ERS SHORT-TERM RESEARCH TRAINING FELLOWSHIP

ZVORISTEANU Anca Valeria

Fellowship Number STRTF 46-2010

ERS SHORT-TERM RESEARCH TRAINING FELLOWSHIPS APPLICATION

No. STRTF 46-2010 – Dr. ANCA VALERIA ZVORISTEANU

Section 1 – Fellowship Sought

Fellowship number: STRTF 46-2010

Title of proposed project: IL-10 produced by macrophages suppresses antiviral innate IFNs in rhinovirus-infected macrophages and epithelial cells

ERS Fellowship programme: STRTF

Keywords: IL-10, macrophages, rhinoviruses, asthma

Expected starting date: 01.01.2011

Expected finishing date: 31.03.2011

Do you intend to return home after the Fellowship?

Yes

What are you plans after the Fellowship?

Clinical Hospital Oradea have got modern laboratory appliance, the main issue remaining is the lack of experience of the medical staff in experimental work. Receiving the ERS STRF fellowship will alow me to learn techniques such cell/virus titration,ELISA,RT-PCR and perform the study. Obtaining the Fellowship will put me in an excellent position to initiate further studies in asthma field ,together with Prof Nanulescu and his group, when I return back to Romania after the completion of this project.

Section 2 – Applicant personal details

Title:

Last name:

Dr.

ZVORISTEANU

First names: ANCA VALERIA

Gender Female

Date of birth: 01.09.1970

Nationality: ROMANIA

Present position: MD Specialist in Allergy and Clinical Immunology

Since when: 2002

Name and address ofthe home institution:

Clinical Hospital Oradea, Allergy and Immunology Department,Republicii Street Nr 37, Oradea, Bihor, Romania

Country: ROMANIA

Telephone: 0040 744 580301

E-mail: ancazvori@yahoo.com

ERS membership number: 133374

Are you now based in your Home country?:

Yes

Are you now based in your Host country?:

No - ROMANIA

Home/Host address: Dacia Street, nr 37, Bl AN 54, ap 16, Oradea, Bihor,Romania

Since

Section 3 – Home supervisor

Title:

Last name:

Prof.

NANULESCU

First names: MIRCEA VICTOR

Present position: Consultant Professor of Pediatrics and Pediatric Respiratory Medicine

Since when: 1993

Name and address ofthe home institution:

Clinical Hospital Oradea, Allergy and Immunology Department,Republicii Street, nr 37, Oradea, Bihor, Romania

Country: ROMANIA

Telephone: 0040 744 510287

Fax:

E-mail: nvmircea@cluj.astral.ro

ERS member? Yes

ERS membership number: 80933

Section 4 – Host supervisor

Title:

Last name:

Prof.

JOHNSTON

First names: SEBASTIAN L.

Present position: Professor of Respiratory Medicine

Since when: 2000

Name and address ofthe home institution:

Department of Respiratory Medicine, National Heart and Lung Institute, MRC & Asthma UK Centre in Allergic Mechanism of Asthma, Centre of Respiratory Infection, Imperial College London, Norfolk Place, London, W2 1PG, United Kingdom

Country: UNITED KINGDOM

Telephone: 44( 0 )2075943764

Fax: 44( 0) 2072628913

E-mail: s.johnston@imperial.ac.uk

ERS member? Yes

ERS membership number: 113528

Section 5 – Professional qualifications and experience of the applicant

Postdoctoral researcher since:

--

PhD holder since: --

MSc holder since --

MD holder since 1996

Other: MD Specialist in Allergoloy and Clinical Immunology 2002 Senior Specialist in Allergy and Clinical Immunology 2008

List degrees/diplomas/field of study/ years in which obtained and name of institutes):

-MD Licence Degree, 1996, University of Medicine and Pharmacy 'V.Papilian', Sibiu, Romania - MD Specialist in Allergology and Clinical Immunology Degree 2002, 'N.Malaxa', Institute of Allergology and Clinical Immunology, Bucharest -Senior Specialist in

Number of years fulltime research experience

0

Please provide other information on your research experience (part time, full time, while working, while studying, etc.)

part time research experience

Number of years professional experience(list: years, position, name of employer)

9 years professional experience as a clinician

Last two positions:

Position 1

Name of employer

From-To:

MD Specialist in Allergy and Clinical Immunology

Clinical Hospital Oradea, Allergy and Immunology Department

2002

Do you have 1 first author publication:

No - -

Main publication as a

co-author:

-P.Chereches,A.Zvoristeanu, M.Nanulescu, Growth Velocity at Children with Asthma Treated with Inhaled Corticosteroids, Allergy-Journal of EAACI,no 57,pag 194, July 2002, vol 57,ISSUE s73 -D.Dumitrascu,A.Zvoristeanu, The Prevalence of Allergic Diseases in

Section 6 – ERS Fellowship application details

Number of publications in international peer-reviewed periodicals as per date of this application

In English: 1

In other language: 3

Professional societies or associations of which you are a member:

ERS-European Respiratory Society EAACI-European Academy of Allergology and Clinical Immunology UCB-Institute of Allergy -WAO -Romanian Society of Allergology and Clinical Immunology

6.1 For International Fellowship applications:

Explain why the objectives of the research project are not attainable in Europe

Are the facilities and skills not available in a European Host unit?

Why is going abroad essential?

6.2 Type of the previous ERS Fellowship for which you are asking for a continuation:

Title of the previous ERS Fellowship for which you are asking for a continuation:

Type of award:

Fellowship number:

Year of award:

Start date:

End date:

Home Supervisor name and unit address:

Host Supervisor name and unit address:

Briefly inform us about the success of the earlier Fellowship. Which skills or objectives did you attain until this point?

Why is an extension essential? What are the new techniques or results to be acquired, and what will be the expected added value it would bring?

6.3 ERS Fellowship application for extension of previous support from other sources

Title of the previous Fellowship or Research project for which you are asking for an extension with ERS support:

Year of award:

Start date:

End date:

Type of the previous Fellowship/Project which you would like to extend with ERS funds:

Home Supervisor name and unit address:

Host Supervisor name and unit address:

Candidate status:

Candidate funded by:

Project’s facilities, resources, materials, operational funds initiated and supported by:

Briefly inform us about the success of the earlier Fellowship/Project. Which skills or objectives did you attain so far?

Why is an extension essential?

6.4 Previous ERS Fellowship recipient applying for a NEW ERS Fellowship

Title of the previous ERS Fellowship:

Year of award:

Type of award:

Fellowship number:

Start date:

End date:

Home Supervisor name and unit address:

Host Supervisor name and unit address:

Briefly inform us about the success of the earlier ERS Fellowship:

What was the impact on your career, on patient care

in your Home unit, etc?

Why are you re-applying for an ERS Fellowship?

What are the new techniques or results to be acquired and what will be the expected added it would bring?

Why is an extension essential? Why do you think you should be reconsidered/refunded?

6.5 ERS Fellowship re-application after a previously rejected project

Re-application with exactly the same title, research project, duration, Host/Home units and application files:

Re-application with the same project and general content but with improved application files, project description, etc.:

What are the minor amendments in this re-application? (methodology, objectives, timelines, etc.):

Re-application with similar project but with major amendments (other title, other duration, other Host unit, etc.):

Yes:

Title of the previously rejected ERS Fellowship application:

Type of the previously rejected ERS Fellowship:

Fellowship number of the previously rejected ERS Fellowship:

Date of negative notification:

Exact duration requested

previously:

Home Supervisor name and unit address:

Host Supervisor name and unit address:

What are the major amendments in this re-application?

CURRICULUM VITAE

PERSONAL DETAILS

Name: Anca Valeria ZVORISTEANU DOB: 1th September 1970

Address: 16, Hegel Street, Sibiu, Romania

Phone No: +40 744 580301; E-mail: ancazvori@yahoo.com

EDUCATION

-1990-1996: Faculty of General Medicine, University of Medicine and Pharmacy “Victor

Papilian ” Sibiu, Romania

-1996 - 2002 Residency in Allergy and Clinical Immunology

-2002 - present: Specialist in Allergy and Clinical Immunology

FOREIGN LANGUAGES

English: written/spoken

WORK EXPERIENCE

-2002 - present -Specialist in Allergy and Clinical Immunology at the Clinical Hospital

Oradea –Ambulatory -Department of Allergy and Immunology, Republicii Street, no 37,

Oradea, Bihor, Romania

COURSES/CONTINIOUS MEDICAL EDUCATION/GRANTS

-EAACI Summer Course”Environment, Sports and Allergy”, Rome 14-16 September

2000-Vilasimius 16-20 September 2000

-EAACI Summer Course” Modern Trends in Allergy and Clinical Immunology” Kiev,

Ukraine 30 august-4 September 2001

-Spirometry Tests Training, 01 –30 September 2001, “M.Nasta” Institute, Bucharest,

2001

-EAACI Summer School” Highlights on Allergic Rhinitis and Its Impact of Asthma-

ARIA”21-25 August, 2002, Sofia, Bulgaria

-EAACI Summer Course “Flowcitometry and Allergy”, Bilbao,7-11 September 2002

-Course “Asthma at children”, Cluj, 10-12 April, 2003

-“Flowcytometry Techniques” Course-Cantacuzino Institute, Bucharest, 15-31 March

2004

-GA2LEN/EAACI Summer School”Dubrovnik, Croatia, 26-30 September, 2004

- CMR Course “The Treatment of Allergic Diseases”, Oradea, 15 December 2004 (lector)

-Medical Management Training, Oradea, 28 January-01 February 2005

-GA2LEN/EAACI Summer Course “Asthma and Allergy- Bringing the Gap between

Basic and Clinical Science”, Rotterdam, The Netherlands, 27-31 Aug 2005

-ERS/ATS Joint School Course on “Basics in Asthma”, Oslo, Norway, June 8-10, 2005

-ERS School Course on “Medical Aerosols” Budapest, Hungary, 25-26 November 2005

-Course “Allergy, Congestion and Inflamation”,Oradea, 01. January-30. Decembre 2006

-EAACI Course “Food Allergy Training” , Hindsgavl Castle, April 8-11, 2006

-The Annual Conference of the Romanian Society of Allergology and Clinical

Immunology, Cluj-Napoca, Romania, 24-26 March 2006

-The Congress of Romanian Society of Allergology and Clinical Immunology, Targu-

Mures, Romania , 26-28 April 2007

-Specific Immunotherapy Training, Bucharest, 11 April 2007

-The Congress of EAACI, Goteborg, Sweden, 9-13 June 2007

-ERS Annual Congress, Stockholm, Sweden, 15-19 September 2007

-Summit Evolving Paradigms in the Treatment of Histamine –Mediated Allergic Disease,

Athens, Greece, 26-28 February 2009

-AAAAI Annual Meeting –Washington, DC, March 13-17, 2009

-CMR Course the Respiratory Pathology, From the Etiology to the Treatment, Oradea, 23

March, 2009

-CMR Course The Role of the Nasal Corticosteroids in the Treatment of Inflammatory

Nasal Obstruction, Oradea, 25 November, 2009 (lector)

-Specific Immunotherapy Training, Brasov, Romania,19-20 March,2010

-ATS International Conference, New Orleans, Louisiana, May 14-19, 2010

-UCB Allergy School “For a Global Management of Respiratory Allergy”, Belgium, 28-

29 May, 2010

-Art Forum 2010 Advances in Respiratory Therapeutics Forum, Rome, 12 June, 2010

-CMR Course The Modern Management of Allergic Rhinitis, Oradea, 15 June, 2010

(lector)

-EAACI/GA2LEN Allergy School, Lifestyle Intervention in Allergy and Asthma,

Sardinia, Italy, September 9-12, 2010

RESEARCH / PUBLICATIONS

1. Diana Dumitrascu, Anca Zvoristeanu, Dana Ranta: The Influence of the

Environmental Factors, the Increased Prevalence of Allergic Diseases in Children

Age 10-13 Years Old in Cluj County - presentation at National Conference of the

Allergy and Clinical Immunology Society 1998, UMF Medicine Journal Cluj

University,1998

2. I Fodor, V. Cristea, A. Zvoristeanu: The Association of Benign Monoclonal

Gammapathy in Collagen Diseases, Sibiul Medical, vol. 9, no4, 1998, pag. 367-368,

ISSN 1221-4990.

3. D. Dumitrascu, A. Zvoristeanu, D. Ranta: The Prevalence of Allergic Diseases in

Cluj County - ISAAC Study, Journal Medicina Moderna” - 1999, vol. VI, no7, pag 227,

ISSN 1223-0472.

4. A. Zvoristeanu, I. Fodor: Sclerodermia-Case Presentation, Journal Acta Medica

Transilvania, vol. 1, no 1, 2000, pag. 54, ISSN 1453-1968.

5. L. Marzan, A. Zvoristeanu: The Direct and Indirect Costs of Allergic Diseases,

Medicina Moderna, 2000, vol. VII, no 6, pag 327-330.

6. P. Chereches, A. Zvoristeanu, M. Nanulescu: Asthma and Bronchial Tests at

Children, UMF Medicine Journal Cluj University, 2001

7. P. Chereches, A. Zvoristeanu, M. Nanulescu: Growth Velocity at Children With

Asthma Treated With Inhaled Corticosteroids, ALLERGY-Journal of the EAACI ,

no 57, pag 194, July 2002, vol 57, ISSUE s73.

8. P. Chereches, M. Farcau, A. Zvoristeanu: Risk Factors for Skin Hypersensitivity in

Children With Asthma, poster presentation at national Conference of the Romanian

Allergy and Clinical Immunology Society, Cluj-Napoca 2003, pag 115

9. A. Zvoristeanu: Ambrosia Allergy-local project research, 2005-2008 Allergy and

Allergenic Plants, www.aspbihor.ro

10. Clinical Study, A Randomised Double-Blind Placebo-Controlled study To Assess

The Safety of Oral Microincapsulated Ragweed Pollen Extract Administered

For One Year, 2007, Principal Investigator,

11. A Multicenter, Randomized, Double-Blind, Placebo-Controlled, Parallel-Group

Study to Evaluate the Efficacy ,Safety and Tolerability of Two Doses of ACT-

128800, an Oral SIP1 Receptor Agonist, administered Up To Twenty-Eight

Weeks in Patient With Moderate to Severe Chronic Plaque Psoriasis, 2010,

Pulmonary Function Tests, Co-investigator

PROJECT: IL-10 produced by macrophages suppresses antiviral innate IFNs in rhinovirus-infected macrophages and epithelial cells

SummaryOne current view on allergic asthma pathogenesis is that IL-10 treatment may prove to be a successful therapeutic strategy. However, in relation to virus-induced asthma, there are data that IL-10 downregulates mediators associated with Th1 responses and antiviral activity. The aim of this study is to determine if one cause of the deficient IFN production in asthmatic airway cells could be increased production of the suppressor cytokine IL-10 by alternatively activated macrophages.

Work planTo study in vitro if:1. Upon rhinovirus infection alternatively activated macrophages produce less antiviral IFN-α/β/λ and more suppressor cytokine IL-10, type 2 chemokines, and fibrotic factors (arginase 1, FIZZ1) and harbor more virus as compared to normal subjects.2. IL-10 treatment of RV-infected epithelial cells and macrophages decreases IFNs and increases virus load.

Reasons for selecting the HostProf Johnston’s group has a long interest in studying the role of monocytes/macrophages in virus-induced asthma exacerbations, previously demonstrating the increase of IL-10 production by in vitro RV infection in peripheral blood mononuclear cells from asthmatics. He has recently reported that macrophages sustain limited rhinovirus replication and that macrophages from asthmatic produce less IFN-α/β/λ as compared to normal subjects ex vivo upon rhinovirus infection. All methods to be employed are available and daily used in Prof Johnston’s laboratory.

The value of the project for my careerMy main interest is Allergology and Immunology and I treat many patients with asthma, children and adults. In the last 5 years, in this part of country we have been confronted with an increase of incidence of ragweed allergic diseases. I have been involved in a project initiated by the Local administration and Health authorities which aim is to prevent, detect, treat and monitor this disease. I was actively involved in research activities in asthma field in Romania, at the level available here and I am interested in studying the laboratory immunology technique (in the asthma field, in particular). In the last years, in Romania, there is a constant progress in asthma therapy, therefore the Clinical Hospital Oradea have got modern laboratory appliances, the main issue remaining is the lack of experience of the medical staff in the experimental research. Receiving the ERS STRF fellowship will allow me to learn techniques such cell/virus culture, virus titration, ELISA, RT-PCR and perform the study. I hope that by working hard I will generate interesting data which will be published. Obtaining the Fellowship will put me in an excellent position to initiate further studies in this field, together with Prof Nanulescu and his group, in order to personalized treatments at the state of the art, when I return back to Romania after the completion of this project.

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Background of the project Activation via different signaling pathways results in MØ with different phenotype and functions: classically activated MØ (caMØ), type II activated MØ (MØ-II), and alternatively activated MØ (aaMØ)1. aaMØ are induced by IL-4, IL-13, TGF- and ingestion of apoptotic material. aaMØ have increased anti-inflammatory, phagocytic and profibrotic functions. aaMØ produce large amounts of Th2 attracting chemokines (MDC/CCL22, TARC/CCL17 and PARC/CCL18), arginase-1 and FIZZ1 (found in inflammatory zone 1)2. aaMØ produce also high levels of IL-10, thatvia autocrine activity mediated through binding to IL-10 heterotetramer receptor (IL-10R, composed of two of each of the receptor chains IL-10Rα and IL-10R), inhibits antigen-presenting function, type I IFN, IL-12 production and microbicidal activity of macrophages3. Also, by acting on T cells IL-10 induces regulatory T cells4 thereby creating an immunosuppressive environment. It was reported that TARC/CCL17 and IL-10 inhibit caMØ generation from resident MØ5. It was recently suggested that IL-10 and regulatory T cells could be of therapeutic benefit in the treatment of allergic diseases. There are however data suggesting that IL-10 activities could be detrimental in virus-induced asthma.

Importance of the topic and the reason for pursuing the research now Respiratory viral infections are the major cause of asthma exacerbations in adults and children6,7. Alveolar MØ play a key role in the responses to virus infection8 and alternative activation of MØ by excess IL-4 may diminish antiviral responses9,10. In RSV-infected children, IL-10-production by peripheral blood monuclear cells (PBMC) correlated with recurrent wheezing on follow-up10,11 and RSV bronchiolitis12. Rhinovirus uptake by MØ has been demonstrated13,14 and Prof Johnston’s group has previously investigated the potential interactions between rhinoviruses and monocytes in terms of intracellular replication, surface costimulatory molecule expression and cytokine production15,16 and reported limited rhinovirus replication in monocyte-derived macrophages (MDM)17. He also reported that in vitro rhinovirus infection of PBMC induces a shift towards the type-2 phenotype (increased IL-10 and decreased IFN- and IL-12) in atopic asthmatic compared to normal volunteers18; RV inactivation decreased levels of IL-10, suggesting that RV-replication in monocytes induced the major part of IL-10 production. IL-10 is involved in dowregulation of mediators associated with Th1 responses and antiviral activity3,19,20. Type-III IFN (interferon lambda 1/2/3 also referred to as IL-29/IL-28A/IL-28B) are related to both type-I interferons and IL-10, have antiviral activity and signal through a heterodimeric receptor composed of CRF2-12 (IFN--R1/IL-28Rα) and CRF2-4 (IL-10-R-/IL-10-R2) chains21,22. In one study of human monocytes IL-10 acted as an antagonist to IFN-1 functions, suggesting a feedback mechanism witch regulates the function of IFN-23. There are data suggesting that the dominant macrophage phenotype in atopic asthmatic subjects is alternatively activated characterized by increased production of IL-10. It was previous shown that IL-10 decreases mediators with antiviral activity implying that IL-10 effects might be unfavorable in virus-induced asthma24,25. Johnston et al reported deficient induction of innate IFN-α/β/λ by RV16 in bronchoalveolar cells, which was correlated with severity of RV16-induced asthma

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exacerbation and virus load in experimentally infected asthmatic volunteers26,(Laza-Stanca et al submitted). We think IL-10 might play a role in decreased IFN-α/β/λ and increased viral susceptibility in asthmatics. To support our hypothesis, we investigated whether RV increases MØ IL-10 (Fig. 1 and Fig. 2) and type 2 chemokine MDC (data not shown) production in MDM and whether this is further increased by IL-4-treatment. RV16 induced IL-10 production in a dose dependent manner; inoculum from which virus had been filtered27 and soluble-ICAM-1 coated or UV-inactivated RV16 did not induce IL-10, confirming that RV16 replication induces IL-10 production, and not virus receptor binding, phagocytosis of the virus or soluble factors in the virus inoculum (Fig. 1& 2).

Furthermore, RV16-induction of IL-10 was significantly amplified in IL-4-pretreated MDM (data not shown). Our preliminary data confirm that macrophage RV16 infection induces IL-10 production, which is further increased by IL-4-pretreatment of macrophages. These data support our hypothesis that macrophages in asthmatic subjects produce more suppressor IL-10 and less antiviral IFN-α/β/λ and that IL-10 may play a role in the impaired antiviral immune responses in asthmatic subjects.

Material and methods Due to the large number of macrophages necessary for the in vitro studies we will use blood monocyte-derived macrophages (MDM). Monocytes will be prepared from PBMC by positive sorting using anti-CD14-conjugated magnetic beads and differentiated to macrophages by culturing with GM-CSF17. Stock solutions of RV16, a major group and RV1b, a minor group, are available and are kept at –70 oC.

1. Do upon RV infection aaMØ produce less IFN- α/β/λ and more IL-10, type 2 chemokines and fibrotic factors and harbor more virus as compared to caMØ?MDM preteated with IL-4 (aaMØ) or IFN-γ (caMØ) will be exposed to RV16 at MOI from 0.1 to 10 RV16/cell for 1h, washed with PBS and cultured in RPMI 1640 supplemented with 2% FCS for 72 h. Cell lysates and supernatants will be collected at various time points after the infection (6h/12h/24h/48h/72h). Supernatants will be used to titrate RV16 release and to measured cytokine/chemokine production (IL-10, IFN- α/β/λ, PARC, TARC, MDC) and arginase-1 by ELISA.

Fig 1. RV16 increases MDM IL-10 production in a dose-dependent manner.MDM were exposed to increasing concentrations of live RV16 [0.1-5 multiplicity of infection (MOI)] and IL-10 measured by ELISA at 24h. Values are meanSEM of 4 experiments. *P<0.05; **P<0.01; *** P<0.001, versus medium

Fig 2. RV16-replication dependent induction of IL-10 in MDM.MDM were exposed to medium, filtered RV16, UV-inactivated RV16 (UV RV16), sICAM coated RV16 (sICAM RV16) and live RV16 (1 MOI) and IL-10 measured by ELISA at 24h. Values are meanSEM of 4 experiments. **P<0.01

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RNA will be extracted from cells and used to assess viral RNA and IL-10, IFN- α/β/λ, IFNL-R1 and IL10-R2, PARC, TARC, MDC, arginase-1 and FIZZ1 mRNA expression by qPCR Taqman. In order to assess whether the observed changes are specifically due to RV-replication, purified as well as UV-inactivated RV preparations will be also used. Virus persistence and replication at macrophage level and differences between susceptibility to virus infection between caMØ and aaMØ will be assessed by measuring virus titre in culture supernatants and cell mRNA levels by RT-PCR. Soluble factors levels will be correlated with the level of virus replication and persistence. RV serotype specificity and responses to RSV and influenza will be investigated to determine the generalisabilty of the findings to other virus types.

2. Does IL-10 decreases IFN-α/β/λ and increases virus load in epithelial cells and macrophages?In order to examine if IL-10 negatively modulates IFN-α/β/λ, RV-infected epithelial cells or MDM will be treated with IL-10 up to 72h and innate IFN levels and virus titre measured in supernatants.We will also use blocking anti-IL-10 Ab (neutralizing anti-IL-10, BD) and measure IFN-α/β/λ and virus levels in MDM infected with RV16.

Statistical analysis Testing for statistical significance in the time-course and dose-response studies will be undertaken by analysis of variance (ANOVA). Correlations between production of cytokine mRNA/protein and virus titre will be investigated by Spearman’s rank correlations.

Conclusion Other scientists are investigating whether treatments that raise the level of IL-10 could be used to treat asthma, as IL-10 seems to help some people with severe asthma who experience ongoing symptoms. It is therefore incredibly important to know how such treatments might affect people in other situations, such as those with milder asthma who know that colds make their symptoms worse. This research should provide important information as to whether people whose asthma is made worse by virus infections should avoid any treatment likely to increase the level of IL-10.

References to recent publications in the project field from the host and others1. Gordon, S. Alternative activation of macrophages. Nat Rev Immunol 3, 23-35

(2003).2. Chung, M.J., Liu, T., Ullenbruch, M. & Phan, S.H. Antiapoptotic effect of

found in inflammatory zone (FIZZ)1 on mouse lung fibroblasts. J Pathol 212, 180-187 (2007).

3. Gary-Gouy H et al. Type I interferon production by plasmacytoid dendritic cells and monocytes is triggered by viruses, but the level of production is controlled by distinct cytokines. J Interferon Cytokine Res 22, 653-659 (2002).

4. Moore, K.W., de Waal Malefyt, R., Coffman, R.L. & O'Garra, A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19, 683-765 (2001).

5. Katakura, T., Miyazaki, M., Kobayashi, M., Herndon, D.N. & Suzuki, F. CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. J Immunol 172, 1407-1413 (2004).

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6. Johnston, S.L., et al. Community study of role of viral infections in exacerbations of asthma in 9-11 year old children. Bmj 310, 1225-1229 (1995).

7. Wark, P.A., et al. Neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma. Eur Respir J 19, 68-75 (2002).

8. Kumagai Y et al. Alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with RNA viruses. Immunity 27, 240-252 (2007).

9. Aandahl, E.M., Michaelsson, J., Moretto, W.J., Hecht, F.M. & Nixon, D.F. Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol 78, 2454-2459 (2004).

10. Panuska JR et al. Respiratory syncytial virus induces interleukin-10 by human alveolar macrophages. Suppression of early cytokine production and implications for incomplete immunity. J Clin Invest 96, 2445-2453 (1995).

11. Bont L et al. Monocyte IL-10 production during respiratory syncytial virus bronchiolitis is associated with recurrent wheezing in a one-year follow-up study. Am J Respir Crit Care Med 161, 1518-1523 (2000).

12. Legg JP et al. Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med 168, 633-639 (2003).

13. Gern, J.E., et al. Rhinovirus enters but does not replicate inside monocytes and airway macrophages. J Immunol 156, 621-627 (1996).

14. Parry DE et al. Rhinovirus-induced PBMC responses and outcome of experimental infection in allergic subjects. J Allergy Clin Immunol 105, 692-698 (2000).

15. Johnston SL et al. Rhinoviruses induce interleukin-8 mRNA and protein production in human monocytes. J Infect Dis 175, 323-329 (1997).

16. Papadopoulos NG et al. Rhinovirus-induced alterations on peripheral blood mononuclear cell phenotype and costimulatory molecule expression in normal and atopic asthmatic subjects. Clin Exp Allergy 32, 537-542 (2002).

17. Laza-Stanca V et al. Rhinovirus replication in human macrophages induces NF-kB-dependent tumor necrosis factor alpha production. J Virol 80, 8248-8258 (2006).

18. Papadopoulos, N.G., et al. A defective type 1 response to rhinovirus in atopic asthma. Thorax 57, 328-332 (2002).

19. Tebo JM et al. Interleukin-10 suppresses IP-10 gene transcription by inhibiting the production of class I interferon. Blood 92, 4742-4749 (1998).

20. Maris, C.H., Chappell, C.P. & Jacob, J. Interleukin-10 plays an early role in generating virus-specific T cell anergy. BMC Immunol 8, 8 (2007).

21. Kotenko, S.V., et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex. Nat Immunol 4, 69-77 (2003).

22. Jordan, W.J., et al. Human interferon lambda-1 (IFN-lambda1/IL-29) modulates the Th1/Th2 response. Genes Immun 8, 254-261 (2007).

23. Jordan, W.J., et al. Modulation of the human cytokine response by interferon lambda-1 (IFN-lambda1/IL-29). Genes Immun 8, 13-20 (2007).

24. Payvandi, F., et al. Exogenous and endogenous IL-10 regulate IFN-alpha production by peripheral blood mononuclear cells in response to viral stimulation. J Immunol 160, 5861-5868 (1998).

25. Busse, W.W. & Gern, J.E. Is interleukin-10 a "10" in virus-provoked asthma? Am J Respir Crit Care Med 172, 405-406 (2005).

26. Contoli, M., et al. Role of deficient type III interferon-lambda production in asthma exacerbations. Nat Med 12, 1023-1026 (2006).

27. Papi, A. & Johnston, S.L. Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (ICAM-1) via increased NF-kappaB-mediated transcription. J Biol Chem 274, 9707-9720 (1999).

CURRICULUM VITAEName Mircea V. Nanulescu Date of birth 14th August 1940Address 3 rd Paediatric Clinic University of Medicine and Pharmacy –Cluj Campeni Street, no 2-4, 3400, Cluj-Napoca Tel/Fax 0040 64 432018 Tel 0040 74 Email nvmircea@cluj.astral.ro Qualifications

1965 MD- University of Medicine and Pharmacy Bucharest1972 Paediatric Degree, University of Medicine and Pharmacy Cluj1974 Ph.D Degree, University of Medicine and Pharmacy Cluj 1979 Consultant in Paediatrics, University of Medicine and Pharmacy,

Bucharest, Romania1994 Consultant in Nephrology, Board of Health Romania1999 Paediatric Respiratory Medicine Degree2000 Consultant in Paediatric Respiratory Medicine, Health Ministry

Present post Consultant Professor 2003- The President of the Romanian Society of Paediatric Respiratory

Medicine

Previous Appointments1980-1990 Lecturer in Paediatrics, University of Medicine and Pharmacy Cluj1990-1993 Associate Professor University of Medicine and Pharmacy, Cluj1991-2005 Director of 3rd Pediatric Clinic, Cluj, Romania1993-2005 Professor of Paediatrics University of Medicine and Pharmacy Cluj1997-2002 Professor of Paediatrics- University “Vasile Goldis”, Arad1998-1999 Professor in Public Health University “ Babes-Bolyai”, Cluj

Other Position Held1985-1989 Secretary of the Paediatrics Department within the Union of Medical Scientific Association - Cluj branch1996-2004 Secretary of the Professional Association of Physicians in Cluj County 1997-2006 Member of the Faculty Board University of Medicine and Pharmacy Cluj 1998-2005 Director of the Nursing College University of Medicine and Pharmacy Cluj2000-2004 Vice-President of the Paediatric Nephrology Association Romania2000-2004 Member in the Paediatric Board of the Health Ministry Romania

AchievementsFoundation of Medical Centres and Editor of National Guidelines

1987-1988 “National Guide for Management of Acute Respiratory Infection in Children”1992 AIDS Centre for Medical Assistance and Screening for the New-born”

Cluj 1998 Coordinator of the Paediatric Expert Group for the Implementation in

Romania of GINA Guideline Establishment of Foundations and Associations

1997 Establisher and President of the Foundation and Association “The protection of Asthmatic Children”, Cluj-Napoca

1998 Establisher and Vice-President of Paediatric Nephrology Association

Romania 1999 Coordinator of the Regional Centre for derulation the program “ The

prophylaxis of Asthma in Children”2003 Establisher and President of the Romanian Society of Paediatric Respiratory Medicine2008 Coordinator of the Medical Practical Guide “Allergic Rhinitis in

Children”

Collaboration Programmes with Centres Abroad1992 Cooperation Program with Healthcare Leadership Council-

USA(Humana Inc Louisville and Baylor University, Dallas). This assistance program offered for 3rd Paediatric Clinic: medical equipment (value $50.000), medical literature (25 monographies, 10 subscriptions to paediatric journals1996, 1997, 1998, 1999) grants for training in Louisville and Dallas USA (11 physicians, 6 nurses, 3 psychologists).

1996-1997 Cooperation Program with “Amsterdam Medical Centre”. Thisprogram offered 7 grants for training and congress participation

First Implementation in Paediatrics Practice in Romania prolonged artificial ventilation in infants prolonged laryngeal intubation for management of obstructive laryngitis aerosol therapy by intermittent positive pressure peritoneal dialysis in children Creation and Assessment of Medical Technologies transcutaneous oxymeter monitor for respiratory control device for oxygen humidification

Organization of National and International Conferences2000 Lectures in collaboration with University Louisville, Kentucky, USA2001 Workshop “The Asthma Therapy in Children”, Cluj2002 Workshop “ The Chronic Nasal Obstruction”Cluj2003 The first National Conference of the Romanian Society of Paediatric

Respiratory Medicine, “Asthma in Children”, Cluj-Napoca 2005 Workshop on Paediatrics in collaboration with University Louisville(Kentucky, USA),

Cluj2005 The second National Conference of the Romanian Society of Paediatric Respiratory

Medicine “Asthma in Children. Viral Acute Respiratory Infections”2007 The 3rd National Conference of the Romanian Society of Paediatric Respiratory Medicine “Asthma in Children. Tuberculosis in Children”2010 National Conference with International Participations of the Romanian Society of Paediatric Respiratory Medicine

Invited Lecturer International 2 National 68

Honours and AwardsTitle Associate Distinguished Professor 1992, 1989Diploma: 75 Years of Romanian-Pharmaceutical Education in Transilvania 1994Excellency Diploma given by the Romanian Society of Respiratory Medicine 2010

1

Mircea Nanulescu

Publications List

1. M. Nanulescu, D. Dumitrascu, M. Popa, D. Iacob, P.Chereches-Panta, M. Muresan, The Epidemiological Study of Asthma, Allergic Rhinitis, Atopic Dermatitis in 7 years old Children in Cluj County, The Journal of Romanian Society of Pediatrics, Pediatria, vol XLIX, nr 1, pag 96-102, 2000

2. M.Nanulescu, P.Chereches-Panta, C.Valeanu, Asthma in Adolescent, Journal Medical Aradean, vol III, no 6, pag 64-66, 2000

3. S.Gheorghe, M.Nanulescu, P.Chereches-Panta, M.Popa, The Skin Hypersensitivity ofPneumoallergens in School Children With Asthma, Pediatria Journal, vol XLIX, No,2000

4. P.Chereches-Panta, D. Campeanu, M. Popa, C. Valeanu, M. Nanulescu, Inhaled Beclomethasone Treatment Efficacy in Children With Asthma, Acta Medica Transilvania Journal, vol I, no 1, pag 76-80, 2000

5. M.Culea, N. Palibroda, P.Chereches-Panta, M. Nanulescu, Comparison of Isotopic Dilution Methods for Determination of Theophylline in the Plasma and Saliva in Infants and Children, Chromatographia Supplement, vol 53, 2001

6. .S. Man, S .Hodor, M. Nanulescu, Respiratory Resistance Measured by the Interrupter Technique: Feasibility Versus Spirometry in 7-8 Years Children, Journal of Romanian Pneumology Society , Pneumologia , vol 52, no 3-4, pag 217-220, 2003

7. M.Nanulescu, Recurrent Wheeze in Infants and Toddlers Children, Pneumologia Journal, vol 52, no 2, pag 141-144, 2003

8. I .Susa , M. Nanulescu, Induced Sputum-Method for Showing Bronchial Inflammation and Monitoring Treatment in Children With Atopic Asthma, Pneumologia Journal, vol 53, no 3, pag 79-84, 2004

9. D. Farcau, D. Draghiciu, P. Chereches-Panta, M. Nanulescu, The Pathological Effect of Gastroesophagial Reflux in Children With Asthma, The Prevalence and Pathogenesis Role, Pneumologia Journal, vol 53, no 3, pag 127-131, 2004

10. M.Nanulescu, P.Chereches-Panta, The Prevalence of Asthma in 13-14 years Children in 1994/1995 –2000/2001 in Cluj County, Journal of Romanian Society of Allergology and Clinical Immunology, vol II, no 3, pag 26-29, 2005

11. C. Pop, P. Chereches-Panta, M. Nanulescu, The Life Quality In Children With Asthma and Allergic Rhinitis, Journal of Romanian Society of Pneumology, Pneumologia, vol 55, no2, pag 74-79, 2006

12. M. Nanulescu, I. Crisan, The Association Between Asthma and Allergic Rhinitis, Journal of Romanian Society of Allergology and Clinical Immunology, no 2, pag 16-18, 2007

13. C. Valeanu, S. Tatar, M. Nanulescu, A. Leucuta, G. Ichim, The Relation between Asthma and Obesity in School Children, Journal of Romanian Society of Pneumology, Pneumologia, Vol 58, no 1, pag 55-58, 2009

CURRICULUM VITAE

Name Sebastian L Johnston Date of birth 30th March 1959

Address Department of Respiratory MedicineNational Heart and Lung Institute andWright Fleming Institute of Infection & ImmunityImperial College of Science, Technology & Medicine Norfolk Place, London W2 1PGTel 020 7594 3764 Fax 020 7262 8913Email s.johnston@ic.ac.uk

Qualifications 1982 MB BS (Guys Hospital, London UK)1993 PhD (University of Southampton UK)2000 FRCP (London UK)

GMC registration number 2620176

Present PostSept 1999 - Professor of Respiratory Medicine

National Heart and Lung InstituteImperial College London

Previous AppointmentsJuly 1996 – Aug 1999 Senior Lecturer in Medicine, Respiratory Medicine &

Respiratory Infection, University of Southampton

Jun 1992 - Jul 1996 Lecturer in Medicine and Respiratory MedicineUniversity of Southampton

Jan 1994 - Jan 1995 MRC Travelling Research FellowUniversity of Iowa, Iowa, USA.

Research GrantsDate Awarding Body Purpose of Award Duration Amount

2005 Asthma UK Role of macrophage-type 1 interferon 3 yr £168,992Grant number 05/067 production in rhinovirus induced

asthma exacerbation.

2006 Firestone Institute for The Diary of Asthma and Viral 1 yr £66,150Respiratory Health Infections Study

2006 Asthma UK Regulation of MUC5B expression 3 yr £196,153Grant number 06/050 by respiratory virus infection: role in

the pathogenesis of asthma

2006 British Lung Foundation Regulation of rhinovirus induced 3 yr £119,979Grant number PO6/13 INF- and pro-inflammatory cytokine

production in bronchial epithelial cellsfrom asthmatic and normal individuals

2006 Medical Research Council Role of oxidative and nitrative 4 yr £991,076Grant number G0600879 stress and histone de-acetylation

in rhinovirus induced acuteexacerbations of COPD

2007 Medical Research Council Mechanisms of deficient innate 3 yr £1,219,732Grant number G0601236 immune responses in asthma

2007 Medical Research Council Biomarkers to target antibiotic and 3 yr £1,063,284Grant number G0601369 steroid therapy in COPD exacerbations

2007 Wellcome Trust Grant Centre for Respiratory Infection 3 yr £3,400,000number 083567/Z/07/Z

2008 Asthma UK Alveolar macrophage IL-10/IFN- 3 yr £156,085Grant number 08/048 production in rhinovirus

induced asthma exacerbations

2008 European Research Council Human and mouse models of 5yr €2,497,762Advanced Research Grant rhinovirus induced acute 233015 asthma exacerbations

2008 Biomedical Research Centre Respiratory Theme Award 1 yr £36,000

2009 NIHR Senior Investigator Award 5 yr £60,000

2009 Biomedical Research Centre Translational studies in a human 2 yr £143,418Project Grant P26095 model of rhinovirus induced acute

exacerbations of asthma.

2009 Wellcome Trust Grant Immune Regulation of Viral 5 yr £1,500,000Lung Disease

2010 NH-MRC grant 631075 The role of microRNAs in antiviral 3yr A$583,500and inflammatory responses during experimental rhinovirus infection

Sebastian L Johnston

Major Publications

1. Papadopoulos NG, Stanciu LA, Papi A, Holgate ST and Johnston SL. A defective

type 1 response to rhinovirus in atopic asthma. Thorax 2002;57:328-32

2. Wark PAB*, Johnston SL*, Bucchieri F, Powell R, Puddicombe S, Laza-Stanca V,

Holgate ST, Davies DE. Asthmatic bronchial epithelial cells have a deficient innate

immune response to infection with rhinovirus. J Exp Med 2005;201:937-947.

*=equal contributors.

3. Contoli M, Message SD, Laza-Stanca V, Edwards MR, Wark PAB, Bartlett NW,

Mallia P, Stanciu LA, Parker HL, Slater L, Kon OM, Holgate ST, Davies DE,

Kotenko SV, Papi A, Johnston SL. Role of deficient type III interferon-λ production

in asthma exacerbations. Nature Med 2006;12:1023-1026.

4. Laza-Stanca V, Stanciu LA, Message SD, Edwards MR, Gern JE, Johnston SL.

Rhinovirus replication in human macrophages induces NF-kappaB-dependent tumor

necrosis factor alpha production. J Virol 2006 Aug;80(16):8248-58.

5. Khaitov MR, Laza-Stanca V, Edwards MR, Walton RP, Rohde G, Contoli M, Papi A,

Stanciu LA, Kotenko SV, Johnston SL. Respiratory virus induction of alpha-, beta-

and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear

cells. Allergy. 2009 Mar;64(3):375-86. Epub 2009 Jan 28

6. Corne JM, Marshall C, Smith S, Schreiber J, Sanderson G, Holgate ST, Johnston SL. Frequency, severity and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study. Lancet 2002;359:831-4.

7. Hewson CA, Jardine A, Edwards MR, Laza-Stanca V, Johnston SL. Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells. J Virol. 2005;79:12273-9.

8. Johnston SL, Blasi F, Black PN, Martin RJ, Farrell DJ, Nieman RB. The Effect of Telithromycin in Acute Exacerbations of Asthma. N Engl J Med 2006;354:1589-600.

9. Bartlett NW, Walton RP, Edwards MR, Aniscenko J, Caramori G, Zhu J, Glanville N, Choy KJ, Jourdan P, Burnet J, Tuthill TJ, Pedrick MS, Hurle MJ, Plumpton C, Sharp NA, Bussell JN, Swallow DM, Schwarze J, Guy B, Almond JW, Jeffery PK, Lloyd CM, Papi A, Killington RA, Rowlands DJ, Blair ED, Clarke NJ, Johnston SL. Mouse models of rhinovirus-induced disease & exacerbation of allergic airway inflammation. Nature Med 2008 Feb;14(2):199-204. Epub 2008 Feb 3.

10. Message SD, Laza-Stanca V, Mallia P, Parker HL, Zhu J, Kebadze T, Contoli M, Sanderson G, Kon OM, Papi A, Jeffery PK, Stanciu LA, Johnston SL. Rhinovirus induced lower respiratory illness is increased in asthma and related to virus load and Th1/2 cytokine and IL-10 production. Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13562-7.