Parenchymal Cell Loss Research Articles

Targeting the progression of chronic kidney disease.

Chronic kidney disease (CKD) is a devastating condition that is reaching epidemic levels owing to the increasing prevalence of diabetes mellitus, hypertension and obesity, as well as ageing of the population. Regardless of the underlying aetiology, CKD is slowly progressive and leads to irreversible nephron loss, end-stage renal disease and/or premature death. Factors that contribute to CKD progression include parenchymal cell loss, chronic inflammation, fibrosis andreduced regenerative capacity of the kidney. Current therapies have limited effectiveness and only delay disease progression, underscoring the need to develop novel therapeutic approaches to either stop or reverse progression. Preclinical studies have identified several approaches that reduce fibrosis in experimental models, including targeting cytokines, transcription factors, developmental and signalling pathways and epigenetic modulators, particularly microRNAs. Some of these nephroprotective strategies are now being tested in clinical trials. Lessons learned from the failure of clinical studies of transforming growth factor β1 (TGFβ1) blockade underscore the need for alternative approaches to CKD therapy, as strategies that target a single pathogenic process may result in unexpected negative effects on simultaneously occurring processes. Additional promising avenues include preventing tubular cell injury and anti-fibrotic therapies that target activated myofibroblasts, the main collagen-producing cells.

Cell Death in the Kidney.

Apoptotic cell death is usually a response to the cell’s microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.

Apoptosis and necroptosis in the liver: a matter of life and death.

Cell death represents a basic biological paradigm that governs outcomes and long-term sequelae in almost every hepatic disease condition. Acute liver failure is characterized by massive loss of parenchymal cells but is usually followed by restitution ad integrum. By contrast, cell death in chronic liver diseases often occurs at a lesser extent but leads to long-term alterations in organ architecture and function, contributing to chronic hepatocyte turnover, the recruitment of immune cells and activation of hepatic stellate cells. These chronic cell death responses contribute to the development of liver fibrosis, cirrhosis and cancer. It has become evident that, besides apoptosis, necroptosis is a highly relevant form of programmed cell death in the liver. Differential activation of specific forms of programmed cell death might not only affect outcomes in liver diseases but also offer novel opportunities for therapeutic intervention. Here, we summarize the underlying molecular mechanisms and open questions about disease-specific activation and roles of programmed cell death forms, their contribution to response signatures and their detection. We focus on the role of apoptosis and necroptosis in acute liver injury, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and liver cancer, and possible translations into clinical applications.

Ductular Reaction and the K7-19 Progenitor Cell Sequence in Vascular Liver Disease: A New Perspective to Pathogenetic Mechanisms of Injury and Repair

Background:Ductular reaction (DR), a process intricately connected with hepatocyte progenitor cells (HPCs) at the canal of Hering evokes considerable scientific interest. The present study was undertaken to (a) identify DR patterns in different subsets of hepatic vascular disease, (b) localize intermediate hepatobiliary progenitors activated in response to stresses to vascular flow while studying their K7/19 immunoexpression profiles, and (c) possibly provide new insights to pathogenesis. Methods: Forty-six cases of hepatic vasculopathy including Budd-Chiari syndrome [BCS] (11), acute hemorrhagic necrosis [AHN] (6), veno-occlusive disease [VOD] (7), non-cirrhotic portal fibrosis [NCPF] (10), extrahepatic portal vein obstruction [EHPVO] (10) and chronic venous congestion [CVC] (2) were randomly selected. Mean DR was scored as 1: 2–5, 2: 6–8, 3 > 8 after counting ductules per portal tract (D/ PT) separately on K7 and 19 immunostains. Extraportal ‘‘progenitors’’ were semiquantitatively graded as 0: None, 1: Occasional, 2: Significant number. This was done separately for hepatocytic and ductular cells on K7 & 19. Zonation effect, if any was noted. Results: DR, intermediate hepatocytic progenitors and extraportal ductules were seen in 69.5/45.6%, 84.7/30.4% and 65.2/50% cases respectively on K7/ 19 stain. DR was seenmost frequently in the subset of AHN (100%) and least in EHPVO (40%). All cases of BCS, AHN and CVC showed intermediate K7+ hepatocytic cells. Extraportal ductular cells (K7+/K19 ) were most common in AHN (100%) and showed a distinct centrilobular pattern. Conclusions: Derangements in vascular flow trigger prominent ductular reaction in all subsets of hepatic vasculopathy barring EHPVO. Severe parenchymal loss as seen in AHN activates both biliary and hepatocytic intermediate cells possibly as a response to extensive parenchymal cell loss. Vascular liver diseases show heterogeneity in K7/19 expression profiles of extraportal progenitors across all subsets, emphasizing that as a group vasculopathies may be trickier to reverse. Timely diagnosis hence is paramount tominimize damage. Corresponding author Tel: +91 9718599073: Puja Sakhuja. E-mail: pujasak@gmail.com

Methods for defining vascular and parenchymal leukocyte populations: implications for characterizing innate and adaptive immune responses in non-lymphoid tissue (P3281)

Abstract Defining the anatomic location of cells of the innate and adaptive immune system is critical for understanding immune responses to infection. Recently, we published that intravascular staining can distinguish CD8 T cells located in the lung tissue from those confined to capillary vasculature. Remarkably, up to 95% of CD8 T cells isolated from lung were present within capillaries, despite extensive perfusion, and removing these cells from analysis resulted in revised interpretations of lung T cell homing and differentiation. We now show that perfusion results in a disruption of lung architecture and a loss of parenchymal cells of interest. Moreover, we expand our analysis to other leukocyte populations (lymphoid and myeloid) in additional non-lymphoid tissues and in the context of a variety of disease models, including graft versus host disease and infection with influenza or Mycobacterium tuberculosis. We have defined revised methodologies that, if applied, will have a pronounced impact on the interpretation of studies examining numerous leukocyte subsets in many non-lymphoid compartments. We are now utilizing these methods to examine the dynamics of primary and anamnestic responses to, and mechanisms of protection against, respiratory infections.

Mesenchymal stem cell–conditioned medium reduces liver injury and enhances regeneration in reduced-size rat liver transplantation

BackgroundMesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promotes tissue repair through the action of trophic, secreted molecules. In this study, we investigated whether MSC-conditioned medium (MSC-CM) could protect hepatocytes and sinusoidal endothelial cells (SECs) and stimulate their regeneration in 50% reduced-size liver transplantation (RSLT). Materials and methodsRats were randomly divided into three groups: sham-operated group, MSC-CM group (rats with 50% RSLT receiving MSC-CM infusion), and medium group (rats with 50% RSLT receiving medium therapy). Graft function, proinflammatory cytokines, incidence of apoptosis, proliferation of hepatocytes and SECs, and the expression of vascular endothelial growth factor and matrix metallopeptidase 9 were assessed in this study. ResultsSystemic infusion of MSC-CM prevented the release of liver injury biomarkers and provided a significant survival benefit. Furthermore, MSC-CM therapy resulted in reduction of apoptosis of hepatocytes and SECs. The number of proliferating hepatocytes and SECs increased 1.2- and 1.6-fold, respectively, accompanied by a decrease in the expression levels of several proinflammatory cytokines and a noticeable decrease in infiltration of neutrophils and activation of Kupffer cells. Also, increased expression of vascular endothelial growth factor and matrix metallopeptidase 9 in the grafts was observed after MSC-CM therapy. ConclusionsThese data suggest that MSC-CM therapy in RSLT provides trophic support to the injured liver by inhibiting SEC and hepatocellular death and stimulating their regeneration.

Mesenchymal Stem Cells Overexpressing C-X-C Chemokine Receptor Type 4 Improve Early Liver Regeneration of Small-for-Size Liver Grafts

Mesenchymal stem cell (MSC) therapy can prevent hepatic parenchymal cell loss and promote tissue repair. However, poor MSC engraftment is one of the primary barriers to the effectiveness of cell therapy because culture-expanded MSCs progressively down-regulate C-X-C chemokine receptor type 4 (CXCR4) expression and lose their ability to migrate toward a concentration gradient of stromal cell-derived factor 1a (SDF1a). In this study, we investigated whether a CXCR4-MSC infusion could protect hepatocytes and stimulate regeneration in 50% reduced size liver transplantation (RSLT). Rats that underwent 50% RSLT were randomly divided into 3 groups: a phosphate-buffered solution group (PBS), a green fluorescent protein (GFP)-MSC group, and a CXCR4-MSC group. Rats received 1 mL of PBS with or without a resuspension of GFP-MSCs or CXCR4-MSCs. The factors secreted by MSCs, the graft function, the apoptosis and proliferation of hepatocytes, the efficacy of MSC engraftment, and the expression of SDF1α, albumin (Alb), and cytokeratin 18 (CK18) in engrafted GFP-positive MSCs were assessed. A systemic infusion of GFP-MSCs led to a reduction of the release of liver injury biomarkers and apoptosis of hepatocytes; CXCR4 overexpression did not further reduce the liver injury. However, CXCR4 overexpression enhanced MSC engraftment in liver grafts, improved the effect on the proliferation of hepatocytes, and thus provided a significant 1-week survival benefit. SDF1α expression in grafts was elevated after transplanted CXCR4-MSCs were recruited to the remnant liver. However, engrafted MSCs did not express the markers of hepatocytes, including Alb and CK18, in vivo 168 hours after transplantation. CXCR4 overexpression enhanced the mobilization and engraftment of MSCs into small-for-size liver grafts, in which these cells promoted the early regeneration of the remnant liver not by direct differentiation but perhaps by a paracrine mechanism.

Sphingolipid-mediated Inhibition of Apoptotic Cell Clearance by Alveolar Macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Potential role of CXCL10 in the induction of cell injury and mitochondrial dysfunction

Chemokines have been known to play a critical role in pathogenesis of chronic pancreatitis and acinar cell death. However, the role played by one of the CXC chemokines: CXCL10 in regulation of acinar cell death has remained unexplored. Hence, this study was designed to assess the role of CXCL10 promoting apoptosis in ex vivo cultured acinar cells. Primary human pancreatic acinar cell cultures were established and exposed to varying doses of CXCL10 for different time intervals. Apoptotic induction was evaluated by both qualitative as well as quantitative analyses. Various mediators of apoptosis were also studied by Western blotting, membrane potential (Psim) and ATP depletion in acinar cells. Analysis of apoptosis via DNA ladder and cell death detection - ELISA demonstrated that CXCL10 induced 3.9-fold apoptosis when administrated at an optimal dose of 0.1 mug of recombinant CXCL10 for 8 h. Quantitative analysis using FACS and dual staining by PI-annexin showed increased apoptosis (48.98 and 53.78% respectively). The involvement of upstream apoptotic regulators like pJNK, p38 and Bax was established on the basis of their increased expression of CXCL10. The change of Psim by 50% was observed in the presence of CXCL10 in treated acinar cells along with enhanced expression of Cytochrome C, apaf-1 and caspase 9/3 activation. In addition, ATP depletion was also noticed in CXCL10 stimulated acinar cells. CXCL10 induces cell death in human cultured pancreatic cells leading to apoptosis and DNA fragmentation via CXCR3 signalling. These signalling mechanisms may play an important role in parenchymal cell loss and injury in pancreatitis.

Targeting the TGF-β1 Pathway to Prevent Normal Tissue Injury After Cancer Therapy

With >10,000,000 cancer survivors in the U.S. alone, the late effects of cancer treatment are a significant public health issue. Over the past 15 years, much work has been done that has led to an improvement in our understanding of the molecular mechanisms underlying the development of normal tissue injury after cancer therapy. In many cases, these injuries are characterized at the histologic level by loss of parenchymal cells, excessive fibrosis, and tissue atrophy. Among the many cytokines involved in this process, transforming growth factor (TGF)-beta1 is thought to play a pivotal role. TGF-beta1 has a multitude of functions, including both promoting the formation and inhibiting the breakdown of connective tissue. It also inhibits epithelial cell proliferation. TGF-beta1 is overexpressed at sites of injury after radiation and chemotherapy. Thus, TGF-beta1 represents a logical target for molecular therapies designed to prevent or reduce normal tissue injury after cancer therapy. Herein, the evidence supporting the critical role of TGF-beta1 in the development of normal tissue injury after cancer therapy is reviewed and the results of recent research aimed at preventing normal tissue injury by targeting the TGF-beta1 pathway are presented.

Cell death serum biomarkers are early predictors for survival in severe septic patients with hepatic dysfunction

IntroductionSevere sepsis, septic shock, and resulting organ failure represent the most common cause of death in intensive care medicine, with mortality ranging from 40% to 70%. It is still unclear whether necrosis or apoptosis plays the predominant role in severe sepsis. Determining the prevalent mode of cell death would be valuable, as new therapeutic agents (eg, antiapoptotic drugs such as caspase inhibitors) may improve unsatisfactory outcomes in patients with severe sepsis. Furthermore, the prognostic value of newly developed cell death serum biomarkers is of great interest.MethodsIn total, 147 patients (101 patients with severe sepsis, 28 postoperative patients after major abdominal surgery, 18 healthy volunteers) were enrolled. Baseline and clinical data were evaluated. Blood samples from patients with severe sepsis were collected at the time of sepsis diagnosis, and 48 and 120 hours later; samples from healthy volunteers were collected once, and from postoperative patients, once immediately after surgery. We measured caspase-cleaved and uncleaved cytokeratin-18 (CK-18, intermediate filament protein) as a marker of cell death, isolated CK-18 fragments as a marker of apoptosis, as well as IL-6, soluble vascular cell adhesion molecule, and soluble intercellular adhesion molecule.ResultsAge and sex of patients with severe sepsis and postoperative patients were comparable, whereas healthy volunteers were significantly younger. In healthy volunteers, the mode of cellular turnover was primarily apoptotic cell death. Postoperative patients showed comparable levels of apoptotic activity, but necrotic cell death was markedly increased, probably due to surgical tissue injury. In contrast, patients with severe sepsis, and especially non-survivors of the septic group showed increased levels of markers for both apoptotic and necrotic cell death. In severe septic patients with liver dysfunction, necrosis is increased relative to severe septic patients with intact hepatic function. For severe septic patients with liver dysfunction, a cut-off value for caspase-cleaved and uncleaved cytokeratin-18 could be calculated, in order to identify patients at high risk for death due to severe sepsis.ConclusionsThe measurement of caspase-cleaved and uncleaved cytokeratin-18 appears to be an early predictor for survival in severe septic patients with hepatic dysfunction. Furthermore, the loss of parenchymal cells due to necrosis may be the primary mode of cell death in these patients. This may limit possible therapeutic options.

Mechanisms of Renal Apoptosis in Health and Disease

Apoptotic cell death is usually a response to the cell microenvironment. Apoptosis requires the activation of lethal molecules and the inactivation of prosurvival ones. Both are potential therapeutic targets. Apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury. Apoptotic pathways that are active in glomerular and tubular epithelium include death induced by survival factor deprivation, death receptor activation, mitochondrial injury, endoplasmic reticulum stress, lysosomal destabilization, and caspase cascade activation. These pathways are not mutually exclusive, and stimulus-specific differences in the recruitment of apoptotic pathways have been observed. In some cases, the activation of a certain death pathway is redundant, and its inhibition does not prevent eventual cell death. This review summarizes recent advances in the field and discusses the rational basis to choose from the available tools to target apoptosis therapeutically.

Mesenchymal stem cell–derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo

Orthotopic liver transplantation is the only proven effective treatment for fulminant hepatic failure (FHF), but its use is limited because of organ donor shortage, associated high costs, and the requirement for lifelong immunosuppression. FHF is usually accompanied by massive hepatocellular death with compensatory liver regeneration that fails to meet the cellular losses. Therefore, therapy aimed at inhibiting cell death and stimulating endogenous repair pathways could offer major benefits in the treatment of FHF. Recent studies have demonstrated that mesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promote tissue repair in models of myocardial infarction, acute kidney failure, and stroke through the action of trophic secreted molecules. In this study, we investigated whether MSC therapy can protect the acutely injured liver and stimulate regeneration. In a D-galactosamine-induced rat model of acute liver injury, we show that systemic infusion of MSC-conditioned medium (MSC-CM) provides a significant survival benefit and prevents the release of liver injury biomarkers. Furthermore, MSC-CM therapy resulted in a 90% reduction of apoptotic hepatocellular death and a three-fold increment in the number of proliferating hepatocytes. This was accompanied by a dramatic increase in the expression levels of 10 genes known to be up-regulated during hepatocyte replication. Direct antiapoptotic and promitotic effects of MSC-CM on hepatocytes were demonstrated using in vitro assays. These data provide the first clear evidence that MSC-CM therapy provides trophic support to the injured liver by inhibiting hepatocellular death and stimulating regeneration, potentially creating new avenues for the treatment of FHF.

The Irreversibility of Radiation-Induced Fibrosis: Fact or Folklore?

As the number of long-term cancer survivors increases, late complications of therapy will become an increasingly important concern to both patients and physicians. In administering radiation therapy, the physician typically has attempted to prevent complications primarily by limiting the irradiated dose and volume. Unfortunately, the relationship between radiation dose, volume of tissue irradiated, development of complications, and tumor control is complex and not precisely defined for most normal tissues and malignancies. Unlike medical oncology, there are few formal prospective studies in radiation oncology designed to establish the maximum-tolerated dose for a particular tumor or normal tissue. Commonly accepted normal tissue tolerance doses of radiation, for the most part, have been derived empirically, often with little supporting data. Although these estimates may be reasonably generalized, there remains considerable individual variation in radiation sensitivity. Recently, however, much progress has been made toward better elucidating the molecular mechanisms underlying radiation injury. Late radiation damage in most tissues is characterized by loss of parenchymal cells and excessive formation of fibrous tissue. For years, it was taught that radiation fibrosis is a permanent irreversible condition, but its underlying mechanism remained uncertain. It has been known for decades that the biologic effects of ionizing radiation begin almost instantaneously with the generation of reactive oxygen species. More recently, however, we are beginning to learn that these immediate biochemical events lead to clinically and histologically recognizable injury through a series of genetic and molecular responses. This process is dynamic and involves a number of proinflammatory cytokines, profibrotic cytokines, and chemokines produced by a variety of cell types, including macrophages, epithelial cells, and fibroblasts. Furthermore, these events seem to be sustained for months or even years after therapy is completed. For example, the presence of progressive hypoxia has been noted after radiation of the lung and spinal cord. Because hypoxia itself is known to generate reactive oxygen species, promote inflammation and vascular damage, activate profibrotic cytokines, and promote collagen formation, it has been suggested that postradiation hypoxia may be an important contributor to maintenance of the injured phenotype. These findings have also suggested that, because the process is dynamic, it may not be irreversible. In this issue, Delanian et al contribute significantly to the growing body of knowledge that indicates that, indeed, radiation-induced fibrosis is at least partially reversible. They also demonstrate for the first time that long-term antifibrotic therapy will be needed to sustain benefit. The authors report on a retrospective series of 44 patients with 55 distinct superficial progressive fibrotic lesions induced by radiation therapy for breast cancer. The patients were administered the combination of pentoxifylline and vitamin E twice daily for either 6 to 12 months or 24 to 48 months. Both drugs have antioxidant properties, and pentoxifylline may also have antifibrotic effects, although neither agent is clearly effective as a single agent in reversing radiation-induced fibrosis. The main end points were reduction in the size of the fibrotic region and reduction in the global score of late injury. Delanian et al demonstrated the following: both treatment regimens produced significant improvements in both outcome measures; the maximum regression to be expected is approximately 69%; improvement occurred sooner in younger lesions ( 6 years since radiotherapy); and stopping the drugs resulted in a rebound effect that was more severe in the patients treated for a short duration. Tolerance of the regimen was good, and no patient discontinued therapy as a result of toxicity. Although oncologists should be encouraged by the mounting evidence that radiation-induced fibrosis may be reversible, many questions remain unanswered. First, JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 34 DECEMBER 1 2005

The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and puncture-induced sepsis model.

Apoptotic loss of parenchymal cells may lead to organ dysfunctions in critically ill patients with septic states. As an antioxidant, the protective effects of N-acetylcysteine (NAC) are documented in many experimental and clinical studies. In this experimental study, we investigated the role of chronically used NAC in septic lung injury on a cecal ligation and puncture (CLP) model. To evaluate this, 30 male Wistar rats were randomly divided into four groups as sham (n = 7), CLP (n = 8), sham + NAC (n = 7) and CLP + NAC (n = 8) groups. NAC was administered 150 mg kg(-1) day through intramuscular route beginning 6 h after the operations and lasting for a period of 1 week. One week later, histopathology and epithelial apoptosis were assessed by hematoxylin-eosin and immunohistochemically by M30 and caspase 3 staining to demonstrate septic lung injury. Additionally, lung tissue myeloperoxidase (MPO) activity, malondialdehyde (MDA), and nitrite/nitrate levels were measured. The MPO activity and MDA levels in lung homogenates were found to be increased in CLP group and the administration of NAC prevented their increase significantly (P < 0.05). However, there were no significant differences among the groups regarding nitrite/nitrate levels. The number of apoptotic cells was significantly lower in CLP+NAC group than CLP group, and this finding was supported by M30 and caspase 3 expression in lung (P < 0.05). Lung histopathology was also protected by NAC in CLP-induced sepsis. In conclusion, the chronic use of NAC inhibited MPO activity and lipid peroxidation, which resulted in reduction of apoptosis in lung in this CLP model. Because lung tissue nitrite/nitrate levels did not change significantly, organs other than the lungs may be responsible for producing the increased nitric oxide during sepsis. The chronic use of NAC needs further investigation for its possible antiapoptotic potential in septic states besides its documented antioxidant and antiinflammatory effects.

Biotransformation, genotoxic, and histopathological effects of environmental contaminants in European eel ( Anguilla anguilla L.)

A prolonged toxicity study was carried out in young European eel ( Anguilla anguilla L.) to evaluate the effects of environmental contaminants, namely, two individual standard compounds, benzo[ a]pyrene (BaP) and dehydroabietic acid (DHAA), and a complex mixture, bleached kraft pulp mill effluent (BKPME). Fish were exposed to BaP (0.22, 0.45, and 0.9 μM) and BKPME (3.12%, 6.25%, and 12.5% (v/v)) for 3, 7, and 30 days and to DHAA (0.07, 0.15, and 0.30 μM) for 3, 7, 30, 90, and 180 days. The biomarkers include biotransformation and genotoxicity indicators, such as total ethoxyresorufin O-deethylase (EROD) activity and frequency of erythrocytic nuclear abnormalities (ENAs), respectively. Hematological dynamics was assessed as frequency of immature erythrocytes (IEs). Histopathological examinations were carried out for the highest concentrations and for 30 days and longer exposures. Total EROD increases significantly only after 180 days of DHAA exposure. However, significant ENA induction was generally observed during exposure to all contaminants tested. Nevertheless, some of the ENA results suggest an altered genotoxic response, which may arise either from short-term exposures to the highest contaminant levels or long-term exposures to the lowest contaminant levels. IE frequency decreased significantly after 30 days of exposure to 0.45 μM BaP and 180 days of exposure to the entire DHAA concentration range. Increased density of pigmented macrophage aggregates in 30-day BaP- and BKPME-exposed fish as well as in 90- and 180-day DHAA-exposed fish confirmed histopathological liver alterations. Bile accumulation in hepatocytes after BaP treatment, cytoplasmic vacuolization and cell atrophy following DHAA exposure, as well as liver loss of parenchymal cells in BKPME-exposed fish, were also detected. Dispersed necrosis and focal inflammation were observed in the livers of all treated groups. Fish exposed to DHAA and BKPME showed skin and gill disruption as well as kidney Malpighian corpuscle alterations. All 30-day-treated groups revealed intense spleen hemosiderosis, indicating increased erythrophagia. This splenic effect may be strongly correlated with the observed disappearance of ENAs. Neoplastic lesions were not found. A multibiomarker strategy, which includes EROD, ENA, and IE assays as well as histopathological studies, contributed to a better understanding of the global toxic process.

Doença granulomatosa sistêmica em bovinos no Rio Grande do Sul associada ao pastoreio de ervilhaca (Vicia spp)

Dois surtos de uma doença associada ao pastoreio de duas espécies de ervilhaca (predominantemente Vicia villosa e, em menor grau, V. sativa) foram observados em agosto-setembro de 2001, em vacas Holandesas adultas de duas propriedades rurais do Rio Grande do Sul. Foram afetadas, em uma das propriedades, quatro de 42 vacas (9,5%) e, na outra, uma de oito vacas (12,5%). Os sinais clínicos incluíam, embora não em todos os casos, febre, prurido, espessamento e enrugamento da pele com placas multifocais de alopecia, conjuntivite, corrimento nasal seroso, perda de peso, acentuada queda na produção de leite e diarréia. O curso clínico foi de aproximadamente duas semanas. Todas as vacas afetadas clinicamente morreram, uma foi sacrificada; três foram necropsiadas. Em cada um desses animais havia um padrão de lesões sistêmicas que consistiam de nódulos multifocais ou coalescentes, macios ou moderadamente firmes e branco-acinzentados, que infiltravam vários órgãos, mas eram particularmente proeminentes no miocárdio, nos linfonodos, no baço, na glândula adrenal e no córtex renal. Essas lesões resultavam em aumento de volume e alteração na arquitetura do órgão invadido. Microscopicamente, as lesões consis-tiam de extensa infiltração celular composta de proporções variáveis de macrófagos epitelióides, linfócitos, plasmócitos, células gigantes multinucleadas e eosinófilos. Os números de eosinófilos eram geralmente altos. Essa infiltração granulomatosa causava degeneração e perda de células parenquimatosas no órgão afetado. A intensidade das lesões variou entre as três vacas e entre os vários órgãos de cada animal. Essa é a primeira documentação no Brasil de doença granulomatosa sistêmica em bovinos associada ao pastoreio de ervilhaca.

Pancreatic duct obstruction is an aggravating factor in the canine model of chronic alcoholic pancreatitis

Background & Aims: Chronic alcoholic pancreatitis occurs in only a limited number of heavy drinkers. Other factors than alcohol are necessary for the occurrence of chronic alcoholic pancreatitis. The aim of this study was to examine whether pancreatic duct obstruction resulted in increased alcohol-induced parenchymal cell damage. Methods: Four groups of adult mongrel dogs were used. In group A, 2.0 g · kg −1 · day −1 of ethanol was administered via a gastric cannula. In group O, after ligation of the minor pancreatic duct, a polyethylene tube was inserted transduodenally into the major duct. In group AO, the protocols used in groups A and O were combined. Laparotomy was repeated after 3 months in each group. Results: Three of the 9 dogs in group AO had pancreatic calculi in the main pancreatic duct. Moderate interlobular fibrosis, parenchymal cell loss, and inflammatory cell infiltration resembling human chronic alcoholic pancreatitis were observed in group AO. Little change was observed in groups A and O. Exocrine function assessed by secretin test in group AO was significantly reduced. Total protein, hexosamine, and calcium contents of the pancreatic juice in group AO were significantly increased. Conclusions: Pancreatic duct obstruction is an aggravating factor in chronic alcoholic pancreatitis. GASTROENTEROLOGY 1998;115:1248-1253

An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats.

Quantitative data defining the relationship between the hepatic microcirculation and the development of liver pathological changes could provide a basis for a better understanding of fibrogenic processes, such as cirrhosis. Therefore, we established the technique of intravital fluorescence microscopy and computer-assisted microcirculation analysis systems in developing cirrhosis in rats with the aim of quantitatively assessing the association of hepatic microvascular morphology with its disordered acinar architecture, and nonparenchymal cell transformation with collagen deposition, parenchymal cell loss, and liver dysfunction. In animals chronically exposed to carbon tetrachloride (CCl4), the most significant microvascular changes progressively observed in vivo were the concomitant appearance of 1) sinusoid-free space around dilated postsinusoidal venules with 2) substituting occurrence of yellow-green autofluorescent collagen deposition, 3) reduction in sinusoidal density, but 4) increase of vascular lumen caused by the formation of shunting vessels bypassing the sinusoids. Present on-line analysis further indicated the local coincidence of changed spatial distribution of Ito cells (accumulation of vitamin A ultraviolet autofluorescence in zone 3) with fibrotic autofluorescent septa, causing significant collapse of parenchymal tissue (hepatocellular bis-benzamide fluorescence) and diminution of hepatocellular excretory function (bile flow). Regression analysis revealed strong correlations between loss of parenchymal tissue and both collagen deposition and sinusoidal rarefication, as well as between sinusoidal rarefication and collagen deposition. Thus, sequential in vivo analysis presented herein provides the new information on the concomitant onset of cellular, fibrotic, and microvascular changes in developing fibrosis/cirrhosis, excluding that distinct cellular or fibrotic alterations are a prerequisite for the manifestation of microcirculatory and vascular derangements or vice versa.

Matrix degradation in renal disease

Summary: The histological appearance of the accumulation of glomerular and tubulointerstitial extracellular matrix (ECM) is a characteristic feature of progressive renal disease. the usual processes behind this finding are a combination of relative loss of parenchymal cells with respect to ECM, increased synthesis of ECM and decreased degradation of ECM. the physiology and pathophysiology of matrix degradation form the basis of tissue remodelling in general and have only been specifically studied in relation to renal disease recently. the two major ECM degrading enzyme systems (the matrix metalloproteinase (MMP), tissue inhibitor of the MMP (TIMP) system and the plasminogen activator/plasmin system) and the interaction between these systems and other non‐specific seem to have an important role in the processes causing matrix accumulation in the ageing kidney, focal sclerosis, diabetes, glomerulonephritis with matrix accumulation and tubulointerstitial disease. A pattern of changes in the expression of components of these enzyme‐inhibitor systems marked by increased TIMP‐1, increased plasminogen activator inhibitor‐1, decreased MMP‐1 and MMP‐3, and increased MMP‐2 and MMP‐9 characterized several models of glomerular and tubulointerstitial fibrosis that are associated, and perhaps caused by, transforming growth factor β. A thorough understanding of the processes controlling matrix degradation may not only be necessary to explain the pathogenesis of matrix accumulation but may also be important in attempting to reverse the processes.