Induction of chemokine gene expression from peripheral blood mononuclear cells (PBMCs) stimulated by proinflammatory cytokines plays an important role in both wound repair and response to infectious agents. In the present study, we show that the proinflammatory cytokine interleukin-6 (IL-6) potently induced mRNA expression and secretion of the CC chemokine monocyte chemotactic protein 1 (MCP-1) in PBMCs. In addition, because human immunodeficiency virus (HIV) infection in vivo and in vitro has been shown to dysregulate the production of and/or the response to cytokines, PBMCs from both healthy uninfected and HIV-infected individuals were studied for their constitutive and IL-6-induced expression of MCP-1. No substantial differences were observed between the two groups of individuals. In addition, IL-6 upregulated MCP-1 expression in the promonocytic cell line U937 and in its chronically HIV-infected counterpart, U1. In these cell lines, IL-6 selectively induced MCP-1 and not other chemokines, including regulated upon activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and IL-8. IL-6 induction of MCP-1 was partially inhibited by hydrocortisone in U1 cells. Thus, IL-6 activates PBMCs to secrete MCP-1, a CC chemokine pivotal for monocyte recruitment in tissue and organs in which important inflammatory events occur.

A major difference between "naive" and "memory" or "effector" Th cells is the spectrum of cytokines that they are capable of producing. After stimulation naive cells produce only IL-2, whereas memory cells produce several cytokines including IFN-gamma and IL-4. Using umbilical cord blood-derived CD4 T cells as a source of naive T cells, we first report that these cells are capable of producing large amounts of IFN-gamma when cultured with low concentrations of IL-12. The response is time- and dose-dependent, and it is observed at the protein and mRNA levels. IL-12 also induces neonatal CD4 T cells to produce lymphotoxin but not IL-2, TNF-alpha, or IL-4. The production of IFN-gamma by IL-12-stimulated neonatal T cells is associated with a small but significant T cell activation evidenced by DNA synthesis and by the expression of the activation markers CD25, CD71, and HLA-DR; moreover, it is inhibited by hydrocortisone, cyclosporin A, and transforming growth factor-beta. The response to IL-12 is enhanced and is much more rapid when CD4 T cells are cultured in the presence of accessory cells or of exogenous IL-1, IL-2, or TNF-alpha. Using a three-step culture system, we next show that IL-12 induces the maturation of resting naive CD4 T cells into cells producing both IL-2 and IFN-gamma but not IL-4 upon stimulation with PMA and ionomycin. Endogenously produced IFN-gamma plays a role in this IL-12-induced T cell maturation, as shown by the inhibitory effect of neutralizing IFN-gamma antibodies. Finally, we show that IL-12 supports the production of IFN-gamma during primary stimulation of neonatal T cells via the CD3/TCR complex by means of either immobilized anti-CD3 mAb or superantigen-coated (Staphylococcus enterotoxin B) fixed L cell transfectants expressing HLA-DR. It is suggested that IL-12 is involved in the selection of Th1 type immune responses.

Primary adrenal insufficiency (PAI), or Addison's disease, is a rare, potentially deadly, but treatable disease. Most cases of PAI are caused by autoimmune destruction of the adrenal cortex. Consequently, patients with PAI are at higher risk of developing other autoimmune diseases. The diagnosis of PAI is often delayed by many months, and most patients present with symptoms of acute adrenal insufficiency. Because PAI is rare, even medical specialists in this therapeutic area rarely manage more than a few patients. Currently, the procedures for diagnosis, treatment and follow-up of this rare disease vary greatly within Europe. The common autoimmune form of PAI is characterized by the presence of 21-hydroxylase autoantibodies; other causes should be sought if no autoantibodies are detected. Acute adrenal crisis is a life-threatening condition that requires immediate treatment. Standard replacement therapy consists of multiple daily doses of hydrocortisone or cortisone acetate combined with fludrocortisone. Annual follow-up by an endocrinologist is recommended with the focus on optimization of replacement therapy and detection of new autoimmune diseases. Patient education to enable self-adjustment of dosages of replacement therapy and crisis prevention is particularly important in this disease. The authors of this document have collaborated within an EU project (Euadrenal) to study the pathogenesis, describe the natural course and improve the treatment for Addison's disease. Based on a synthesis of this research, the available literature, and the views and experiences of the consortium's investigators and key experts, we now attempt to provide a European Expert Consensus Statement for diagnosis, treatment and follow-up.

Large forms of Candida are characteristically present in invasive lesions and are often cleared by host defenses. Therefore, an in vitro system was developed to study interactions between leukocytes and pseudohyphae. By light, phase contrast, and electron microscopic observations, in the absence of serum, neutrophils attached to and spread over the surfaces of partially ingested pseudohyphae, which then appeared damaged. Using a new assay which measured neutrophil-induced inhibition of uptake of [(14)C]cytosine by Candida, damage to Candida in the absence of serum was 53.04+/-2.96% by neutrophils from 27 normal subjects. With serum, damage to Candida increased because of opsonization by low levels of anti-Candida immunoglobulin G in normal sera. Damage to Candida was inhibited by colchicine, cytochalasin B, and 2-deoxyglucose, which interfered with spreading of neutrophils over the surfaces of Candida. Dibutyryl cyclic AMP, theophylline, and isoproterenol also inhibited damage to Candida. Hydrocortisone was inhibitory in levels (10 muM) achievable with pharmacologic doses in man. Light, fluorescence, and electron microscopy indicated that neutrophils degranulated after contact with Candida. Quantitative studies revealed only a minimal increase in specific release of lysosomal enzymes from azurophil granules, but much greater release of lysozyme from specific granules. Candida activated neutrophil oxidative microbicidal mechanisms, as shown by iodination of Candida by neutrophils, and chemiluminescence from neutrophils interacting with Candida. Unlike live Candida, killed Candida did not induce chemiluminescence, were not iodinated, and did not attach to neutrophils by microscopy. Like Candida pseudohyphae, contact between neutrophils and hyphal forms of Aspergillus and Rhizopus occurred in the absence of serum. This did not occur with Cryptococcus neoformans, an encapsulated yeast, and was low with Candida yeasts. These findings indicate that neutrophils can recognize and attach to Candida pseudohyphae, then damage the Candida. This may represent a general reaction between neutrophils and large forms of fungi. Though the size of the organisms precludes complete ingestion, neutrophil oxidative microbicidal mechanisms are activated, and preferential release of contents of specific granules appears to occur.

OBJECTIVE

Ghrelin is a gut-derived peptide and an endogenous ligand for the growth hormone (GH) secretagogue receptor. Exogenous ghrelin stimulates the release of GH (potently) and adrenocorticotropic hormone (ACTH) (moderately). Ghrelin is also orexigenic, but its impact on substrate metabolism is controversial. We aimed to study direct effects of ghrelin on substrate metabolism and insulin sensitivity in human subjects.

METHODS

Six healthy men underwent ghrelin (5 pmol . kg(-1) . min(-1)) and saline infusions in a double-blind, cross-over study to study GH signaling proteins in muscle. To circumvent effects of endogenous GH and ACTH, we performed a similar study in eight hypopituitary adults but replaced with GH and hydrocortisone. The methods included a hyperinsulinemic-euglycemic clamp, muscle biopsies, microdialysis, and indirect calorimetry.

RESULTS

In healthy subjects, ghrelin-induced GH secretion translated into acute GH receptor signaling in muscle. In the absence of GH and cortisol secretion, ghrelin acutely decreased peripheral, but not hepatic, insulin sensitivity together with stimulation of lipolysis. These effects occurred without detectable suppression of AMP-activated protein kinase phosphorylation (an alleged second messenger for ghrelin) in skeletal muscle.

CONCLUSIONS

Ghrelin infusion acutely induces lipolysis and insulin resistance independently of GH and cortisol. We hypothesize that the metabolic effects of ghrelin provide a means to partition glucose to glucose-dependent tissues during conditions of energy shortage.

In the present study we have investigated the expression of mRNAs for hyaluronan synthase isoforms (HAS1, HAS2 and HAS3) in different cells in response to various stimuli. Human mesothelial cells, which synthesize large amounts of hyaluronan, express mRNAs encoding all three HAS isoforms, whereas their transformed counterparts, mesothelioma cells, which produce only minute amounts of hyaluronan, express only HAS3 mRNA. Human lung fibroblasts and the glioma cell line U-118 MG express only the HAS2 and HAS3 genes. The expression of the transcripts was higher in subconfluent than in confluent cultures and was well correlated with the production of hyaluronan by the cells. Stimulation of mesothelial cells with platelet-derived growth factor-BB induced an up-regulation of mRNA for HAS2 to a maximum after 6 h of stimulation; HAS1 and HAS3 genes were only induced slightly. Transforming growth factor-beta1 reduced HAS2 mRNA slightly, and hydrocortisone reduced it strongly, within 6 h of stimulation in mesothelial cell cultures but did not significantly affect the expression of mRNAs for HAS1 and HAS3. Induction of HAS1 and HAS2 protein levels in response to the stimuli above correlated with HAS transcript levels. Thus the expression of the three HAS isoforms is more prominent in growing cells than in resting cells and is differentially regulated by various stimuli suggesting distinct functional roles of the three proteins.

In male patients with congenital adrenal hyperplasia, testicular tumors, or so-called adrenal rest tumors, have been described, but their presence in well controlled patients is thought to be rare. In this study, the prevalence of testicular tumors in 17 adolescent and adult male patients with congenital adrenal hyperplasia (age, 16-40 yr) was investigated. In 16 of 17 patients, one or more testicular tumors, ranging in maximal length from 0.2-4.0 cm, were found on ultrasonography. In 6 patients, the testicular tumors were palpable. Undertreatment, defined as the presence of a salivary androstenedione level (mean of 6 saliva samples collected over 24 h with intervals of 4 h) above the upper reference morning level, was found in 5 of 17 patients at the time of investigation. The other 12 patients were treated adequately or even over treated at the time of investigation. Nevertheless, 11 of these 12 patients showed testicular tumors on ultrasonography. Neither the presence of undertreatment at the time of investigation nor characteristics of the therapeutic regimen (daily dose of hydrocortisone equivalents per body surface, the use of glucocorticoid medication either two or three times a day, or the time of taking the highest glucocorticoid dose either in the morning or the evening) could predict tumor size (maximal diameter of largest tumor). In patients who were heterozygous or homozygous for the deletion or conversion of the CYP21 gene, tumor size was significantly larger than in patients who did not have this genotype. Impairment of Leydig cell function as manifested by decreased plasma levels of T was found in 6 of 17 patients. Semen analysis in 11 patients revealed azoospermia in 3 patients and poor semen quality in 4 patients. We conclude that, when carefully sought for, testicular adrenal rest tumors are frequently present in adolescent and adult males with congenital adrenal hyperplasia and are often accompanied by impaired spermatogenesis and Leydig cell failure.

BACKGROUND

Patients with treated adrenal insufficiency (AI) have increased morbidity and mortality rate. Our goal was to improve outcome by developing a once-daily (OD) oral hydrocortisone dual-release tablet with a more physiological exposure-time cortisol profile.

OBJECTIVE

The aim was to compare pharmacokinetics and metabolic outcome between OD and the same daily dose of thrice-daily (TID) dose of conventional hydrocortisone tablets.

METHODS

We conducted an open, randomized, two-period, 12-wk crossover multicenter trial with a 24-wk extension at five university hospital centers.

METHODS

The trial enrolled 64 adults with primary AI; 11 had concomitant diabetes mellitus (DM).

METHODS

The same daily dose of hydrocortisone was administered as OD dual-release or TID.

METHODS

We evaluated cortisol pharmacokinetics.

RESULTS

Compared with conventional TID, OD provided a sustained serum cortisol profile 0-4 h after the morning intake and reduced the late afternoon and the 24-h cortisol exposure. The mean weight (difference = -0.7 kg, P = 0.005), systolic blood pressure (difference = -5.5 mm Hg, P = 0.0001) and diastolic blood pressure (difference: -2.3 mm Hg; P = 0.03), and glycated hemoglobin (absolute difference = -0.1%, P = 0.0006) were all reduced after OD compared with TID at 12 wk. Compared with TID, a reduction in glycated hemoglobin by 0.6% was observed in patients with concomitant DM during OD (P = 0.004).

CONCLUSIONS

The OD dual-release tablet provided a more circadian-based serum cortisol profile. Reduced body weight, reduced blood pressure, and improved glucose metabolism were observed during OD treatment. In particular, glucose metabolism improved in patients with concomitant DM.

A survey of commercially available media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal keratinocytes (HK) when supplemented with 10 micrograms/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 micrograms/ml HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly from NCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal growth of HK occurs at a very low level of Ca2+ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium. However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the Ca2+ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDF 151 with 10 micrograms/ml HC and 1.0 mg/ml FBSP.

For the study of in-vitro skin penetration of candidate drugs, excised animal skin is frequently used as a replacement for human skin. Reconstructed human skin or epidermis equivalents have been proposed as alternatives. We compared the penetration properties of human, pig and rat skin with the Graftskin LSE (living skin equivalent) and the Skinethic HRE (human reconstructed epidermis) models using four topical dermatological drugs (salicylic acid, hydrocortisone, clotrimazole and terbinafine) with widely varying polarity. In agreement with published data, pig skin appeared as the most suitable model for human skin: the fluxes through the skin and concentrations in the skin were of the same order of magnitude for both tissues, with differences of at most two- or fourfold, respectively. Graftskin LSE provided an adequate barrier to salicylic acid, but was very permeable for the more hydrophobic compounds (e.g. about 900-fold higher flux and 50-fold higher skin concentrations of clotrimazole as compared to human skin), even more than rat skin. In the case of the Skinethic HRE, we found similar concentrations of salicylic acid as in human skin and an approximately sevenfold higher flux. In contrast, the permeation of hydrophobic compounds through the epidermal layer was vastly higher than through split-thickness human skin (up to a factor of about 800). To conclude, currently available reconstituted skin models cannot be regarded as generally useful for in-vitro penetration studies.

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

BACKGROUND

Hydrocortisone protects against ischemia-reperfusion injury, reduces paracellular permeability for macromolecules, and is routinely applied in the prevention of interstitial edema. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, suggesting that the glycocalyx is a target for hydrocortisone action.

METHODS

Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer. Hydrocortisone was applied in a stress dose (10 microg/ml) before inducing 20 min of ischemia (37 degrees C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure, 70 cm H2O) with Krebs-Henseleit buffer or Krebs-Henseleit buffer plus 2 g% hydroxyethyl starch (130 kd). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion fixed to visualize the glycocalyx.

RESULTS

Ischemia-induced degradation of the glycocalyx enhanced coronary perfusion pressure (118.8 +/- 17.3 cm H2O) and increased vascular permeability (8 +/- 0.2 microl x min(-1) x cm H2O(-1) at baseline vs. 34 +/- 3.3 microl x min(-1) x cm H2O(-1) after reperfusion). Enzymatic digestion of the glycocalyx (heparinase) elicited similar effects. Hydrocortisone reduced postischemic oxidative stress, perfusion pressure (86.3 +/- 6.4 cm H2O), and transudate formation (11 +/- 0.6 microl x min(-1) x cm H2O(-1)). Applying colloid augmented this (70.6 +/- 5.6 cm H2O and 9 +/- 0.5 microl x min(-1) x cm H2O(-1)). Postischemic shedding of syndecan-1, heparan sulfate, and hyaluronan was inhibited by hydrocortisone, as was release of histamine from resident mast cells. Electron microscopy revealed a mostly intact glycocalyx after hydrocortisone treatment, but not after heparinase treatment.

CONCLUSIONS

Hydrocortisone preserves the endothelial glycocalyx, sustaining the vascular barrier and reducing interstitial edema. The effect of colloids suggests that prevention of postischemic rise in coronary resistance by hydrocortisone could also be based on alleviation of endothelial swelling. Stabilization of myocardial mast cells by hydrocortisone may account for the mitigated inflammatory affect of ischemia-reperfusion.

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

Classical pituitary apoplexy is a medical emergency and rapid replacement with hydrocortisone maybe life saving. It is a clinical syndrome characterized by the sudden onset of headache, vomiting, visual impairment and decreased consciousness caused by haemorrhage and/or infarction of the pituitary gland. It is associated with the sudden onset of headache accompanied or not by neurological symptoms involving the second, third, fourth and sixth cranial nerves. If diagnosed patients should be referred to a multidisciplinary team comprising, amongst others, a neurosurgeon and an endocrinologist. Apart from patients with worsening neurological symptoms in whom surgery is indicated, it is unclear currently for the majority of patients whether conservative or surgical management carries the best outcome. Post apoplexy, there needs to be careful monitoring for recurrence of tumour growth. It is suggested that further trials be carried out into the management of pituitary apoplexy to optimize treatment.

HeLa cells grow in a nutritionally complete synthetic medium (Ham's F12) supplemented with insulin, transferrin, hydrocortisone (aldosterone), fibroblast growth factor, and epidermal growth factor. This hormone-supplemented medium supports clonal growth, long-term cultivation, and a growth rate equal to that of serum-supplemented medium. The omission of any one of the five components results in less than maximal cell growth.

1 Dexamethasone and hydrocortisone induced the release of anti-phospholipase proteins into the peritoneal cavities of rats. 2 Adrenocorticotrophic hormone (ACTH) also releases these proteins in normal but not in adrenalectomized rats. 3 Peritoneal lavage proteins were separated by ion-exchange and size exclusion chromatography. The anti-phospholipase activity occurred in four separate fractions with the major component having an apparent mol. wt. of 40 k. 4 Column fractions containing these anti-phospholipase proteins had anti-inflammatory effects in the rat carrageenin pleurisy model whereas other fractions were inactive. 5 The proteins appear to be identical to macrocortin and lipomodulin, the 'second messengers' of glucocorticoid hormone action on the arachidonate system.

The effect of acute activation of the ACTH-adrenal axis on circulating testosterone (T) levels was investigated. Elevation of circulating cortisol resulting from insulin-induced hypoglycemia or the administration of hydrocortisone was followed by a rapid decrease in serum T levels, without accompanying changes in LH or PRL. These findings suggest that hypercortisolism of endogenous or exogenous sources suppresses T secretion by a direct action on the testis. This adrenal-testicular axis may have biological implications on the reproductive adaptation to stress.

Stromal colonies with fibroblastic morphology grown from rabbit marrow cells in culture supplemented with foetal calf serum. In this study the same marrow cells cultured with autologous rabbit plasma and hydrocortisone form colonies of a single lineage that express the adipocytic phenotype. A comparison of the potential for differentiation of cloned cell populations grown from fibroblastic and adipocytic colonies has been made using an in vivo diffusion chamber assay. The adipocytic colonies differentiated and grew to a limited size in medium with rabbit plasma and hydrocortisone, but attempts to isolate them and expand them in this medium failed. When the serum supplement was changed to foetal calf serum at day 10 the cells in the adipocytic colonies acquired a less differentiated morphology, there was a large increase in colony growth and cells were produced in sufficient numbers for the diffusion chamber assay. Thirty one fibroblastic colonies and twenty one adipocytic colonies were isolated either by limiting dilution or ring cloning and then expanded. Of these, eleven fibroblastic and eight adipocytic colonies provided enough cells (2 x 10(5) to 2 x 10(6] for implantation and culture in the chambers. Four of the eleven fibroblastic and three of the eight adipocytic colonies formed an osteogenic tissue in the chambers. It was concluded that cells that have differentiated in an adipocytic direction are able to revert to a more proliferative stage and subsequently to differentiate along the osteogenic pathway. Adipocytic and fibroblastic cells cultured in vitro from marrow have, with osteogenic cells, a common precursor in adult marrow.

The negative surface charge of human granulocytes was diminished after incubation with the chemotactic factors C5a, dialyzable transfer factor, and the enzymes kallikrein and plasminogen activator. No such change was observed after incubation with human IgG, albumin, horeseradish peroxidase, or a mixture of prekallikrein and plaminogen proactivator. Hydrocortisone inhibited the effect of C5a upon granulocyte surface charge and inhibited its chemotactic activity, suggesting that steroids act at the cell surface. The chemotactic inhibitors cholchicine and cytochalsin B had no effect upon granulocyte surface charge, consistent with their presumed effect upon microtubules and microfilaments, respectively. The data suggest that the decrease in cell surface charge may be a preerequiste for normal cell movement.

Tumor aromatase has been correlated with clinical response to treatment with aminoglutethimide in patients with estrogen receptor-positive advanced breast cancer. There was a significant positive relationship between aromatase status and likelihood of response to therapy, none of five patients with aromatase-negative tumors responding compared with 11 of 18 having aromatase-positive cancers. Measurement of in vitro aromatase in sequential biopsies of large primary tumors before and during treatment with aminoglutethimide-hydrocortisone showed a marked but paradoxical rise in activity following therapy. Assays of aromatase in adipose tissue from the different quadrants of mastectomy specimens from patients with breast cancer indicate that activity was always higher in quadrants associated with tumor as compared with non-involved quadrants. These results emphasize the importance of local estrogen synthesis within the breast in terms of both the natural history and behavior of breast cancers.

The electrophysiology of chemotactic factor interaction with cultured human macrophages was investigated with standard intracellular recording techniques. In initial studies, E. coli endotoxin-activated serum, added to cell cultures during intracellular recordings, caused membrane hyperpolarizations which were greater than 30 s in duration, 10-50 mV in amplitude, and associated with decreased membrane resistance. Control serum produced smaller hyperpolarizations lasting 10-20 s and 5-30 m V in amplitude. Endotoxin-activated human serum deficient in the third complement component (C3) did not produce hyperpolarizations unless the serum was reconstituted with C3 before activation. Fractionation of normal activated serum by molecular seive chromatography (G-75 Sephadex) indicated that only fractions that eluted with an estimated molecular weight of 12,500 produced membrane potential changes. The active material that was chemotactic for the macrophages was identified as the small molecular weight cleavage product of C5, C5a, by heat stability (30 min at 56 degrees C) and inactivation by goat antisera to human C5 but not C3. 17 percent of macrophages stimulated with C5a exhibited a biphasic response characterized by a small (2-6 mV), brief (1-10 s) depolarization associated with a decreased membrane resistance preceding the larger and prolonged hyperpolarizations. Magnesium-ethylene glycol bis[beta-aminoethyl ether]N,N'-tetraacetic acid (Mg [2.5 mM]-EGTA [5.0 mM]) blocked the C5a-evoked potential changes, whereas colchine (10(- 6)M) and cytochalasin B (3.0 mug/ml did not. Hydrocortisone sodium succinate (0.5 mg/ml) decreased the percentage of cells responding to C5a. In related studies, synthetic N-formyl methionyl peptide (f-met-leu-phe), which had chemotactic activity for cultured macrophages, produced similar membrane potential changes. Repeated exposure of macrophages to C5a or f- met-leu-phe resulted in desensitization to the same stimulus. Simultaneous photomicroscope and intracellular recording studies during macrophage stimulation with chemotactic factor demonstrated that the membrane potential changes preceded membrane spreading, ruffling, and pseudopod formation. These observations demonstrate that ion fluxes associated with membrane potential changes are early events in macrophage activation by chemotactic factors