LRR domain of NLRX1 protein delivery by dNP2 inhibits T cell functions and alleviates autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS), which is a chronic progressive disease and is caused by uncontrolled activation of myelin antigen specific T cells. It has high unmet medical needs due to the difficulty of efficient drug delivery into the CNS to control tissue inflammation. In this study, we demonstrate that a fusion protein of NOD-like receptor family member X1 (NLRX1) and blood brain barrier (BBB)-permeable peptide, dNP2 ameliorates experimental autoimmune encephalomyelitis (EAE). Methods: We purified recombinant LRR or NBD regions of NLRX1 protein conjugated with dNP2. To examine intracellular delivery efficiency of the recombinant protein, we incubated the proteins with Jurkat T cells or murine splenic T cells and their delivery efficiency was analyzed by flow cytometry. To investigate the therapeutic efficacy in an EAE model, we injected the recombinant protein into mice with 3 different treatment schemes e.g., prevention, semi-therapeutic, and therapeutic. To analyze their functional roles in T cells, we treated MACS-sorted naïve CD4 T cells with the proteins during their activation and differentiation into Th1, Th17, and Treg cells. Results: dNP2-LRR protein treatment showed significantly higher delivery efficiency than TAT-LRR or LRR alone in Jurkat T cells and mouse splenic T cells. In all three treatment schemes of EAE experiments, dNP2-LRR administration showed ameliorated tissue inflammation and disease severity with reduced number of infiltrating T cells producing inflammatory cytokines such as IFNγ. In addition, dNP2-LRR inhibited T cell activation, cytokine production, and Th1 differentiation. Conclusion: These results suggest that dNP2-LRR is a novel agent, which regulates effector T cell functions and could be a promising molecule for the treatment of CNS autoimmune diseases such as multiple sclerosis.

Meningitic Escherichia coli-induced upregulation of PDGF-B and ICAM-1 aggravates blood-brain barrier disruption and neuroinflammatory response.

BACKGROUND: Blood-brain barrier (BBB) disruption and neuroinflammation are considered key mechanisms of pathogenic Escherichia coli invasion of the brain. However, the specific molecules involved in meningitic E. coli-induced BBB breakdown and neuroinflammatory response remain unclear. Our previous RNA-sequencing data from human brain microvascular endothelial cells (hBMECs) revealed two important host factors: platelet-derived growth factor-B (PDGF-B) and intercellular adhesion molecule-1 (ICAM-1), which were significantly upregulated in hBMECs after meningitic E. coli infection. Whether and how PDGF-B and ICAM-1 contribute to the development of E. coli meningitis are still unclear. METHODS: The western blot, real-time PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence were applied to verify the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in vivo and in vitro. Evan's blue assay and electric cell-substrate impedance sensing assay were combined to identify the effects of PDGF-B on BBB permeability. The CRISPR/Cas9 technology, cell-cell adhesion assay, and electrochemiluminescence assay were used to investigate the role of ICAM-1 in neuroinflammation subversion. RESULTS: We verified the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in mouse as well as monolayer hBMECs models. Functionally, we showed that the increase of PDGF-B may directly enhance the BBB permeability by decreasing the expression of tight junction proteins, and the upregulation of ICAM-1 contributed to neutrophils or monocytes recruitment as well as neuroinflammation subversion in response to meningitic E. coli infection. CONCLUSIONS: Our findings demonstrated the roles of PDGF-B and ICAM-1 in mediating bacterial-induced BBB damage as well as neuroinflammation, providing new concepts and potential targets for future prevention and treatment of bacterial meningitis.

Overexpression of matriptase in tumor stroma is a poor prognostic indicator of extrahepatic bile duct cancer.

Bile duct cancer is known to contain numerous fibroblasts, and reported to recruit cancer- associated fibroblasts by secreting platelet-derived growth factor-D (PDGF-D) which needs serine proteases, such as matriptase, to behave as a ligand. However, their expression pattern, and prognostic value have not been clarified. In this study, we investigated the clinicopathological significance of PDGF-D and matriptase expression in patients with extrahepatic bile duct cancer. The samples were obtained from 256 patients who underwent the surgical resection between 1991 and 2015, and the expression levels of PDGF-D and matriptase were evaluated immunohistochemically. Staining intensities and distribution were scored, and finally classified into low and high expression groups in cancer cells and stroma respectively. High expression of matriptase in the cancer stroma was detected in 91 tumors (40%). The high stromal matriptase expression was significantly associated with shorter recurrence-free survival (RFS) and overall survival (OS) (P = 0.0027 and 0.0023, respectively). Multivariate analyses also demonstrated that the stromal matriptase expression level was an independent influential factor in RFS (P = 0.0050) and OS (P = 0.0093). Our findings suggest that the high stromal matriptase expression was strongly associated with tumor progression, recurrence and poor outcomes in patients with extrahepatic bile duct cancer.

Molecular and Cellular Effect of Angiotensin 1-7 on Hypertensive Kidney Disease.

BACKGROUND: Studies implicate that angiotensin 1-7 (Ang1-7) imparts protective effects in the kidney. However, its relevance in hypertensive kidney disease is not fully understood. The purpose of this study was to explore the role of Ang1-7 on renal damage/remodeling during hypertension and its potential underlying molecular-cellular mechanisms. METHODS: Hypertension was induced in adult Sprague-Dawley rats by infusion of aldosterone (ALDO; 0.75 µg/hour) for 4 weeks with or without co-treatment of Ang1-7 (1 mg/kg/day). Untreated rats served as controls. Systolic blood pressure was monitored by tail-cuff technique. Renal fibrosis was evaluated by picrosirius red staining and renal collagen volume fraction was quantitated using imaging analyzing system. The expression of profibrotic factors [transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor-D (PDGF-D), fibroblast growth factor-1 (FGF-1), vascular endothelial growth factor-D (VEGF-D), and tissue inhibitors of metalloproteinases (TIMPs)] and free radical producing enzymes (inducible nitric oxide synthase and nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in the kidney were examined by reverse transcription-polymerase chain reaction and western blot. Renal oxidative stress was assessed by malondialdehyde (MDA) measurement. RESULTS: Chronic ALDO infusion caused hypertension and hypertensive renal disease represented as glomerular damage/sclerosis. Ang1-7 co-treatment did not affect blood pressure in ALDO-treated rats, but significantly attenuated the glomerular damage/fibrosis. ALDO treatment significantly elevated renal expression of profibrogenic factors, including TGF-ß1, TIMP-1/TIMP-2, FGF-1, PDGF-D, and VEGF-D, whereas Ang1-7 co-treatment significantly reduced renal TGF-ß1, TIMP-1/TIMP-2, and FGF-1, but not PDGF-D and VEGF-D. Furthermore, ALDO infusion elevated NADPH oxidase (gp91phox) and MDA in the kidney, which was attenuated by Ang1-7 co-treatment. CONCLUSIONS: Ang1-7 plays a protective role in the hypertensive kidney disease independent of blood pressure. The beneficial effects of Ang1-7 are likely mediated via suppressing TGF-ß/FGF-1 pathways and oxidative stress.

TIM-3 Engagement Promotes Effector Memory T Cell Differentiation of Human Antigen-Specific CD8 T Cells by Activating mTORC1.

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.

El caseinato de sodio incrementa número de linfocitos B en ratones.

Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento.Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje.Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA.Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml).Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.

PDGF-CC underlies resistance to VEGF-A inhibition and combinatorial targeting of both suppresses pathological angiogenesis more efficiently.

Anti-VEGF-A therapy has proven to be effective for many neovascular diseases. However, drug resistance to anti-VEGF-A treatment can develop. Also, not all patients with neovascular diseases are responsive to anti-VEGF-A treatment. The mechanisms underlying these important issues remain unclear. In this study, using different model systems, we found that inhibition of VEGF-A directly upregulated PDGF-CC and its receptors in multiple cell types in pathological angiogenesis in vitro and in vivo. Importantly, we further revealed that combinatorial targeting of VEGF-A and PDGF-CC suppressed pathological angiogenesis more efficiently than monotherapy. Given the potent angiogenic activity of PDGF-CC, our findings suggest that the development of resistance to anti-VEGF-A treatment may be caused by the compensatory upregulation of PDGF-CC, and combined inhibition of VEGF-A and PDGF-CC may have therapeutic advantages in treating neovascular diseases.

Tadalafil Promotes the Recovery of Peripheral Neuropathy in Type II Diabetic Mice.

We previously demonstrated that treatment of diabetic peripheral neuropathy with the short (4 hours) half-life phosphodiesterase 5 (PDE5) inhibitor, sildenafil, improved functional outcome in diabetic db/db mice. To further examine the effect of PDE5 inhibition on diabetic peripheral neuropathy, we investigated the effect of another potent PDE5 inhibitor, tadalafil, on diabetic peripheral neuropathy. Tadalafil is pharmacokinetically distinct from sildenafil and has a longer half-life (17+hours) than sildenafil. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at age 20 weeks were treated with tadalafil every 48 hours for 8 consecutive weeks. Compared with diabetic mice treated with saline, tadalafil treatment significantly improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal sensitivity. Tadalafil treatment also markedly increased local blood flow and the density of FITC-dextran perfused vessels in the sciatic nerve concomitantly with increased intraepidermal nerve fiber density. Moreover, tadalafil reversed the diabetes-induced reductions of axon diameter and myelin thickness and reversed the diabetes-induced increased g-ratio in the sciatic nerve. Furthermore, tadalafil enhanced diabetes-reduced nerve growth factor (NGF) and platelet-derived growth factor-C (PDGF-C) protein levels in diabetic sciatic nerve tissue. The present study demonstrates that tadalafil increases regional blood flow in the sciatic nerve tissue, which may contribute to the improvement of peripheral nerve function and the amelioration of diabetic peripheral neuropathy.

GABAergic neurons in cerebellar interposed nucleus modulate cellular and humoral immunity via hypothalamic and sympathetic pathways.

Our previous work has shown that cerebellar interposed nucleus (IN) modulates immune function. Herein, we reveal mechanism underlying the immunomodulation. Treatment of bilateral cerebellar IN of rats with 3-mercaptopropionic acid (3-MP), a glutamic acid decarboxylase antagonist that reduces γ-aminobutyric acid (GABA) synthesis, enhanced cellular and humoral immune responses to bovine serum albumin, whereas injection of vigabatrin, a GABA-transaminase inhibitor that inhibits GABA degradation, in bilateral cerebellar IN attenuated the immune responses. The 3-MP or vigabatrin administrations in the cerebellar IN decreased or increased hypothalamic GABA content and lymphoid tissues' norepinephrine content, respectively, but did not alter adrenocortical or thyroid hormone levels in serum. In addition, a direct GABAergic projection from cerebellar IN to hypothalamus was found. These findings suggest that GABAergic neurons in cerebellar IN regulate immune system via hypothalamic and sympathetic pathways.

Ectosomes from neutrophil-like cells down-regulate nickel-induced dendritic cell maturation and promote Th2 polarization.

DCs are the first immune cells to be exposed to allergens, including chemical sensitizers, such as nickel, a human TLR4 agonist that induces DC maturation. In ACD, DCs can interact with PMNs that are recruited and activated, leading, in particular, to ectosome release. The objective of this work was to characterize the effects of PMN-Ect on DC functions in an ACD context. We first developed a standardized protocol to produce, characterize, and quantify ectosomes by use of human PLB-985 cells, differentiated into mature PMN (PLB-Ect). We then studied the in vitro effects of these purified ectosomes on human moDC functions in response to NiSO4 and to LPS, another TLR4 agonist. Confocal fluorescence microscopy showed that PLB-Ect was internalized by moDCs and localized in the lysosomal compartment. We then showed that PLB-Ect down-regulated NiSO4-induced moDC maturation, as witnessed by decreased expression of CD40, CD80, CD83, CD86, PDL-1, and HLA-DR and by decreased levels of IL-1ß, IL-6, TNF-α, and IL-12p40 mRNAs. These effects were related to p38MAPK and NF-κB down-regulation. However, no increase in pan-regulatory DC marker genes (GILZ, CATC, C1QA) was observed; rather, levels of effector DC markers (Mx1, NMES1) were increased. Finally, when these PLB-Ect + NiSO4-treated moDCs were cocultured with CD4(+) T cells, a Th2 cytokine profile seemed to be induced, as shown, in particular, by enhanced IL-13 production. Together, these results suggest that the PMN-Ect can modulate DC maturation in response to nickel, a common chemical sensitizer responsible for ADC.

Functional regulation of adipose-derived stem cells by PDGF-D.

Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-ß are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation.

The polycomb protein Ezh2 regulates differentiation and plasticity of CD4(+) T helper type 1 and type 2 cells.

After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.

Alveolar macrophages are critical for the inhibition of allergic asthma by mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSCs) possess reparative and immunoregulatory properties, making them attractive candidates for cellular therapy. However, the majority of MSCs administered i.v. encounter a pulmonary impasse and soon disappear from the lungs, raising the question of how they induce such durable immunosuppressive effects. Using a mouse model of allergic asthma, we show that administration of MSCs isolated from human bone marrow, umbilical cord, or adipose tissue provoked a pronounced increase in alveolar macrophages and inhibited hallmark features of asthma, including airway hyperresponsiveness, eosinophilic accumulation, and Th2 cytokine production. Importantly, selective depletion of this macrophage compartment reversed the therapeutic benefit of MSC treatment on airway hyperresponsiveness. Our data demonstrate that human MSCs exert cross-species immunosuppressive activity, which is mediated by alveolar macrophages in allergic asthma. As alveolar macrophages are the predominant immune effector cells at the air-tissue interface in the lungs, this study provides a compelling mechanism for durable MSC effects in the absence of sustained engraftment.

Platelet-derived growth factor C and calpain-3 are modulators of human melanoma cell invasiveness.

The molecular mechanisms responsible for the elevated metastatic potential of malignant melanoma are still not fully understood. In order to shed light on the molecules involved in the acquisition by melanoma of a highly aggressive phenotype, we compared the gene expression profiles of two cell clones derived from the human cutaneous metastatic melanoma cell line M14: a highly invasive clone (M14C2/MK18) and a clone (M14C2/C4) with low ability to invade the extracellular matrix (ECM). The highly invasive phenotype of M14C2/MK18 cells was correlated with overexpression of neuropilin-1, activation of a vascular endothelial growth factor (VEGF)-A/VEGFR-2 autocrine loop and secretion of matrix metalloprotease-2. Moreover, in an in vivo murine model, M14C2/MK18 cells displayed a higher growth rate as compared with M14C2/C4 cells, even though in vitro both clones possessed comparable proliferative potential. Microarray analysis in M14C2/MK18 cells showed a strong upregulation of platelet-derived growth factor (PDGF)-C, a cytokine that contributes to angiogenesis, and downregulation of calpain-3, a calcium-dependent thiol-protease that regulates specific signalling cascade components. Inhibition of PDGF-C with a specific antibody resulted in a significant decrease in ECM invasion by M14C2/MK18 cells, confirming the involvement of PDGF-C in melanoma cell invasiveness. Moreover, the PDGF-C transcript was found to be upregulated in a high percentage of human melanoma cell lines (17/20), whereas only low PDGF-C levels were detected in a few melanocytic cultures (2/6). By contrast, inhibition of calpain-3 activity in M14C2/C4 control cells, using a specific chemical inhibitor, markedly increased ECM invasion, strongly suggesting that downregulation of calpain-3 plays a role in the acquisition of a highly invasive phenotype. The results indicate that PDGF-C upregulation and calpain-3 downregulation are involved in the aggressiveness of malignant melanoma and suggest that modulators of these proteins or their downstream effectors may synergise with VEGF­A therapies in combating tumour-associated angiogenesis and melanoma spread.

Impaired function of CTLA-4 in the lungs of patients with chronic beryllium disease contributes to persistent inflammation.

Chronic beryllium disease (CBD) is an occupational lung disorder characterized by granulomatous inflammation and the accumulation of beryllium-responsive CD4(+) T cells in the lung. These differentiated effector memory T cells secrete IL-2, IFN-γ, and TNF-α upon in vitro activation. Beryllium-responsive CD4(+) T cells in the lung are CD28 independent and have increased expression of the coinhibitory receptor, programmed death 1, resulting in Ag-specific T cells that proliferate poorly yet retain the ability to express Th1-type cytokines. To further investigate the role of coinhibitory receptors in the beryllium-induced immune response, we examined the expression of CTLA-4 in blood and bronchoalveolar lavage cells from subjects with CBD. CTLA-4 expression was elevated on CD4(+) T cells from the lungs of study subjects compared with blood. Furthermore, CTLA-4 expression was greatest in the beryllium-responsive subset of CD4(+) T cells that retained the ability to proliferate and express IL-2. Functional assays show that the induction of CTLA-4 signaling in blood cells inhibited beryllium-induced T cell proliferation while having no effect on the proliferative capacity of beryllium-responsive CD4(+) T cells in the lung. Collectively, our findings suggest a dysfunctional CTLA-4 pathway in the lung and its potential contribution to the persistent inflammatory response that characterizes CBD.

Spatial and temporal transcriptomics of Schistosoma japonicum-induced hepatic granuloma formation reveals novel roles for neutrophils.

The severity of schistosome egg-induced hepatic granulomatous pathology depends markedly on the nature of the host immune responses. In this study, we used LMM and microarray analysis to compare gene expression profiles of histologically distinct zones within, and directly proximal to, hepatic granulomas that developed in C57BL/6 mice infected with Schistosoma japonicum. There was significant up-regulation of type-1, type-2, and type-17 immune-associated genes within the granuloma core (adjacent to eggs), followed by increased expression of type-2 and fibrotic genes at the outer zones of granulomas. Neutrophil-associated genes were also found to be expressed differentially in the core and at the peripheral zone of granulomas, present at 7 weeks p.i., demonstrating a significant role of neutrophils in S. japonicum granulomatous pathology. The release of NETs was observed microscopically in granulomas obtained from the livers of infected mice and when human neutrophils were incubated in vitro in the presence of S. japonicum eggs. These finding are the first to suggest a novel, dual role for neutrophils in the mediation of tissue damage and repair in S. japonicum egg-induced hepatic granulomatous lesions. Together, these results provide an overview of the local events occurring within the granuloma microenvironment.

New proteoliposome vaccine formulation from N. meningitidis serogroup B, without aluminum hydroxide, retains its antimeningococcal protectogenic potential as well as Th-1 adjuvant capacity.

Proteoliposomes purified from the Outer Membrane of Neisseria meningitidis B, have been successfully used as core for adjuvants and vaccine formulations. We have tried to increase their structural definition and to conserve their efficacy and stability avoiding the addition of the aluminum hydroxide to the final formulation. Liposomal particle systems were prepared from components of defined molecular structure, such as a Neisseria meningitidis B protein complex, extracted and purified without forming vesicle structures. Liposomes were prepared from a mixture of dioleoyl phosphatidyl serine and cholesterol, using the classical dehydration-rehydration method. Transmission Electron Microscopy (TEM) was used to characterize the liposomes. BALB/c mice were used for animal testing procedures. Analysis of specific IgG response, serum bactericidal activity as well as DTH reaction was carried out. Isolation and purification of mRNA and real-time PCR, was performed to determine the dominating Th lymphokine pattern. The new antimeningococcal formulation without aluminum hydroxide prepared with components of defined molecular structure assembled itself into Neoproteoliposomes (NPL) ranging from 50 to 70 nm in diameter. The extraction and purification of selected membrane proteins to provide the antigen for this new formulation (PD-Tp), as well as the NPL-formulation favors a Th1 response pattern, suggested by the higher percentages of DTH, increased expression of proinflamatory lymphokine mRNAs when administered by intramuscular and intranasal routes. It stimulates a systemic bactericidal antibody response against Neisseria meningitidis B and immunologic memory similar to the Cuban VA-MENGOC-BC vaccine, even at lower dosages and is less reactogenic at the injection site in comparison with the formulation with aluminum hydroxide. This new adjuvant formulation could be applicable to the development of new and improved vaccines against meningococcal disease, and eventually as modulators of the immune response against other diseases.

The role of B cells and humoral immunity in Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a serious threat to public health, causing 2 million deaths annually world-wide. The control of TB has been hindered by the requirement of long duration of treatment involving multiple chemotherapeutic agents, the increased susceptibility to Mycobacterium tuberculosis infection in the HIV-infected population, and the development of multi-drug resistant and extensively resistant strains of tubercle bacilli. An efficacious and cost-efficient way to control TB is the development of effective anti-TB vaccines. This measure requires thorough understanding of the immune response to M. tuberculosis. While the role of cell-mediated immunity in the development of protective immune response to the tubercle bacillus has been well established, the role of B cells in this process is not clearly understood. Emerging evidence suggests that B cells and humoral immunity can modulate the immune response to various intracellular pathogens, including M. tuberculosis. These lymphocytes form conspicuous aggregates in the lungs of tuberculous humans, non-human primates, and mice, which display features of germinal center B cells. In murine TB, it has been shown that B cells can regulate the level of granulomatous reaction, cytokine production, and the T cell response. This chapter discusses the potential mechanisms by which specific functions of B cells and humoral immunity can shape the immune response to intracellular pathogens in general, and to M. tuberculosis in particular. Knowledge of the B cell-mediated immune response to M. tuberculosis may lead to the design of novel strategies, including the development of effective vaccines, to better control TB.

Progenitor cells identified by PDGFR-alpha expression in the developing and diseased human heart.

Platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors play instrumental roles in embryonic organogenesis and diseases of adult organs. In particular, platelet-derived growth factor receptor-alpha (PDGFRα) is expressed by multipotent cardiovascular progenitors in mouse and human embryonic stem cell systems. Although cardiac PDGFRα expression has been studied in multiple species, little is known about its expression in the human heart. Using immunofluorescence, we analyzed PDGFRα expression in both human fetal and diseased adult hearts, finding strong expression in the interstitial cells of the epicardium, myocardium, and endocardium, as well as the coronary smooth muscle. Only rare endothelial cells and cardiomyocytes expressed PDGFRα. This pattern was consistent for both the fetal and adult diseased hearts, although more PDGFRα+ cardiomyocytes were noted in the latter. In vitro differentiation assays were then performed on the PDGFRα+ cell fraction isolated from the cardiomyocyte-depleted human fetal hearts. Protocols previously reported to direct differentiation to a cardiomyocyte (5-azacytidine), smooth muscle (PDGF-BB), or endothelial cell fates (vascular endothelial growth factor [VEGF]) were used. Although no significant cardiomyocyte differentiation was observed, PDGFRα+ cells generated significant numbers of smooth muscle cells (smooth muscle-α-actin+ and smooth muscle myosin+) and endothelial cells (CD31+). These data suggest that a subfraction of the cardiac PDGFRα+ populations are progenitors contributing predominantly to the vascular and mesenchymal compartments of the human heart. It may be possible to control the fate of these progenitors to promote vascularization or limit fibrosis in the injured heart.

Transcription factors GATA-3 and RORγt are important for determining the phenotype of allergic airway inflammation in a murine model of asthma.

In refractory asthma, neutrophils, rather than eosinophils, often predominate in the airways. Neutrophilic airway inflammation appears to be resistant to steroids and may be related to the Th17, rather than the Th2, cytokine milieu. However, the role of GATA-3 and RORγt, transcription factors for Th2 and Th17 cell differentiation, respectively, in the pathogenesis of steroid-insensitive asthma remains unclear. To examine the effect of GATA-3- and RORγt-overexpression backgrounds on airway inflammation and steroid sensitivity, we generated two strains of transgenic mice overexpressing GATA-3 or RORγt. Mice were sensitized and challenged with OVA. Some OVA-sensitized/challenged mice were treated with dexamethasone, anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab to demonstrate their therapeutic effects on airway inflammation. Although Ag-specific airway inflammation and hyperresponsiveness were induced in each mouse, the phenotype of inflammation showed a distinct difference that was dependent upon the genotype. GATA-3-overexpressing mice exhibited steroid-sensitive eosinophilic inflammation with goblet cell hyperplasia and mucus hyperproduction under Th2-biased conditions, and RORγt-overexpressing mice developed steroid-insensitive neutrophilic inflammation under Th17-biased conditions. The levels of keratinocyte-derived chemokine, MIP-2, IL-6, and other neutrophil chemotaxis-related mediators were significantly elevated in OVA-exposed RORγt-overexpressing mice compared with wild-type mice. Interestingly, airway hyperresponsiveness accompanied by neutrophilic airway inflammation in RORγt-overexpressing mice was effectively suppressed by anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab administration. In conclusion, our results suggest that the expression levels of GATA-3 and RORγt may be important for determining the phenotype of asthmatic airway inflammation. Furthermore, blockade of the Th17-signaling pathway may be a treatment option for steroid-insensitive asthma.