Incipient Fibrosis Research Articles

ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD), which still lacks of reliable markers and therapeutic options. Cellular senescence has been considered an important mechanism of intestinal fibrosis, but the underlying molecular link remains elusive. Tissues were stained using α-smooth muscle actin (α-SMA), fibronectin, and collagen I as markers of myofibroblastic differentiation. Cellular senescence was confirmed through Lamin B1 staining, senescence-associated β-galactosidase staining, and the expression of senescence-associated secretory phenotype (SASP) factors. We explored the relationship between senescence of intestinal epithelial cells (IECs) and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. The effects of irisin on cellular senescence and fibrosis were determined. Here, we identify engulfment and cell motility protein 1 (ELMO1) as a novel biomarker for intestinal cellular senescence and fibrosis. In fibrostrictured tissues from patients and murine models with IBD, significantly high levels of cellular senescence score and factors were noted, which positively correlated with the fibrotic regulator fibronectin. Senescent IECs, not fibroblast itself, released SASP factors to regulate fibroblast activation. Prolonging exposure to severe and persistent injurious stimuli decreased ELMO1 expression, which dampened SIRT1 deacetylase activity, enhanced NF-κB (p65) acetylation, and thereby accelerated cellular senescence. Deletion of ELMO1 led to senescent IECs accumulation and triggered premature fibrosis in murine colitis. Furthermore, irisin, inhibiting the degradation of ELMO1, could downregulate p65 acetylation, reduce IECs senescence, and prevent incipient intestinal fibrosis in murine colitis models. This study reveals ELMO1 downregulation is an early symbol of intestinal senescence and fibrosis, and the altered ELMO1-SIRT1-p65 pathway plays an important role in intestinal cellular senescence and IBD-related fibrosis.

Highlighting Fibroblasts Activation in Fibrosis: The State-of-The-Art Fibroblast Activation Protein Inhibitor PET Imaging in Cardiovascular Diseases.

Fibrosis is a common healing process that occurs during stress and injury in cardiovascular diseases. The evolution of fibrosis is associated with cardiovascular disease states and causes adverse effects. Fibroblast activation is responsible for the formation and progression of fibrosis. The incipient detection of activated fibroblasts is important for patient management and prognosis. Fibroblast activation protein (FAP), a membrane-bound serine protease, is almost specifically expressed in activated fibroblasts. The development of targeted FAP-inhibitor (FAPI) positron emission tomography (PET) imaging enabled the visualisation of FAP, that is, incipient fibrosis. Recently, research on FAPI PET imaging in cardiovascular diseases increased and is highly sought. Hence, we comprehensively reviewed the application of FAPI PET imaging in cardiovascular diseases based on the state-of-the-art published research. These studies provided some insights into the value of FAPI PET imaging in the early detection of cardiovascular fibrosis, risk stratification, response evaluation, and prediction of the evolution of left ventricular function. Future studies should be conducted with larger populations and multicentre patterns, especially for response evaluation and outcome prediction.

Ekspresija neuralnog ćelijskog adhezionog molekula u intersticijumu bubrega sa različitim stepenom fibroze

Introduction: In healthy adult kidneys, differentiated tubular epithelial cells do not express the neural cell adhesion molecule (NCAM), while a small number of NCAM-expressing cells can be detected in the renal interstitium. The role and the significance of these cells have not yet been clarified, but it has been observed that the number of NCAM-expressing cells increases in the initial stage of interstitial fibrosis. Aim: The aim of the study is to examine the significance of the expression of NCAM molecules in the renal interstitium, in etiologically different diseases, with varying degrees of interstitial fibrosis, as well as to define the pathohistological and clinical indicators (predictors) of impaired kidney function. Materials and methods: The study included 69 patients who underwent needle biopsies of the kidneys in 2011 and 2012. Clinical and laboratory data were collected at the time of the biopsy and at the time of the latest follow-up examination. Pathohistological characteristics were defined optically-microscopically, while NCAM-expressing interstitial cells were detected with immunohistochemical staining, using the primary NCAM antibody (1:50, clone 123C3.D5). Results: NCAM-expressing interstitial cells were detected in 59.4% of kidney biopsies, the presence of these cells was significantly more frequent in the initial stages of interstitial fibrosis than in the remaining stages (p < 0.001), and it did not depend on the pathohistological diagnosis (p = 0.995). Patients in whom NCAM cells were detected had significantly lower proteinuria levels at the time of biopsy, as compared to patients without NCAM interstitial cells (p = 0.024). The levels of serum creatinine (p < 0.001) and urea (p = 0.007) significantly influenced the probability of the deterioration of renal function. Conclusion: The presence of NCAM cells in the kidney interstitium is a characteristic of the early stages of chronic kidney disease with incipient interstitial fibrosis and a lesser degree of proteinuria

Dual roles of B lymphocytes in mouse models of diet-induced nonalcoholic fatty liver disease.

Growing evidence suggests an important role of B cells in the development of NAFLD. However, a detailed functional analysis of B cell subsets in NAFLD pathogenesis is lacking. In wild-type mice, 21 weeks of high fat diet (HFD) feeding resulted in NAFLD with massive macrovesicular steatosis, modest hepatic and adipose tissue inflammation, insulin resistance, and incipient fibrosis. Remarkably, Bnull (JHT) mice were partially protected whereas B cell harboring but antibody-deficient IgMi mice were completely protected from the development of hepatic steatosis, inflammation, and fibrosis. The common feature of JHT and IgMi mice is that they do not secrete antibodies, whereas HFD feeding in wild-type mice led to increased levels of serum IgG2c. Whereas JHT mice have no B cells at all, regulatory B cells were found in the liver of both wild-type and IgMi mice. HFD reduced the number of regulatory B cells and IL-10 production in the liver of wild-type mice, whereas these increased in IgMi mice. Livers of patients with advanced liver fibrosis showed abundant deposition of IgG and stromal B cells and low numbers of IL-10 expressing cells, compatible with our experimental data. B lymphocytes have both detrimental and protective effects in HFD-induced NAFLD. The lack of secreted pathogenic antibodies protects partially from NAFLD, whereas the presence of certain B cell subsets provides additional protection. IL-10-producing regulatory B cells may represent such a protective B cell subset.

Liver Tumor Post Liver Transplantation for Hepatitis C Cirrhosis: An Unusual Cause

Question: A 60-year-old African American man with a history of hepatitis C virus (HCV) cirrhosis underwent orthotopic liver transplantation in 2009. The explant showed chronic HCV with mild activity and cirrhosis (grade 2, stage 4) without evidence of malignancy. The post-transplant course was complicated by recurrent HCV treated 5 years later with simeprevir and sofosbuvir with a sustained viral response. However, the patient developed fibrosing cholestatic hepatitis with rapid progression to fibrosis and portal hypertension demonstrated by small varices and ascites. A liver biopsy in August 2010 revealed mild to moderated inflammatory activity and incipient bridging fibrosis grades 2–3, stages 2–3. Due to worsening renal function and new-onset proteinuria, the dose of the calcineurin inhibitor was reduced and mycophenolate mofetil was added. He was lost to follow-up from 2014 to 2016. Magnetic resonance imaging in February 2017 for screening showed numerous masses in the liver with arterial enhancement and incomplete but progressive washout, some with an enhancing capsule (Figure A–C). At the time, his laboratory tests showed excellent graft function. What is the diagnosis and strategy for treatment? See the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Given the history of HCV infection and transplantation, there was concern for hepatocellular carcinoma. An ultrasound-guided core biopsy showed a vascular tumor positive for CD34 and negative for pancytokeratin and CK7, consistent with epithelioid hemangioendothelioma (HEHE) (Figure D-F). Low-power image on with hematoxylin and eosin staining (Figure D) shows upper core with normal liver, and lower core with replacement by vascular lesion (black arrow) with hemorrhage (thick black arrows). Figure E shows high power image with multiple diminutive vascular channels replacing liver parenchyma (black arrowheads), and Figure F with vascular channels replacing liver parenchyma on CD34 stain (black arrows). Figures A-C demonstrate HEHE lesions as target shaped lesion on a T1-weighted volumetric interpolated breath-hold examination sequence (Figure A), with largest lesion in a postcontrast sequence (Figure B), and multiple lesions seen on venous phase sequence (Figure C). Retransplantation was considered; however, given the indolent nature of the lesions, he was referred to oncology and interventional radiology for treatment. He did not make those appointments, and the latest blood tests in August 2020 showed stable graft function with no decompensation. He developed dementia and has not been able to follow-up with further imaging. HEHE was first described in 1984 and is a rare indolent malignant tumor with unclear etiology with vague presentations including abdominal discomfort, nausea, and vomitting.1Ishak K.G. Sesterhenn I.A. Goodman M.Z.D. et al.Epithelioid hemangioendothelioma of the liver: A clinicopathologic and follow-up study of 32 cases.Hum Pathol. 1984; 15: 839-852Crossref PubMed Scopus (382) Google Scholar HEHE rarely involves the vasculature, causing Budd–Chiari Syndrome, veno-occlusive disease, and portal hypertension. At diagnosis, extrahepatic involvement is reported in 36.6% of patients involving lungs, regional lymph nodes, and peritoneum.2Mehrabi A. Kashfi A. Fonouni H. et al.Primary malignant hepatic epithelioid hemangioendothelioma: a comprehensive review of the literature with emphasis on the surgical therapy.Cancer. 2006; 107: 2108-2121Crossref PubMed Scopus (343) Google Scholar Although the etiology is unknown, reported associations include oral contraceptive use, alcohol consumption, liver trauma, sarcoidosis, Crohn’s disease, viral hepatitis, and vinyl chloride or asbestos exposure.2Mehrabi A. Kashfi A. Fonouni H. et al.Primary malignant hepatic epithelioid hemangioendothelioma: a comprehensive review of the literature with emphasis on the surgical therapy.Cancer. 2006; 107: 2108-2121Crossref PubMed Scopus (343) Google Scholar Treatment options include a combination of liver resection, transarterial chemoembolization, liver transplantation, and the possible addition of chemotherapy.2Mehrabi A. Kashfi A. Fonouni H. et al.Primary malignant hepatic epithelioid hemangioendothelioma: a comprehensive review of the literature with emphasis on the surgical therapy.Cancer. 2006; 107: 2108-2121Crossref PubMed Scopus (343) Google Scholar,3Brahmbhatt M. Prenner S. Bittermann T. Liver transplantation for hepatic epithelioid hemangioendothelioma is facilitated by exception points with acceptable long-term outcomes.Transplantation. 2020; 104: 1187-1192Crossref PubMed Scopus (13) Google Scholar HEHE should be considered in the differential diagnosis of de novo liver masses after liver transplantation.

Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney.

ABSTRACTGlomerulosclerosis and tubulointerstitial fibrosis are pathological features of chronic kidney disease. Transforming growth factor β (TGFβ) is a key player in the development of fibrosis. However, of the three known TGFβ isoforms, only TGFβ1 has an established role in fibrosis, and the pathophysiological relevance of TGFβ2 and TGFβ3 is unknown. Because Tgfb3 deficiency in mice results in early postnatal lethality, we analyzed the kidney phenotype of heterozygous Tgfb3-knockout mice (Tgfb3+/−) and compared it with that of matched wild-type mice. Four-month-old Tgfb3+/− mice exhibited incipient renal fibrosis with epithelial–mesenchymal transition, in addition to glomerular basement membrane thickening and podocyte foot process effacement associated with albuminuria. Also evident was insulin resistance and oxidative stress at the renal level, together with aberrant renal lipid metabolism and mitochondrial function. Omics analysis revealed toxic species, such as diacylglycerides and ceramides, and dysregulated mitochondrial metabolism in Tgfb3+/− mice. Kidneys of Tgfb3+/− mice showed morphological alterations of mitochondria and overactivation of non-canonical MAPK ERK1/2 and JNK cascades. Our study indicates that renal TGFβ3 might have antifibrotic and renoprotective properties, opposing or counteracting the activity of TGFβ1. This article has an associated First Person interview with the first author of the paper.

Serum hemoglobin concentration and risk of renal function decline in early stages of diabetic kidney disease: a nationwide, biopsy-based cohort study.

Prognosticating disease progression in patients with diabetic kidney disease (DKD) is challenging, especially in the early stages of kidney disease. Anemia can occur in the early stages of kidney disease in diabetes. We therefore postulated that serum hemoglobin (Hb) concentration, as a reflection of incipient renal tubulointerstitial impairment, can be used as a marker to predict DKD progression. Drawing on nationally representative data of patients with biopsy-proven DKD, 246 patients who had an estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 at renal biopsy were identified: age 56 (45-63) years; 62.6% men; Hb 13.3 (12.0-14.5) g/dL; eGFR 76.2 (66.6-88.6) mL/min/1.73 m2; urine albumin-to-creatinine ratio 534 (100-1480) mg/g Crea. Serum Hb concentration was divided into quartiles: ≤12, 12.1-13.3, 13.4-14.5 and ≥14.6 g/dL. The association between serum Hb concentration and the severity of renal pathological lesions was explored. A multivariable Cox regression model was used to estimate the risk of DKD progression (new onset of end-stage kidney disease, 50% reduction of eGFR or doubling of serum creatinine). The incremental prognostic value of DKD progression by adding serum Hb concentration to the known risk factors of DKD was assessed. Serum Hb levels negatively correlated with all renal pathological features, especially with the severity of interstitial fibrosis (ρ = -0.52; P < 0.001). During a median follow-up of 4.1 years, 95 developed DKD progression. Adjusting for known risk factors of DKD progression, the hazard ratio in the first, second and third quartile (the fourth quartile was reference) were 2.74 [95% confidence interval (CI) 1.26-5.97], 2.33 (95% CI 1.07-5.75) and 1.46 (95% CI 0.71-3.64), respectively. Addition of the serum Hb concentration to the known risk factors of DKD progression improved the prognostic value of DKD progression (the global Chi-statistics increased from 55.1 to 60.8; P < 0.001). Serum Hb concentration, which reflects incipient renal fibrosis, can be useful for predicting DKD progression in the early stages of kidney disease.

Phenotypic Characterization of CD4+ T Lymphocytes in Periportal Fibrosis Secondary to Schistosomiasis.

Schistosomiasis is a parasitic disease that affects about 166 million people around the world. It is estimated that 5%–10% of individuals with schistosomiasis develop severe forms of the disease, which are characterized by pulmonary hypertension, ascites, periportal fibrosis, and other significant complications. The chronic phase of the disease is associated with a Th2 type immune response, but evidence also suggests there are roles for Th1 and Th17 in the development of severe disease. The aim of this study was to evaluate the CD4+ T lymphocyte profile of patients with different degrees of periportal fibrosis secondary to schistosomiasis. These individuals had been treated for schistosomiasis, but since they live in a S. mansoni endemic area, they are at risk of reinfection. They were evaluated in relation to the degree of periportal fibrosis and classified into three groups: without fibrosis or with incipient fibrosis (WF/IFNE), n=12, possible periportal fibrosis/periportal fibrosis, n=13, and advanced periportal fibrosis/advanced periportal fibrosis with portal hypertension, n=4. We observed in the group without fibrosis a balance between the low expression of Th2 cytokines and high expression of T reg cells. As has already been described in the literature, we found an increase of the Th2 cytokines IL-4, IL-5, and IL-13 in the group with periportal fibrosis. In addition, this group showed higher expression of IL-17 and IL-10 but lower IL-10/IL-13 ratio than patients in the WF/IFNE group. Cells from individuals who present any level of fibrosis expressed more TGF-β compared to the WF/IFNE group and a positive correlation with left lobe enlargement and portal vein wall thickness. There was a negative correlation between IL-17 and the thickness of the portal vein wall, but more studies are necessary in order to explore the possible protective role of this cytokine. Despite the fibrosis group having presented a higher expression of pro-fibrotic molecules compared to WF/IFNE patients, it seems there is a regulation through IL-10 and T reg cells that is able to maintain the low morbidity of this group.

NASH-inducing Diets in Göttingen Minipigs

BackgroundOwing to the human-like physiology, a minipig model of nonalcoholic steatohepatitis (NASH) could be valuable. Pigs, however, rarely develop substantial hepatic steatosis, even when fed diets with high fat, fructose, and cholesterol (FFC) content. The potential of choline-deficient, amino acid–defined high-fat diets (CDAHFD) was therefore evaluated in Göttingen Minipigs.MethodsCastrated male Göttingen Minipigs were fed either chow (n = 5) or one of the three NASH diets: FFC (n = 5), CDAHFD with sucrose (CDAHFD-S; n = 4), or fructose (CDAHFD-F; n = 4) for 8 weeks. Liver and blood samples were collected after 2 weeks and at termination.ResultsCompared with chow, the body weight was higher after FFC (9.8 ± 0.4 versus 8.5 ± 1.2 kg; mean ± SD) and less after CDAHFD-S (6.4 ± 0.8 kg) and CDAHFD-F (6.9 ± 0.8 kg). Liver weight per kg body weight was significantly increased in all 3 NASH groups (FFC 2.1 times; and both CDAHFD diets 3.1 times). Histologically, pronounced macrovesicular steatosis developed only in the CDAHFD groups. Inflammation was present in all three NASH groups. In the CDAHFD groups, inflammatory cells formed crown-like structures around steatotic hepatocytes. Sirius red staining revealed mild fibrosis in the two CDAHFD groups with the fibrotic potential being further supported by immunohistochemical staining for activated stellate cells and gene expression analyses. No noticeable differences were found between CDAHFD-S and CDAHFD-F.ConclusionsGöttingen Minipigs fed CDAHFD developed pronounced steatosis with inflammation around steatotic hepatocytes and incipient fibrosis, thereby showing potential as a model for human NASH. Further studies are needed to investigate the period needed for marked fibrosis to develop.

Diet-Induced Pulmonary Inflammation and Incipient Fibrosis in Mice: a Possible Role of Neutrophilic Inflammation.

Chicken fat and fructose are added into food-processing to reduce costs and enhance acceptability; however, these additives turn food into unhealthy and hypercaloric meals. Herein we havehypothesized that chronic feeding with chicken fat and fructose, together or by separate, can cause pulmonary redox and inflammatory changes. These changes are particularly related to neutrophils and myeloperoxidase, with consequent changes in the organ histophysiology. To test this hypothesis, we fed mice for 16weeks with either control food (low-fat diet, LFD) or control food supplemented with 22% chicken fat and with or without 10% fructose in the drinking water. At the end of the feeding regimen, we measured redox and inflammatory changes in the lung with particular emphasis on neutrophil accumulation/activation and molecular-histological markers of fibrosis. Our results suggest that a diet supplemented with chicken fat and fructose causes additive effects on pulmonary oxidative stress, inflammation, and a pro-fibrotic status. Neutrophilic inflammation may play a critical role in pulmonary pathology associated with metabolic syndrome.

PM2.5 induced pulmonary fibrosis in vivo and in vitro

Epidemiological studies have revealed positive correlation between particulate matter with an aerodynamic diameter of < 2.5 µm (PM2.5) and pulmonary fibrosis (PF). As etiology and pathogenesis of PF have not been fully elucidated, this study was to investigate the potential mechanism by which PM2.5 exposure adversely induced PF in vivo and in vitro. In the present study, 6-week-old C57/BL6J mice were intranasally administrated with PM2.5 (100 μg/day) for 4 weeks. Micro-CT and hematoxylin-eosin (HE) staining analysis showed that lung inflammation and incipient fibrosis symptoms were induced after PM2.5 exposure. The expression of Transforming growth factor-β1 (TGF-β1), α-Smooth muscle actin (α-SMA), and Collagen type I (COL1) in mice lung was increased. Upregulation of TGF-β1 in mice serum was also detected by ELISA after exposure to PM2.5. Moreover, chronic PM2.5 exposure on human bronchial epithelial cell line BEAS-2B cells led to activation of TGF-β1/SMAD3 pathway, TGF-β1 excretion and epithelial-mesenchymal transition (EMT), while PM2.5 also triggered the activation of TGF-β1/SMAD3 pathway, TGF-β1 excretion as well as differentiation of human pulmonary fibroblast cell line HFL-1 cells, and TGF-β1 production in mouse macrophage cell line RAW264.7 cells. Furthermore, cell culture medium of PM2.5-treated BEAS-2B and RAW264.7 cells could both activate TGF-β1/SMAD3 signaling, α-SMA and COL1 upregulation in HFL-1 cells. Therefore, we concluded that PM2.5 could induce PF by targeting pulmonary epithelium, macrophages and fibroblasts, suggesting that PM2.5 was a potent initiator of PF.

The Value of Using a SkinFibroMeter for Diagnosis and Assessment of Secondary Lymphedema and Associated Fibrosis of Lower Limb Skin.

Skin fibrosis is a clinically serious pathological process of secondary lymphedema (SLE). The detection of latent or incipient skin fibrosis, including early tissue changes, using sensitive diagnostic equipment is essential. The SkinFibroMeter has been used in skin care; however, its ability to assess skin fibrosis in patients with SLE of the lower limb has not been explored. In this study, 45 female patients with secondary unilateral lower limb LE and 15 healthy female volunteers were enrolled. The skin stiffness values of affected and healthy limbs were measured by using the SkinFibroMeter, and skin samples were assayed by Masson staining. The circumference and interstitial fluid of limbs were also measured and analyzed. Increasing skin stiffness with increasing severity of SLE of the lower limb and skin stiffness significantly positively correlated with stage of lower limb SLE. Our results demonstrate that the SkinFibroMeter is very sensitive and accurate for detecting skin fibrosis of edematous limb. Therefore, this new instrument is a promising prospect for diagnosis and assessment of skin fibrosis in patients with lower limb SLE.

Beneficial Effects of Silymarin After the Discontinuation of CCl4-Induced Liver Fibrosis.

Silymarin (Si) is a herbal product with hepatoprotective potential, well-known for its antioxidant, anti-inflammatory, and immunomodulatory properties. We have recently demonstrated that the usual therapeutic doses of Si are capable of inhibiting the progression of incipient liver fibrosis. We aimed at further investigating the benefits of Si administration upon liver alterations after the hepatotoxin discontinuation, using CCl4 to induce liver injuries on rats. CCl4 administration induces first of all oxidative stress, but other mechanisms, such as inflammation and liver fibrosis are also triggered. Fifty Wistar rats were randomly divided into five groups (n = 10). The control group received sunflower oil twice a week for 8 weeks. Carboxymethyl cellulose group received sunflower oil twice a week, for 8 weeks and CMC daily, for the next 2 weeks. CCl4 group received CCl4 in sunflower oil, by gavage, twice a week, for 8 weeks. CCl4 + Si 50 group received CCl4 twice a week, for 8 weeks, and then 50 mg/body weight (b.w.) Silymarin for the next 2 weeks. CCl4 + Si 200 group was similar to the previous group, but with Si 200 mg/b.w. Ten weeks after the experiment had begun, we assessed inflammation (IL-6, MAPK, NF-κB, pNF-κB), fibrosis (hyaluronic acid), TGF-β1, MMP-9, markers of hepatic stellate cell activation (α-SMA expression), and proliferative capacity (proliferating cell nuclear antigen). Our data showed that Silymarin administered after the toxic liver injury is capable of reducing inflammation and liver fibrosis. The benefits were more important for the higher dose than for the usual therapeutic dose.

Sarcoïdose cutanée à type de livédo

The cutaneous signs of sarcoidosis are extremely polymorphous and may be classified under several different headings. Herein, we report the case of a 51-year-old female patient presenting bilateral livedo of the knees accompanied by systemic signs with polyarthralgia, impaired general state of health, weight loss, and a sensation of dyspnoea. Skin biopsy revealed giant-cell granuloma around the dermal vessels, with no caseous necrosis. Histopathological examination of the patient's blood vessels revealed no abnormalities. Laboratory tests showed high levels of angiotensin converting enzyme (1.5×ULN), bilateral mediastinal adenomegaly and incipient pulmonary fibrosis. Systemic sarcoidosis was diagnosed on the basis of the overall clinical and laboratory picture. The patient subsequently presented hepatic involvement and hypercalcaemia associated with the sarcoidosis; systemic corticosteroid therapy (prednisone) was initiated, with slow dose-reduction. The outcome was favourable with a return to normal laboratory values, regression of the adenomegaly and subsidence of the livedo. The literature contains reports of a number of cases of livedo heralding sarcoidosis. The majority of such cases involved young or middle-aged female patients of Asian origin presenting systemic sarcoidosis, with a high frequency of ocular and neural involvement. Livedo could be accounted for by the specific disposition of granulomas around the arterioles, resulting in disturbance of local blood flow, which was probably associated with the mechanical compression of vessels and with the micro-thrombi noted at histology. Livedo may be considered a clinical sign of cutaneous sarcoidosis. Laboratory tests to screen for sarcoidosis may be proposed in the exploration of atypical livedo.

Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis.

Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms’ RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys.

Convenient formulation and application of a supramolecular ureido-pyrimidinone modified poly(ethylene glycol) carrier for intrarenal growth factor delivery

The development of local, intrarenal drug delivery therapies is imperative to induce a therapeutic effect without the requirement of high concentrations of drugs, thereby diminishing systemic side effects. Hydrogels are eminently suitable as drug delivery carriers in soft tissues. Here, we show that a supramolecular hydrogel carrier based on ureido-pyrimidinone (UPy) modified poly(ethylene glycol) can be easily formulated and conveniently be applied to deliver anti-inflammatory and anti-fibrotic growth factor protein BMP7 to the kidney. Short-term, immediate modulation of renal inflammation and extracellular matrix remodelling is shown in a rat model of acute kidney injury. Induction of ischemia/reperfusion injury was followed by renal subcapsular implantation of pristine and BMP7-loaded supramolecular hydrogels. The cortical area under the site of implantation was studied after 3 and 7days. Subcapsular delivery of only 0.30μg BMP7 from these supramolecular hydrogels led to a significant reduction in interstitial inflammatory and myofibroblast cell numbers at the site of implantation. These findings show that local, intrarenal delivery of an anti-inflammatory and anti-fibrotic drug from a supramolecular hydrogel carrier can be effective in the reduction of acute inflammation and incipient fibrosis.

Histopathological and biochemical assessment of d-limonene-induced liver injury in rats

The aim of the present work was to develop a biochemical, histologic and immunohistochemical study about the potential hepatotoxic effect of d-limonene – a component of volatile oils extracted from citrus plants. Blood alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from d-limonene-treated animals were determined and compared to morphologic hepatic lesions in order to investigate the possible physiopathologic mechanisms involved in the liver toxicity, in experimental animals treated with d-limonene. Wistar rats were randomly divided into seven groups: two control groups (untreated or receiving only vehicle, tween-80); one positive control (vehicle); two experimental groups treated with d-limonene at doses of 25mg/kg/day and 75mg/kg/day for 45 days, and two other groups treated with the same doses for 30 days and kept under observation during 30 more days. Biochemical data showed significant reduction in ALT levels in the animals treated with 75mg/kg of d-limonene. Histological analysis revealed some hepatocyte morphological lesions, including hydropic degeneration, microvesicular steatosis and necrosis, Kupffer cell hyperplasia and incipient fibrosis. By immunohistochemistry, influx of T (CD3+) and cytotoxic (CD8+) lymphocytes was observed in the rats treated with d-limonene at both dose levels. In conclusion, it is possible that d-limonene has been directly responsible for hepatic parenchymal and matrix damage following subchronic treatment with d-limonene.

The WHO ultrasonography protocol for assessing hepatic morbidity due to Schistosoma mansoni. Acceptance and evolution over 12 years.

The aim of this study is to review the worldwide acceptance of the World Health Organization (WHO) ultrasound protocol for assessing hepatosplenic morbidity due to Schistosoma mansoni since its publication in 2000. A PubMed literature research using the keywords "schistosomiasis and ultrasound," "schistosomiasis and ultrasonography," and "S. mansoni and ultrasound" from 2001 to 2012 was performed. Case reports, reviews, reports on abnormalities due to parasites other than S. mansoni, organ involvement other than the human liver, and reports where ultrasound method was not described were excluded. Six studies were retrieved from other Brazilian sources. Sixty studies on 37,424 patients from 15 countries were analyzed. The WHO protocol was applied with increasing frequency from 43.75% in the years 2001 to 2004 to 84.61% in 2009 to 2012. Results obtained using the pictorial image pattern approach of the protocol are reported in 38/41 studies, whereas measurements of portal branch walls were applied in 19/41 and results reported in 2/41 studies only. The practical usefulness of the pictorial approach of the WHO protocol is confirmed by its wide acceptance. This approach alone proved satisfactory in terms of reproducibility, assessment of evolution of pathology, and comparability between different settings. The measurements of portal branches, also part of the protocol, may be omitted without losing relevant information since results obtained by these measurements are nonspecific. This would save resources by reducing the time required for each examination. It is also more feasible for examiners who are not specialized in medical imaging. As with all protocols, incipient liver fibrosis is difficult to distinguish from normal ultrasound findings of the liver. The ability of this protocol to predict complications in severe cases should be further evaluated in a higher number of patients.

Impaired Lymphocyte Profile in Schistosomiasis Patients with Periportal Fibrosis

The Th2 immune response in chronic schistosomiasis is associated with the development of periportal fibrosis. However, little is known about the phenotype and activation status of T cells in the process. Objective. To evaluate the profile of T cells in schistosomiasis patients with periportal fibrosis. Methods. It was a cross-sectional study, conducted in the village of Agua Preta, Bahia, Brazil, which included 37 subjects with periportal fibrosis determined by ultrasound. Peripheral blood mononuclear cells were obtained by the Ficcol-hypaque gradient and the frequency of T cells expressing the surface markers CD28, CD69, CD25, and CTLA-4 was determined by flow cytometry. Results. The frequency of CD4+CD28+ T lymphocytes was higher in individuals with moderate to severe fibrosis compared to patients with incipient fibrosis. We did not observe any significant difference in the frequency of CD4+ T cells expressing CD69 among groups of individuals. There was also no significant difference in the frequency of CD8+ T cells expressing CD28 or CD69 among the studied groups. Individuals with moderate to severe fibrosis presented a lower frequency of CD8+ T cells, CD4+CD25high T cells, and CD4+CTLA-4+ T cells when compared to patients without fibrosis or incipient fibrosis. The frequency of CD4+CD25low cells did not differ between groups. Conclusion. The high frequency of activated T cells coinciding with a low frequency of putative Treg cells may account for the development of periportal fibrosis in human schistosomiasis.

Immunomodulatory effect of R848 on cytokine production associated with Schistosoma mansoni infection

This work aimed to determine the effect of resiquimod (R848), a low molecular weight imidazoquinolinamine compound with properties as immune response modifiers, on cytokine production in patients infected with Schistosoma mansoni with various degrees of hepatic fibrosis. Schistosoma-infected patients were classified into four groups according to the degree of hepatic fibrosis as examined by ultrasonography group 0 (no fibrosis), group I (incipient fibrosis), group II (moderate fibrosis), and group III (severe fibrosis). Levels of interferon-γ (IFN-γ), interleukin 4 (IL-4), IL-10, and IL-13 were measured by ELISA in the supernatants of cultured peripheral blood mononuclear cells (PBMC) in response to schistosoma egg soluble antigen (SEA) in the presence and absence of R848. R848 induced a significantly higher level of IL-10 in supernatants of SEA-stimulated PBMC of patients in all groups of hepatic fibrosis except group III. Moreover, we significantly found higher levels of IFN-γ in group 0 and group I and significantly lower level of IL-4 in group 0 after R848 stimulation. However, R848 had no effect on IL-13. Taken together, these results suggest that R848 might be used as an immunopotentiating agent in view of novel therapy or vaccine for S. mansoni infection.