Removal of hydrophilic, hydrophobic, and charged xenobiotic organic compounds from greywater using green wall media.

Green walls offer a novel on-site approach for greywater treatment and reuse in densely build urban environments. However, they need to be engineered for effective removal of a wide range of emerging contaminants such as xenobiotic organic compounds (XOCs), which may be present in greywater due to extensive use of personal care products and household chemicals. This study used laboratory column design and batch experiments to investigate the performance of three lightweight green wall media (coco coir, zeolite, and perlite) and their mixture in three different combinations for the removal of twelve XOCs, covering wide range of hydrophilic, hydrophobic, and charged pollutants in greywater. The experiments were designed to assess the removal of targeted XOCs under different operational condition (i.e., hydraulic loading, infiltration rate, drying) and uncover the dominant mechanisms of their removal. Results showed excellent removal (>90%) of all XOCs in coco coir and media mix columns at the start of the experiment (i.e., fresh media and initial 2 pore volume (PV) of greywater dosing). The removal of highly hydrophobic and positively charged XOCs remained high (>90%) under all operational conditions, while hydrophilic and negatively charged XOCs exhibited significant reduction in removal after 25 PV and 50 PV, possibly due to their low adsorption affinity and electrostatic repulsion from negatively charged media. The effect of infiltration rate on the removal of XOCs was not significant; however, higher removal was achieved after 2-weeks of drying in coco coir and media mix columns. The dominant removal mechanism for most XOCs was found to be adsorption, however, a few hydrophilic XOCs (i.e., acetaminophen and atrazine) exhibited both adsorption and biodegradation removal processes. While findings showed promising prospects of unvegetated media for removing XOCs from greywater, long term studies on vegetated green wall systems are needed to understand any synergetic contribution of plants and media in removing these XOCs.

Assembly of extracellular matrix.

A great challenge in understanding how different extracellular matrices assemble is to sort through the vast number of possible interactions between and among matrix molecules. The most profound insights are likely to come from patients with defined defects of matrix molecules and the use of transgenic mice or other experimental technologies that mimic the complexity of the human system.

Chemical monitoring strategy for the assessment of advanced water treatment plant performance.

A pilot-scale plant was employed to validate the performance of a proposed full-scale advanced water treatment plant (AWTP) in Sydney, Australia. The primary aim of this study was to develop a chemical monitoring program that can demonstrate proper plant operation resulting in the removal of priority chemical constituents in the product water. The feed water quality to the pilot plant was tertiary-treated effluent from a wastewater treatment plant. The unit processes of the AWTP were comprised of an integrated membrane system (ultrafiltration, reverse osmosis) followed by final chlorination generating a water quality that does not present a source of human or environmental health concern. The chemical monitoring program was undertaken over 6 weeks during pilot plant operation and involved the quantitative analysis of pharmaceuticals and personal care products, steroidal hormones, industrial chemicals, pesticides, N-nitrosamines and halomethanes. The first phase consisted of baseline monitoring of target compounds to quantify influent concentrations in feed waters to the plant. This was followed by a period of validation monitoring utilising indicator chemicals and surrogate measures suitable to assess proper process performance at various stages of the AWTP. This effort was supported by challenge testing experiments to further validate removal of a series of indicator chemicals by reverse osmosis. This pilot-scale study demonstrated a simplified analytical approach that can be employed to assure proper operation of advanced water treatment processes and the absence of trace organic chemicals.

Molecular cloning of a murine fibronectin receptor and its expression during inflammation. Expression of VLA-5 is increased in activated peritoneal macrophages in a manner discordant from major histocompatibility complex class II.

Human fibronectin receptor (VLA-5) alpha and beta chain probes were used to identify their mouse homologues in a thioglycollate-elicited peritoneal exudate cell cDNA library. Sequence analysis of both alpha and beta chain-related murine clones revealed approximately 90% homology to their human counterparts by both nucleotide and derived amino acid sequence comparisons. Detectable alpha chain transcripts were seen predominantly in total RNA of peritoneal macrophages. beta chain expression, however, was detected at higher levels in lung, heart, brain, and kidney, suggesting the presence of a large murine VLA family similar to the human family. Analysis of levels of expression comparing resting peritoneal macrophages with macrophages elicited using inflammatory stimuli indicated that alpha chain message and surface VLA-5 expression were significantly increased using thioglycollate or Listeria monocytogenes as stimuli to elicit cells. Interestingly, beta chain message was unaffected by these inflammatory stimuli, suggesting that VLA-5 expression is regulated by VLA-5 alpha chain message levels. These results indicate that macrophage VLA-5 expression can be modulated in vivo and may provide an important mechanism by which macrophages are recruited to or adhere to fibronectin in inflammatory foci.

Quantifying human exposure to contaminants for multiple-barrier water reuse systems.

Reliance upon advanced water treatment processes to provide safe drinking water from relatively compromised sources is rapidly increasing in Australia and other parts of the world. Advanced treatment processes such as reverse osmosis have the ability to provide very effective treatment for a wide range of chemicals when operated under optimal conditions. However, techniques are required to comprehensively validate the performance of these treatment processes in the field. This paper provides a discussion and demonstration of some effective statistical techniques for the assessment and description of advanced water treatment plant performance. New data is provided, focusing on disinfection byproducts including trihalomethanes and N-nitrosamines from a recent comprehensive quantitative exposure assessment for an advanced water recycling scheme in Australia.

Metabonomics and the Gut Microbiome Associated With Primary Response to Anti-TNF Therapy in Crohn's Disease.

BACKGROUND AND AIMS: Anti-tumour necrosis factor [anti-TNF] therapy is indicated for treatment of moderate to severe inflammatory bowel disease [IBD], but has a primary non-response rate of around 30%. We aim to use metabonomic and metataxonomic profiling to identify predictive biomarkers of anti-TNF response in Crohn's disease. METHODS: Patients with luminal Crohn's disease, commencing anti-TNF therapy, were recruited with urine, faeces, and serum samples being collected at baseline and 3-monthly. Primary response was defined according to a combination of clinical and objective markers of inflammation. Samples were measured using three UPLC-MS assays: lipid, bile acid, and Hydrophillic Interaction Liquid Chromatography [HILIC] profiling with 16S rRNA gene sequencing of faeces. RESULTS: Samples were collected from 76 Crohn's disease patients who were anti-TNF naïve and from 13 healthy controls. There were 11 responders, 37 non-responders, and 28 partial responders in anti-TNF-treated Crohn's patients. Histidine and cysteine were identified as biomarkers of response from polar metabolite profiling [HILIC] of serum and urine. Lipid profiling of serum and faeces found phosphocholines, ceramides, sphingomyelins, and triglycerides, and bile acid profiling identified primary bile acids to be associated with non-response to anti-TNF therapy, with higher levels of phase 2 conjugates in non-responders. Receiver operating curves for treatment response demonstrated 0.94 +/ -0.10 [faecal lipid], 0.81 +/- 0.17 [faecal bile acid], and 0.74 +/- 0.15 [serum bile acid] predictive ability for anti-TNF response in Crohn's disease. CONCLUSIONS: This prospective, longitudinal cohort study of metabonomic and 16S rRNA gene sequencing analysis demonstrates that a range of metabolic biomarkers involving lipid, bile acid, and amino acid pathways may contribute to prediction of response to anti-TNF therapy in Crohn's disease. PODCAST: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.

A randomised controlled study shows supplementation of overweight and obese adults with lactobacilli and bifidobacteria reduces bodyweight and improves well-being.

In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25-34.9 kg/m2 received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p < 0.0001), BMI (0.045 kg/m2, p < 0.0001), WC (0.94 cm, p < 0.0001) and WtHR (0.006, p < 0.0001) were in favour of the probiotic. Stratification identified greater body weight reductions in overweight subjects (1.88%, p < 0.0001) and in females (1.62%, p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (-2.5%, p < 0.0001) alongside a significant between group reduction in small dense LDL-cholesterol (0.2 mmol/L, p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p < 0.0001) were recorded for the probiotic group. No adverse events were recorded. Six months supplementation with Lab4P probiotic resulted in significant weight reduction and improved small dense low-density lipoprotein-cholesterol (sdLDL-C) profiles, QoL and URTI incidence outcomes in overweight/obese individuals.

Specific binding of fibronectin--antifibronectin immune complexes to procollagen: a new pitfall in immunostaining.

We observed intense intracellular immunofluorescence of rat lung fibroblasts stained with hybridoma culture supernatant containing monoclonal antibodies to human plasma fibronectin, but no pericellular matrix staining. Immunoprecipitation and absorption experiments revealed that this intracellular staining by hybridoma-conditioned medium was due to binding of fibronectin-antifibronectin immune complexes via the fibronectin to intracellular procollagen. The anomalous staining patterns we encountered were not revealed by the usual controls for immunohistochemical specificity, and also occurred in rat tissue sections. This general phenomena--binding of serum antigens present in hybridoma medium to cellular components--could in principle result in artifactual staining with monoclonal antibodies to other serum components, so investigators using monoclonal antibodies should be aware of this new artifact. Our results also demonstrate that fibronectin binds specifically to native procollagen. Monoclonal antibodies may be useful for studying fibronectin-procollagen and other macromolecular interactions.

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix.

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding.

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab')(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.

The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices.

Cells interact with extracellular fibronectin (FN) via adhesive fibronectin receptors (FNRs) that are members of the very late antigens (VLAs) subgroup of the integrin family. In stationary fibroblasts, the FNR is highly organized and distributed identically to extracellular FN fibrils. However, in highly migratory neural crest cells and embryonic somatic fibroblasts, this organization is lost and the FNR appears diffuse. Similarly, oncogenic transformation typically leads to disorganization of the FN receptor and loss of matrix FN. Two models can account for these observations. First, the FN matrix may organize the FN receptor at extracellular matrix contacts on the cell surface. Motile cells not depositing FN matrices thus lack organized receptors. Alternatively, as the FNR is required for optimal FN matrix assembly, (McDonald, J. A., B. J. Quade, T. J. Broekelmann, R. LaChance, K. Forseman, K. Hasegawa, and S. Akiyama. 1987. J. Biol. Chem. 272:2957-2967; Roman, J. R. M. LaChance, T. J. Broekelmann, C. J. R. Kennedy, E. A. Wayner, W. G. Carter, J. A. McDonald. 1989. J. Cell Biol. 108:2529-2543) and has putative cytoskeletal links, it could be organized from within the cell helping to position newly forming FN fibrils. To study this question, we developed peptide antibodies specifically recognizing the alpha 5 subunit of the FNR. Using these antibodies, we examined the organization of FN and of the FNR in normal, matrix assembly inhibited, and SV40-transformed human fibroblasts. On FN-coated substrates, the FNR is found in focal contacts rather than diffusely on the basal cell surface, suggesting FNR interaction with intracellular components. However, when FN fibrils are deposited, the FNR is co-distributed with these fibrils. Preventing FN matrix assembly prevents organization of the FNR. Moreover, when fibroblasts with well established FN matrices and co-distributed FNR are incubated briefly with monoclonal antibodies that block FNR binding to FN, the FNR is no longer co-distributed with the FN matrix. Thus, the FN receptor is organized in fibrils on the cell surface in response to extracellular FN. Because exogenous FN restores a FN matrix and receptor organization to SV40-transformed cells, the diffuse FN receptor phenotype appears to be related to loss of the FN matrix rather than to impaired FNR function. These results explain diffusely distributed FNRs in migratory neural crest and embryonic fibroblasts lacking well organized FN matrices and emphasize the existence of separate but related systems controlling FN deposition and recognition by receptor-armed cells.

Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin.

Using mAb technology (Wayner, E. A., W. G. Carter, R. Piotrowicz, and T. J. Kunicki. 1988. J. Cell Biol. 107:1881-1891), we have identified a new fibronectin receptor that is identical to the integrin receptor alpha 4 beta 1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the alpha 4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IIICS for alpha 4 beta 1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to alpha 4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-gly-asp) adhesion sequence. mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, alpha 5 beta 1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both alpha 4 beta 1 and alpha 5 beta 1 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts. In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) alpha 5 beta 1 is the receptor for the RGD containing cell adhesion domain, and (b) alpha 4 beta 1 is the receptor for a carboxy-terminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that alpha 4 beta 1 is the receptor for this adhesion site.

Lesch-Nyhan syndrome: altered kinetic properties of mutant enzyme.

Hypoxanthine-guanine phosphoribosyltransferase is virtually inactive in erythrocytes from patients with the classical Lesch-Nyhan syndrome. In one such patient, activity of this enzyme ranged from 8 to 34 percent of normal in erythrocytes when assayed with a very high concentration of magnesium 5-phosphoribosyl-1-pyrophosphate. In addition, the mutant enzyme exhibited sigmoidal kinetics with this substrate as well as an increased Michaelis constant for both guanine and hypoxanthine. These findings provide the first evidence for genetic heterogeneity within the group of patients with the Lesch-Nyhan syndrome.

Orality, preference behavior, and reinforcement value of nonfood object in monkeys with orbital frontal lesions.

Monkeys with orbital frontal ablation, compared with sham-operated controls, showed enhancement of oral tendencies toward nonfood items. Further, unlike the controls, they persistently performed an instrumental response for one of these nonfood items. On the other hand, the lesioned monkeys did not show altered preferences for food versus nonfood items. These findings suggest that reinforcement value and preferential ordering are dissociated by orbital frontal ablation.

Fibronectin mediates adherence of rat alveolar type II epithelial cells via the fibroblastic cell-attachment domain.

The lung alveolar surface is composed of types I and II epithelial cells. Extremely attenuated type I cells cover 90% of the surface and are prone to necrosis during acute lung injury. After denudation of type I cells, the alveolar epithelium is restored by proliferation of type II cells. During reepithelialization in vivo the type II cells have been observed to reorganize on an extracellular matrix that contains fibronectin. We thus sought to determine whether type II cells would adhere to purified fibronectin. Adherence assays of primary rat type II cells were performed in protein-coated bacteriologic microtiter wells for 24 h at 37 degrees C. Concentrations of fibronectin from 1 to 300 micrograms/ml mediated type II cell adherence, 10 micrograms/ml gave maximal adherence, and 4 micrograms/ml gave 50% maximal adherence. Adherence progressively increased from 1 to 72 h. Adherence on fibronectin was at least 50% greater than adherence on laminin, types I and III collagen, or IV collagen. Little or no adherence was observed on bacteriologic plastic or albumin. Spreading on these various substrata paralleled adherence. Adherence to fibronectin, laminin, and fibrinogen was specifically blocked by their respective polyclonal antibodies. Monoclonal antibodies (MoAb) to the fibronectin cell-attachment domain blocked adherence to fibronectin, whereas MoAb to other domains did not. From the data reported here and the previously mentioned in vivo study we propose that fibronectin is an important functional component of the extracellular matrix that supports type II cells during alveolar reepithelialization.

Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro.

Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, (125)I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by alpha(1)-proteinase inhibitor or alpha(2)-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render alpha(1)-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8+/-6.3 and 73.5+/-12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that alpha(1)-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of alpha(1)-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.

Inosinic acid dehydrogenase activity in the Lesch-Nyhan syndrome.

Inosinic acid dehydrogenase was evaluated in normal subjects and in patients with the Lesch-Nyhan syndrome. A significant difference in activity was found between erythrocytes derived from normal controls (1.21+/-0.47 pmoles/hr per mg protein) and from 15 patients with the Lesch-Nyhan syndrome (6.72+/-6.23 pmoles/hr per mg protein). However, no difference in activity was demonstrable in muscle or leukocytes derived from normal and Lesch-Nyhan patients. The increased activity of inosinic acid dehydrogenase in erythrocytes from patients with the Lesch-Nyhan syndrome is due to stabilization of the enzyme in vivo as well as the absence of an inhibitor which is present in erythrocytes from normal subjects.

Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease.

A monoclonal antibody to the carboxyterminal domain of procollagen type I visualizes collagen-synthesizing fibroblasts. Detection of an altered fibroblast phenotype in lungs of patients with pulmonary fibrosis.

Excessive collagen deposition plays a critical role in the development of fibrosis, and early or active fibrosis may be more susceptible to therapeutic intervention than later stages of scarring. However, at present there is no simple method for assessing the collagen-synthesizing and secreting activity of fibroblasts in human tissues. Type I procollagen carboxyterminal domains are proteolytically removed during collagen secretion. Thus, antibodies to these domains should stain fibroblasts synthesizing type I collagen but not extracellular collagen fibrils which could mask the signal from the cells. We developed and characterized a monoclonal antibody (Anti-pC) specific for the carboxyterminal propeptide of type I procollagen. To determine the relationship between Anti-pC staining and collagen synthesis, we stained embryonic and adult chicken tendon. Embryonic chick tendon fibroblasts actively synthesizing type I collagen stained heavily with Anti-pC, while quiescent adult tendon fibroblasts did not stain with Anti-pC. Wounded adult tendons developed fibroblasts that stained with Anti-pC at the wound site. Thus, Anti-pC specifically visualized fibroblasts actively synthesizing collagen. Lung biopsies from patients with fibrotic lung disease were stained with Anti-pC. Interstitial and intraalveolar fibroblasts in biopsies from patients with active fibrosis stained intensely with Anti-pC, while normal human lung was unstained. The absence of staining in normal lung supports the hypothesis that fibrosis is associated with an altered collagen-synthesizing phenotype of tissue fibroblasts. Anti-pC may provide a useful clinical tool for assessing fibrogenic activity at sites of tissue injury.

Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.

We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.