Co-occurrence and seasonal and environmental distributions of the sandflies Lutzomyia longipalpis and Nyssomyia whitmani in the city of Puerto Iguazú, northeastern Argentina.

The aim of this work was to study the distribution of Phlebotominae (Diptera: Psycodidade) abundance in time and space in an area in northeastern Argentina with vector transmission of visceral and tegumentary leishmaniasis. For this, 51 households were selected using a 'worst scenario' criterion where one light trap was set during two consecutive nights in peridomiciles in the transitions between the four seasons, and the environment was surveyed simultaneously. The relationships of phlebotomine assemblage structure and the most abundant species with seasonality and environmental variables were evaluated using a canonical correspondence analysis and generalized linear mixed models, respectively. A total of 5110 individuals were captured. Lutzomyia longipalpis (Lutz & Neiva, 1912) and Nyssomyia whitmani (Antunes & Coutinho, 1939) were the most abundant species captured in all samplings (98.3% of the total capture). The period of highest abundance of Lu. longipalpis was early autumn, and it was distributed in the most urbanized areas. Nyssomyia whitmani occupied mainly the less urbanized areas, showing peaks of abundance in early spring and summer. Other species were captured in low numbers and showed seasonal-spatial variations similar to those of Ny. whitmani. We confirmed Leishmania spp. vector persistence throughout the year in spatial patches of high abundance even during the less favorable season.

Evaluation of a multilayered chitosan-hydroxy-apatite porous composite enriched with fibronectin or an in vitro-generated bone-like extracellular matrix on proliferation and diferentiation of osteoblasts.

The use of extracellular matrix (ECM) molecules from tissues is an interesting way to induce specific responses of cells grown onto composite scaffolds to promote adhesion, proliferation and differentiation. There have been several studies on the effects on cell proliferation and differentiation of osteoprogenitor cells cultured onto composites, either adding some ECM molecules or grown in the presence of growth factors. Other studies involve the use of osteoblasts cultured on a three-dimensional (3D) matrix, enriched with ECM molecules produced by the same cells grown previously inside the composite. Here, the effect of enrichment of a novel multilayered chitosan-hydroxyapatite composite with ECM molecules produced by osteoblasts, or the addition of 25 or 50 µg/ml fibronectin to the composite, on proliferation and differentiation of osteoblasts cultured on these composites was studied. The results showed an increase in the number of osteoblasts from day 1 of culture, which was higher in the group grown onto composites enriched with the highest concentration of fibronectin or with ECM molecules produced naturally by osteoblasts cultured previously on them, when compared with the control group. However, this increment tended to decline in all groups after day 7 of culture, the day when they reached the highest peak of proliferation. Differentiation expressed as alkaline phosphatase activity followed the proliferation pattern of the cells cultivated on the scaffolds. The results demonstrate the potential offered by these enriched 3D multilayered composites for improving their ability as bone grafting material.

Eggshell membrane as a biodegradable bone regeneration inhibitor.

The efficiency of chicken eggshell membranes combined with a minimally invasive small osteotomy procedure of the ulna to accomplish an efficient release of the radius so that it can continue to grow in an unstressed manner was tested in rabbits. Eggshell membranes were extracted from chicken eggs, rinsed, dried and sterilized with ethylene oxide for 24 h. For reactivity testing, four separate subcutaneous pockets were created in 10 rats in the paravertebral region by blunt dissection and eggshell membranes were implanted in two of them. After 1-16 weeks, the implants were retrieved with the surrounding soft tissues and submitted to histological examination. Subsequently, 10 rabbits were anaesthetized and a complete 0.5 mm wide osteotomy was performed in both the right and the left distal ulna. A piece of eggshell membranes was interposed in the osteotomy site of one ulna. The opposite osteotomized ulna was left as a negative control. The rabbits were injected with oxytetracycline at the time of surgery and this was repeated every 7 days for labelling new bone formation. After 1-16 weeks, ulnar osteotomized regions were histologically examined. After histological, fluorescence microscopy and radiological evaluation, we demonstrate here for the first time that eggshell membranes as interpositional material in rabbit osteotomized ulnar experiments acted as an active barrier against bone bridging. The degradation of the eggshell membrane, due to host reaction, appeared sufficiently late to cause the desirable delay of bone healing that is compatible with the time needed for a corrective response.

Localization of osteopontin in oviduct tissue and eggshell during different stages of the avian egg laying cycle.

The avian eggshell is an acellular bioceramic containing organic and inorganic phases that are sequentially assembled during the time the egg moves along the oviduct. As it has been demonstrated in other mineralized tissues, mineralization of the eggshell is regulated by extracellular matrix proteins especially the anionic side chains of proteoglycans. Among them, osteopontin has been found in the avian eggshell and oviduct. However, its precise localization in the eggshell or in different oviduct regions during eggshell formation, nor its function have been established. By using anti-osteopontin antibody (OPN 1), we studied its immunolocalization in the isthmus, red isthmus and shell gland of the oviduct, and in the eggshell during formation. In the eggshell, osteopontin was localized in the core of the non-mineralized shell membrane fibers, in the base of the mammillae and in the outermost part of the palisade. In the oviduct, OPN 1 was localized in the ciliated epithelial but not in the tubular gland cells of the isthmus, in the ciliated epithelial cells of the red isthmus, and in the non-ciliated epithelial cells of the shell gland. The occurrence of osteopontin in each of the oviduct regions, coincided with the concomitant presence of the egg in such region. Considering the reported inhibitory function of osteopontin in other mineralized systems, together with its main occurrence in the non-mineralized parts of the eggshell and at the outermost part of the shell, suggests that this molecule could be part of the mechanism regulating the eggshell calcification.

Prospective comparison of clinical and echocardiographic diagnosis of rheumatic carditis: long term follow up of patients with subclinical disease.

OBJECTIVE: To determine the frequency of occurrence and long term evolution of subclinical carditis in patients with acute rheumatic fever. DESIGN: Valvar incompetence was detected by clinical examination and Doppler echocardiographic imaging during the acute and quiescent phases of rheumatic fever. Patients were followed prospectively and submitted to repeat examinations at one and five years after the acute attack. Persistence of acute mitral and aortic lesions detected solely by echocardiography (subclinical disease) was compared with that of disease detected by clinical examination as well (thereby fulfilling the latest 1992 Jones criteria for rheumatic carditis). SETTING: Three general hospitals with a university affiliation in Chile. PATIENTS: 35 consecutive patients fulfilling the revised Jones criteria for rheumatic fever. Clinical and echocardiographic examination was repeated in 32 patients after one year and in 17 after five years. Ten patients had subclinical carditis on admission, six of whom were followed for five years. MAIN OUTCOME MEASURES: Auscultatory and echocardiographic evidence of mitral or aortic regurgitation during the acute attack or at follow up. RESULTS: Mitral or aortic regurgitation was detected by Doppler echocardiographic imaging in 25/35 rheumatic fever patients as opposed to 5/35 by clinical examination (p = 0.03). Doppler echocardiography revealed acute valvar lesions in 10 of 20 rheumatic fever patients who had no auscultatory evidence of rheumatic carditis (subclinical carditis). Three of these subclinical lesions and three of the clinical or auscultatory lesions detected on admission were still present after five years of follow up, emphasising that subclinical lesions are not necessarily transient. CONCLUSIONS: Doppler echocardiographic imaging improves the detection of rheumatic carditis. Subclinical valve lesions, detected only by Doppler imaging, can persist. Echocardiographic findings should be accepted as a major criterion for the diagnosis of rheumatic fever.

The interaction of phospholipase A2 with liposomes: an immunological approach to its study.

Porcine pancreatic phospholipase A2 (PLA2) has been incubated for 2 hours at 34 degrees C with rabbit anti-pig pancreatic PLA2 polyclonal antiserum in the absence or presence of phosphatidylcholine liposomes in different physical states. Subsequent assay of hydrolysis -triggered through addition of 5 mM Ca2+- at 34 degrees C, shows that preincubation with antiserum in the presence of 1,2-dimyristoyl-3-sn-phosphatidylcholine liquid-crystalline vesicles, renders the PLA2 activity undetectable, similarly to what is found if preincubation is carried out in the absence of liposomes. In contrast, 1,2-dipalmitoyl-3-sn-phosphatidylcholine liposomes, which at 34 degrees C are in the gel-phase, protect the enzyme from the antiserum effect. The results are consistent with a stronger binding of PLA2 to gel phase, as compared to liquid crystalline vesicles and suggest that through the physical interaction with liposomes in the gel state, the enzyme is shielded from reaction with the antibodies. Taking into account the characteristic hydrolysis profiles of vesicles in different physical states, it can be concluded that the above interpretation agrees with the proposal that PLA2-membrane association promotes the interfacial activation of the enzyme.

Effect of beta-aminoproprionitrile on eggshell formation.

1. Eggshells are bioceramic-biopolymer composites made by a cell-mediated deposition of an extracellular matrix which drives the organisation of the inorganic phase. Ultrastructurally, eggshells are composed of shell membranes, mammillary knobs, palisade, and cuticle. Shell membranes are two nets of type X collagen-containing fibrils. On to these membranes, the mammillary knobs, that is, the crystal nucleation sites, are deposited. Type X collagen is highly cross-linked and insoluble. 2. In order to evaluate the role of type X collagen cross-linking on eggshell formation, hens were injected with different doses of beta-aminoproprionitrile, which specifically interferes with cross-link formation. 3. Changes in egg size and shape were observed. Scanning electron micrographs analysis of these eggs demonstrated marked changes in crystal growth and shell membrane structure and arrangement. A dot-blot analysis, using a monoclonal antibody against chicken type X collagen, shows a dose-dependent increase in shell membrane collagen extractability. 4. It is concluded that the formation of beta-aminoproprionitrile-sensitive cross-links among the type X collagen molecules of the shell membranes play an essential role in normal eggshell formation.

Eggshells are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules.

The avian eggshell is a composite bioceramic which is formed by a controlled interaction of an organic and an inorganic phase. The organic phase contains, among other constituents, type X collagen and proteoglycans, mainly keratan and dermatan sulfate. Understanding the principles governing the synthesis and temporo-spatial distribution of such macromolecules, and their influence on the organization of the crystalline phase, is an essential aspect of establishing the biological basis of the quality of eggshell, both as an embryonic chamber and as a natural food package. In the present study, we have examined the process of eggshell formation by immunohistochemistry, scanning electron microscopy and energy dispersive X-ray microanalysis. Precise sites and timing of secretion were established for the deposition of particular macromolecules. Type X collagen is detected at the very first moment of shell membrane formation. The appearance of keratan sulfate coincides with the appearance of mammillae, while dermatan sulfate is deposited later, coincident with shell matrix deposition. We propose that keratan sulfate, due to its precise localization, temporal appearance and calcium-binding affinity, relates to the maintenance of calcium reserve bodies, the primary source of calcium for the embryo. On the other hand, dermatan sulfate may control crystal growth, resulting in a preferential orientation of calcite crystals within the palisade layer.

Role of type X collagen on experimental mineralization of eggshell membranes.

Type X collagen is a transient and developmentally regulated collagen that has been postulated to be involved in controlling the later stages of endochondral bone formation. However, the role of this collagen in these events is not yet known. In order to understand the function of type X collagen, if any, in the process of biomineralization, the properties of type X collagen in eggshell membranes were further investigated. Specifically, calvaria-derived osteogenic cells were tested for their ability to mineralize eggshell membranes in vitro. Immunohistochemistry with specific monoclonal antibodies was used to correlate the presence or absence of type X collagen or its propeptide domains with the ability of shell membranes to be mineralized. The extent of mineralization was assessed by Von Kossa staining, scanning electron microscopy and energy-dispersive spectroscopy. The results indicate that the non-helical domains of type X collagen must be removed to facilitate the cell-mediated mineralization of eggshell membranes. In this tissue, intact type X collagen does not appear to stimulate or support cell-mediated mineralization. We postulate that the non-helical domains of type X collagen function in vivo to inhibit mineralization and thereby establish boundaries which are protected from mineral deposition.

Membrane fluidity of Escherichia coli during heat-shock.

The excimer-forming fluorophore dipyrenylpropane has been used to measure the relative fluidity of total membranes isolated from Escherichia coli grown at 30 or 45 degrees C, or exposed to a heat-shock from 30 to 45 degrees C for various periods of time. Parallel experiments were performed using [35S]methionine pulse-labeling of cells, to study the induction of heat-shock proteins (HSPs) at different times after the sudden change in E. coli growth-temperature from 30 to 45 degrees C. Results suggest that upon an abrupt temperature upshift from 30 to 45 degrees C, membrane fluidity adjustment to the steady-state level at the high temperature, takes place during the E. coli heat-shock response.

Activity of phospholipase A2 on a fluorescent substrate incorporated into non-hydrolyzable phospholipid liposomes.

The activity of phospholipase A2 (PLA2) on phospholipid liposomes depends on the physicochemical properties of the aggregated substrate, which are subject to continuous modification by the products released during hydrolysis. We propose here an experimental design that, by means of the incorporation of a fluorescent substrate at very low molar ratio (< or = 1:500) into a nonhydrolizable liposomal matrix of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC), allows the study of hydrolysis by porcine pancreatic phospholipase A2, in virtual absence of physical perturbations of the lamellar phase, by the released products. We have been able to measure immediate hydrolysis of the fluorescent substrate 1,2-di-[omega(1'-pyreno)-decanoyl]-sn- glycero-3-phosphocholine when the sonicated liposomal matrix is in the gel phase. In the liquid crystalline state, in contrast, hydrolysis is very poor even after 80 min of adding the enzyme. Both in the gel and liquid-crystalline phases, incorporation of unlabeled PLA2 products activates the hydrolysis rate to comparable levels. It appears that the conformation adopted by the substrate immersed in the gel or liquid crystalline matrix is especially important in determining its susceptibility to hydrolysis in the absence of products.

[Renal scintigraphy with captopril in the evaluation of renovascular hypertension]. / Cintigrafía renal con captopril en la evaluación de hipertensión renovascular.

The administration of an angiotensin-converting enzyme (ACE) inhibitor transitorily reduces the GFR in a kidney with renal artery stenosis, effect that can be ascertained with scintigraphic studies using ACE inhibitors. We evaluated the clinical usefulness of captopril renography (CR) in the diagnosis of renovascular hypertension (RVH) in 51 hypertensive patients in which this diagnosis was suspected. All subjects underwent angiography and RVH was diagnosed when renal artery stenosis exceeded 60%, there was lateralization of renal vein renin or there was a concordant clinical outcome. Renography was performed 15 minutes after Lasix administration, using Tc-99m DTPA, before and 60 minutes after the oral administration of 50 mg of captopril. The scintigraphic criteria for a positive test were a decreased split renal function, a delayed peak uptake, a decreased excretion of DTPA and a prolonged transit time. In three of 28 patients in whom angiography discarded RVH, CR was positive. In the 23 patients with confirmed RVH, CR was positive in 10 of 12 with unilateral stenosis, in 3 of 8 with bilateral stenosis and 1 of 3 with stenosis in a transplanted kidney. The overall sensitivity and specificity of the test for RVH was 60.9% and 89.2% respectively. There were no changes in blood pressure or adverse effects after captopril administration. We conclude that CR in RVH is useful to select patients for further studies (angiography) and to perform a functional interpretation of angiographic alterations.

[Sexually transmitted diseases: notification in Chile, in the V region and in the Vinã Del Mar-Quillota health service (1977-1988)]. / Enfermedades de transmision sexual: notificacion en Chile, en la V region y en el servicio de salud Viña Del Mar-Quillota (1977-1988).

Reports of sexually transmitted diseases in the Vth Region of Chile from 1977 to 1988 were analyzed and compared to nationwide reports and to figures for the Viña del Mar-Quillota area. Overall rates of syphilis decreased during the period. In contrast both number of cases and risk of disease increased for gonorrhoea. Risk of syphilis and gonorrhoea was highest in the Vth Region. HIV infection is increasing, especially in the area covered by the Health Service in Viña del Mar-Quillota. Sexually transmitted diseases not subject to reports were screened along with routine examinations for cancer of the cervix. Infection rates of 9.9% for trichomona and 1.09% for candida were thus detected.

[Sexually transmitted diseases: epidemiological characteristics of cases notified in the health service of Viña del Mar-Quillota]. / Enfermedades de transmisión sexual: características epidemiológicas de los casos notificados en el Servicio Salud Viña del Mar-Quillota.

During 1987 and 1988 sexually transmitted diseases affected 87.2 per 100,000 inhabitants of the Viña del Mar-Quillota area. At all ages rates were 3.8 times higher in males compared to females. Gonorrhoea, syphilis and non gonorrheal urethritis accounted for most cases. In males, 82% of cases corresponded to gonorrhoea, in females 50% corresponded to gonorrhoea and 50% to syphilis. Rural and urban figures were not significantly different, except for HIV infection which was predominantly urban. Rates of different infections peaked at different ages: HIV between 30 and 39, gonorrhoea from 15 to 29, syphilis from 40 to 49 and after 60 and non gonorrhoeal urethritis from 15 to 59. In a survey of affected subjects 85% manifested to be heterosexual, 10% omitted a response.

[Value of 2-dimensional echocardiography in infective endocarditis]. / Valor de la ecocardiografía bidimensional en la endocarditis infecciosa.

We analyzed the two dimensional echocardiograms (2D echo) of 41 patients with infective endocarditis (IE). The diagnosis of endocarditis was based on typical clinical findings, positive blood cultures or anatomical findings at operation or necropsy. Age ranged from 21 to 73 years. Endocarditis was localized in a native valve in 30 patients (aortic 18, mitral 11 and tricuspid 1), a biologic prosthesis in 9 (mitral 4, aortic 4, unknown 1) and a mechanical valve in 4 (aortic 3, mitral 1). The echocardiogram showed vegetations, valve rupture or abscess in 83% of IE in native valves, 56% of IE in biologic prosthesis and 25% of IE in mechanical valves (p less than 0.05, native vs prosthetic valves). The sensitivity of 2D echo was studied in 27 subjects with known anatomical findings. It was 79% for vegetations (mitral 91%, aortic 72%, NS), 53% for valve rupture (mitral 83%, aortic 33%, p less than 0.05) and 70% for abscess. Specificity was over 89% for all lesions. Systemic embolism occurred in 41% of patients with vegetations and in 25% of those without (p = 0.07).

Induction of a latency period in the time-course of phospholipase A2 action on dipalmitoylphosphatidylcholine liposomes in the gel phase.

The hydrolysis of dipalmitoylphosphatidylcholine liposomes by porcine pancreatic phospholipase A2 was studied at 31 degrees C, i.e., with the substrate in the gel phase. Addition of delipidated bovine serum albumin to the assay medium induces the appearance of a latency phase in the time course of the enzymatic action. The lag period can be abolished by addition of free palmitic acid whereas no reversal by lysolecithin is found. The generation of a latency period by albumin appears to be due to its ability to sequester the palmitic acid newly released by the phospholipase A2 catalysis. Thus, the nascent fatty acid seems to be an essential activator of the enzymatic process.

Effect of LiCl on differentiation of mouse embryos beyond the blastocyst stage.

In order to test the effect of Li+ on cell differentiation, 2-cell embryos were cultured in vitro with or without Li+ to the blastocyst stage and then, in a "implantation" culture medium, with or without Li+, up the early egg-cylinder stage. Our results show: 1) that Li+ in the preimplantation medium diminishes "implantation" success significantly; 2) that trophoblastic vesicles, produced by culturing preimplantation embryos with Li+, do not generate an inner cell mass when further cultured in "implantation" medium; 3) that the ultrastructure of "implanted" embryos is similar in all experimental series.