Transforming growth factor-beta stimulation of lung fibroblast prostaglandin E2 production.

Transforming growth factor-beta (TGF beta) stimulated the production of total protein, collagen, and fibronectin by normal human lung fibroblasts. The stimulatory response was maximal at 100 pM TGF beta and reversed toward control at higher concentrations. Inhibition of fibroblast prostaglandin (PG) synthesis enhanced TGF beta-induced stimulation of total protein, collagen, and fibronectin production and reversed the negative slope of the dose-response curve at high concentrations of TGF beta. Determination of the steady-state levels of Types I and III procollagens and fibronectin mRNAs employing specific cDNA probes demonstrated that inhibition of fibroblast PG production increased the stimulatory effect of TGF beta on the levels of these transcripts. Exogenous PGE2 abrogated the stimulatory effects of TGF beta. These findings suggest that fibroblast stimulation by TGF beta may be down-regulated by endogenous PG synthesized in response to TGF beta. This notion was supported by the demonstration that TGF beta markedly stimulated fibroblast PGE2 production. These results indicate that TGF beta-induced stimulation of fibroblast PGE2 production may be an autoregulatory control mechanism to limit the effects of TGF beta on connective tissue protein synthesis

Interstitial lung disease in connective tissue diseases: evolving concepts of pathogenesis and management

Interstitial lung disease (ILD) is a challenging clinical entity associated with multiple connective tissue diseases, and is a significant cause of morbidity and mortality. Effective therapies for connective tissue disease-associated interstitial lung disease (CTD-ILD) are still lacking. Multidisciplinary clinics dedicated to the early diagnosis and improved management of patients with CTD-ILD are now being established. There is rapid progress in understanding and identifying the effector cells, the proinflammatory and profibrotic mediators, and the pathways involved in the pathogenesis of CTD-ILD. Serum biomarkers may provide new insights as risk factors for pulmonary fibrosis and as measures of disease progression. Despite these recent advances, the management of patients with CTD-ILD remains suboptimal. Further studies are therefore urgently needed to better understand these conditions, and to develop effective therapeutic interventions

The Impact of Disease-Modifying Therapy Access Barriers on People With Multiple Sclerosis: Mixed-Methods Study.

BACKGROUND: In the United States, people with relapsing-remitting multiple sclerosis (RRMS) can face difficulty accessing disease-modifying therapies (DMTs) because of insurance, pharmacy, or provider policies. These barriers have been associated with poor adherence and negative health outcomes. OBJECTIVE: The goals of this study were to describe the overall occurrence of difficulties and delays associated with gaining access to DMTs among people with RRMS, to assess DMT adherence during periods of reduced access, and to contextualize the patients\u27 journey from receipt of a prescription for DMT to obtaining and taking their medication when faced with access barriers. METHODS: We recruited US-based adults self-reporting RRMS from a Web-based health data-sharing social network, PatientsLikeMe. Individuals were invited to complete a Web-based survey if they reported a diagnosis of RRMS and were prescribed a DMT for MS. Follow-up phone interviews were conducted with 10 respondents who reported experiencing an MS-related relapse during the time they had experienced challenges accessing DMTs. RESULTS: Among 507 survey completers, nearly half were either currently experiencing an issue related to DMT assess or had difficulty accessing a DMT in the past (233/507, 46.0%). The most frequently reported reasons for access difficulty were authorization requirements by insurance companies (past issues: 78/182, 42.9%; current issues: 9/42, 21%) and high out-of-pocket costs (past issues: 54/182, 29.7%; current issues: 13/42, 31%). About half (20/39, 51%) of participants with current access issues and over a third (68/165, 41.2%) of those with past issues went without their medication until they could access their prescribed DMT. Relapses were reported during periods of reduced DMT access for almost half (56/118, 47.5%) of those with past issues and nearly half (22/45, 49%) of those with current issues. Resolving access issues involved multiple stakeholder agents often coordinated in a patient-led effort. Among those who had resolved issues, about half (57/119, 47.9%) reported that doctors or office staff were involved, under half (48/119, 40.3%) were involved themselves, and about a third (39/119, 32.8%) reported the drug manufacturer was involved in resolving the issue. Follow-up interviews revealed that the financial burden associated with obtaining a prescribed DMT led to nonadherence. Additionally, participants felt that DMT treatment delays and stress associated with obtaining the DMT triggered relapses or worsened their MS. CONCLUSIONS: This study expands current research by using a patient-centered, mixed-methods approach to describe barriers to MS treatment, the process to resolve barriers, and the perceived impact of treatment barriers on outcomes. Issues related to DMT access occur frequently, with individuals often serving as their own agents when navigating access difficulties to obtain their medication(s). Support for resolution of DMT access is needed to prevent undue stress and nonadherence

Elevated expression of type VII collagen in the skin of patients with systemic sclerosis. Regulation by transforming growth factor-beta.

A hallmark of systemic sclerosis (SSc) is the development of tissue fibrosis. Excessive production of several connective tissue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteoglycans, is a consistent finding in the skin of SSc patients. Type VII collagen is a major constituent of anchoring fibrils, present in the skin at the dermal-epidermal basement membrane zone. TGF-beta has been shown to upregulate the expression of the type VII collagen gene. In this study, we assessed the expression of type VII collagen and TGF-beta in the skin of patients with SSc. Indirect immunofluorescence showed an abundance of type VII collagen in the patients\u27 skin, including the dermis. Ultrastructural analysis of SSc skin revealed an abundance of fibrillar material, possibly representing type VII collagen. The increased expression of type VII collagen epitopes was accompanied by the elevated expression of immunodetectable TGF-beta 1 and TGF-beta 2. Dermal fibroblasts cultured from the affected individuals showed a statistically significant (P \u3c 0.02) increase in the expression of type VII collagen at the mRNA level, as detected by reverse transcription-PCR with a mutated cDNA as an internal standard, and increased deposition of the protein as assessed by indirect immunofluorescence. Thus, type VII collagen is abundantly present in SSc patients\u27 dermis, a location not characteristic of its normal distribution, and its aberrant expression may relate to the presence of TGF-beta in the same topographic distribution. The presence of type VII collagen in the dermis may contribute to the tightly bound and indurated appearance of the affected skin in SSc patients

Functional analysis of human alpha 1(I) procollagen gene promoter. Differential activity in collagen-producing and -nonproducing cells and response to transforming growth factor beta 1.

To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(I) procollagen 5\u27-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5\u27 end points from -804 to -174 bp. A further 5\u27 deletion to -84 bp caused a marked reduction in CAT activity. Cells transfected with plasmids containing longer 5\u27-flanking fragments of the alpha 1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the alpha 1(I) procollagen promoter was much lower in cells that do not normally express type I collagen (HeLa cells) compared with collagen-producing NIH/3T3 cells. The CAT activity of deletion constructs containing longer 5\u27 regions than -84 bp was increased by approximately 2-fold in NIH/3T3 cells treated with transforming growth factor beta 1 (TGF beta 1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the alpha 1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGF beta 1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGF beta 1 was specific for an Sp1-like sequence at positions -164 to -142 bp in the promoter. These results demonstrate that 1) there are both positive and negative cis-acting regulatory elements in the human alpha 1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and -nonproducing cells, 3) the alpha 1(I) procollagen promoter contains TGF beta 1-responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGF beta 1 caused marked stimulation of the DNA binding activity of a nuclear factor interacting with an Sp1-like binding site located within a region encompassing -164 to -142 bp of the alpha 1(I) procollagen promoter

Cations Affect the Rate of Gating Charge Recovery in Wild-type and W434F Shaker Channels through a Variety of Mechanisms

In this study we examine the effects of ionic conditions on the gating charge movement in the fast inactivation–removed wild-type Shaker channel and its W434F mutant. Our results show that various ionic conditions influence the rate at which gating charge returns during repolarization following a depolarizing pulse. These effects are realized through different mechanisms, which include the regulation of channel closing by occupying the cavity, the modulation of transitions into inactivated states, and effects on transitions between closed states via a direct interaction with the channel's gating charges. In generating these effects the cations act from the different binding sites within the pore. Ionic conditions, in which conducting wild-type channels close at different rates, do not significantly affect the rate of charge recovery upon repolarization. In these conditions, channel closing is fast enough not to be rate-limiting in the charge recovery process. In the permanently P-inactivated mutant channel, however, channel closing becomes the rate-limiting step, presumably due to weakened ion–ion interactions inside the pore and a slower intrinsic rate of gate closure. Thus, variations in closing rate induced by different ions are reflected as variations in the rate of charge recovery. In 115 mM internal Tris+ and external K+, Cs+, or Rb+, low inward permeation of these ions can be observed through the mutant channel. In these instances, channel closing becomes slower than in Tris+O//Tris+I solutions showing resemblance to the wild-type channel, where higher inward ionic fluxes also retard channel closing. Our data indicate that cations regulate the transition into the inactivated states from the external lock-in site and possibly the deep site. The direct action of barium on charge movement is probably exerted from the deep site, but this effect is not very significant for monovalent cations

Three-Tone Coherent Microwave Electromechanical Measurement of a Superfluid Helmholtz Resonator

We demonstrate electromechanical coupling between a superfluid mechanical mode and a microwave mode formed by a patterned microfluidic chip and a 3D cavity. The electric field of the chip-cavity microwave resonator can be used to both drive and detect the motion of a pure superflow Helmholtz mode, which is dictated by geometric confinement. The coupling is characterized using a coherent measurement technique developed for measuring weak couplings deep in the sideband unresolved regime. The technique is based on two-probe optomechanically induced transparency/amplification using amplitude modulation. Instead of measuring two probe tones separately, they are interfered to retain only a signal coherent with the mechanical motion. With this method, we measure a vacuum electromechanical coupling strength of $g_0 = 2\pi \times 23.3$ $\mathrm{\mu}$Hz, three orders of magnitude larger than previous superfluid electromechanical experiments.Comment: 13 pages, 6 figures, submitted to Appl. Phys. Lett., out for peer review, revte

Elevated Expression of the Genes for Transforming Growth Factor-β1 and Type VI Collagen in Diffuse Fasciitis Associated with the Eosinophilia-Myalgia Syndrome

Full-thickness skin biopsies obtained from four patients with rapidly progressive diffuse fasciitis associated with the Eosinophilia-Myalgia syndrome (EMS) were examined for the ex- pression of transforming growth factor-β1 (TGF-β1), type VI collagen, and fibronectin genes employing immunohistochemistry and in situ hybridizations. The immunohistochemical studies demonstrated increased deposition of TGF-β, type VI collagen, and fibronectin epitopes in the extracellular matrix of the fascia in comparison to the adjacent dermis in the same specimens. Increased levels of type VI collagen mRNA, as evidenced by positive in situ hybridization signals with an α2(VI) collagen cDNA, were also found in the fascia in comparison with the dermis. In situ hybridizations of affected fascia with a human sequence-specific TGF-β1 cDNA demonstrated numerous fibroblasts displaying positive hybridization signals indicative of high levels of transcripts for this cytokine. In contrast, no hybridization signal for TGF-β1 was detected in fibroblasts in the adjacent dermis. These findings suggest that TGF-β1 may play an important role in the development of the connective tissue alterations present in EMS-associated diffuse fasciitis

Stimulation of Type I Collagen Transcription in Human Skin Fibroblasts by TGF-β: Involvement of Smad 3

Transforming growth factor-β (TGF-β) stimulates the transcription of the α2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-β. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-β. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-β response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-β. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-β-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-β-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-β treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-β-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-β signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-β; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-β

Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice

Indoleamine 2,3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. In cultured cells, the induction of IDO leads to depletion of tryptophan and tryptophan starvation. Recent studies suggest that modulation of tryptophan concentration via IDO plays a fundamental role in innate immune responses. Induction of IDO by interferon-γ in macrophages and dendritic cells results in tryptophan depletion and suppresses the immune-mediated activation of fibroblasts and T, B, and natural killer cells. To assess the role of IDO in collagen-induced arthritis (CIA), a model of rheumatoid arthritis characterized by a primarily Th1-like immune response, activity of IDO was inhibited by 1-methyl-tryptophan (1-MT) in vivo. The results showed significantly increased incidence and severity of CIA in mice treated with 1-MT. Activity of IDO, as determined by measuring the levels of kynurenine/tryptophan ratio in the sera, was increased in the acute phase of arthritis and was higher in collagen-immunized mice that did not develop arthritis. Treatment with 1-MT resulted in an enhanced cellular and humoral immune response and a more dominant polarization to Th1 in mice with arthritis compared with vehicle-treated arthritic mice. The results demonstrated that development of CIA was associated with increased IDO activity and enhanced tryptophan catabolism in mice. Blocking IDO with 1-MT aggravated the severity of arthritis and enhanced the immune responses. These findings suggest that IDO may play an important and novel role in the negative feedback of CIA and possibly in the pathogenesis of rheumatoid arthritis