Molekulska i stanična svojstva imunopatofiziološkog odgovora na klicu i cjepivo Bordetellae pertussis u mišjem modelu

doctoral thesis

• Title (english): Molecular and cellular properties of immunopathophysiologic response to Bordetella pertussis bacterium and vaccine in the mouse model]
• Author: Jerko Barbić; MBZ: 258420
• Mentor: Zdenko Kovač (mentor)
• Committee member: nije dostupno (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2003-12-08, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences
• Universal decimal classification (UDC): 61 - Medical sciences

Abstract

U ovoj disertaciji analizirana su svojstva staničnog i molekulskog odgovora na infekciju i imunizaciju u mišjem modelu infekcije B. pertussis. Dobiveni rezultati pokazuju da klica B. pertussis uspješno kolonizira respiratorni epitel, odnosno da se uzročnik može uzgojiti iz tkiva pluća i dušnika nakon aerosolne infekcije eksperimentalnih životinja. Nakon 5 tjedana infekcije odrasli imunodostatni miševi otklanjaju klice iz respiratornog tkiva. Neonatalni miševi ugibaju, jer zbog svoje imunonedostatnosti ne mogu nadzirati infekciju. Kada nastupi uklanjanje klice iz plućnog tkiva, tada se u serumu i bronhoalveolarnom ispirku javljaju protutijela specifična za virulentne sastavnice B. pertussis. U područnim traheobronhalnim limfnim čvorovima inficiranih životinja može se dokazati nazočnost limfocita T CD4+ specifičnih za B. pertussis koji proliferiraju u uvjetima ex vivo pri pobudi s antigenima klice fiksirane u formalinu. U nadtalogu tako pobuđenih limfocita T može se dokazati proizvodnja IFN- ali ne i IL-4, odnosno IL-10. U tijeku praćenja infekcije nazočnost serumskih i sluzničkih protutijela specifičnih za B. pertussis može se dokazati samo u razdoblju kada dolazi do uklanjanja klica iz respiratornog tkiva. Suprotno tomu, nakon infekcije izražaj mRNK upalotvornih citokina u plućnom tkivu kolerira s količinom klica B. pertussis uzgojenih iz tkiva. Imunonedostatne odrasle životinje s nedostatkom limfocita T i B ( miševi SCID) i one samo s nedostatkom limfocita T (atimični miševi nude) koje ugibaju 3-5 tjedana nakon infekcije pokazuju važnost stanica specifične imunosti u nadzoru infekcije B. pertussis. Ove imunonedostatne životinje u trenutku ugibanja pokazuju pojačani izražaj mRNK za upalotvorne citokine. Nakon tjedan dana infekcije u plućnom tkivu imunodostatnih miševa javlja se izražaj mRNK za IFN-. Važnost IFN- pokazana je pokusima neutralizacije protutijelima ovog citokina in vivo, odnosno tijekom infekcije kod IFN--/-KO životinja koje pokazuju veći broj uzgojenih klica iz plućnog tkiva. Suprotno tomu, neutralizacija IL-4 nema učinak na broj uzgojenih klica.

Intranazalna imunizacija odraslih miševa staničnim odnosno bestaničnim cjepivom B. pertussis ima zaštitni učinak u aerosolnom modelu infekcije. Ova dva oblika imunizacije se razlikuju u zaštitnom mehanizmu što se ogleda u činjenici da se kod imunizacije cijelim klicama ne može utvrditi korelacija između broja kolonija klica u tkivima s razinom protutijela specifičnih za B. pertussis. Limfociti T dobiveni iz područnih limfnih čvorova proizvode IFN- ako su dobiveni iz životinja imuniziranih staničnim cjepivom. Suprotno njima, limfociti T dobiveni iz miševa imuniziranih bestaničnim cjepivom proizvode IL-4 i IL-10. Miševi s izbačenim limfocitima B ne mogu se u potpunosti zaštititi imunizacijom staničnim cjepivom. Rekonstitucija imunim limfocitima B ima zaštitni učinak bez postojanja specifičnih protutijela u serumu rekonstituiranih životinja. Imunizacija miševa koji nemaju limfocite T ili samo limfocite T CD4+ nije moguća. Suprotno tomu, miševi s nedostatkom limfocita T CD8+ mogu se uspješno imunizirati staničnim cjepivom za B. pertussis. Prema tomu, imunizacija cijelom klicom B. pertussis zahtijeva prisutnost limfocita T CD4+ i limfocita B. Ovi rezultati pokazuju kako postoje dva različita mehanizma zaštite kod imunizacije protiv infekcije B. pertussis. Jedan oblik imunizacije pokazuje povezanost s razinom protutijela specifičnih za B. pertussis, a kod drugoga ova veza nije dokazana. Rezultati prikazani u ovoj disertaciji mogu doprinijeti razumijevanju mehanizma infekcije B. pertussis i zaštitnih mehanizama imunizacije za ovu klicu, što može imati praktičnu važnost u stvaranju novih oblika cjepiva protiv ove zarazne bolesti.

Abstract (english)

Cellular and molecular response to infection and immunisation in mouse model of B. pertussis infection were studied in the disertation. The results show that B. pertussis microorganisms successfully colonize respiratory epithelium, i.e. the bacteria could be cultured from the lung and tracheal tissue taken from previously infected experimental animals. Five weeks after infection adult immunocompetent mice clear away microorganisms from respiratory tissue. Neonatal mice die due to immunological incompetence that makes them unable to control infection. When elimination of microorganisms from lung tissue ensues, specific antibodies against virulent components of B. pertussis appear in serum and bronchoalveolar lavage fluid. The presence of CD4+ T lymphocytes specific for B. pertussis that proliferate after ex vivo stimulation by antigens of microorganisms fixated in formalin could be detected in regional tracheobronchal lymph nodes of the infected animals. The production of IFN-γ, but not IL-4 nor IL-10, can be demonstrated in the supernatant of thus stimulated T lymphocytes. During the follow up of the infection, specific serum and mucosal antibodies against B. pertussis could be demonstrated only in the period of elimination of the microorganisms from respiratory tissue. Unlike this, the expression of inflammatory cytokines mRNK in lung tissue after infection correlates with the amount of B. pertussis bacteria cultured from the tissues. Immunologically defective adult animals lacking in T and B lymphocytes (SCID mice), as well as those lacking only in T lymphocytes (atymic mice, nude) which die 3-5 weeks after infection, show the importance of cells of specific immunity in the control of B. pertussis infection. The immunodeficient animals show enhanced expression of inflammatory cytokines mRNK at the death time. The expression of IFN-γ mRNK in lung tissue of immunocompetent mice appear a week after the infection. The importance of IFN-γ has been demonstrated by the in vivo neutralization of the cytokines by antibodies and by the demonstration of more microorganisms bred from lung tissue of infected IFN-γ-/-KO animals. Unlike this, IL-4 neutralization does not affect the number of bred microorganisms.

Intranasal immunization of adult mice by cellular or acellular B. pertussis vaccine has protective effect in the aerosol model of infection. These two forms of immunization differ in the protective mechanism, which reflects in the lack of correlation with titer of antibodies against B. pertussis when whole microorganisms are applied. T lymphocytes from regional lymph nodes produce IFN-γ if taken from animals immunized with cellular vaccine. Unlike this, T lymphocytes from mice immunized with acellular vaccine produce IL-4 and IL-10. Mice knocked out for B lymphocytes cannot be completely immunized with cellular vaccine. Reconstitution with immune B lymphocytes has protective effect without presence of specific antibodies in the serum of reconstituted animals. Immunization of mice lacking in T lymphocytes or lacking only in CD4+ T lymphocytes is not possible. Unlike this, mice deficient in CD8+ T lymphocytes can be successfully immunized with cellular B. pertussis vaccine. Based on these evidences, immunization with whole B. pertussis microorganism requires presence of CD4+ T lymphocytes and B lymphocytes. The results indicate the presence of two different protective mechanisms of immunization against B. pertussis infection. One of them demonstrates a correlation with the titer of specific B. pertussis antibodies, while the other lacks in that relation. The results presented in the thesis could contribute in comprehension of the mechanisms of B. pertussis infection and protective mechanisms of immunisation against that bacterium, which could be practically important for production of new forms of vaccines against that infective disease.

• Keywords: medicina
• Keywords (english): medicine
• Language: croatian
• URN:NBN: urn:nbn:hr:105:247920
• Study programme: Title: Biomedicine and Health Sciences; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstva (doktor/doktorica znanosti, područje biomedicine i zdravstva)
• Type of resource: Text
• Extent: 148 str.
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-05-05 06:50:01