Evaluation of photo-crosslinked fibrinogen as a rapid and strong tissue adhesive.

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Heterozygosity for ABCA1 gene mutations: effects on enzymes, apolipoproteins and lipoprotein particle size.

A cohort of 13 female and 14 male heterozygotes for ATP binding cassette A1 (ABCA1) gene defects was directly compared with 13 and 14 unaffected female and male family members of almost exact same age. The activities of three proteins that play key roles in HDL metabolism were measured in addition to extensive lipid and (apo) lipoprotein subfraction analysis. Compared to controls, LCAT activity was reduced by 15% in affected subjects (P < 0.001) while PLTP activity was unaffected. Interestingly, CETP activity was elevated by 50% in the heterozygote siblings of one kindred but was unaffected in heterozygotes of the three other families. With respect to lipids, the heterozygotes had normal total cholesterol (TC), and LDL-cholesterol concentrations but presented with a trend towards increased triglyceride levels (13%; P = 0.08). HDL metabolism, by contrast, was severely affected as illustrated by 40% reductions in HDL-cholesterol (P < 0.001) with concomitant reductions in apoAI (25%; P < 0.001) levels and in lipoprotein subfraction LpAI (28%; P < 0.001), LpAI:AII (24%; P=0.014), and LpCIII:nonB (34%; P < 0.001) concentrations. We furthermore observed reduced average HDL particle size (5%; P = 0.004; 16% in female and 3.6% in male) and reduced plasma apoCIII concentration (15%; P = 0.006) while apoAII, apoAIV, apoE and apoB levels were unchanged. In conclusion, heterozygosity for ABCA1 defects was associated with reduced LCAT activity in absence of effects on PLTP activity. Of special interest was our finding that the effects of compromised ABCA1 function on HDL were more pronounced in women than in men.

A taxonomy of obstacles to breast examinations in African American women.

The purpose of this study was to describe and classify the barriers to breast self-examinations (BSE) and mammography in African American women. A total of 125 African American women were recruited from historically black colleges, churches and community organizations in Nashville, Tennessee. Their responses to a comprehensive open- and closed-ended questionnaire about barriers to BSE and mammography were coded using a hierarchical coding system and analyzed according to participants' stage of behavior change assignment. On the average, each woman reported 3.1 barriers to BSE (2.5 psychological and 0.6 environmental) and 2.5 barriers to mammography (1.5 psychological and 1.0 environmental). Barriers cited included fear of finding cancer, forgetting, lack of time, lack of knowledge, competing demands, costs, pain, emotional consequences, cultural attitudes towards medicine, uncertainty about benefits and laziness. For BSE, the number of psychological barriers exceeded environmental barriers, while for mammography, the number of psychological and environmental barriers was similar. For BSE, but not mammography, psychological barriers appeared most important for women in the precontemplation, contemplation and preparation stages of behavior change. Overcoming barriers to BSE and mammography could increase early detection rates in African American women. Interventions based on stage of change theory may be especially applicable.

IgA myeloma presenting as Henoch-Schönlein purpura with nephritis.

IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are both characterized by IgA-mediated tissue injury, including mesangial proliferative glomerulonephritis. Abnormalities of IgA1 glycosylation are described in IgA nephropathy and HSP nephritis. IgA-antineutrophil cytoplasmic antibodies (ANCA) have been inconsistently described in the serum of patients with HSP. In IgA myeloma, the paraprotein-mediated renal lesion is typically cast nephropathy; IgAN or HSP have only rarely been reported in myeloma even when an IgA paraprotein is circulating in large concentrations. We report the case of a 50-year-old man with IgA myeloma who presented with HSP including nephritis and rapidly progressive renal failure. His IgA1 had altered O-glycosylation in the pattern seen in IgAN and also contained an IgA-ANCA. This case adds further weight to the evidence that IgA1 O-glycosylation abnormalities predispose to mesangial IgA deposition and also that IgA-ANCA may have a pathogenic role in the development of HSP.

Heat treatment of normal human sera reveals antibodies to bactericidal permeability-inducing protein (BPI).

Heat treatment of normal sera to 56 degrees C for 30 min, a common procedure for the inactivation of viruses, e.g. HIV, reveals the presence of antibodies to neutrophil cytoplasm antigens (ANCA), as detected by indirect immunofluorescence on ethanol-fixed human neutrophils and by antigen-specific ELISA for BPI. Reactivity was not seen to the other common vasculitis-associated antigens proteinase 3 (PR3) or myeloperoxidase (MPO). The effect of temperature was maximal at 56 degrees C, with substantial antibody demonstrable after only 5 min at this temperature. In experiments using polyethylene glycol (PEG)6000 to remove immune complexes, the effect of heating could be abrogated by preincubation with 8% PEG, which suggested that these anti BPI antibodies might be complexed in sera. After passage of normal plasma over a protein G column, the acid-eluted fraction contained elevated levels of antibodies to BPI but not to other vasculitis-associated antigens such as PR3 or MPO, nor to glomerular basement membrane (GBM), the Goodpasture antigen which is recognized by the pathogenically important human antibodies shown to mediate nephritis in transfer experiments. Moreover the levels of anti-BPI in the IgG fraction could be augmented by preincubation with glycine pH 2.5 for 30 min. This anti-BPI activity could be inhibited by addition of the unbound material from the protein G column and this inhibitory material was not heat-labile at 56 degrees C. The molecular specificity of this autoreactivity was confirmed using recombinant BPI in coincubation experiments and the epitope localized to the C or N terminal moieties by the use of recombinant fusion proteins.

Antineutrophil cytoplasm autoantibodies against bactericidal/permeability-increasing protein in inflammatory bowel disease.

BACKGROUND: Bactericidal/permeability-increasing protein (BPI), a constituent of primary neutrophil granules, is a potent natural antibiotic and an antineutrophil cytoplasm antibody (ANCA) antigen in cases of vasculitis in which the target antigen is neither myeloperoxidase (MPO) nor proteinase-3 (PR3). AIM: To investigate BPI as a possible target antigen for ANCAs in inflammatory bowel disease. METHODS: ANCAs were detected by routine immunofluorescence (IIF) and solid phase enzyme linked immunosorbent assay (ELISA) performed for antibodies to the purified neutrophil granule proteins; MPO, PR3, cathepsin-G, lactoferrin, and BPI in serum samples from 88 patients with inflammatory bowel disease (36 with Crohn's disease, 52 with ulcerative colitis). Thirty patients with bacterial enteritis acted as controls. RESULTS: Significantly more patients with ulcerative colitis were ANCA positive by IIF (60%) than patients with Crohn's disease (28%) or infectious enteritis (23%) (p < 0.001). IgG anti-BPI antibodies were present in 29% of patients with ulcerative colitis, 14% of patients with Crohn's disease, and 23% of patients with infectious enteritis, occurring in 44% of those patients with inflammatory bowel disease who were ANCA positive by IIF. Antibodies to other ANCA antigens were rare. The presence of ANCAs was not related to either disease activity or extent; presence of anti-BPI antibodies was significantly related to both a lower serum albumin concentration (p = 0.001) and a higher erythrocyte sedimentation rate (p = 0.02) in patients with ulcerative colitis, and to colonic involvement in patients with Crohn's disease (p = 0.01). CONCLUSION: BPI is a significant minority target antigen for ANCAs in inflammatory bowel disease that seems related to colonic Crohn's disease and disease activity in ulcerative colitis. Anti-BPI antibodies occur in infectious enteritis.

Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice.

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-ace B gene-ovine growth hormone (GH) gene (3' GH sequence) construct was fused to the ovine, MT-Ia promoter-ace A gene-ovine GH gene (3' GH sequence). Therefore, in this single DNA sequence, both ace A and ace B are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of the ace B-ace A gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressed Escherichia coli cells.

Characterization of Cryptococcus neoformans by random DNA amplification.

Random amplified polymorphic DNA (RAPD) was optimized and used to distinguish between the varieties and serotypes of Cryptococcus neoformans. The RAPD technique distinguished between serotypes A, D or AD within C. neoformans var. neoformans, and revealed further differentiation within each serotype. Four RAPD profiles were clearly recognizable within C. neoformans var. gattii, although its two serotypes, B and C, were only differentiated with one primer combination out of seven.

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting.

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.

Natural environmental sources of Cryptococcus neoformans var. gattii.

We sought evidence for new environmental sources of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA (RAPD) analysis of isolates from 29 animals with a restricted territorial range in five Australian states. Twenty-three of the 29 isolates and 45 of 45 eucalypt isolates tested previously exhibited one RAPD profile, VGI. RAPD profile VGII was identified in 6 of 17 isolates from domesticated species but in none of 12 native species. Four VGII isolates originated from an area of Western Australia with no natural stands of known eucalypt host, indicating the existence of at least one unrecognized natural source of C. neoformans var. gattii.

Identification by random amplification of polymorphic DNA of a common molecular type of Cryptococcus neoformans var. neoformans in patients with AIDS or other immunosuppressive conditions.

Sixty clinical isolates of Cryptococcus neoformans var. neoformans were analyzed by random amplification of polymorphic DNA (RAPD) using 12- to 22-mer primers in pairs. Five major profiles, which clearly distinguished between serotypes A (profiles I-III), AD (profile IV), and D (profile V), were identified. Forty-two of 58 serotype A isolates were assigned to profile I, 13 to profile II, and 3 to profile III. Profile I compromised 5 subtypes (profiles Ia-Ie), with 37 to 42 isolates in profile Ia. Twenty-seven of 28 isolates from patients with AIDS belonged to profile Ia (P<.001), as did 7 of 10 isolates from otherwise immunocompromised patients. Isolates from immunocompetent hosts were broadly distributed (profile I, 8 isolates; profile II, 10 isolates; profile III, 2 isolates). RAPD profiles were independent of body site and geographic origin of isolates. Isolates pairs from 3 patients produced identical profiles. A predominant genetic profile among serotype A strains from AIDS patients has not been reported previously.

Cloning and sequencing of a cDNA encoding an ovine oestrus-associated oviducal protein.

Ovine oestrus-associated oviducal glycoprotein (oEGP) is synthesized and secreted specifically by the ampullary region of the ovine oviduct during the peri-ovulatory stages of the oestrous cycle. A cDNA that encodes oEGP was isolated and sequenced. Isolation of oEGP was achieved using the polymerase chain reaction (PCR) with primers based on a bovine oestrus-associated oviducal glycoprotein cDNA (bOGP) sequence. A 1599-bp cDNA encodes, in part, a deduced 519-amino acid sequence of mature protein which carries two potential N-linked glycosylation sites. The deduced amino acid sequence is more than 95% identical to that of bOGP and more than 74% identical to the first 491 amino acids of human oestrogen-dependent oviducal glycoprotein (hOGP). Northern blot hybridizations of RNA from several sheep tissues detected mRNA (2.4 kb) only in an ampulla oviduct sample.

Urinary kallikrein in the rat: stimulation with angiotensin infusion but depression with increasing sodium concentration.

1. The kallikrein response to angiotensin II infusion in the conscious rat was studied to compare it with the response in the dog. 2. Active kallikrein was measured by the aprotinin-suppressible esterase technique in 20 min periods. Angiotensin (5 x 10(-9) to 5 x 10(-2) micrograms min-1) was infused in 10 mM saline in period 10 (group A), or in 90 mM saline in periods 10-12 (group B). 3. In group A, no dose of angiotensin was antinatriuretic. Natriuresis and urinary sodium concentration were dose dependent. 4. Kallikrein excretion was dose dependent with angiotensin (P < 0.0001) and inversely correlated with urinary sodium concentration (P = 0.011). In natriuretic and non-natriuretic rats, kallikrein excretion after angiotensin was inversely correlated with urinary sodium concentration in the preceding period. 5. In group B, natriuresis and urinary sodium concentration were dose dependent. Kallikrein excretion in periods 10-13 was inversely correlated with urinary sodium concentration in the preceding period (P = 0.0001) and inversely correlated with urinary osmolality in periods 9-13. 6. Infusion of angiotensin II at 5 x 10(-6) micrograms min-1 led to antinatriuresis. 7. Formulae were derived which enabled the opposing effects of angiotensin and urinary sodium concentration on kallikrein excretion to be separated. In group A both these effects were statistically significant only in the natriuretic rats (natriuresis > 20 mumols per period). In group B the formulae showed a dose-dependent rise in kallikrein excretion, which was counteracted by the decrease in kallikrein excretion associated with the increasing urinary sodium concentration. 8. With infusions of 0.9% saline, kallikrein excretion in periods 10-13 was inversely correlated with urinary sodium concentration in the preceding period (P = 0.001). 9. The overall effect in the rat differs from that in the dog, where kallikrein increases with angiotensin natriuresis and dilution of the urine occurs.

Prospective study of the aetiology of adult community acquired bacterial pneumonia needing hospitalisation in Singapore.

A prospective survey of 96 consecutive adult patients with community acquired pneumonia requiring hospitalisation was carried out at National University Hospital, Singapore. Causative pathogens were identified in 58% of patients. Mycobacterium tuberculosis was the most common pathogen (21%), followed by Streptococcus pneumoniae (12%), Haemophilus influenzae (5.2%), Mycoplasma pneumoniae (5.2%) and Staphylococcus aureus (4.2%). Gram-negative organisms (apart from Haemophilus influenzae) were found in 10% of pneumonia patients. More than half of the patients had pre-existing illness, the most common was diabetes mellitus (21%).

The 5S ribosomal RNA gene is linked to large and small subunit ribosomal RNA genes in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax.

Southern hybridization and polymerase chain reaction data indicate that the 5S ribosomal RNA gene is linked to the ribosomal RNA gene repeat unit in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax, and is apparently transcribed in the same direction as the large and small subunit ribosomal RNA genes. The polymerase chain reaction has been used to amplify all components of the entire ribosomal RNA gene repeat unit for each of these oomycetes. The total size of all amplified products is identical to the size of the ribosomal RNA gene repeat unit, as determined by Southern analysis.

Association of IgM with IgG ANCA in patients presenting with pulmonary hemorrhage.

ANCA are markers for systemic vasculitis such as Wegener's granulomatosis (WG) and microscopic polyarteritis (MPA) and are usually of the IgG isotype. However, IgM ANCA may rarely occur in isolation, and in these circumstances, we have found that they are associated clinically with a syndrome of pulmonary hemorrhage and systemic vasculitis. How frequently IgM ANCA may occur in conjunction with IgG has not previously been investigated. We report here a study of 24 consecutive patients with IgG ANCA-positive systemic vasculitis (14 WG, 10 MPA) in whom we determined whether IgM ANCA occurred in association with IgG ANCA, and if so, whether this had clinical importance. Eight patients were found to have IgM ANCA as well as IgG ANCA, and of these, seven presented with severe pulmonary hemorrhage. None of the IgM ANCA-negative patients presented with pulmonary hemorrhage. Although the occurrence of pulmonary hemorrhage in ANCA positive vasculitis was closely correlated with the presence of IgM ANCA, the antigen specificity of these IgM autoantibodies was variable, since both myeloperoxidase (4 patients), PR3 (3 patients), and an unknown ANCA antigen (1 patient) were found to be targets. We conclude that knowledge of ANCA isotype may have important clinical and therapeutic implications for the management of patients with systemic vasculitis.

Molecular evolution of the fungi: relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes.

Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.