Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae

Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity

Functional Memory B Cells and Long-Lived Plasma Cells Are Generated after a Single Plasmodium chabaudi Infection in Mice

Antibodies have long been shown to play a critical role in naturally acquired immunity to malaria, but it has been suggested that Plasmodium-specific antibodies in humans may not be long lived. The cellular mechanisms underlying B cell and antibody responses are difficult to study in human infections; therefore, we have investigated the kinetics, duration and characteristics of the Plasmodium-specific memory B cell response in an infection of P. chabaudi in mice. Memory B cells and plasma cells specific for the C-terminal region of Merozoite Surface Protein 1 were detectable for more than eight months following primary infection. Furthermore, a classical memory response comprised predominantly of the T-cell dependent isotypes IgG2c, IgG2b and IgG1 was elicited upon rechallenge with the homologous parasite, confirming the generation of functional memory B cells. Using cyclophosphamide treatment to discriminate between long-lived and short-lived plasma cells, we demonstrated long-lived cells secreting Plasmodium-specific IgG in both bone marrow and in spleens of infected mice. The presence of these long-lived cells was independent of the presence of chronic infection, as removal of parasites with anti-malarial drugs had no impact on their numbers. Thus, in this model of malaria, both functional Plasmodium-specific memory B cells and long-lived plasma cells can be generated, suggesting that defects in generating these cell populations may not be the reason for generating short-lived antibody responses

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

Alterations of Splenic Architecture in Malaria Are Induced Independently of Toll-Like Receptors 2, 4, and 9 or MyD88 and May Affect Antibody Affinity▿ †

Splenic microarchitecture is substantially altered during acute malaria infections, which may affect the development and regulation of immune responses. Here we investigated whether engagement of host Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adaptor protein MyD88 is required for induction of the changes and whether antibody responses are modified when immunization takes place during the period of splenic disruption. The alterations in splenic microarchitecture were maximal shortly after the peak of parasitemia and were not dependent on engagement of TLR2, TLR4, or TLR9, and they were only minimally affected by the absence of the MyD88 adaptor molecule. Although germinal centers were formed in infected mice, they did not contain the usual light and dark zones. Immunization of mice with chicken gamma globulin 2 weeks prior to acute Plasmodium chabaudi infection did not affect the quantity or avidity of the immunoglobulin G antibody response to this antigen. However, immunization at the same time as the primary P. chabaudi infection resulted in a clear transient reduction in antibody avidity in the month following immunization. These data suggest that the alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibody response during a malaria infection and could impact the development of immunity to malaria or to other infections or immunizations given during a malaria infection

Eosinophils are required for clearance of <i>B. malayi</i> Mf during primary (1°), but not challenge (2°) infection.

<p><b>A.</b> Mf survival in groups of PHIL (black squares) and C57Bl/6 (open circles) mice, following primary (dotted line) and secondary (solid line) infection. Mf were counted in blood from the tail vein of mice on days 6 and 12 p.i. or in cardiac blood on day 21 p.i. (d21 PHIL mice 1° versus C57Bl/6 1° p = 0.0018) <b>B–D.</b> Cell recruitment into BALF of naïve PHIL and C57Bl/6 mice and both mouse strains mice given primary or challenge Mf infections on day 21 p.i. <b>B.</b> Mean total and differential cell recruitment into BALF. <b>C.</b> Numbers of mast cells recruited into BALF (Mean ± S.E) of naïve and infected PHIL and C57Bl/6 mice. <b>D.</b> Numbers of eosinophils recruited into BALF (Mean ± S.E) of naïve and infected PHIL and C57Bl/6 mice. <b>E.</b> Mf-specific IgG1 antibody in serum during infection of PHIL and C57Bl/6 mice (Mean ± S.E.) (dotted line represents naïve levels in both PHIL and C57Bl/6). <b>F.</b> Total IgE antibody in serum during infection of PHIL and C57Bl/6 mice (Mean ± S.E.). <b>G–I.</b> Cytokines produced by splenocytes upon stimulation with Mf antigen and measured in cell culture supernatants by ELISA 72 h later. Graphs show mean ± S.E. cytokine concentration of naïve PHIL and C57Bl/6 mice and mice 21 days post primary (1°) and challenge (2°) infections of live Mf. <b>G.</b> IFN-γ responses <b>H.</b> IL-5 responses. <b>I.</b> IL-13 responses. This figure represents data from two independent experiments with 6 individual mice per group. *represents a significant difference at p<0.05, ** p<0.01 ****p<0.0001 between groups of PHIL mice and C57Bl/6 mice given the same infection regimen.</p

Lung function and levels of mucus-secreting goblet cells in eosinophil-less mice given <i>B. malayi</i> Mf infection.

<p><b>A.</b> Penh values were measured in conscious, unrestrained mice administered with increasing doses of the aerosolized bronchoconstrictor, methacholine. Mean Penh ± S.E. is shown during Mf infection. <b>B–C.</b> Lungs were embedded in wax, sections were cut at 6 µm and stained with periodic acid Schiff (magnification ×40). <b>B.</b> Mean ± S.E. percentage of positively staining goblet cells per airway are shown for each infection group. <b>C.</b> The arrows show positive PAS staining at day 6, 12 and 21 post infection. This figure represents data from two independent experiments with 6 individual mice per group. *represents a significant difference at p<0.05, ** p<0.01 ****p<0.0001 between groups of PHIL mice and C57Bl/6 mice given the same infection regimen.</p

MBP-1 is not required for Mf survival but contributes to pulmonary eosinophil recruitment and goblet cell mucus production following Mf infection.

<p><b>A.</b> Mf survival in MBP-1^−/− (black squares) and C57Bl/6 (open circles) mice following primary (dotted line) and challenge (solid line) infection. <b>B.</b> Mean total and differential cell recruitment to BALF in MBP-1^−/− and C57Bl/6 mice given primary or challenge Mf infections. <b>C.</b> Lung function (Penh) on day 12 post live Mf challenge showing mean ± S.E. Penh. <b>D.</b> Mucus-secreting goblet cells in 6 µm sections of lung, stained with Periodic Acid Schiff (magnification ×40). The graph shows mean ± S.E. percentage of positively staining cells per airway at day 20 post live Mf infection. <b>A–D.</b> These graphs represent data from two independent experiments with 4 individual mice per group. *represents a significant difference at p<0.05, ** p<0.01 between groups of gene-targeted mice and C57Bl/6 mice given the same infection regimen.</p

Mouse infection regimen.

<p>In each experiment, groups of four to six male C57Bl/6 wild-type mice and/or gene-targeted mice were either left uninfected (naïve), or they were injected with 200,000 <i>B. malayi</i> Mf i.v. (primary (1°) infection) or they were immunised on three occasions with 200 µg soluble Mf extract prior to challenge with 200,000 <i>B. malayi</i> Mf i.v. (challenge (2°) infection). At days 5–6, 10–12 and 20–21 post infection with Mf, immunological and pathological parameters of mice were measured. This figure shows the exact regimen used in the PHIL mice experiments.</p

EPO is not required for Mf survival but contributes to pulmonary eosinophil recruitment, total IgE production and nematode-induced in lung physiology following Mf infection.

<p><b>A.</b> Mf survival in EPO^−/− (black squares) and C57Bl/6 (open circles) mice following primary (dotted line) and challenge (solid line) infection. <b>B.</b> Numbers of eosinophils recruited to BALF (mean ± S.E.) in EPO^−/− and C57Bl/6 mice given primary or challenge Mf infections. <b>C.</b> Total serum IgE in µg/ml (mean ± S.E) as measured by ELISA on day 20 post live Mf infection during infection of C57Bl/6, EPO^−/− and MBP^−/− mice (Mean ± S.E.). <b>D.</b> EPO contributes to lung hyper-responsiveness following primary, but not challenge infection. Penh values were measured in conscious, unrestrained mice administered with increasing doses of the aerosolized bronchoconstrictor, methacholine. Mean Penh ± S.E. measurement at day 5, 10 and 20 post live Mf challenge. Graphs show mean Penh ± S.E. <b>E.</b> The number of breaths per minute of naïve and infected EPO^−/− and WT mice This figure represents data from two independent experiments with 4–6 individual mice per group. *represents a significant difference at p<0.05, ** p<0.01 ****p<0.0001 between groups of gene-targeted mice and C57Bl/6 mice given the same infection regimen.</p

Parenting a child with Phenylketonuria (PKU): An interpretative phenomenological analysis (IPA) of the experiences of parents

Phenylketonuria (PKU) is a rare inherited metabolic disorder which can cause neurological damage if left untreated. PKU is identified through newborn screening in developed countries, and treatment begins immediately to prevent these severe consequences. When a child is diagnosed, parents must assume immediate responsibility for the management of PKU and prevention of neurological damage. Quantitative studies have identified significant psychosocial stressors for parents, but little is known about how the parents experience this process. This study aimed to explore the experiences of parents of children with PKU under the age of two. It is the first study to examine these experiences in this way. Seven parents were interviewed about their experiences, and interpretative phenomenological analysis was used to analyse the data. Three main themes were identified: control, striving for normality and acceptance of PKU as a continuum. Links between the themes and processes underpinning the results were explored with relation to existing literature and theories from a clinical psychology perspective. The role of acceptance of PKU was central to the parent’s experiences. Clinical implications and suggestions for further research are discussed