The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.

Role of CD4+ T cells in the regulation of the immune response against encapsulated Group B Streptococcus

Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1. En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène.

Inhibition of 125I-epidermal growth factor binding to murine fibroblasts and keratinocytes by irradiation with ultraviolet-B light

Treatment of murine Swiss 3T3 fibroblasts and XB/2 keratinocytes with UV-B light (302 nm) resulted in a dose-dependent inhibition of [125I] epidermal growth factor (EGF) binding. The light dose required to achieve 50% inhibition of binding in both cell types was 80–85 J/m2 Decreased [125I] platelet-derived growth factor binding was not evoked even by light doses of up to 280 J/m2 UV-B irradiation did not stimultate phosphorylation of the 80 kd protein substrate for protein kinase C. Furthermore, its effect on [125I]EGF binding was not altered as a consequence of protein kinase C down-regulation following prolonged exposure of cells to phorbol esters. These results indicate that UV-B-induced transmodulation of the epidermal growth factor receptor is a specific event mediated through a protein kinase C-indepen dent pathway. Transfer of culture medium from irradiated cells to untreated control cells showed this effect was not induced as a result of transforming growth factor α release and subsequent binding to the EGF receptor in these cells.

Bradykinin B(1) receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-beta and VEGF in a murine model of asthma

In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-beta and VEGF were determined. The antagonists of the constitutive B(2) (HOE 140) and inducible B(1) (R954) receptors were administered intraperitoneally 30 min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B(1) receptor antagonist. The B(2) antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-beta significantly increased and the B(1) receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-beta was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B(1) receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B(1) receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B(1) antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis. (C) 2009 Elsevier Ltd. All rights reserved.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Involvement of proteinase-activated receptors 1 and 2 in spreading and phagocytosis by murine adherent peritoneal cells: Modulation by the C-terminal of S100A9 protein

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the pepticle corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function. (C) 2009 Elsevier B.V. All rights reserved.

Relationship between B-Cell-specific moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase III beta (topoIII beta) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIII beta, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA1 (p=0.003), H2AX (p=0.024) and topoIII beta (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a protooncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.

RAG2 is regulated differentially in B and T cells by elements 5′ of the promoter

To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5′ of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5′ region starting just upstream of the RAG2 promoter, as well as the region from 2–7 kb 5′, could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5′ region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5′ elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5′ region.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells

A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.

The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.

To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression in vivo. Murine bone marrow cells were transduced by each of the recombinant viruses and subsequently used to reconstitute the hematopoietic system of lethally irradiated recipients. Five to seven months after transplantation, analysis of the peripheral blood of animals transplanted with cells transduced by vectors which employ viral long terminal repeats (LTRs) for gene expression indicated that in 83% (77/93) of these animals, the level of human enzyme was equal to or greater than the level of endogenous murine enzyme. Even in bone marrow transplant recipients reconstituted for over 1 year, significant levels of gene expression were observed for each of the vectors in their bone marrow, spleen, macrophages, and B and T lymphocytes. However, derivatives of the parental MFG-ADA vector which possess either a single base mutation (termed B2 mutation) or myeloproliferative sarcoma virus LTRs rather than the Moloney murine leukemia virus LTRs led to significantly improved gene expression in all lineages. These studies indicate that retroviral vectors which employ viral LTRs for the expression of inserted sequences make it possible to obtain high levels of a desired gene product in most hematopoietic cell lineages for close to the lifetime of bone marrow transplant recipients.