Prediction of HLA-DQ3.2 beta ligands: evidence of multiple registers in class II binding peptides

Motivation: While processing of MHC class II antigens for presentation to helper T-cells is essential for normal immune response, it is also implicated in the pathogenesis of autoimmune disorders and hypersensitivity reactions. Sequence-based computational techniques for predicting HLA-DQ binding peptides have encountered limited success, with few prediction techniques developed using three-dimensional models. Methods: We describe a structure-based prediction model for modeling peptide-DQ3.2 beta complexes. We have developed a rapid and accurate protocol for docking candidate peptides into the DQ3.2 beta receptor and a scoring function to discriminate binders from the background. The scoring function was rigorously trained, tested and validated using experimentally verified DQ3.2 beta binding and non-binding peptides obtained from biochemical and functional studies. Results: Our model predicts DQ3.2 beta binding peptides with high accuracy [area under the receiver operating characteristic (ROC) curve A(ROC) > 0.90], compared with experimental data. We investigated the binding patterns of DQ3.2 beta peptides and illustrate that several registers exist within a candidate binding peptide. Further analysis reveals that peptides with multiple registers occur predominantly for high-affinity binders.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

ESTUDO COMPARATIVO DA POSIÇÃO ÂNTERO POSTERIOR DOS PRIMEIROS MOLARES INFERIORES NO TRATAMENTO ORTODÔNTICO, UTILIZANDO AS TÉCNICAS STRAIGHT-WIRE E EDGEWISE, EM CASOS DE MÁ OCLUSÃO DE CLASSE I DE ANGLE, COM EXTRAÇÃO DE PRIMEIROS PRÉ-MOLARES

Este estudo avaliou o posicionamento ântero posterior dos primeiros molares inferiores, durante o tratamento ortodôntico, utilizando o arco lingual inferior como acessório de ancoragem na técnica Straight-Wire, em comparação aos casos tratados pela técnica Edgewise, sem a utilização do arco lingual. Dois grupos foram selecionados, ambos apresentando má oclusão de Classe I de Angle7, tratados com extração dos primeiros pré-molares superiores e inferiores. Foi utilizada uma amostra de 255 telerradiografias em norma lateral, obtidas de pacientes brasileiros, de ambos os sexos, com média de idade de 13 anos e 6 meses e com diferentes padrões de crescimento facial. Embasado na análise e discussão dos resultados, concluiu-se que: 1) do início do tratamento ao fim da fase de nivelamento, a perda de ancoragem coronária do primeiro molar inferior foi maior nos casos tratados com a técnica Straight-Wire; 2) do fim da fase de nivelamento ao fim do tratamento, a perda de ancoragem coronária e radicular do primeiro molar inferior foi maior na técnica Edgewise; 3) do início ao fim tratamento a perda de ancoragem radicular foi maior nos pacientes tratados com a técnica Edgewise; e 4) o deslocamento ântero-posterior dos incisivos inferiores não apresentou diferença estatisticamente significante para ambas as técnicas, em todas as etapas observadas.(AU)

AVALIAÇÃO DA PROPORÇÃO DENTAL DE BOLTON EM INDIVÍDUOS COM OCLUSÃO NORMAL NATURAL E MALOCLUSÕES DE CLASSE I E CLASSE II DIVISÃO 1 DE ANGLE.

Introdução: A análise de Bolton, análise que quantifica o tamanho dentário, é uma referência importante para profissionais que buscam finalizações ortodônticas adequadas. Objetivo: O objetivo deste trabalho é verificar se há discrepância entre os indivíduos com oclusão normal natural e maloclusões de Classe I e de Classe II divisão 1 de Angle pertencentes a amostra selecionada, em relação aos valores encontrados por Bolton, bem como verificar também se há dimorfismo sexual. Metodologia: 3 grupos contendo 35 pares e modelos em gesso cada, separados pelo tipo de oclusão, pertencentes ao acervo do programa de pós-graduação em Ortodontia da Universidade Metodista de São Paulo foram medidos com paquímetro digital em sua maior distância mésiodistal desde 1º molar direito a 1º molar esquerdo, dos arcos superiores e inferiores, com dentição permanente. Os valores foram tabulados e a proporção de Bolton foi aplicada. Resultados: Respectivamente para os grupos 1, 2 e 3, a proporção total encontrada foi de 90,36 (DP±1,70), 91,17 (DP±2,58) e 90,76 (DP±2,45), e a proporção anterior foi de 77,73 (DP±2,39), 78,01 (DP±2,66) e 77,30 (DP±2,65). Conclusão: não houve dimorfismo sexual nem diferença estatisticamente significante comparando os valores aos sugeridos por Bolton.

HLA-DP2 binding prediction by molecular dynamics simulations

Major histocompatibility complex (MHC) II proteins bind peptide fragments derived from pathogen antigens and present them at the cell surface for recognition by T cells. MHC proteins are divided into Class I and Class II. Human MHC Class II alleles are grouped into three loci: HLA-DP, HLA-DQ, and HLA-DR. They are involved in many autoimmune diseases. In contrast to HLA-DR and HLA-DQ proteins, the X-ray structure of the HLA-DP2 protein has been solved quite recently. In this study, we have used structure-based molecular dynamics simulation to derive a tool for rapid and accurate virtual screening for the prediction of HLA-DP2-peptide binding. A combinatorial library of 247 peptides was built using the "single amino acid substitution" approach and docked into the HLA-DP2 binding site. The complexes were simulated for 1 ns and the short range interaction energies (Lennard-Jones and Coulumb) were used as binding scores after normalization. The normalized values were collected into quantitative matrices (QMs) and their predictive abilities were validated on a large external test set. The validation shows that the best performing QM consisted of Lennard-Jones energies normalized over all positions for anchor residues only plus cross terms between anchor-residues.

Large-scale molecular dynamics simulations of HLA-A*0201 complexed with a tumor-specific antigenic peptide:can the α3 and β2m domains be neglected?

Large-scale massively parallel molecular dynamics (MD) simulations of the human class I major histo-compatibility complex (MHC) protein HLA-A*0201 bound to a decameric tumor-specific antigenic peptide GVY-DGREHTV were performed using a scalable MD code on high-performance computing platforms. Such computational capabilities put us in reach of simulations of various scales and complexities. The supercomputing resources available Large-scale massively parallel molecular dynamics (MD) simulations of the human class I major histocompatibility complex (MHC) protein HLA-A*0201 bound to a decameric tumor-specific antigenic peptide GVYDGREHTV were performed using a scalable MD code on high-performance computing platforms. Such computational capabilities put us in reach of simulations of various scales and complexities. The supercomputing resources available for this study allow us to compare directly differences in the behavior of very large molecular models; in this case, the entire extracellular portion of the peptide–MHC complex vs. the isolated peptide binding domain. Comparison of the results from the partial and the whole system simulations indicates that the peptide is less tightly bound in the partial system than in the whole system. From a detailed study of conformations, solvent-accessible surface area, the nature of the water network structure, and the binding energies, we conclude that, when considering the conformation of the α1–α2 domain, the α3 and β2m domains cannot be neglected. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 1803–1813, 2004

Histidine hydrogen bonding in MHC at pH 5 and pH 7 modeled by molecular docking and molecular dynamics simulations

Hydrogen bonds play important roles in maintaining the structure of proteins and in the formation of most biomolecular protein-ligand complexes. All amino acids can act as hydrogen bond donors and acceptors. Among amino acids, Histidine is unique, as it can exist in neutral or positively charged forms within the physiological pH range of 5.0 to 7.0. Histidine can thus interact with other aromatic residues as well as forming hydrogen bonds with polar and charged residues. The ability of His to exchange a proton lies at the heart of many important functional biomolecular interactions, including immunological ones. By using molecular docking and molecular dynamics simulation, we examine the influence of His protonation/deprotonation on peptide binding affinity to MHC class II proteins from locus HLA-DP. Peptide-MHC interaction underlies the adaptive cellular immune response, upon which the next generation of commercially-important vaccines will depend. Consistent with experiment, we find that peptides containing protonated His residues bind better to HLA-DP proteins than those with unprotonated His. Enhanced binding at pH 5.0 is due, in part, to additional hydrogen bonds formed between peptide His+ and DP proteins. In acidic endosomes, protein His79β is predominantly protonated. As a result, the peptide binding cleft narrows in the vicinity of His79β, which stabilizes the peptide - HLA-DP protein complex. © 2014 Bentham Science Publishers.

EPIPOX:immunoinformatic characterization of the shared T-cell epitome between variola virus and related pathogenic orthopoxviruses

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Evaluación de la posición hioidea en radiografías de pacientes clase I, II y III esqueletales entre 9 y 18 años de edad registrados en el período 2012 y 2015, de un Centro Radiológico de la Ciudad de Cuenca

Objetivo: El objetivo del presente estudio descriptivo, fue evaluar la posición del hueso hioides en los diferentes patrones esqueletales de Clase I, II y III mediante el trazado cefalométrico del triángulo hioideo propuesto por Bibby y Preston, estableciendo diferencias entre cada clase esqueletal. Materiales y métodos: La muestra consistió en 161 radiografías cefálicas laterales digitales, correspondientes a individuos de ambos sexos (75 hombres y 86 mujeres), entre edades de 9 y 18 años, las mismas que fueron divididas en tres subgrupos (Clase I, clase II y clase III) de acuerdo a los ángulos ANB y APDI. Se determinó la posición anteroposterior, vertical y angular del hueso hioides mediante el trazado cefalométrico del triángulo hioideo siendo el mentón, la tercera vértebra cervical y el hueso hioides las estructuras anatómicas utilizadas para el trazado del mismo. Se obtuvieron medidas estándar para cada clase esqueletal. Resultados: Se observaron diferencias estadísticamente significativas en la medida de H-Rgn entre clase I y II y entre clase II y III (p<0,005). El valor del ángulo del plano hioidal presentó diferencias estadísticamente significativas entre clase I y III y entre clase II y III (p<0,005). Se evidenciaron diferencias estadísticamente significativas entre hombres y mujeres con clase I esqueletal en la medida H-Rgn (p<0,005). Conclusiones: La posición del hueso hioides varía en los diferentes patrones esqueletales. Sin embargo, su posición en relación a la columna cervical presenta menos variabilidad que su relación con la mandíbula

Relación cráneo cervical en pacientes clase I, II, y III esqueletal entre 9 y 18 años de edad, mediante radiografías cefálicas laterales de un centro radiológico de la ciudad de cuenca.

El objetivo de este estudio fue determinar la relación cráneo cervical en pacientes clases I, II y III esqueletal entre 9 y 18 años de edad, mediante radiografías cefálicas laterales de un centro radiológico de la ciudad de Cuenca, utilizando el análisis cráneo cervical propuesto por Rocabado. Materiales y métodos: Fueron analizadas 161 radiografías cefálicas laterales digitales, de ambos sexos, con edad promedio de 12.3 años (DE± 2.4). Se incluyeron radiografías de individuos con dentición mixta y permanente, sin tratamiento ortodóncico y en donde se observe hasta la sexta vértebra cervical. Fueron excluidas las radiografías de pacientes con mordida abierta, traumatismos maxilofaciales y radiografías de mala calidad. Las telerradiografías fueron analizadas mediante el programa cefalométrico Nemoceph NX, donde se determinó el patrón esqueletal mediante los ángulos SNA, SNB, ANB y APDI. La evaluación de la postura cervical, se realizó mediante el análisis cráneo cervical propuesto por Rocabado. Se obtuvo el índice de concordancia (ICC=0.94). Mediante estadística descriptiva se analizaron las relaciones entre variables usando la prueba de Chi cuadrado y T de Student. Resultados: Se encontró mayor rotación posterior de cráneo en clase I y II esqueletal, encontrándose diferencias estadísticamente significativas respecto al ángulo cráneo vertebral entre hombres y mujeres en individuos clase II esqueletal. Las mujeres presentaron mayor rotación posterior de cráneo a diferencia de los hombres. (p=0.004). En clase III se encontró una relación normal. El espacio suboccipital en las tres clases esqueletales se presento con normalidad. No se encontró diferencia significativa respecto a la edad. Conclusiones: La relación cráneo cervical se presenta con una tendencia a la rotación posterior de cráneo, influida fuertemente por el sexo del individuo. El espacio suboccipital es normal en clase I y II esqueletal y con tendencia al aumento en clase III.

Access and Quality of Heart Failure Management Programs in Australia

Background/aim In response to the high burden of disease associated with chronic heart failure (CHF), in particular the high rates of hospital admissions, dedicated CHF management programs (CHF-MP) have been developed. Over the past five years there has been a rapid growth of CHF-MPs in Australia. Given the apparent mismatch between the demand for, and availability of CHF-MPs, this paper has been designed to discuss the accessibility to and quality of current CHF-MPs in Australia. Methods The data presented in this report has been combined from the research of the co-authors, in particular a review of the inequities in access to chronic heart failure which utilised geographical information systems (GIS) and the survey of heterogeneity in quality and service provision in Australian. Results Of the 62 CHF-MPs surveyed in this study 93% (58) centres had been located areas that are rated as Highly Accessible. This result indicated that most of the CHF-MPs have been located in capital cities or large regional cities. Six percent (4 CHF-MPs) had been located in Accessible areas which were country towns or cities. No CHF-MPs had been established outside of cities to service the estimated 72,000 individuals with CHF living in rural and remote areas. 16% of programs recruited NYHA Class I patients and of these 20% lacked confirmation (echocardiogram) of their diagnosis. Conclusion Overall, these data highlight the urgent need to provide equitable access to CHF-MP's. When establishing CHF-MPs consideration of current evidence based models to ensure quality in practice.

Drugs for cardiac arrhythmmias, antithrombotic drugs and lipid modulating drugs

13.1 Drugs for cardiac arrhythmias 13.1.1 Introduction to cardiac arrhythmias 13.1.2 Cardiac action potentials 13.1.3 Mechanisms of cardiac arrhythmias 13.1.3 Class I 13.1.4 Class II 13.1.5 Class III 12.1.6 Class IV 13.1.7 Amiodarone 13.1.8 Adenosine 13.2 Antithrombotic drugs 13.2.1 Thrombus formation 13.2.2 Platelet aggregation and anti-platelet drugs 13.2.3 Coagulation 13.2.4 Anticoagulants 13.2.5 Fibrinolysis and fibrinolytics 13.3. Lipid modulating drugs 13.3.1 Cholesterol 13.3.2 Statins 13.3.3 Fibric acid derivatives 13.3.4 Ezetimibe

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Some major problems with existing models and terminology associated with kimberlite pipes from a volcanological perspective, and some suggestions

Five significant problems hinder advances in understanding of the volcanology of kimberlites: (1) kimberlite geology is very model driven; (2) a highly genetic terminology drives deposit or facies interpretation; (3) the effects of alteration on preserved depositional textures have been grossly underestimated; (4) the level of understanding of the physical process significance of preserved textures is limited; and, (5) some inferred processes and deposits are not based on actual, modern volcanological processes. These issues need to be addressed in order to advance understanding of kimberlite volcanological pipe forming processes and deposits. The traditional, steep-sided southern African pipe model (Class I) consists of a steep tapering pipe with a deep root zone, a middle diatreme zone and an upper crater zone (if preserved). Each zone is thought to be dominated by distinctive facies, respectively: hypabyssal kimberlite (HK, descriptively called here massive coherent porphyritic kimberlite), tuffisitic kimberlite breccia (TKB, descriptively here called massive, poorly sorted lapilli tuff) and crater zone facies, which include variably bedded pyroclastic kimberlite and resedimented and reworked volcaniclastic kimberlite (RVK). Porphyritic coherent kimberlite may, however, also be emplaced at different levels in the pipe, as later stage intrusions, as well as dykes in the surrounding country rock. The relationship between HK and TKB is not always clear. Sub-terranean fluidisation as an emplacement process is a largely unsubstantiated hypothesis; modern in-vent volcanological processes should initially be considered to explain observed deposits. Crater zone volcaniclastic deposits can occur within the diatreme zone of some pipes, indicating that the pipe was largely empty at the end of the eruption, and subsequently began to fill-in largely through resedimentation and sourcing of pyroclastic deposits from nearby vents. Classes II and III Canadian kimberlite models have a more factual, descriptive basis, but are still inadequately documented given the recency of their discovery. The diversity amongst kimberlite bodies suggests that a three-model classification is an over-simplification. Every kimberlite is altered to varying degrees, which is an intrinsic consequence of the ultrabasic composition of kimberlite and the in-vent context; few preserve original textures. The effects of syn- to post-emplacement alteration on original textures have not been adequately considered to date, and should be back-stripped to identify original textural elements and configurations. Applying sedimentological textural configurations as a guide to emplacement processes would be useful. The traditional terminology has many connotations about spatial position in pipe and of process. Perhaps the traditional terminology can be retained in the industrial situation as a general lithofacies-mining terminological scheme because it is so entrenched. However, for research purposes a more descriptive lithofacies terminology should be adopted to facilitate detailed understanding of deposit characteristics, important variations in these, and the process origins. For example every deposit of TKB is different in componentry, texture, or depositional structure. However, because so many deposits in many different pipes are called TKB, there is an implication that they are all similar and that similar processes were involved, which is far from clear.

The effect of HLA-DR genes on susceptibility to and severity of ankylosing spondylitis

Objective. To analyze the effect of HLA-DR genes on susceptibility to and severity of ankylosing spondylitis (AS). Methods. Three hundred sixty- three white British AS patients were studied; 149 were carefully assessed for a range of clinical manifestations, and disease severity was assessed using a structured questionnaire. Limited HLA class I typing and complete HLA-DR typing were performed using DNA-based methods. HLA data from 13,634 healthy white British bone marrow donors were used for comparison. Results. A significant association between DR1 and AS was found, independent of HLA-B27 (overall odds ratio [OR] 1.4, 95% confidence interval [95% CI] 1.1-1.8, P = 0.02; relative risk [RR] 2.7, 95% CI 1.5-4.8, P = 6 x 10-4 among homozygotes; RR 2.1, 95% CI 1.5-2.8, P = 5 x 10-6 among heterozygotes). A large but weakly significant association between DR8 and AS was noted, particularly among DR8 homozygotes (RR 6.8, 95% CI 1.6-29.2, P = 0.01 among homozygotes; RR 1.6, 95% CI 1.0-2.7, P = 0.07 among heterozygotes). A negative association with DR12 (OR 0.22, 95% CI 0.09-0.5, P = 0.001) was noted. HLA-DR7 was associated with younger age at onset of disease (mean age at onset 18 years for DR7-positive patients and 23 years for DR7-negative patients; Z score 3.21, P = 0.001). No other HLA class I or class H associations with disease severity or with different clinical manifestations of AS were found. Conclusion. The results of this study suggest that HLA-DR genes may have a weak effect on susceptibility to AS independent of HLA-B27, but do not support suggestions that they affect disease severity or different clinical manifestations.