DynaDom: structure-based prediction of T cell receptor inter-domain and T cell receptor-peptide-MHC (class I) association angles

Table S3. Per residue flip states using Reduce, Protoss and DynaDom comparing single domains and TCR complexes. (PDF 145 kb

Fosfolipidit hevosen kesäihottumassa ja sen hoidossa

Summer eczema is one of the most common diseases that causes discomfort and impairs the quality of life of horses worldwide. The lack of a feasible treatment has made this recurrent, insect hypersensitivity-linked allergic pruritus a challenge for veterinary medicine. The first aim of this study was to examine serum phospholipids and their use in the therapy of summer eczema by using an autologous serum preparation. The second aim was to delineate clinical features of summer eczema among Finnhorses. The efficacy of the therapy was investigated in 28 horses in a placebo-controlled study and was also evaluated according to long-term information collected from the owners of the 343 horses treated with this therapy over 12 years. Serum phospholipids and their changes after autoserum therapy were analysed in 10 horses with summer eczema and 10 matched healthy controls by LC-MS. Content of phospholipids in autoserum preparations of 10 affected and 6 healthy horses were analysed by ESI-MS. Horses in the placebo group showed significant aggravation in their clinical signs compared with horses treated with autoserum therapy at the same time (P=0.0329). According to long-term data, 70% of the horses benefited from this autoserum therapy (95% CI 0.64-0.75, P<0.0001) and 16% did not, and 14% of the owners did not provide a clear opinion. No harmful side effects were observed. Horses with summer eczema displayed significantly lower concentrations of phosphatidylcholine (P<0.0001) and sphingomyelin (P=0.0115) in their sera than healthy horses. After a 4-week autoserum therapy, no significant difference between these horses could be demonstrated. The change in clinical signs correlated significantly with the alterations in sphingomyelin concentrations (P=0.0047). Analysis of autoserum preparations revealed that these preparations contained major serum phospholipids, however, in significantly differing concentrations between eczema and healthy horses. Affected horses showed more abundant concentrations of phosphatidylcholine (P=0.042) and sphingomyelin (P=0.0017). In addition, concentrations of these phospholipids correlated significantly with the clinical status. Finnhorses formed the largest group. Most Finnhorses had become affected before the age of 5 years and showed moderate signs. Severity of the signs was not related to age at onset. No significant correlation existed between duration and severity of the disease. This study showed that an autoserum preparation containing serum phospholipids was a favourable method to treat summer eczema. Affected horses displayed significant differentiations in their serum phospholipid profiles and these alterations seemed to change according to the clinical status. Applications of this autoserum therapy for other allergic manifestations of horses should be explored.Kesäihottuma on yleisin hevosen elämänlaatua heikentävä allerginen ihosairaus. Kesäihottumaa tavataan kaikkialta maailmasta siellä, missä hevoset altistuvat polttiaisten pistoille. Suomessa tämä aika ulottuu yleensä toukokuusta lokakuuhun ja sen takia sairaudesta käytetään meillä nykyisin nimitystä kesäihottuma entisten kutka tai hankuri sijaan. Tähän kesäisin toistuvaan kutinaan ei ole toistaiseksi ollut sopivaa hoitoa. Tämän tutkimuksen tarkoituksena oli selvittää seerumin rasvoihin kuuluvien, ns. fosfolipidien, eroja kesäihottumaa sairastavilla ja terveillä hevosilla sekä näiden lipidien käyttöä kesäihottuman hoitoon. Fosfolipidit ovat rasvoja, jotka osallistuvat useisiin elimistön puolustusjärjestelmän reaktioihin. Lisäksi kartoitettiin kesäihottuman kliinistä kuvaa omalla kansallisella hevosrodullamme, suomenhevosella. Seerumin ja kunkin hevosen omasta seerumista tehdyn hoitovalmisteen, ns. seerumivalmisteen, fosfolipidien laatua ja pitoisuutta analysoitiin massaspektrometrisin menetelmin. Seerumivalmisteen tehoa selvitettiin kaksoissokkotutkimuksella ja kokemuksia seerumivalmisteen käytöstä kerättiin 12 vuoden ajalta. Koska kyseessä oli maailmanlaajuisestikin uusi hoitomuoto, oli tarpeellista koota hoitoon liittyvää tietoa pitkältä ajanjaksolta. Hevosen seerumissa runsaimmin esiintyvien fosfolipidien pitoisuudet olivat kesäihottumaa sairastavilla hevosilla merkitsevästi alhaisempia verrattuna terveisiin. Neljän viikon seerumivalmistehoidon jälkeen ei eroa ollut enää. Toisin kuin seerumi, sairaiden hevosten seerumivalmiste sisälsi merkitsevästi enemmän näitä fosfolipidejä verrattuna terveiden seerumista tehtyyn valmisteeseen. Lisäksi seerumivalmisteen fosfolipidipitoisuudella ja oireiden voimakkuudella havaittiin selvä yhteys. Kaksoissokkotutkimuksessa 4 viikon tutkimusjakson jälkeen lumevalmisteella hoidettujen hevosten oireet pahenivat merkitsevästi verrattuna seerumivalmisteella hoidettuihin. Omistajilta kerätyn tiedon perusteella seerumivalmistetta saaneista 343 hevosesta 70 % oli saanut apua, 16 % ei ja 14 % omistajista ei ilmaissut selvää kantaa. Haitallisia sivuvaikutuksia ei todettu. Suomenhevosilla kesäihottuma puhkesi tavallisesti 5 ikävuoteen mennessä, ja useimmilla oireet olivat kohtalaisen voimakkaita, vain 11 % kärsi kaikkein vaikeimmista oireista. Merkitsevää yhteyttä oireiden voimakkuudella ja sairastumisiällä ei havaittu eikä myöskään sairastettujen kesien määrällä ja sairauden voimakkuudella. Tämä tutkimus osoitti, että kesäihottumaa sairastavilla hevosilla on poikkeava seerumin fosfolipidiprofiili, joka muuttuu oireiden lievenemisen myötä, ja että fosfolipidejä sisältävää valmistetta voidaan käyttää allergisen ihosairauden hoitoon ilman sivuvaikutuksia. Hoitoa voisi soveltaa myös muihin hevosen allergisiin sairauksiin

Human myeloid cell and innate lymphocyte responses to mycobacterial vaccination or infection

We investigated immune responses beyond conventional T cells in the context of BCG vaccination and tuberculosis disease

Mitochondrial respiration contributes to the interferon gamma response in antigen presenting cells [preprint]

The immunological synapse allows antigen presenting cells (APC) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While toll-like receptor ligation relies on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFNγ). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in macrophages to identify the regulators of IFNγ-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multi-screen approach enabled us to identify novel pathways that control these functionally distinct markers. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFNγ signaling pathway. We report that the IFNγ response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFNγ and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers

ELUCIDATION OF THE MECHANISM OF ACTION OF A RESPIRATORY SYNCYTIAL VIRUS SUBUNIT VACCINE CANDIDATE CONTAINING A POLYMER-BASED COMBINATION ADJUVANT

Human respiratory syncytial virus (RSV) is the primary cause of respiratory illnesses in infants, young children, elderly and immunocompromised individuals. Supportive care is the mainstay of RSV treatment. Currently no licensed vaccine against RSV is available. We have developed a subunit RSV vaccine candidate (ΔF/TriAdj) consisting of a truncated version of the RSV fusion protein (ΔF) formulated with a combination adjuvant (TriAdj) comprised of low molecular weight (LMW) polyinosinic:polycytidylic acid [poly(I:C)], an innate defense regulator (IDR) peptide and poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP). We previously demonstrated the safety and protective efficacy of ΔF/TriAdj in several animal models. The overall objective of this thesis was to elucidate the mechanism of action of ΔF/TriAdj in BALB/c mice. First, we determined that ΔF/TriAdj when delivered intranasally plays a crucial role in stimulating innate immune responses in both upper and lower respiratory tracts of immunized mice as demonstrated by local production of cytokines, chemokines and interferons, as well as infiltration and activation of immune cells. Innate activation subsequently led to robust adaptive immunity and protection against RSV. Next, we elucidated the mechanisms of action of ΔF/TriAdj at the cell-signaling level in macrophages. Macrophages responded directly to in vitro stimulation with ΔF/TriAdj with induction of both endosomal and cytosolic pattern recognition receptors (PRRs). Based on inhibition studies, we determined that multiple signal transduction pathways are involved in ΔF/TriAdj-mediated activation of macrophages. Finally, we conducted a comprehensive chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS) analysis of the lung tissues from vaccinated and unvaccinated, RSV-infected mice as well as healthy controls, to understand the underlying mechanisms of action of ΔF/TriAdj at the further downstream metabolomic level. Metabolomic profiling revealed alterations of tryptophan metabolism (including kynurenine pathway), biosynthesis of amino acids (including arginine biosynthesis), urea cycle and tyrosine metabolism due to RSV infection. Interestingly, ΔF/TriAdj was found to a play a critical role in modulating alterations in the concentrations of the metabolites of the above-mentioned pathways in response to RSV infection. Ultimately, information on the mechanism of action of this RSV vaccine candidate may serve to identify potential biomarkers for immunogenicity and protective efficacy of ΔF/TriAdj in future

Studies of immune response in human tuberculosis

TB (tuberculosis), caused by Mycobacterium tuberculosis (Mtb), continues to be a world-leading killer and a serious global health problem primarily affecting poor people in many developing countries. The difficult situation with TB/HIV co-infection is also a major key challenge to public health. Despite recent advances in TB research, the host- and pathogen-specific factors that lead to protective immunity, particularly in humans, remain unclear. While it is well-established that cell-mediated immunity is required to control TB infection, the role of humoral immunity including Th2 immune responses is debated. This thesis aimed to explore immune responses in human TB and the immunopathogenic mechanisms involved in the progression of clinical TB in both HIV-negative and HIV-positive individuals. To address the aims of this thesis work, well-defined study cohorts of active TB patients were obtained in close collaboration with the Black Lion University Hospital in Addis Ababa, Ethiopia. We used a novel immunodiagnostic test, Antibodies in Lymphocyte Supernatants (ALS), to demonstrate that IgG-secreting plasmablasts were significantly higher in the peripheral circulation of patients with active TB compared to latent TB cases and non-TB controls. Interestingly, BCG-specific IgG titers were particularly high in blood samples from TB/HIV co-infected patients with CD4 T cell counts <200 cells/ml who produced low levels of Mtb-specific IFN-γ in vitro. A technological platform including quantitative assessments of mRNA and protein expression in tissue (lymph nodes), fluids (bronchoalveolar lavage (BAL) and pleura fluid) and peripheral blood, was also used to investigate antimicrobial effector pathways and adverse immune responses in unique clinical samples obtained from the local site of Mtb infection. The results from these studies revealed that TB disease was associated with extensive tissue remodelling including an altered cellular composition, collagen deposition and granuloma formation. Here, the degree of necrotic granuloma formation was particularly prominent in TB/HIV-co-infected patients. Despite granuloma enrichment of activated CD68+ macrophages containing Mtb-antigens, mRNA levels of IFN-γ, TNF-α and IL-17 remained low in Mtb-infected lymph nodes. Accordingly, CD8+ T cells expressing cytolytic and antimicrobial effector molecules perforin and granulysin were low inside the TB lesions, while CD4+FoxP3+ regulatory T cells (Treg) and Th2/Treg cell cytokines IL-13 and TGF-β were up-regulated in the Mtb-infected tissues. The observed shift of the immune response from a Th1/Th17 towards an immunoregulatory phenotype was supported by our finding of a Th2 polarized response in the lung of patients with pulmonary TB. Here, multiplex protein analysis of BAL and plasma samples demonstrated low levels of Th1/Th17 cytokines and the T cell-chemoattractant CCL5, but significantly up-regulated levels of pro-inflammatory cytokines and the Th2 cytokine IL-4. The enhanced Th2 response was associated with increased levels of CCL4, suppressors of cytokine signaling-3 (SOCS3) and mycobacteria-specific IgG in BAL fluid from patients with active pulmonary TB. Contrary, IL-4, CCL4, SOCS3 and IgG-responses remained low in patients with less severe extrapulmonary pleural TB disease, who demonstrated up-regulated levels of both IFN-γ and CCL5. Taken together, our results provide evidence that human TB is associated with impaired Th1 immunity but elevated Th2/immunoregulatory responses and induction of antibody-mediated immunity. Importantly, enhanced Th2 and/or plasmablast responses may be used as relevant biomarkers or immune response signatures of active progressive TB disease that could be explored as potential targets for clinical TB management in the future