‐19 convalescent patients: n = 40; SARS‐CoV‐2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS‐CoV‐2‐specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation‐induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS‐CoV‐2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin).ResultsN‐peptide mix stimulation of WB identified the combination of IL‐2 and IL‐13 secretion as superior to IFN‐γ secretion to discriminate between COVID‐19‐convalescent patients and healthy controls (p < .0001). Comparable results were obtained with M‐ or S‐peptides, the latter almost comparably recalled IL‐2, IFN‐γ, and IL‐13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID‐19, but not allergic disease status, positively correlated with IL‐13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen‐induced neo‐expression of the C‐type lectin CD69 on CD4+ (p < .0001) and CD8+ (p = .0002) T cells informed best about the SARS‐CoV‐2 exposure status with additional benefit coming from CD25 upregulation.ConclusionAlong with N‐ and S‐peptide‐induced IL‐2 and CD69 neo‐expression, we suggest to include the type 2 cytokine IL‐13 as T‐cellular recall marker for SARS‐CoV‐2 specific T‐cellular immune responses after infection and vaccination.

Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis

Immunocomplex capture fluorescence analysis (ICFA) which basic principle is same as Luminex crossmatch (LXM), could detect donor-specific HLA antibody (DSA). The advantages of ICFA are (i) detection of DSA and (ii) no requirement of viable cells over the flow cytometry crossmatch (FCXM). However, FCXM has been widely used because of its higher sensitivity than ICFA, in particular HLA-class II antibody detection. In this study the accuracy of DSA detection against HLA-class II was investigated by modifying the original method of ICFA. Increment of the sensitivity was found when purified peripheral blood mononuclear cells (PBMCs) were used instead of whole blood. An ICFA-PBMC in addition to FCXM-T/B was conducted for 118 patients before kidney transplantation and 13 patients with de novo DSA against HLA-class II after transplantation. Significantly positive correlation was observed between the values of ICFA-PBMC and DSA mean fluorescence intensity (MFI) targeting class II (p < 0.0001). When the cutoff level of 1.4 was determined by receiver operating characteristic curve analysis, the average DSA MFI was found to be significantly higher in the ICFA-PBMC (class II) positive group comparing to that in the negative group (12,217 vs 3885, p = 0.0027). ICFA-PBMC and optimized cutoff level could provide valid information in cases of suspected DSA.

Pathogenic effector TH2 cells discriminate between allergic and eosinophilic inflammation in food allergy and eosinophilic gastrointestinal disease

Abstract IgE-mediated food allergies (FA) and delayed food-triggered (FT) gastrointestinal tissue eosinophilia (EGID) have distinct clinical manifestations and are treated differently. Pathogenic effector TH2 (peTH2) cells are implicated in inappropriate TH2 responses in both of these diseases. Furthermore, EGID patients can have concomitant FA, and some EGID patients do not report any food triggers. To better understand the role of peTH2 cells in the context of FA and EGID and the overlap between these two conditions, peTH2 cells (CD3+CD4+CD27-CD49d+CRTH2+CD161+) from purified peripheral blood mononuclear cells (PBMC) were profiled by intracellular cytokine flow cytometry in these deeply-characterized patient cohorts: patients with FA alone (FA, n=5), EGID with FA and FT (EGID+FA+FT, n=7), EGID with FT (EGID+FT, n=7) and EGID without FA or FT (EGID, n=9). Patients with any food allergy or food trigger (FA, EGID+FA+FT, and EGID+FT) have increased peTH2 cells compared to healthy volunteers (HV). Interestingly, a greater percentage of peTH2 cells in patients with food triggers (EGID+FA+FT, EGID+FT) exhibit a trend toward IL5+IL13+ double positivity (P = 0.0556) than those from patients without food triggers (FA, EGID). In contrast, peTH2 cells from patients with FA had a greater percentage that were IL17A+ (P=0.009) or IFNγ+ (P=0.0444) than patients with eosinophilic disease (EGID+FA+FT, EGID+FT, EGID). Our studies indicate that while elevated levels of peTH2 cells are indicative of allergic inflammation of both immediate hypersensitivity and eosinophilic disease, the cytokine profile of each disorder may be distinct, where IL17A+ and IFNγ+ predominate in those without eosinophilic disease.

Interleukin-27 inhibits helper T cell type-2 response in allergic rhinitis

ObjectiveInterleukin-27 (IL-27) is a recently identified cytokine which plays both pro-inflammatory and anti-inflammatory effect in different diseases. However, its function in the pathogenesis of allergic rhinitis (AR) was not clear so far. MethodsQuantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect the expression of interleukin 27 (IL-27) and Th2 cytokines (IL-4, IL-5, IL-13) from 22 patients diagnosed with AR and 20 normal controls. Purified peripheral blood mononuclear cells (PBMCs) were prepared for in vitro experiment. ResultsSerum mRNA and protein levels of IL-27 were down-regulated in AR patients compared with normal controls. The expression of IL-27 showed negative correlation with Th2 cytokines. In vitro study showed that IL-27 significantly inhibited Th2 cytokines expression from PBMCs. ConclusionOur results suggested that decreased IL-27 expression in AR were correlated with Th2 response. IL-27 may be used as potential target in the future treatment of AR.

330 - Analysis of t cell proliferation using different methods of activation and seeding densities in serum-free media

T cell based immunotherapy applications have gained tremendous attention in the past few years due to their advancing clinical potential as the next life-saving treatment option for cancer patients. In order to ensure a successful manufacturing process for adoptive cell therapy applications, a great number of functional T cells need to be generated. Important factors involved in successful cell culture production such as media composition, method of activation, initial seeding density, and expansion platforms, need to be optimized in order to generate high-quality T cells that can be used clinically. In this study, we evaluated the T cell expansion capability in various conditions such as using different serum-free media, culture vessels, and seeding densities. All tested conditions involved the use of anti-human CD3 and CD28 soluble, plate-bound, and dynabead-coupled antibodies to induce T cell stimulation for expansion. Purified peripheral blood mononuclear cells (PBMCs) were inoculated at various seeding densities in PRIME-XV® T Cell CDM, PRIME-XV® T Cell Expansion XSFM, and another commercially available xeno-free expansion medium. All conditions were supplemented with 200U/mL of IL-2 and expanded for up to three weeks, in vitro. In addition to using traditional tissue culture plates, we also evaluated the T cell expansion potential in the G-Rex® Cell Culture Device. Characterization was performed by determining cell density, viability, morphology and expression of T cell markers by flow cytometry. The results demonstrated the scalability and user-friendliness of generating proliferative T cells using soluble antibodies. Furthermore, the chemically-defined, animal component-free medium generated the highest cell density throughout the process and also illustrated its suitability for use in the manufacture of adoptive T cell therapy products. Altogether, our study indicates the enhancement in the growth of desired T cell populations can be achieved when the right method and medium are paired together.

Cross reactive immune responses in cattle arising from exposure to Mycobacterium bovis and non-tuberculous mycobacteria

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-ɣ). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-ɣ) responses were measured in whole blood cultures using the IFN-ɣ assay and the IFN-ɣ elispot assay on purified peripheral blood mononuclear cells (PBMC).We observed the expected strong IFN-ɣ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-ɣ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-ɣ immune responses to the 10 R-Mag measured by the IFN-ɣ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests.

Staphylococcus aureus SaeR/S-Regulated Factors Decrease Monocyte-Derived Tumor Necrosis Factor-α to Reduce Neutrophil Bactericidal Activity.

The ability of Staphylococcus aureus to evade killing by human neutrophils significantly contributes to disease progression. In this study, we characterize an influential role for the S. aureus SaeR/S 2-component gene regulatory system in suppressing monocyte production of tumor necrosis factor alpha (TNF-α) to subsequently influence human neutrophil priming. Using flow cytometry and TNF-α specific enzyme-linked immunosorbent assays we identify the primary cellular source of TNF-α in human blood and in purified peripheral blood mononuclear cells (PBMCs) during interaction with USA300 and an isogenic saeR/S deletion mutant (USA300∆saeR/S). Assays with conditioned media from USA300 and USA300∆saeR/S exposed PBMCs were used to investigate priming on neutrophil bactericidal activity. TNF-α production from monocytes was significantly reduced following challenge with USA300 compared to USA300∆saeR/S. We observed that priming of neutrophils using conditioned medium from peripheral blood mononuclear cells stimulated with USA300∆saeR/S significantly increased neutrophil bactericidal activity against USA300 relative to unprimed neutrophils and neutrophils primed with USA300 conditioned medium. The increased neutrophil bactericidal activity was associated with enhanced reactive oxygen species production that was significantly influenced by elevated TNF-α concentrations. Our findings identify an immune evasion strategy used by S. aureus to impede neutrophil priming and subsequent bactericidal activity.

AKAP4 is a circulating biomarker for non-small cell lung cancer.

Cancer testis antigens (CTAs) are widely expressed in tumor tissues, circulating tumor cells (CTCs) and in cancer derived exosomes that are frequently engulfed by lymphoid cells. To determine whether tumor derived CTA mRNAs could be detected in RNA from purified peripheral blood mononuclear cells (PBMC) of non-small cell lung cancer (NSCLC) patients, we assayed for the expression of 116 CTAs in PBMC RNA in a discovery set and identified AKAP4 as a potential NSCLC biomarker. We validated AKAP4 as a highly accurate biomarker in a cohort of 264 NSCLCs and 135 controls from 2 different sites including a subset of controls with high risk lung nodules. When all (264) lung cancers were compared with all (135) controls the area under the ROC curve (AUC) was 0.9714. When 136 stage I NSCLC lung cancers are compared with all controls the AUC is 0.9795 and when all lung cancer patients were compared to 27 controls with histologically confirmed benign lung nodules, a comparison of significant clinical importance, the AUC was 0.9825. AKAP4 expression increases significantly with tumor stage, but independent of age, gender, smoking history or cancer subtype. Follow-up studies in a small number of resected NSCLC patients revealed a decrease of AKAP4 expression post-surgical resection that remained low in patients in remission and increased with tumor recurrence. AKAP4 is a highly accurate biomarker for the detection of early stage lung cancer.

Expression of CTLA-4 and Foxp3 in peripheral blood T cells of patients with squamous cell laryngeal carcinoma

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) and Foxp3 (forkhead box P3) are receptors present on T cells which play a critical role in the down-regulation of antigen-activated immune responses. To evaluate the potential influences of CTLA-4 and Foxp3 on cancer invasiveness, a case-control study was conducted in 86 patients treated for squamous cell laryngeal carcinoma. The abundance of CTLA-4 and Foxp3 gene transcripts in the purified peripheral blood mononuclear cells (PBMCs) by quantitative real-time PCR (qRT-PCR) was determined. The analysis of proteins by Western blot was performed. The relationships between CTLA-4 and Foxp3 gene and protein expression as well as the aggressiveness of tumor determined on pT, type and depth of invasion were investigated. Our work revealed a significant dependence of mRNA CTLA-4 on tumor front grading (TFG) total score (p = 0.04) as well as CTLA-4 protein expression on pT (p = = 0.03) and type of invasion (p = 0.03). Advanced pT3-pT4 tumors with diffuse infiltration and > 14 TFG points were characterized by higher average values of CTLA-4 protein in PBMCs. Our data also demonstrated significant differences between Foxp3 protein levels in relation to pT (p = 0.04), depth of invasion (p = = 0.02) and type of invasion (p = 0.03). In tumors with the highest invasiveness identified by the pT3-pT4 status, deep invasion with involvement of cartilage and diffuse infiltration, the highest Foxp3 protein level was observed. In conclusion, these results suggest an impact of CTLA-4 and Foxp3 in determining proliferative and aggressive potential of laryngeal carcinoma, highlighting the significance of CTLA-4 and Foxp3 as potential predictive indicators.

Potent B-Cell Depletion by IMGN529, a CD37-Targeting Antibody-Maytansinoid Conjugate for the Treatment of B-Cell Malignancies,

Abstract 3726CD37 is a B-cell surface antigen which is widely expressed on malignant B cells in non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues CD37 expression is limited to blood cells and lymphoid tissues. This restricted expression profile makes CD37 an attractive therapeutic target for antibodies and antibody-drug conjugates.We developed a novel anti-CD37 antibody, K7153A, which provides a unique combination of functional properties: it demonstrated strong pro-apoptotic and direct cell killing activity against NHL cell lines and could mediate effector activity such as CDC and ADCC. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. The direct cytotoxic potency of the K7153A antibody was superior to that of the CD20-directed rituximab and was further enhanced with maytansinoid conjugation in IMGN529. In vivo, IMGN529 demonstrated better anti-tumor activity than the K7153A antibody in established subcutaneous follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and CLL xenograft models in SCID mice. A single administration of IMGN529 showed similar or improved efficacy compared to anti-CD20 antibodies or standard chemotherapy where tested.Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissue sections was performed to evaluate CD37 expression. CD37 exhibited a similar prevalence to CD20 in subtypes of NHL such as FL, DLBCL, Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL).B-cell depletion is an important measure of efficacy for targeted therapies, such as CD20-directed antibodies, in B-cell malignancies. CD37 expression in blood cells from healthy human donors was measured by quantitative flow cytometry in comparison to CD20. The greatest CD37 expression was found in B cells at approximately 77,000 antibodies bound per cell (ABC), which was similar to CD20 expression in B cells at 95,000 ABC. In other blood cell types CD37 staining was seen at low levels, about 2,000 – 5,000 ABC, in monocytes, NK cells and T cells. In vitro depletion experiments were performed with purified peripheral blood mononuclear cells (PBMCs) and with whole blood, both derived from several healthy donors. Cells were incubated for 1 hr with 10 μg/mL of either K7153A, IMGN529, CD37-targeting TRU-016, rituximab or the anti-CD52 antibody alemtuzumab, with cell depletion determined relative to counting beads by flow cytometry. The K7153A antibody and the IMGN529 conjugate efficiently and specifically depleted B-cells in a dose-dependent manner in the context of purified PBMCs and whole blood. With purified PBMCs, both K7153A and IMGN529 caused 50–60% depletion of B cells, with little to no depletion of T cells or monocytes. IMGN529 was more potent than rituximab, which led to 30–40% B-cell depletion, or TRU-016, which caused 20–30% B-cell depletion. IMGN529 also was more specific than alemtuzumab, which depleted T-cells and monocytes as well as B cells. With whole blood samples, both K7153A and IMGN529 resulted in 30–40% B-cell depletion with no effect on T cells, NK cells or monocytes. IMGN529 was again more potent than rituximab or TRU-016, which caused approximately 10% B-cell depletion, and was more specific than alemtuzumab, which depleted the majority of T cells in addition to 40% of B cells.IMGN529 embodies a unique B-cell targeted agent as it combines the intrinsic pro-apoptotic, CDC and ADCC activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by the targeted delivery of its maytansinoid payload. It is highly active in vitro and in vivo against B-cell lymphoma and CLL cell lines. In addition, it mediates specific B-cell depletion in vitro that is greater than B-cell depletion by CD20-directed rituximab. Together, these findings indicate that IMGN529 is a promising therapeutic candidate for the treatment of B-cell malignancies. Disclosures:Deckert:ImmunoGen, Inc.: Employment. Chicklas:ImmunoGen, Inc.: Employment. Yi:ImmunoGen, Inc.: Employment. Li:ImmunoGen, Inc.: Employment. Pinkas:ImmunoGen, Inc.: Employment. Chittenden:ImmunoGen, Inc.: Employment. Lutz:ImmunoGen, Inc.: Employment. Park:ImmunoGen, Inc.: Employment.

Impact of EGFR immunoexpression on STAT3 activation and association with proinflammatory/regulatory cytokine pattern in laryngeal squamous cell carcinoma

Stimulation of epidermal growth factor receptor (EGFR) results in the activation of signal transducer and activator of transcription-3 (STAT3), a transcriptional factor associated with carcinogenesis. Proinflammatory cytokines are capable of activating a tumor cell death program by reducing EGFR tyrosine phosphorylation. This study aimed to identify EGFR expression in laryngeal carcinoma and determine the relationship with STAT3 and proinflammatory/regulatory cytokine secretion. An analysis of EGFR expression (membranous EGFR-m and cytoplasmic EGFR-c) was performed in tumor tissues by immunohistochemical (IHC) staining in 45 medical cases of laryngeal carcinoma. STAT3 expression in freshly isolated tumor and non-cancerous normal epithelial cells by RT-PCR was analyzed in 24 patients after total larynx resection. The concentrations of TNFalpha, IL-2, IL-6, IL-8 and IFNgamma secreted by purified peripheral blood mononuclear cells (PBMCs) or contained in whole blood samples were measured by ELISA. The relationship between EGFR and mRNA STAT3 expression as well as the level of secreted cytokines was investigated. In our study, 93.3% tumors expressed EGFR-m and 37.8% EGFR-c. It also revealed a statistically significant dependence of the EGFR status on STAT3 expression in neoplastic tissues. Tumors with IHC EGFR-m positive staining >50% of the total number of cells, as well as with EGFR-c positive staining, were characterized by the most frequent presence of STAT3 expression. Our data demonstrate a significant negative relationship between EGFR-m expression and TNFalpha concentration, and a positive connection between membranous EGFR and IL-8 or IFNgamma levels recorded in isolated PBMCs. Furthermore, this study revealed a significant relationship between EGFR-c immunoexpression and IL-8 or IFNgamma concentration. Our findings have confirmed a key role of EGFR in determining the proliferative and malignant potential of laryngeal carcinoma.

Characterization of IFNλ1 (IL-29) gene regulation and analysis of inter-individual variation of IFNλ1 levels (38.18)

Abstract Type III interferons are a relatively new class of anti-viral cytokines comprised of three members: IFNλ1, 2 and 3. Through an uncharacterized mechanism, levels of IFNλ1 are reduced in bronchial epithelial cells of rhinovirus-infected asthmatics. In this study, we have examined the regulation of IFNλ1 transcription by utilizing BEAS-2B bronchial epithelial cells as a model system. We have identified the IFNλ1 minimal promoter region that is responsive to viral infection. Using fluorescent DNA footprinting of epithelial cells and purified peripheral blood mononuclear cells (PBMCs) stimulated with various mitogens, we are mapping the occupancy of transcription factor binding sites up to 4kb from the IFNλ1 transcription start site. Furthermore, we have identified key regulators such as NFκ-B that are involved in control of IFNλ1 expression in epithelial cells. Data obtained from a sample set of PBMCs stimulated with HSV has shown inter-individual variation in IFNλ1, both at the level of mRNA and protein production. These findings have been correlated with analysis of selected SNP haplotypes within the IFN-λ1 promoter. Genotyping of the IFNλ1promoter has led to identification of three major haplotypes, which may relate to variation in IFNλ1 levels. The data presented in this study represent an initial characterization of IFNλ1 gene regulation that can now be applied to understanding pathogenic regulation of IFNλ1.

Characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile

The influence of interferon (IFN)-α on the in vitro differentiation of myeloid porcine dendritic cells (DC) was evaluated as the ability of the DC to stimulate to cell proliferation in a mixed leukocyte reaction (MLR), and as their ability to produce cytokines at exposure to bacterial and viral preparations. Porcine monocytes were enriched from purified peripheral blood mononuclear cells (PBMC) by plastic adherence and cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 or in GM-CSF, IL-4 and IFN-α. After 5 days of culture, the cells developed a dendritic morphology and the proportion of cells expressing MHC class II and B7 molecules was increased as determined by flow cytometry. Dendritic cells, differentiated for 5 days in GM-CSF, IL-4 and IFN-α, were able to stimulate both allogeneic and syngeneic PBMC to proliferation in an MLR. The DC produced the Th1 associated cytokines IFN-α at Sendai virus stimulation, and IL-12 at stimulation with plasmid DNA (pre-incubated in the presence of lipofectin), heat-inactivated Actinobacillus pleuropneumoniae, UV-inactivated Aujeszky’s disease virus and live Sendai virus. The heat-inactivated bacteria and Sendai virus also induced production of the Th2 associated cytokines IL-10 and IL-6. The addition of IFN-α during differentiation of DC in GM-CSF and IL-4 enhanced their ability to stimulate allogeneic and syngeneic MLR, but did not alter their ability to produce cytokines.

Effect of selection for increased body weight on mitogenic responses in turkeys

Mitogenic responses were examined for purified peripheral blood mononuclear cells (PBMC) and whole blood from individuals in a line (F) of turkeys selected for increased 16-wk BW and its corresponding randombred control (RBC2). The PBMC were isolated by centrifugation over Histopaque-1077 density gradient and tested for mitogenic responses to concanavalin A (Con A; 25 microg/mL) and phytohemagglutinin (PHA)-M; 100 microg/mL). For the whole blood assay, 6-wk-old poults from both lines were injected with inactivated Pasteurella multocida. Heparinized blood samples were collected prior to injection (0 d) and at 2, 4, 7, and 14 d postinjection. The diluted whole blood was then tested for the mitogenic responses to Con A (25 microg/mL) and PHA-M (25 microg/mL). The cultures were then pulsed with 3H-thymidine, and incorporation was measured using a liquid scintillation counter. There was a line difference in the mitogenic responses to Con A for PBMC and whole blood assays, but no line difference was observed in the response to PHA-M for both assays. For the purified PBMC assay, the F line had a lower response than its randombred control line (P < or = 0.05) to Con A expressed as either cpm or a stimulation index (SI; ratio of cpm for stimulated cells to the cpm for unstimulated cells). For the whole blood assay, the F line had generally lower SI values in the responses to Con A than the RBC2 line, with differences being significant at 0 and 2 d postinjection (P < or = 0.01) and at 14 d postinjection (P < or = 0.05). Genetic selection for increased BW might have affected the lymphoblastogenic potential of Line F that could affect disease resistance.

Effects of maximal exercise on natural killer (NK) cell cytotoxicity and responsiveness to interferon-alpha in the young and old.

We investigated the effects of a graded maximal exercise treadmill test on natural killer (NK) cell number, activity, and responsiveness to interferon-alpha (IFN-alpha) in young (22+/-0.7 yrs) and elderly (65+/-0.8 yrs) sedentary subjects. NK cell cytotoxicity (NKCC) was determined using Ficoll purified peripheral blood mononuclear cells (PBMCs) by a 51Cr release assay against NK-sensitive (K562) and NK-insensitive (Daudi) target cells at various effector:target (E:T) ratios before and immediately after exercise. PBMCs were incubated with rhu IFN-alpha (125 and 250u/10(6) PBMCs) or without for 2 hrs before addition to the 51Cr release assay. There were no differences in unstimulated NKCC against K562 or Daudi targets between the old and the young despite significantly (p=.01) higher percentages of CD56+ NK cells (21.1+/-2.3% in old vs 12.5+/-2.5% in young, pre-exercise). IFN-alpha increased NKCC versus both targets, and NK cells from old subjects were hyporesponsive to IFN-alpha stimulation; this was especially evident at low E:T ratios versus Daudi cells. Maximal exercise significantly increased (50-200%) unstimulated NKCC versus K562 and Daudi targets similarly in both young and old and increased the percentage of CD56+ cells in the PBMC fraction to 33.3+/-3.7% and 23.3+/-3.6% in old and young, respectively. We found a significant correlation between %CD56+ and basal NKCC versus K562s and Daudi cells in the young (i.e., r=.55; p=.02 vs K562s), but not the old (i.e., r=.20; p=.29 vs K562s) subjects. This indicates that, in the young, part of the exercise-induced increase in NKCC is due to an increase in NK cell number. Maximal exercise did not affect unstimulated per cell killing of K562s, but tended to increase per cell killing of Daudis. These results indicate that CD56+ cells from old subjects have an intrinsic defect in their ability to perform cytolysis and respond to IFN-alpha. Furthermore, a single bout of maximal exercise increases NKCC and CD56+ cell number similarly in both young and old subjects regardless of the target cell used.

Interferon-alpha production and tissue localization of interferon-alpha/beta producing cells after intradermal administration of Aujeszky's disease virus-infected cells in pigs.

Intradermal administration of glutaraldehyde–fixed Aujeszky's disease virus (ADV) infected autologous or allogeneic cells resulted in the induction of an interferon(IFN)–α/β response in pigs. Using a sensitive dissociation–enhanced lanthanide fluoroimmunoassay (DELFIA), IFN–α/β was detected in blood at 8 and 24 h after injection of ADV–infected cells. In parallel, by means of in situ hybridization, IFN–α/β mRNA containing cells were demonstrated in regional lymph nodes. Occasional IFN–α/β mRNA positive cells were also seen in injected dermal areas, but not in contralateral lymph nodes, spleen, bone marrow, blood or liver. The ability of leucocytes in whole blood cultures to produce IFN–α/β upon stimulation by ADV was markedly diminished 3–7 days after intradermal injection of ADV–infected cells. In contrast, cultures of purified peripheral blood mononuclear cells (PBMC) had intact IFN–α/β responses. Further, serum from ADV–injected pigs inhibited the in vitro ADV–induced IFN‐α/β responses in PBMC from control pigs, most likely due to the demonstrated presence of anti–ADV antibodies. We suggest that the IFN‐α/β producing cells in lymph nodes may participate in the development of antiviral immunity and could be equivalent to Natural IFN–α/β producing (NIP) cells.

Influence of experimentally induced endogenous production of cortisol on the immune capacity in swine

Field studies have suggested that ‘stressors’, such as transportation and mixing, might interfere with the immune competence of pigs. Therefore, an experimental model was established to study the influence of elevated concentrations of circulating cortisol on the immune capacity in swine. Three experimental groups, with six pigs in each, were immunized twice, 4 weeks apart, with Mycoplasma hyopneumoniae antigen. Endogenous production of cortisol was induced by intramuscular injection of adrenocorticotropic hormone (ACTH) twice daily. One group received ACTH during the week before and after the second immunization, one group during the week after the second immunization only, while one group served as untreated controls.The treatment with ACTH induced high, but physiological, concentrations of cortisol in plasma. Simultaneously, the number of lymphocytes per milliliter blood decreased while the neutrophil number increased. The elevated concentrations of cortisol also coincided with reduced proliferation and interleukin-2 production by blood lymphocytes stimulated with the mitogens concanavalin A and phytohemagglutinin in vitro, while the responses to pokeweed mitogen were less affected. The suppression of mitogen responses was more pronounced in cultures of whole blood than in cultures of purified peripheral blood mononuclear cells (PBMC).Antibody production, induced by M. hyopneumoniae in cultures of purified PBMC was also inhibited by ACTH treatment. Both the rate of increase and the magnitude of the antibody production induced by the primary immunization were reduced. In contrast, no effects of ACTH treatment were recorded for the response to the second immunization or on the serum levels of antibodies to M. hyopneumoniae.The ability of blood leukocytes to produce interferon-α (IFN-α) at exposure in vitro to fixed pseudorabies virus adsorbed to porcine kidney cells increased in all animals shortly after the second immunization with M. hyopneumoniae. The influence of cortisol on the IFN-α-producing capacity was dependent on whether the test was carried out in whole blood cultures or in cultures with purified PBMC. This finding further emphasizes that the relevance of in vitro assays for measuring in vivo phenomena must be carefully scrutinized.

Cytokine profile of viral and autoimmune chronic active hepatitis

Background: Patients with hepatitis have multiple immunologic abnormalities, which may be related to cytokine production. Methods: We examined the in vitro production of interleukins (IL-2, IL-4, IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in purified peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus positive (HBV), acute viral hepatitis (A-HBV), HBV + chronic active hepatitis (HBV-CAH), and autoimmune-type chronic active hepatitis (AI-ACH). Results: IFN-γ and TNF-α production were characteristically higher in patients with A-HBV than in healthy control subjects ( p < 0.001). However, patients with AI-CAH produced highly elevated levels of IL-4 and IL-6 compared with patients with A-HBV and HBV-CAH and healthy control subjects. The cytokine profile (PBMC-induced IL-2, IL-4, IL-6, IFN-γ, and TNF-α production) is different in A-HBV, HBV-CAH, and AI-CAH disease. The increased cytokine secretion (IFN-γ and TNF-α in A-HBV and IL-4 and IL-6 in AI-CAH) could reflect altered relative frequencies of different cell phenotypes in these diseases. Conclusion: Specific eytokine production may be important in the pathophysiology associated with diverse inflammatory states in patients with hepatitis.