The potential of anti-infectives and immunomodulators as therapies for asthma and asthma exacerbations.

Asthma is responsible for approximately 25,000 deaths annually in Europe despite available medicines that maintain asthma control and reduce asthma exacerbations. Better treatments are urgently needed for the control of chronic asthma and reduction in asthma exacerbations, the major cause of asthma mortality. Much research spanning >20 years shows a strong association between microorganisms including pathogens in asthma onset, severity and exacerbation, yet with the exception of antibiotics, few treatments are available that specifically target the offending pathogens. Recent insights into the microbiome suggest that modulating commensal organisms within the gut or lung may also be a possible way to treat/prevent asthma. The European Academy of Allergy & Clinical Immunology Task Force on Anti-infectives in Asthma was initiated to investigate the potential of anti-infectives and immunomodulators in asthma. This review provides a concise summary of the current literature and aimed to identify and address key questions that concern the use of anti-infectives and both microbe- and host-based immunomodulators and their feasibility for use in asthma.

Association of ORMDL3 with rhinovirus-induced endoplasmic reticulum stress and type I Interferon responses in human leucocytes.

BACKGROUND: Children with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus illnesses have a greatly increased likelihood of developing childhood asthma. In mice, overexpression of the 17q21 gene ORMDL3 leads to airway remodelling and hyperresponsiveness. However, the mechanisms by which ORMDL3 predisposes to asthma are unclear. Previous studies have suggested that ORMDL3 induces endoplasmic reticulum (ER) stress and production of the type I interferon (IFN)-regulated chemokine CXCL10. OBJECTIVE: The purpose of this study was to determine the relationship between ORMDL3 and rhinovirus-induced ER stress and type I IFN in human leucocytes. METHODS: ER stress was monitored by measuring HSPA5, CHOP and spliced XBP1 gene expression, and type I IFN by measuring IFNB1 (IFN-ß) and CXCL10 expression in human cell lines and primary leucocytes following treatment with rhinovirus. Requirements for cell contact and specific cell type in ORMDL3 induction were examined by transwell assay and depletion experiments, respectively. Finally, the effects of 17q21 genotype on the expression of ORMDL3, IFNB1 and ER stress genes were assessed. RESULTS: THP-1 monocytes overexpressing ORMDL3 responded to rhinovirus with increased IFNB1 and HSPA5. Rhinovirus-induced ORMDL3 expression in primary leucocytes required cell-cell contact, and induction was suppressed by plasmacytoid dendritic cell depletion. The degree of rhinovirus-induced ORMDL3, HSPA5 and IFNB1 expression varied by leucocyte type and 17q21 genotype, with the highest expression of these genes in the asthma-associated genotype. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple lines of evidence support an association between higher ORMDL3 and increased rhinovirus-induced HSPA5 and type I IFN gene expression. These associations with ORMDL3 are cell type specific, with the most significant 17q21 genotype effects on ORMDL3 expression and HSPA5 induction evident in B cells. Together, these findings have implications for how the interaction of increased ORMDL3 and rhinovirus may predispose to asthma.

Genetic associations with viral respiratory illnesses and asthma control in children.

BACKGROUND: Viral respiratory infections can cause acute wheezing illnesses in children and exacerbations of asthma. OBJECTIVE: We sought to identify variation in genes with known antiviral and pro-inflammatory functions to identify specific associations with more severe viral respiratory illnesses and the risk of virus-induced exacerbations during the peak fall season. METHODS: The associations between genetic variation at 326 SNPs in 63 candidate genes and 10 phenotypes related to viral respiratory infection and asthma control were examined in 226 children enrolled in the RhinoGen study. Replication of asthma control phenotypes was performed in 2128 children in the Copenhagen Prospective Study on Asthma in Childhood (COPSAC). Significant associations in RhinoGen were further validated using virus-induced wheezing illness and asthma phenotypes in an independent sample of 122 children enrolled in the Childhood Origins of Asthma (COAST) birth cohort study. RESULTS: A significant excess of P values smaller than 0.05 was observed in the analysis of the 10 RhinoGen phenotypes. Polymorphisms in 12 genes were significantly associated with variation in the four phenotypes showing a significant enrichment of small P values. Six of those genes (STAT4, JAK2, MX1, VDR, DDX58, and EIF2AK2) also showed significant associations with asthma exacerbations in the COPSAC study or with asthma or virus-induced wheezing phenotypes in the COAST study. CONCLUSIONS: We identified genetic factors contributing to individual differences in childhood viral respiratory illnesses and virus-induced exacerbations of asthma. Defining mechanisms of these associations may provide insight into the pathogenesis of viral respiratory infections and virus-induced exacerbations of asthma.

A role for interleukins in ochronosis in a chondrocyte in vitro model of alkaptonuria.

Alkaptonuria is a rare autosomal recessive condition resulting from inability to breakdown homogentisic acid (HGA), an intermediate in tyrosine degradation. The condition has a triad of clinical features, the most damaging of which is ochronotic osteoarthropathy. HGA is elevated from birth, but pigmentation takes many years. We hypothesise that interleukins play a role in initiation and progression of ochronotic osteoarthropathy. C20/A4 cells were cultured and maintained in 9-cm petri dishes containing either HGA at 0.33 mM, a single interleukin (IL-1ß, IL-6 or IL-10) at 1 ng/ml or a combination of HGA and a single interleukin. Statistical analysis of pigment deposits and cell viability was performed using analysis of variance with Newman-Keuls post-test. All cultures containing HGA showed a significant increase in pigment deposition compared to control and IL cultures alone. The cultures containing HGA and IL-6 showed a significant increase in pigment deposits compared to HGA alone. The cell viability counts across all cultures on day 10 demonstrated a significant decrease in cultures containing HGA compared to those which did not. There was no significant difference between cultures containing just HGA or those combined with an interleukin. This work demonstrates a role for cytokines present in the joint(s) in the pigmentation process, particularly IL-6, and that the presence of HGA in joint tissues appears more detrimental to chondrocytes than the presence of any of the interleukins found in response to joint injury, trauma and osteoarthritis (OA). This further supports the evidence that the arthropathy in alkaptonuria is much more severe and rapidly progressing.

Osteoarticular cells tolerate short-term exposure to nitisinone-implications in alkaptonuria.

Alkaptonuria (AKU) is a rare genetic disease resulting in severe, rapidly progressing, early onset multi-joint osteoarthropathy. A potential therapy, nitisinone, is being trialled that reduces the causative agent; homogentisic acid (HGA) and in a murine model has shown to prevent ochronosis. Little is currently known about the effect nitisinone has on osteoarticular cells; these cells suffer most from the presence of HGA and its polymeric derivatives. This led us to investigate nitisinone's effect on chondrocytes and osteoblast-like cells in an in vitro model. Human C20/A4 immortalized chondrocytes, and osteosarcoma cells MG63 cultured in DMEM, as previously described. Confluent cells were then plated into 24-well plates at 4 × 10(4) cells per well in varying concentrations of nitisinone. Cells were cultured for 7 days with medium changes every third day. Trypan blue assay was used to determine viability and the effect of nitisinone concentration on cells. Statistical analysis was performed using analysis of variance, and differences between groups were determined by Newman-Keuls post-test. Analysis of C20/A4 chondrocyte and MG63 osteoblast-like cell viability when cultured in different concentrations of nitisinone demonstrates that there is no statistically significant difference in cell viability compared to control cultures. There is currently no literature surrounding the use of nitisinone in human in vitro models, or its effect on chondrocytes or osteoblast like cells. Our results show that nitisinone does not appear detrimental to cell viability of chondrocytes or osteoblast-like cells, which adds to the evidence that this therapy could be useful in treating AKU.

Late outcomes in adults with coarctation of the aorta.

AIMS: Previous cohort studies of patients with coarctation of the aorta (CoA) demonstrate reduced long-term survival. Improved surgical outcomes in children and evolution of adult congenital heart disease (ACHD) services have resulted in improved survival in patients with other CHDs. We hypothesise that for young adult patients with CoA long-term outcomes have improved in the contemporary era. METHODS: 151 patients (58% men) with simple CoA followed up at a tertiary ACHD service in Sydney, Australia, from 1993 to 2013 were included. We documented mortality and major morbidity such as the need for re-intervention for re-coarctation or aneurysms. RESULTS: 140 patients (mean age 35±15 years) underwent CoA repair at median age of 5 (IQR 0-10) years. Initial surgical strategy included end-to-end repair in 43, subclavian flap aortoplasty in 28 and patch aortoplasty in 31 patients (and was not documented in 28 cases). 6 patients had endovascular repair, 4 had interposition tube grafts and 11 were unrepaired. There were a total of seven deaths at a median age of 60 years. Actuarial survival was 98% at 40, 98% at 50 and 89% at 60 years of age. Re-coarctation occurred in 34% and descending aortic aneurysms were noted in 18%. Patients with end-to-end repair had lower rates of significant re-coarctation or descending aortic aneurysms (p=0.026 and <0.001, respectively). 66% had bicuspid aortic valve and 44% were hypertensive. CONCLUSIONS: Patients with CoA who reach adolescence demonstrate very good long-term survival up to age 60 years. Long-term morbidity is common, however, related largely to aortic complications and late hypertension.

Dielectric, calorimetric and mesophase properties of 1''-(2',4-difluorobiphenyl-4'-yloxy)-9''-(4-cyanobiphenyl-4'-yloxy) nonane: an odd liquid crystal dimer with a monotropic mesophase having the characteristics of a twist-bend nematic phase.

This paper reports a novel liquid crystal phase having the characteristics of a twist-bend nematic phase formed by a non-symmetric ether-linked liquid crystal dimer. The dimer 1''-(2',4-difluorobiphenyl-4'-yloxy)-9''-(4-cyanobiphenyl-4'-yloxy) nonane (FFO9OCB) exhibits two liquid-crystalline phases on cooling at a sufficiently high rate from the isotropic phase. The high temperature mesophase has been reported in the literature as nematic and confirmed in this study. The other mesophase is metastable and can be supercooled giving rise to a glassy state. Its identification and characterization are based on optical textures, broadband dielectric spectroscopy, calorimetry, measurements of both splay and bend elastic constants in the nematic phase and miscibility studies. It is concluded that the low temperature mesophase exhibits the characteristics of a twist-bend nematic phase. Dielectric measurements enable us to obtain the static permittivity and information about the molecular dynamics in the isotropic phase, in the nematic mesophase and across the isotropic-to-nematic phase transition. Two orientations, parallel and perpendicular to the director, have been investigated. In the high temperature nematic mesophase, the dielectric anisotropy is found to be positive. Measurements of the parallel component of the dielectric permittivity are well-explained by the molecular theory of dielectric relaxation in nematic dimers (M. Stocchero, A. Ferrarini, G. J. Moro, D. A. Dunmur and G. R. Luckhurst, J. Chem. Phys., 2004, 121, 8079). The dimer is modelled as a mixture of cis and trans conformers and the model allows an estimate of their relative populations at each temperature. The nematic-to-isotropic phase transition has been exhaustively studied from the accurate evolution of the heat capacity and the static dielectric permittivity data. It has been concluded that the transition is first order in nature, but close to tricritical. The nature of the nematic-to-the novel liquid crystal phase transition is difficult to analyze to the same extent because of insufficient precision. Only observations at cooling rates of 10 K min(-1) or higher were possible because on heating from the glassy state, the twist-bend nematic mesophase crystallizes at temperatures far below the nematic-nematic phase transition.

IL-15 complexes induce NK- and T-cell responses independent of type I IFN signaling during rhinovirus infection.

Rhinoviruses are among the most common viruses to infect man, causing a range of serious respiratory diseases including exacerbations of asthma and COPD. Type I IFN and IL-15 are thought to be required for antiviral immunity; however, their function during rhinovirus infection in vivo is undefined. In RV-infected human volunteers, IL-15 protein expression in fluid from the nasal mucosa and in bronchial biopsies was increased. In mice, RV induced type I IFN-dependent expressions of IL-15 and IL-15Rα, which in turn were required for NK- and CD8(+) T-cell responses. Treatment with IL-15-IL-15Rα complexes (IL-15c) boosted RV-induced expression of IL-15, IL-15Rα, IFN-γ, CXCL9, and CXCL10 followed by recruitment of activated, IFN-γ-expressing NK, CD8(+), and CD4(+) T cells. Treating infected IFNAR1(-/-) mice with IL-15c similarly increased IL-15, IL-15Rα, IFN-γ, and CXCL9 (but not CXCL10) expression also followed by NK-, CD8(+)-, and CD4(+)-T-cell recruitment and activation. We have demonstrated that type I IFN-induced IFN-γ and cellular immunity to RV was mediated by IL-15 and IL-15Rα. Importantly, we also show that IL-15 could be induced via a type I IFN-independent mechanism by IL-15 complex treatment, which in turn was sufficient to drive IFN-γ expression and lymphocyte responses.

Human platelet antigen (HPA)-1a peptides do not reliably suppress anti-HPA-1a responses using a humanized severe combined immunodeficiency (SCID) mouse model.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)-1a-positive fetal platelets are destroyed by maternal HPA-1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high-dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA-1a/1b polymorphism is Leu/Pro33 on ß3 integrin (CD61), and the anti-HPA-1a response is restricted to HPA-1b1b and HLA-DRB3*0101-positive pregnant women with an HPA-1a-positive fetus. We investigated whether or not HPA-1a antigen-specific peptides that formed the T cell epitope could reduce IgG anti-HPA-1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA-1a-immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA-1a-positive platelets. Human anti-HPA-1a in murine plasma was quantitated at intervals up to 15 weeks. HPA-1a-specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA-1a peptides in vitro, stimulation of anti-HPA-1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA-1a-specific T cell proliferation in vitro, marked suppression of the anti-HPA-1a response by HPA-1a peptides occurred in vivo. HPA-1a peptide immunotherapy in this model depended upon reactivation of HPA-1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen-specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies.

Characterisation and expression of the biomineralising gene Lustrin A during shell formation of the European abalone Haliotis tuberculata.

The molluscan shell is a remarkable product of a highly coordinated biomineralisation process, and is composed of calcium carbonate most commonly in the form of calcite or aragonite. The exceptional mechanical properties of this biomaterial are imparted by the embedded organic matrix which is secreted by the underlying mantle tissue. While many shell-matrix proteins have already been identified within adult molluscan shell, their presence and role in the early developmental stages of larval shell formation are not well understood. In the European abalone Haliotis tuberculata, the shell first forms in the early trochophore larva and develops into a mineralised protoconch in the veliger. Following metamorphosis, the juvenile shell rapidly changes as it becomes flattened and develops a more complex crystallographic profile including an external granular layer and an internal nacreous layer. Amongst the matrix proteins involved in abalone shell formation, Lustrin A is thought to participate in the formation of the nacreous layer. Here we have identified a partial cDNA coding for the Lustrin A gene in H. tuberculata and have analysed its spatial and temporal expression during abalone development. RT-PCR experiments indicate that Lustrin A is first expressed in juvenile (post-metamorphosis) stages, suggesting that Lustrin A is a component of the juvenile shell, but not of the larval shell. We also detect Lustrin A mRNAs in non-nacre forming cells at the distal-most edge of the juvenile mantle as well as in the nacre-forming region of the mantle. Lustrin A was also expressed in 7-day-old post-larvae, prior to the formation of nacre. These results suggest that Lustrin A plays multiple roles in the shell-forming process and further highlight the dynamic ontogenic nature of molluscan shell formation.

Specific patterns of allergic sensitization in early childhood and asthma & rhinitis risk.

BACKGROUND: Specific patterns of allergic sensitization as well as quantification of the in vitro IgE response in early life may provide relevant clinical insight into future rhinitis and asthma risk. OBJECTIVE: To define relationships among established sensitization to particular aeroallergens, quantitative analyses of allergen-specific IgE levels, pet exposure and sensitization, and asthma and rhinitis risk. METHODS: Children at high-risk for the development of asthma and allergic diseases were enrolled at birth into the Childhood Origins of ASThma (COAST) study. Allergen-specific IgE was assessed at ages 1, 3, 6, and 9 years by fluoroenzyme immunoassay (Unicap(®) 100; Pharmacia Diagnostics). Current asthma and rhinitis were diagnosed at age 6 and 8 years. RESULTS: Sensitization to dog was strongly associated with increased asthma risk (P < 0.0001). Sensitization to perennial compared with seasonal allergens was more strongly associated with asthma risk, while sensitization to seasonal allergens was more closely associated with rhinitis risk. Increased levels of specific IgE to perennial allergens were associated with an increased asthma risk (P = 0.05), while any detectable level of IgE to seasonal allergens was associated with increased rhinitis risk (P = 0.0009). While dog and cat sensitization were both independently associated with increased asthma and rhinitis risk, dog exposure at birth was associated with a reduced risk of asthma, regardless of dog sensitization status during the first 6 years of life (P = 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: Analysing specific patterns of an individual's allergic sensitization profile reveals additional relevant associations with asthma and rhinitis risk as opposed to the information gained from characterizing an individual as 'atopic' by the presence of any demonstrable sensitization alone. Furthermore, protective mechanisms of dog exposure with regards to asthma risk appear to be unrelated to the prevention of sensitization.

International consensus on (ICON) pediatric asthma.

Asthma is the most common chronic lower respiratory disease in childhood throughout the world. Several guidelines and/or consensus documents are available to support medical decisions on pediatric asthma. Although there is no doubt that the use of common systematic approaches for management can considerably improve outcomes, dissemination and implementation of these are still major challenges. Consequently, the International Collaboration in Asthma, Allergy and Immunology (iCAALL), recently formed by the EAACI, AAAAI, ACAAI, and WAO, has decided to propose an International Consensus on (ICON) Pediatric Asthma. The purpose of this document is to highlight the key messages that are common to many of the existing guidelines, while critically reviewing and commenting on any differences, thus providing a concise reference. The principles of pediatric asthma management are generally accepted. Overall, the treatment goal is disease control. To achieve this, patients and their parents should be educated to optimally manage the disease, in collaboration with healthcare professionals. Identification and avoidance of triggers is also of significant importance. Assessment and monitoring should be performed regularly to re-evaluate and fine-tune treatment. Pharmacotherapy is the cornerstone of treatment. The optimal use of medication can, in most cases, help patients control symptoms and reduce the risk for future morbidity. The management of exacerbations is a major consideration, independent of chronic treatment. There is a trend toward considering phenotype-specific treatment choices; however, this goal has not yet been achieved.

Phase behavior and properties of the liquid-crystal dimer 1'',7''-bis(4-cyanobiphenyl-4'-yl) heptane: a twist-bend nematic liquid crystal.

The liquid-crystal dimer 1'',7''-bis(4-cyanobiphenyl-4'-yl)heptane (CB7CB) exhibits two liquid-crystalline mesophases on cooling from the isotropic phase. The high-temperature phase is nematic; the identification and characterization of the other liquid-crystal phase is reported in this paper. It is concluded that the low-temperature mesophase of CB7CB is a new type of uniaxial nematic phase having a nonuniform director distribution composed of twist-bend deformations. The techniques of small-angle x-ray scattering, modulated differential scanning calorimetry, and dielectric spectroscopy have been applied to establish the nature of the nematic-nematic phase transition and the structural features of the twist-bend nematic phase. In addition, magnetic resonance studies (electron-spin resonance and (2)H nuclear magnetic resonance) have been used to investigate the orientational order and director distribution in the liquid-crystalline phases of CB7CB. The synthesis of a specifically deuterated sample of CB7CB is reported, and measurements showed a bifurcation of the quadrupolar splitting on entering the low-temperature mesophase from the high-temperature nematic phase. This splitting could be interpreted in terms of the chirality of the twist-bend structure of the director. Calculations using an atomistic model and the surface interaction potential with Monte Carlo sampling have been carried out to determine the conformational distribution and predict dielectric and elastic properties in the nematic phase. The former are in agreement with experimental measurements, while the latter are consistent with the formation of a twist-bend nematic phase.

Improved phylogenomic taxon sampling noticeably affects nonbilaterian relationships.

Despite expanding data sets and advances in phylogenomic methods, deep-level metazoan relationships remain highly controversial. Recent phylogenomic analyses depart from classical concepts in recovering ctenophores as the earliest branching metazoan taxon and propose a sister-group relationship between sponges and cnidarians (e.g., Dunn CW, Hejnol A, Matus DQ, et al. (18 co-authors). 2008. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452:745-749). Here, we argue that these results are artifacts stemming from insufficient taxon sampling and long-branch attraction (LBA). By increasing taxon sampling from previously unsampled nonbilaterians and using an identical gene set to that reported by Dunn et al., we recover monophyletic Porifera as the sister group to all other Metazoa. This suggests that the basal position of the fast-evolving Ctenophora proposed by Dunn et al. was due to LBA and that broad taxon sampling is of fundamental importance to metazoan phylogenomic analyses. Additionally, saturation in the Dunn et al. character set is comparatively high, possibly contributing to the poor support for some nonbilaterian nodes.

An evolutionary fast-track to biocalcification.

The ability to construct mineralized shells, spicules, spines and skeletons is thought to be a key factor that fuelled the expansion of multicellular animal life during the early Cambrian. The genes and molecular mechanisms that control the process of biomineralization in disparate phyla are gradually being revealed, and it is broadly recognized that an insoluble matrix of proteins, carbohydrates and other organic molecules are required for the initiation, regulation and inhibition of crystal growth. Here, we show that Astrosclera willeyana, a living representative of the now largely extinct stromatoporid sponges (a polyphyletic grade of poriferan bauplan), has apparently bypassed the requirement to evolve many of these mineral-regulating matrix proteins by using the degraded remains of bacteria to seed CaCO(3) crystal growth. Because stromatoporid sponges formed extensive reefs during the Paelozoic and Mesozoic eras (fulfilling the role that stony corals play in modern coral reefs), and fossil evidence suggests that the same process of bacterial skeleton formation occurred in these stromatoporid ancestors, we infer that some ancient reef ecosystems might have been founded on this microbial-metazoan relationship.

Inhibition of HPV-16 E7 oncogenic activity by HPV-16 E2.

Human papillomavirus (HPV) E7 is essential in inducing S-phase progression in differentiating epithelial cells. We have previously shown that HPV-16 E7 activity can be controlled by a direct interaction with the viral transcriptional activator E2, thereby inhibiting transforming potential of E7. We have extended these analyses to show that E2 induces a generalized re-localization of E7 within the cell nucleus, one potential consequence of which is the inhibition of E7-induced degradation of pRb. Most importantly, we show that E2 can also inhibit the ability of E7 to induce centrosome abnormalities, thus preventing aberrant mitoses. Taken together, these studies highlight the central importance of E2 in controlling the functions of E7, independently of the ability of E2 to regulate transcription.

Knowledge, attitudes and practices related to healthy childbearing in the West Coast/Winelands.

INTRODUCTION: Many of the known risk factors associated with low birth weight (LBW) infants, such as socio-economic status, ethnicity, genetic makeup, and obstetric history, are not within a woman's immediate control. However, there are many things that a woman can do to improve her chances of having a normal healthy child. Lifestyle behaviours, such as cigarette smoking, nutrition and the use of alcohol, play an important role in determining the growth of the foetus. There is a high rate of low birth weight infants born to women living and working on the farms in the Western Cape. Very little is known about the knowledge, attitudes and practices of the women living and working on the farms that may be influencing their pregnancy outcomes. The aim of this qualitative exploratory study was to establish the knowledge, attitudes and practices of reproductive age women related to lifestyle factors such as alcohol use, smoking and nutrition, and the perceptions of these factors by health care workers, in Stellenbosch and Vredendal areas (small towns in the Western Cape). METHODS: Four methods of data collection were employed: focus groups and individual interviews with women on farms, and focus groups and semi-structured interviews with health workers. All focus groups and interviews were recorded, transcribed, and then coded to form themes. Findings were then triangulated across data collection methods. RESULTS: Participants described high levels of use of alcohol and cigarettes by women living on the farms in general, and in pregnancy, despite reasonable levels of awareness of the dangers to the foetus. Regarding nutrition, women have a fairly good sense of eating in a balanced way during pregnancy, but affording this on very low wages is difficult. Many ideas regarding how to increase healthy lifestyles were offered, ranging from environmental improvements, such as access to recreational facilities and handwork classes, to more contact with health services, and improvement in conditions of employment. CONCLUSION: This study highlights the lifestyle factors related to LBW infants on farms, and proposes that these should be addressed collectively by all the relevant sectors in the community. Although some of these processes have been initiated, there are gaps in the health services, which should be addressed immediately to provide women with opportunities to ensure acceptable pregnancy outcomes.

Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism.

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20-39 and 24-45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by (3)H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.

Correlation between blood and milk serum leptin in goats and growth of their offspring.

Boer and Boer crossbred meat-type does were used in two experiments to determine whether goat milk serum contains leptin and to investigate possible correlations of milk and serum leptin in does and subsequent growth of their offspring. Blood and milk samples were collected within 2 h of kidding (d 0) from 20 (Exp. 1; spring) or 22 does (Exp. 2; the following fall). Blood milk samples were then collected again on d 0.5, 1, 3, 5, 7, 14, 21, 28, 35, 42, 49, and 56 (Exp. 1) or d 0.5, 1, 2, 3, 4, 5, 6, 7, 14, and 21 (Exp. 2). Body weights of kids were recorded on d 0, and BW of kids and does were recorded weekly beginning on d 7 (kids) or 21 (does), with BCS also recorded for does beginning on d 28 for Exp. 1 and on d 0.5, 1, 2, 3, 4, 5, 6, 7, 14, and 21 for Exp. 2. Leptin was detected in colostral milk and was influenced by days postpartum, decreasing (P < 0.001) over time with an average of 4.4 +/- 0.3 ng/mL (Exp. 1) and 18.1 +/- 1.0 ng/mL (Exp. 2) on d 0 compared with 1.0 +/- 0.3 ng/mL on d 56 (Exp. 1) and 2.9 +/- 0.2 ng/mL on d 21 (Exp. 2). Day postpartum and milk serum leptin were negatively correlated (P < 0.001) for Exp. 1 (r = -0.27) and Exp. 2 (r = -0.46). For Exp. 1 only, blood serum leptin tended (P = 0.09) to be influenced by day, with a weak positive correlation (r = 0.15; P < 0.02). Weak positive correlations (P < 0.01) were found between blood serum leptin and doe BCS (r = 0.42 in Exp. 1, and r = 0.13 in Exp. 2) and doe BW (r = 0.44 in Exp. 1, and r = 0.26 in Exp. 2), with the absence of a stronger relationship likely due in part to the short time period measured and the lack of significant changes in BCS and BW during that time. In conclusion, leptin was present in milk and blood serum of does, and blood serum leptin was weakly correlated with doe BW and BCS, but it was not related to kid BW. Therefore, further studies are needed to clarify the relationships involving milk and serum leptin in goats.

Flow cytometric characterisation of cells of differing densities isolated from human term placentae and enrichment of villous trophoblast cells.

Cells were isolated from human term placentae by trypsinisation of fragments of chorionic villi and fractionation of cells on a Percoll density gradient into six layers. A panel of 10 monoclonal antibodies to antigens on or in trophoblast cells (placental alkaline phosphatase (PLAP), cytokeratin-7, beta-human chorionic gonadotrophin (beta-hCG), human leucocyte antigen-G (HLA-G)), leucocytes (CD45), monocytes, macrophages, dendritic cells, B cells (HLA class II), mesenchyme cells (vimentin), fibroblasts (fibroblast antigen) and nucleated cells excluding villous trophoblast (HLA class I, CD9) was used to characterise the cells by flow cytometry. For staining intracellular antigens (cytokeratin, vimentin, beta-hCG) the cells were first fixed and permeabilised. The upper two layers from the gradient (density 1.013-1.039 g/ml) contained predominantly PLAP-positive cells or fragments, probably derived from the syncytiotrophoblast. Cytokeratin-positive cells accumulated mainly in the layer of density 1.039-1.052 g/ml and comprised the majority of the cell types identified in this fraction. Few or no cells reactive with antibodies to beta-hCG or HLA-G were identified in any layer. Non-trophoblast cells were heavier, being present mainly at densities 1.052-1.079 g/ml (CD45, HLA class I, vimentin) and 1.066-1.092 g/ml (fibroblast). Fewer than 10% of cells in any layer were HLA class II- or CD9-positive. Further purification of trophoblast cells was by negative immunomagnetic separation with removal of CD45-positive cells and HLA class II-positive cells to less than 1%. On culture of the cells from each layer, those of density 1.039-1.066 g/ml exhibited characteristics of cytotrophoblast cells; they secreted high levels of human chorionic gonadotrophin and formed adherent multinucleate cells. This procedure enabled the selection and enrichment of cytotrophoblast cells and/or syncytiotrophoblast fragments that are suitable for cellular and molecular studies.