Histological Evidence Of Necrosis Research Articles

Diffusion histogram profiles predict molecular features of grade 4 in histologically lower-grade adult diffuse gliomas following WHO classification 2021

ObjectivesIn the latest World Health Organization classification 2021, grade 4 adult diffuse gliomas can be diagnosed with several molecular features even without histological evidence of necrosis or microvascular proliferation. We aimed to explore whole tumor histogram-derived apparent diffusion coefficient (ADC) histogram profiles for differentiating between the presence (Mol-4) and absence (Mol-2/3) of grade 4 molecular features in histologically lower-grade gliomas.MethodsBetween June 2019 and October 2022, 184 adult patients with diffuse gliomas underwent MRI. After excluding 121 patients, 18 (median age, 64.5 [range, 37–84 years]) Mol-4 and 45 (median 40 [range, 18–73] years) Mol-2/3 patients with histologically lower-grade gliomas were enrolled. Whole tumor volume-of-interest-derived ADC histogram profiles were calculated and compared between the two groups. Stepwise logistic regression analysis with Akaike’s information criterion using the ADC histogram profiles with p values < 0.01 and age at diagnosis was used to identify independent variables for predicting the Mol-4 group.ResultsThe 90th percentile (p < 0.001), median (p < 0.001), mean (p < 0.001), 10th percentile (p = 0.014), and entropy (p < 0.001) of normalized ADC were lower, and kurtosis (p < 0.001) and skewness (p = 0.046) were higher in the Mol-4 group than in the Mol-2/3 group. Multivariate logistic regression analysis revealed that the entropy of normalized ADC and age at diagnosis were independent predictive parameters for the Mol-4 group with an area under the curve of 0.92.ConclusionADC histogram profiles may be promising preoperative imaging biomarkers to predict molecular grade 4 among histologically lower-grade adult diffuse gliomas.Clinical relevance statementThis study highlighted the diagnostic usefulness of ADC histogram profiles to differentiate histologically lower grade adult diffuse gliomas with the presence of molecular grade 4 features and those without.Key Points• ADC histogram profiles to predict molecular CNS WHO grade 4 status among histologically lower-grade adult diffuse gliomas were evaluated.• Entropy of ADC and age were independent predictive parameters for molecular grade 4 status.• ADC histogram analysis is useful for predicting molecular grade 4 among histologically lower-grade gliomas.

Histopathologic and MR Imaging Appearance of Spontaneous and Radiation-Induced Necrosis in Uveal Melanomas: Initial Results.

Simple SummaryUveal melanomas may undergo necrosis, both spontaneously or following radiotherapy. Nowadays radiotherapy is the preferred treatment, whereas enucleation of the eye is used in selected cases. In order to differentiate the effects of radiotherapy from spontaneous degenerative changes in uveal melanomas, we compared the appearance of necrosis, both from a histopathological point of view and from the perspective of MR imaging, in two groups of patients with uveal melanoma: a group who had undergone previous proton beam radiotherapy (secondary enucleation); a control group who had undergone enucleation without any previous radiotherapy treatment (primary enucleation). Irradiated and nonirradiated uveal melanomas differ on the basis of the histological appearance, the MR imaging appearance and the distribution of necrosis. We hope that the findings we observed could be extended to all patients with uveal melanomas treated with radiotherapy, and may enhance the accuracy of radiologists in evaluating MR examinations after radiotherapy.Necrosis in uveal melanomas can be spontaneous or induced by radiotherapy. The purpose of our study was to compare the histopathologic and MRI findings of radiation-induced necrosis of a group of proton beam-irradiated uveal melanomas with those of spontaneous necrosis of a control group of patients undergoing primary enucleation. 11 uveal melanomas who had undergone proton beam radiotherapy, MRI and secondary enucleation, and a control group of 15 untreated uveal melanomas who had undergone MRI and primary enucleation were retrospectively identified. Within the irradiated and nonirradiated group, 7 and 6 eyes with histological evidence of necrosis respectively, were furtherly selected for the final analysis; the appearance of necrosis was assessed at histopathologic examination and MRI. Irradiated melanomas showed a higher degree of necrosis as compared with nonirradiated tumors. Irradiated and nonirradiated lesions differed based on the appearance and distribution of necrosis. Irradiated tumors showed large necrotic foci, sharply demarcated from the viable neoplastic tissue; nonirradiated tumors demonstrated small, distinct foci of necrosis. Radiation-induced necrosis, more pigmented than surrounding viable tumor, displayed high signal intensity on T1-weighted and low signal intensity on T2-weighted images. The hemorrhagic/coagulative necrosis, more prevalent in nonirradiated tumors (4 out of 6 vs. 1 out of 7 cases), appeared hyperintense on T2-weighted and hypointense on T1-weighted images. Our study boosts the capability to recognize radiation-induced alterations in uveal melanomas at MRI and may improve the accuracy of radiologists in the evaluation of follow-up MR examination after radiotherapy.

Neonatal necrotizing enterocolitis: a case series examining clinical diagnosis with discrepant versus concordant autopsy results.

The objective of this study is to determine whether rapidity of death in necrotizing enterocolitis (NEC) increased odds of discordance between clinical and pathological diagnosis. Retrospective case-series study including preterm infants admitted to the NICU. Twenty-two infants met the selection criteria. Gross pathologic evidence of NEC was present in 1/6 cases (17%) where demise occurred <12 h after onset of symptoms, 3/5 cases (60%) within 12-24 h, and 8/11 cases (73%) in >24 h. Histological evidence of necrosis was present in 4/6 (67%) cases when death occurred <12 h, 4/5 (80%) in 12-24 h, and 9/11 (82%) in >24 h. The percentage with gross pathologic evidence showed a monotonic trend (P = 0.031), while the trend was less clear for histologic findings (P = 0.496). Pathologic features of cell death may not have had sufficient time to develop. This study could reassure both healthcare providers and families when pathologic and clinical diagnoses are not consistent.

Abstract 4489: IL-10 blockade reactivates antitumor immunity in human colorectal cancer liver metastases

Abstract Background: Colorectal cancer (CRC) is the 4th most common cancer in the US, and the liver is the most common site of metastatic disease. Immune checkpoint inhibitor therapy has not been successful in achieving a clinical response in most patients with CRC liver metastases (CRCLM). The liver is known to induce tolerance to foreign antigens as a result of immunosuppressive cytokines including IL-10. We hypothesized that blockade of IL-10 signaling in CRCLM would potentiate tumor infiltrating lymphocyte (TIL)-mediated tumor cell death. Methods: We performed single-cell RNA sequencing (scRNAseq) of CRCLM using the 10x platform to evaluate for expression of IL-10 or IL-10 receptor (IL-10R) RNA within the tumor (n=8). To confirm if the IL-10R protein was present within the tumor microenvironment (TME), we also performed immunohistochemistry (IHC) (n=3). In order to study the functional effects of IL-10 blockade, we utilized a tumor slice culture (TSC) model, which allows for the study of cancers with their intact TME including immune cells. For TSCs, cores (6 mm diameter) were taken from freshly resected sterile human CRCLM and cut to 250 µm thick slices using a vibratome (n=3). Duplicate slices were treated with either IgG control or anti-IL-10 monoclonal antibodies and cultured for up to 6 days. To evaluate for histological evidence of necrosis and cell apoptosis within the tumor slice, we stained slides with either hematoxylin and eosin (H&amp;E) or cleaved-Caspase-3 (CC3). To gain insight into the activation state of TIL after treatment, we measured levels of cytokines within the culture supernatants. Results: We found by scRNAseq that that IL-10 was expressed by a subset of tumor-associated macrophages, and IL-10R was expressed by both CD4+ and CD8+ T cells as well as macrophages. We confirmed that IL-10R protein was present within the CRCLM TME by IHC, and IL-10R expression was distributed throughout the stroma in non-tumor cells. In TSC treated with anti-IL-10 antibody, CC3+ cells were found to be 82.8% of total cells, compared to 36.1% of control (p = 1 x 10-6) at day 6. These findings were consistent across all human tumor samples treated with IL-10 blockade versus control at all time points examined. Furthermore, IL-10 blockade led to histologic evidence of generalized necrosis compared to an intact TME seen in the control group. Analysis of cytokines released into the media confirmed that IL-10 was present in controls, but absent in slices blocked with anti-IL-10 antibody. We also found increased levels of granzyme B, IL-2, GM-CSF, and IL-18, as well as a reduction in the immune checkpoint receptor TIM3, after one day of IL-10 blockade in culture. Conclusion: Treatment of human CRCLM TSCs with anti-IL-10 antibody leads to a marked increase in immune-mediated cell death within the tumor. Our data suggest that IL-10 serves as a critical regulator of anti-tumor immunity in the CRCLM TME and may serve as an important immunotherapeutic target. Citation Format: Kevin M. Sullivan, Xiuyun Jiang, Yongwoo David Seo, Heidi L. Kenerson, Xiaowei Yan, Chris Lausted, Changting Meng, Neda Jabbari, Kevin P. Labadie, Sara K. Daniel, Qiang Tian, Teresa S. Kim, Raymond S. Yeung, Venu G. Pillarisetty. IL-10 blockade reactivates antitumor immunity in human colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4489.

Effects of alcohol injection in rat sciatic nerve as a model for Morton's neuroma treatment.

Previous studies have shown that the injection of dehydrated alcohol has been successful for the treatment of Morton's neuroma in the foot. In this study, we determined the cellular effect of injection of alcohol into and around the sciatic nerve of rats and measured the extent of cell necrosis and/or any associated histologic or inflammatory changes. Twenty-two male (~375 g) Wistar rats were randomized into 2 groups each receiving alcohol injections into or around the sciatic nerve after nerve exposure under sterile technique. Group 1 rats were injected with a 0.5 ml solution of 0.5% Marcaine in the left sciatic nerve as a control group. In the right sciatic nerve a 0.5 ml solution of 4% ethanol with 0.5% Marcaine was injected. Group 2 rats received 0.5 ml of 20% ethanol with 0.5% Marcaine injected into the left sciatic nerve and 0.5 ml of 30% ethanol with 0.5% Marcaine injected into the right sciatic nerve. In each group, the rats were placed in 3 subgroups: intraneural, perineural, perimuscular injections. All rats were sacrificed and tissue harvested for histologic evaluation at day 10 post injection. No evidence of alcohol-associated cell necrosis, apoptosis, or apparent inflammation was observed in histologic specimens of any injected nerves, perineural tissue, or muscles in controls or experimental groups regardless of concentration of ethanol injected on day 10. We concluded that alcohol injection (≤30% ethanol) into and/or around the sciatic nerve or the adjacent muscle of rats has no histologic evidence of necrosis or inflammation to the nerve or surrounding tissue. There was no observable histological change in apoptosis, or cell number, in response to the alcohol injection. The lack of any measureable changes in nerve or adjacent muscle histology with ethanol injection into the rat sciatic nerve (and surrounding tissues) raises questions about the efficacy of using ethanol injections in the treatment of Morton's neuroma in human clinical practice.

Characterization of intratumor magnetic nanoparticle distribution and heating in a rat model of metastatic spine disease

The goal of this study was to optimize local delivery of magnetic nanoparticles in a rat model of metastatic breast cancer in the spine for tumor hyperthermia while minimizing systemic exposure. A syngeneic mammary adenocarcinoma was implanted into the L-6 vertebral body of 69 female Fischer rats. Suspensions of 100-nm starch-coated iron oxide magnetic nanoparticles (micromod Partikeltechnologie GmbH) were injected into tumors 9 or 13 days after implantation. For nanoparticle distribution studies, tissues were harvested from a cohort of 36 rats, and inductively coupled plasma mass spectrometry and histopathological studies with Prussian blue staining were used to analyze the samples. Intratumor heating was tested in 4 anesthetized animals with a 20-minute exposure to an alternating magnetic field (AMF) at a frequency of 150 kHz and an amplitude of 48 kA/m or 63.3 kA/m. Intratumor and rectal temperatures were measured, and functional assessments of AMF-exposed animals and histopathological studies of heated tumor samples were examined. Rectal temperatures alone were tested in a cohort of 29 rats during AMF exposure with or without nanoparticle administration. Animal studies were completed in accordance with the protocols of the University Animal Care and Use Committee. Nanoparticles remained within the tumor mass within 3 hours of injection and migrated into the bone at 6, 12, and 24 hours. Subarachnoid accumulation of nanoparticles was noted at 48 hours. No evidence of lymphoreticular nanoparticle exposure was found on histological investigation or via inductively coupled plasma mass spectrometry. The mean intratumor temperatures were 43.2°C and 40.6°C on exposure to 63.3 kA/m and 48 kA/m, respectively, with histological evidence of necrosis. All animals were ambulatory at 24 hours after treatment with no evidence of neurological dysfunction. Locally delivered magnetic nanoparticles activated by an AMF can generate hyperthermia in spinal tumors without accumulating in the lymphoreticular system and without damaging the spinal cord, thereby limiting neurological dysfunction and minimizing systemic exposure. Magnetic nanoparticle hyperthermia may be a viable option for palliative therapy of spinal tumors.

Idiopathic granulomatous hypophysitis: a systematic review of 82 cases in the literature

Idiopathic granulomatous hypophysitis (IGH) is a rare inflammatory disease of the pituitary. There is debate in the scientific literature as to whether IGH represents a continuum of disease with lymphocytic hypophysitis or has a distinct pathogenesis. Due to the rare nature of the disease, previous descriptions have been limited to single case reports or small series. In the present study, a systematic review of the literature was performed for cases of IGH. 82 cases met inclusion criteria. Data was gathered on IGH clinical aspects, in order to elucidate any associations useful in determining pathogenesis, appropriate clinical treatment, or prognosis. Univariate and multivariate analysis was performed on available data. Female sex was significantly associated with IGH (p < 0.0001). Fever (p = 0.002), nausea or vomiting at presentation (p = 0.031), and histological evidence of necrosis (p = 0.022) correlated with reduced time to presentation. Panhypopituitarism at presentation predicted need for long term hormone replacement (p = 0.014). Hyperprolactinaemia (p = 0.032), normal gonadal (p = 0.037) and thyroid axes (p = 0.001) were associated with reduced likelihood of long-term hormone replacement. Anorexia (p = 0.017), cold intolerance (p = 0.046), and fatigue (p = 0.0033) were associated with death from IGH. Patients who had excisional surgery alone trended towards increased rates of symptom resolution, compared with patients who received corticosteroids as an adjunct to excisional surgery (p = 0.11). This article details the first systematic review of IGH, and presents evidence for a female predilection of the disease. Implications for pathogenesis, and a suggested clinical approach are discussed. An online disease registry has been established to facilitate further IGH research.

Impact through time of different sized titanium dioxide particles on biochemical and histopathological parameters

Due to corrosion, a titanium implant surface can be a potential source for the release of micro (MPs) and nano-sized particles (NPs) into the biological environment. This work sought to evaluate the biokinetics of different sized titanium dioxide particles (TiO2 ) and their potential to cause cell damage. Wistar rats were intraperitoneally injected with 150 nm, 10 nm, or 5nm TiO2 particles. The presence of TiO2 particles was evaluated in histologic sections of the liver, lung, and kidney and in blood cells at 3 and 12 months. Ultrastructural analysis of liver and lung tissue was performed by TEM, deposit concentration in tissues was determined spectroscopically, and oxidative metabolism was assessed by determining oxidative membrane damage, generation of superoxide anion (O2(-)), and enzymatic and non-enzymatic antioxidants. TiO2 particles were observed inside mononuclear blood cells and in organ parenchyma at 3 and 12 months. TiO2 deposits were consistently larger in liver than in lung tissue. Alveolar macrophage O2(-) generation and average particle size correlated negatively (p < 0.05). NPs were more reactive and biopersistent in lung tissue than MPs. Antioxidant activity, particularly in the case of 5 nm particles, failed to compensate for membrane damage in liver cells; the damage was consistent with histological evidence of necrosis.

Rattus model utilizing selective pulmonary ischemia induces bronchiolitis obliterans organizing pneumonia.

Bronchiolitis obliterans organizing pneumonia (BOOP), a morbid condition when associated with lung transplant and chronic lung disease, is believed to be a complication of ischemia. Our goal was to develop a simple and reliable model of lung ischemia in the Sprague-Dawley rat that would produce BOOP. Unilateral ischemia without airway occlusion was produced by an occlusive slipknot placed around the left main pulmonary artery. Studies were performed 7 days later. Relative pulmonary and systemic flow to each lung was measured by injection of technetium Tc 99m macroaggregated albumin. Histological sections were examined for structure and necrosis and scored for BOOP. Apoptosis was detected by immunohistochemistry with an antibody against cleaved caspase 3. Pulmonary artery blood flow to left lungs was less than 0.1% of the cardiac output, and bronchial artery circulation was ∼2% of aortic artery flow. Histological sections from ischemic left lungs consistently showed Masson bodies, inflammation, and young fibroblasts filling the distal airways and alveoli, consistent with BOOP. In quantitative evaluation of BOOP using epithelial changes, inflammation and fibrosis were higher in ischemic left lungs than right or sham-operated left lungs. Apoptosis was increased in areas exhibiting histological BOOP, but there was no histological evidence of necrosis. Toll-like receptor 4 expression was increased in ischemic left lungs over right. An occlusive slipknot around the main left pulmonary artery in rats produces BOOP, providing direct evidence that ischemia without immunomodulation or coinfection is sufficient to initiate this injury. It also affords an excellent model to study signaling and genetic mechanisms underlying BOOP.

Immuno-histochemistry in the detection of early myocardial infarction (a post-mortem study)

BackgroundImmunohistochemical techniques have proven to be effective for use in forensic pathology, as they can be applied to tissue samples fixed in formalin and embedded in paraffin. Histologic sections, including ischaemic and non-ischaemic myocardia, were studied via immunohistochemistry with three different antibodies to human cardiac troponin (cTnT), myoglobin (MB), and caspase 3, using a standard avidin–biotin–peroxidase system. ResultsAll antibodies detected cTnT or MB in the normal myocardium as well as its loss from the necrotic myocardium, in some cases before histologic evidence of necrosis was present. Loss was non-uniform and greater at the periphery of the infarcts than at their central regions. Usually, loss of cTnT appeared to be better than loss of MB; however, MB was more sensitive, and caspase 3 was the most sensitive. Caspase 3 was detected in areas with established ischaemia and in cases with coronary occlusion where there was no histologic evidence of necrosis, as well as in cases with latent infarction. ConclusionsImmunohistochemical staining using antibodies to human cTnT, MB, and caspase 3 can be used to visualize myocardial infarctions in histologic sections of the myocardium during forensic autopsy. Loss of cTnT and MB, as well as positive staining for caspase 3, occurs very early following ischaemic injury and may precede histologic evidence of necrosis. Immunohistochemical staining of hearts for cTnT, MB, and caspase 3 can assist in the recognition of myocardial necrosis at autopsy in patients suspected of dying from acute myocardial ischaemia.

Cardiac Myosin Light Chain Kinase is Essential for Myosin Regulatory Light Chain Phosphorylation and Normal Cardiac Function in vivo

In contrast to studies on skeletal and smooth muscles, protein kinases that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) in the heart are not known. Expression of a Ca2+/calmodulin-activated myosin light chain kinase found only in cardiac muscle (cMLCK) was ablated in mice. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Lack of cMLCK and RLC phosphorylation led to (1) ventricular hypertrophy demonstrated by increased heart weight to tibial length ratios, (2) myocyte hypertrophy and (3) histological evidence of necrosis and fibrosis. Loss of MLC2v phosphorylation led to compromised cardiac function evidenced by echocardiography showing a proportional decrease in systolic performance assessed as percent fractional shortening from 71% in wildtype mice to 34% for hearts with no cMLCK. Declines in cardiac function were associated with ventricular dilation and progressive increases in left ventricular end-systolic and end-diastolic dimensions. Hearts from female mice showed similar responses to loss of cMLCK including diminished RLC phosphorylation and significant ventricular hypertrophy. Prolonged isoproterenol infusion elicited hypertrophic cardiac responses in wildtype mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation which is required for normal physiological cardiac performance in vivo. Supported by NIH NHLBI.

Cardiac Myosin Light Chain Kinase Is Necessary for Myosin Regulatory Light Chain Phosphorylation and Cardiac Performance in Vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.

Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L

Background Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis. Materials and Methods Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO 4. Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined. Results The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis. Conclusions DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.

A pilot study of EUS-guided hot saline injection for induction of pancreatic tissue necrosis

Hot saline may be potentially useful for inducing necrosis of pancreatic tissue. However, the local and systemic effects are largely unknown. This pilot study aimed to evaluate the feasibility and safety of EUS-guided injection of hot saline into the pancreas in the porcine model. Boiling hot saline was injected into the tail of normal porcine pancreas under EUS guidance in six pigs via a transgastric approach. Three pigs were killed 4 hours later to study the acute effect of the hot saline injection (acute study). The remaining three pigs were killed after 7 days of clinical observation (survival study). Injection of 5 mL, 2 mL and 1 mL of hot saline produced localized necrosis (7 - 10 mm) of pancreatic tissue in the acute study. However, there was pooling of hot saline on the surface of the pancreas when 5mL was injected. On the basis of the results of the acute study, the volume of hot saline injected in the survival study was 1 mL. One milliliter of hot saline produced localized or sporadic necrosis of pancreatic tissue without any signs of pancreatitis in all three pigs in the survival study; hot saline was observed to pool on the pancreatic surface of one pig. There was no histological evidence of necrosis in the pancreatic tissue adjacent to the pooled hot saline in either the acute or the survival study. EUS-guided hot saline injection of pancreatic tissue in the porcine model was technically successful and led to localized necrosis of pancreatic tissue without any sign of pancreatitis.

Replication-deficient Adenovirus Induces Host Topoisomerase I Activity: Implications for Adenovirus-mediated Gene Expression

Replication-deficient adenoviruses are useful vectors for the transfer of therapeutic transgenes to malignant and non-malignant tissues. Yet their clinical application is limited by the potential toxicity of viral infection and the transient nature of transgene expression. Although transgene expression from adenovirus vectors is initially higher than expression of transgenes transduced by other viral or non-viral vectors, it is often insufficient to generate a significant therapeutic effect. We addressed this issue by searching for DNA-targeted viral-induced host responses potentially restricting transgene expression. Nuclear protein extracts from livers of rats systemically infected with replication-deficient adenovirus exhibited enhanced topoisomerase I activity compared with extracts from uninfected animals. Consequently, the inhibition of topoisomerase I by the anti-cancer drug topotecan greatly enhanced transgene expression in adenovirus-infected hepatic cells, colon cancer and prostate cancer cell cultures, mouse liver, human ex vivo tumor specimens, and mouse tumor in vivo. The enhancement could not be ascribed to non-specific genotoxic stress, cell death, or cell-cycle perturbation. These findings are significant for gene therapy as they reveal novel aspects of the host anti-adenovirus response and set the stage for the development of a rational molecular-pharmacological approach to increase the effectiveness, and safety, of adenovirus-mediated cancer therapeutics.

Modulation of mitochondrial calcium management attenuates hepatic warm ischemia-reperfusion injury

Hepatic warm ischemia and reperfusion (IR) injury occurs in many clinical situations and has an important link to subsequent hepatic failure. The pathogenesis of this injury involves numerous pathways, including mitochondrial-associated apoptosis. We studied the effect of mitochondrial calcium uptake inhibition on hepatic IR injury using the specific mitochondrial calcium uptake inhibitor, ruthenium red (RR). Rats were subjected to 1 hour of 70% warm hepatic ischemia following RR pretreatment or vehicle injection. Sham-operated animals served as controls. Analysis was performed at 15 minutes, 1 hour, 3 hours, or 6 hours after reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were determined. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to assess apoptosis, and hepatocellular necrosis was semiquantitated from hematoxylin and eosin-stained tissue sections. RR pretreatment significantly decreased both AST and ALT serum levels after 6 hours of reperfusion (AST: 1,556 +/- 181 U/L vs. 597 +/- 121 U/L, P = 0.005; ALT: 1,118 +/- 187 U/L vs. 294 +/- 39 U/L, P = 0.005). Apoptosis was observed within 15 minutes of reperfusion in vehicle-pretreated animals and peaked after 3 hours of reperfusion (98 +/- 21 cells/high-power field [hpf]). Apoptosis was inhibited at all time points by RR pretreatment. Histologic evidence of necrosis was not observed prior to 3 hours of reperfusion (23% +/- 4%), and maximal necrosis was observed after 6 hours of reperfusion (26% +/- 1% percent area). RR pretreatment significantly decreased the necrotic percent area at both the 3-hour and the 6-hour time points (4.2% +/- 2%; 3.7% +/- 1%, respectively). Hepatic IR injury resulted in both apoptotic and necrotic cell death, which were attenuated by RR pretreatment. In conclusion, these observations implicate mitochondrial calcium uptake in the pathogenesis of hepatic IR injury.

A study of unfractionated and low molecular weight heparins in a model of cholestatic liver injury in the rat

Forty-eight rats with biliary obstruction induced by double ligation and section of the common bile duct were randomly and blindly assigned to receive subcutaneous injection of either conventional heparin sodium (1000 IU kg −1), three already marketed low molecular weight heparin (LMWH) preparations: nadroparin (1000 anti-Xa IU kg −1), tinzaparin (1000 anti-Xa IU kg −1), enoxaparin (180 anti-Xa IU kg −1) or saline. Drugs were administered once a day, starting 7 days after surgery and continued for 3 weeks. At the end of the treatment period, rats were killed and analyzed for blood biochemistry and liver pathology. Liver fibrosis was assessed by image analysis. Data indicated that treatment with nadroparin decreased plasma total bilirubin, serum alkaline phosphatase (ALP) and gamma glutamyltransferase (GGT) levels by 80.3, 70.7 and 42%, compared with bile duct ligated (BDL) control values. The reduction in plasma total protein observed in BDL controls was prevented by nadroparin. Enoxaparin-treated rats showed significant reduction in plasma total bilirubin and alanine aminotransferase levels by 32.5 and 38.4% versus BDL controls. Liver necrosis evaluated histologically was significantly reduced in the nadroparin- and enoxaparin-treated rats. Morphometric analysis showed significant reduction in fibrosis on nadroparin and enoxaparin treatment: area of fibrosis: 1.66±0.17% and 14.03±1.1% versus 18.94±2.4% ( P<0.05); nadroparin and enoxaparin versus BDL control. By contrast, neither conventional heparin nor tinzaparin prevented the bile duct ligation-induced liver damage as indicated by increased plasma aminotransferases, ALP and GGT concentrations and the histological evidence of necrosis. Total serum bilirubin was increased by 27.5% in rats treated with conventional heparin, while ALP and GGT levels were 38.6 and 31.4% higher after tinzaparin treatment versus BDL controls. Significant increase in the area of fibrosis was observed after tinzaparin treatment compared to BDL control group. Results suggest a beneficial effect for nadroparin and enoxaparin in the therapy of patients with obstructive jaundice or cholestatic liver disorders. The present data from bile duct ligated rats suggest an antifibrotic effect for nadroparin and enoxaparin.

Effects of endotoxin tolerance on Propionibacterium acnes-primed lipopolysaccharide hepatic injury 1,2

Background. Prior administration of the Gram-positive bacteria Propionibacterium acnes (PA) results in hypersensitivity and hepatocyte necrosis to a subsequent low dose of lipopolysaccharide (LPS). Endotoxin tolerance has been shown to prevent lethality after ischemia/reperfusion injuries, sepsis, and endotoxic shock. We investigated whether prior induction of LPS tolerance could prevent subsequent PA-priming and LPS-induced death. The levels of known effector cytokines possibly responsible for these changes were measured. Methods. C57BL/6 (B6) mice were given heat-killed PA (0.5 mg/mouse) followed 7 days later by LPS (20 μg/mouse). In parallel experiments, B6 mice were pretreated with a single 20 μg/mouse dose of LPS (lethal dose = 800 μg/mouse) 7 days prior to PA priming. Animal survival, liver and spleen weights, and histology were examined. Cytokine levels of the inflammatory cytokines interferon-α, tumor necrosis factor-γ, interleukin (IL)-6, and IL-12 and the anti-inflammatory cytokines IL-4 and IL-10 were measured by enzyme-linked immunosorbent assay and by reverse-transcription polymerase chain reaction. Results. Hepatomegaly, splenomegaly, and hepatocyte necrosis with death developed in all PA-primed B6 mice challenged with LPS. However, 83% of B6 mice given a tolerizing dose of LPS prior to PA survived ( P < 0.01) without any increase in liver or spleen weights and without histological evidence of necrosis. Markedly decreased in vivo and in vitro inflammatory (interferon-α, tumor necrosis factor-γ, IL-6, and IL-12) cytokine levels corresponded with survival in the LPS-tolerant mice. Endotoxin tolerance and subsequent survival were also associated with an increase in anti-inflammatory (IL-4 and IL-10) mRNA expression. Conclusions. Lethal PA-primed LPS-induced hepatic injury can be prevented by administering a tolerizing dose of LPS prior to PA-priming. LPS protects the liver by preventing hepatic mononuclear cellular infiltration, reducing the production of the toxic proinflammatory cytokines, and inducing the production of endogenous anti-inflammatory cytokines.

Phase II study of accelerated fractionation radiation therapy with carboplatin followed by PCV chemotherapy for the treatment of anaplastic gliomas

Purpose: To conduct a Phase II one-arm study to evaluate the long-term efficacy and safety of accelerated fractionated radiotherapy combined with i.v. carboplatin for patients with previously untreated anaplastic gliomas. Methods and Materials: Between 1988 and 1992, 90 patients received 1.9–2.0-Gy radiation 3 times a day with 2-h infusions of 33 g/m 2 carboplatin for two 5-day cycles separated by 2 weeks. After radiotherapy, patients received procarbazine, lomustine (CCNU), and vincristine (PCV) for 1 year or until the tumor progressed. Results: Ninety patients were evaluable for analysis. Histologically, 69 had anaplastic astrocytoma; 14, anaplastic oligoastrocytoma; and 7, anaplastic oligodendroglioma. Gross total resection was performed in 20 (22%), subtotal resection in 45 (50%), and biopsy in 25 (28%); reoperation (total or subtotal resection) was performed in 50 (56%) patients. A multivariate analysis showed that a younger age ( p = 0.026), Karnofsky performance score (KPS; p = 0.009), and brain necrosis ( p = 0.0002) were predictive of a better survival. Results from analysis of extent of surgery (biopsy, subtotal resection, gross total resection) approached significance ( p = 0.058). Radiation dose, irradiated tumor volume, and techniques used (boost and fields) were not significant variables. The median survival (MS) of all anaplastic glioma patients was 28.1 months; for anaplastic astrocytoma patients, MS was 28.7 months and 40.8 months for the combined anaplastic oligodendroglioma/oligoastrocytoma patients. Long-term survival occurred in 25% of anaplastic glioma patients who were alive 8.6 years after treatment was initiated. Treatment-induced necrosis was documented by surgery or autopsy in 19 (21%) patients; 21 (23%) had a mixed pattern of necrosis and tumor; and an additional 13 (14%) patients who did not have surgical or autopsy demonstration of predominant radiation necrosis had magnetic resonance imaging (MRI) evidence of radiation necrosis. Serious clinical neurologic deterioration and/or dementia requiring full-time caregiver attention were observed in 9 (10%) patients. Conclusion: When comparable selection criteria are applied, the rate of MS in this study is inferior to results attainable with current radiation and chemotherapy approaches, although the rates of long-term survival are comparable. Theoretically, patients failing therapy and dying earlier than anticipated may be because of excessive central nervous system (CNS) toxicity resulting from the combination of accelerated fractionated irradiation, intensive carboplatin chemotherapy before each radiation fraction, and postirradiation PCV chemotherapy. On the other hand, patients with treatment-induced necrosis survived significantly longer than patients who did not demonstrate MRI or histologic evidence of necrosis (MS, 106 months vs. 18–33 months).

Combination of preconditioning and delayed flap elevation: evidence for improved perfusion and oxygenation of the latissimus dorsi muscle for cardiomyoplasty

Background. Atrophy and fibrosis of the distal part of the latissimus dorsi muscle (LDM) wrap is a recognized complication of cardiomyoplasty that has been attributed to ischemia. Failure of the muscle wrap contributes to the late attrition seen in clinical cardiomyoplasty. In this study we examined the role of two-staged mobilization and of preconditioning by electrical stimulation on the regional perfusion and oxygenation of the LDM. Methods. In a rabbit model (n = 36) the LDM was preconditioned as follows: group A muscles received preconditioning in situ; group B muscles were partially mobilized by dividing the intercostal perforators and then preconditioned; and group C muscles were completely mobilized and wrapped around a silicone-rubber mandrel before conditioning. Controls received no conditioning. The preconditioning regimen consisted of 2 weeks of continuous stimulation at 2.5 Hz. At completion of preconditioning the muscles were fully mobilized and mounted on a muscle-testing apparatus. Purpose-built microelectrodes measured regional Po 2 and perfusion using a diffusible gas tracer technique. Muscles were weighed and processed for fiber typing and capillary counting. Results. All preconditioned muscles demonstrated fiber transformation, with increased fatigue resistance. Perfusion of preconditioned muscles both at rest and during contraction was higher than control in the proximal part of the muscle. Distal regions of group B muscles had higher perfusion and capillary density than any other group ( p < 0.05). Distal regions of group C had the lowest perfusion and capillary density, and showed muscle atrophy and histologic evidence of necrosis. During fatigue testing there was a decrease in the Po 2 in the distal regions of the control and group C muscles ( p < 0.05), whereas it was maintained at resting levels in both group A and B muscles. Conclusions. Conditioning in situ improves perfusion of the distal LDM and prevents a fall in tissue Po 2 during contraction. Two-stage mobilization further improves distal perfusion and capillary density. In contrast, short-term elevation followed by conditioning produces impaired distal perfusion, decrease in Po 2, and fiber necrosis in the distal muscle. The present study suggests that partial mobilization of the LDM performed at the same time as placement of electrodes for preconditioning may prepare the LDM better for the demands of cardiomyoplasty.