Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell–mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR–HLA-B*4405^EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.     

Enhancement of MHC-I Antigen Presentation via Architectural Control of pH-Responsive,Endosomolytic Polymer Nanoparticles

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8⁺ T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8⁺ T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8⁺ T cell responses to protein-based vaccines.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9697-1) contains supplementary material, which is available to authorized users.KEY WORDS: MHC-I antigen presentation, pH-responsive nanoparticle, polymer architecture, RAFT polymerization, vaccine     

CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation

Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti‐cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra‐pelvic). None of the biomarkers showed any correlation with overall or disease‐free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours.     

Apparent improvement in diabetic autonomic neuropathy induced by captopril

Eight diabetic subjects with moderately elevated blood pressure (BP) (systolic (SBP) greater than 140 and/or diastolic (DBP) greater than 90 mmHg) were studied. Each had evidence of mild asymptomatic autonomic neuropathy (impairment of forced sinus arrhythmia). The effect of a single oral 25 mg dose of captopril on BP and some aspects of autonomic function was compared with matched placebo in a double-blind, cross-over study. Resting supine SBP and DBP fell significantly (P less than 0.01) over 90 minutes following captopril, indicating that the hypotensive effect of the drug was not dependent on intact autonomic function. There was no significant change in resting heart rate. The bradycardic response to apnoeic face immersion was significantly (P less than 0.01) enhanced following captopril. Sinus arrhythmia did not change. The BP responses to standing and to the cold pressor test were unaffected. There was no exacerbation of postural hypotension. The ingestion of a single dose of captopril appears to increase vagal function, without affecting sympathetic nervous function, in diabetics with evidence of mild vagal impairment.     

Biochemical and clinical manifestations of dopamine-producing paragangliomas: utility of plasma methoxytyramine

Measurements of plasma-free normetanephrine and metanephrine provide a sensitive test for diagnosis of pheochromocytoma but may fail to detect tumors that produce predominantly dopamine. Such tumors are extremely rare, usually found as extraadrenal paragangliomas. This report describes measurements of plasma concentrations of free methoxytyramine, the O-methylated metabolite of dopamine, in 120 patients with catecholamine-producing tumors, including nine with extraadrenal paragangliomas secreting predominantly dopamine. In seven of these nine patients, tumors were found incidentally or secondary to the space-occupying complications of the lesions. Plasma concentrations of free methoxytyramine and dopamine were increased in all nine patients, including two with normal plasma and urinary normetanephrine and metanephrine and normal urinary outputs of dopamine. Relative increases above normal for plasma methoxytyramine (104-fold) and dopamine (56-fold) were much greater (P < 0.001) than those for urinary dopamine (3-fold). Insensitivity of the latter for identification of dopamine-secreting tumors was due to dependence of the urinary amine on renal extraction and decarboxylation of circulating 3,4-dihydroxyphenylalanine. Measurements of plasma-free methoxytyramine, in addition to normetanephrine and metanephrine, are unlikely to improve diagnosis of pheochromocytomas in hypertensive patients with symptoms of catecholamine excess but may be useful in selected patients for identification of tumors that produce predominantly dopamine.     

Narrative inquiry as a research methodology exploring person centred care in nursing

Background

Although, person centred care has for a long time been an important approach to nursing care, it is often not a reality in the clinical environment. The focus of health research has, until recently, been on the physical aspects of a persons’ illness and this has influenced how care is delivered. There is a need to broaden the focus from the illness to the person who is ill. A holistic approach to the persons’ social and cultural experience of their illness will aid health care professions to provide person centred care.This paper will make the argument that narrative inquiry is a well suited to health care research in general and nursing research in particular as it focuses its inquiry on the individual person's experience of their illness – ‘what matters’ from the person's point of view. Narrative inquiry explores the narrative from a temporal, social and spatial view.