Borrelia burgdorferi sensu lato (s.l.) and tick-borne encephalitis (TBE) are two of the most common tick-borne diseases in Sweden. Varicella-zoster-virus (VZV) is a contagious, airborne herpesvirus with nearly 100% prevalence in individuals over 30 years old. At the Laboratorymedicine, County Hospital Ryhov, Jönköping, IgG-antibody analysis against B. burgdorferi s.l., TBE and VZV is performed on serum. The aim of the study was to compare ethylenediaminetetraacetic-acid-plasma and lithium-heparin-plasma with serum for thesea nalyses. The goal was to investigate whether it was possible to switch the sample material from serum to plasma for these analyses. Samples from 500 blood donors were collected for analysis in serum, ethylenediaminetetraacetic-acid-plasma and lithium-heparin-plasma. The qualitative data from the study indicated that there was no statistically significant difference in the interpretations (positive and negative) between the sample materials. The quantitative analysis of antibody levels showed a statistically significant difference between the sample materials. Due to the study´s limited sample size, no general conclusions could be drawn about whether plasma could replace serum. Further studies on the subject could provide increased understanding of the relationship between serum and plasma for analysis of Borrelia, TBE and VZV and how results are affected in the different sample materials. / Borrelia burgdorferi sensu lato (s.l.) och tick-borne encefalit (TBE) är två av de vanligaste fästingöverförda sjukdomarna i Sverige. Varicella-zoster-virus (VZV) är ett smittsamt, luftburet herpesvirus med nära 100% prevalens hos personer över 30 år. På Laboratoriemedicin, Länssjukhuset Ryhov, Jönköping utförs analys av IgG-antikroppar mot B. burgdorferi s.l. TBE och VZV på serum. Syftet med studien var att jämföra etylendiamintetraättiksyra-plasma och litiumheparinplasma mot serum för dessa analyser. Målet var att undersöka om det var möjligt att byta provmaterial från serum till plasma för nämnda analyser. Prover från 500 blodgivare samlades in för analys i serum, etylendiamintetraättiksyra- och litiumheparin-plasma. Resultatet från studiens kvalitativa data indikerade att ingen statistisk signifikant skillnad förelåg vad det gäller tolkningarna (positiv och negativ) mellan provmaterialen. Kvantitativa analysen av antikroppsnivåerna visade en statistisk signifikant skillnad mellan provmaterialen. På grund av studiens begränsadeprovstorlek kunde inga generella slutsatser dras kring huruvida plasma kunde ersätta serum. Vidare studier kring ämnet kan ge ökad förståelse kring sambandet mellan serum och plasma för analys av Borrelia, TBE och VZV samt hur resultaten påverkas i de olika provmaterialen.

<p>Background: The reasons why some individuals become IgE-sensitised and allergic are largely unknown, though genetic- and early life environmental factors seem to be of importance.</p><p>Objective: The overall aim of this thesis was to investigate the relationship between IgE-sensitisation and allergic disease, viral infections, genetic markers and early life cytokines.</p><p>Results: IgE-sensitised children were found to have reduced numbers of IL-12 producing cord blood mononuclear cells (CBMC), whereas children diagnosed with eczema were found to have reduced numbers of IFN-γ producing CBMC. When dividing the children into early onset of IgE-sensitisation and late onset of IgE-sensitisation we found that the children with an early onset had low numbers of PHA-induced IL-4, IL-12 and IFN-γ secreting CBMC. At the age of two there was a general exacerbation of cytokine responses in the IgE-sensitised children, and the results were similar for the children with early onset IgE-sensitisation. Children with a late onset IgE-sensitisation were more similar to the non-sensitised children, but with a specific increase in the response to cat allergen (IL-4 and IFN-γ). The mothers of IgE-sensitised children, were just as their children, found to have an exaggerated cytokine response as compared to mothers of non-sensitised children. Maternal responses correlated well to the responses seen in the child, though the samples were taken two years after delivery.</p><p>Cytomegalovirus (CMV) infection in early life was associated to reduced numbers of IL-4, and increased numbers of IFN-γ producing cells at the age of two. No association between CMV seropositivity and IgE-sensitisation was seen. Epstein-Barr virus (EBV) infection, on the other hand, was inversely correlated with IgE –sensitisation, whereas no statistically significant association to cytokine production could be seen.</p><p>We also showed that the IL12B 1188 C-allele was associated to having a positive skin prick test at the age of two. The rare alleles of the three SNPs investigated (IL12B 1188C, IL12RB1132C and IRF1 1688A) were all associated to low IL-12 production at birth.</p><p>Conclusions: Our results indicate that allergic diseases are complex traits, and that both the genetic and the cytokine background differ between the different allergic diseases. We can also conclude that the time of onset seem to play a role when investigating IgE-sensitisation, and that perhaps early and late onset IgE-sensitisation have partly different causes. CMV and EBV infection early in life are associated to a protective cytokine profile and to protection from IgE-sensitisation, respectively, again indicating the heterogeneity and the complexity of allergic diseases.</p>

<p>Malaria is one of the major infectious diseases in the world with regard to mortality and morbidity, and the development of a vaccine against the malaria parasite <i>Plasmodium falciparum</i> is considered of high priority. The aim of the work presented in this thesis was to develop and characterize recombinant vaccine constructs based on the <i>P. falciparum</i> asexual blood-stage antigen Pf332. We have studied the humoral responses in mice elicited by various types of constructs, including naked DNA plasmids, naked mRNA, alphavirus, and peptides. Immunological memory was successfully induced against the repetitive EB200 fragment of Pf332, although the antibody titers were generally low and the highest titers were unexpectedly obtained with a conventional DNA plasmid. In another study, we also demonstrated the ability to circumvent genetically restricted immune responses in mice against two malaria epitopes, one of them derived from Pf332, by inclusion of universal T-cell epitopes into multiple antigen peptide constructs. However, the overall variability of the responses stressed the importance of including several epitopes in a future malaria vaccine. Further, the recent completion of sequencing of Pf332 enabled us to identify and characterize the immunogenic properties of a non-repeat fragment of the Pf332, termed C231. Our analyses of C231 showed that antibodies raised against the recombinant protein possess an <i>in vitro</i> parasite inhibitory capacity similar to that of antibodies against recombinant EB200. Furthermore, the recognition of C231 by antibodies in sera from individuals naturally primed to <i>P. falciparum</i>, correlated well with that previously observed for the corresponding sera and EB200. When analyzing the IgG subclass distribution of anti-C231 antibodies, we noted a bias towards IgG2 and IgG3 relative to IgG1, differing from the subclass profiles of IgG binding crude <i>P. falciparum</i> antigen, which were dominated by IgG1. Taken together, the work presented herein is likely to facilitate further studies on Pf332 as a potential target for protective immune responses, and amounts to a small step towards the realization of a malaria vaccine.</p>

Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis. Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.

Background: Idiopathic inflammatory myopathies (IIM) constitute a heterogeneous group of diseases with severe consequences for the life of affected patients. Dermatomyositis, polymyositis and inclusion body myositis (IBM) are the classical representatives of this group. The treatments given today often have limited effects, and are taken at the cost of side effects. Major obstacles in the search for more effective treatments are; (1) an incomplete understanding of the disease mechanisms, (2) difficulties to delineate homogeneous disease groups for clinical studies and (3) the sometimes challenging task to diagnose these diseases. Aims: We addressed a number of “loose ends” in the areas of pathogenesis, classification and diagnosis; mechanisms of muscle fiber degeneration in IIM, with a focus of programmed cell death (apoptosis) and invasion of muscle  fibers by inflammatory cells (partial invasion); protecting and mediating factors present in muscle; the association of other diseases with IIM, in particular celiac disease ; the evaluation of two classification systems and laboratory methods for increased diagnostic performance. The studies: We included 106 patients, diagnosed at the Neuromuscular unit in Linköping, Sweden, with pathological muscle findings consistent with IIM. The incidence in the county of Östergötland (during 5 years) was 7.3 per million/year (3 patients each year). Of 88 patients with confirmed IIM 4 (4.5 %) had celiac disease, 33 (38%) had an associated systemic inflammatory disease and 5 (5.7 %) had a malignancy. Ninety-nine patients were included for a comparison of two classification systems using criteria of the European Neuromuscle Centre (Amato/ENMC), and the widely used Bohan and Peter classification, both with the addition of IBM according to Griggs et al. Using the Amato/ENMC criteria the most prevalent diagnostic group after IBM (30%) was nonspecific myositis (23%), followed by polymyositis (20%) and dermatomyositis 17%). A substantial number of patients meeting Bohan and Peter (or Griggs) criteria were excluded by Amato/ENMC criteria, most (21/23) due to lack of detectable muscle weakness. Extended muscle sectioning increased the sensitivity of a muscle biopsy by 15 % and the specificity by 22%, and showed an overlap between disease groups. Muscle biopsies from patients with IIM and controls were used to investigate pathological findings considered specific for disease groups, and for the presence of programmed cell death (apoptosis) and disease protecting and mediating factors in muscle. The presence of apoptotic muscle fiber nuclei was detected in muscle with partial invasion (however not in the invaded fibers) in the presence of granzyme B and CD8+ cytotoxic T cells. The major apoptosis inhibiting protein Bcl-2 was shown to be constitutionally expressed in healthy muscle but weakened in IIM. Conclusion: We present apoptosis as a possible disease mechanism in parallel with partial invasion of fibers. Furthermore, partial invasion may not be a suitable distinguishing feature in the pathogenesis, or for classification and diagnosis of IIM. We also introduce the anti-apoptotic Bcl-2 as a possible relevant muscle fiber protecting factor. A more extensive pathological work-up improves classification and diagnosis of IIM. The proposed Amato/ENMC creates a substantial portion of patients with non-specific or unclassified myositis. Associated diseases are common in IIM, and also include celiac disease.

Humana leukocytantigen (HLA) är en typ av proteiner som uttrycks på alla cellers yta, de karakteriseras som antigenpresenterande och är en mekanism som ryggradsdjur utvecklat för att lokalisera infekterade eller defekta celler. HLA presenterar peptider från cellens insida för immunförsvaret, vilket i sin tur aktiverar andra delar av immunförsvaret eller inducerar apoptos om främmande peptider presenteras. HIV är ett virus som infekterar celler som uttrycker CD4 på ytan, så som makrofager och T-hjälparceller som ingår i immunförsvaret. Dessa sjunker i antal och immunförsvaret blir försvagat. Obehandlad HIV leder till AIDS, därför är bromsmediciner som Abacavir viktigt. Abacavir har visat goda resultat, dock drabbas 5-8% av hypersensitivitetsreaktioner. Forskare har visat att dessa reaktioner är starkt relaterade till HLA-B*57:01 allelen. Genom screening av denna allel kan behandling med Abacavir undvikas för denna grupp patienter. Syftet med denna studie var att jämföra två olika kit, Olerup SSP® HLA-B*57:01 och Inno-train HLA-READY GENE B57, för typning av HLA-B*57 i 20 olika DNA prover. Dessa jämfördes med den befintliga metoden för HLA-typning som används på klinisk immunologi och transfusionsmedicin, Universitetssjukhuset, Lund. Jämförelsen grundade sig på skillnader mellan kiten, både vad gäller resultat, kostnader och analystider.  Proverna analyserades med PCR innan de separerades på agarosgel med gelelektrofores. Resultaten tolkades enligt tillverkarnas anvisningar. Resultaten visade att båda metoderna kan typa HLA korrekt. Alla resultat med Olerup stämde. Däremot blev 8 av 20 prover fel vid första körningen med Inno-train men resultaten stämde efter omkörning. Avseende kostnader är Inno-train billigare per prov men Olerup skulle bli billigare långsiktigt. För att verkligen fastställa vilket kit som är mest lämpligt behövs fler analyser utföras. / Human leokocyte antigen (HLA), a protein present on the surface of our cells, is characterized as antigen-presenting a mechanism vertebrates have developed to locate infected or defect cells. HLA presents peptides which are brought from the cell's inside to be presented for the immune system. HIV is a virus that infects cells that express CD4 on the surface, such as macrophages and T-helper cells. When these are decreasing, the immune system gets weakened. Untreated HIV leads to AIDS, therefore are inhibiting pharmaceuticals like Abacavir important. Abacavir has shown good results, unfortunately 5-8% gets hypersensitivity-reactions. Scientists have shown that these reactions are strongly related to the HLA-B*57:01 allele. By screening for this allele, treatment with Abacavir could be avoided for this group of patients. The purpose of this study was to compare two different kits, Olerup SSP® HLA-B*57:01 and Inno-train HLA-READY GENE B57, for screening of HLA-B*57 in 20 different DNA samples. These have previously been typed with the established method at the clinical immunology and transfusion medicine, Lund University Hospital. The comparison was based on differences between the kits, both in terms of results, costs and analytical times. PCR was run before the samples were separated on gel with electrophoresis. The results were interpreted in accordance to the manufactures instructions. The results showed that both methods could type HLA correctly. All results from Olerup was correct. However, 8 out of 20 samples showed wrong results at the first run with Inno-train. These were correct after a second run. In terms of costs, Inno-train is cheaper per test, but Olerup would be cheaper in the long term. To really determine which kit is most suitable, more analyzes are required.

Att förstå olika tumörers biologi är en viktig förutsättning för att kunna utveckla nya cancerbehandlingsmetoder. Ett nytt verktyg inom cancerterapin, både för att förstå tumörers uppkomst samt hitta nya läkemedelsmål och behandlingar, är det mycket potenta genredigeringsverktyget Clustered Regularly Interspaced Short Palindromic Repeats med CRISPR-Associerade proteiner, CRISPR-Cas9. Det är ett adaptivt immunförsvar funnet hos prokaryoter. CRISPR är ett programmerbart RNA-guidat system som har DNA som mål. Tekniken går att tillämpa inom cancerimmunologin genom att t ex manipulera T-celler på olika sätt. Syftet med den här litteraturstudien var att: 1) undersöka vilka idag pågående kliniska prövningar, med cancerimmunologisk inriktning, som använder sig av CRISPR-Cas9 samt 2) vilka resultat som framkommit; 3) hur CRISPR-Cas9 ska kunna levereras till celler in vivo; 4) vilka problem har stötts på samt 5) hur framtiden kan se ut med CRISPR inom cancerforskningen. Information inhämtades under tidsperioden januari till maj 2019 på främst PubMed, clinicaltrials.gov och Google. Idag pågår 8 kliniska prövningar men studierna har ännu inte publicerat några resultat. Att finna en lämplig leveransmetod för leverans av CRISPR till målcellen är en av de stora utmaningarna med CRISPR där virala metoder är den leveransmetod som hittills har använts mest. Många studier undersöker möjligheterna med lipida nanobärare men ingen leveransmetod överträffar någon annan i dagsläget. Problem som framkommit med CRISPR-Cas9-tekniken är att metoden kan orsaka cancer vid redigering i celler som saknar p53 (ett viktigt tumör-supressor-protein); det kan orsaka patogena konsekvenser pga långa deletioner då Cas9 klyvt DNA; samt Cas9 kan klyva på andra ställen i genomet än det önskade. I framtiden kan CRISPR, med olika Cas-proteiner, komma att användas för att bland annat tillverka universella T-celler med chimär antigenreceptor (CAR-T-celler), förstå tumörers uppkomst och utveckling, finna nya läkemedelsmål, radera DNA-sekvenser från virus som inkorporerat sitt genom i vårt, och kanske även för redigering i könscellslinjen för att minska risker för bland annat cancerutveckling. / Understanding the biologi of tumors is an important prerequisite to develop new methods for cancer treatment. The gene editing tool Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with CRISPR-associated proteins (Cas9), CRISPR-Cas9 is a new tool in cancer therapy, both for understanding the origins of the tumors and for finding treatments and drug targets. It is an adaptive immune system in prokaryotes. CRISPR is a programmable RNA- guided system which targets DNA. The technology is applicable in cancer immunology by e.g. manipulating T-cells. The purpose of this literature study was to: 1) investigate which cancer immunotherapy clinical trials, using CRISPR-Cas9, that are ongoing today and 2) which results have emerged so far; 3) how CRISPR-Cas9 can be delivered to cells in vivo; 4) which problems have arisen, and 5) what the future might hold for CRISPR within cancer research. The information was gathered from January to May 2019 from primarily PubMed, clinicaltrials.gov, and Google. Today, 8 clinical trials are ongoing but the studies have not yet published any results. One of the main challenges of CRISPR is finding a suitable methodology of delivery where viral methods is the one that has mainly been used. Many studies investigate possibility of lipid nanocarriers but as of today, no one single delivery system is superior to the others. The problems with CRISPR-Cas9 that have emerged is that it can cause cancer when editing cells that are missing p53 (an important tumor suppressing protein); it has pathogenic consequenses due to long deletions as Cas9 cuts DNA; and Cas9 can cut the DNA at off-targeted sites. In the future, CRISPR with different Cas-proteins, can be used to manufacture universal Chimeric antigen receptor T cells (CAR-T-cells), understanding the origin and development of tumors, finding new drug targets, deleting DNA-sequences from viruses that have incorporated their genome into ours, and maybe also for editing the germ line in order to reduce the risk of e.g. developing cancer.

<p>Protective immunity against <i>Plasmodium falciparum</i> may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the <i>P. Falciparum</i> infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to <i>P. falciparum</i> infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure.</p><p>Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an <i>in vitro</i> assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes.</p><p>The <i>P. falciparum</i> parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated<i> P. falciparum </i>parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition <i>in vitro</i> by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition <i>in vitro</i>, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses.</p><p>Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area.</p><p>There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against<i> P. falciparum</i> asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.</p>

<p>The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion.</p><p>In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2^d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes.</p><p>We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2^s, H-2^q and H-2^f) mouse strains, silver induced only ANolA responses in H-2^s and H-2^q mice, but not in H-2^fmice. Further studies showed that the resistance to silver-induced ANolA production in H-2^fmice was inherited as a dominant trait.</p><p>We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice.</p><p>Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.</p>

<p>Transition metals such as nickel (Ni), cobalt (Co), palladium (Pd), chromium (Cr) and gold (Au) are widely used as alloys in jewelry and biomaterials such as orthodontic and orthopaedic appliances. These metals also cause cell-mediated allergic contact dermatitis (ACD) reactions in a significant proportion of the population upon prolonged direct exposure. The immune mechanisms underlying the response to these metals are not yet well defined. In the studies described in this thesis we therefore investigated the profile of cytokine responses to various metal ions <i>in vitro</i> and the relationship with the ACD reaction <i>in vivo</i>. In the first study, we investigated the relationship between the profile and magnitude of Ni²⁺-induced cytokine responses <i>in vitro</i> and the degree of <i>in vivo</i> reactivity to Ni²⁺. PBMC from Ni²⁺-reactive (ACD) and non-reactive control subjects were cultured with or without NiCl₂. The numbers of IL-4-, IL-5- and IL-13-producing cells and the concentrations of IFN-γ, IL-10 and IL-13 produced were analysed by ELISpot and ELISA respectively. Ni²⁺ elicited a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects with a positive correlation observed between the levels of the elicited cytokines and the degree of patch test reactivity. Hence, suggesting an involvement of both Th1- and Th2-type cytokines in ACD to Ni²⁺ and a direct association between the magnitude of the Ni²⁺-induced cytokine response overall and the <i>in vivo</i> reactivity to Ni²⁺. The impact of the regulatory cytokine IL-10 on Ni²⁺-induced Th1- and Th2-type cytokine responses in human PBMC was investigated in the next study. PBMC from blood donors with a history of Ni²⁺ reactivity and non-reactive control donors were stimulated with Ni²⁺ <i>ex vivo</i> with or without addition of human recombinant IL-10 (rIL-10) or neutralising mAb to IL-10. Depletion/enrichment experiments were performed to phenotype the Ni²⁺-specific cytokine producing cells. Exogenous rIL-10 significantly down-regulated the production of all cytokines but with a more pronounced effect on IFN-γ. IL-10 neutralisation, on the other hand, enhanced the levels of Ni²⁺-induced IFN-γ only. Ni²⁺-specific cytokine-producing cells in PBMC were found to be predominantly CD4⁺ T cells. Thus, IL-10 may play a regulatory role <i>in vivo</i> by counteracting the ACD reactions mediated by CD4⁺ T cells producing Th1-type cytokines. In the third study, we investigated the relationship between <i>in vivo</i> patch test reactivity to a number of metals (Ni, Co, Pd, Cr and Au) included in the standard and/or dental patch test series and <i>in vitro</i> responses to the metals in question. PBMC from metal patch test positive and negative control subjects were stimulated with a panel of eight metal salts and cytokine responses analysed by ELISpot and/or ELISA. A mixed Th1- (IL-2 and/or IFN-γ) and Th2-type (IL-4 and/or IL-13) cytokine profile was observed in PBMC from most metal allergic subjects upon <i>in vitro</i> stimulation with the metal(s) to which the subject was patch test positive. Our data suggest that other metals included in the standard and dental patch test series, just like Ni2⁺, induce a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects <i>in vitro</i>. We further developed a simplified ELISpot protocol utilising plates precoated with capture monoclonal antibodies (mAb) and subsequent detection in one step using enzyme-labelled mAb, for enumerating the frequency of allergen (Ni²⁺)-specific cytokine producing cells. This was compared with a regular ELISpot protocol, with an overnight incubation for capture mAb adsorbtion and detection with biotinylated mAb followed by enzyme-labelled streptavidin. PBMC from Ni²⁺-reactive and non-reactive subjects were incubated with or without NiCl₂ and the enumeration of cells producing IFN-γ, IL-4 or IL-13 by the two protocols were compared. PBMC from Ni²⁺-reactive subjects showed significantly higher Ni²⁺-induced IL-4 and IL-13 responses and the number of antigen-specific cytokine-producing cells determined by the two ELISpot protocols correlated well. In a nutshell, our data point to the potential use of <i>in vitro</i> cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.</p>