Murine Peripheral Blood Mononuclear Cells Research Articles

Determination of the autophagic flux in murine and human peripheral blood mononuclear cells.

The autophagy lysosomal system (ALS) is crucial for cellular homeostasis, contributing to maintain whole body health and alterations are associated with diseases like cancer or cardiovascular diseases. For determining the autophagic flux, inhibition of lysosomal degradation is mandatory, highly complicating autophagy measurement in vivo. To overcome this, herein blood cells were used as they are easy and routinely to isolate. Within this study we provide detailed protocols for determination of the autophagic flux in peripheral blood mononuclear cells (PBMCs) isolated from human and, to our knowledge the first time, also from murine whole blood, extensively discussing advantages and disadvantages of both methods. Isolation of PBMCs was performed using density gradient centrifugation. To minimize changes on the autophagic flux through experimental conditions, cells were directly treated with concanamycin A (ConA) for 2h at 37°C in their serum or for murine cells in serum filled up with NaCl. ConA treatment decreased lysosomal cathepsins activity and increased Sequestosome 1 (SQSTM1) protein and LC3A/B-II:LC3A/B-I ratio in murine PBMCs, while transcription factor EB was not altered yet. Aging further enhanced ConA-associated increase in SQSTM1 protein in murine PBMCs but not in cardiomyocytes, indicating tissue-specific differences in autophagic flux. In human PBMCs, ConA treatment also decreased lysosomal activity and increased LC3A/B-II protein levels, demonstrating successful autophagic flux detection in human subjects. In summary, both protocols are suitable to determine the autophagic flux in murine and human samples and may facilitate a better mechanistic understanding of altered autophagy in aging and disease models and to further develop novel treatment strategies.

Dietary obesity and glycemic excursions cause a parallel increase in STEAP4 and pro-inflammatory gene expression in murine PBMCs.

The balance between pro-atherogenic and anti-atherogenic factors is very crucial in thedevelopmentof atherosclerotic lesions. Although the expression of the six-transmembrane epithelial antigen of the prostate 4 (STEAP4) in myeloid cells is known to be atheroprotective,there is not a single study reporting on the status of STEAP4 expression in circulating monocytes in the early stages of diet-induced obesity or in events of glycemic excursions. We induced glycemic spikes twice daily for a 1-week duration to rats fed on regular chow and western diet, and analyzed gene expression changes in the peripheral blood mononuclear cells (PBMCs).We also conducted experiments on RAW 264.7 cells to gain insight into some of our in vivo findings. Diet-induced obesity and glycemic excursions independently caused a significant increase in STEAP4 mRNA expression in PBMCs.This was also accompanied by an induction of a substantial number of pro-inflammatory cytokines, chemokines, and chemokine receptors.However, the combined effect of western diet and hyperglycemic spikes was subtle and non-additive.In the in vitro setting, either glucose spikes, persistent hyperglycemia,or a combination of palmitic acid and insulin resulted in a parallel increase in expression of STEAP4 and pro-inflammatory genes.This was, however, significantly abrogated with 4-octyl itaconate or attenuated by inhibitors of p38MAPK and NF-kB. STEAP4 expression in mononuclear cells is induced by increasing inflammation or oxidative stress.The observed increase in STEAP4 expression in circulating monocytes due to visceral obesity or glycemic excursions is a compensatory response. The online version contains supplementary material available at 10.1007/s13340-021-00542-1.

Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells.

Simple SummaryAlmost all human cells release extracellular vesicles participating in intercellular communication. Extracellular vesicles are rounded structures surrounded by the cytoplasmic membrane, which embody cytoplasmic contents of the parental cells, which makes extracellular vesicles a promising therapeutic tool for cell-free cancer therapy. In this study, human mesenchymal stem cells were genetically modified to overexpress human interleukin-2 (IL2), a cytokine which regulates the proliferation and activation of immune cells. Membrane vesicle release from native and genetically modified stem cells was induced by cytochalasin B treatment to increase the yield of membrane vesicles. To evaluate the immunomodulating properties of isolated membrane vesicles, immune cells were isolated from human peripheral blood and co-cultured with membrane vesicles from native or IL2 overexpressing stem cells. To analyze the anti-tumor activity of immune cells after interaction with IL2-enriched membrane vesicles, immune cells were co-cultured with triple negative breast cancer cells. As a result, IL2-enriched membrane vesicles were able to activate and stimulate the proliferation of immune cells, which in turn were able to induce apoptosis in breast cancer cells. Therefore, the production of IL2-enriched membrane vesicles represents a unique opportunity to meet the potential of extracellular vesicles to be used in clinical applications for cancer therapy.Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

Selective inhibition of anti-MAG IgM autoantibody binding to myelin by an antigen-specific glycopolymer.

Anti‐myelin‐associated glycoprotein (MAG) neuropathy is a disabling autoimmune peripheral neuropathy that is caused by circulating monoclonal IgM autoantibodies directed against the human natural killer‐1 (HNK‐1) epitope. This carbohydrate epitope is highly expressed on adhesion molecules such as MAG, a glycoprotein present in myelinated nerves. We previously showed the therapeutic potential of the glycopolymer poly(phenyl disodium 3‐O‐sulfo‐β‐d‐glucopyranuronate)‐(1→3)‐β‐d‐galactopyranoside (PPSGG) in selectively neutralizing anti‐MAG IgM antibodies in an immunological mouse model and ex vivo with sera from anti‐MAG neuropathy patients. PPSGG is composed of a biodegradable backbone that multivalently presents a mimetic of the HNK‐1 epitope. In this study, we further explored the pharmacodynamic properties of the glycopolymer and its ability to inhibit the binding of anti‐MAG IgM to peripheral nerves. The polymer selectively bound anti‐MAG IgM autoantibodies and prevented the binding of patients’ anti‐MAG IgM antibodies to myelin of non‐human primate sciatic nerves. Upon PPSGG treatment, neither activation nor inhibition of human and murine peripheral blood mononuclear cells nor alteration of systemic inflammatory markers was observed in mice or ex vivo in human peripheral blood mononuclear cells. Intravenous injections of PPSGG to mice immunized against the HNK‐1 epitope removed anti‐MAG IgM antibodies within less than 1 hr, indicating a fast and efficient mechanism of action as compared to a B‐cell depletion with anti‐CD20. In conclusion, these observations corroborate the therapeutic potential of PPSGG for an antigen‐specific treatment of anti‐MAG neuropathy. Read the Editorial Highlight for this article on page 465.

Extracellular vesicles derived from Echinococcus granulosus hydatid cyst fluid from patients: isolation, characterization and evaluation of immunomodulatory functions on T cells

Cystic echinococcosis is a chronic and complex zoonotic disease. The mechanisms underlying the parasite’s establishment, growth and persistence are not completely understood, and are thought be modulated by a crosstalk through extracellular vesicles. Here, EVs were isolated from the hydatid cyst fluid of patients with cystic echinococcosis and protoscolex culture supernatant. Proteomic analysis of these EVs revealed several parasite- and human-derived proteins. Very few studies have performed proteomic analysis of EVs isolated from HCF and PCS. Our proteomic analysis of the EVs derived from HCF and PCS facilitated identification of 1175 proteins, wherein 1026 and 38 proteins were exclusively identified in the EVs derived from HCF (HCF-EVs) and PCS (PCS-EVs), respectively, and 111 proteins were shared in both. The results of co-culture of PCS-EVs with murine peripheral blood mononuclear cells showed that PCS-EVs significantly regulated T lymphocyte functions in a dose-dependent manner. Collectively, our results provide valuable information on parasite survival strategies and new insights into the role of these EVs in the establishment and persistence of hydatid cysts.

Abstract 5576: BPM 31510, a clinical stage candidate demonstrates potent anti-tumor effect in an immune-competent syngeneic pancreatic cancer model

Abstract BPM 31510, a clinical stage nanodispersion of ubidecarenone, demonstrates anti-tumor effects by eliciting an anti-Warburg metabolic switch in cancer. Previous studies in an immune compromised PaCa2 xenograft model has unequivocally demonstrated significant efficacy of BPM 31510 on tumor volume and survival. The fundamental property of BPM 31510 to influence mitochondrial bioenergetics and the recognized interplay between T cell metabolism and maturation prompted investigation into the effects of BPM 31510 on T lymphocyte functions in eliciting anti-cancer effects. In this study BPM 31510 selectively influenced activation and maturation of T cells in murine peripheral blood mononuclear cells (PBMCs). Moreover, in addition to determining changes in the CD3+ population, changes in surface expression of PD-1 and CTLA4 along with the IFN-γ secretion were examined. Murine cancer cell lines exposed to BPM 31510 were associated with variable sensitivity with highly metabolic tumor types being most sensitive. Next, the anti-cancer activity of BPM 31510 in an in vivo immunocompetent syngeneic Pan02 rodent model was investigated. Murine Pan02 pancreatic cancer cells were implanted subcutaneously into C57BL/6 mice. Tumors with mean volume of 80 mm3 were treated twice a day with vehicle control or BPM 31510 at 25, 50, 100 mg/kg, administered intraperitoneal. Tumor volumes were measured every 4 days. At day 21 post treatment, tumors were harvested and analyzed for the level of infiltrating immune cells by immunofluorescent staining with CD8+ for T cells and F4/80 for tumor macrophages. These results demonstrate a dose-dependent reduction in tumor volume following 21 days of BPM 31510 treatment. In summary, BPM 31510 exerts potent anti-tumor effects through its dual function of modulating tumor cell metabolism and potentially influencing immune check-point to improve overall survival outcomes. Citation Format: Shiva Kazerounian, Aishwarya Sarma, Nidhi Gaur, Maria D. Nastke, Tulin Dadali, Anne R. Diers, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510, a clinical stage candidate demonstrates potent anti-tumor effect in an immune-competent syngeneic pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5576. doi:10.1158/1538-7445.AM2017-5576

Desipramine decreases expression of human and murine indoleamine-2,3-dioxygenases

Abundant evidence connects depression symptomology with immune system activation, stress and subsequently elevated levels of kynurenine. Anti-depressants, such as the tricyclic norepinephrine/serotonin reuptake inhibitor desipramine (Desip), were developed under the premise that increasing extracellular neurotransmitter level was the sole mechanism by which they alleviate depressive symptomologies. However, evidence suggests that anti-depressants have additional actions that contribute to their therapeutic potential. The Kynurenine Pathway produces tryptophan metabolites that modulate neurotransmitter activity. This recognition identified another putative pathway for anti-depressant targeting. Considering a recognized role of the Kynurenine Pathway in depression, we investigated the potential for Desip to alter expression of rate-limiting enzymes of this pathway: indoleamine-2,3-dioxygenases (Ido1 and Ido2). Mice were administered lipopolysaccharide (LPS) or synthetic glucocorticoid dexamethasone (Dex) with Desip to determine if Desip alters indoleamine-dioxygenase (DO) expression in vivo following a modeled immune and stress response. This work was followed by treating murine and human peripheral blood mononuclear cells (PBMCs) with interferon-gamma (IFNγ) and Desip. In vivo: Desip blocked LPS-induced Ido1 expression in hippocampi, astrocytes, microglia and PBMCs and Ido2 expression by PBMCs. Ex vivo: Desip decreased IFNγ-induced Ido1 and Ido2 expression in murine PBMCs. This effect was directly translatable to the human system as Desip decreased IDO1 and IDO2 expression by human PBMCs. These data demonstrate for the first time that an anti-depressant alters expression of Ido1 and Ido2, identifying a possible new mechanism behind anti-depressant activity. Furthermore, we propose the assessment of PBMCs for anti-depressant responsiveness using IDO expression as a biomarker.

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 μm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-β transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-β-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-β transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

Molecular cloning and functional characterization of murine toll-like receptor 8

Toll-like receptors (TLRs) are a large family of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns and evoke the relevant innate immune responses. TLR8 is a member of several endosome nucleic acid-sensing TLRs; however little attention has been paid to murine TLR8 (mTLR8) compared with other endosome nucleic acid-sensing TLRs. In the present study, mTLR8 was cloned using reverse transcription-polymerase chain reaction from murine peripheral blood mononuclear cells and its function in regulating innate immune response was characterized. The open reading frame of mTLR8 consists of 3,099 bps and encodes 1,032 amino acids. It contains typical leucine-rich repeats, a transmembrane domain and a Toll/interleukin-1 receptor domain, and it shares a high level of identity with other mammalian species. The expression of mTLR8 has been widely observed in different tissues, and higher expression levels of mTLR8 have mainly been detected in the heart, spleen and lung. Overexpression of mTLR8 is required for the activation of transcription factor nuclear factor-κB and the production of tumor necrosis factor-α. However, mTLR8 is not able to activate interferon regulatory factor 3 or activator protein 1, nor can it induce interferon-α in HEK293T cells. These results indicate that mTLR8, as an important PRR, is indeed functional and is vital role in the activation of innate immune responses. This study may aid in determining the molecular basis of the interactions between mTLR8 and pathogens.

Effects of Fetal and Neonatal Murine Peripheral Blood Mononuclear Cells Infusion on MicroRNA-145 Expression in Renal Vascular Smooth Muscle Cells in MRL/lpr Mice

For patients with refractory systemic lupus erythematosus, current medications are insufficient to control their condition, and new treatments are necessary. We aimed to evaluate the therapeutic effect of fetal and neonatal murine peripheral blood (FNPB) mononuclear cells and their impact on microRNA-145 (miR-145) in renal vascular smooth muscle cells (VSMCs) of MRL/lpr lupus-prone mice. MRL/lpr mice aged 20 weeks were randomized to 3 groups of 15 (control group, radiation group, infusion group). The renal tissues were subjected to pathological examination. In situ hybridization assay was applied to measure miR-145 expression in renal vessels of MRL/lpr mice. The infusion group had significantly better results for pathological renal tissue lesions than either the control or radiation group. In MRL/lpr mice, there was positive expression of miR-145 in renal VSMCs, although the expression of miR-145 was not discernible in renal vascular intima and adventitia. The miR-145 expression in renal VSMCs in the infusion group was significantly higher than in the control or radiation group, and higher in the radiation group than in the control group; however, the difference was not statistically significant. The increased expression of miR-145 in renal VSMCs might be one of the mechanisms supporting FNPB as a therapy for lupus nephritis; it also suggests that the miR-145 in renal vessels might be a new target for treatment of lupus nephritis.

Effect of nonmyeloablative unrelated fetal and neonatal murine peripheral blood mononuclear cell infusion on MRL/lpr mice.

For patients with refractory systemic lupus erythematosus (SLE), current medications are insufficient to control their condition, and new treatments are necessary. We investigated the effect of fetal and neonatal murine peripheral blood (FNPB) mononuclear cells on MRL/lpr lupus-prone mice. Female MRL/lpr mice were randomized to three groups (control, radiation and infusion groups). The infusion group had significantly better results for survival rate, body weight increase, reduction of spleen index, serum anti-ds-DNA antibody, antinuclear antibodies (ANA) and creatinine (Cr), 24 h urine protein and pathological renal tissue lesions than either the control or radiation group. Graft versus host disease (GVHD) was not observed in MRL/lpr mice in the infusion group. The infusion group also had better hematogenesis reconstruction than the radiation group. Flow cytometry analysis revealed a significant increase in Th1, CD8+ T and T reg cells and a reduction in Th2, CD4+ T and Th17 cells in the peripheral blood of the radiation and infusion groups compared with the control group. Immunocytochemical assay revealed a significant increase in serum transforming growth factor (TGF)-β and a significant reduction in interleukin (IL)-17 in the radiation and infusion groups compared with the control group. Therefore, our study showed that FNPB mononuclear cell infusion has a significant role in regulating CD4+ T cells, Th1/Th2, Th17/T reg balance and their corresponding cytokines in MRL/lpr mice. The FNPB mononuclear cell infusion provided evidence in animals and suggested a potential clinical application for umbilical cord blood transplantation to treat SLE patients.

Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion

Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals.Our results showed that, in our model the endotoxin decreased PBMC’s FAAH activity and this condition was reverted by P in a receptor-mediated fashion.

Effects of sterigmatocystin on TNF-α, IL-6 and IL-12 expression in murine peripheral blood mononuclear cells and peritoneal macrophages in vivo

Sterigmatocystin (ST) is a toxic metabolite mainly produced by the fungi Aspergillus nidulans and Aspergillus versicolor. ST is considered a potent carcinogen, mutagen and teratogen. However, over the past few years, it has been demonstrated that it is less acutely toxic to rodents in vivo. In this study, we evaluated the putative effects of ST on the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-12 at mRNA levels in murine peripheral blood mononuclear cells (mPBMCs) and peritoneal macrophage cells and on the serum TNF-α and IL-6 levels in BALB/c mice. Our results show the downregulation of TNF-α, IL-6 and IL-12 mRNA expression in mPBMCs and peritoneal macrophage cells using semi-quantitative reverse transcription-polymerase chain reaction following ST treatment by intraperitoneal injection. Additionally, serum TNF-α and IL-6 levels were also decreased as shown by enzyme-linked immunosorbent assay (ELISA). These results suggest that ST contamination has negative immunomodulatory effects through the downregulation of cytokine expression and secretion.

MicroRNAs in Angiogenesis and Vascular Smooth Muscle Cell Function

The Human Genome Project was barely completed in 2003 before a new quest was launched: this time, in a search for short stretches of RNA called microRNA. The belief that these miniature molecules, which had only been discovered a decade earlier, were major regulators of genetic function gained momentum in the intervening years and sparked a worldwide interest in the physiologic and pathologic roles of microRNAs in the human body. These single-stranded, noncoding molecules of RNA, spanning to lengths of only 25 nucleotides, appear to modulate a range of cellular events by modifying the primary function of messenger RNA (mRNA): protein synthesis. The first regulatory RNA, lin-4, was discovered by Victor Ambros in 1993. Later, Andy Fire, Craig Mello, and David Baulcombe discovered small interfering RNA (siRNAs) and established the role of small noncoding mRNA in regulating cell function under a variety of conditions. The field has exploded in directions that no one could have predicted. We now know that small RNAs are expressed in most eukaryotic cells and regulate a remarkable number of cellular functions.1 The exact mechanism of action of microRNAs is unclear; however, it is believed that these molecules bind to select, noncoding regions of mRNA through traditional Watson and Crick base pairing, repressing or increasing expression of the genetic transcript and its corresponding protein. It is theorized that each molecule of microRNA regulates more than 100 distinct molecules of mRNA, with unknown but potentially far-reaching effects in the human body.2 Already multiple reports implicate an array of distinct microRNAs in cardiac and skeletal muscle myogenesis, apoptosis, regeneration, hypertrophy, fibrosis, and cardiac function. Investigators use microRNA expression profiling to identify specific microRNAs in a host of human and animal cells. Quantitative mass spectrometry measures protein levels in cells following the introduction of a specific microRNA …

Chicken intestine defensins activated murine peripheral blood mononuclear cells through the TLR4-NF-κB pathway

Defensins serve as alarm signals in mobilizing the immune system and activating the innate and adaptive immune responses. In order to investigate whether avian defensins could activate monocytes of another species, and whether chicken defensins could modulate or amplify the adaptive immune responses of murine through the TLR-NF-κB pathway, the relationship between the chicken intestinal defensin AvBD13 and TLR4 in murine peripheral blood mononuclear cells (PBMCs) was explored in vitro. Monocytes were stimulated with AvBD13 (1 μg/mL). The levels of NF-κB p65, CD80, CD86, IL-12 and IFN-α were measured by immunohistochemical analysis of the cells or enzyme-linked immunosorbent assay (ELISA), and the TLR4 levels in monocytes were measured by flow-cytometry. We found that AvBD13 can activate NF-κB, induce the inflammatory cytokines IL-12 and IFN-α, and upregulate costimulatory molecules like CD80 and monocyte proliferation, which was clearly inhibited by the anti-TLR4 antibody. TLR4 expression was rapidly downregulated in the presence of AvBD13. AvBD13 could modulate monocytes directly and serve as an endogenous ligand for TLR4 and upregulate costimulatory molecules and monocyte proliferation. Thus, TLR4 is involved in AvBD13-mediated activation of adaptive immune responses.

Infection of Endothelial Cells with VirulentRickettsia prowazekiiIncreases the Transmigration of Leukocytes

Rickettsia prowazekii, the etiologic agent of epidemic typhus, infects vascular endothelium, leading to vasculitis and tissue infiltration of leukocytes. Murine and human endothelial cells (ECs) were infected with R. prowazekii, including the virulent Breinl strain and the attenuated Madrid E strain. The transendothelial migration (TM) of murine and human peripheral blood mononuclear cells (PBMCs) across ECs infected with Breinl organisms was significantly increased compared with that for uninfected ECs or for ECs infected with attenuated organisms, demonstrating that increased TM was related to R. prowazekii virulence. Increased TM was associated with a specific inflammatory pattern. Indeed, only Breinl organisms induced the expression of transcripts for inflammatory cytokines and chemokines by ECs. Murine PBMCs that had transmigrated across ECs infected with Breinl organisms overexpressed inflammatory cytokines and chemokines as well as tissue factor, whereas interleukin-10 expression was down-regulated. The impact of R. prowazekii infection on the TM of PBMCs may play a prominent role in the development of lesions in epidemic typhus.

Anti-tumoural activity of peripheral blood mononuclear cells against melanoma cells: discrepant in-vitro and in-vivo effects

As tumour cells use multiple mechanisms to escape from chemotherapeutic drugs, the anti-tumoural activity of naive mouse peripheral blood mononuclear cells was examined in this study, using a mouse melanoma cell subline resistant to doxorubicin (B16R). Multicellular spheroids are known to be the most adapted in-vitro model to mimic solid tumours in vivo and are used to investigate many aspects of tumour biology. For in-vitro studies, murine peripheral blood mononuclear cells recovered by Ficoll gradient centrifugation after caudal puncture were co-cultured with multicellular tumour spheroids of B16R cells. Morphological investigations show that peripheral blood mononuclear cells were gathered and focused around the spheroids after 14 h of co-culture and contacts were established within 32 h. Between 38 and 62 h of co-culture, the size of the spheroids decreased significantly. The peripheral blood mononuclear cells exerted cytolytic effects that correlated with the induction of cell death in spheroids of B16R melanoma cells. Immunological investigations to localize and identify peripheral blood mononuclear cells that exerted anti-tumoural effects have shown that spheroids were deeply infiltrated by monocytes/macrophages at a stage in which a significant cytolytic activity and a strong cell death rate were observed. For in-vivo studies, intratumoural injections of syngeneic naive peripheral blood mononuclear cells were administered. A weak potential in-vivo anti-tumoural effect of these cells was observed (inhibition of B16R melanoma growth by 20-25%) but the median survival time of mice treated with peripheral blood mononuclear cells did not increase compared with untreated control mice. Thus, despite anti-tumoural activities of peripheral blood mononuclear cells against the poorly immunogenic and highly metastatic chemoresistant B16 melanoma cells in vitro, a potential anti-melanoma effect in vivo, if present, did not increase the life span of B16R melanoma-bearing mice.

Soluble Expression of Recombinant Human Secondary Lymphoid Chemokine (SLC) in E. coli and Research on Its In Vitro and In Vivo Bioactivity

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that plays an important role in leukocytes homing to lymphoid tissues. The ability of SLC to co-localize both T cells and dendritic cells formed the rationale to evaluate its utility in cancer immunotherapy. The in vivo antitumor effect of murine SLC (mSLC) has been well documented, but little is known about that of human SLC (hSLC). To investigate the antitumor efficiency in vivo of hSLC, the hSLC gene was artificially synthesized and induced to express as a soluble form in Escherichia coli. After purification, the purity of the recombinant human SLC (rhSLC) protein was above 95% by SDS-PAGE analysis. The K(d) of rhSLC binding to peripheral blood lymphocytes (PBLs) was 0.2186 +/- 0.02675 microM as assessed by FACS, and the maximal chemotactic index of rhSLC was 9.49 at 100 nM as assessed by in vitro chemotaxis assay. Then genomic sequences of hSLC and mSLC, and of human CCR7 (hCCR7) and murine CCR7 (mCCR7), the receptor for SLC, were aligned. It was found that hSLC and mSLC share 70.72% identity and hCCR7 and mCCR7share 86.77% identity. Furthermore, we found that rhSLC could chemoattract murine peripheral blood mononuclear cells (PBMCs) in vitro. On the basis of these facts, immune competent mice inoculated with S180 sarcoma cells were chosen as an in vivo model. Intratumoral injections of rhSLC inhibited tumor growth and increased survival. These findings suggest that, despite its incapability to bind to either human or murine CXCR3, which is related to angiostasis, rhSLC can induce an antitumor response in vivo by another route. This report proves that rhSLC has a potent tumor-inhibition ability that makes it a promising candidate agent in cancer immunotherapy.

Early steps in interferon-gamma production: possible involvement of Ca2+-calmodulin-dependent enzymes.

A23187 in combination with phorbol myristate acetate (PMA) strongly induces production of interferon-gamma (IFN-gamma) by human peripheral blood mononuclear cells (PBMC) and even by murine PBMC, which respond poorly to A23187 alone. Macrophage depletion of PBMC strongly reduces IFN-gamma production induced by several mitogens, but does not affect IFN-gamma production induced by A23187 and PMA. In addition the same stimuli are able in combination to induce strong amounts of IFN-gamma, even in the Jurkat T cell line. The protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and the calmodulin antagonist N-(6-aminoehexyl)-5-chloro-1-naphthalenesulfonamide (W-7) were examined for their ability to inhibit IFN-gamma production induced by PMA and A23187. At concentrations near the Ki for protein kinase C, H-7 failed to inhibit PMA- and A23187-induced IFN-gamma production. In contrast, W-7 at low concentrations inhibited IFN-gamma production induced by the same stimuli. In addition OAG, which is known to directly activate protein kinase C, failed to act synergistically with A23187 in the induction of IFN-gamma. On the basis of these results we propose that A23187 and PMA may mimic the early steps of lymphocyte activation, without the requirement of macrophage, bypassing antigen-, or lectin-induced signal. Our results suggest that Ca2+-calmodulin-dependent reactions other than protein kinase C activation may be essential for IFN-gamma production, at least at level of the producing cells.

A method to assess the proliferative activity of small numbers of murine peripheral blood mononuclear cells

An economical, reproducible method was developed to assess the mitogenic response of small numbers of murine peripheral blood mononuclear cells (PBMC, 10 3/ml) to phytohemagglutinin (PHA) stimulation in the presence of feeder layer cells (FLC) and interleukin-2 (IL-2). A linear cell dose-mitogenic response relationship was then demonstrated between the activity of PBMC and spleen cells of individual mice (slope = 1.054±0.037; r = 0.989±0.003). This would suggest that mice can be used for longitudinal studies because small blood volumes can be obtained frequently over a period of time from individual mice and the PBMC can be assessed for their T cell proliferative activity.