Inflammation dynamically regulates steroid hormone metabolism and action within macrophages in rheumatoid arthritis.

RATIONALE: In inflammatory diseases such as rheumatoid arthritis (RA), steroid metabolism is a central component mediating the actions of immuno-modulatory glucocorticoids and sex steroids. However, the regulation and function of cellular steroid metabolism within key leukocyte populations such as macrophages remain poorly defined. In this study, the inflammatory regulation of global steroid metabolism was assessed in RA macrophages. METHODS: Bulk RNA-seq data from RA synovial macrophages was used to assess transcripts encoding key enzymes in steroid metabolism and signalling. Changes in metabolism were assessed in synovial fluids, correlated to measures of disease activity and functionally validated in primary macrophage cultures. RESULTS: RNA-seq revealed a unique pattern of differentially expressed genes, including changes in genes encoding the enzymes 11ß-HSD1, SRD5A1, AKR1C2 and AKR1C3. These correlated with disease activity, favouring increased glucocorticoid and androgen levels. Synovial fluid 11ß-HSD1 activity correlated with local inflammatory mediators (TNFα, IL-6, IL-17), whilst 11ß-HSD1, SRD5A1 and AKR1C3 activity correlated with systemic measures of disease and patient pain (ESR, DAS28 ESR, global disease activity). Changes in enzyme activity were evident in inflammatory activated macrophages in vitro and revealed a novel androgen activating role for 11ß-HSD1. Together, increased glucocorticoids and androgens were able to suppress inflammation in macrophages and fibroblast-like-synoviocytes. CONCLUSIONS: This study underscores the significant increase in androgen and glucocorticoid activation within inflammatory polarized macrophages of the synovium, contributing to local suppression of inflammation. The diminished profile of inactive steroid precursors in postmenopausal women may contribute to disturbances in this process, leading to increased disease incidence and severity.

Dirofilariasis mouse models for heartworm preclinical research.

Introduction: Dirofilariasis, including heartworm disease, is a major emergent veterinary parasitic infection and a human zoonosis. Currently, experimental infections of cats and dogs are used in veterinary heartworm preclinical drug research. Methods: As a refined alternative in vivo heartworm preventative drug screen, we assessed lymphopenic mouse strains with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase of Dirofilaria immitis. Results: Non-obese diabetic (NOD) severe combined immunodeficiency (SCID)γc-/- (NSG and NXG) and recombination-activating gene (RAG)2-/-γc-/- mouse strains yielded viable D. immitis larvae at 2-4 weeks post-infection, including the use of different batches of D. immitis infectious larvae, different D. immitis isolates, and at different laboratories. Mice did not display any clinical signs associated with infection for up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, which is the natural site of this stage of heartworm in dogs. Compared with in vitro-propagated larvae at day 14, in vivo-derived larvae had completed the L4 molt, were significantly larger, and contained expanded Wolbachia endobacteria titres. We established an ex vivo L4 paralytic screening system whereby assays with moxidectin or levamisole highlighted discrepancies in relative drug sensitivities in comparison with in vitro-reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70%-90% in D. immitis L4 following 2- to 7-day oral in vivo exposures of NSG- or NXG-infected mice with doxycycline or the rapid-acting investigational drug, AWZ1066S. We validated NSG and NXG D. immitis mouse models as a filaricide screen by in vivo treatments with single injections of moxidectin, which mediated a 60%-88% reduction in L4 larvae at 14-28 days. Discussion: Future adoption of these mouse models will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround, and reduced costs and may simultaneously decrease the need for experimental cat or dog use.

Early Release Science of the exoplanet WASP-39b with JWST NIRSpec PRISM.

Transmission spectroscopy1-3 of exoplanets has revealed signatures of water vapour, aerosols and alkali metals in a few dozen exoplanet atmospheres4,5. However, these previous inferences with the Hubble and Spitzer Space Telescopes were hindered by the observations' relatively narrow wavelength range and spectral resolving power, which precluded the unambiguous identification of other chemical species-in particular the primary carbon-bearing molecules6,7. Here we report a broad-wavelength 0.5-5.5 µm atmospheric transmission spectrum of WASP-39b8, a 1,200 K, roughly Saturn-mass, Jupiter-radius exoplanet, measured with the JWST NIRSpec's PRISM mode9 as part of the JWST Transiting Exoplanet Community Early Release Science Team Program10-12. We robustly detect several chemical species at high significance, including Na (19σ), H2O (33σ), CO2 (28σ) and CO (7σ). The non-detection of CH4, combined with a strong CO2 feature, favours atmospheric models with a super-solar atmospheric metallicity. An unanticipated absorption feature at 4 µm is best explained by SO2 (2.7σ), which could be a tracer of atmospheric photochemistry. These observations demonstrate JWST's sensitivity to a rich diversity of exoplanet compositions and chemical processes.

Intravenous dexamethasone fails to prolong psoas compartment block when assessed by objective pinprick sensory testing: a prospective, randomised, dose-dependent, placebo-controlled equivalency trial.

BACKGROUND: Recent studies have concluded that i.v. dexamethasone can prolong the duration of peripheral nerve blockade. We hypothesized that a 4 mg dose would equally prolong the duration of psoas compartment blocks (PCBs) when compared with 8 mg, and that both doses would prolong the duration when compared with placebo. METHODS: This was a prospective, randomized, placebo-controlled, dose-dependent, equivalency trial with 115 patients undergoing total hip arthroplasty. The patients received a PCB. Subsequently, 15 patients received i.v. normal saline (placebo), 50 patients received i.v. dexamethasone 4 mg, and 50 patients received i.v. dexamethasone 8 mg. The primary outcome was the duration in hours of PCB, determined by serial pinprick assessments. Secondary outcomes included pain scores, time to first analgesic, and opioid consumption. An intention-to-treat-analysis (ITA) and per-protocol analysis (PPA) were performed. RESULTS: The ITA showed that block duration in the 4 and 8 mg groups was equivalent [mean (standard deviation), 18.5 h (8.0) vs 18.1 h (7.1)]. However, neither group differed from placebo [19.6 h (6.7), (4 mg vs placebo), P=0.97; (8 mg vs placebo), P=0.77)]. Postoperative pain scores and opioid consumption were not different between groups. Time to first analgesic was not different between the 4 and 8 mg groups, or the 4 mg and placebo groups. The 8 mg group, however, had a longer time to first analgesic (median of 533 vs 432 min, P=0.047) when compared with placebo, although the significance was not observed in the PPA (P=0.058). CONCLUSIONS: I.V. dexamethasone did not prolong PCB when duration was objectively assessed, or decrease total opioid consumption. However, dexamethasone 8 mg prolonged the time to first analgesic. CLINICAL TRIAL REGISTRATION: NCT 02464176.

Impact of physical activity on fatigue and quality of life in people with advanced lung cancer: a randomized controlled trial.

BACKGROUND: Physical activity (PA) improves fatigue and quality of life (QOL) in cancer survivors. Our aim was to assess whether a 2-month PA intervention improves fatigue and QOL for people with advanced lung cancer. METHODS: Participants with advanced lung cancer, Eastern Cooperative Oncology Group performance status (PS) ≤2, >6 months life expectancy, and ability to complete six-min walk test, were stratified (disease stage, PS 0-1 versus 2, centre) and randomized (1:1) in an open-label study to usual care (UC) (nutrition and PA education materials) or experimental intervention (EX): UC plus 2-month supervised weekly PA and behaviour change sessions. Assessments occurred at baseline, 2, 4, and 6 months. The primary endpoint was fatigue [Functional Assessment of Cancer Therapy-Fatigue (FACT-F) questionnaire] at 2 months. The study was designed to detect a difference in mean FACT-F subscale score of 6. Analysis was intention-to-treat using linear mixed models. RESULTS: We recruited 112 patients: 56 (50.4%) were randomized to EX, 55(49.5%) to UC; 1 ineligible. Male 55%; median age 64 years (34-80); 106 (96%) non-small cell lung cancer; 106 (95.5%) stage IV. At 2, 4 and 6 months, 90, 73 and 62 participants were assessed, respectively, with no difference in attrition between groups. There were no significant differences in fatigue between the groups at 2, 4 or 6 months: mean scores at 2 months EX 37.5, UC 36.4 (difference 1.2, 95% CI - 3.5, 5.8, P = 0.62). There were no significant differences in QOL, symptoms, physical or functional status, or survival. CONCLUSIONS: Adherence to the intervention was good but the intervention group did not increase their PA enough compared to the control group, and no difference was seen in fatigue or QOL. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry No. ACTRN12609000971235.

Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression.

OBJECTIVES: Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. METHODS: The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. RESULTS: TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. CONCLUSIONS: The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism.

Schistosome infection is associated with enhanced whole-blood IL-10 secretion in response to cercarial excretory/secretory products.

Infection of the human host by schistosome parasites follows exposure of skin to free-swimming cercariae and is aided by the release of excretory/secretory (E/S) material, which is rich in proteases and glycoconjugates. This material provides the initial stimulus to cells of the innate immune system. The study presented here is the first to examine human innate/early immune responsiveness to cercarial E/S in subjects from an area co-endemic for Schistosoma mansoni and S. haematobium. We report that in infected participants, stimulation of whole-blood cultures with cercarial E/S material (termed 0-3 hRP) caused the early (within 24 h) release of greater quantities of regulatory IL-10, compared with uninfected controls. Elevated levels of IL-10 but not pro-inflammatory TNFα or IL-8 were most evident in participants co-infected with S. mansoni and S. haematobium and were accompanied by a higher 0-3 h RP-specific IL-10: TNFα ratio. We also report that glycosylated components within 0-3 h RP appear to be important factors in the stimulation of IL-8, TNFα and IL-10 production by whole-blood cells.

Granulocytes in helminth infection -- who is calling the shots?

Helminths are parasitic organisms that can be broadly described as "worms" due to their elongated body plan, but which otherwise differ in shape, development, migratory routes and the predilection site of the adults and larvae. They are divided into three major groups: trematodes (flukes), which are leaf-shaped, hermaphroditic (except for blood flukes) flatworms with oral and ventral suckers; cestodes (tapeworms), which are segmented, hermaphroditic flatworms that inhabit the intestinal lumen; and nematodes (roundworms), which are dioecious, cylindrical parasites that inhabit intestinal and peripheral tissue sites. Helminths exhibit a sublime co-evolution with the host's immune system that has enabled them to successfully colonize almost all multicellular species present in every geographical environment, including over two billion humans. In the face of this challenge, the host immune system has evolved to strike a delicate balance between attempts to neutralize the infectious assault versus limitation of damage to host tissues. Among the most important cell types during helminthic invasion are granulocytes: eosinophils, neutrophils and basophils. Depending on the specific context, these leukocytes may have pivotal roles in host protection, immunopathology, or facilitation of helminth establishment. This review provides an overview of the function of granulocytes in helminthic infections.

Atopic dermatitis. A clinical challenge.

Atopic dermatitis has a significant impact on both the pediatric and adult population worldwide, which has triggered extensive research on the topic. However, various limitations have created difficulties both in making accurate diagnoses and effectively managing atopic dermatitis patients. This review summarizes the current knowledge in the field, providing an overview of the pathophysiology, disease progression, clinical presentation, and diagnosis and treatment of atopic dermatitis.

Cytokine response profiles predict species-specific infection patterns in human GI nematodes.

This study investigated associations between pre-treatment cytokine expression and infection patterns, before and after de-worming, in humans exposed to two gastrointestinal nematode species. Quantitative measures of Ascaris lumbricoides and Trichuris trichiura infection (based on faecal egg counts) were estimated immediately before and 8-9 months after treatment in a Cameroonian population. Whole blood cytokine responses to parasite-derived antigens were assayed immediately pre-treatment. An overall measure of the tendency towards species-specific infection (increasing with A. lumbricoides faecal egg counts and decreasing with T. trichiura faecal egg counts) was significantly positively related to IL-10 levels in older (14-57 year) hosts. There was a significant negative influence of IL-5 on reinfection probability in T. trichiura but not A. lumbricoides. This effect coincided with reduced reinfection success in T. trichiura compared to A. lumbricoides. T(H)2 cytokine expression by younger hosts (4-13 year) was negatively associated with contemporary A. lumbricoides faecal egg counts before treatment. Following treatment, the pre-treatment T(H)2 cytokine expression data for younger hosts (now reflecting responsiveness 8-9 months in the past) were negatively associated with T. trichiura faecal egg counts. Taken together, these observations suggest a successional interaction between T(H)2-driven immune responses and species infection over time. However, any differential effects of the measured immune responses on species-specific recruitment, maturation and mortality were superimposed upon (and outweighed by) the effects of other factors favouring coinfection.

Microrough implant surface topographies increase osteogenesis by reducing osteoclast formation and activity.

Titanium implant surfaces with rough microtopographies exhibit increased pullout strength in vivo suggesting increased bone-to-implant contact. This is supported by in vitro studies showing that as surface microroughness increases, osteoblast proliferation decreases whereas differentiation increases. Differentiation is further enhanced on microrough surfaces by factors stimulating osteogenesis including 1alpha,25(OH)2D3. Levels of PGE2 and TGF-beta1 are increased in cultures grown on rough microtopographies; this surface effect is enhanced synergistically by 1alpha,25(OH)2D3-treatment. PGE2 and TGF-beta1 regulate osteoclasts as well as osteoblasts, suggesting that surface microtopography may modulate release of other factors from osteoblasts that regulate osteoclasts. To test this hypothesis, we examined the effects of substrate microarchitecture on production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL), which have been identified as a key regulatory system of bone remodeling. We also examined the production of 1alpha,25(OH)2D3, which regulates osteoblast differentiation and osteoclastogenesis. MG63 osteoblast-like cells were grown on either tissue culture plastic or titanium disks of different surface microtopographies: PT (Ra < 0.2 microm), SLA (Ra = 4 microm), and TPS (Ra = 5 microm). At confluence, cultures were treated for 24 h with 0, 10(-8) M or 10(-7) M 1alpha,25(OH)2D3. RANKL and OPG were determined at the transcriptional level by RT-PCR and real time PCR and soluble RANKL, OPG and 1alpha,25(OH)2D3 in the conditioned media were measured using immunoassay kits. Cell number was reduced on SLA and TPS surfaces and 1alpha,25(OH)2D3 caused further decreases. OPG mRNA levels increased on rougher surfaces and 1alpha,25(OH)2D3 treatment caused a further synergistic increase. While the cells expressed RANKL mRNA, levels were low and independent of surface microtopography. OPG protein was greater when cells were grown on SLA and TPS. 1alpha,25(OH)2D3 increased OPG by 50% on the smooth Ti surface but on SLA, 10(-8) M 1alpha,25(OH)2D3 caused a 100% increase and 10(-7) M 1alpha,25(OH)2D3 increased OPG by 200%. On TPS 10(-7) M 1alpha,25(OH)2D3 increased OPG 350%. Soluble RANKL was not detected in the conditioned media of any of the cultures. 1alpha,25(OH)2D3 was produced endogenously and levels were positively correlated with surface roughness. Thus, on surfaces with rough microtopographies, osteoblasts secrete factors that enhance osteoblast differentiation while decreasing osteoclast formation and activity.

Recreational ecstasy/MDMA and other drug users from the UK and Italy: psychiatric symptoms and psychobiological problems.

RATIONALE: Recreational drug use is increasingly widespread amongst young people, but there are concerns that psychoactive drugs may be associated with psychiatric symptoms or psychobiological problems. OBJECTIVES: To assess the psychiatric health status of a large, non-clinical sample of young adults from Italy and the UK, and relate it to their use of ecstasy/MDMA and other recreational drugs. METHODS: The UEL Recreational Drug Use Questionnaire was completed by 768 young people (mean age 21.7 years) from four European cities. The subjects comprised 150 non-drug users, 185 alcohol/tobacco users, 97 cannabis and alcohol/tobacco users, 102 illicit polydrug but not ecstasy users, 115 light (<20 times) ecstasy polydrug users, and 119 heavy (>20 times) ecstasy polydrug users. The unpaid volunteers completed the SCL-90 self-rating inventory for psychiatric symptoms when off drug, with 30 additional questions covering positive moods and life experiences. RESULTS: Heavy ecstasy polydrug users reported significantly higher scores than non-drug users on several SCL-90 factors, including phobic anxiety, obsessive-compulsive behaviour, anxiety, psychoticism, somatisation, and significantly higher rates of 'loss of sex interest or pleasure'. Self-rated symptom scores increased in line with greater drug use, so that polydrug users who had never taken ecstasy also reported a variety of psychobiological impairments. In contrast, positive moods and life experiences were broadly similar across subgroups. CONCLUSIONS: The recreational use of ecstasy/MDMA is associated with a range of psychiatric symptoms and psychobiological problems. However, these problems are not specific to ecstasy users but are also evident in other recreational polydrug users.

Lipid peroxidation is associated with the inhibitory action of all-trans-retinoic acid on mammary cell transformation.

BACKGROUND: Retinoids are effective in reducing transformation of various cell types; however the role of lipid peroxidation has not been studied in this regard. MATERIALS AND METHODS: Retinoic acid (RA) and retinol were tested on several SV-40 large-T-antigen transformed bovine mammary fibroblast (MFB) lines that form foci on plastic with long-term culture. RESULTS: Dose response studies revealed that RA at 10(-6) M was the most potent dose in delaying the onset of foci formation and reducing total foci number. RA was always more effective than retinol. Addition of RA (10(-6) M) to MFB cells increased lipid peroxide (LPO) concentrations by approximately three-fold relative to untreated MFB cells or to RA or control treated normal mammary fibroblasts. The lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), acted synergistically with RA to increase LPO and cell death in MFB cells. The combination of the cyclooxygenase inhibitor, indomethacin, at 10(-4) M with 10(-6) M RA lowered MFB fibroblast cell numbers when compared to fibroblasts cultured singly with either RA or indomethacin. CONCLUSIONS: These data indicate that an increase in lipid peroxidation occurs specifically in tumor cells treated with RA and this may play a role in RA-mediated suppression of cellular transformation in the mammary gland. Additionally, eicosanoid inhibitors may have an additive or synergistic effect with RA on the inhibition of mammary tumor cell transformation and proliferation.

Quiescent mammary epithelial cells have reduced connexin43 but maintain a high level of gap junction intercellular communication.

Gap junctions have been implicated in growth control, but it remains unclear whether cells that enter a quiescent state continue to express connexins and maintain a high level of gap junction intercellular communication (GJIC). To this end, MAC-T cells, a bovine mammary epithelial cell line, were serum starved for 48 h to induce a quiescent (G0) state. In quiescent cells, [3H]thymidine incorporation was reduced by 97.3% from serum-fed controls. Western blotting in conjunction with Phosphorlmager analysis revealed up to a 20-fold decrease in the expression of the gap junction protein connexin43 (Cx43 or alpha 1) and a shift toward the unphosphorylated form in quiescent cells. However, cell-to-cell transfer of the gap junction-permeable fluorescent tracer, Lucifer yellow, was only moderately reduced in quiescent cells. In control cells, Cx43 was predominantly perinuclear, although it was also present at sites of cell-cell apposition. In quiescent cells, intracellular labeling for Cx43 decreased without a corresponding reduction at areas of cell-cell contact. Recovery from serum deprivation resulted in increased thymidine incorporation that corresponded with an elevation in Cx43 protein expression and phosphorylation. In parallel studies, MAC-T cells were also induced to enter a quiescent state through contact inhibition. Despite a 20-fold reduction in 5-bromo-2'-deoxyuridine and a substantial reduction in intracellular Cx43, contact inhibited MAC-T cells also maintained gap junctions and GJIC. These experiments demonstrate that the maintenance of dye coupling in quiescent mammary cells is correlated with a redistribution of intracellular stores of Cx43.

Identification of CXCR4 domains that support coreceptor and chemokine receptor functions.

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.

Deficient epithelial-fibroblast heterocellular gap junction communication can be overcome by co-culture with an intermediate cell type but not by E-cadherin transgene expression.

Epithelial, fibroblast and intermediate cell lines were employed to examine the mechanism(s) essential for heterocellular gap junction intercellular communication in vitro. These cell lines were characterized extensively for cell type based on morphology, intermediate cytoskeletal proteins, cell adhesion molecules and their associated proteins, tight junction proteins as well as functional differentiation. All cell types expressed connexin43 and were dye-coupled in homocellular culture. Epithelial and intermediate cells or fibroblasts and intermediate cells readily assembled heterocellular connexin43-positive gap junction plaques when co-cultured, while gap junction plaques in mixed cultures of epithelial cells and fibroblasts were rare. Dye microinjection studies were used to show that there was little gap junction intercellular communication between epithelial cells and fibroblasts. However, intermediate cells were able to communicate with epithelial cells and, to a lesser extent, fibroblasts and could transfer dye to both epithelial cells and fibroblasts when all three cell types were cultured together. Fibroblasts that were stably transfected with a cDNA encoding E-cadherin had a greater tendency to aggregate and exhibited a more epithelial-like phenotype but heterocellular gap junction intercellular communication with epithelial cells, which endogenously express E-cadherin, was not enhanced. These results suggest that mutual expression of E-cadherin is insufficient to stimulate gap junction formation between epithelial cells and fibroblasts. Moreover, our results also demonstrate that communication gaps between epithelial cells and fibroblasts can be bridged by intermediate cells, a process that may be important in mammary gland development, growth, differentiation and cancer.

Regulation of IGF binding protein synthesis by a bovine mammary epithelial cell line.

IGF-I has been proposed as a key regulator of mammary epithelial cell (MEC) growth and differentiation. As IGF-I bioactivity is modulated by specific, high-affinity binding proteins (IGFBP), the forms of IGFBP that are secreted by the bovine MEC line, MAC-T, were identified. Media conditioned by MAC-T cells contained four forms of IGFBP that were identified, by western blotting with specific antibodies, as IGFBP-2, -3, -4 and -6. The amounts of IGFBP-3 in conditioned media were relatively low under basal conditions when analyzed by ligand blotting with 125I-IGF-II, but were increased dramatically relative to serum-free controls by exposure to IGF-I (100 ng/ml) or IGF-II (100 ng/ml) for 24 h. These increases in IGFBP-3 protein corresponded with dose-dependent increases in IGFBP-3 mRNA, with IGF-II eliciting a smaller response than was elicited by IGF-I at each concentration. Leu-IGF-I, which has reduced affinity for the IGF-I receptor but normal affinity for IGFBPs, failed to increase IGFBP-3 protein and mRNA levels, whereas B-chain IGF-I (normal affinity for the receptor but reduced affinity for IGFBPs) elicited the response, thus implying an IGF-I receptor-mediated event. Time-course studies indicated that IGFBP-3 mRNA was increased fourfold by 3 h of IGF-I treatment, with maximal increases of eightfold above serum-free controls observed between 8 and 13 h of treatment. By 24 h of treatment, IGFBP-3 mRNA levels had declined and were approximately threefold above controls in cells exposed to IGF-I. Amounts of messenger RNA of IGFBP-6 and IGFBP-2 were not increased by IGF treatment. However, retinoic acid (10(-6) M) stimulated both IGFBP-2 and IGFBP-6 protein and mRNA levels, but it decreased IGFBP-3 mRNA levels relative to controls. The combination of retinoic acid plus IGF-I had no additional effect on IGFBP-6 or -2 above that observed with retinoic acid alone, whereas IGF-I together with retinoic acid attenuated the decrease in IGFBP-3 observed with retinoic acid alone. Protein kinase A-mediated pathways were also shown to alter IGFBP synthesis. Forskolin, which increases cAMP, increased IGFBP-3 protein and mRNA levels. The combination of IGF-I plus forskolin resulted in greater increases in both protein and mRNA than were observed with either treatment alone. In contrast, forskolin decreased IGFBP-6 mRNA relative to controls, but had no effect on IGFBP-2. The decrease in IGFBP-6 was less marked when cells were treated with a combination of IGF-I and forskolin. Forskolin had no effect on IGFBP-2 mRNA levels. In summary, the ability of IGF-I specifically to regulate IGFBP-3 synthesis represents a mechanism whereby IGF-I may regulate its own bioactivity. In addition, the differential regulation of IGFBP-2, -3 and -6 by retinoic acid (which inhibits proliferation) and IGF-I (which stimulates proliferation) suggests that these forms of IGFBP have different roles in regulating mammary epithelial cell physiology.