Using the past to contextualize anthropogenic impacts on the present and future distribution of an endemic Caribbean mammal.

Island species are difficult to conserve because they face the synergy of climate change, invasive species, deforestation, and increasing human population densities in areas where land mass is shrinking. The Caribbean island of Hispaniola presents particular challenges because of geopolitical complexities that span 2 countries and hinder coordinated management of species across the island. We employed species distribution modeling to evaluate the impacts of climatic change and anthropogenic activities on the distribution of an endemic mammal of conservation concern, the Hispaniolan solenodon (Solenodon paradoxus). We aggregated occurrence points for this poorly known species for the Last Glacial Maximum (LGM) and the present (1975-2016) based on museum collections, online biodiversity databases, and new field surveys. We quantified degree of overlap between periods and scenarios with Schoener's D. Through a conservation paleobiology lens, we found that over time humans played an increasing role in shaping the distribution of S. paradoxus, thus, providing a foundation for developing conservation strategies on appropriate spatiotemporal scales. Human population density was the single most important predictor of S. paradoxus occurrence. Densities >166 people/km2 corresponded to a near-zero probability of occurrence. Models that accounted for climate but not anthropogenic variables falsely identified suitable habitat in Haiti, where on-the-ground surveys confirm habitat is unavailable. Climate-only models also significantly overestimated the potential for habitat connectivity between isolated populations. Our work highlights that alternative fates for S. paradoxus in the Anthropocene exist across the political border between the Dominican Republic and Haiti due to the fundamentally different economic and political realities of each country. Relationships in the fossil record confirm that Hispaniola's sociopolitical boundary is not biologically significant but instead represents one imposed on the island's fauna in the past 500 years by colonial activity. Our approach reveals how a paleontological perspective can contribute to concrete management insights.

Uso del Pasado para Contextualizar los Impactos Antropogénicos en la Distribución Presente y Futura de un Mamífero Endémico del Caribe Resumen Las especies insulares son difíciles de conservar ya que enfrentan la sinergia del cambio climático, las especies invasoras, la deforestación y la densidad creciente de la población humana en áreas en donde la masa de tierra se está encogiendo. La isla caribeña de La Española representa un reto particular debido a las complejidades geopolíticas que abarcan a dos países y obstaculizan el manejo coordinado de las especies en toda la isla. Empleamos el modelado de distribución de especies para evaluar los impactos del cambio climático y las actividades antropogénicas sobre la distribución de un mamífero endémico de importancia para la conservación: el solenodonte de La Española (Solenodon paradoxus). Agregamos puntos de presencia para esta especie muy poco conocida durante el Último Máximo Glacial (LGM, en inglés) y durante el presente (1975-2016) con base en colecciones de museos, bases de datos de biodiversidad en línea y nuevos censos de campo. A través de este lente de paleobiología de la conservación encontramos que con el tiempo los humanos tuvieron un papel cada vez mayor en la distribución de S. paradoxus, proporcionando así los cimientos para el desarrollo de estrategias de conservación a escalas espacio-temporales adecuadas. La densidad de la población humana fue el pronosticador más importante de la presencia de S. paradoxus. Las densidades mayores a 166 personas/km2 correspondieron con una probabilidad cercana a cero de la presencia de este mamífero. Los modelos que consideraron al cambio climático pero no a las variables antropogénicas identificaron falsamente hábitats aptos en Haití, en donde los censos de campo confirman que no hay hábitat disponible. Los modelos que sólo consideraron el clima también sobreestimaron significativamente el potencial para la conectividad de hábitat entre poblaciones aisladas. Nuestro trabajo resalta que existen destinos alternativos para S. paradoxus en el Antropoceno, que además traspasan la frontera política entre Haití y la República Dominicana causada por las realidades económica y política fundamentalmente diferentes de cada país. Las relaciones en el registro fósil confirman que la frontera socio-política de La Española no es significativa biológicamente, sino que representa una frontera impuesta sobre la fauna de la isla durante los últimos 500 años por la actividad colonial. Nuestra estrategia revela cómo la perspectiva paleontológica puede contribuir para concretar la percepción del manejo.

Beyond palliative mastectomy in inflammatory breast cancer--a reassessment of margin status.

BACKGROUND: Inflammatory breast cancer is a locally advanced tumor with an aggressive local and systemic course. Treatment of this disease has been evolving over the last several decades. The aim of this study was to assess whether current therapies, both surgical and chemotherapeutic, are providing better local control (LC) and overall survival (OS). We also attempted to identify clinical and pathologic factors that may be associated with improved OS, disease-free survival (DFS), and LC. METHODS: A 25-year retrospective review performed at the City of Hope National Medical Center identified 90 patients with the diagnosis of inflammatory breast cancer. RESULTS: Of the 90 patients identified with inflammatory breast cancer, 33 received neoadjuvant therapy (NEO) consisting of chemotherapy followed by surgery with radiation (n = 26) and without radiation (n = 7). Fifty-seven patients received other therapies (nonNEO). Treatments received by the nonNEO group consisted of chemotherapy, radiation, mastectomy, adrenalectomy, and oophorectomy, alone or in combination. The median follow-up was 28.9 months for the NEO group and 17.6 months for the nonNEO group. Borderline significant differences in the OS distributions between the two groups were found (P = .10), with 3- and 5-year OS for the NEO group of 40.0% and 29.9% and for the nonNEO group of 24.7% and 16.5%, respectively. DFS and LC were comparable in the two groups. Lower stage was associated with an improved OS (P < .05). The 5-year OS for stage IIIB was 30.9%, compared to 7.8% for stage IV. In those patients with stage III disease who were treated with mastectomy and rendered free of disease, margin status was identified by univariate analysis to be a prognostic indicator for OS (P < .05). The 3-year OS, DFS, and LC for patients with negative margins were 47.4%, 37.5%, and 60.3%, respectively, compared to 0%, 16.7%, and 31.3% in patients with positive margins. CONCLUSIONS: This study suggests that in patients with inflammatory breast cancer and nonmetastatic disease, an aggressive surgical approach may be justified with the goal of a negative surgical margin. Achievement of this local control is associated with a better overall outcome for this subset of patients. The ability to obtain negative margins may further identify a group of patients with a less aggressive tumor biology that may be more responsive to other modalities of therapy.

High-dose oral dexamethasone therapy for chronic childhood idiopathic thrombocytopenic purpura.

Because high-dose oral dexamethasone therapy has been reported to be effective for adults with idiopathic thrombocytopenic purpura, we assessed the short-term efficacy and toxicity of dexamethasone in seven children with chronic or refractory idiopathic thrombocytopenic purpura. Dexamethasone therapy was effective and well tolerated; further long-term studies are warranted.

Demonstration of functionally different interactions between phospholipase C-gamma and the two types of platelet-derived growth factor receptors.

Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC-gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma.

A unique autophosphorylation site in the platelet-derived growth factor alpha receptor from a heterodimeric receptor complex.

The platelet-derived growth factor (PDGF) alpha and beta receptors undergo dimerization as a consequence of ligand binding. Depending on the PDGF isoform (PDGF-AA, -AB or -BB), homodimers or heterodimers of receptors are formed. In this study, we have used transfected porcine aortic endothelial cells, coexpressing cDNAs for the alpha receptor and the beta receptor at comparable levels, to investigate the properties of the alpha beta-heterodimeric receptor complex. PDGF-AB, which mainly induced alpha beta-heterodimeric complexes, was the most efficient isoform for stimulating mitogenicity. Actin reorganization, in the form of circular membrane ruffling and chemotaxis, was induced by PDGF-AB and PDGF-BB, but not by PDGF-AA, thus indicating that the beta receptor in the homodimeric or heterodimeric configuration was required for induction of motility responses. The molecular basis for the apparent receptor dimer-specific properties was examined by analyzing receptor autophosphorylation and phosphorylation of substrates. The alpha receptor was found to be phosphorylated at an additional tyrosine residue, Tyr754, in the heterodimeric complex as compared to the alpha alpha receptor homodimer. Phosphorylation of this tyrosine residue could permit the binding of a specific signal-tranducing protein. A candidate is a 134,000-M(r) protein, which was shown to associate preferentially with the alpha receptor in the heterodimeric receptor complex. It is possible that phosphorylated Tyr754 in the alpha receptor mediates activation of specific signal-tranducing molecules like the 134,000-M(r) substrate, and thereby initiates signal-tranduction pathways from the alpha beta receptor heterodimer, which are distinct from those initiated via homodimeric receptor complexes.

Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

Differential NPY mRNA expression in granule cells and interneurons of the rat dentate gyrus after kainic acid injection.

Using in situ hybridization histochemistry neuropeptide Y (NPY) mRNA expression was investigated after intraperitoneal injection of kainic acid (KA) and after local application of KA or quinolinic acid into the dentate gyrus of the rat. Enhanced concentrations of NPY mRNA were observed in interneurons of the hilus, including presumptive fusiform neurons and pyramidal-shaped basket cells already 4 hours after initiation of limbic seizures by KA (10 mg/kg, i.p.). Increased NPY expression persisted in neurons resistant to seizure-induced cell death (6-48 h after i.p. KA). Exceptionally high hybridization signals were found in interneurons of the hilus and the CA1 and CA3 sectors 8 months after KA-induced limbic seizures. In the granule cell layer only a transient but pronounced increase in NPY mRNA was observed 12-24 h after injection. Only moderate changes were observed in this cell layer at later intervals. Anticonvulsant treatment with thiopental, after a brief period of generalized seizures, prevented the increase in NPY mRNA in granule cells but not in interneurons. No change in NPY message was found also in granule cells of rats which responded with mild "wet dog shake" behavior but not with motor seizures to KA injection. Local injections of low doses of KA (0.05-0.2 nmol) or quinolinic acid (6.5-100 nmol) into the dentate gyrus of the hippocampus under deep thiopental anesthesia, after 24 h, resulted in increased concentrations of NPY message in interneurons of the ipsilateral, but not of the contralateral hilus and not in granule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of MHC class I synthesis and expression by human neutrophils.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

Recombinant human retinoic acid receptor alpha. Binding of DNA and synthetic retinoids to the protein expressed in Escherichia coli.

The human retinoic acid receptor alpha was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA binding activities in gel-retardation assays. Ligand binding properties were measured in a charcoal absorption assay. The dissociation constant for highly specific bound retinoic acid was found to be 0.67 nM. The affinity of several synthetic retinoids to the recombinant protein was determined and compared to their biological activity. Some of the values presented here differ significantly from those published earlier for the receptor or its isolated hormone-binding domain.

The promoter of the human parathyroid hormone gene contains a functional cyclic AMP-response element.

We have screened the sequence of the 394 base pairs upstream of the main transcriptional start site of the promoter of the human parathyroid hormone (PTH) gene for well-known protein recognition motifs with the aim to identify potential positive or negative regulatory elements. Within this region we found a potential cAMP-response element (CRE) besides several other putative binding sites for transcription factors. We fused promoter regions that contain this element and extend beyond the transcription start site to an appropriate reporter gene (CAT) and transfected different cell lines with these constructs. Transient expression of the CAT gene from these hybrid genes could be shown to be significantly stimulated by forskolin or isoproterenol thus proving the responsiveness of the whole promoter region towards elevated cAMP levels. DNase I protection studies revealed protein binding around the putative CRE (PTH-CRE) and an adjacent CCAAT element. Gel retardation assays with the PTH-CRE as well as the well-characterized CRE from the rat somatostatin promoter indicated specific binding of the same protein to both elements, although with a slightly reduced affinity of the PTH-CRE. Both of these elements were also able to confer cAMP-responsiveness to a heterologous promoter.

Intravenous infusions of nitroprusside, dobutamine, and nitroglycerin are compatible.

It is now customary practice to couple separately metered infusions via a manifold to a common catheter that enters the patient. Nitroprusside, however, is considered incompatible with all other medications. Critically ill patients who require multiple infusions of vasoactive and inotropic medications would benefit if physicians had additional information regarding compatibility of nitroprusside with other commonly used infusions. Utilizing high-performance liquid chromatography, the authors investigated the physical and chemical compatibility of nitroprusside, dobutamine, and nitroglycerin in solutions of 5% dextrose or 0.9% NaCl at clinically relevant concentrations. All drugs were present within the guidelines of the U.S. Pharmacopeia (+/- 10%) over 24 h in NaCl, but nitroglycerin degraded over 24 h when the three drugs were mixed in dextrose. We recommend diluting these medicines in NaCl when mixtures of them would exist for greater than 4 h.

Cytoplasmic pH and anchorage-independent growth induced by v-Ki-ras, v-src or polyoma middle T.

We had previously shown that spreading of normal cells on tissue culture plastic coated with extracellular matrix (ECM) proteins led to an increase in cytoplasmic pH (pHi). Since alkalinization of the cytoplasm is associated with activation of a number of signaling pathways that control growth, and is itself required for cell growth, we proposed that this phenomenon could explain, at least in part, why growth of normal cells is anchorage-dependent. Preliminary results showed that pHi in cells transformed by the ras or src oncogenes had an alkaline pHi even when completely round. To further explore the relationship between pHi and anchorage-independent growth, a series of cells transformed by mutants of the polyoma middle T oncogene, and a series of ras-transformed cells and revertants were examined. Growth in methyl cellulose was assayed, and pHi in both maximally spread and completely round cells was measured for each cell line. We found that all of the normal cells required spreading to maintain an alkaline pHi, whereas transformed cell lines had an alkaline pHi independent of spreading. There was a strong correlation between pHi in round cells and anchorage-independent growth. Thus, some plasma membrane oncogenes can substitute for cell spreading on EMC to raise pHi as well as to promote growth. These results may be relevant to understanding why transformation leads to changes not only in cellular requirements for growth factors, but also for anchorage.

Ivermectin in loiasis and concomitant O. volvulus and M. perstans infections.

Two clinical studies were carried out in Gabon, Africa to evaluate the efficacy, safety, and tolerability of ivermectin in the treatment of patients with Loa loa infection. In the first study, 35 patients received single oral doses of ivermectin, 5-200 mcg/kg body weight. Blood microfilariae levels did not decrease after a single oral 5, 10, 30, or 50 mcg/kg dose of ivermectin, but levels did decrease after doses of 100, 150, and 200 mcg/kg. The most efficacious dose was 200 mcg/kg; mean blood microfilariae levels decreased to 12% of mean pretreatment values by day 15 and remained decreased for 28 days. A second study evaluated the safety and efficacy of ivermectin in patients with multifilarial infections. All 17 patients had concomitant Loa loa and O. volvulus infection. M. perstans affected 5 of the patients. Sixteen patients also had infections due to intestinal nematodes. The patients each received single oral doses of 200 mcg/kg ivermectin. Ten days later, the mean Loa loa blood microfilariae level had decreased to 20% of the mean pretreatment level. O. volvulus dermal microfilariae densities were reduced to 2% of the pretreatment levels. A minimal increase in blood microfilaria levels was observed on day 28. In contrast, dermal microfilariae levels remained near zero for the duration of the study. Intestinal infection due to Ascaris was eradicated in all of the affected patients by day 23; efficacy against Trichuris and hookworm infections, however, was poor. All patients tolerated ivermectin well including those with multiple infections.

Identification of a high-affinity receptor for interleukin 1 alpha and interleukin 1 beta on cultured human rheumatoid synovial cells.

In this report the binding of recombinant human interleukins 1 alpha and 1 beta (rIL-1 alpha and rIL-1 beta) to primary cultures of human rheumatoid synovial cells is measured and compared to the concentrations of these mediators required for stimulation of PGE2 production by these same cells. The average concentration of IL-1 alpha required for half-maximal stimulation of PGE2 was 4.6 +/- 1.5 pM (+/- SEM) (n = 6), whereas for IL-1 beta half-maximal stimulation was observed at a concentration of 1.3 +/- 0.24 pM (n = 6). Both direct and competitive binding experiments were performed. In direct binding experiments, IL-1 alpha bound with a Kd of 66 pM (n = 1), while IL-1 beta bound with a Kd of 4 pM (n = 2). In competitive binding experiments, IL-1 alpha inhibited binding of 125I-IL-1 alpha with a Ki of 33-36 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 51-63 pM (n = 2). IL-1 beta inhibited binding of 125I-IL-1 alpha with a Ki of 2-3 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 7 pM (n = 2). The binding data were best fit by a model specifying a single class of receptors with homogeneous affinity for either IL-1 alpha or IL-1 beta and with an abundance of 3,000-14,000 sites per cell. Autoradiography showed that the vast majority of the synoviocytes within the cultures possessed IL-1 receptors. Comparison of biological response curves with the binding curves indicates that the observed receptors exhibit sufficiently high affinity to mediate the response of human synoviocytes to low picomolar concentrations of IL-1 alpha and IL-1 beta.

Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.

Identification of a high-affinity receptor for native human interleukin 1 beta and interleukin 1 alpha on normal human lung fibroblasts.

Native human IL-1 beta and IL-1 alpha stimulated prostaglandin E2 secretion by human embryonic lung fibroblasts at half-maximal concentrations of 3 +/- 1.2 pM (+/- SEM) and 10 +/- 2.3 pM, respectively. In contrast to the 20-50-fold lower affinities previously found for IL-1-R on 3T3 cells as well as murine and human lymphoblastoid lines, monoiodo 125I-IL-1 beta bound to normal human fibroblasts with a Kd of 8.4 +/- 4.1 pM in direct binding experiments, and with a Ki of 11.2 +/- 2.8 pM in competitive binding experiments. IL-1 alpha bound to the receptor identified by 125I-IL-1 beta with a Ki of 50 +/- 18 pM. The receptor exhibited homogeneous affinity for IL-1 beta or IL-1 alpha. The receptor did not recognize IL-2, IFN-gamma, tumor necrosis factor alpha, a functionally related monokine, or bovine acidic fibroblast growth factor, a structurally related mediator. Comparison of the biological response curves and binding curves obtained for IL-1 alpha and IL-1 beta showed that they were parallel and that 10-15% occupancy of the estimated 3,000 sites by either species of IL-1 was sufficient to give half-maximal stimulation of prostaglandin E2 secretion. Thus, the amount of apparent signal amplification observed on fibroblasts was considerably lower than the 100-100,000 fold amplification previously reported for lymphoid lines. Crosslinking experiments revealed a major band with a corrected molecular mass of approximately 80 kD and a minor band of approximately 200 kD. Labeling of these bands was blocked by IL-1 beta and IL-1 alpha but not by IL-2, IFN-gamma, or tumor necrosis factor alpha. These results demonstrate that normal human embryonic lung fibroblasts bear IL-1-R of sufficiently high affinity to mediate their biological responsiveness to low picomolar concentrations of IL-1 beta and IL-1 alpha and are consistent with the existence of a single receptor mediating the biological properties of both human IL-1 species.

Specific bioactivities of monocyte-derived interleukin 1 alpha and interleukin 1 beta are similar to each other on cultured murine thymocytes and on cultured human connective tissue cells.

In this report we compare the bioactivities of pure, human monocyte-derived interleukin 1 (IL-1) alpha and beta in the standard murine thymocyte proliferation assay, a human dermal fibroblast proliferation assay, and in an assay measuring stimulation of prostaglandin E2 (PGE2) release from human rheumatoid synoviocytes. In each case the different species of IL-1 produced saturable stimulation and gave similar dose response curves. Half-maximal stimulation was observed at average IL-1 concentrations of 29 pM in the thymocyte assay, 2 pM in the dermal fibroblast proliferation assay, and 5 pM in the synovial cell assay. Our results show that native, monocyte-derived IL-1 alpha and IL-1 beta are both potent stimulators of connective tissue cells and that the specific bioactivities of these molecules are similar to each other in tests on human connective tissue cells, as well as on murine lymphoid cells.

Immunocytochemical detection of interleukin 1 within stimulated human monocytes.

We have used synthetic peptides coupled to KLH to raise high titer antisera to human IL-1 beta, and in the present report show the usefulness of these sera for immunocytochemical analyses of IL-1 production. Using indirect immunofluorescence, we have been able to specifically identify IL-1 within human monocytes and to monitor its accumulation with time. After indirect immunofluorescent staining of LPS- and PHA-stimulated mononuclear cell cultures, intense cytoplasmic fluorescence was observed in 93% of the monocytes, but not in lymphocytes or platelets present in the same preparation. Unstimulated monocytes did not contain immunocytochemically detectable IL-1. When put into culture, however, some of the otherwise unstimulated monocytes subsequently showed a transient accumulation of intracellular IL-1. Monocytes cultured in the presence of LPS and PHA exhibited detectable fluorescence after 2.5 h, and the fluorescent intensity of these cells continued to increase over the course of 21 h. Fluorescent staining was abolished by preincubation of the sera with relevant but not irrelevant peptide, and while preimmune or anti-KLH serum produced no staining, antisera against either the amino terminus or an internal region of IL-1 beta produced identical staining patterns. Immunoblot analyses of lysates from stimulated monocytes showed that the antisera against IL-1 recognize a single intracellular species with an apparent molecular weight (33 kD) similar to that predicted for IL-1 precursor from the nucleotide sequence of IL-1 cDNA. The ability to specifically identify and immunocytochemically localize IL-1 within producing cells should prove extremely useful for studying the in situ production of IL-1 in immune-based and inflammatory diseases.