Going Pro to enhance T‐cell immunogenicity: Easy as π?

MHC class I molecules bind intracellular oligopeptides and present them on the cell surface for CD8⁺ T‐cell activation and recognition. Strong peptide/MHC class I (pMHC) interactions typically induce the best CD8⁺ T‐cell responses; however, many immunotherapeutic tumor‐specific peptides bind MHC with low affinity. To overcome this, immunologists can carefully alter peptides for enhanced MHC affinity but often at the cost of decreased T‐cell recognition. A new report published in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43:3051–3060] shows that the substitution of proline at the third residue (p3P) of a common tumor peptide increases pMHC affinity and complex stability while enhancing T‐cell receptor recognition. X‐ray crystallography indicates that stability is generated through newly introduced CH‐π bonding between p3P and a conserved residue (Y159) in the MHC heavy chain. This finding highlights a previously unappreciated role for CH‐π bonding in MHC peptide binding, and importantly, arms immunologists with a novel and possibly general approach for increasing pMHC stability without compromising T‐cell recognition.     

Borrelia burgdorferi bba66 Gene Inactivation Results in Attenuated Mouse Infection by Tick Transmission

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb^ΔA66, remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb^ΔA66 following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb^ΔA66-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb^ΔA66 with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.     

VEGF-A165b Is an Endogenous Neuroprotective Splice Isoform of Vascular Endothelial Growth Factor A in?Vivo and in?Vitro

Vascular endothelial growth factor (VEGF) A is generated as two isoform families by alternative RNA splicing, represented by VEGF-A₁₆₅a and VEGF-A₁₆₅b. These isoforms have opposing actions on vascular permeability, angiogenesis, and vasodilatation. The proangiogenic VEGF-A₁₆₅a isoform is neuroprotective in hippocampal, dorsal root ganglia, and retinal neurons, but its propermeability, vasodilatatory, and angiogenic properties limit its therapeutic usefulness. In contrast, a neuroprotective effect of endogenous VEGF-A₁₆₅b on neurons would be advantageous for neurodegenerative pathologies. Endogenous expression of human and rat VEGF-A₁₆₅b was detected in hippocampal and cortical neurons. VEGF-A₁₆₅b formed a significant proportion of total VEGF-A in rat brain. Recombinant human VEGF-A₁₆₅b exerted neuroprotective effects in response to multiple insults, including glutamatergic excitotoxicity in hippocampal neurons, chemotherapy-induced cytotoxicity of dorsal root ganglion neurons, and retinal ganglion cells (RGCs) in rat retinal ischemia-reperfusion injury in vivo. Neuroprotection was dependent on VEGFR2 and MEK1/2 activation but not on p38 or phosphatidylinositol 3–kinase activation. Recombinant human VEGF-A₁₆₅b is a neuroprotective agent that effectively protects both peripheral and central neurons in vivo and in vitro through VEGFR2, MEK1/2, and inhibition of caspase-3 induction. VEGF-A₁₆₅b may be therapeutically useful for pathologies that involve neuronal damage, including hippocampal neurodegeneration, glaucoma diabetic retinopathy, and peripheral neuropathy. The endogenous nature of VEGF-A₁₆₅b expression suggests that non–isoform-specific inhibition of VEGF-A (for antiangiogenic reasons) may be damaging to retinal and sensory neurons.Vascular endothelial growth factor (VEGF) A, originally described as a potent vascular permeability and growth factor for endothelial cells, is up-regulated in the brain during stroke and ischemic episodes¹ and has been linked with many neuronal diseases. The most widely studied isoform of VEGF-A, VEGF-A₁₆₅a, is up-regulated in hypoxia, induces increased vascular permeability in neuronal vasculature, and can stimulate angiogenesis after ischemic episodes. The resulting edema and hyperemia can be damaging, but VEGF-A₁₆₅a has also been found to have direct anticytotoxic effects on neurons, raising the possibility that it may act as an endogenous neuroprotective agent in neurodegenerative pathologies. VEGF-A exerts neurotrophic (survival) and neurotropic (neurogenesis and axon outgrowth) actions, which, although initially thought to be a function of increased angiogenesis and perfusion after neuronal injury,² are now appreciated as direct effects of VEGF-A on neurons.The vegfa gene encodes numerous products by differential splicing, but not all isoforms exert the same effects.³ Alternative splicing of exon 8 leads to two functionally distinct families: the proangiogenic VEGF-A_xxxa family and the counteracting VEGF-A_xxxb family.^4,5 VEGF-A₁₆₅b prevents the VEGF-A₁₆₅a effects on increased vascular permeability, blood vessel growth, and vasodilatation.^4–7The therapeutic potential of VEGF-A and anti–VEGF-A treatments are now widely recognized, and effective anti–VEGF-A treatments are available in ophthalmology⁸ and oncology.⁹ The finding that VEGF-A is implicated in neuronal disorders (eg, Alzheimer disease, Parkinson disease, Huntington disease, diabetic neuropathy, and amyotrophic lateral sclerosis¹⁰) provides a rationale for the use of VEGF-A as a therapeutic agent in neurodegenerative conditions. Although this rationale is supported by preclinical evidence,¹¹ the identification of the VEGF-A_xxxb family requires reexamination of VEGF-A isoforms in these contexts to allow for the clear evidence that VEGF-A splicing variants are not functionally equivalent³ and to determine whether augmentation of the proangiogenic isoform family (VEGF-A_xxxa) alone may have deleterious effects (eg, in occult malignancy and carcinoma in situ).The neuroprotective profile of the exon 8 alternatively spliced isoforms VEGF-A_xxxb remains unexplored. Interestingly, VEGF-A_xxxb isoforms do not exhibit the vascular effects seen with VEGF-A_xxxa isoforms, such as a sustained increase in capillary permeability or hypotension.^5,12 The lack of these potential adverse effects may make VEGF-A_xxxb isoforms more amenable as therapeutic agents in neurodegenerative diseases.We therefore tested the hypothesis that VEGF-A₁₆₅b is neuroprotective for central and peripheral neurons. We found that VEGF-A₁₆₅b is expressed in central neurons and is neuroprotective in vitro and in vivo. This finding indicates that VEGF-A₁₆₅b may prove to be a suitable therapeutic agent in neurodegenerative disorders, exhibiting fewer adverse effects than VEGF-A₁₆₅a.     

Slaying the dragon myth: an ethnographic study of receptionists in UK general practice

Background

General practice receptionists fulfil an essential role in UK primary care, shaping patient access to health professionals. They are often portrayed as powerful ‘gatekeepers’. Existing literature and management initiatives advocate more training to improve their performance and, consequently, the patient experience.

Aim

To explore the complexity of the role of general practice receptionists by considering the wider practice context in which they work.

Design and setting

Ethnographic observation in seven urban general practices in the north-west of England.

Method

Seven researchers conducted 200 hours of ethnographic observation, predominantly in the reception areas of each practice. Forty-five receptionists were involved in the study and were asked about their work as they carried out their activities. Observational notes were taken. Analysis involved ascribing codes to incidents considered relevant to the role and organising these into related clusters.

Results

Receptionists were faced with the difficult task of prioritising patients, despite having little time, information, and training. They felt responsible for protecting those patients who were most vulnerable, however this was sometimes made difficult by protocols set by the GPs and by patients trying to ‘play’ the system.

Conclusion

Framing the receptionist–patient encounter as one between the ‘powerful’ and the ‘vulnerable’ gets in the way of fully understanding the complex tasks receptionists perform and the contradictions that are inherent in their role. Calls for more training, without reflective attention to practice dynamics, risk failing to address systemic problems, portraying them instead as individual failings.     

Impact of maternal trauma-related psychopathology and life stress on HPA axis stress response

To understand and curb intergenerational transmission of stress-related disorder, it is important to identify how trauma-related psychopathology in mothers impacts their psychophysiological stress regulation, particularly in the context of parenting their infants. In this study we investigated associations between mothers’ trauma-related psychopathology and life stress and HPA axis response to a personally relevant stressor (infant separation stress) in a non-clinical sample followed longitudinally postpartum. A community sample of low-income mothers (n = 73) and their infants completed laboratory sessions at 3, 6, 12, and 18 months postnatal, and salivary cortisol samples collected before and after dyadic stress tasks at the latter three sessions. These tasks were used to assess HPA function. A three-level hierarchical linear model of repeated cortisol measures nested within sessions within mother-infant dyads did not reveal significant main effects of trauma-related psychopathology on maternal cortisol response, but there was evidence that both a clinical interviewer-rated diagnosis of PTSD and ongoing self-reported trauma symptoms blunted effects of life events on cortisol reactivity. Region of significance analyses indicated that current life stress predicted more pronounced cortisol reactivity only among mothers without trauma-related psychopathology; for those with trauma-related psychopathology, life stress did not relate to cortisol response. Effects held when controlling for childhood trauma and previous (prenatal) maternal distress symptoms, suggesting they did not reflect ongoing impacts of past trauma exposure and/or psychopathology. Blunting effects of trauma-related psychopathology on maternal life stress responsiveness may help clarify how stress sensitivities and mental health are transmitted from parent to child.

     

Pregnancy during the COVID-19 pandemic: a qualitative examination of ways of coping

Archives of Women's Mental Health - The COVID-19 pandemic and related public health restrictions have impacted the mental health and coping strategies of many population groups, including...     

Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains

CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant Δ 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca²⁺ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca²⁺ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.     

Expert opinion on the management and follow-up of uveitis patients during SARS-CoV-2 outbreak

ABSTRACT

Introduction

Routine medical and ophthalmic care is being drastically curtailed in the context of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Uveitis patients require particular attention because of their theoretical risk of viral infection, in the context of therapeutic immunosuppression.     

The Clinical Genome Resource (ClinGen) Familial Hypercholesterolemia Variant Curation Expert Panel consensus guidelines for LDLR variant classification

PurposeIn 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published consensus standardized guidelines for sequence-level variant classification in Mendelian disorders. To increase accuracy and consistency, the Clinical Genome Resource Familial Hypercholesterolemia (FH) Variant Curation Expert Panel was tasked with optimizing the existing ACMG/AMP framework for disease-specific classification in FH. In this study, we provide consensus recommendations for the most common FH-associated gene, LDLR, where >2300 unique FH-associated variants have been identified.MethodsThe multidisciplinary FH Variant Curation Expert Panel met in person and through frequent emails and conference calls to develop LDLR-specific modifications of ACMG/AMP guidelines. Through iteration, pilot testing, debate, and commentary, consensus among experts was reached.ResultsThe consensus LDLR variant modifications to existing ACMG/AMP guidelines include (1) alteration of population frequency thresholds, (2) delineation of loss-of-function variant types, (3) functional study criteria specifications, (4) cosegregation criteria specifications, and (5) specific use and thresholds for in silico prediction tools, among others.ConclusionEstablishment of these guidelines as the new standard in the clinical laboratory setting will result in a more evidence-based, harmonized method for LDLR variant classification worldwide, thereby improving the care of patients with FH.