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The genus Borrelia is a widespread, pathogenic pest and the causative of among others borreliosis or Lyme disease. The vector for the bacteria is the hard tick, Ixodes ricinus and the infection is transferred through a bite. Untreated, Borrelia may cause arthritis, heart damage or neuroborreliosis. Infection is made possible through different strategies for avoiding the body’s complement system. One such strategy involves the binding of factor H to specific bacterial membrane proteins and, thus, no complement activation and lysis. Another defence mechanism, phagocytosis, acts in cooperation with the complement and is subsequently retarded. The present study includes optimizing of Borrelia culturing, characterisation of different Borrelia strains in regard to sensitivity against the complement including culture counting and the analysis of free C3a and the Terminal Complement Complex (TCC). Further, tagging with FITC in order to study morphology as well as phagocytosis with the aid of microscopy and FACS was performed. The culturing experiments showed Borrelia to be rather easy to culture, although a strict sterile technique has to be applied. Concerning sensitivity to complement, the B.afzelii strains showed to be resistant to complement action, while most of the B. garninii are sensitive. Analysis of C3a and TCC after incubation with normal human blood serum as well as human whole blood, showed that complement activation demands rather or very high cell densities. Tagging with FITC followed by microscopic analysis resulted in good illustrations of the bacterial morphology. The FACS analysis resulted in findings of phagocytosis in both monocytes and granulocytes and, further, the different stages of phagocytosis were visualised through nuclear staining followed by microscopy. The genus Borrelia is a widespread, pathogenic pest and the causative of among others borreliosis or Lyme disease. The vector for the bacteria is the hard tick, Ixodes ricinus and the infection is transferred through a bite. Untreated, Borrelia may cause arthritis, heart damage or neuroborreliosis. Infection is made possible through different strategies for avoiding the body’s complement system. One such strategy involves the binding of factor H to specific bacterial membrane proteins and, thus, no complement activation and lysis. Another defence mechanism, phagocytosis, acts in cooperation with the complement and is subsequently retarded. The present study includes optimizing of Borrelia culturing, characterisation of different Borrelia strains in regard to sensitivity against the complement including culture counting and the analysis of free C3a and the Terminal Complement Complex (TCC). Further, tagging with FITC in order to study morphology as well as phagocytosis with the aid of microscopy and FACS was performed. The culturing experiments showed Borrelia to be rather easy to culture, although a strict sterile technique has to be applied. Concerning sensitivity to complement, the B.afzelii strains showed to be resistant to complement action, while most of the B. garninii are sensitive. Analysis of C3a and TCC after incubation with normal human blood serum as well as human whole blood, showed that complement activation demands rather or very high cell densities. Tagging with FITC followed by microscopic analysis resulted in good illustrations of the bacterial morphology. The FACS analysis resulted in findings of phagocytosis in both monocytes and granulocytes and, further, the different stages of phagocytosis were visualised through nuclear staining followed by microscopy. / Bakteriegruppen Borrelia burgdorferi har en stor utbredning och förorsakar sjukdomen borreliosis, även kallad Lyme disease, som orsakar mycket lidande. Sjukdomen är en zoonos och smittämnet, bakterien, sprids via en vektor, fästingen Ixodes ricinus. Både människor och djur kan smittas i samband med bett från fästingen. Sjukdomen borreliosis börjar oftast som en lokal hudinfektion med ett karakteristiskt utslag, erythema migrans. Utslaget uppvisar en blek mittregion som omges av en rodnad, 4 – 5 cm i diameter. Antibiotikabehandlig är ofta framgångsrik. Utan behandling kan sjukdomen få allvarliga konsekvenser med påverkan på leder, hjärta, andra organ och centrala nervsystemet (CNS). Hur bakterierna lyckas sprida sig i kroppen och orsaka kroniska tillstånd är ännu inte kartlagt. Det har dock visats att en del av borreliaarterna har en utvecklad strategi för att undgå en del av kroppens immunförsvar genom inaktivering av komponenter i komplementsystemet. Komplementsystemet utgör en del av kroppens immunförsvar och består av ett 30-tal lösliga proteiner i inaktiv form. Vid aktivering till exempel vid invasion av mikrober interagerar proteinerna med varandra och generar produkter, som i sin tur resulterar i olika effektorfunktioner. I samband med proteolytisk aktivitet frisätts mindre produkter, som kan inducera kärlvidgning, agera som kemotaktiskt stimuli för fagocyterande celler och inducera opsonisering. Det är sedan tidigare känt att förhållandet till komplementsystemet skiljer sig åt hos olika borreliaarter. Arter och stammar inom gruppen Borrelia burgdorferi sensu lato interagerar med proteiner från värdens komplementsystem för att på så sätt undgå komplementattack. Faktor H är exempel på ett sådant protein. Borreliabakterien kan uttrycka receptorer i sitt yttermembran, som binder in denna faktor. Vid medverkan av faktor H dämpas komplementaktiveringen och bakterien kan undgå lys. Målet med föreliggande studie var att optimera odlingsbetingelserna för Borrelia följt av kartläggning av olika stammar av Borrelia burgdorferi sensu lato avseende känslighet respektive resistens mot serumkomplement och dess beroende av serumkoncentration respektive bakterietäthet. Vidare skulle grad av komplementaktivering för olika stammar bedömas, liksom inmärkning med FITC följt av mikroskopisk och/ eller FACS - analys för studium av morfologi och fagocytos. För bedömning av känslighet respektive resistens för olika stammar gentemot komplementsystemet, genomfördes räkning av levande bakterier genom faskontrastmikroskopi. Resultaten avseende bakteriehalter kontra komplementaktivering analyserades med hjälp av ELISA-teknik och analys av lösligt C3a respektive lösligt Terminal Complement Complex (TCC). Samtliga i projektet ingående borreliastammar, med ett undantag Borrelia garninii Pbi, befanns vara lättodlade under förutsättning av sträng sterilteknik och upprepad subkultivering. En förklaring till problemet med B. garninii Pbi kan vara en observerad längre lagfas.  Observation är subjektiv utan experimentell studie. Resultaten av räkning av överlevande bakterier som mått på känslighet/resistens mot komplementsystemet i humant normalserum visade att samtliga Borrelia afzelii – stammar var resistenta medan flertalet Borrelia garninii - stammar var känsliga. Resultaten från analyser av fritt C3a och TCC efter inkubering med NHS visar att komplementaktivering sker först vid ökande och mycket höga bakterietätheter. Försök med inkubering med NHS respektive hirudinbehandlat helblod visade inte på någon nämnvärd skillnad i utfall. FITC-inmärkning av B. afzelii K78 och B. garninii Lu59 resulterade i goda illustrationer av spiroketernas morfologi. FACS -analys av fagocyterade inmärkta bakterier visade att både monocyter och granulocyter har förmåga att fagocytera borreliabakterier och att EDTA märkbart hämmade fagocytosen. Vidare tycks B. afzelii K78 fagocyteras i något större omfattning än B. garninii Lu59. De  olika stadierna i fagocytosförloppet framgår av den mikroskopiska analysen av kärninfärgade preparat / Borrelia, Faktor H, Odling, Bakterier

The Enterococcus genus consists of natural members of the gastrointestinal tract but they are also opportunistic pathogens. They are a common cause of urinary tract infections but can also cause sepsis and other infections. Enterococcus faecalis and Enterococcus faecium are the most abundant in clinical specimens. Enterococci are a leading cause of nosocomial infections and they have developed resistance against a number of antibiotics e.g. vancomycin. E. faecalis V583 was the first vancomycin resistant isolate reported in the U.S. Movable genetic elements such as pathogenicity islands, PAI, are important for bacterial evolution. PAI:s are large chromosomal fragments mostly seen in pathogenic strains and carry regions such as transposons and insertion elements along with virulence factors and transfer genes. A PAI has been detected in the chromosome of E. faecalis. Excision of PAI:s has been studied for uropathogenic E. coli and frequencies of 10-5 and 10-6 have been reported. In this study the excision rate and excision patterns of E. faecalis V583 was studied using the counterselectable marker pheS*, causing p-Cl-phe sensitivity, and a chloramphenicol resistance gene, cat, inserted at two different positions of the PAI and selecting for excisions by growth on p-Cl-phe. Excision rates of 10-6 and 10-8 were seen based on the p-Cl-phe resistance and chloramphenicol sensitivity. Mutation rate in the pheS* gene was high compared to excision rate which made the method difficult to work with. No obvious excision patterns were detected but the excisions seemed to be limited to the close surroundings of the pheS*/cat insertion. / Bakterier finns överallt i vår omgivning och hos oss människor, exempelvis på huden och i vår mag-tarmkanal. Flertalet av dessa är apatogena, d.v.s. orsakar inte sjukdom. Vissa normalt goda bakterier kan dock orsaka sjukdom när de hamnar på fel plats och får tillfälle att orsaka sjukdom s.k. opportunistiska patogener. Ett exempel på detta är bakterien Genetiska egenskaper hos såväl människor som bakterier styrs av arvsmassan, DNA. Hos människor är arvsmassan samlad i 46 kromosomer medan bakterier har en. På senare år har vi lärt oss hur man kan klippa och klistra i exempelvis bakteriers DNA för att introducera egenskaper eller ta bort. Detta används inom forskning för att studera t.ex. bakteriers förmåga att orsaka sjukdom eller anpassning till sin omgivning. Bakterier är mycket duktiga på just anpassning vilket beror på deras förmåga att snabbt förändra sitt DNA ofta genom utbyte med andra bakterier, detta kan bl.a. leda till utveckling av antibiotikaresistens eller nyvunnen förmåga att orsaka sjukdom. Största delen av en bakteries arvsmassa består av konserverade regioner medan andra är mycket föränderliga exempelvis s.k. isertions element, tansposoner och patogenicitetsöar, som har visat sig kunna lämna kromosomen via excision. En patogenicitetsö har hittas hos I den här studien klistras en gen in i patogenicitetsön hos Bakteriekloner där excision förekommit erhölls och excisionsfrekvensen bestämdes till 10 bakterier. Inga kloner där hela patogenicitetsön lämnat kromosomen kunde detekteras, dock visade det sig att områden precis intill området där genen klistrats in hade försvunnit. Inga tydliga excisionsmönster kunde bestämmas. En hög frekvens av mutationer i den insatta genen gjorde metoden svår att arbeta med. Enterococcus faecalis som finns i mag-tarmkanalen hos friska människor men som när den hamnar på fel plats kan orsaka bl.a. urinvägsinfektion, sårinfektioner och i svåra fall blodförgiftning, s.k. sepsis. E. faecalis. Patogenicitetsöar är delar av kromosomen som ofta innehåller virulensfaktorer, som gör bakterien patogen och hos vissa bakterier har man påvisat gener för antibiotikaresistens på sådana öar. E. faecalis som gör att bakterien inte kan växa på ett speciellt selektivt media. Detta gör det möjligt att välja bakterier där ön lämnat kromosomen för vidare studier och en excisionsfrekvens bestämmas. -6 till 10-8. Detta är låga frekvenser jämfört med vad man kommit fram till hos andra 3

Malaria is one of the world’s most threatening diseases. About half the world’s population is at risk of infection and the infection claims a million lives each year. A vast majority of the deaths occur in children below the age of 5 in sub-Saharan Africa. Survivors typically acquire immunity only after long time of repeated exposure and immunity is rapidly lost. Immunity is created by the activation of naive T cells and their differentiation into effector cells. The most potent activators of naive T cells are dendritic cells (DCs). The life cycle of DCs is adapted to find and process microbes in order to be able to present their antigens to T cells and thereby activate them. Antigen presentation typically takes place in the lymph nodes and that is why migration to these areas is an essential part of the DC life cycle. Various studies have shown that DC function may be hampered by the malaria parasite or its components. We have investigated activation and migratory capacities of DCs upon in vitro exposure of the malarial pigment hemozoin and Plasmodium falciparum infected red blood cells. Furthermore, we have assessed the activation status of blood DCs in the Fulani, a traditionally nomadic population that respond better to malaria infection and exhibit less clinical symptoms than other ethnicities living under similar conditions, and a neighbouring ethnic group, the Dogon, in Mali. Our results indicate that DCs are semi-activated upon malaria exposure in vitro, including enhanced migratory capacity, partial up-regulation of co-stimulatory markers and no IL-12, which may lead to inappropriate T-cell priming. We also observed that DCs from the Fulani have a higher degree of activation than DCs from the Dogon upon malaria exposure in vivo. We hypothesize that this increased DC activation may be the reason for the relatively increased protection against malaria. Taken together, our findings suggest that improper DC activation may contribute to poor immunity in Malaria.

More than a century after the identification of the tubercle bacillus and the first attempts at vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious diseases. TB is typically a disease of the lung, which serves both as port of entry and as the major site of disease manifestation. The currently used vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is administered parentally and induces a systemic immune response. However, it fails to protect against pulmonary TB, thereby raising the question whether vaccination targeting the mucosal immunity in the lungs could be favourable. The respiratory mucosal surfaces represent the first line of defence against a multitude of pathogens. Secretory IgA (sIgA) in mucosal secretions has an important function by blocking entrance of pathogenic organisms and preventing infections. Yet, another role for IgA in protection against intracellular pathogens has lately been appreciated, when sIgA was demonstrated to neutralize viruses intracellulary. We aimed to investigate the relevance of sIgA in protection against mycobacterial infections using mice deficient for IgA and the polymeric Ig receptor. Mice were immunized intranasally with a mycobacterial antigen which elicited, in wild-type mice, a strong IgA response in mucosal secretions in the respiratory tract. Gene-targeted mice failed to induce the same response and more importantly, were more susceptible to mycobacterial infections in the respiratory tract, as demonstrated by higher bacterial loads in the lungs than wild-type mice. Analysis of immune responses after infection revealed reduced production of proinflammatory, and protective, factors such as IFN-γ and TNF-α in the lungs of deficient mice, which was in concordance with the higher bacterial burden seen in the lungs of these mice. The mechanisms explaining the defective proinflammatory responses in the lungs of deficient mice are not clear but might involve impaired signalling through Fcα receptors, or homologous receptors, which could lead to inadequate activation of pulmonary macrophages. This could subsequently result in suboptimal induction and production of cytokines and chemokines important for attraction and migration of cells to sites of infection in the lungs. Our results demonstrate a role for IgA in protection against mycobacterial infection in the respiratory tract by blocking the entrance of the mycobacterium into the lungs, and/or by modulating the locally induced proinflammatory immune responses.

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Immunotherapies for cancer are widely studied at present. We are currently studying a specific form of “Vγ9Vδ2 T cells” found in the peripheral blood of healthy donors that can be used for the killing of HT-29 colon cancer cells. In order to determine the cytotoxicity of effectors, Vγ9Vδ2 T cells towards target cells, HT-29, it is important to first evaluate the absolute number of Vγ9Vδ2 T cells in a mixed cell population, and next to determine the phenotypic characterization, their activity and cytotoxicity in the presence of target cells. A flow cytometry and bead based assay was developed to evaluate the absolute number of Vγ9Vδ2 T cells in a mixed cell population. Peripheral Blood Mononuclear Cells (PBMCs) were surface stained with monoclonal antibodies (MoAbs) conjugated to fluorochromes that are cross reactive to cell surface markers such as CD3 (T Lymphocytes), γδ2 and were mixed with fluorophore beads. In these assays, no washes and centrifugation steps were performed after the cell surface staining and bead addition. The absolute cell counts were evaluated based on referencing a known concentration of beads. In addition, quantification assays were also performed to measure the cell and bead loss on surface staining that included washes and centrifugation steps and thus found a higher percentage loss of cells than beads. Immunophenotyping assays with four color staining were performed to monitor the phenotypic differentiation of effector cells based on cell surface markers CD27 and CD45RA. Only the naïve (CD27+CDRA+) and terminally differentiated effector memory (CD27-CD45RA+) were identified on the assays performed using Vγ9Vδ2 T cells of different donors. A flow cytometry based cytotoxicity (FCC) assay was completed to monitor the effector cell activity (CD69+) in the presence and absence of target cells and also the cytotoxicity was measured based on % specific lysis of target cells at four different effector to target (E:T) cell ratios. Only preliminary data were obtained for the FCC assay and the development is still in progress.

This review is a dissertation and contains a summary of the following publications: I. J.-O Jeppsson and J. Sjöquist: Separation of Normal Human Transferrin into Two Fractions. Biochim. Biophys. Actay 78 (1963) 658 II. J.-O. Jeppsson: Isolation and Partial Characterization of Three Human Transferrin Variants. Biochim. Biophys. Acta, 1967, in press III. J.-O. Jeppsson: Subunits of Human Transferrin. Acta Chem. Scand.1967, in press IV. J.-O. Jeppsson and J. Sjöquist: Thin-layer Chromatography of PTH Amino Acids. Analyt. Biochem. 18 (1967) 264 V. J.-O. Jeppsson: Structural Studies of Fragments Resulting from Cyanogen Bromide Degradation of Human Transferrin. Biochim. Biophys. Acta, 1967, in press. In addition the dissertation contains some hitherto unpublished results. In the text the above mentioned papers will be referred to by the Roman figures I — V, other references are indicated by Arabic figures. / digitalisering@umu.se