Introduction: Waldenström's macroglobulinemia is a hematological neoplasm belonging to the group of monoclonal gammopathies, which includes systemic symptoms and those related to an increase in M paraprotein. Objective: To describe a case of cardiac amyloidosis associated with macroglobulinemia. Clinical case: Male patient who was admitted for asthenia, dysphonia, and who, during his evolution, developed progressive dyspnea, heart failure and pleural effusion. Additionally, echocardiography showed myocardial granular pattern, while pleural biopsy was positive for Congo red staining. Subsequently, he received treatment with bortezomib, dexamethasone and rituximab, with favorable evolution. Conclusions: In this disease, early diagnosis is an important advantage for survival. Therefore, its management is palliative of cardiac manifestations. The present case shows a diagnostic challenge, in which the less frequent etiologies of heart failure must be taken into account. / Revisión por pares

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I den här studien har metoder för att separera immunoglobulin (IgG) utvecklats med hjälp av kapillärelektrofores (CE) och Ultra högupplösande vätskekromatografi-Mass spektrometri (UHPLC-MS). Vid analysen av peptider från IgG spjälkat av trypsin (IgG TD) detekterades 26 glykopepeptider med UHPLC-MS genom att använda olika gradienter och mobilfaser. Dessutom analyserades fyra olika koncentrations av IgG TD för att testa detektionsgränsen. Resultatet visade att glykopeptider kunde detekteras i alla fyra koncentrationer men antalet detekterade glykopeptider minskade med minskande koncentration. Den sista delen av UHPLC-MS analysen var en selektivitetsanalys där en blandning av bovint serumalbumin spjälkat av trypsin (BSA TD) och IgG TD analyserades i olika koncentrationer. Selektivitetsanalysen visade att ungefär samma antal IgG glykopeptider detekterades som vid analys av IgG TD. Vid CE-analysen användes tre olika kapillärer, varav två av dem var polyvinylalkohol-coated och olika buffertlösningar (BGE) användes. De slutgiltiga resultaten visade 5 toppar som skulle kunna representera IgG gykopeptider men eftersom ingen MS utfördes kunde inga slutsatser dras. Slutligen, användes Matrix-Assisted Laser desorption/ionisation-time-of-flight (MALDI-TOF) för att analysera proverna innan analys och för att jämföra resultaten med UHPLC-MS. / In this study, methods to separate immunoglobulin G (IgG) glycopeptides were developed using capillary electrophoresis (CE) and ultra-high-performance liquid chromatography-Mass spectrometry (UHPLC-MS). In the UHPLC-MS- analysis, 26 glycopeptides were detected during the analysis of trypsin digested IgG (IgG TD) using different gradients and mobile phases. Moreover, a limit of detection study with four different concentrations (0.01, 0.1, 0.5 and 2mg/mL) was performed. Although the number of detected glycopeptides decreased with decreasing concentration, glycopeptides were detected in the 0.01 mg/mL IgG TD sample. Lastly, a selectivity study was performed, in which two trypsin digested Bovine serum albumin (BSA TD) and IgG TD mixtures with different concentrations were analysed. The results showed that approximately the same number glycopeptides were detected as in the IgG sample suggesting that the BSA sample did not interfere with the detection of glycopeptides. Moreover, the CE-analysis included three different capillaries and several background electrolytes (BGEs). Two of the capillaries were polyvinyl alcohol (PVA) coated. The final electropherogram showed five peaks possibly representing IgG glycopeptides but no MS were performed on the CE part leading to no conclusions for the CE-part. Matrix-Assisted Laser Desorption/Ionisation- time-of flight (MALDI-TOF) was performed throughout the project to control the samples and for comparison to the UHPLC-MS analysis.

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<p>UVOD:&nbsp; Citopenija je jedna od glavnih&nbsp; karakteristika mnogih hematolo&scaron;kih bolesti. U&nbsp; rutinskoj transfuziolo&scaron;koj upotrebi su metode&nbsp; detekcije prisustva antitela u serumu ili na&nbsp; eritrocitima bolesnika. Primena direktnog testa utro&scaron;ka antihumanog globulina predstavlja&nbsp; efikasan način da se stekne kompletan uvid u imunolo&scaron;ka zbivanja na svim krvnim lozama, prati dinamika razvoja antitela i toka bolesti. MATERIJAL I METODE: Svim pacijentima su se iz uzoraka periferne&nbsp; krvi vr&scaron;ile sledeće analize krvi: 1) direktni antiglobulinski test mikrometodom&nbsp; aglutinacije u gel karticama (LISS)/ Coombs ID. Dobijeni rezultat aglutinacije mikrometodom na gelu moţe biti negativan ili pozitivan i 2) direktni test utro&scaron;ka antihumanog globulina metodom aglutinacije u epruveti. Očitavanje se vr&scaron;ilo određivanjem razlike u titru antihumanog globuli na i očitavanjem postojeće reakcije aglutinacije dobijene u uzorcima pacijenta u odnosu na rezultate reakcije aglutinacije dobijene sa uzorcima zdrave kontrolne osobe. Test se smatra o pozitivnim ukoliko se dobijala razlika u titru AHG - a za bar dva razređen ja sa pacijentovim ćelijama u odnosu na ćelije zdrave kontrolne osobe. Statistička značajnost&nbsp; je analizirana t - testom, Spirmanovim&nbsp; koeficijentom korelacije REZULTATI: Analizirano je 100 pacijenata sa dijagnozom anemije, leukopenije,&nbsp; limfoproliferativnih bolesti,&nbsp; trombocitopenije, trombotične trombocitopenijske purpure, idiopatske trombocitopenične purpure, mijelodisplastičnog sindroma, miastenije gravis i sistemskog eritematoznog lupusa pre i nakon primljene terapije. Direktni antiglobulinski test je biopozitivan u 20% slučajeva dok je direktni test utro&scaron;ka antihumanog globulina bio u 51%, odnosno za 31% vi&scaron;e. Nakon primljene terapije direktni antiglobulinski test je ostao pozitivan u 18% slučajeva a direktni test utro&scaron;ka antihumanog globulina u 46% &scaron;to je za 28% vi&scaron;e. Utvrđivanjem povezanosti između citopenije i stepena utro&scaron;ka antihumanog globulina dokazano je da svi praćeni parametri utiču na stepen utro&scaron;ka AHG-a: hemoglobin (&beta;=-0,579, p=0,000), hematokrit (&beta;=-0,568, p=0,000), eritrociti (&beta;=-0,519, p=0,000), trombociti (&beta;=-0,617, p=0,000) i leukociti(&beta;=-0,119, p=0,237). Takođe je dokazano da &scaron;to su vrednosti posmatranih parametara veće, razlika u titru direktnog testa utro&scaron;ka antihumanog globulina je manja &scaron;to bi i&scaron;lo u prilog boljoj prognozi posmatranog oboljenja. ZAKLJUČAK: Direktni test utro&scaron;ka antihumanog globulina je značajno osetljiviji test u odnosu na direktni antihumani globulinski test. Postoji pozitivna korelacija između citopenije i stepena konzumacije antihumanog globulina. Smanjenje titra antitela direktnog testa utro&scaron;ka antihumanog globulina je jedan od pokazatelja bolje prognoze bolesti.</p> / <p>INTRODUCTION: Cytopenia is one of the main characteristics of many hematologic diseases. In routine use are methods of detecting the presence of antibodies in the serum or on red blood cells of patients. The application of direct consumption test of antihuman globulin is an efficient way to gain complete insight into the immunological events at all bloodlines, monitor the dynamics of the development of antibodies and disease progression. MATERIALS AND METHODS: All patients samples were tested for: 1) direct antiglobulin test by micro agglutination method in the gel card (LISS) / Coombs ID. The result obtained by micro agglutination gel can be negative or positive, 2) direct consumption test of antihuman globulin in a test tube. Interpretation is performed by determining differences in titer of antihuman globulin by reading existing reactions of agglutination in samples of the patient and compare it to the results obtained with the samples of the healthy control persons. The test is considered positive if the difference in titres obtained AHG differs for at least two dilutions of a patient&#39;s cells compared to cells of healthy control persons. Statistical significance was analyzed by t-test, Spearman correlation coefficient. RESULTS: A total of 100 patients diagnosed withanemia, leukopenia, lymphoproliferative disease, thrombocytopenia, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, myelodysplastic syndrome, myasthenia gravis and systemic lupus erythematosus were analyzed before and after receiving treatment. Direct antiglobulin test was positive in 20% cases, while the direct consumption test of anti-human globulin was 51%, that is the difference of 31%. After treatment direct antiglobulin test remained<br />positive in 18% of cases and direct consumption test of antihuman globulin was in 46%, which is 28% higher. Determining the relationship between the degree of cytopenia and consumption of anti-human globulin showed that all monitored parameters affect the level of consumption: hemoglobin (&beta; = -0.579, p = 0.000), hematocrit (&beta; = -0.568, p = 0.000), erythrocytes (&beta; = -0.519 , p = 0.000), platelets (&beta; = -0.617, p = 0.000) and leukocytes (&beta; = -0.119, p = 0.237). It was also proved that if the values of observed parameters are higher, difference in titer of direct consumption test of antihuman globulin is lower, which can indicate better prognosis of disease. CONCLUSION: Direct consumption test of antihuman globulin was significantly more&nbsp; sensitive test than the direct anti-human globulin test. There is a positive correlation between the degree of cytopenia and consumption of anti-human globulin. Decrease in antibody titer in direct consumption test of antihuman globulinis an indicator of a better prognosis of the disease.</p>

Allergic reactions are mainly mediated by the interactions between the IgE and its ligands, amongst them CD23 and CD21 in what is termed the IgE network. CD23 is involved in upregulating IgE expression by forming a trimolecular complex with CD21 and IgE on the B-cell surface, resulting in the specific activation of IgE-positive B cells. CD21 also interacts with C3d and is a bridge between the innate and the immune system. A crystal structure of the interaction has been solved (Szakonyi et al., 2001) but was controversial because it contradicted previous biochemical analyses. The aims of this thesis were to use various biophysical techniques to study the interactions between the molecules in the IgE network and its possible inhibition. Part 1: Characterisation of a phage display-derived peptide that inhibit IgE binding to CD23 A peptide was previously derived using phage display technology and tested for binding ability to CD23 using SPR and ITC. Subsequent NMR experiments were performed to identify the binding site, followed by characterization of its derivatives. Crystallisation of CD23 with the peptide and soaking with its truncated tripeptide, NWP, were also attempted. Part 2: Characterisation of CD23 and its interaction with its ligands X-ray crystallography was undertaken to solve the structure of derCD23 in complex with a phage display-derived peptide (Part1) followed by crystal soaking with a truncated tripeptide, NWP. However, a reproducible, high-resolution wild type derCD23 structure was determined at 1.9 Å. A comparison of the binding behaviour between the monomeric derCD23 and a trimeric CD23 construct was carried out in order to see the effect of oligomerisation upon IgE binding. Using the known interaction map as well as a crystal structure, the possible interacting residues between CD23 and IgE were examined. The characterisation of the CD23/CD21 interaction was continued from previous efforts in order to confirm that the binding epitope of CD23 for CD21 lies within the C-terminus of CD23. Characterisation of the interactions of CD23/IgE/FcεRI was performed to examine these multimolecular interactions and possible regulatory mechanisms in mast cell degranulation. It was shown that CD23 can form multimeric complexes with IgE-Fc that bind to FcεRI with higher apparent affinity than IgE-Fc alone, which may lead to increases in mast cell degranulation. It was also found that the IgE bound on FcεRI still binds to CD23 although with a lower binding capacity, presumably due allosteric changes. The binding of CD23 with a monoclonal antibody IDEC-152 was also characterised using SPR and NMR spectroscopy. It was proposed that IDEC-152 might interfere with the trimerisation site of CD23 thus reducing its affinity for IgE. A thermofluor assay was developed and optimised for potential screening of compounds that bind to derCD23 using a qPCR machine, which may be useful to screen compounds that bind to CD23 as part of future drug discovery project. Crystallisation of the derCD23/CD21 and IgE/triCD23/CD21 complexes was also attempted as part of ongoing crystallisation projects. Part 3: The interaction between C3d and CD21 The interaction between C3d and CD21 is believed to be a bridge between the innate and adaptive immune response, and is thought to be pivotal in the initiation of autoimmune disease. Following from previous studies on this interaction, further characterisations were performed using NMR and ITC to confirm the involved sites on CD21 (SCR1-2) in binding to C3d. Several potential salt bridges have been identified so far, allowing a high-resolution docked structure of the C3d/CD21 complex.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.