Concepciones y prácticas de salud de las familias de la comunidad mapuche Payla Menuko a partir de la implementación del plan de salud rural / Conceptions and health practices of the families of the Mapuche community Payla Menuko from the implementation of the rural health plan

INTRODUCCIÓN El Programa de Salud Rural de la provincia de Neuquén funciona desde 1970 y atiende a las comunidades mapuche asentadas en su mayoría en áreas rurales. OBJETIVOS Analizar las concepciones y prácticas de salud de las familias de la comunidad mapuche Payla Menuko de San Martín de los Andes, Neuquén, a partir de la implementación del Plan de Salud Rural desarrollado por los agentes sanitarios del Hospital Ramón Carrillo. MÉTODOS Estudio cualitativo de carácter exploratorio de corte transversal basado en fuentes de información primaria. Se realizaron 20 entrevistas en profundidad y observación participante. Se trabajó con una muestra intencional, no probabilística, por cuotas según sexo. RESULTADOS Las relaciones interculturales entre las familias mapuche de Payla Menuko y los agentes sanitarios han posibilitado una apropiación y resignificación de saberes y prácticas biomédicas, así como también una mejor accesibilidad de esta población al sistema de salud. A su vez, las familias mapuche han desarrollado a lo largo del tiempo un proceso de reactualización de la concepción de salud mediante la recreación de prácticas de medicina tradicional mapuche que complementan con el uso de servicios de salud pública. DISCUSIÓN Desde la perspectiva de la comunidad mapuche Payla Menuko, los procesos de salud-enfermedad-atención están interrelacionados con el concepto de territorio, que incluye todos los otros ámbitos de la vida: la economía, la religiosidad, la política, la educación, la memoria histórica, entre otros. Esta concepción mapuche de la salud como indisociable del territorio se reactualiza en la comunidad Payla Menuko, a partir de ceremonias de curación colectivas y familiares y la utilización de remedios realizados con plantas medicinales (lawenes). Sin embargo, también son complementadas con prácticas de medicina occidental, introducidas sistemáticamente por los agentes sanitarios.

Analysis of mast cell activation using diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry.

We review a new technique--diamine oxidase (DAO)-gold ultrastructural enzyme-affinity labeling--which we developed to localize histamine in subcellular sites of mast cells. The DAO-gold method showed that isolated human lung mast cells contained abundant histamine in their cytoplasmic granules, a conclusion which was verified by a large number of specificity controls. We also studied mast-cell-rich eyelid lesions which developed in interleukin-4 transgenic mice. The DAO-gold method demonstrated histamine in the electron-dense granules of mast cells in these lesions, but little or no histamine was detected in the swollen, empty granules of mast cells undergoing piecemeal degranulation. This new enzyme-affinity-gold method has permitted the first ultrastructural localization of histamine in subcellular sites of routinely prepared electron microscopy samples. The method has also permitted the first morphological studies of histamine secretion in vivo and has demonstrated that such secretion can be associated with the ultrastructural changes of piecemeal degranulation.

Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro.

We examined the ultrastructural localization of (a) a secondary granule matrix protein--eosinophil peroxidase (EPO)--by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMs) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhIL-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMs, as well as identification of unicompartmental, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.

Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry.

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.

Vesicular transport of peroxidase in human eosinophilic myelocytes.

We performed ultrastructural cytochemistry to detect peroxidase in developmentally arrested human eosinophilic myelocytes. Human umbilical cord blood mononuclear cells were cultured for 21 days in the presence of murine-derived conditioned media, resulting in the development of eosinophilic myelocytes. Unlike normally developing eosinophilic myelocytes, which contain peroxidase in synthetic organelles (i.e. cisterns surrounding the nucleus and bounded by the rough endoplasmic reticulum and Golgi structures) and in immature and mature granules, the developmentally arrested cells showed ultrastructural evidence of decreased synthesis and secretory transport of peroxidase. Thus, peroxidase was generally absent in the perinuclear and rough endoplasmic cisterns, in Golgi structures, in immature granules and in the matrix compartment of most mature granules. Rather, biocompartmental specific granules displayed empty, peroxidase-negative matrix and central, peroxidase-negative core material. Peroxidase was present in perigranular vesicles, some of which were attached to granules. Such peroxidase-loaded transport vesicles are similar to those that effect piecemeal degranulation of mature human eosinophils cultured in rhIL-5-containing media [1]. These findings establish vesicle-mediated piecemeal degranulation in the secretory repertoire of immature human eosinophils and suggest the possibility that eosinophilic myelocytes may participate in vivo in important physiological and/or pathological events that require selective secretion from the specific granule matrix compartment.

C-kit ligand induction of immature neutrophils in cultures of human umbilical cord blood.

Human cord blood cells cultured in suspension with soluble c-kit ligand produce immature mast cells from their agranular precursors; cocultures of cord blood and mouse 3T3 fibroblasts produce fully mature human mast cells. We noted cells of the neutrophil lineage in the c-kit ligand-supplemented suspension cultures. Similar cultures were prepared from individual cord bloods with several sources of the c-kit ligand, including mouse fibroblast conditioned media, a partially purified mouse 3T3 fibroblast factor(s), recombinant human stem cell factor, recombinant murine mast cell growth factor, and were sampled sequentially for routine and cytochemical ultrastructural studies. These studies show that peroxidase-positive azurophilic granule-containing neutrophilic myelocytes develop in quantity from their agranular precursors in cord blood when the c-kit ligand is present, but little to no maturation to mature neutrophils with specific granules occurs. Specific granules were also absent in the neutrophil precursors. The effect of c-kit ligand in vitro on two cell lineages in man is similar--i.e., it permits the development of immature cells to differentiate from their agranular precursors in cord blood, but complete maturation to fully mature mast cells or neutrophils does not occur.

Axonal necrosis of enteric autonomic nerves in continent ileal pouches. Possible implications for pathogenesis of Crohn's disease.

OBJECTIVE: Axonal necrosis was first described in samples of small intestine from patients with Crohn's disease (A.M. Dvorak et al. Hum Pathol 1980; 11:620-634). Clinically evident inflammation of continent ileal reservoirs (pouches) has clinical features that resemble Crohn's disease. Possible similarities in the pathogenesis of Crohn's disease and pouchitis were sought using ultrastructural and microbiologic tools to identify damaged enteric nerves and tissue bacteria. METHODS: An encoded ultrastructural and microbiologic study of replicate biopsies from 114 samples of human intestine was done. Biopsies from ileum, colon, conventional ileostomy or continent pouch were obtained from patients with ulcerative colitis, Crohn's disease, or familial polyposis and grouped into three clinical study groups (control, normal pouch, pouchitis), based on clinical and endoscopic criteria. Biopsies were prepared for electron microscopy with standard methods; replicate biopsy samples were washed extensively before preparing cultures designed to identify aerobic as well as facultative and obligate anaerobic bacteria (Onderdonk et al. J Clin Microbiol 1992; 30:312-317). The ultrastructural diagnosis of damaged enteric nerves was based on previously published criteria for axonal necrosis (A.M. Dvorak and W. Silen. Ann Surg 1985; 201:53-63). Intergroup comparisons were tested for significance using Chi-square analysis. RESULTS: The highest incidence of axonal necrosis was present in Crohn's disease control biopsies (53%), regardless of whether bacteria were present (or not) in cultures of replicate biopsies. Axonal necrosis also occurred in more ulcerative colitis and familial polyposis biopsies (regardless of biopsy site) that had positive bacterial cultures than in those that did not (p < 0.001). In addition, axonal necrosis was documented in 42% of the pouch biopsies from ulcerative colitis and familial polyposis patients, particularly in those pouches that were found to be inflamed by clinical criteria and that also had positive bacterial cultures of the biopsied tissues. Control biopsies from patients with ulcerative colitis and familial polyposis had significantly less nerve damage than pouch biopsies in the presence of positive cultures (p < 0.01). Among the clinically inflamed pouches biopsied in ulcerative colitis or familial polyposis patients, we found that none had damaged enteric nerves when bacterial cultures were negative (p < 0.005). If the presence of axonal necrosis alone was compared with the presence of undamaged enteric nerves in all biopsies from patients with ulcerative colitis, a highly significant number of ulcerative colitis biopsies with axonal necrosis occurred in pouches (72%) compared with controls (p < 0.001). CONCLUSIONS: The ultrastructural finding of axonal necrosis in Crohn's disease confirms previous studies. The presence of damaged enteric nerves in patients with pouchitis provides ultrastructural support to the clinical impression of similarities between pouchitis and Crohn's disease. The association of damaged nerves and invasive bacteria in pouchitis suggests mechanistic similarities for the pathogenesis of Crohn's disease that requires further investigation.

Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation.

Ultrastructural evidence for piecemeal and anaphylactic degranulation of human gut mucosal mast cells in vivo.

One hundred and seventeen coded intestinal biopsies were examined by electron microscopy and evaluated for morphological evidence of mast cell and basophil secretion in situ. Sixty percent of the biopsies had evidence of secretion. Mast cell secretion was evident in control biopsies, many of which were obtained from uninvolved tissues of patients with inflammatory bowel disease. Biopsies of inflamed continent pouches from ulcerative colitis (UC) patients showed more mast cell secretion than noninflamed UC pouch biopsies. This evidence of mast cell secretion supports recent work that documents high constitutive levels of histamine in jejunal fluids of Crohn's disease patients and suggests a proinflammatory role for mast cells in inflammation associated with pouchitis.

Human gut mucosal mast cells: ultrastructural observations and anatomic variation in mast cell-nerve associations in vivo.

One hundred and seventeen coded intestinal biopsy specimens were examined by electron microscopy. All surgical biopsies were obtained from uninvolved sites of patients with two inflammatory bowel diseases (ulcerative colitis or Crohn's disease) and from patients with preneoplastic and neoplastic diseases (adenocarcinoma, rectal polyp, familial polyposis). Biopsy sites included normal ileum, colon, and rectum as well as conventional ileostomies and continent pouches constructed from the ileum. The data reported here describe the ultrastructural anatomy of human gastrointestinal tract mucosal mast cells in vivo and their anatomic associations with enteric nerves.

Human lung-derived mature mast cells cultured alone or with mouse 3T3 fibroblasts maintain an ultrastructural phenotype different from that of human mast cells that develop from human cord blood cells cultured with 3T3 fibroblasts.

Culture systems designed to maintain or develop human mast cells have proved difficult, yet these systems would provide valuable resources for future investigations of human mast cell biology. Cocultures of either isolated mature human lung mast cells (Levi-Schaffer et al., J Immunol 1987, 139:494-500) or human cord blood mononuclear cells (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043) with 3T3 embryonic mouse skin fibroblasts have implicated fibroblasts as an important factor in the successful maintenance and development of human mast cells in vitro. The authors cultured isolated, mature human lung mast cells either with or without 3T3 cells for 1 month and examined their ultrastructural phenotype. Mast cell viability in each circumstance was equivalent, but mast cell yield was improved in the presence of 3T3 cells. The ultrastructural phenotype was identical in both culture systems. Mast cells were shown to maintain the phenotype of their in vivo lung counterparts (ie, scroll granules predominanted, and numerous lipid bodies were present). This ultrastructural phenotype differs from that of mast cells that develop in cocultures of human cord blood cells and 3T3 cells, where developing mast cells with crystalline granules and few lipid bodies prevail, a phenotype much like that of human skin mast cells in vivo (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043).

Ultrastructure of monkey peripheral blood basophils stimulated to develop in vivo by recombinant human interleukin 3.

Ultrastructural and cytochemical studies of peripheral blood samples from a monkey continuously infused with recombinant human interleukin 3 were performed. Recombinant human interleukin 3 stimulated a delayed granulocytosis primarily characterized by numerous mature basophils and fewer eosinophils and neutrophils. Basophilic leukocytes were identified by ultrastructural analysis. They were found to be typical granulocytes with polylobed nuclei containing condensed chromatin and numerous cytoplasmic granules. Basophil secretory granules were filled with homogeneous dense contents and were larger than eosinophil and neutrophil secretory granules. Evidence of increased basophil production was accompanied by interleukin 3-associated activation morphologies. These included increased numbers of cytoplasmic and granule-associated vesicles, as are routinely present in a non-IgE-mediated basophil release reaction, termed piecemeal degranulation, and focal perigranular matrix swelling and granule membrane fusion which accompanies anaphylactic degranulation of basophils in other species. Monkey basophils were shown to have a different ultrastructural morphology than that published for monkey mast cells, but exhibited general morphologic criteria for the identification of circulating mature basophils in a number of species. Like human and guinea pig basophils, monkey basophils did not display endogenous peroxidase or peroxidatic activity in a cytochemical assay which simultaneously identified peroxidase-positive granules in neutrophils and eosinophils as well as in synthetic structures in eosinophils. In summary, these studies have identified monkey basophils in an in vivo recombinant human interleukin 3-stimulated model. Interleukin-3 induction of basophilia clearly allowed differentiation of activated mature basophils from eosinophils and neutrophils and mast cells in this species using ultrastructural morphologic criteria.

Ultrastructure of eosinophils and basophils stimulated to develop in human cord blood mononuclear cell cultures containing recombinant human interleukin-5 or interleukin-3.

We used recombinant human interleukins (IL) and cultured human cord mononuclear cells to determine the cell lineages supported by the inclusion of the individual interleukins-5, -3, or -4 in the cultured media. Cultures were examined by electron microscopy at 2-, 3-, and 5-week culture intervals. We found that IL-5 primarily supported the eosinophilic lineage with lesser numbers of basophils, that IL-3 initially supported all granulocyte lineages but eventually supported the eosinophilic lineage with lesser numbers of basophils, and that IL-4 did not support the development of granulocyte lineages in these cultures. At early culture intervals in either IL-5 or IL-3, eosinophilic and basophilic myelocytes were seen in variable proportions. Mature cells of these lineages developed later. Both mature eosinophils and basophils showed morphologic evidence of activation similar to those described for tissue eosinophils and basophils in various disorders. Mast cells were absent in all cultures. Recognition of the variable morphologies associated with maturation and activation of human eosinophils and basophils is important for the correct identification of cell lineages that develop in cultures containing human recombinant interleukins.

Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing.

Fibrin deposition is a consistent early event in solid tumors and healing wounds and precedes new blood vessel ingrowth in both. We now demonstrate that fibrin gels of themselves induce an angiogenic response in the absence of tumor cells or platelets. Angiogenesis was enhanced when certain chemoattractants or mitogens were included in the fibrin gel. Newly devised, inert plastic chambers with one porous surface were filled with varying contents and were implanted in the subcutaneous space of guinea pigs. Chambers filled with cross-linked homologous fibrin or plasma induced an angiogenic response within 4 days. Vessels entered chambers through the surface pores and flared out radially; angiogenesis was quantitated by point counting. Vessels were functional and matured along a gradient that proceeded from distal (least mature) to proximal. The intensity of the angiogenic response was enhanced when zymosan activated serum, an N-formylmethionine tripeptide, or platelet-derived growth factor was included in the fibrin matrix. Prior aldehyde fixation or boiling of fibrin-filled chambers inhibited angiogenesis, as did high concentrations of hyaluronic acid. Chambers filled with type I collagen or agarose did not induce new blood vessel formation, but addition of collagen did not reduce fibrin's capacity to initiate angiogenesis. The novel assay introduced here offers several advantages that should facilitate the study of angiogenesis. These include reproducibility, low background, objective and quantitative scoring, and the capacity to evaluate native molecules in animals of several species. Taken together, our findings strongly implicate fibrin or related proteins in the pathogenesis of angiogenesis and offer a new approach for elucidating the underlying molecular mechanisms.