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851

and pathogen-derived factors on DCs to find out which reagent can

efficiently induce tolDCs. BMDCs were stimulated with those factors

and the expressions of co-stimulatory, co-inhibitory, and MHC

molecules on BMDCs were observed. In addition, we investigated

cytokine profiles of BMDCs by ELISA to further estimate tolerogenic

potential of BMDCs. Notably, treatment of inflammatory cytokine,

IFN-γ, up-regulated the expression of inhibitory molecules (PD-L1

and PD-L2) on DCs. Another interesting finding was that treatment

with one of the inhibitors for glycogen synthase kinase 3 beta (GSK-

3β) slightly up-regulated PD-L1.

Moreover, upon stimulation with lipopolysaccharide (LPS), the

GSK-3β inhibitor-treated DCs showed significantly increased IL-10

production. Finally, we performed functional assay using OT-I and

OT-II cells to test the abilities for antigen uptake, processing and T

cell priming of induced tolDCs. Through our investigation, we could

take another step for searching tolerogenic agent and establishing

protocols for generating tolDCs, which can be further applied into

clinical trialswhere immune suppression is needed.

Histamine contributes to the resistance against tick-blood-

feeding during the re-infestation

Ohta,T.

1

,Yoshikawa,S.

1

,Ishiwata,K.

2

,Yamaji,K.

2

,Okayama,N.

1

,

Yamanishi, Y.

1

, Kanuka, H.

2

, Watanabe, N.

3

, Ohtsu, H.

4

, Karasuyama, H.

1

1

TokyoMedicalandDentalUniversity,DepartmentofImmune

Regulation,Tokyo,Japan,

2

JikeiUniversitySchoolofMedicine,

DepartmentofTropicalMedicine,Tokyo,Japan,

3

JikeiUniversity

SchoolofMedicine,DepartmentofAllergology,Tokyo,Japan,

4

Tohoku

University,DepartmentofEngineering,Miyagi,Japan

Ticks transmit a variety of pathogens from reservoir host to humans

and animals during the tick-blood-feeding, leading to tick- borne

infectionssuchaslymedisease.Somestudieshavereportedthattick-

infested animals acquired resistance against tick- blood-feeding and

furthermoreprevent transmissionof pathogens fromtick to animals.

We previously reported that basophils infiltrate around the tick-

bite-sites during the re-infestation but not primary infestation, and

basophil-depletion before re-infestation can abolish the resistance

againsttick-blood-feeding.Moreover,adoptivetransferexperiments

elucidated the importance of Ig-Fc receptors on basophils for the

resistance against tick-blood-feeding.These findings suggested that

basophil-activation through the Ig-Fc receptors are important for

the resistance against tick-blood-feeding during the re-infestation.

Basophils are well known for their release inflammatory molecules

such as histamine and protease from theirs secretary granules

in response to IgE/antigen stimulation. We examined whether

histamine is involved in resistance against tick-blood-feeding

using histamine (

HDC

)-deficient mice. We confirmed that immune

reactions such as the levels of tick antigen reactive IgE, basophil-

infiltration and their-activation in tick-feeding-sitewere comparable

betweenwild type and

HDC

-deficientmiceduring the re-infestation.

However,

HDC

-deficient mice failed to develop resistance against

tick-blood-feeding. These findings implied that histamine released

by basophils upon IgE plus tick antigens is important for the

resistance to tick-blood-feedingduring the re-infestation.

Blood gene expression profiles from distinct outcomes of

infection with Leishmania infantum

Gardinassi,L.G.

1

,RochaGarcia,G.

1

,CostaSilva,V.

2

,Costa,C.H.N.

2

,de

MirandaSantos,I.K.F.

1

1

UniversityofSaoPaulo-RibeiraoPretoMedicalSchool,Biochemistry

andImmunology,RibeiraoPreto,Brazil,

2

Federal UniversityofPiauí/

NatanPortellaTropicalDiseasesInstitute,Teresina,Brazil

Visceral leishmaniasis (VL) canbe lethal if left untreated; however, the

majority of human infections with the etiological agent

Leishmania

infantum

,areasymptomatic.UsingBeadChipmicroarraytechnology

(Illumina), we investigated the profiles of gene expression from

blood of VL patients, patients that received therapy, asymptomatic

individuals and controls. Our data demonstrate that each group

included in the study presents a unique transcriptional signature.

By employing computational analysis based on diverse functional

methods as differential gene expression, gene set enrichment

analysis, weighted gene co-expression network analysis and

immune cell deconvolution, our findings support that VL patients

present transcriptional profiles associated to an activation of T

lymphocytes via MHC class I. Of interest, VL patients also presented

negative associations with transcriptional modules related to cell

adhesion and chemotaxis, monocytes, neutrophils and B cells.

Treated patients presented with a mixed transcriptional profile,

sharing several features of untreated patients or healthy individuals.

This group of study presented a positive regulation of cell cycle and

proliferation of T cells, as well as transcriptional activity correlated

with a modulation of cell adhesion and chemotaxis and the Notch

signaling pathway. Asymptomatic and uninfected individuals

present similar patterns of gene expression. Nevertheless,

asymptomatic individuals present subtle particularities, such as a

strong associationwith type I interferon

response.We

conclude that

VL patients present dysfunctions on several compartments of the

immune response and suggest that an efficient global regulation of

the immune response is associated with protection to development

of clinical symptoms.

Financialsupport:FAPESP

A multi-color Natural Killer-cell mediated cytotoxicity

detection of tumor cells using fluorescence and direct cell

imaging

Sundararaman,S.,Roen,D.,Karulin,A.,Caspell,R.,Lehmann,P.

CellularTechnologyLtd,ShakerHeights,UnitedStates

The most essential role of effector immune cells such as CD8+ cells

andNatural Killer (NK) cells is to identify and lyse target cells. NK Cell -

and Antibody Dependent (ADCC) Cell - Cytotoxicity has traditionally

been assessed by the release of radioactive Chromium from

target cells following lysis. These assays are laborious and require

substantialquantitiesofpatientbloodtodetect minorchangesincell

lysis.We

have previously shown that we can achieve similar results as

the Chromium Release Assay by the direct imaging of fluorescently

labelled target cells and visualizing the loss of fluorescence signal

upon cytolytic activity. We showed that when performed in a 96-

well plate, the % of target cell lysis is inversely proportional to the

number of effector cells in each well. In order to further reduce the

amount of cell material required and detect the effect of NK cells

on different target cell lines we have now developed a multi-color