Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and β-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to β-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic.

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Lineage Tracing Reveals the Dynamic Contribution of Pericytes to the Blood Vessel Remodeling in Pulmonary Hypertension

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.119.313715 ◽

2020 ◽

Vol 40 (3) ◽

pp. 766-782 ◽

Cited By ~ 9

Author(s):

Jennifer Bordenave ◽

Ly Tu ◽

Nihel Berrebeh ◽

Raphaël Thuillet ◽

Amélie Cumont ◽

...

Keyword(s):

Smooth Muscle ◽

Structural Changes ◽

Chronic Hypoxia ◽

Transforming Growth Factor ◽

Primary Cultures ◽

Lineage Tracing ◽

In Vivo Model ◽

Dependent Manner

Objective: Excessive accumulation of resident cells within the pulmonary vascular wall represents the hallmark feature of the remodeling occurring in pulmonary arterial hypertension (PAH). Furthermore, we have previously demonstrated that pulmonary arterioles are excessively covered by pericytes in PAH, but this process is not fully understood. The aim of our study was to investigate the dynamic contribution of pericytes in PAH vascular remodeling. Approach and Results: In this study, we performed in situ, in vivo, and in vitro experiments. We isolated primary cultures of human pericytes from controls and PAH lung specimens then performed functional studies (cell migration, proliferation, and differentiation). In addition, to follow up pericyte number and fate, a genetic fate-mapping approach was used with an NG2CreER;mT/mG transgenic mice in a model of pulmonary arteriole muscularization occurring during chronic hypoxia. We identified phenotypic and functional abnormalities of PAH pericytes in vitro, as they overexpress CXCR (C-X-C motif chemokine receptor)-7 and TGF (transforming growth factor)-βRII and, thereby, display a higher capacity to migrate, proliferate, and differentiate into smooth muscle-like cells than controls. In an in vivo model of chronic hypoxia, we found an early increase in pericyte number in a CXCL (C-X-C motif chemokine ligand)-12-dependent manner whereas later, from day 7, activation of the canonical TGF-β signaling pathway induces pericytes to differentiate into smooth muscle-like cells. Conclusions: Our findings reveal a pivotal role of pulmonary pericytes in PAH and identify CXCR-7 and TGF-βRII as 2 intrinsic abnormalities in these resident progenitor vascular cells that foster the onset and maintenance of PAH structural changes in blood lung vessels.

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Monocytes control natural killer cell differentiation to effector phenotypes

Blood ◽

10.1182/blood-2010-10-312264 ◽

2011 ◽

Vol 117 (17) ◽

pp. 4511-4518 ◽

Cited By ~ 59

Author(s):

Katrina Soderquest ◽

Nick Powell ◽

Carmelo Luci ◽

Nico van Rooijen ◽

Andrés Hidalgo ◽

...

Keyword(s):

Cell Differentiation ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

In Vivo Model ◽

Dependent Manner ◽

Nk Cell Differentiation

Abstract Natural killer (NK) cells play a major role in immunologic surveillance of cancer. Whether NK-cell subsets have specific roles during antitumor responses and what the signals are that drive their terminal maturation remain unclear. Using an in vivo model of tumor immunity, we show here that CD11bhiCD27low NK cells migrate to the tumor site to reject major histocompatibility complex class I negative tumors, a response that is severely impaired in Txb21−/− mice. The phenotypical analysis of Txb21-deficient mice shows that, in the absence of Txb21, NK-cell differentiation is arrested specifically at the CD11bhiCD27hi stage, resulting in the complete absence of terminally differentiated CD11bhiCD27low NK cells. Adoptive transfer experiments and radiation bone marrow chimera reveal that a Txb21+/+ environment rescues the CD11bhiCD27hi to CD11bhiCD27low transition of Txb21−/− NK cells. Furthermore, in vivo depletion of myeloid cells and in vitro coculture experiments demonstrate that spleen monocytes mediate the terminal differentiation of peripheral NK cells in a Txb21- and IL-15Rα–dependent manner. Together, these data reveal a novel, unrecognized role for Txb21 expression in monocytes in promoting NK-cell development and help appreciate how various NK-cell subsets are generated and participate in antitumor immunity.

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The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y99-068 ◽

1999 ◽

Vol 77 (11) ◽

pp. 886-895 ◽

Cited By ~ 1

Author(s):

Gordon Bolger ◽

Jean-Claude Vigeant ◽

Francine Liard ◽

Bruno Simoneau ◽

Diane Thibeault ◽

...

Keyword(s):

Oral Administration ◽

Pressor Response ◽

Biological Evaluation ◽

In Vivo Model ◽

Dependent Manner ◽

Renin Inhibitors ◽

Human Renin ◽

Dose Dependent

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 µg·kg-1·min-1) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47 ± 3 mmHg (1 mmHg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3 ± 0.1, 2.5 ± 0.9, and 5.2 ± 1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 µg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.Key words: renin-angiotensin system, recombinant human renin, rat, renin inhibitors.

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Cholecystokinin Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0430 ◽

2008 ◽

Vol 19 (1) ◽

pp. 198-206 ◽

Cited By ~ 29

Author(s):

Grzegorz T. Gurda ◽

LiLi Guo ◽

Sae-Hong Lee ◽

Jeffery D. Molkentin ◽

John A. Williams

Keyword(s):

Calcium Ionophore ◽

Confocal Imaging ◽

Luciferase Reporter ◽

In Vivo Model ◽

Dependent Manner ◽

Activated T Cells ◽

Pancreatic Acini ◽

Pancreatic Growth

Elevated endogenous cholecystokinin (CCK) release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated nuclear factor of activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5–1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo.

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PAX5-ELN oncoprotein promotes multistep B-cell acute lymphoblastic leukemia in mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721678115 ◽

2018 ◽

Vol 115 (41) ◽

pp. 10357-10362 ◽

Cited By ~ 4

Author(s):

Laura Jamrog ◽

Guillaume Chemin ◽

Vincent Fregona ◽

Lucie Coster ◽

Marlène Pasquet ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Fusion Proteins ◽

Lymphoblastic Leukemia ◽

Chromosomal Translocation ◽

Suppressor Gene ◽

In Vivo Model ◽

Functional Studies

PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (B-ALL) and is involved in various chromosomal translocations that fuse a part of PAX5 with other partners. However, the role of PAX5 fusion proteins in B-ALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human B-ALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in B-ALL development, we generated a knockin mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN–expressing mice efficiently developed B-ALL with an incidence of 80%. Leukemic transformation was associated with recurrent secondary mutations on Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrate that PAX5-ELN affected B-cell development in vitro and in vivo featuring an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Finally, our molecular and computational approaches identified PAX5-ELN–regulated gene candidates that establish the molecular bases of the preleukemic state to drive B-ALL initiation. Hence, our study provides a new in vivo model of human B-ALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development.

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Effects of nerve growth factor antagonist K252a on peritoneal mast cell degranulation: implications for rat postoperative ileus

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00152.2015 ◽

2015 ◽

Vol 309 (10) ◽

pp. G801-G806 ◽

Cited By ~ 6

Author(s):

Sergio Berdún ◽

Jakub Rychter ◽

Patri Vergara

Keyword(s):

Mast Cell ◽

Nerve Growth Factor ◽

Growth Factor ◽

Postoperative Ileus ◽

Peritoneal Mast Cell ◽

In Vivo Model ◽

Dependent Manner ◽

Nerve Growth

Stabilization of mast cell (MC) degranulation has been proposed to prevent postoperative ileus (POI). Nerve growth factor (NGF) mediates MC degranulation. The aim of the study was to evaluate whether NGF receptor antagonist K252a acts as a MC stabilizer in vitro and in vivo model of POI. Peritoneal mast cells (PMCs) were obtained from Sprague-Dawley rats and were incubated with K252a and exposed to NGF or Compound 48/80 (C48/80). MC degranulation was assessed by β-hexosaminidase assay. POI was induced in rats by intestinal manipulation (IM). Rats were pretreated with K252a (100 μg/kg sc) 20 min prior to POI induction. At 20 min after IM, release of rat mast cell protease 6 (RMCP-6) was evaluated in peritoneal lavage. At 24 h, intestinal transit (IT) and gastric emptying (GE) were evaluated. Ileal inflammation was assessed by myeloperoxidase (MPO) activity, expression of IL-6, NGF, TrkA, RMCP-2 and 6, and MC density within the full-thickness ileum. C48/80 and NGF evoked degranulation of PMCs in a dose-dependent manner. K252a prevented NGF-evoked, but not C48/80-evoked, MC degranulation. IM evoked the release of peritoneal RMCP-6 and subsequently delayed IT and GE. IM increased MPO activity and expression of IL-6. In IM rats, K252a prevented upregulation of IL-6 expression and reduced TrkA. IT, GE, and inflammation were not affected by K252a. K252a inhibited NGF-evoked degranulation of PMCs in vitro. In vivo, K252a decreased IL-6 and PMC degranulation. This may be of relevance for the development of new therapeutic targets for POI.

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RSV Downregulates IL-21/IL-21R On TFH Cells Via PD-L1 Induction In APCs Impairing Protective Humoral Responses

10.1101/203133 ◽

2017 ◽

Cited By ~ 1

Author(s):

Rodrigo Benedetti Gassen ◽

Tiago Fazolo ◽

Deise Nascimento de Freitas ◽

Thiago Jesus Borges ◽

Fabio Maito ◽

...

Keyword(s):

B Cells ◽

Passive Immunization ◽

Affinity Maturation ◽

Dependent Manner ◽

Neutralization Capacity ◽

Tfh Cells ◽

Rsv Infection ◽

Syncytial Virus

Respiratory syncytial virus (RSV) is the major cause of hospitalization for children under two years of age. RSV vaccines are currently unavailable, and children suffering from multiple reinfections by the same viral strain, fail to develop protective memory responses. Follicular helper T (TFH) cells specialize in providing B cell help to antibody production and affinity maturation, mainly via IL-21 secretion. Although RSV-specific antibodies can be detected upon infection, how they are generated and their relevance against disease protection has not been fully examined. Here, we observed that RSV expands a functionally impaired murine TFH cell population in vitro and vivo, with downregulated IL-21R expression and IL-21 production. IL-21 treatment of RSV-infected mice, however, increased TFH cells frequency, enhanced the germinal center reaction and improved protective humoral immune responses by increasing viral protein F specific antibody avidity and neutralization capacity. In vivo, it protected from RSV infection, decreasing lung inflammation. Passive immunization with purified IgG from IL-21 treated RSV-infected mice protected against RSV infection. Both viable and UV-inactivated RSV induced PD-L1 expression on B cells and DCs, however, only in DCs a direct effect of RSV was detected. Blocking PD-L1 during infection recovered IL-21R expression in TFH and B cells and increased secretion of IL-21 by TFH cells in a DC-dependent manner. Our results unveil a novel pathway by which RSV affects TFH cells activity, reducing levels of IL-21 and its receptor, by increasing PD-L1 expression on APCs. These results highlight the PD-L1/IL-21 axis importance for the generation of protective responses to RSV infection.

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The Yeast-Phase Virulence Requirement for α-Glucan Synthase Differs among Histoplasma capsulatum Chemotypes

Eukaryotic Cell ◽

10.1128/ec.00214-10 ◽

2010 ◽

Vol 10 (1) ◽

pp. 87-97 ◽

Cited By ~ 52

Author(s):

Jessica A. Edwards ◽

Elizabeth A. Alore ◽

Chad A. Rappleye

Keyword(s):

Cell Wall ◽

Histoplasma Capsulatum ◽

Yeast Cells ◽

Dependent Manner ◽

Glucan Synthase ◽

Content Type ◽

I Factor ◽

Yeast Phase

ABSTRACTHistoplasma capsulatumstrains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype IIHistoplasmavirulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucanin vitro, yet they remain fully virulentin vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishesAGS1expression. Nonetheless,AGS1mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced duringin vivogrowth despite its absencein vitro. To directly test whetherAGS1contributes to chemotype I strain virulence, we preventedAGS1function by RNA interference and by insertional mutation. Loss ofAGS1function in chemotype I does not impair the cytotoxicity ofags1(−) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, theags1(−) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate thatAGS1expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a uniqueHistoplasmachemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

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Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611776114 ◽

2017 ◽

Vol 114 (19) ◽

pp. 5023-5028 ◽

Cited By ~ 49

Author(s):

Hao Li ◽

Xing-xing Wang ◽

Bin Wang ◽

Lei Fu ◽

Guan Liu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Healthcare Workers ◽

Protective Efficacy ◽

Passive Immunization ◽

Antibody Responses ◽

Protective Antibody ◽

Protective Antibodies ◽

Protection Against Infection

The role of Igs in natural protection against infection by Mycobacterium tuberculosis (Mtb), the causative agent of TB, is controversial. Although passive immunization with mAbs generated against mycobacterial antigens has shown protective efficacy in murine models of infection, studies in B cell-depleted animals only showed modest phenotypes. We do not know if humans make protective antibody responses. Here, we investigated whether healthcare workers in a Beijing TB hospital—who, although exposed to suprainfectious doses of pathogenic Mtb, remain healthy—make antibody responses that are effective in protecting against infection by Mtb. We tested antibodies isolated from 48 healthcare workers and compared these with 12 patients with active TB. We found that antibodies from 7 of 48 healthcare workers but none from active TB patients showed moderate protection against Mtb in an aerosol mouse challenge model. Intriguingly, three of seven healthcare workers who made protective antibody responses had no evidence of prior TB infection by IFN-γ release assay. There was also good correlation between protection observed in vivo and neutralization of Mtb in an in vitro human whole-blood assay. Antibodies mediating protection were directed against the surface of Mtb and depended on both immune complexes and CD4+ T cells for efficacy. Our results indicate that certain individuals make protective antibodies against Mtb and challenge paradigms about the nature of an effective immune response to TB.

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E3 Ubiquitin Ligase ASB17 Promotes Apoptosis by Ubiquitylating and Degrading BCLW and MCL1

Biology ◽

10.3390/biology10030234 ◽

2021 ◽

Vol 10 (3) ◽

pp. 234

Author(s):

Ge Yang ◽

Pin Wan ◽

Qi Xiang ◽

Shanyu Huang ◽

Siyu Huang ◽

...

Keyword(s):

Cell Death ◽

Ubiquitin Ligase ◽

In Vivo Model ◽

Dependent Manner ◽

Important Process ◽

Bcl2 Family ◽

Socs Box ◽

Deficient Mice

Apoptosis is a very important process of cell death controlled by multiple genes during which cells undergo certain events before dying. Apoptosis helps to clean the unnecessary cells and has critical physiological significance. Altered apoptosis results in a disorder of cell death and is associated with many diseases such as neurodegenerative diseases and cancers. Here, we reported that the ankyrin repeat and SOCS box protein 17 (ASB17) was mainly expressed in the testis and promoted apoptosis both in vivo and in vitro. Analyzing ASB17-deficient mice generated by using the CRISPR/Cas9 system, we demonstrated that ASB17 deficiency resulted in the reduction of apoptosis in spermatogenic cells, but it did not affect the development of spermatozoa or normal fertility. Next, in an in vivo model, ASB17 deficiency prevented the apoptosis of spermatogonia induced by etoposide in male mice. We noted that ASB17 promoted apoptosis in a caspase-dependent manner in vitro. Moreover, ASB17 interacted with the members of the BCL2 family, including BCL2, BCLX, BCLW, and MCL1. Interestingly, ASB17 specifically degraded the two anti-apoptotic factors, BCLW and MCL1, in a ubiquitylation-dependent fashion. Collectively, our findings suggested that ASB17 acted as a distinct positive regulator of cell apoptosis.

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