Fibrocyte Counts Research Articles

Pravastatin prevents colitis-associated carcinogenesis by reducing CX3CR1high M2-like fibrocyte counts in the inflamed colon

Colorectal cancer (CRC) resulting from chronic inflammation is a crucial issue in patients with inflammatory bowel disease (IBD). Although many reports established that intestinal resident CX3CR1high macrophages play an essential role in suppressing intestinal inflammation, their function in colitis-related CRC remains unclear. In this study, we found that colonic CX3CR1high macrophages, which were positive for MHC-II, F4/80 and CD319, promoted colitis-associated CRC. They highly expressed Col1a1, Tgfb, II10, and II4, and were considered to be fibrocytes with an immunosuppressive M2-like phenotype. CX3CR1 deficiency led to reductions in the absolute numbers of CX3CR1high fibrocytes through increased apoptosis, thereby preventing the development of colitis-associated CRC. We next focused statins as drugs targeting CX3CR1high fibrocytes. Statins have been actively discussed for patients with IBD and reported to suppress the CX3CL1/CX3CR1 axis. Statin treatment after azoxymethane/dextran sulfate sodium-induced inflammation reduced CX3CR1high fibrocyte counts and suppressed colitis-associated CRC. Therefore, CX3CR1high fibrocytes represent a potential target for carcinogenesis-preventing therapy, and statins could be safe therapeutic candidates for IBD.

Neoplastic Monocyte-Derived Fibrocytes Induce Bone Marrow Fibrosis in Patients with Primary Myelofibrosis

Progressive bone marrow (BM) fibrosis is a defining feature of primary myelofibrosis (PMF). The extent of BM fibrosis has been closely associated with the clinical manifestations of advanced disease, including anemia and thrombocytopenia, rendering it an important prognostic indicator. Mesenchymal stromal cells (MSCs) have long been regarded as the exclusive inducer of BM fibrosis. However, using xenograft and transgenic animal models, we and other investigators demonstrated that neoplastic monocyte-derived fibrocytes independently contribute to the induction of BM fibrosis in PMF. Notably, our data revealed that PMF fibrocytes but not MSCs, carry pro-fibrotic signaling pathways that are absent in healthy individuals' fibrocytes or MSCs. Despite these findings, the precise contribution of BM fibrocytes and MSCs to the induction of BM fibrosis in patients with PMF remains elusive. To determine which of two subsets of collagen-producing cells are the main contributors to the induction of BM fibrosis in PMF, we analyzed BM biopsies of patients with PMF and assessed whether their distribution spatially correlated with areas of fibrosis. We analyzed formalin-fixed paraffin-embedded BM trephine biopsies obtained from previously untreated patients with PMF (n = 85) and age-matched healthy donors (n = 6). Using a 7-plexed fluorescence immunohistochemistry (IHC) in conjunction with hematoxylin/eosin, Masson's trichrome, and Gomorri's silver stains we analyzed those BM biopsy specimens. To ensure comprehensive assessment, we imaged serial whole-tissue sections from each patient at 400x resolution, meticulously aligning and carefully correcting for bone trabeculae and any artifacts. To quantitate the spatial distribution of individual cells, collagen fibers, and reticulin fibers, we developed a set of analytical algorithms based on a U-Net neural network in Visiopharm software. Our analysis revealed a 6-fold higher density of fibrocytes, defined as CD45 +/CD68 +/procollagen type I + cells, in the BM of patients with PMF ( P=0.0003) compared to normal controls. By contrast, the number of MSCs, defined as CD45 -/CD90 +/CD105 + cells, in the PMF BM was not different from the number of MSCs in normal BM. After dividing the area of each BM biopsy into a 100 μm regular hexagonal grid, the observed spatial distribution of fibrocytes strongly and positively correlated with the areas covered by reticulin and collagen fibers ( P<0.0001 in both). Furthermore, spatial analysis showed that increased densities of fibrocytes were associated with an increased number of fibrotic cellular zones in the BM tissue of PMF patients. To investigate the clonality of PMF BM fibrocytes, we conducted DNA in situ hybridization (ISH) analysis, employing two sets of locus-specific probe pairs. The DNA-ISH analysis provided valuable insights, revealing that fibrocytes obtained from PMF patients with trisomy 9 or 20q deletion carried the same corresponding cytogenetic abnormalities, indicating that PMF fibrocytes indeed originate from the neoplastic PMF clone. Because increased peripheral blood monocyte counts, commonly detected in patients with PMF, are associated with a poor prognosis, we sought to determine the potential correlation between BM fibrocyte and circulating monocyte counts. As expected, we found a significant positive correlation between BM fibrocyte counts and the percentage of circulating monocytes ( P=0.001). Moreover, we also found that fibrocyte numbers correlated with the levels of the plasma cytokines pentraxin 3, known to induce the differentiation of monocytes into fibrocytes, and matrix metalloproteinase 2, as assessed by using magnetic immunoassay, suggesting that neoplastic fibrocytes induce BM fibrosis in PMF and that fibrocyte quantification could serve as a valuable indicator of disease severity and prognosis. Taken together, our quantitative imaging analysis of BM biopsy tissue from treatment naïve PMF patients revealed that neoplastic fibrocytes, rather than MSCs, are the main contributors to the induction of BM fibrosis in PMF. Our findings underscore the importance of BM fibrocytes in the pathogenesis of PMF and warrant further investigation to identify novel therapeutic agents that will either inhibit fibrocyte differentiation or block their pro-fibrotic function.

Circulating fibrocyte level in children with cystic fibrosis.

This study aimed to evaluate circulating fibrocyte levels in cystic fibrosis (CF) patients during stable and exacerbation periods of the condition. The study group consisted of 39 patients diagnosed with CF and 20 healthy controls. Individuals included in the study were divided into three groups: CF, CF exacerbated, and a healthy control group. Their circulating fibrocyte levels were compared. Findings from a pulmonary function test and high-resolution computed tomography of the lung were evaluated and compared. The circulating fibrocyte count was found to be significantly higher in patients with CF compared with the exacerbated and control groups. No correlation was found between the forced expiratory volume in 1 s and forced vital capacity values in the pulmonary function test and the circulating fibrocyte count. The circulating fibrocyte count in patients (in the CF group) with positive findings in the high-resolution computed tomography was statistically significantly lower. The circulating fibrocyte level in the peripheral blood of the patients with CF was increased.

In vitro effect of biological and conventional disease-modifying antirheumatic drugs on fibrocyte differentiation in patients with rheumatoid arthritis and healthy controls.

Fibrocytes are circulating bone-marrow-derived cells that migrate to organs with ongoing repair or inflammation. In the target organ, the cells differentiate, become long and spindle-shaped, and are able to produce extracellular matrix components. In fibrotic diseases, the levels of fibrocytes are increased, both in circulation and the diseased tissue. In rheumatoid arthritis (RA), fibrocytes have been proposed to be involved in the spread of the disease and possibly in RA fibrotic manifestations, as can be seen in RA interstitial lung disease (RA-ILD). Therefore, we aimed to investigate a range of current RA treatment modalities (corticosteroids and conventional and biological disease-modifying antirheumatic drugs (DMARDs)) regarding their effect on in vitro fibrocyte differentiation. A total of 10 participants were included (5 patients with RA and 5 healthy controls). Peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 5 days with prednisolone, conventional DMARDs (methotrexate, sulfasalazine, and hydroxychloroquine), and biological DMARDs (etanercept, tocilizumab, adalimumab, abatacept, and rituximab). The numbers of fibrocytes were counted. Dose-response data for abatacept and tocilizumab were collected. Abatacept and prednisolone significantly suppressed differentiation of PBMC into fibrocytes compared with control (p=0.02 and p<0.01, respectively) (n=10). In overall analysis (n=10), abatacept reduced fibrocyte levels with an average of 44% overall and 71% in the RA group compared with the control wells. Tocilizumab reduced the fibrocyte count by 63% overall and 45% in the RA group, although it was not significant (p=0.07 and p=0.06, respectively). Both tocilizumab and abatacept display a dose-response relationship. Abatacept and prednisolone suppress the differentiation of mononuclear cells to mature fibrocytes in vitro in patients with RA, and data indicate a similar effect of tocilizumab; this was further supported by the observed dose-response relationship. Clinical trials are needed to compare the effect of these drugs on fibrotic RA manifestations, for example, RA-ILD.

Monocyte M1/M2 profile is altered in paediatric burn patients with hypertrophic scarring

Hypertrophic scars (HTS) remain a common outcome of burn injury, particularly in children. They can arise from variations in the wound healing stages, such as an excessive inflammatory response or inefficient remodelling. Of the cells contributing to these healing stages, macrophages and fibrocytes are crucial. Specifically, the inflammatory phase is dominated by M1 macrophages, the proliferation/remodelling stages by M2 macrophages, and scar tissue contains numerous fibrocytes. As the progenitors to these cells, monocytes, can also exhibit M1- and M2-skewing, we proposed that their profile, or circulating fibrocyte counts, could be used to predict poor healing outcomes. To investigate this, we obtained blood samples from paediatric controls and burns patients, which were then divided into HTS and NoHTS groups upon scar assessment at 12 months. The samples were assessed by whole blood flow cytometry to quantify fibrocytes and monocyte subset proportions and to determine monocyte levels of M1 (CD86, CD120b, CD319) and M2 (CD93, CD163, CD200R) markers. Both burns groups had higher proportions of classical monocytes compared to controls, indicating increased cell turnover and/or entry of other subsets into the wound. In burns patients who took more than 21 days to heal, the HTS group had lower M2 (CD200R) expression with the ratio of M1/M2 (CD86/CD200R) being significantly higher. These results suggest an elevated early inflammatory monocyte response contributes to development of HTS. Correlations of marker expression with remaining healing time revealed a significant positive correlation with M1 (CD120b) and M1/M2 (CD120b/CD200R), suggesting a potential role for CD120b as an indicator of healing delay. Fibrocytes did not significantly differ between the groups. In conclusion, increased monocyte inflammation likely contributes to slower healing and development of scarring, but further studies are needed to determine the predictive power of monocyte inflammatory profile.

The effects of human amniotic membrane on silicone related capsule formation in rats

Silicone breast implants are commonly used materials in plastic surgery for breast augmentation and reconstruction and the most severe complication of silicone implants are capsule contraction which occurs in 40% of patients. The aim of our study is to evaluate how the amniotic membrane alters the capsule formation effects of silicone 24 wistar rats were used in the study. We placed a bare silicone block into the left side (Subgroup A) and single layer amniotic membrane coated silicone block into the right side (Subgroup B) of the rats back. The rats were then separated into three groups and in group 1 rats were euthanized after 3 weeks, in group 2 after 12 weeks and in group 3 after 24 weeks. Then capsule thickness, fibroblast and lymphocyte cell counts were evaluated for each sample. In Group 2 and group 3, the capsule thickness in Subgroup B was detected to be statistically significantly lower than that in Subgroup A. In Group 1, 2, and 3, the lymphocyte count in the capsule tissue taken from Subgroup B was lower than Subgroup A but the difference was not statistically significant. In Group 2 and 3, the fibrocyte count detected in the capsule tissue in Subgroup B was found to be statistically significantly lower than Subgroup A. the amniotic membrane was demonstrated to reduce capsule thickness by the antifibrinolytic effect in our study.

Biochemical, histopathological, and pharmacological evaluations of cutaneous wound healing properties of Quercus brantii ethanolic extract ointment in male rats

The recent experiments have revealed the property of ethnomedicinal plants on the treatment of the cutaneous wound. The purpose of the experiment was to investigate the wound healing and antioxidant potentials of the internal layer of Quercus brantii ethanolic extract ointment. DPPH free radical scavenging test was carried out to examine the antioxidant effect of the internal layer of Q. brantii ethanolic extract, which indicated similar antioxidant activity with butylated hydroxy toluene (BHT) as a positive control. In vivo design, 120 Sprague Dawley male rats were used. After creating the cutaneous wound, the animals were randomly divided into four groups: untreated control, treatment with Eucerin ointment, treatment with 3% tetracycline ointment, and treatment with 3% Q. brantii ethanolic extract ointment (3 g of Q. brantii ethanolic extract + 97 g base ointment). At days 10, 20, and 30 after creating the wound, for histopathological and biochemical analysis of the cutaneous wound healing trend, a section was prepared from all dermal thicknesses. At days 10, 20, and 30, Q. brantii ethanolic extract ointment significantly (p > 0.05) enhanced wound contraction, fibrocyte count, hexuronic acid, and hydroxyproline levels and decreased the wound area, all cell, macrophage, lymphocyte, and neutrophil counts as compared with the basal ointment and control groups. In conclusion, the acquired findings showed the cutaneous wound healing potential of Q. brantii ethanolic extract ointment.

PRM-151 in Myelofibrosis: Efficacy and Safety in an Open Label Extension Study

Introduction: PRM-151, a recombinant human pentraxin-2 molecule, has been shown to prevent and reverse fibrosis in animal models of myelofibrosis (MF) by postulated targeting differentiation of fibrocytes (essential cells in fibrotic process) from monocytes. In the first stage of a two-stage trial, 27 patients (pts) with primary myelofibrosis (PMF), post-essential thrombocythemia/polycythemia vera (post-ET/PV) MF, and Grade 2 or 3 bone marrow fibrosis (BMF) received PRM-151 10 mg/kg IV ± ruxolitinib (RUX) for 6 cycles (24 weeks). A reduction in BMF, decrease in symptoms (MPN-SAF Total Symptom Score [TSS]), and a reduction of palpable splenomegaly were observed, along with a favorable safety profile. Pts experiencing clinical benefit were allowed to continue beyond 24 weeks in an open label extension (OLE) study. Here, we report interim efficacy and safety data for 18 pts enrolled in the OLE, who had been treated for up to 35 cycles (140 weeks). Methods: All pts in the OLE received a monthly infusion of PRM-151 10 mg/kg IV. Pts receiving PRM-151 alone or in combination with RUX in the main study continued with their respective drugs. Safety and hematology parameters were assessed monthly, and MPN-SAF TSS Q3 months. BM biopsies were obtained at baseline, end of main study, and at physician discretion in the OLE. These were evaluated centrally by two independent, blinded hematopathologists for the degree of reticulin and collagen fibrosis. Immunostaining for fibrocytes was performed on available biopsies. The primary objective of the OLE was to assess safety and efficacy of long-term administration of PRM-151. Results: Of the 18 pts enrolled in the OLE, 9 received PRM-151 as single agent, and 9 in conjunction with RUX. Baseline characteristics included median age of 66 years (51-78); 8 pts were DIPSS Int-1, 9 Int-2, and 1 high-risk; 6 (33%) had Hgb Conclusions: This long-term follow-up study of 18 pts with MF who received PRM-151 for a median of 31 months showed the drug to be well tolerated. An overall improvement in BM reticulin and collagen fibrosis grade, as well as reductions in MPN-SAF TSS and splenomegaly were observed. In a subset of pts for whom bone marrow fibrocyte immunostaining data were available, a mean reduction in fibrocyte counts was observed, as well as improved bone marrow reticulin and collagen fibrosis grade. These data warrant confirmation in a larger controlled study. Disclosures Verstovsek:Incyte: Consultancy; Italfarmaco: Membership on an entity9s Board of Directors or advisory committees; Novartis: Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity9s Board of Directors or advisory committees. Pozdnyakova:Promedior, Inc.: Consultancy. Mesa:Promedior: Research Funding; Incyte: Research Funding; Ariad: Consultancy; Novartis: Consultancy; Celgene: Research Funding; Galena: Consultancy; Gilead: Research Funding; CTI: Research Funding. Foltz:Incyte: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Mascarenhas:Incyte: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Celgene: Membership on an entity9s Board of Directors or advisory committees; Merck: Research Funding; Roche: Research Funding; CTI Biopharma: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Promedior: Research Funding. Ritchie:Astellas Pharma: Research Funding; NS Pharma: Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; ARIAD Pharmaceuticals: Speakers Bureau; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Incyte: Consultancy, Speakers Bureau. Palmer:Novartis: Research Funding. Kremyanskaya:Incyte: Research Funding. van den Blink:Promedior, Inc.: Employment. Gupta:Promedior, Inc.: Employment. Gotlib:Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Promedior: Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Circulating Fibrocytes Are Increased in Neonates with Bronchopulmonary Dysplasia.

BackgroundBronchopulmonary dysplasia (BPD) is characterized by the aberrant remodeling of the lung parenchyma, resulting from accumulation of fibroblasts or myofibroblasts. Circulating fibrocytes are implied in pulmonary fibrosis, but whether these cells are associated with the development of BPD or the progressive fibrosis is unknown. The aim of the present study was to investigate the occurrence of fibrocytes in peripheral venous blood and explore whether these cells might be associated with severity of BPD.MethodsWe investigated circulating fibrocytes in 66 patients with BPD, 23 patients with acute respiratory distress syndrome(ARDS) and 11 normal subjects. Circulating fibrocytes were defined and quantified as cells positive for CD45 andcollagen-1 by flow cytometry. Furthermore, serum SDF-1/CXCL12 and TGF-β1 were evaluated using ELISA methods. We also investigated the clinical value of fibrocyte counts by comparison with standard clinical parameters.ResultsThe patients with BPD had significantly increased numbers of fibrocytes compared to the controls (p < 0.01). Patients with ARDS were not different from healthy control subjects. There was a correlation between the number of fibrocytes and pulmonary hypertension or oxygen saturation (p < 0.05). Fibrocyte numbers were not correlated with other clinical or functional variables or radiologic severity scores. The fibrocyte attractant chemokine CXCL12 increased in plasma (p < 0.05) and was detectable in the bronchoalveolar lavage fluid of 40% of the patients but not in controls.ConclusionThese findings indicate that circulating fibrocytes are increased in patients with BPD and may contribute to pulmonary fibrosis in BPD. Circulating fibrocytes, likely recruited through the CXCR4/CXCL12 axis, might contribute to the production of TGF-β1 for the expansion of fibroblast/myofibroblast population in BPD.

Involvement of fibrocytes in asthma and clinical implications.

Bloodborne fibrocytes are bone marrow-derived cells that participate in immune responses and exhibit pro-inflammatory and matrix remodelling properties. In patients with asthma receiving an adequate treatment, the blood fibrocyte count is very low and comparable to that obtained in healthy individuals. In these patients, a transient increase in fibrocyte numbers in the peripheral blood and in the airways occurs in concomitance with increased bronchial inflammation and reflects disease worsening and the need for more intensive treatment. Persistently elevated numbers of fibrocytes in the peripheral blood and in the bronchial mucosa are observed in chronically undertreated or corticosteroid-resistant asthma and are associated with persistent airway inflammation and ongoing remodelling of the bronchial wall. The asthmatic bronchial epithelium is the main source of fibrocyte chemoattractants in asthma and contributes with T helper type 2 lymphocytes and eosinophils to promote the proliferation and pro-remodelling function of recruited fibrocytes. The presence of elevated numbers of fibrocytes in the bronchial mucosa of allergic patients with undertreated or treatment-resistant asthma may also increase the risk of acute exacerbations because these cells can amplify T helper type 2 lymphocyte-driven inflammation on every exposure to the clinically relevant allergen and can promote further inflammation on rhinovirus infections by allowing viral replication and releasing additional pro-inflammatory factors. Improved methods for the isolation and functional analysis of pure populations of viable circulating fibrocytes have allowed a better understanding of the effector role of these cells. A reliable and clinically applicable assay has been developed to measure blood fibrocyte counts as outcome measure in future clinical trials. New therapeutic agents are needed to block both persistent inflammation and fibrocytosis in corticosteroid-resistant asthma.

Pathogenetic and prognostic roles of bloodborne fibrocytes in asthma

Bloodborne fibrocytes are cells mobilized from the bone marrow, which express surface antigens commonly ascribed to hematopoietic progenitors and have phenotypic and functional characteristics similar to those of immature mesenchymal cells. They exhibit predominant proinflammatory or profibrotic activities at tissue sites, depending on the host's response to environmental insults and on the characteristics of the cell infiltrate and cytokine milieu. In patients with allergic asthma, fibrocytes egress from the bone marrow and are recruited into the airways after every allergen exposure and during viral infections. Recruited fibrocytes amplify the inflammatory responses driven by T helper type 2 lymphokines and favor viral replication and further inflammation on respiratory virus infections. Persistently elevated blood fibrocyte counts and persisting airway fibrocytosis are present in patients with chronically undertreated or corticosteroid-insensitive asthma, and are linked to an enhanced risk of adverse outcomes because of the major involvement of fibrocytes in the development of structural abnormalities that lead to chronic airflow obstruction in these patients. Consequently, blood fibrocyte count is an emerging biomarker of asthma control and disease progression and its clinical applicability as a new outcome measure deserves further evaluation in large clinical trials.

Circulating fibrocytes as biomarker of prognosis in Hermansky-Pudlak syndrome.

The rate of progression of most interstitial lung diseases (ILD) is unpredictable. Fibrocytes are circulating bone marrow-derived cells that have been implicated in the pathogenesis of lung fibrosis. Hermansky-Pudlak syndrome (HPS), a genetic cause of ILD in early adulthood, allows for study of biomarkers of ILD in a homogeneous population at near-certain risk of developing fibrotic lung disease. To test the hypothesis that, in subjects with HPS, the number or phenotype of circulating fibrocytes predicts progression and outcome of ILD. We measured circulating fibrocyte counts and chemokine levels in a cohort of subjects with HPS and healthy control subjects and correlated the results to disease outcome. In a cross-sectional analysis, peripheral blood fibrocyte concentrations were markedly elevated in a subset of subjects with HPS who had ILD but not subjects without lung disease or normal control subjects. The blood concentration of fibrocytes expressing the chemokine receptor CXCR4 correlated significantly with the plasma concentration of the CXCR4 ligand, CXCL12. In a longitudinal study, we found marked episodic elevations in circulating fibrocyte counts over a median follow-up period of 614 days. Elevations in both maximal values and final values of peripheral blood CXCR4(+) fibrocyte concentration were strongly associated with death from ILD. CXCR4(+) fibrocyte concentration may be useful as a biomarker for outcome of ILD in subjects with HPS.

Circulating fibrocytes are an indicator of severity and exacerbation in chronic obstructive pulmonary disease

RationaleChronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation that is associated with an enhanced inflammatory response in the airways and the lung. The remodeling process in COPD is greatly under the influence of growth factors. Lung fibroblasts in COPD demonstrated alterations in its functional capacity that is mediated by TGF-β1, therefore, could play a role in the pathogenesis of COPD. Fibrocytes are bone marrow derived cells that migrate to the injured sites and differentiate into fibroblast-like cells. ObjectivesTo test the hypothesis that assay of circulating fibrocytes may provide a biomarker for exacerbation and severity of COPD. MethodsFibrocytes were defined by flow cytometry and quantified in fifty male patients with stable COPD and during exacerbation. We investigated the clinical and prognostic value of fibrocytes by comparison with standard clinical parameters. Thirteen healthy subjects were selected as control. ResultsFibrocytes were significantly elevated in stable COPD patients (n=25), with a further increase during exacerbation (n=25; P<0.001 vs. control subjects n=13). Correlation analysis between fibrocyte counts and mMRC score, 6-MWT, BODE index, arterial oxygen saturation, pre- and post-bronchodilator FEV1/FVC, FEV1, FVC and FEF25–75 showed a direct relationship in COPD patients. There was a direct correlation between fibrocytes with the mMRC score and the serum levels of TGF-β1 only in COPD patients in exacerbations (n=25). ConclusionsFibrocytes are an indicator of COPD exacerbation and might be useful as a clinical (bio) marker for disease progression.

Enumeration of circulating fibrocytes for clinical use in asthma by an optimized single-platform flow cytometry assay

BackgroundElevated numbers of circulating fibrocytes are associated with inadequately controlled asthma, poor response to available therapies, and increased risk of adverse outcomes. The lack of reliable and clinically-applicable assays precludes a proper evaluation of blood fibrocyte count as a prognostic biomarker in asthma. This report concerns the use of a multiparameter flow cytometry assay for the enumeration of fibrocytes in the whole blood. MethodsConsenting fibrocyte donors were 19 patients with asthma well controlled by current treatment, 16 patients with treatment-resistant asthma, 9 patients with transiently uncontrolled asthma and 14 age-matched normal individuals. Blood sampling was performed once in patients with transiently uncontrolled asthma and twice, at an interval of one week, in the other subjects. The assay was performed in 100μl of whole blood and involved a sequential gating strategy and absolute fibrocyte counting with a single instrument (single-platform assay). ResultsThe quantification of circulating fibrocytes by this assay was analytically and clinically valid. In individuals with stable clinical conditions, the repeatability of blood fibrocyte counts over one week was good. The intraclass correlation coefficient was 0.939 and 96.88% of the total variability reflected on-average differences among the tested subjects. Stabilized blood samples could be stored at 4°C for up to 96h before processing. ConclusionsThe novel assay for the enumeration of fibrocytes in the whole blood is reliable and clinically applicable. General significanceThis report demonstrates the validity and reliability of the first optimized assay for the enumeration of circulating fibrocytes in multicenter clinical trials.

Elevation in Peripheral Fibrocyte Counts Correlates With Presence of Interstitial Lung Disease in Patients With Hermansky-Pudlak Syndrome

SESSION TITLE: Biomarkers in ILD

Korean red ginseng ameliorates acute 3-nitropropionic acid-induced cochlear damage in mice

3-Nitropropionic acid (3-NP), a mitochondrial toxin, has been reported to induce an acute cochlear damage. Korean red ginseng (KRG) is known to have protective effects from some types of hearing loss. This study aimed to observe the protective effect of KRG in an ototoxic animal model using 3-NP intratympanic injection. BALB/c mice were classified into 5 groups (n=15) and dose-dependent toxic effects after intratympanic injection with 3-NP (300–5000mM) on the left ear were investigated to determine the appropriate toxicity level of 3-NP. For observation of the protective effects of KRG, 23 mice were grouped into 3-NP (500mM, n=12) and KRG+3-NP groups (300mg/kg KRG for 7 days before 500mM 3-NP administration, n=11). Auditory brain response (ABR) and cochlear morphological evaluations were performed before and after drug administration.The ABR thresholds in the 800–5000mM groups exceeded the maximum recording limit at 16 and 32kHz 1 day after 3-NP administration. The ABR threshold in the 500mM 3-NP+KRG group was significantly lower than that in the 500mM 3-NP group from post 1 week to 1 month. The mean type II fibrocyte counts significantly differed between the control and 3-NP groups and between the 3-NP and 3-NP+KRG groups. Spiral ganglion cell degeneration in the 3-NP group was more severe than that in the 3-NP+KRG group. This animal model exhibited a dose-dependent hearing loss with histological changes. KRG administration ameliorated the deterioration of hearing by 3-NP.

Tendinosis-like Histologic and Molecular Changes of the Achilles Tendon to Repetitive Stress: A Pilot Study in Rats

Tendinopathy (pain and tendon degeneration) is associated with repetitive use and mechanical overload. However, the etiology of tendinopathy remains unclear. Clarification of histologic and molecular changes of tendon to repetitive stress could provide better understanding of Achilles tendon disorders related to repetitive stress. We asked whether repetitive stress simulating overuse of the Achilles tendon induced (1) histologic changes in rats similar to tendinosis (increased cellularity of fibrocytes, increased disorganization of collagen fiber, and increased roundness of the nucleus of the fibrocyte), (2) increased collagen Type III occurrence, and (3) increased inducible nitric oxide synthase (iNOS) expression. We used an exercise protocol simulating repetitive, jerky, eccentric contraction of the triceps surae in 15 rats. We conducted the exercise for 2 hours per day, three times per week using the right rear legs only and the left legs as internal controls. We harvested Achilles tendons after either 2, 4, or 6 weeks of exercise, and evaluated changes in tendon thickness, fibrocyte count, collagen fiber arrangement, collagen fiber type, and occurrence of iNOS. Exercised Achilles tendons showed increased cellularity of fibrocytes at 4 and 6 weeks of exercise, and disorganized collagen fiber arrangement at 6 weeks of exercise. There was a trend for Type III collagen occurrence being greater in experimental groups. Expression of iNOS increased after 2 and 4 weeks of exercise when compared with that of the controls, but decreased after 6 weeks. These observations suggest repetitive, synchronized, passive, and jerky exercise induced by electrical stimulation can lead to the tendinosis-like changes in the Achilles tendons in rats with imbalance between synthesis and degeneration after 4 weeks of exercise. This newly designed exercise protocol may be used to design an animal model of Achilles tendon overuse. With this model, therapeutic interventions of tendinopathy could be analyzed by investigation of tendon biology and response in terms of histologic and molecular changes.

Non‐invasive biomarkers in pulmonary fibrosis

The practise of modern medicine is unthinkable without biological markers, and their adoption is now established in various clinical settings. Pulmonary fibrosis occurs in a heterogeneous group of diseases. IPF is the most frequent fibrotic lung disease and has a poor prognosis. Despite the heterogeneity in underlying diseases multiple common mechanism resulting in pulmonary fibrosis are documented. In this context, several biomarkers for pulmonary fibrosis have been described. Some substances are produced by alveolar epithelial cells such as surfactant proteins A and D, the glycoprotein Krebs von den Lungen 6 Antigen, while others are derived from macrophages such as CCL18. Most recently, increased fibrocyte counts were proposed as a marker of acute exacerbation in IPF. None of these markers is established in clinical practise so far. Studies regarding the clinical value of biomarkers in pulmonary fibrosis are hampered by the fact that fibrotic lung diseases are uncommon and variable in course. However, some markers are clearly promising, and as a spin-off the analysis of some of these in samples from recently finished pharmacological multicentric studies might give urgently needed information regarding their clinical value.

Circulating Fibrocytes Are an Indicator of Poor Prognosis in Idiopathic Pulmonary Fibrosis

The clinical management of idiopathic pulmonary fibrosis (IPF) remains a major challenge due to lack of effective drug therapy or accurate indicators for disease progression. Fibrocytes are circulating mesenchymal cell progenitors that are involved in tissue repair and fibrosis. To test the hypothesis that assay of these cells may provide a biomarker for activity and progression of IPF. Fibrocytes were defined as cells positive for CD45 and collagen-1 by flow cytometry and quantified in patients with stable IPF and during acute exacerbation of the disease. We investigated the clinical and prognostic value of fibrocyte counts by comparison with standard clinical parameters and survival. We used healthy age-matched volunteers and patients with acute respiratory distress syndrome as control subjects. Fibrocytes were significantly elevated in patients with stable IPF (n = 51), with a further increase during acute disease exacerbation (n = 7; P < 0.001 vs. control subjects). Patients with acute respiratory distress syndrome (n = 10) were not different from healthy control subjects or stable patients with IPF. Fibrocyte numbers were not correlated with lung function or radiologic severity scores, but they were an independent predictor of early mortality. The mean survival of patients with fibrocytes higher than 5% of total blood leukocytes was 7.5 months compared with 27 months for patients with less than 5% (P < 0.0001). Fibrocytes are an indicator for disease activity of IPF and might be useful as a clinical marker for disease progression. This study suggests that quantification of circulating fibrocytes may allow prediction of early mortality in patients with IPF.