Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Incidence and risk factors for pulmonary exacerbation treatment failures in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa

Background: Pulmonary exacerbations (PEx) are responsible for much of the morbidity and mortality associated with cystic fibrosis (CF). However, there is a paucity of data on outcomes in CF PEx and factors influencing outcomes.

Methods: We reviewed all PEx in patients infected with Pseudomonas aeruginosa treated with parenteral antibiotics over 4 years at our center. Treatment failures were categorized a priori as those PEx requiring antibiotic regimen change, prolongation of therapy > 20 days because of failure to respond, an early recurrent event within < 45 days, or failure to recover lung function to > 90% of baseline FEV1.

Results: A total of 101 patients were followed for 452 PEx. Treatment failures were observed in 125 (28%) of PEx; antibiotic regimen change was observed in 27 (6%), prolongation of therapy in 29 (6%), early recurrent events in 63 (14%), and failure to recover lung function to > 90% of baseline FEV1 in 66 (15%). Demographic factors associated with one or more treatment failures per year included advanced airways disease, use of enteric feeds, CF-related diabetes, and CF liver disease but did not include female sex or F508del homozygosity. Increased treatment failure risk was associated with lower admission FEV1 and increased markers of inflammation. At therapeutic completion, increased inflammatory markers correlated with treatment failure. Failure rates decreased with increasing number of active antimicrobial agents used based on in vitro susceptibility (zero, 28/65 [43%]; one, 38/140 [27%]; two, 59/245 [24%]; three, 0/2 [0%]; P = .02).

Conclusions: One-fourth of PEx fail to respond adequately to initial management. Patient demographic and episode-specific clinical information can be used to identify individuals at increased risk of initial management failure.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

Recent Innovations in Antibody-Mediated, Targeted Particulate Nanotechnology and Implications for Advanced Visualisation and Drug Delivery

Research into the targeting of drug substances to a specific disease site has enjoyed sustained activity for many decades. The reason for such fervent activity is the considerable clinical advantages that can be gained when the delivery system plays a pivotal role in determining where the drug is deposited. When compared to conventional formulations where no such control exists, such as parenteral and oral systems, the sophisticated targeting device can reduce side effects and limit collateral damage to surrounding normal tissue. No more so is this important than in the area of oncology when dose-limiting side effects are often encountered as an ever present difficulty. In this review, the types of colloidal carrier commonly used in targeted drug delivery are discussed, such as gold and polymeric colloids. In particular, the process of attaching targeting capabilities is considered, with reference to antibody technologies used as the targeting motifs. Nanotechnology has brought together a means to carry both a drug and targeting ligand in self-contained constructs and their applications to both clinical therapy and diagnosis are discussed.

Service User and Carer Involvement in Role-Plays to Assess Readiness for Practice

Service user and carer involvement in social work education is now well established since its inception as a compulsory requirement in the social work curriculum in the United Kingdom in 2003. Since then, there have been many examples of how such involvement has been approached by education providers. Nevertheless, one of the key obstacles and challenges in this field continues to centre on the need to achieve non-tokenistic user involvement which cements the engagement of service users and carers at the heart of social work education. This paper describes one such initiative where service user and carer colleagues in a university in Northern Ireland have been actively involved in the assessment of first year social work students’ preparation for their first period of practice learning. The paper presents the background to this initiative explaining how the project unfolded; the detailed preparations that were involved and the evidence gathered from evaluations undertaken with the students, service users and carers, and academic colleagues who were all involved. We believe that the findings from this project can contribute to the advancement of existing knowledge in the field in exploring and recommending creative methodologies for user involvement in social work education.

Social Work Students Learn about Social Work Values from Service Users and Carers

Teaching on social work values is centrally important in social work education as a core aspect of underpinning knowledge in preparing students for practice. This paper describes an innovative project occurring within the first year of the degree in social work, where service users and carers have assisted students with their understanding of social work values. The positive contribution of service users and carers in facilitating students to make links between theory and practice is now well documented. Applying this user perspective to the educational domain of values, however, is relatively uncharted territory given the challenges that have traditionally accompanied the teaching of values. Importantly, this paper describes the ‘value talk’ which occurred when first-year students sought further meaning from service-user and carer groups in their community settings following classroom teaching on values. The paper not only discusses the detailed preparations involved in the project but also the learning which resulted, drawing on the evaluation findings from the students and participating groups. Whilst the findings show that the students' understanding of social work values has been most significantly influenced by the contributions from service users and carers, it is recognized that further research is needed to monitor the longer term impact on social work students' practice after they qualify.

Antimicrobial prescribing in residential homes.

Objectives: Research in residential homes has been limited to date and the extent of systemic and topical antimicrobial prescribing is largely unknown. The aim of this study was to investigate antimicrobial prescribing in residential homes in Northern Ireland (NI).

Methods: Point prevalence studies (PPSs) were completed in November 2010 (PPS1) and April 2011 (PPS2) in 30 residential homes. Data were obtained from care plans, medication administration records and staff in relation to antimicrobial prescribing and facility and resident characteristics, and analysed descriptively.

Results: The point prevalence of systemic antimicrobial prescribing was 9.4% in PPS1 and 9.2% in PPS2 (range 0.0%–33.3% during both PPSs). Trimethoprim was the most commonly prescribed systemic antimicrobial and the main indication was the prevention of urinary tract infections. Almost 25% of systemic antimicrobials were prescribed at inappropriate doses. The point prevalence of topical antimicrobial prescribing was 6.4% (range 0.0%–22.2%) in PPS1 and 5.9% (range 0.0%–21.1%) in PPS2. The most commonly prescribed topical antimicrobials were chloramphenicol eye preparations in PPS1 and fusidic acid skin preparations in PPS2; treatment with these topical antimicrobials was generally prolonged. More than 25% of all systemic and 55% of all topical antimicrobials were initiated following telephone consultations as opposed to face-to-face consultations.

Conclusions: The prevalence of systemic antimicrobial prescribing in residential homes in NI is relatively high compared with care homes (particularly nursing homes) in other countries. Systemic and topical antimicrobial prescribing is not always appropriate in terms of the doses prescribed and the duration of use. It is apparent that current strategies employed in NI are insuf?cient to ensure prudent antimicrobial prescribing within this environment.

THE ACTION OF SEROTONIN AND THE NEMATODE NEUROPEPTIDE KSAYMRFAMIDE ON THE PHARYNGEAL MUSCLE OF THE PARASITIC NEMATODE, ASCARIS-SUUM

The pharyngeal component of the enteric nervous system of the parasitic nematode, Ascaris suum exhibits immunoreactivity for serotonin (5-hydroxytryptamine or 5-HT) and for FMRFamide-like peptides. This paper describes the application of an in vitro pharmacological approach to investigate the functional role of 5-HT and FMRFamide-like peptides. The pharyngeal pumping behaviour of Ascaris suum was monitored using a modified pressure transducer system which measures pharyngeal pressure changes and therefore pumping. The pharynx did not contract spontaneously; however, 5-HT (10-1000 mu M) stimulated pumping at a frequency of 0 . 5 Hz. FMRFamide had no apparent effect on pharyngeal pumping. The native nematode FMRFamide-related peptide (FaRP), KSAYMRFamide inhibited the pumping elicited by 5-HT. The duration of inhibition was dose-dependent (0 . 1-1000 nM) with a threshold of 0 . 1 nM. In 4 preparations, the inhibition of the pharyngeal muscle was preceded by an initial excitation and increase in the amplitude of pharyngeal pressure changes. The pharynx is involved in various nematode processes, including feeding, regulation of hydrostatic pressure and excretion. The role of 5-HT and KSAYMRFamide in the pharyngeal function of nematodes is discussed.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF PEPTIDERGIC AND SEROTONINERGIC COMPONENTS IN THE ENTERIC NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides and an indoleamine (serotonin or 5-hydroxytryptamine) in the enteric nervous system (ENS) of the pig roundworm, Ascaris suum, have been determined by the application of an indirect immunofluorescence technique in conjunction with confocal scanning laser microscopy. Whole-mount preparations of pharyngeal, intestinal and rectal regions were screened with antisera to 23 vertebrate peptides, 2 invertebrate peptides and serotonin(= 5-HT). Positive immunoreactivity (IR) was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), FMRFamide, gastrin and serotonin. The only IR observed in the ENS was that evident in the nerve supply to the pharynx and rectal region; no IR was associated with any region of the intestine. The most extensive patterns of IR occurred with antisera to PW, FMRFamide and serotonin. In the pharyngeal component of the ENS, IR was evident in the lateral and dorsal longitudinal pharyngeal nerves, pharyngeal commissures, nerve plexus, and associated nerve cells and fibres. In contrast, the distribution of IR to the PP and gastrin antisera was more restricted and displayed a lower intensity of immunostaining. The other component of the ENS, the rectal enteric system, only yielded immunostaining to FMRFamide. The possible role of neuropeptides and serotonin in the nutritional biology of nematodes is discussed.

IMMUNOCYTOCHEMICAL AND RADIOIMMUNOMETRICAL DEMONSTRATION OF SEROTONIN-IMMUNOREACTIVITIES AND NEUROPEPTIDE-IMMUNOREACTIVITIES IN THE ADULT-RAT TAPEWORM, HYMENOLEPIS-DIMINUTA (CESTODA, CYCLOPHYLLIDEA)

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PHI-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

CYTOCHEMICAL DEMONSTRATION OF CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN GORGODERINA-VITELLILOBA (TREMATODA, DIGENEA)

Standard enzyme cytochemical and indirect immunocytochemical techniques have been used in conjunction with light and confocal scanning laser microscopy (CSLM) to visualize cholinergic, serotoninergic and peptidergic nerve elements in whole-mount preparations of the amphibian urinary-bladder fluke, Gorgoderina vitelliloba. Cholinesterase (ChE) activity was localized in paired anterior ganglia, a connecting dorsal commissure and in the origins of the ventral nerve cords. Cholinergic ganglia were also evident in shelled embryos in the uterus. Serotonin-immunoreactivity (IR) was more extensive than ChE activity and was identified in both the central and peripheral nervous systems. Serotoninergic nerve fibres were associated with the somatic musculature and female reproductive ducts. Antisera to nine mammalian peptides and one invertebrate (FMRFamide) peptide have been used to investigate the peptidergic nervous system in the parasite. Immunoreactivity was obtained to five peptides, namely pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP) and FMRFamide. Peptidergic nerve fibres were found to be more abundant than demonstrable cholinergic or serotoninergic nerve fibres. NPY-IR was identified only in the main components of the central nervous system. However, PP- and PYY-IR occurred in the anterior ganglia, dorsal commissure, main nerve cords and in numerous small varicose fibres that ramified throughout the worm. Additionally, PP-immunoreactive nerve fibres were found to innervate the musculature of the female reproductive tracts. Six sites of IR were found in the acetabulum, using antisera directed towards the C-terminal end of PP and PYY, and these matched with the distribution of six non-ciliated rosette-like papillae observed by scanning electron microscopy. SP- and FMRFamide-IR were identified in the CNS, and FMRFamide-immunopositive nerve fibres were also evident in association with the gonopore/cirrus region and with the terminal excretory pore. Results are discussed with respect to possible roles for each of the neurochemical types.

Spontaneous intramedullary apoptosis is present in disorders other than myelodysplasia

Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders characterized by peripheral cytopenias in the presence of normo- or hypercellular dysplastic marrow. It has been suggested that premature intramedullary apoptosis may contribute to this phenomenon. We used terminal dUTP nick-end labeling (TUNEL) of bone marrow biopsy specimens and cytocentrifuge preparations from patients with MDS and a variety of other hematopoietic disorders to determine whether there is increased intramedullary apoptosis in MDS and whether any such effect is specific to MDS. TUNEL labeling of bone marrow from 24 patients with MDS revealed significant positivity in 10 of 11 patients with refractory anemia (RA), five of seven with RA and excess of blasts (RAEB), all three patients with RAEB in transformation (RAEB-t), and all three patients with RA with ring sideroblasts (RARS). The percent of positive cells ranged from 5 to 50% but showed no apparent correlation with morphological subtype. In a series of 29 patients with acute leukemia, 17 showed significant positivity (13 of 13 with myeloid disease: three M1, seven M2, one M3, two M4; four of 16 patients with lymphoid disease: one Burkitt-type lymphoma, two null acute leukemia, and one common acute lymphoid leukemia). Intramedullary apoptosis was associated with myeloid or early committed progenitor cells and was highest in secondary acute myeloid leukemia (AML). Normal bone marrow samples from 12 individuals showed no evidence of apoptosis. Our results suggest that an increased level of intramedullary apoptosis is apparent in both patients with MDS and those with AML; those with secondary AML have the highest levels. The relative absence of such findings in lymphoid malignancy suggests that the apoptotic pathways are different in this lineage.

Isolation, pharmacology and gene organization of KPSFVRFamide: A neuropeptide from Caenorhabditis elegans

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH](+)) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected-in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, greater than or equal to 0.1 mu M) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, greater than or equal to 10 nM). (C) 1999 Academic Press.

Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.