Escherichia coli O115 forms fewer attaching and effacing lesions in the ovine colon in the presence of E. coli O157:H7.

Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.

Time dependent expression of cytokines in Mycobacterium bovis infected cattle lymph nodes.

Advancements in the current diagnostic and vaccination protocols employed against bovine tuberculosis rely heavily upon a sound knowledge of the bovine immunological response. Central to this is the importance of timing in the cellular immune profile and how this dynamic process evolves post-Mycobacterium bovis challenge. In the present study, we quantitatively analysed mRNA expression of interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α) and interleukins (IL) 4 and 10 within select thoracic lymph nodes of cattle infected with M. bovis for 5, 12 and 19 weeks as compared to non-infected bovine tissues. The M. bovis infected lymph nodes displayed significantly higher expression levels of IFN-γ and TNF-α as compared to the non-infected lymph node tissues. This, in conjunction with undetectable levels of IL4, suggests a pro-inflammatory cytokine response. However a significant increase was also detected in IL10 mRNA which is consistent with a described aspect of T(H)1 type T cells in Leishmania infection, a 'self-limiting' process in which cells produced both IFN-γ and IL10 with the aim of controlling the heightened immunopathological responses. This was further reflected when comparing the cytokine profiles of the individual lymph node types, as those displaying a higher IFN-γ/IL10 ratio also had a greater level of gross pathology. This data highlights the important role of IL10 in the bovine response to M. bovis infection and supports its involvement as an immunological marker of disease progression.

Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers.

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.

Advanced granulomatous lesions in Mycobacterium bovis-infected cattle are associated with increased expression of type I procollagen, gammadelta (WC1+) T cells and CD 68+ cells.

The pathognomonic characteristic of tuberculosis (TB) is the formation of a tuberculous granuloma. The objective of this study was to classify lymph node granulomas from experimentally infected calves into different histopathological stages and characterize them further by studying cell types and markers of fibrosis associated with each of the stages. Four stages of granuloma were identified and mRNA and protein expression for cell markers, cytokines and pro-fibrotic markers were studied by immunohistochemistry (IHC) and in-situ hybridization (ISH). In advanced stage granulomas, there was an increase in the expression of TGF-beta, and of type I procollagen as demonstrated by IHC and ISH. As the granulomas advanced, there were fewer CD3+T cells and they tended to be more prominent towards the periphery of the lesions, with a steady increase in the number of CD68+ cells and gammadelta (WC1+) T cells. Granuloma classification and application of cell cytokine markers will assist in improving understanding of the pathogenesis of bovine TB and may help to identify the immunopathology of active disease versus contained or inactive disease. Such disease correlates may help to inform the development of improved diagnostic methods and support vaccine development programmes.

Enhanced antimycobacterial response to recombinant Mycobacterium bovis BCG expressing latency-associated peptide.

With a view to exploring the role of transforming growth factor beta (TGF-beta) during mycobacterial infection, recombinant clones of bacillus Calmette-Guérin (BCG) were engineered to express the natural antagonist of TGF-beta, latency-activated peptide (LAP). Induction of TGF-beta activity was reduced when macrophages were infected with BCG expressing the LAP construct (LAP-BCG). There was a significant reduction in the growth of LAP-BCG in comparison to that of control BCG following intravenous infection in a mouse model. The enhanced control of mycobacterial replication was associated with an increase in the production of gamma interferon by splenocytes challenged during the acute stage of infection but with a diminished recall response assessed after 13 weeks. Organ weight and hydroxyproline content, representing tissue pathology, were also lower in mice infected with LAP-BCG. The results are consistent with the hypothesis that TGF-beta has a detrimental effect on mycobacterial immunity. While a reduction in TGF-beta activity augments the initial response to BCG vaccination, early bacterial clearance may adversely affect the induction of a long-term memory response by LAP-BCG.

Contribution of Th1 and Th2 cells to protection and pathology in experimental models of granulomatous lung disease.

Mice that had received adoptive transfer of DO11.10 TCR transgenic T cells polarized toward a Th1 or a Th2 phenotype were challenged with Ag-coated beads or with recombinant Mycobacterium tuberculosis expressing the OVA determinant. The resulting bead-induced pulmonary granulomas reflected the phenotype of the adoptively transferred T cells, with the Th2 cells promoting a fibrotic reaction. Mice receiving Th1 cells mounted an epitope-specific protective response to challenge with recombinant M. tuberculosis. Th2 recipients were characterized by enhanced weight loss and lung fibrosis during acute high-dose infection. The combination of TCR transgenic T cells and epitope-tagged mycobacteria provides a novel experimental model for investigation of the pathogenesis of tuberculosis.

Tumour necrosis factor-alpha production in human alveolar macrophages: modulation by inhaled corticosteroid.

Using an ex vivo alveolar macrophage model, the hypothesis that inhaled preparations of corticosteroids might have important anti-inflammatory effects on cells of the peripheral airway was tested. The tumour necrosis factor (TNF)-alpha-inducing potential of three glycolipid preparations from nonpathogenic (arabinofuranasyl lipoarabinomannan (LAM (Ara-LAM)) and virulent (mannase LAM (ManLAM)) mycobacteria and Gram-negative bacteria (lipopolysaccharide (LPS)), in primary alveolar macrophage preparations was investigated. A novel inhaled chlorofluorocarbon (CFC)-free preparation of beclomethasone dipropionate (hydrofluoroalkane 134a (HFA)-BDP) with increased peripheral lung deposition was investigated for its ability to modulate glycolipidinduced TNF-alpha production by human alveolar macrophages, in comparison with a CFC-containing preparation and placebo. Compared to the basal TNF-alpha bioactivity of 0.72 ng x mL-1 (geometric mean), the TNF-alpha bioactivity in the macrophage preparation increased following incubation with LPS (138 ng x mL-1, p<0.001), AraLAM (12.6 ng-mL-1, p<0.001) and ManLAM (1.42 ng x mL-1, p=0.02). HFA-BDP, administered in vivo, significantly reduced LPS- and ManLAM-induced TNF-alpha production by alveolar macrophages cultured ex vivo. No change in glycolipid-induced TNF-alpha production was observed following in vivo administration of CFC-BDP or HFA-placebo. This is the first demonstration of an immunomodulatory effect on alveolar cells of corticosteroid delivered via metered dose inhaler. The present findings suggest that alveolar deposition of beclomethasone dipropionate is capable of modulating the inflammatory potential of the alveolar macrophage population.

Bacille Calmette-Guérin (BCG)-associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-gamma).

Immunization with existing BCG vaccines has failed to confer consistent protection against tuberculosis. One of the ways to improve the efficacy of BCG is by enhancing its ability to induce a type-1 T cell response. However, this approach carries the risk that enhanced immunoreactivity may exacerbate tissue pathology associated with vaccination. The aim of the present study was to determine whether use of a recombinant BCG expressing IFN-gamma (BCG-IFN) would result in an alteration in the pattern of inflammation and local tissue fibrosis. A murine intravenous BCG infection model was used in which there was a time- and dose-dependent increase in the weight and number of granulomas in the liver. Infection was associated with increased inflammatory activity in the liver, as shown by the increase in expression of inducible nitric oxide synthase (iNOS) assessed by immunochemistry and by measurement of specific mRNA, and in fibrosis measured by hydroxyproline content of the liver and percentage of granuloma cells staining positively for type 1 procollagen. Infection with BCG-IFN resulted in a reduction in organ weight and bacterial load on day 21 compared with infection with control BCG transformed with vector alone (BCG-plasmid). By day 21, there was also a reduction in iNOS mRNA and iNOS+ cells in granulomas in mice infected with BCG-IFN compared with infection with BCG-plasmid, and a similar reduction in both total number of granulomas and liver hydroxyproline content. These results demonstrate that the granulomas in the areas of mycobacterial infection are active sites of both inflammation and fibrosis, and that the local expression of IFN-gamma by the recombinant BCG results in more efficient bacterial clearance which is accompanied by a reduction in tissue pathology.

Cryptogenic fibrosing alveolitis: lack of association with Epstein-Barr virus infection.

BACKGROUND: Cryptogenic fibrosing alveolitis (CFA) is a well defined clinical entity of unknown aetiology. An association between CFA and the presence of protein indicating Epstein-Barr virus (EBV) replication within epithelial cells of the respiratory tract has recently been suggested, leading to speculation for a role for EBV in the pathogenesis of CFA. METHODS: Lung tissue was obtained from patients in three groups: those with cryptogenic fibrosing alveolitis, either lone or associated with systemic sclerosis; patients with other pulmonary disorders; and patients with normal lung. Paraffin blocks were stained using three antibodies raised against well defined EBV antigens. In addition, EBER-1 and EBER-2 anti-sense nucleotide probes were used in an attempt to identify EBV RNA. DNA was also extracted from the tissue sections and evaluated for evidence of EBV DNA using the polymerase chain reaction. RESULTS: Immunohistochemistry showed inconsistent focal positive staining with anti-EBV antibodies in all three groups, but there was no evidence of EBV RNA using in situ hybridisation. None of the samples from patients with pulmonary fibrotic disorders was found to contain EBV DNA following gene amplification. CONCLUSION: Contrary to an earlier report, these results do not support the hypothesis that EBV has a role in the pathogenesis of CFA.

Production of tumor necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and their suppression by an interleukin-10-secreting recombinant.

We have analyzed mycobacterium-induced cytokine secretion in the J774A.1 macrophage-like cell line. Tumor necrosis factor alpha (TNF-alpha) was preferentially induced by live organisms, both slow and rapid growing. Expression of interleukin-10 by a recombinant strain of Mycobacterium smegmatis caused reduced production of TNF-alpha and nitric oxide during the early stages of infection.

Interleukin-10- and corticosteroid-induced reduction in type I procollagen in a human ex vivo scar culture.

Fibroblasts act as the effector cells of the fibrotic response via production of collagen. In an attempt to understand the regulation of fibroblasts from areas of active human tissue fibrosis, we have developed an ex vivo model in which biopsies of scars from patients 6 weeks post thoracotomy were cultured. This model has been used to investigate whether interleukin-10 (IL-10) and triamcinolone acetonide modulate the expression of type I procollagen mRNA and protein. In situ hybridization and a quantitative competitive RT-PCR were used to measure type I procollagen mRNA. Type I procollagen protein was evaluated by immunochemistry. Viability of biopsies in culture using 3H-uridine incorporation into RNA was observed to be > 80% for at least 96 hours. Following addition of either IL-10 or triamcinolone acetonide there was a modest but significant decrease (P < 0.05) in type I procollagen mRNA expression. Similarly, each agent added individually to biopsies reduced the proportion of cells staining positively for type I procollagen when compared to biopsies treated with medium alone (P < 0.05). These results extend in vitro data that IL-10 and corticosteroids down-regulate collagen synthesis in skin fibroblast cell lines and suggest that this ex vivo model may offer a closer approximation to the post-operative scarring process when testing new therapeutic agents for reducing an over-exuberent fibrotic response.

Differential responses to challenge with live and dead Mycobacterium bovis Bacillus Calmette-Guérin.

Bacillus Calmette-Guérin (BCG) vaccination has been shown to protect against challenge with virulent Mycobacterium tuberculosis in a range of experimental animal models: in each case, protective efficacy requires vaccination with live bacteria. With the goal of moving to a new generation of safer, nonliving vaccines, efforts have been made to identify the factors that determine the efficacy of live vaccination. We show that injection of live, but not dead, BCG induces localized swelling in the mouse footpad model. Live and dead bacteria induce similar responses during the first week after vaccination as determined by immunohistochemical analysis of the site of injection and of the draining lymph node. The subsequent differential response is characterized by migration of acid-fast bacilli to the draining lymph node in the case of the live vaccine. This is accompanied by an increase in mononuclear cells in the lymph node and by expression of inducible nitric oxide synthase (iNOS) both in the lymph node and at the site of injection. The ability of the bacteria to migrate to the lymph node may be an important element in the efficacy of live BCG vaccination.

Thalidomide reduces tumour necrosis factor-alpha production by human alveolar macrophages.

Overexuberant production of tumour necrosis factor-alpha (TNF-alpha) by macrophages and other cells is thought to contribute to the development of permanent lung damage in many inflammatory conditions. There is a need for an agent, without the side-effects of corticosteroids, which can reduce the production of TNF-alpha by macrophages activated by disease. This study evaluated the effect of thalidomide on lipopolysaccharide (LPS)-induced TNF-alpha production by human alveolar macrophages obtained from patients with tuberculosis and a group of other diseases associated with macrophage activation. Alveolar macrophages obtained by bronchoalveolar lavage from 31 patients (tuberculosis = 12, sarcoidosis = 3, lung cancer = 5, chronic bronchitis = 5, pneumonia = 6) were stimulated with LPS alone or LPS in combination with either thalidomide or dexamethasone. Cell-associated TNF-alpha, as measured by immunochemistry, and TNF-alpha released by macrophages, as assessed by ELISA, were markedly increased when cells were incubated with LPS (P < 0.05), and both were decreased following addition of thalidomide (P < 0.05) or dexamethasone (P < 0.05) to amounts similar to those observed when macrophages were incubated with medium alone. Similarly, TNF-alpha mRNA as measured by in situ hybridization was increased following incubation with LPS (P < 0.05), but this increase was prevented by addition of thalidomide (P < 0.05) or dexamethasone (P < 0.05). The ability of thalidomide to reduce LPS-induced TNF-alpha production by alveolar macrophages was the same when cells from patients with tuberculosis (a disease associated with TNF-alpha production) and cells from patients with the other conditions were compared. The ability of thalidomide to reduce TNF-alpha production by human alveolar macrophages from patients with active lung disease suggests that thalidomide and its analogues may have potential as drugs to reduce TNF-alpha production in disease.

Increased inflammatory cytokines and new collagen formation in cutaneous tuberculosis and sarcoidosis.

BACKGROUND: Interactions between mononuclear cells, vascular endothelium, fibroblasts, and cytokines during the inflammatory reaction within a granuloma have the potential to contribute to the progression to fibrosis. METHODS: Biopsy specimens of six tuberculous and eight sarcoidosis skin lesions were examined by immunohistochemistry to seek evidence for the presence of inflammatory and fibrotic reactions in human granulomatous disease. Additionally, to understand how a T cell mediated delayed type hypersensitivity reaction--a component of chronic granulomatous inflammation--could progress to fibrosis, the human in vivo model of the cutaneous tuberculin Heaf reaction to purified protein derivative (PPD) was studied in a group of 48 subjects. RESULTS: Granulomas from tuberculous and sarcoidosis skin biopsy specimens were seen to contain cells with marked staining by antibodies to fibronectin, transforming growth factor beta (pan TGF-beta), and type 1 procollagen (PCP-1). Accentuated staining of extracellular matrix was seen both in the granulomas and in the peri-granulomatous regions. Less prominent staining was observed using antibodies against interleukin 1 beta (IL-1 beta) and alpha-smooth muscle actin (alpha-SMA). Biopsies of Heaf reactions revealed cells staining for IL-1 beta, tumour necrosis factor alpha (TNF-alpha), platelet derived growth factor B (PDGF-B), and fibronectin which were detected as early as day 1 and persisted throughout the 14 day study period. Cells staining for PCP-1 increased to greatest abundance at day 14. All these cytokines were present in low abundance in biopsy specimens from sites inoculated with saline only. CONCLUSIONS: Evidence is provided that granulomas in tuberculosis and sarcoidosis behave as active centres of fibrogenesis. Using the Heaf model, the temporal relationship between the early appearance of cytokines and the later increase in the collagen precursor PCP-1 linked the immune mediated chronic inflammatory response with subsequent fibrosis and suggested that the tuberculin Heaf reaction will serve as a model for studying the early events of granuloma formation in patients with tuberculosis and sarcoidosis.

Increase in interleukin (IL)-1 beta and IL-6 in bronchoalveolar lavage fluid obtained from infants with chronic lung disease of prematurity.

Chronic lung disease (CLD) of prematurity is associated with an initial increase in pulmonary neutrophils followed by pulmonary fibrosis. We determined whether the proinflammatory cytokines, IL-1 beta and IL-6, were increased in the bronchoalveolar lavage fluid obtained from nine infants (median gestation 25 wk, birthweight 820 g) who developed CLD, seven (28 wk, 1110 g) who recovered from the respiratory distress syndrome (RDS), and four (38 wk, 2690 g) control infants. IL-1 beta and IL-6 protein were both increased in the bronchoalveolar lavage fluid from the CLD groups when compared with the RDS and control groups. This difference for both the cytokines was most marked on d 10 of age, when results from infants with and without CLD were compared (IL-1 beta, 4.6 versus 1.1 ng/mL, p < 0.05; and IL-6, 9.5 versus 1.5 ng/mL, p < 0.05). Immunocytochemistry of lavage cells for IL-1 beta, IL-6, and IL-8 protein showed alveolar macrophages to contain all three cytokines, with lesser staining evident in neutrophils, and in epithelial cells occasionally obtained by lavage. The contribution of alveolar macrophages and luminal cells to the increase in IL-6 and IL-1 was determined by performing semiquantitative reverse transcription-polymerase chain reactions on RNA extracted from lavage cells. IL-6 mRNA expression was increased in lavage cells from the CLD infants when compared with the RDS group. However, the expression for IL-1 beta and IL-8 mRNA was similar in both groups. These results suggest that IL-1 beta, IL-6, and IL-8 may contribute to the pathogenesis of CLD, and that, in CLD, IL-6 may be produced by cells within the air spaces.

Increase in the concentration of transforming growth factor beta-1 in bronchoalveolar lavage fluid before development of chronic lung disease of prematurity.

OBJECTIVE: Pulmonary fibrosis is a prominent feature of chronic lung disease of prematurity (CLD). We sought to determine the influence of the potent profibrotic cytokine transforming growth factor beta-1 (TGF-Beta 1) on the development of CLD. METHODS: We determined the concentration of active and total TGF-Beta 1 in bronchoalveolar lavage fluid obtained from 18 infants who subsequently had CLD (mean gestation, 25.7 weeks; birth weight, 816 gm) 15 (29.8 weeks, 1493 gm) who recovered from the respiratory distress syndrome, and 7 (35.1 weeks, 2441 gm) control infants. RESULTS: The concentration of both active and total TGF-Beta 1 was increased in the infants with CLD when compared with the respiratory distress syndrome and control groups. The increase in active and total TGF-Beta 1 was greatest on day 4 of age, when infants who eventually had CLD were compared with those who did not progress to CLD (active TGF-Beta 1, 39.5 vs 4.6 ng/ml; total TGF-Beta 1, 43.8 vs 13.8 ng/ml). In addition, immunocytochemistry studies localized pan-TGF-Beta to alveolar macrophages obtained by bronchoalveolar lavage. CONCLUSIONS: These observations indicate that TGF-Beta 1 may contribute to the fibrotic response that is observed in the lungs of infants who have CLD.

Enhanced expression of type 1 procollagen and transforming growth factor-beta in tuberculin induced delayed type hypersensitivity.

AIMS: Tissue fibrosis is a common and serious consequence of chronic inflammation. The mechanism linking these two processes is poorly understood. The present study has utilised a human in vivo model of a delayed type hypersensitivity (DTH) reaction, the tuberculin Heaf reaction, induced by intradermal tuberculin in BCG immunised subjects, to dissect the relation between these two processes. METHODS: Punch skin biopsy specimens were obtained on day 5, day 13 and six to 16 weeks following the tuberculin Heaf test in 18 subjects with grade 3 or 4 responses. Skin biopsy specimens from six subjects served as controls. The specimens were examined using immunohistochemical staining for type 1 procollagen and transforming growth factor-beta (TGF-beta), as well as in situ hybridisation for type 1 procollagen messenger RNA (mRNA). RESULTS: Immunohistochemical analysis revealed increased deposition of TGF-beta in tissue matrix in the biopsy specimens obtained on day 5 following the tuberculin Heaf test. There was also extensive type 1 procollagen staining in the biopsy specimens obtained as early as day 5. Procollagen-1 staining was maximal on day 13, and was present in biopsy specimens from tuberculin Heaf test sites up to eight weeks after the tuberculin inoculation. The type 1 procollagen was localised within cells surrounding areas of inflammatory infiltrate and in perivascular tissues. The presence of new collagen formation was confirmed by in situ hybridisation using oligonucleotide probes for type 1 procollagen mRNA in cells in sections from biopsy specimens obtained on day 13. CONCLUSIONS: These data from a human in vivo model of a DTH response indicate that the immune response is intimately associated with an increase in the production of growth factors and the initiation of a fibrotic response.

Platelet-derived growth factor-beta mRNA in human alveolar macrophages in vivo in asthma.

Collagen deposition and myofibroblast proliferation beneath the epithelial basement membrane in patients with asthma is now increasingly recognized, although the molecular pathogenesis remains obscure. We have evaluated messenger ribonucleic acid (mRNA) expression of the profibrotic cytokine, platelet-derived growth factor-beta (PDGF-beta), in alveolar macrophages obtained following fibreoptic bronchoscopy and bronchoalveolar lavage in patients with asthma. Three subject groups were studied: 1) asthmatics using regular inhaled glucocorticoid medication (ASTST, n = 9), 2) asthmatics using intermittent inhaled beta 2-agonist therapy only (ASTBR, n = 10); 3) nonasthmatic control volunteers (n = 10). Alveolar macrophage mRNA was extracted and PDGF-beta mRNA quantified by reverse-transcriptase polymerase chain reaction (PCR) (RT-PCR) and expressed as the ratio to that of a control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). There were no significant differences in PDGF-beta mRNA expression between the groups, or between all asthmatic (n = 19) and control subjects. Furthermore, there was no correlation between alveolar macrophage PDGF-beta mRNA expression and airway spirometry, or duration of glucocorticoid usage or dose. Thus, in contrast to other fibrotic lung diseases, we found little evidence of enhanced expression of PDGF-beta mRNA in alveolar macrophages in clinically stable bronchial asthma.

Type 1 procollagen as a marker of severity of scarring after sternotomy: effects of topical corticosteroids.

AIMS: To determine whether the abundance of newly formed collagen in healing surgical wounds correlated with scar severity, and whether topical application of steroid cream reduced new collagen formation in patients who have undergone median sternotomy. METHODS: Thirty three patients six weeks after sternotomy, and 12 controls were studied. Scars were photographed, and biopsy specimens from scars at sites treated or untreated with topical corticosteroids (clobetasol proprionate 0.5%) were examined using immunohistochemical staining for type 1 procollagen (PCP 1) and transforming growth factor beta (TGF-beta), and in situ hybridisation for type 1 procollagen messenger RNA (mRNA). RESULTS: The degree of hypertrophy of the scar and the abundance of PCP 1 immunostaining were ranked independently, blind, and a correlation between these two variables was observed (r = 0.604, p < 0.001). The PCP 1 immunostaining was accompanied by a great abundance of PCP 1 mRNA and only a slight increase in TGF-beta immunostaining, when compared with normal skin or mature scars. Following the application of topical corticosteroids, for either 48 hours or twice daily for seven days, there was no reduction in PCP 1 immunostaining nor the abundance of PCP 1 mRNA. CONCLUSIONS: These data suggest that the extent of new collagen formation as assessed by PCP 1 immunohistochemistry may be a useful marker of the exuberance of the scarring process following sternotomy, and that topical corticosteroids are ineffective in reducing this component of the fibrotic response.