Genotypes and haplotypes in the IL-1 gene cluster: analysis of two genetically and diagnostically distinct groups of Alzheimer patients

Increased risk of Alzheimer's disease (AD) has been associated with polymorphisms in the IL-1 gene cluster, and in particular with the IL-1alpha-889 T/T genotype. However, this association is still unclear, and needs further investigation. In order to clarify the role of these polymorphisms in the complex pathogenesis of AD we examined genotype and haplotype frequencies of the two C-to-T SNPs at position -889 and -551 in the IL-1alpha and IL-1beta genes, respectively, and of the 86 bp VNTR intron-2 polymorphisms in the IL-1Ra gene. The analysis was performed in two genetically and diagnostically distinct groups of sporadic AD from Italy and the USA. In the Italian group a significant association between the IL-1alpha-889 T/T genotype and AD (OR=3.022, 95% CI: 1.001-9.119) was found, whereas no difference was found in the group from the USA. Results were also compared with previously published studies that analyzed the same IL-1 polymorphisms in AD. In both groups, the analysis of the estimated haplotypes shows that AD patients and controls who carry the IL-1beta-511 C allele, were also more frequently carriers of the IL-1Ra 1 allele (haplotypes -C-1). The total frequency of the two -C-1 haplotypes (C-C-1 plus T-C-1) was about one half of the total frequency of the eight estimated haplotypes. This was confirmed by significant linkage disequilibrium between these two loci in both the Italian and USA groups. In the Italian group a weak association of the T-C-2 haplotype with the disease (OR=1.648, 95% CI: 1.519-1.788) was also found, whereas in the USA group no difference was found. Although ours and other published data on different samples of Caucasian and non-Caucasian AD show a great heterogeneity in the frequencies of the IL-1alpha-889, the IL-1beta-511 and the IL-1Ra VNTR gene polymorphisms, we confirm the role of the IL-1alpha-889 T/T genotype as a risk factor for sporadic AD, and show the presence of an allelic association between IL-1beta C and IL-1Ra 1 alleles in both the Italian and the USA groups, confirmed by the presence of significant levels of linkage disequilibrium between these two loci.     

Antifungal coating by biofunctionalized polyelectrolyte multilayered films

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a chromogranin A-derived antifungal peptide (CGA 47-66, chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. In vitro studies demonstrate that such an antifungal coating is able to inhibit the growth of yeast Candida albicans by 65% and completely stop the proliferation of filamentous fungus Neurospora crassa. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly(methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an in vivo oral candidiasis rat model. Taken together, these results assessed the functionalized multilayer films containing a new potent antifungal non-toxic peptide, as a novel and promising technique for local antifungal protection.     

Impact of intranasal budesonide on immune inflammatory responses and epithelial remodeling in chronic upper airway inflammation

BACKGROUND: Histologic and immunohistologic features of nasal polyps (NP) are similar to those observed in asthma, thus suggesting a similar immunopathology. OBJECTIVE: The primary objective of this study was to further understand the anti-inflammatory and immunoregulatory effects of locally delivered corticosteroids. To this end, the effect of intranasal budesonide on the expression of specific cytokines, lymphocyte subsets, and epithelial remodeling in this model of airway tissue inflammation were studied. METHODS: We used immunohistochemical techniques to examine nasal mucosae (NM) from healthy individuals and nasal polyp (NP) tissues from patients with nasal polyposis obtained before and after intranasal budesonide treatment. RESULTS: First, the density of CD8(+) cells was markedly increased in NP tissues after intranasal budesonide treatment from 16.1 +/- 8.4 (M +/- SEM) per mm(2) to 39.9 +/- 24.1. Second, the density of cells immunoreactive for IL-4, IL-5, IFN-gamma, IL-12, and TGF-beta in NP was significantly greater than in control NM tissues. The density of IL-4(+) and IL-5(+) cells in NP tissues significantly decreased after budesonide treatment from 40 +/- 12 to 17.8 +/- 8 and from 19.3 +/- 11 to 10.4 +/- 7, respectively. In contrast, the density of IFN-gamma(+) and IL-12(+) cells remained unchanged. In addition, we found that the density of TGF-beta(+) cells significantly increased after intranasal budesonide from 18 +/- 5 to 41 +/- 9. Third, damage to the entire length of the NP epithelium was quantified using a grading system. The epithelium of untreated NP was substantially damaged; remarkable epithelial restitution with no apparent changes in stromal collagen deposition was observed after intranasal budesonide treatment. CONCLUSIONS: These findings demonstrate that intranasal budesonide induced an increase in CD8 population and a selective regulatory effect on tissue cytokine expression. Furthermore, intranasal budesonide promoted epithelial remodeling. We hypothesize that these immunoregulatory and remodeling effects elicited by steroids might be, at least in part, mediated by the induction of TGF-beta.     

Effecting Change in Maltreating Fathers: Critical Principles for Intervention Planning

Although fathers perpetrate a significant proportion of child maltreatment, the intervention needs of abusive and neglectful fathers have not been adequately addressed or researched. This paper argues that well-designed treatment has the potential to benefit men, their children, and their families. However, the treatment needs of maltreating and at-risk fathers are unique, and programs must be designed accordingly. Based on the integration of parenting, child abuse, change promotion, and batterer treatment literatures, five principles to guide intervention with maltreating fathers are advanced: (a) overly controlling behavior, a sense of entitlement, and self-centered attitudes are primary problems of abusive fathers; thus, the development of child-management skills should not be an initial focus of intervention; (b) abusive fathers are seldom initially ready to make changes in their parenting; (c) fathers' adherence to gender-role stereotypes also contributes to their maltreatment of children; (d) the relationship between abusive fathers and the mothers of their children requires special attention; and (e) because abusive fathers have eroded children's emotional security, the need to rebuild trust will affect the pace of change and potential impact of relapse on the child. These principles are contrasted with the supportive and child-management goals of conventional group parenting programs, and the implications for providing service to fathers are considered.     

Fibrolamellar Carcinoma of Liver: A Primary Malignant Oncocytic Carcinoid?

Immunohistochemical and ultrastructural findings in two cases of fibrolamellar carcinoma of the liver and two cases of hepatocellular carcinoma of the common histologic type are described. Ultrastructural examination of both cases of fibrolamellar carcinoma revealed the presence of neurosecretory (NS) granules which were sparse in some cells and abundant in others. Many of the tumor cells had a distinct oncocytic appearance with abundant mitochondria. A portion of the glutaraldehyde-fixed neoplasm was processed for the uranaffin reaction (an ultrastructural cytochemical stain specific for the NS granules of neuroendocrine tissue). Abundant uranaffin-positive granules were found in the neoplastic cells of both cases of fibrolamellar carcinoma, whereas no uranaffin-positive granules were found in hepatocellular carcinoma of the common histologic type. There was no statistical difference in the mean diameter of the uranaffin-positive granules measured from both cases. Immunohisto-chemistry revealed the presence of neuron-specific enolase (NSE) and serotonin in one of the two cases of fibrolamellar carcinoma and no NSE staining in two cases of hepatocellular carcinoma of the common histologic type. These findings suggest that some liver tumors presenting histologically as fibrolamellar carcinoma may be neuroendocrine in nature.     

Efficient generation of monoclonal antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions

Monoclonal antibody production (mAb) first requires the availability of large amounts of pure immunogen for animal immunisation and fusion screening procedures. To overcome this obstacle, we have developed a simple method for rapid generation of pure antigen by generation of recombinant protein containing the antigen of interest fused to the hinge and Fc domains of human immunoglobulin (Ig). The Fc domain forms a convenient 'tag' to enable detection of the protein in supernatant of transfected cells and for purification of immunogen by protein A affinity chromatography. The only requirement for immunogen preparation using this methodology is that a DNA sequence encoding a portion of the molecule of interest is known and that a suitable PCR template is available. Antibody production can be tailored to specific protein domains, for example functional domains, by expressing solely those domains in the fusion protein. We illustrate the technique with two different fusions used to raise antibodies against the porcine and human analogues of a complement (C) regulatory protein, decay accelerating factor (DAF) (CD55). Use of the specific Ig-fusion protein and a control protein facilitated screening of fusions by ELISA. We demonstrate two expression systems used to generate Ig fusion proteins, the first utilised a commercial vector to incorporate an amino terminal leader sequence and carboxy terminal Ig domains. Low levels of expression required subcloning into a high expression vector and resulted in yields of fusion protein at between 2 and 10 mg per litre of supernatant. The second expression system utilised the high expression vector directly, Ig domains of the chosen immunoglobulin isotype were amplified from peripheral blood mononuclear cell (PBMC) RNA and ligated into the vector in frame with DNA encoding the antigen. We describe potential pitfalls that may be encountered while using Ig fusion proteins as immunogen and demonstrate ways in which to tailor their design for optimal mAb production.     

A review of the phenotypic variation due to the Denys-Drash syndrome-associated germline WT1 mutation R362X

The gene WT1 is required for the normal development and function of the urogenital tract. Constitutional mutations are associated with familial Wilms tumor and syndromes such as Denys-Drash syndrome (DDS) characterized by nephropathy, genital anomalies and often a predisposition to Wilms tumor. We report a case of constitutional WT1 mutation in an XX female with multifocal Wilms tumor but no genital anomalies or renal dysfunction and, for the first time, review patients previously reported with this germline mutation. The mutation (1084C>T) changes the amino acid arginine at position 362 to the translation stop codon TGA (R362X) resulting in a predicted truncated protein lacking three of the four zinc finger domains necessary for correct functioning of the gene. This constitutional mutation has been reported to cause a variety of phenotypes in eleven different patients, including the classical Denys-Drash phenotype of diffuse mesangial sclerosis which leads to early renal failure, genital anomalies in XY individuals and Wilms tumors. The absence of mesangial sclerosis and renal failure in our patient excludes DDS. Our case differs from those previously described as the normal kidney tissue shows some small subcapsular glomeruli indicating that the WT1 mutation has impaired nephron development. This patient extends the range and variation of phenotypes that may arise from a specific germline mutation in WT1.     

Expression of luminal and basal cytokeratins in human breast carcinoma

We have examined basal and luminal cell cytokeratin expression in 1944 cases of invasive breast carcinoma, using tissue microarray (TMA) technology, to determine the frequency of expression of each cytokeratin subtype, their relationships and prognostic relevance, if any. Expression was determined by immunocytochemistry staining using antibodies to the luminal cytokeratins (CKs) 7/8, 18 and 19 and the basal markers CK 5/6 and CK 14. Additionally, assessment of alpha-smooth muscle actin (SMA) and oestrogen receptor status (ER) was performed. The vast majority of the cases showed positivity for CK 7/8, 18 and 19 indicating a differentiated glandular phenotype, a finding associated with good prognosis, ER positivity and older patient age. In contrast, basal marker expression was significantly related to poor prognosis, ER negativity and younger patient age. Multivariate analysis showed that CK 5/6 was an independent indicator for relapse free interval. We were able to subgroup the cases into four distinct phenotype categories (pure luminal, mixed luminal/basal, pure basal and null), which had significant differences in relation to the biological features and the clinical course of the disease. Tumours classified as expressing a basal phenotype (the combined luminal plus basal and the pure basal) were in a poor prognostic subgroup, typically ER negative in most cases. These findings provide further evidence that breast cancer has distinct differentiation subclasses that have both biological and clinical relevance.     

Primary care funding, contract status, and outcomes: an observational study

BACKGROUND: The introduction of the Quality and Outcomes Framework (QOF) provides a quantitative way of assessing quality of care in general practice. We explore the achievements of general practice in the first year of the QOF, with specific reference to practice funding and contract status. AIM: To determine the extent to which differences in funding and contract status affect quality in primary care. DESIGN OF STUDY: Cross-sectional observational study using practice data obtained under the Freedom of Information Act 2000. SETTING: One hundred and sixty-four practices from six primary care trusts (PCTs) in England. METHOD: Practice data for all 164 practices were collated for income and contract status. The outcome measure was QOF score for the year 2004-2005. All data were analysed statistically. RESULTS: Contract status has an impact on practice funding, with Employed Medical Services (EMS) and Personal Medical Services (PMS) practices receiving higher levels of funding than General Medical Services (GMS) practices (P<0.001). QOF scores also vary according to contract status. Higher funding levels in EMS practices are associated with lower QOF scores (P=0.04); while GMS practices exhibited the opposite trend, with higher-funded practices achieving better quality scores (P<0.001). CONCLUSION: GMS practices are the most efficient contract status, achieving high quality scores for an average of pound 62.51 per patient per year. By contrast, EMS practices are underperforming, achieving low quality scores for an average of pound 105.37 per patient per year. Funding and contract status are therefore important factors in determining achievement in the QOF.