Epithelial proliferative potential of organ cultured corneoscleral rims; implications for allo-limbal transplantation and eye banking.

AIMS: To determine the epithelial proliferative capacity of organ cultured limbal tissue and correlate this with various donor and eye banking factors. METHODS: 24 corneoscleral limbal (CSL) rims left over from penetrating keratoplasty were split in half and set up as in vitro explant cultures. Corneal epithelial proliferative potential (CEPP) was assessed by the number of "cycles" of growth achieved before explants underwent exhaustion and failure to generate an epithelium to subconfluence. The dependence of CEPP on the age of the donor, time of death to enucleation, time of enucleation to organ culture, and time in organ culture in the eye bank was determined. RESULTS: CSL rims were capable of up to four cycles of culture with a wide variation between tissue samples. Of the various factors examined, death to enucleation time was the only statistically significant factor affecting the CEPP (regression coefficient: -0.062 (cycles/hour), CI -0.119 to -0.004, p = 0.037). Time in organ culture had little effect on CEPP. CONCLUSIONS: Preselected organ cultured CSL rims from eye banks may offer a viable alternative tissue source for use in allo-limbal transplantation.

Evidence that the macrophage-granulocyte inducer (MGI) is produced during cell proliferation, stored in G0, released in G1, cell specific, and induces the secretion of other colony-stimulating activities (CSA).

The secretion of the macrophage and granulocyte inducer (MGI), also known as colony-stimulating factor (CSF), by epithelial cells from lungs and kidneys, and by fibroblasts from lungs, was determined as a function of time in culture; it was found to be secreted during the initial exponential proliferation period, and not when the cells approached saturation density. When the cells were again induced to proliferate, large amounts of CSF were released after 3 h, thus hinting at the existence of a reserve pool. A CSF activity of 70,000 daltons was found in cultures of fibroblasts from lungs, kidneys, and the peritoneal cavity, a 45,000-dalton CSF was obtained from mouse peritoneal macrophages, and from bone marrow cells when activated for macrophage proliferation, and a 22,000-dalton CSF was found from epithelial cells, thus suggesting that the different CSFs are cell specific. When fibroblast CSF was used to induce bone marrow cells, three new molecules with colony-stimulating activity were produced, of 45,000, 30,000, and 17,000 daltons. The fraction with the 17,000-dalton activity also contained interleukin 1 activity, hinting at an indirect induction of colony formation by this factor. Finally the possible existence of a cascade reaction in which one CSF induces the appearance of other CSFs during the normal regulation of myeloid cell differentiation is discussed.

Evidence that G-CSF is a fibroblast growth factor that induces granulocytes to increase phagocytosis and to present a mature morphology, and that macrophages secrete 45-kd molecules with these activities as well as with G-CSF-like activity.

Evidence is provided that conditioned medium from a macrophage-like cell line contains molecules of approximately 45 kd molecular weight with granulocyte colony-stimulating factor (G-CSF)-like activity as well as with the property of inducing granulocytes to phagocytose latex particles and to mature morphologically. This type of differentiation was found to be induced on either bone marrow or induced granulocytes, but not on resident or induced macrophages. On the other hand, resident but not induced macrophages are shown to induce these types of activities when challenged by bacterial lipopolysaccharides. Evidence that macrophages produce a factor that is mitogenic for fibroblasts is also provided. This activity was measured by the induction of increased proliferation by either low-density or saturated cultures of fibroblasts. Human recombinant G-CSF was employed and found also to possess these dual capabilities of inducing both the proliferation and differentiation of granulocytes as well as the proliferation of fibroblasts. Finally, a mechanism for the regulation of myeloid cell production and differentiation is described in which G-CSF produced by macrophages not only induces granulocytes to differentiate but induces fibroblasts to proliferate and secrete macrophage colony-stimulating factor (M-CSF), which in turn makes myeloid monocyte precursors proliferate and secrete more G-CSF.

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases.

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, Mössbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.

Evidences that fibroblasts and epithelial cells produce a specific type of macrophage and granulocyte inducer, also known as colony-stimulating factor, and that monocyte-macrophages can produce another factor with proliferative inducing activity on myeloid cells and differentiative activity on macrophages.

Molecules with the property to induce proliferation of bone marrow cells in liquid cultures, and with colony-stimulating activity, were found on media conditioned (MC) by lung fibroblasts and kidney epithelial cells. These factors presented an apparent mol wt of 70,000 and 22,000 d respectively. Also when MC by epithelial cells from lungs was tested for the induction of proliferation of bone marrow cells a molecule with 22,000 d was detected. These molecules are thought to be CSF because they induce colony formation, and they are also similar in mol wt to two of the already known CSF. In fact the GM-CSF obtained from endotoxic lungs with a large epithelial cell content has a mot wt of 22,000 d, and the CSF-1 produced by a fibroblast cell line had 70,000. When the MC by fibroblast was used to induce bone marrow cells to proliferate, three new molecules with colony-stimulating activity were secreted. These molecules with apparent mol wts of 45,000, 30,000 and 17,000 d were also found in the MC by bone marrow cells when induced to proliferate with MC by epithelial cells. When the 45,000-d molecules was used in induced bone marrow cells to proliferate, once again the 30,000- and the 17,000-d molecules were secreted. Evidence is also provided that the 45,000-d molecule is produced by the monocyte-macrophage cells, and that it can induce Fc receptors or resident and elicited peritoneal macrophages. The possibility that the production of CSF is cell specific is discussed together with two models to explain the way in which these molecules can participate as proliferative (MGI-1) and differentiative (MGI-2) function in normal myeloid cell differentiation. Finally, a new terminology is proposed to classify this family of molecules.

[Sodium caseinate induces differentiation of 32D pluripotential hematopoietic cells]. / El caseinato de sodio induce la diferenciación de las células hematopoyéticas multipotenciales 32D.

AIM: To determine the role of sodium caseinate (CasNa) in the modulation of hemopoiesis. MATERIALS AND METHODS: 32D cells, a murine hemopoietic multipotential cell line dependent on interleukin-3 (IL-3) for proliferation and survival, were used. These cells were cultured with 0.5 ng/mL of IL-3, together with different concentrations of CasNa. We evaluated: proliferation (direct counting under the microscope and use of thymidine 3H), morphological differentiation (giemsa staining), cytochemistry (specific staining for monocytes and granulocytes), and function (presence of Fc receptors and reduction of nitro-blue tetrazolium). In addition, we determined cell viability through trypan blue exclusion and apoptosis using the TUNEL assay in situ. RESULTS: We showed that CasNa induced a decrease in cell proliferation, which is dose dependent, and is neither a result of a diminished cell viability, nor due to an increase in cell death through apoptosis. In addition, CasNa induces cell differentiation towards the monocytic lineage. CONCLUSIONS: CasNa has the capacity to differentiate 32D cells towards the monocytic lineage, and, importantly, has a potent differentiating activity on 32D cells being able to promote differentiation in a shorter time than the well known factors G-CSF and GM-CSF.

The impact of a hospital and clinic-based breastfeeding promotion programme in a middle class urban environment.

Hospital interventions in support of breastfeeding have been highly successful in areas where the indigenous population has a well established environment of breastfeeding. However, programmes designed to improve breastfeeding patterns in urban populations have met with mixed success. This paper presents a prospective intervention study with a control group in which a health system-based breastfeeding promotion programme was initiated to support optimal breastfeeding for both child health and child spacing. Following collection of control data, a four-step intervention programme (Breastfeeding Promotion Program) was instituted. This paper reports the process of the development of the intervention programme as well as the comparison of the control and study populations. Major findings include significant increases in duration of full breastfeeding from 31.6 per cent at 6 months in the control group to 66.8 per cent in the intervention group. The duration of lactational amenorrhea was similarly increased: 22 per cent of the control mothers and 56 per cent of the intervention group women were in amenorrhoea at 180 days. The cost-effectiveness of the hospital changes is illustrated.

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum.

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.

Low temperature magnetic circular dichroism spectroscopy as a probe for the optical transitions of paramagnetic nickel in hydrogenase.

A partially-purified sample of hydrogenase from Methanobacterium thermoautotrophicum (delta H strain) has been investigated by optical absorption, magnetic circular dichroism and electron paramagnetic resonance spectroscopy. Variable temperature magnetic circular dichroism studies reveal, for the first time, the optical transitions associated with the Ni(III) center in the oxidized enzyme. Low temperature magnetic circular dichroism spectroscopy provides a new method of assessing both the coordination environment of Ni in hydrogenase and the appropriateness of inorganic model complexes.

Granulocyte colony-stimulating factor induces neutrophils to secrete macrophage colony-stimulating factor.

In this work we provide evidence showing that granulocytes produce macrophage colony-stimulating factor (M-CSF) from the band cell stage and secrete this factor when induced to differentiate into polymorphonuclear cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF). Using an enriched population of myeloid band cells from murine bone marrow, we identified the presence of M-CSF with a chromophore-labelled monoclonal anti-M-CSF antibody. Using ELISA we detected the secretion of M-CSF in the supernatants of cultures of enriched band cells when induced with rhG-CSF to differentiate into mature neutrophils. We also found that M-CSF is the only factor responsible for the colony forming activity in the supernatants and lysates of band cells treated with rhG-CSF.

Valor diagnóstico y pronóstico de la inmunoglobulina E en la cardiopatía isquémica aguda / Diagnosis and prognotic value of the inmunoglobin E at the acute ischemic cardiopathy

Se realizó un estudio de tipo descriptivo y transversal donde se evaluaron 38 pacientes con diagnóstico de infarto de miocardio (IM) o angina inestable, basado en los criterios clínicos, electrocardiográficos y enzimáticos preestablecidos que ingresaron a la unidad de cuidados coronarios del Hospital Dr. Domingo Luciani desde julio hasta septiembre de 1992, descartándose en todos ellos a través del interrogatorio enfermedades alérgicas, asma bronquial, transfunsiones sanguíneas recientes, uso de glucocorticoides, presencia de mieloma IgE o un síndrome de hiper IgE; A través de un frotis de sangre periférica la presencia de eosinofília y mediante examen de heces especial la presencia de parasitosis intestinal. A todos los pacientes se les hizo una determinación de colesterol y triglicéridos a la semana del evento isquémico y de IgE sérica durante los días 1, 3, 8, 15 y 21 desde el ingreso del paciente, tomándose la primera muestra en un tiempo promedio de 11 horas, demostrándose una evidente asociación entre valores elevados IgE séricas y cardiopatía isquémica del tipo IM o angina inestable. Los valores séricos de IgE en nuestros pacientes isquémicos fueron superiores a los descritos en poblaciones normales tanto de estudios internacionales como de nuestro país; además, se demostró una diferencia estadísticamente significativa entre valores IgE sérica del grupo de paciente con IM y el grupo de pacientes con angina inestable estando más elevada en los primeros. En presencia de hábitos tabaquicos y edad superior a los 53 años, la tendencia a presentar niveles elevados de IgE sérica aumenta. De manera que niveles elevados de IgE, al igual que el tabaquismo y la edad pudieran representar un factor de riesgo adicional para cardiopatía isquémica