Deciphering systemic lupus erythematosus-associated serum biomarkers reflecting apoptosis and disease activity.

Systemic lupus erythematosus (SLE) is a severe chronic inflammatory autoimmune connective tissue disease. Despite major efforts, SLE remains a poorly understood disease with unpredictable course, unknown etiology and complex pathogenesis. Apoptosis combined with deficiency in clearing apoptotic cells is an important etiopathogenic event in SLE, which could contribute to the increased load of potential autoantigen(s); however, the lack of disease-specific protein signatures deciphering SLE and the underlying biological processes is striking and represents a key limitation. In this retrospective pilot study, we explored the immune system as a specific sensor for disease, in order to advance our understanding of SLE. To this end, we determined multiplexed serum protein expression profiles of crude SLE serum samples, using antibody microarrays. The aim was to identify differential immunoprofiles, or snapshots of the immune response modulated by the disease, reflecting apoptosis, a key process in the etiology of SLE and disease activity. The results showed that multiplexed panels of SLE-associated serum biomarkers could be decoded, in particular reflecting disease activity, but potentially the apoptosis process as well. While the former biomarkers could display a potential future use for prognosis, the latter biomarkers might help shed further light on the apoptosis process taking place in SLE.

Socio-economic status and major trauma in a Scandinavian urban city: A population-based case-control study.

AIMS: Epidemiological studies of patients with major trauma, including both hospitalized and immediately deceased whom are undergoing medico-legal autopsy, are very rare. We studied the incidence and mortality of major trauma in all 10 districts in the Scandinavian city of Malmö, Sweden, and the association between socio-economic status and major trauma. METHODS: Major trauma was defined as a New Injury Severity Score > 15, or a lethal outcome due to trauma. Cases with a registration address in Malmö between 1 January 2011 and 31 December 2013 were identified from the red trauma alarm list in the hospital and the autopsy register in the Forensic Department. Statistics Sweden matched each case with four randomly selected age-, gender- and district-matched controls. Social assistance within the household, level of education, income and capital income were compared. RESULTS: We identified 117 cases (80 men and 37 women) with a median age of 48.0 years (IQR 28.5-65.0). The incidence of major trauma in Malmö was 12.7 (95% CI 10.4-15.0) per 100,000 person-years; and 69 died due to major trauma, with 8.4 (95% CI 6.4-10.4) per 1000 deaths. Lower income (p = 0.024), no income (OR 1.6; 95% CI 1.0-2.4; p = 0.037) and social assistance (OR 2.3; 95% CI 1.3-4.1; p = 0.003) were associated with major trauma. The level of education was not found to be related to major trauma (p = 0.47). CONCLUSIONS: Low income and social assistance within the household were associated with major trauma in the city of Malmö, but not the level of education; in this age-, gender- and district-matched case-control study of major trauma.

Crystal structure of a gammadelta T cell receptor ligand T22: a truncated MHC-like fold.

Murine T10 and T22 are highly related nonclassical major histocompatibility complex (MHC) class Ib proteins that bind to certain gammadelta T cell receptors (TCRs) in the absence of other components. The crystal structure of T22b at 3.1 angstroms reveals similarities to MHC class I molecules, but one side of the normal peptide-binding groove is severely truncated, which allows direct access to the beta-sheet floor. Potential gammadelta TCR-binding sites can be inferred from functional mapping of T10 and T22 point mutants and allelic variants. Thus, T22 represents an unusual variant of the MHC-like fold and indicates that gammadelta and alphabeta TCRs interact differently with their respective MHC ligands.

Drugs in fall versus non-fall accidents with major trauma - A population-based clinical and medico-legal autopsy study.

BACKGROUND: The main aim of the present population-based study was to compare drugs in fall versus non-fall accidents causing major trauma, including both clinical and medico-legal autopsy data. METHODS: All individuals with accidents resulting in major trauma, a new injury severity score (NISS)>15 or lethal outcome was identified at hospital and/or the Department of Forensic Medicine between 2011 and 2013. Modified Downton Fall Risk Index ranged from 0 to 7, and was based on specific pharmaceuticals (max 5 points), previous fall (1 point) and cognitive impairment (1 point). RESULTS: One hundred and four individuals with major traumatic accidents were identified, 38 (36.5%) died. The median modified Downton Fall Risk Index was 2 for fall accidents and 0 for non-fall accidents (p < 0.001). Modified Downton Fall Risk Index was an age-independent factor associated with fall accident (p < 0.001). The medico-legal autopsy rate for in-hospital patients was 50% (6/12) for fatal fall accidents in comparison with 92.3% (12/13) for fatal non-fall accidents (p = 0.03). In individuals undergoing medico-legal autopsy, the proportion of individuals with any detected drug was 77% in fall accidents compared to 39% in non-fall accidents (p = 0.036). The presence of sedatives (p = 0.002) and bensodiazepines (p = 0.023) were higher for fall accidents compared to non-fall accidents. CONCLUSION: This population-based study on accidents with major trauma showed that drugs had high impact on fall accidents with major trauma. It seems warranted from a public health perspective to study if implementation of medication review guidelines at hospital managing polypharmacy issues may prevent fall accident recidivism.

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Multivariate linear regression modelling of lung weight in 24,056 Swedish medico-legal autopsy cases.

Heavy combined lung weight at autopsy is a non-specific autopsy finding associated with certain causes of death such as intoxication. There is however no clear definition of what constitutes "heavy" lung weight. Different reference values have been suggested but previous studies have been limited by small select populations and only univariate regression has been attempted. The aim of this study was to create a model to estimate lung weight from decedent parameters. We identified all cases >18 years age autopsied at the Swedish National Board of Forensic Medicine from 2000 through 2013, excluding cases with a post-mortem interval >5 days as well as cases with extreme values, totalling 24,056 cases. We analysed body weight, body height, sex, age, BMI, BSA as well as untransformed and transformed lung weight. The analysis was stratified for sex. We evaluated the fit of the models and that the model assumptions were not violated. We set out to apply the model with the highest residual sum of squares to derive limits for heavy lungs. In univariate regression BSA and height showed best performance. The final model included height, weight and age group. After excluding large standardized residuals (>3, <-3) the final model achieved R2 of 0.132 and 0.106 for women and men respectively. While we managed to create a multivariate model its performance was poor, possibly a fact reflective of the physiological nature of the lungs and in turn its variability in fluid content. Linear regression is a poor model for estimating lung weight in an unselected population.

A new approach to examine conformational changes occurring upon binding of ligand by biomolecules.

Liquid-liquid partition chromatography in an aqueous poly(ethylene glycol)/dextran two-phase system (LLPC) is shown to be a quick and sensitive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an example, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand-dependent equilibrium between two forms and that conformational changes occurred when monoclonal antibodies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded proteins that appeared to be homogeneous when analysed by other techniques.

Conformational isomerism of IgG antibodies.

The purpose of this study was to determine why apparently homogeneous IgG antibodies were, in some cases, fractionated into at least two components by liquid-liquid partition chromatography (LLPC) in an aqueous two-phase system. Four mouse monoclonal IgG antibodies, two against albumin, one against IgG and one against thyroxine, were shown to adopt different conformational isomeric forms. The four antibodies existed in an equilibrium between two or three conformational forms, the proportion of which could also be estimated by LLPC. Since LLPC detects mainly conformational differences within the antigen-binding sites of IgG antibodies, it could be concluded that the conformational forms differed with respect to their combining sites. Moreover, the isomeric forms of an antibody directed against a protein antigen, formed antigen-antibody complexes with almost identical surface properties. In contrast, complexes with different surface properties were formed when the hapten or hapten conjugated to BSA was bound. Thus, both the conformational isomers could bind antigen, at least when the antigen was a small hapten or a hapten conjugated to a carrier protein. Our results suggest that six out of 57 monoclonal IgG antibodies exist in equilibrium between at least two conformational forms and the biological significance of this isomerism is discussed.

Antigen-binding sites dominate the surface properties of IgG antibodies.

A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system, was used to detect differences in surface properties of antibodies with different antigen-binding sites. Employing well-characterized monoclonal IgG antibodies and Fab and Fc fragments thereof as well as chimeric IgG antibodies we found a remarkable relationship between structure of the antibody combining site and chromatographic behaviour. The surface properties of the IgG antibodies were dominated by those of its antigen-binding regions. In addition, our results indicated that the constant parts of the IgGs form similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties.

Necessity of including medico-legal autopsy data in epidemiological surveys of individuals with major trauma.

BACKGROUND: It is rare that epidemiological surveys of patients with major trauma include both those admitted to the emergency department and those sent for medico-legal autopsy. The main aim of the present population-based study of major trauma was to examine the importance of medico-legal autopsy data. METHODS: A new injury severity score (NISS)>15 or lethal outcome was used as criteria for major trauma and to identify patients at the emergency, anaesthesiology and forensic departments and/or being within the jurisdiction of the Malmö police authority and subjected to a medico-legal autopsy between 2011 and 2013. According to Swedish legislation all trauma related deaths should be reported to the police who refer these cases for medico-legal autopsy. RESULTS: Among the 174 individuals included, 92 (53%) died and 81 (47%) underwent medico-legal autopsy. One hundred twenty-six patients were primarily admitted to hospital and 48 died before admission to hospital and were sent directly for medico-legal autopsy. Forty-four in-hospital deaths occurred, of whom 33 (75%) were sent to medico-legal autopsy. In those sent directly to the department of forensic medicine the proportion of accidents was lower (p<0.001), self-inflicted injuries higher (p<0.001) and gunshot wounds higher (p=0.002) in comparison with those sent to hospital. The most prevalent drugs detected by forensic toxicology screening in the 81 fatalities were ethanol (20%), sedatives (16%), anti-depressives (15%) and illicit narcotics (9%). Forty-four cases (54%) were positive for at least one drug, and twenty-eight cases (35%) were positive for two or more drugs. Factors associated with a lower rate of medico-legal autopsies among trauma-related deaths at hospital were high age (p<0.001), lower NISS (p<0.001), a longer duration between trauma and death (p<0.001), falls (p=0.030) and trauma-related infections (p<0.001). CONCLUSION: This population based study covering clinical and forensic data shows that more than half of the individuals sustaining major trauma died. An additional 25% of the in-hospital fatalities should have undergone medico-legal autopsy according to legislation, but did not. The high proportion of positive toxicological findings among fatalities examined at medico-legal autopsy implies that toxicology screening should be routine in major trauma patients, in order to improve treatment and prevention.

Differential gene expression analysis of placentas with increased vascular resistance and pre-eclampsia using whole-genome microarrays.

Pre-eclampsia is a pregnancy complication characterized by hypertension and proteinuria. There are several factors associated with an increased risk of developing pre-eclampsia, one of which is increased uterine artery resistance, referred to as "notching". However, some women do not progress into pre-eclampsia whereas others may have a higher risk of doing so. The placenta, central in pre-eclampsia pathology, may express genes associated with either protection or progression into pre-eclampsia. In order to search for genes associated with protection or progression, whole-genome profiling was performed. Placental tissue from 15 controls, 10 pre-eclamptic, 5 pre-eclampsia with notching, and 5 with notching only were analyzed using microarray and antibody microarrays to study some of the same gene product and functionally related ones. The microarray showed 148 genes to be significantly altered between the four groups. In the preeclamptic group compared to notch only, there was increased expression of genes related to chemotaxis and the NF-kappa B pathway and decreased expression of genes related to antigen processing and presentation, such as human leukocyte antigen B. Our results indicate that progression of pre-eclampsia from notching may involve the development of inflammation. Increased expression of antigen-presenting genes, as seen in the notch-only placenta, may prevent this inflammatory response and, thereby, protect the patient from developing pre-eclampsia.

Enzyme conformational alterations detected by partition column chromatography.

In this paper, we demonstrate the ability of liquid-liquid partition chromatography (LLPC) to detect conformational alterations occurring in well-characterized enzymes. The conformational changes induced in dehydrogenases such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases (LDH) and malate dehydrogenase (MDH) upon binding of ligand(s) were detectable by LLPC. The ligand-dependent equilibrium between two forms of citrate synthase (CS), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK) and 3-phosphoglycerate kinase (PGK) could also be demonstrated. Furthermore, different conformational forms of some of the apoenzymes could also be detected and separated by LLPC. The results obtained here are discussed in relation to those obtained by other methods.

Surface properties of antigen-antibody complexes.

In this paper, the authors show that liquid-liquid partition chromatography in an aqueous two-phase system offers unique possibilities of comparing the overall surface properties of intact antibodies in solution before and after binding of antigen. The authors demonstrate that the surface properties of antigen-antibody complexes are dependent on the variable regions of the antibodies, the nature of the antigen and/or possible conformational changes induced by antigen binding. Thus, each antigen-IgG antibody pair formed one type of complex with respect to the exposed dominant surface. The antigen-binding sites of IgG antibodies were exposed and dominant even after binding of hapten or hapten-carrier. In contrast, the antibody-combining sites were concealed upon protein binding and the exposed surfaces of the protein-antibody complexes were related mainly to those of the antigen. IgA1, IgA2, IgE and IgM formed, in comparison to the IgG, hapten-antibody complexes which exhibited surface properties that could be related to both the antigen-binding sites and Fc parts of the antibodies. Moreover, the results indicated that antigen-induced conformational changes occurred in either IgA1, IgA2, IgE, or IgM, but not in IgG1, -2, -3 and -4, making the surfaces of their heavy chain constant regions more similar.

Liquid-liquid partition chromatography as a method to examine surface properties of antibodies and antigen-antibody complexes.

We demonstrate liquid-liquid partition chromatography in aqueous two-phase systems (LLPC) as a simple method for examining the surface properties of immunoglobulins and antigen-antibody complexes in solution. LLPC separates molecules with respect to the properties of the exposed surfaces. As an example, the method may be used to detect changes in the conformation of IgG following chemical modification like acylation or iodination. We have studied the partitioning of antibodies and antigen-antibody complexes, modelled by rabbit antibodies against three human serum proteins, in aqueous polyethylene glycol/dextran two-phase systems at pH 7. Analysis of both polyclonal and monoclonal antibodies against various antigens suggested that the partition properties of immunoglobulins are related mainly to their antigen specificity and not to subclass-specific structures. Furthermore, experiments indicated that changes in the surface properties of antigen and/or antibody following complexation may be detected. Thus, LLPC may prove to be a new way of studying the relation between antibody structure and function in solution.

Comparison of surface properties of human IgA, IgE, IgG and IgM antibodies with identical and different specificities.

In this paper, the authors report the use of liquid-liquid partition chromatography (LLPC) in an aqueous polyethylene glycol (PEG)/dextran two-phase system to compare the surface properties (partition properties) of human antibodies and fragments thereof. The surface properties of all the monoclonal antibodies of different classes and subclasses investigated were within the same broad range as that observed for the polyclonal antibodies and no relationship was found between the exposed surfaces of the immunoglobulins (Ig) and their heavy chain isotype. Moreover, Fc fragments from various IgG1, 2 and 4 myeloma proteins were found to exhibit similar surface properties. Employing chimeric antibodies with identical variable regions the authors found that intact IgG1, 2 and 4 displayed identical surface properties, while the corresponding IgA1, IgA2, IgG3, IgE and IgM antibodies differed both from each other and from the IgGs. The surface properties of chimeric IgG3 could be made similar to those of the IgG1, 2 and 4 chimers by partially reducing the length of the hinge section, but new differences in surface properties appeared when their hinges were of similar length. Thus, LLPC can be used to detect differences or similarities in the surface properties of the antigen-binding regions as well as the Fc part in the various isotypes. This can shed light on biological activities such as antigen binding and effector function.