Epidemiological Approach to Identifying Genetic Predispositions for Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is caused by several susceptibility genes. A registry including analyses of susceptibility genes, familial occurrence and genotype-phenotype correlation should provide classification insights.
Registry data of 187 unrelated index patients included age at onset, gender, family history, relapse of aHUS and potentially triggering conditions. Mutation analyses were performed in the genes CFH , CD46 and CFI and in the six potential susceptibility genes, FHR1 to FHR5 and C4BP .
Germline mutations were identified in 17% of the index cases; 12% in CFH , 3% in CD46 and 2% in CFI . Twenty-nine patients had heterozygous mutations and one each had a homozygous and compound heterozygous mutation. Mutations were not found in the genes FHR1-5 and C4BP . In 40% of the patients with familial HUS a mutation was found. Penetrance by age 45 was 50% among carriers of any mutation including results of relatives of mutation-positive index cases. The only risk factor for a mutation was family history of HUS (p = 0.02).
P enetrance of aHUS in carriers of mutations is not complete. Occurrence of homo- and heterozygous mutations in the same gene suggests that the number of necessary DNA variants remains unclear. Among clinical information only familial occurrence predicts a mutation.     

The outcome of T-cell costimulation through intercellular adhesion molecule-1 differs from costimulation through leucocyte function-associated antigen-1

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. The outcome of T-cell activation can be influenced by the nature of the costimulatory signal the T cell receives. We recently demonstrated the ability of stimulation through intercellular adhesion molecule-1 (ICAM-1), resident on the T-cell surface, to provide a second signal for T-cell activation, and have extended that work here to begin an examination of the functional outcome of this set of signals. Costimulation through ICAM-1 resulted in a greater percentage of cells having undergone more than three divisions when compared to costimulation through leucocyte function-associated antigen-1 (LFA-1). Costimulation through ICAM-1 also had an effect similar to costimulation through CD28 in its ability to down-regulate the cyclin dependent kinase inhibitor p27kip1. Costimulation through ICAM-1 provided greater protection from apoptosis than costimulation through LFA-1, especially in cells having divided more than three times. This was supported by the ability of costimulation through ICAM-1 to up-regulate the anti-apoptotic protein Bcl-2. Finally, costimulation through ICAM-1 or CD28 produced a greater number of T cells with a memory phenotype than costimulation through LFA-1.     

Alanine Mutagenesis of the Primary Antigenic Escape Residue Cluster,C1, of Apical Membrane Antigen 1

Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion of Plasmodium merozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strain Plasmodium falciparum AMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.The merozoite stage of Plasmodium falciparum is a highly specialized form of the parasite that selectively invades human red blood cells. Although the exact mechanism of invasion is still under investigation, the expression of apical membrane antigen 1 (AMA1) appears to be an absolute requirement for successful invasion (31). Monoclonal and polyclonal antibodies against AMA1 inhibit invasion in vitro, and immunization with recombinant AMA1 protects against live parasite challenge in animal models of malaria (10a, 18, 28).P. falciparum AMA1 vaccines based on two laboratory strains, 3D7 and FVO, are being studied for efficacy in human trials (23, 27, 30). One of the major concerns in further development of the AMA1 vaccine is that ∼10% of its 622 amino acids are polymorphic (2). Strain specificity of vaccine-induced AMA1 antibodies has been observed by enzyme-linked immunosorbent assay (ELISA) and in a functional assay of parasite growth and invasion inhibition (growth inhibition assay [GIA]) (15, 24). Allelic replacement experiments show that sequence polymorphism within AMA1 causes antigenic escape (14), and the extent of escape correlates with sequence distance between the vaccine and target strain (18).The crystal structure of AMA1 shows that it contains two PAN domains, with loops extending outwards from its central core (1, 22). The loops contain the majority of the polymorphic residues of AMA1 and surround a highly conserved hydrophobic trough. Residues within the trough have been implicated in the binding of AMA1 to AMA1-associated proteins on the merozoite (5). The location of the polymorphic loops surrounding the trough is highly suggestive of its role of providing diversity to a functional region of AMA1, preventing the binding of inhibitory antibodies. Using chimeric proteins to specifically deplete strain-specific antibodies against 3D7 strain AMA1, in a growth inhibition assay (GIA), we showed that certain polymorphic sites conferred escape upon the FVO strain parasite from invasion inhibitory anti-3D7 AMA1 antibodies. These polymorphic residues were termed “antigenic escape residues” (AER). The majority of AER in the 3D7-FVO model mapped to domain 1 (10), and within domain 1, the highest escape per residue was conferred by 7 polymorphic sites located on a linear stretch of sequence between residues 187 and 207. This polymorphic cluster was termed the C1′ cluster (10). It contains the three most polymorphic residues of AMA1, as follows: residue 187 is located on loop Ic, and residues 197 and 200 are located on the adjacent C1-L loop (also termed loop Id).Given that the polymorphic residues within the C1 cluster are the primary determinants of strain specificity of AMA1, it is likely that protein engineering strategies targeted to C1 AER could be used to modulate the cross-reactivity of AMA1 antibodies. We hypothesized that if the immunogenicity of the C1 AER could be reduced, this might result in an increased antibody response to non-strain-specific cross-reactive epitopes. In this study, seven 3D7-FVO polymorphic differences within the C1 cluster were replaced on the FVO AMA1 protein with alanine residues. The 3D7-FVO escape model was then used to study the effect of alanine mutagenesis on the quantity and quality of the induced antibodies. This strategy resulted in a measurable shift in the immune response away from the C1 region but did not enhance the generation of cross-strain-reactive antibodies that were inhibitory to parasite invasion.     

Patient–provider communication and low-income adults: Age,race, literacy,and optimism predict communication satisfaction

Objective

To assess whether literacy, numeracy, and optimism are related to low-income adults’ satisfaction with their healthcare provider's communication skills.

Methods

Low-income adults (N = 131) were recruited from seven counties in Indiana through University extension programs. To achieve research triangulation, participants were surveyed and interviewed about their communication satisfaction with health providers.

Results

Survey data revealed that four variables significantly predicted satisfaction: age, race, literacy, and optimism. Low-income adults in the current study were more critical of their healthcare provider's communication skills if they were younger, White, functionally literate, and pessimistic. Follow-up interviews confirmed this pattern and suggested it was a byproduct of patient activism.

Conclusion

In low-income populations, communication satisfaction may be lower for groups that are traditionally active in doctor–patient interactions (e.g., younger patients, patients with higher literacy skills).

Practice implications

Healthcare providers should be aware that older, non-White, optimistic, and literacy deficient patients report greater communication satisfaction than their younger, White, pessimistic, and functionally literate peers. Both groups may be coping with their situation, the former by withdrawing and the latter by actively pushing for a higher standard of care. Healthcare providers should continue to seek out ways to facilitate dialogue with these underserved groups.     

Low- and high-risk human papillomavirus E7 proteins regulate p130 differently

The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.     

Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interactions with myeloid antigen-presenting cells

Natural killer T (NKT) cells are innate T lymphocytes that are restricted by CD1d antigen‐presenting molecules and recognize lipids and glycolipids as antigens. NKT cells have attracted attention for their potent immunoregulatory effects. Like other types of regulatory lymphocytes, a high proportion of NKT cells appear to be autoreactive to self antigens. Thus, as myeloid antigen‐presenting cells (APCs) such as monocytes, dendritic cells (DCs) and myeloid‐derived suppressor cells (MDSCs) constitutively express CD1d, NKT cells are able to interact with these APCs not only during times of immune activation but also in immunologically quiescent periods. The interactions of NKT cells with myeloid APCs can have either pro‐inflammatory or tolerizing outcomes, and a central question is how the ensuing response is determined. Here we bring together published results from a variety of model systems to highlight three critical factors that influence the outcome of the NKT–APC interaction: (i) the strength of the antigenic signal delivered to the NKT cell, as determined by antigen abundance and/or T‐cell receptor (TCR) affinity; (ii) the presence or absence of cytokines that costimulate NKT cells [e.g. interleukin (IL)‐12, IL‐18 and interferon (IFN)‐α]; (iii) APC intrinsic factors such as differentiation state (e.g. monocyte versus DC) and Toll‐like receptor (TLR) stimulation. Together with recent findings that demonstrate new links between NKT cell activation and endogenous lipid metabolism, these results outline a picture in which the functions of NKT cells are closely attuned to the existing biological context. Thus, NKT cells may actively promote tolerance until a critical level of danger signals arises, at which point they switch to activating pro‐inflammatory immune responses.