Sampling variability of computer-aided fractal-corrected measures of liver fibrosis in needle biopsy specimens

~~Sampling variability of computer-aided fractal-corrected measures of liver fibrosis in needle biopsy specimens@Fabio Grizzi$Laboralori di Medicina Quanlitativa, Islituto Clinico Humanitas IRCCS, Rozzano, Milan, Italy @Carlo Russo$Laboralori di Medicin…     

Liver fibrosis and tissue architectural change measurement using fractal-rectified metrics and Hurst＇s exponent

AIM: To provide the accurate alternative metrical means of monitoring the effects of new antiviral drugs on the reversal of newly formed collagen. METHODS: Digitized histological biopsy sections taken from 209 patients with chronic C virus hepatitis with different grade of fibrosis or cirrhosis, were measured by means of a new, rapid, user-friendly, fully computer-aided method based on the international system meter rectified using fractal principles. RESULTS: The following were described: geometric perimeter, area and wrinkledness of fibrosis; the collation of the Knodell, Sheuer, Ishak and METAVIR scores with fractal-rectified metric measurements; the meaning of the physical composition of fibrosis in relation to the magnitude of collagen islets; the intra- and inter-biopsy sample variability of these parameters; the "staging" of biopsy sections indicating the pathway covered by fibrosis formation towards its maximum known value; the quantitative liver tissue architectural changes with the Hurst exponent. CONCLUSION: Our model provides the first metrical evaluations of the geometric properties of fibrosis and the quantitative architectural changes of the liver tissue. The representativeness of histological sections of the whole liver is also discussed in the light of the results obtained with the Hurst coefficient.     

Suppressive effects of cyclosporine A on neutrophils and T cells may be related to therapeutic benefits in patients with steroid-resistant ulcerative colitis

An intravenous infusion of cyclosporine A (CsA) shows clinical benefits in patients with steroid-resistant ulcerative colitis (UC). To clarify its mechanisms, we investigated the ability of CsA to inhibit the functions of neutrophils and T cells. The cytotoxic activity by mucosal T cells was analyzed by anti-CD3-triggered cytotoxicity after lamina propria mononuclear cells were cultured with recombinant interleukin (IL)-2. The chemotactic response, the generation of superoxide, and the production of chemokines, IL-8, and macrophage inflammatory protein-1alpha by neutrophils were examined using a multiple-well chamber assay, a chemiluminescence method, and an enzyme-linked immunosorbent assay (ELISA), respectively. Mucosal chemokine activity was determined by an ELISA using the organ culture supernatant of mucosal biopsy tissues. Pretreatment with CsA caused consistent inhibitions of cytotoxic activity by mucosal T cells and chemotactic migration, superoxide generation, and chemokine production by neutrophils mostly in a dose-dependent manner. In patients who received an intravenous infusion of CsA, mucosal chemokine activity decreased after therapy in parallel with decreases in the numbers of neutrophils and mononuclear cells in the biopsy tissues. These results suggest that suppressive effects of CsA on neutrophils and T cells may be related to therapeutic benefits in patients with steroid-resistant UC.     

In vitro effects of oxpentifylline on inflammatory cytokine release in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta, have been implicated as primary mediators of intestinal inflammation in inflammatory bowel disease. AIM: To investigate the in vitro effects of oxpentifylline (pentoxifylline; PTX; a phosphodiesterase inhibitor) on inflammatory cytokine production (1) by peripheral mononuclear cells (PBMCs) and (2) by inflamed intestinal mucosa cultures from patients with Crohn's disease and patients with ulcerative colitis. METHODS: PBMCs and mucosal biopsy specimens were cultured for 24 hours in the absence or presence of PTX (up to 100 micrograms/ml), and the secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 determined by enzyme linked immunosorbent assays (ELISAs). RESULTS: PTX inhibited the release of TNF-alpha by PBMCs from patients with inflammatory bowel disease and the secretion of TNF-alpha and IL-1 beta by organ cultures of inflamed mucosa from the same patients. Secretion of TNF-alpha by PBMCs was inhibited by about 50% at a PTX concentration of 25 micrograms/ml (IC50). PTX was equally potent in cultures from controls, patients with Crohn's disease, and those with ulcerative colitis. The concentrations of IL-6 and IL-8 were not significantly modified in PBMCs, but IL-6 increased slightly in organ culture supernatants. CONCLUSIONS: PTX or more potent related compounds may represent a new family of cytokine inhibitors, potentially interesting for treatment of inflammatory bowel disease.     

MAGNIFYING COLONOSCOPY FOR THE DIAGNOSIS OF INFLAMMATORY CHANGES IN ULCERATIVE COLITIS

Background: Endoscopic observation is the most effective method for the evaluation of staging in ulcerative colitis (UC). However, in cases with very mild inflammatory activity, histopathological diagnosis may also be required. Unfortunately, biopsy‐related accidents are not uncommon. As an alternative, we have used a magnifying colonoscope commonly used for tumor diagnosis to examine in detail the colon mucosa of UC patients in clinical remission, and then compared these findings relative to conventional endoscopy using histopathological diagnosis. Subjects and Methods: Among UC cases examined by colonoscopy between April 2000 and April 2005, 27 cases without hematochezia for at least 1 month were enrolled in this study. Following observations of inflammatory changes using conventional colonoscopy, magnifying observation and biopsies at a total of 144 sites were evaluated. Using histopathological standards, acute‐phase inflammation was indicated by the presence of neutrophil infiltration, whereas chronic‐phase inflammation was indicated by infiltration of lymphocytes, plasma cells and eosinophils. Results: Indicators of significant inflammation by conventional observation was erosion. Under magnification, inflammation appears as superficial defects in mucosa and small whitish spots. When the presence of infiltrating neutrophils was used as a positive histological marker for inflammation, there was no difference in the accuracy of diagnosis by conventional observation (95.1%) versus magnifying observation (97.2%). In contrast, when lymphocyte infiltration was used as a marker, the accuracy of diagnosis increased significantly (88.2%) using magnifying observation relative to conventional observation (61.1%). Conclusions: Magnifying endoscopy can be used effectively in the evaluation of minute mucosal changes in cases of UC remission.     

Successful immunoglobulin treatment for fulminant myocarditis and serial analysis of serum thioredoxin: a case report.

A 31-year-old woman suspected to have acute myocarditis was admitted to hospital and was managed with intra-aortic balloon pumping and a percutaneous cardiopulmonary support system because of sustained ventricular tachycardia. After immunoglobulin treatment, cardiac function and systematic inflammation were improved. The left ventricular endomyocardial biopsy revealed massive necrosis and degeneration of myocardial cells, and extensive infiltration of inflammatory cells. The clinicopathology of this patient was thought to be fulminant myocarditis. Serial serum thioredoxin (TRX) analysis showed that the serum level was high during the acute phase, and decreased during the chronic phase. Immunohistochemistry for TRX in the biopsy samples showed that inflammatory cells and cardiomyocytes were positively stained.     

Effects of progestins and menstrual cycle on fatty acid synthetase and progesterone receptor in human mammary glands

Fatty acid synthetase (FAS) is induced by progestins in human breast cancer cell lines. To study its regulation in normal mammary glands, the FAS level was estimated by immunohistochemistry, using the biotin-streptavidin method, in ducts and lobules of normal tissues adjacent to nonproliferative benign breast lesions collected by biopsy. Rabbit polyclonal antibodies to human FAS specifically recognized the 250-kDa FAS from MCF7 cells, as shown by Western immunoblotting. An excess of purified FAS totally switched off FAS immunostaining of R5020-treated MCF7 cells, demonstrating the validity of FAS immunocytochemical detection. FAS labeling was quantified using a computer-aided image analyzer (SAMBA 2005) in 18 patients receiving progestin therapy from the 15th to the 25th day of the menstrual cycle and 26 untreated patients. In the 2 groups, FAS staining, absent of fibroblasts, was observed in the cytoplasm of epithelial cells. It was higher in lobules than in ducts and increased significantly from the follicular to the luteal phase in both structures. Progestin treatment increased FAS expression in both structures. Using monoclonal antibodies, progesterone receptor expression was measured in frozen serial sections. In patients receiving progestin treatment, the progesterone receptor level increased from the beginning of the cycle to day 14 and then decreased during the second part of the menstrual cycle, probably down-regulated by progestin, indicating a regulation similar to that in the endometrium. We conclude that FAS is induced by progestins in the ducts and lobules of human normal mammary glands as it is in human breast cancer cells. FAS may, therefore, be useful for studying the effect of progesterone in normal human mammary glands.     

老年人巨细胞动脉炎的炎性细胞浸润类型及其分布

目的　研究老年人巨细胞动脉炎的炎性细胞浸润类型及其分布特征。方法　用免疫组织化学技术检测 2 0例巨细胞动脉炎患者颞动脉活检标本T淋巴细胞、B淋巴细胞、巨噬细胞、多核巨细胞和粒细胞表达的情况。以常规HE、美蓝染色及铅铀双重染色 ,在光镜和电镜下观察病理变化 ,比较病理变化与炎性细胞类型及分布的关系。结果　2 0例颞动脉活检标本中 ,浸润的炎性细胞由T淋巴细胞、巨噬细胞和粒细胞构成 ,构成比分别为 40 .5 %、33.5 %和 2 6 .0 %,未见B淋巴细胞和多核巨细胞。炎性细胞分布于颞动脉壁及其周围组织 ,以外膜最明显、内膜次之、中膜居末 ;有明显跳跃现象。炎性细胞分布与病理变化为非比例关系 ,病理损害以内膜明显。结论　老年人巨细胞动脉炎其颞动脉活检标本中可能有较多的粒细胞浸润 ,无多核巨细胞。     

Giant cell granulamatous hypophysitis with remarkable uptake on Gallium-67 scintigraphy

We treated a man with giant cell granulomatous hypophysitis with pituitary enlargement, as seen on magnetic resonance imaging. Endocrinological examination revealed panhypopituitarism and diabetes insipidus. Microscopic examination of the specimen obtained by transsphenoidal pituitary biopsy revealed a granulomatous lesion, composed of epitheliod cells, Langhans' multinucleated giant cells, lymphocytes and other chronic inflammatory cells. On whole body gallium-67 scintigraphy, there was extensive uptake in the pituitary gland. Gallium-67 scintigraphy may greatly aid in the diagnosis of granulomatous hypophysitis.     

Quantitative evaluation of long-term liver repopulation and the reconstitution of bile ductules after hepatocellular transplantation

AIM: The treatment of liver disease is severely limited by a shortage of donor livers. In trying to address this growing problem, hepatocellular transplantation (HTx) has received much attention as an alternative to whole organ transplant. However, the expansion of transplanted cells is at low level, and the reconstitution of functional liver tissue is limited by this cellular property. We set up an animal model to better understand cell dose effect and the kinetics of liver repopulation following HTx. METHODS: Dipeptidyl peptidase Ⅳ (DPPⅣ)-deficient rats treated with retrorsine and subjected to partial hepatectomy were infused with DPPⅣ-positive hepatocytes. Rats were injected with varying numbers of donor hepatocytes down to 100 cells low, and liver repopulation was examined at different time points up to 20 mo long. Repopulation was assessed by computer-aided quantitative detection. RESULTS: Transplanted hepatocytes underwent multiple rounds of proliferation and stably repopulated the injured livers after 20 mo and at all cell doses. Transplanted cells divided 14 times within the 3-mo time period following infusion, and the liver repopulation reached a plateau between 3 and 20 mo. Approximately 90% replacement occurred. Donor-derived cells also reconstituted the bile ductules of the recipients. CONCLUSION: The ability of transplanted hepatocytes to fully reconstitute injured livers strongly supports further investigation into the clinical potential of HTx. Additionally, the observation that transplanted hepatocytes also form components of the biliary system suggests that these cells may have bi-potential property of the stem cells.     

Hepatocyte proliferation and cell cycle phase fractions in chronic viral hepatitis C by image analysis method

OBJECTIVE: Chronic hepatitis is characterized by necrosis of liver cells, accompanied by an inflammatory reaction and compensatory cell proliferation. The interaction of the core and non-structural proteins of hepatitis C virus (HCV) with several cellular factors suggests that cell proliferation may be influenced by HCV. The aim of this study was to investigate hepatocyte proliferation and DNA ploidy patterns in patients with chronic viral hepatitis C (CH-C) compared with chronic non-viral hepatitis (CH-N), using a TV image analysis method. METHODS: The DNA index (DI) and cell phase fractions (G1, S, G2) were measured by means of digital picture analysis method on nuclear suspensions of Feulgen stained hepatocytes. Cells were taken from the liver biopsy specimens of 71 patients with CH-C and 24 patients with CH-N. Twenty-six normal liver samples were used as controls. RESULTS: Significantly higher G1 (94 +/- 4) and lower S (3.56 +/- 3.16) phase fractions were measured in CH-C compared with CH-N (G1, 90 +/- 6; S, 6.4 +/- 5.99). The DI of moderate (1.12 +/- 0.05) and severe (1.12 +/- 0.05) CH-C showed near-aneuploid DNA content, while diploidy (DI < 1.10) was detected in cases of CH-N. CONCLUSION: The higher G1 and lower S cell cycle phase fractions in CH-C reflect decreased hepatocyte proliferation compared with CH-N. The near-aneuploid DNA content of the HCV-infected liver samples may be a sign of increased genetic instability, which may contribute to the carcinogenic potential of HCV.     

Phase 2 prolongation, in the absence of instability and triangulation, antagonizes class III proarrhythmia

OBJECTIVE: To evaluate whether prolongation of the plateau of the action potential duration, in the absence of instability and triangulation, can reverse the proarrhythmia elicited by a class III antiarrhythmic agent. METHODS: The effects of almokalant, erythromycin and their combination, on cardiac electrophysiological parameters (action potential duration (APD), instability, triangulation and ectopics) were evaluated in isolated hearts from female albino rabbits. In this study, proarrhythmia was estimated quantitatively by number of ectopic beats. RESULTS: Erythromycin lengthened the APD primarily by a prolongation of the plateau, while having only minor effects upon phase 3 repolarization. The prolongation did not induce much instability, triangulation or reverse use dependence and, as expected, erythromycin did not induce significant proarrhythmia. Almokalant also lengthened APD, but it did not lengthen the plateau; instead, it prolonged phase 3 repolarization. The prolongation markedly triangulated the action potential, elicited much instability and marked reverse use dependence. This combination of effects induced very marked proarrhythmia. When almokalant and erythromycin were combined, their effects upon APD appeared additive: both the plateau and the repolarization phase were prolonged. However, the larger prolongation of APD did not lead to more proarrhythmia; this suggests that a prolongation of APD is not proarrhythmic per se. On the contrary, proarrhythmia as a function of APD prolongation was reduced in the presence of erythromycin (P<0.05). CONCLUSION: Instability plus triangulation consistently lead to serious proarrhythmia especially when combined with reverse use dependence, but prolongation of APD in itself is not necessarily proarrhythmic. In fact, APD prolongation in the absence of instability and triangulation can be antiarrhythmic.     

The role of MRI in the assessment of polymyositis and dermatomyositis

OBJECTIVES: Acute inflammation in idiopathic inflammatory myopathies (IIM) causes oedema that can be visualized by magnetic resonance imaging (MRI). The inflammatory infiltrate in IIM is thought to be frequently in a focal distribution. The aim of this study is to better evaluate the relationship of MR image of thigh muscles to clinical and histological parameters in patients with IIM. METHODS: MRI-short tau inversion recovery (STIR) technique was used to distinguish between affected and non-affected muscles. Computer tomography (CT)-controlled targeted needle biopsy was used for sampling. The intensity of muscle oedema, its extent and total assessment on MRI were evaluated with 10 cm visual analogue scale. The intensity of inflammatory infiltrate was assessed using 5-point grading system. The second MRI and muscle biopsy were performed after the time interval of treatment. RESULTS: MR scans, muscle biopsy and clinical examination were performed in 29 patients with polymyositis (PM) and dermatomyositis (DM). Paired MRI-affected and MRI-non-affected biopsy samples were obtained from 17 cases. In six cases, the biopsy was available for comparison before and after period of treatment. At the initial examination, it was the intensity of oedema on MRI that was associated with clinical status. Mean intensity of MRI findings significantly decreased in 10 patients where the MRI was available also after treatment. The mean intensity of inflammatory infiltrate in PM/DM patients was 2.5 +/- 0.7 for MRI-affected and 1.7 +/- 0.6 for MRI-non-affected muscles (P < 0.001). Mean intensity of inflammatory infiltrate in the MRI-affected muscles in the first examination (n = 6) was 2.2 +/- 0.8 and did not significantly decrease in the second examination in samples taken after the treatment (2.0 +/- 0.9). CONCLUSION: It is mainly the signal intensity in MR scan, which is associated with disease activity in the acute presentation of PM/DM. Muscle biopsy guided by positive MRI finding contains significantly more inflammatory cells than the biopsy taken from MRI non-affected sites. However, even in parts of muscles, which look unaffected on MR scan, the inflammatory cells can be found. The intensity on MR scans decreases significantly after the treatment, but the histologically detected inflammation does not change substantially.     

Pathology of interstitial lung disease

A large and diverse group of pathologic conditions manifests clinically and radiologically as diffuse parenchymal lung disease. Diffuse interstitial lung diseases (ILDs) encompass mainly inflammatory processes that involve the structural elements of this organ. Some ILDs are caused by infections, but most are the result of immunologic, environmental, or toxic mechanisms. Currently, less morbid sampling techniques have increased dramatically the probability that pulmonologists will be faced with establishing a specific and clinically relevant diagnosis using surgical lung biopsy material. Most of the concepts presented in this article have been established using this type of specimen. In the early years of surgical lung biopsy, a small number of diffuse inflammatory conditions came to light that exclusively involved the lungs and did not seem to be caused by infection, toxin, sarcoidosis, pneumoconiosis, or neoplasm. In this article, these idiopathic disorders are discussed in the context of their dominant pathologic findings rather than presented as a separate group of entities.     

Washing of cord blood grafts after thawing: high cell recovery using an automated and closed system

BACKGROUND AND OBJECTIVES: Cord blood (CB) progenitor cells are an alternative source of haematopoietic stem cells for bone marrow reconstitution. The critical importance of cell dose in the clinical outcome has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. This aim of this study was to evaluate an automated CB washing protocol of thawed cord blood units using the Sepax device. MATERIALS AND METHODS: After an initial 1:1 dilution using a dextran/albumin-containing buffer, the cells were washed in order to obtain a final product ready for transplantation. The automatic method was compared with the conventional manual washing procedure. Blood samples were taken after thawing and after washing. The processing time, viability and mean recovery of nucleated cells (TNC) and progenitors were determined. RESULTS: The automatic procedure resulted in a median recovery of 93% CD34+ cells and 89% TNC; no significant differences were observed between methods. In addition, median viability, as assessed by annexin V and 7-aminoactinomycin D (7-AAD), was 98% and 94%, respectively, within CD34+ cells. CONCLUSIONS: The automatic washing method described is as effective as the manual method in terms of viability and progenitor cells recovery, but faster and easier for the operators to perform. Overall, our data suggest that the automatic method is safe and suitable for the routine washing of thawed CB grafts in the clinic.     

Organ donor pancreases for the study of human islet cell histology and pathophysiology: a precious and valuable resource

Direct in vivo assessment of pancreatic islet-cells for the study of the pathophysiology of diabetes in humans is hampered by anatomical and technological hurdles. To date, most of the information that has been generated is derived from histological studies performed on pancreatic tissue from autopsy, surgery, in vivo biopsy or organ donation. Each approach has its advantages and disadvantages (as summarised in this commentary); however, in this edition of Diabetologia, Kusmartseva et al ( https://doi.org/10.1007/s00125-017-4494-x) provide further evidence to support the use of organ donor pancreases for the study of human diabetes. They show that length of terminal hospitalisation of organ donors prior to death does not seem to influence the frequency of inflammatory cells infiltrating the pancreas and the replication of beta cells. These findings are reassuring, demonstrating the reliability of this precious and valuable resource for human islet cells research.     

Suppression of the inflammatory response to cardiopulmonary bypass

The inflammatory response to major surgery, especially cardiac surgery using cardiopulmonary bypass (CPB) is now a well established entity. A whole body inflammatory response can lead to severe organ dysfunction, postoperative bleeding disorders, respiratory distress syndrome and sometimes death. There is, however, controversy over various methods and their efficacy towards suppression of this response. We studied forty consecutive patients undergoing coronary artery bypass grafting (CABG) using CPB. Ten patients in group A served as control while ten patients in group B received piroxicam, a non steroidal anti-inflammatory drug (NSAID). Ten patients in group C received aprotinin, a kallikrein inhibitor and ten patients in group D underwent haemofiltration during CPB. Inflammatory response by way of increase in total white blood cell (WBC) count (p<0.007), decrease in lymphocyte count (p<0.005), increase in C-reactive protein (CRP, p <0.005) was observed in all four groups at 24 hour after CPB. A decrease in complement C3 and C4 (p<0.01) was observed in groups A and C at 24 hours after CPB. The response observed was not severe enough to cause any organ damage in any group. None of the methods studied could effectively suppress the inflammatory response to CPB but the response was altered in some way by each method.     

Vascular endothelial growth factor is highly expressed in muscle tissue of patients with polymyositis and patients with dermatomyositis

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) in muscle biopsy specimens and serum from patients with polymyositis and patients with dermatomyositis compared with that in healthy control subjects. METHODS: Muscle biopsy specimens from 33 patients with polymyositis or dermatomyositis and 15 healthy control subjects and serum samples from 56 patients and 56 healthy control subjects were analyzed. Patients were categorized into 3 groups, depending on disease duration and the presence or absence of inflammatory infiltrates. The expression of VEGF and the vessel marker CD31 in muscle was analyzed by immunohistochemistry, the expression of VEGF messenger RNA (mRNA) was analyzed by in situ hybridization, and serum levels of VEGF were determined by enzyme-linked immunosorbent assay. RESULTS: Patients with polymyositis or dermatomyositis in the early or chronic phase without inflammatory infiltrates had a decreased total number of capillaries compared with healthy individuals. In patients with early disease without inflammatory infiltrates, the number of VEGF-expressing muscle fibers was increased compared with that in control subjects, whereas VEGF expression was unchanged in the chronic phase of disease. In patients with established disease with inflammatory infiltrates, total VEGF expression was high compared with that in healthy control subjects. In healthy control subjects, VEGF was expressed in endothelial cells and in occasional muscle fibers. VEGF mRNA was expressed in muscle fibers in both healthy individuals and patients. The level of serum VEGF was significantly increased in patients compared with control subjects. CONCLUSION: Our observations support a role of VEGF in the early phases of polymyositis and dermatomyositis. A reduced number of capillaries could lead to induction of VEGF expression in muscle fibers. Furthermore, differences in molecular expression during certain phases of disease may help in the development of specific therapeutic algorithms in the treatment of myositis.     

超细支气管镜肺活检并刷检结合经皮肺自动弹性活检对肺周边病灶的诊断

目的评价经X线引导超细支气管镜活检并刷检,结合B超引导经皮肺自动弹性穿刺活检在肺周围型病变诊断中的价值。方法89例肺外周病变患者先进行X线引导超细支气管镜肺活检并刷检共110次,对于经超细支气管镜检未获得诊断的26例患者,再进行B超引导经皮肺自动弹性穿刺活检共32次。结果经超细支气管镜肺活检并刷检获得诊断者63例,诊断率70.8%。经皮肺自动弹性穿刺活检获得诊断者21例,诊断率80.08%。二者结合后的诊断率明显提高为94.4%。结论经X线引导超细支气管镜肺活检并刷检,结合B超引导经皮肺自动弹性穿刺活检,可明显提高肺外周病变的诊断率,值得临床推广应用。