Search

Supplementary Data from Orthovoltage X-Rays Exhibit Increased Efficacy Compared with γ-Rays in Preclinical Irradiation

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects.Significance:Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects. Significance: Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

PurposeRadionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. There are institutional initiatives to replace radionuclide sources with lower-energy X-ray sources amidst concerns of radiological terrorism. As researchers transition, there are questions whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays, since different energy may cause different biological effects. We, therefore, sought to compare the effects of orthovoltage X-rays and 137Cs γ-rays using mouse models of acute radiation syndrome.Experimental Design137Cs γ-rays were compared with Orthovoltage X-rays, generated at 300 kVp, 10 mA with 1 mm Cu or Thoraeus filtration. We assessed 30-day overall survival following whole-body irradiation and calculated LD50 by logistic regression. Comparing equivalent doses delivered with different average energies (Ē), we assessed bone marrow, spleen, and intestinal histology and flow cytometry.ResultsThe LD50 doses are 6.7 Gy, 7.4 Gy and 8.1 Gy with 1 mm Cu filtered (Ē=120 keV), and Thoraeus filtered X-rays (Ē=160 keV), and 137Cs (E=662 keV), respectively. At constant dose, hematopoietic injury was most severe with 1 mm Cu filtered X-rays with the greatest reduction in bone marrow cellularity, stem and progenitor populations, and intestinal crypts and OLFM4+ intestinal stem cells. Thoraeus filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage.ConclusionsOur study reveals a dichotomy between physical dose and biological effect relevant as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose-reduction to achieve comparable biological effects.Statement of Translational RelevanceRadiation is used in translational studies in fields ranging from hematopoiesis and stem cell biology to cancer radiotherapy, with 137Cs and 60Co radionuclide sources serving as the most common irradiators. Due to the threat of radiological terrorism using stolen radionuclides, there are institutional initiatives to replace these sources with orthovoltage X-ray irradiators. Yet, as shown in this study, the biological effects of radiation are highly dependent on radiation energy. Lower energy orthovoltage X-rays are absorbed differently than higher energy radionuclide γ-rays, provoking more severe hematopoietic, immunologic, and gastrointestinal radiation injury. Thus, an identical physical dose delivered with beams of differing energy does not produce the same biologic effect. As researchers transition between these sources, it is critical that we appreciate that radiation doses are not interchangeable between them. Understanding the significance of physical dose delivered using different methods will allow us to contextualize past results with future studies.

Many gallbladder adenocarcinomas (ACs) are detected incidentally in routine cholecystectomy specimens, yet sampling practices vary when intestinal metaplasia (IM) or dysplasia are found via routine sampling. Our practice has been to submit 5 additional sections when IM is found, but cases with dysplasia are entirely submitted. We sought to determine an appropriate sampling protocol when encountering these findings. We retrospectively identified cholecystectomy specimens with these features over a 26-month period, yielding 48 of 4059 (1%) cases. Four pathologists independently classified the (2 longitudinal and 1 cystic duct margin) original sections into 1 of 3 categories (IM, low-grade dysplasia [LGD] or high-grade dysplasia [HGD]); initial findings were correlated with final diagnoses. Sixteen (33%) cases had additional findings upon further sampling, including LGD (n=10) or HGD (n=4) and AC (n=2). HGD always accompanied malignancy. We prospectively analyzed 39 of 3133 (1%) additional cholecystectomy specimens, initially submitting the same routine sections. We submitted 5 random sections from cases with IM. Cases with LGD were first examined with 1 additional section per centimeter. All remaining tissue was submitted in all of these cases and separately reviewed. Cases with HGD were entirely submitted as both test cases with HGD in initial sections ultimately showed carcinoma. This protocol detected all cases of HGD and AC. Patients with clear cystic duct margins did not experience neoplastic progression, even if dysplasia was present elsewhere. We conclude gallbladders with HGD should be entirely submitted, LGD may be representatively sampled, and routine sampling is adequate for IM.

Background One mechanism underlying the development of alcoholic liver disease is overactivation of the innate immune response. Recent investigations indicate that the lysosomal pathway of autophagy down-regulates the inflammatory state of hepatic macrophages, suggesting that macrophage autophagy may regulate innate immunity in alcoholic liver disease. The function of macrophage autophagy in the development of alcoholic liver disease was examined in studies employing mice with a myeloid-specific decrease in autophagy. Methods Littermate control and Atg5Δmye mice lacking Atg5-dependent myeloid autophagy were administered a Lieber-DeCarli control (CD) or ethanol diet (ED) alone or together with lipopolysaccharide (LPS) and examined for the degree of liver injury and inflammation. Results Knockout mice with decreased macrophage autophagy had equivalent steatosis but increased mortality and liver injury from ED alone. Increased liver injury and hepatocyte death also occurred in Atg5Δmye mice administered ED and LPS in association with systemic inflammation as indicated by elevated serum levels of proinflammatory cytokines. Hepatic macrophage and neutrophil infiltration were unaffected by decreased autophagy, but levels of proinflammatory cytokine gene induction were significantly increased in the livers but not adipose tissue of knockout mice treated with ED and LPS. Inflammasome activation was increased in ED/LPS-treated knockout mice resulting in elevated interleukin (IL)-1β production. Increased IL-1β promoted alcoholic liver disease as liver injury was decreased by the administration of an IL-1 receptor antagonist. Conclusions Macrophage autophagy functions to prevent liver injury from alcohol. This protection is mediated in part by down-regulation of inflammasome-dependent and inflammasome-independent hepatic inflammation. Therapies to increase autophagy may be effective in this disease through anti-inflammatory effects on macrophages.

Toxin-induced liver diseases lack effective therapies despite increased understanding of the role factors such as an overactive innate immune response play in the pathogenesis of this form of hepatic injury. Pentamidine is an effective antimicrobial agent against several human pathogens, but studies have also suggested that this drug inhibits inflammation. This potential anti-inflammatory mechanism of action, together with the development of a new oral form of pentamidine isethionate VLX103, led to investigations of the effectiveness of this drug in the prevention and treatment of hepatotoxic liver injury. Pretreatment with a single injection of VLX103 in the D-galactosamine (GalN) and lipopolysaccharide (LPS) model of acute, fulminant liver injury dramatically decreased serum alanine aminotransferase levels, histological injury, the number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells and mortality as compared to vehicle-injected controls. VLX103 decreased GalN/LPS induction of TNF but had no effect on other proinflammatory cytokines. VLX103 prevented the proinflammatory activation of cultured hepatic macrophages and partially blocked liver injury from GalN/TNF. In GalN/LPS-treated mice, VLX103 decreased activation of both the mitochondrial death pathway and downstream effector caspases 3 and 7 which resulted from reduced c-Jun N-terminal kinase activation and initiator caspase 8 cleavage. Delaying VLX103 treatment for up to 3 h after GalN/LPS administration was still remarkably effective in blocking liver injury in this model. Oral administration of VLX103 also decreased hepatotoxic injury in a second more chronic model of alcohol-induced liver injury, as demonstrated by decreased serum alanine and aspartate aminotransferase levels and numbers of TUNEL-positive cells. Conclusion: VLX103 effectively decreases toxin-induced liver injury in mice and may be an effective therapy for this and other forms of human liver disease. This article is protected by copyright. All rights reserved.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Background & Aims Overactivation of the innate immune response underlies many forms of liver injury including that caused by hepatotoxins. Recent studies have demonstrated that macrophage autophagy regulates innate immunity and resultant tissue inflammation. Although hepatocyte autophagy has been shown to modulate hepatic injury, little is known about the role of autophagy in hepatic macrophages during the inflammatory response to acute toxic liver injury. Our aim therefore was to determine whether macrophage autophagy functions to down regulate hepatic inflammation. Methods Mice with a LysM- CRE -mediated macrophage knockout of the autophagy gene ATG5 were examined for their response to toxin-induced liver injury from D-galactosamine/lipopolysaccharide (GalN/LPS). Results Knockout mice had increased liver injury from GalN/LPS as determined by significant increases in serum alanine aminotransferase, histological evidence of liver injury, positive terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, caspase activation and mortality as compared to littermate controls. Levels of proinflammatory tumor necrosis factor and interleukin (IL)-6 hepatic mRNA and serum protein were unchanged, but serum IL-1β was significantly increased in knockout mice. The increase in serum IL-1β was secondary to elevated hepatic caspase 1 activation and inflammasome-mediated cleavage of pro-IL-1β to its active form. Cultured hepatic macrophages from GalN/LPS-treated knockout mice had similarly increased IL-1β production. Dysregulation of IL-1β was the mechanism of increased liver injury as an IL-1 receptor antagonist prevented injury in knockout mice in concert with decreased neutrophil activation. Conclusions Macrophage autophagy functions to limit acute toxin-induced liver injury and death by inhibiting the generation of inflammasome-dependent IL-1β.

Purpose To identify novel genetic markers predictive of clinical benefit from epidermal growth factor receptor–directed antibody therapy in patients with metastatic colorectal cancer. Patients and Methods Seventy-six consecutive patients who received cetuximab or panitumumab, either alone or in combination with chemotherapy and with available tumor tissue were included. Tumor tissue was tested by pyrosequencing for mutations at known hot spots in the KRAS, BRAF, PIK3CA, PIK3R1, AKT1, and PTEN genes. PTEN promoter methylation status was analyzed by methylation-specific polymerase chain reaction, and expression was determined by immunohistochemistry (IHC). Forty-four patients had 4 weeks of therapy and were considered for clinical correlates. Results Consistent with previous studies, KRAS gene mutations were associated with a shorter progression-free survival (PFS) and overall survival (OS). Among the patients with wild-type KRAS, preservation of PTEN expression and PIK3CA wild-type status was associated with improved OS (median OS, 80.4 vs. 32.5 weeks; hazard ratio, 0.33; P = .0008) and a trend toward improved PFS (median PFS, 24.8 vs. 15.2 weeks; hazard ratio, 0.51; P = .06), compared with PTEN-negative or PIK3CA-mutant tumors. PTEN methylation was more common in the metastatic samples than in the primary samples ( P = .02). The simultaneous presence of methylation and mutation in the PTEN gene was associated with IHC negativity ( P = .026). Conclusion In addition to KRAS mutation, loss of PTEN expression (by IHC) and PIK3CA mutation is likely to be predictive of a lack of benefit to anti-EGFR therapy in metastatic colorectal cancer. PTEN promoter methylation and mutation status was predictive of PTEN expression and may be used as an alternative means of predicting response to EGFR-targeted therapy.

The hepatocellular mechanisms underlying the development of steatosis and the progression to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) remain unclear.1 A critical factor in the pathogenesis of this disease is thought to be the existence of insulin resistance, and NAFLD can be considered a component of the metabolic syndrome whose manifestations also include obesity and diabetes.2,3 Recent investigations have suggested that activation of the mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) is a central mechanism for the development of obesity and insulin resistance as well as for steatohepatitis.4-7 JNK may therefore represent a unique common target for the therapy of a variety of diseases that comprise the metabolic syndrome, but whether an inhibition of JNK function will decrease or prevent further progression of established steatohepatitis is currently unknown. Most cells including hepatocytes express two JNK genes, jnk1 and jnk2, which are alternatively spliced to yield α and β forms of both a p54 and p46 protein.8 Recent studies have demonstrated that the JNK isoforms differ in function as exemplified by the ability of JNK1 to phosphorylate and activate the transcription factor c-Jun, whereas JNK2 lacks this activity and may even oppose this action of JNK1.9 Our prior studies have implicated JNK signaling in the development of steatohepatitis. First, it was demonstrated that hepatocyte overexpression of the prooxidant enzyme cytochrome P450 2E1 as occurs in human NAFLD induced sustained JNK activation and insulin resistance.10 Second, the development of murine steatohepatitis induced by a methionine- and choline-deficient diet was associated with increased JNK activation.6 The development of steatosis and liver injury was prevented in jnk1 but not jnk2 null mice, indicating that JNK1 specifically functions in the development of this disease.6 JNK1 has also been demonstrated to mediate the development of obesity and insulin resistance in both high fat diet (HFD)-fed and genetically obese mouse models.4 JNK2 was reportedly not involved in these processes, but subsequent studies in mice haploinsufficient for jnk1 and null for jnk2 suggested that JNK2 may also promote the development of obesity and insulin resistance.7 These mice and jnk1-/- mice also failed to develop steatosis from a HFD.7 Thus, JNK1 function is clearly linked to the development of steatohepatitis, obesity and insulin resistance whereas the function of JNK2 in these conditions is less clear. To more clearly establish the function of JNK1 and JNK2 in steatohepatitis occurring in the setting of obesity and insulin resistance, the effect of JNK1 or JNK2 ablation on the development of steatohepatitis was examined in HFD-fed mice. Although such studies are important to our understanding of the pathophysiology of NAFLD, findings derived from investigations of developing disease do not define JNK function in the progression or perpetuation of established steatohepatitis. Studies to examine the role of JNK in existing steatohepatitis have not been performed and are critical to determine whether JNK is a viable therapeutic target in this disease. The development of antisense oligonucleotides (ASO) that are effective in inhibiting JNK1 or JNK2 expression in the liver,11 led us to examine the effects of JNK inhibition in established as well as developing steatohepatitis in HFD-fed mice. The studies demonstrate that JNK1 mediates insulin resistance and both the development and progression of steatohepatitis. In contrast, although JNK2 promoted insulin resistance, this JNK isoform did not mediate the development or progression of steatohepatitis. These findings indicate that the JNK isoforms have distinct functions that differ among the various manifestations of the metabolic syndrome and have important implications for JNK as a therapeutic target in this disorder.

Dengue infections are caused by a single-stranded RNA virus, which has four serotypes (DEN 1-4); mosquitoes of the genus Aedes serve as vectors of transmission. Risk factors for dengue infection are related to both the host and virus. Age, gender, immune status, and genetic background of the host all contribute to the severity of dengue infection. Recently, international travel to endemic areas has also been identified as a major risk factor for both primary and secondary dengue infection. Dengue remains a diagnostic challenge, given its protean nature, ranging from mild febrile illness to profound shock. The most severe manifestation of dengue infection is dengue shock syndrome, which has an estimated mortality rate close to 50%. Dengue shock syndrome typically presents with increased anion gap metabolic acidosis, disseminated intravascular coagulation, severe hypotension, and jaundice. Liver involvement appears to occur more frequently when infections involve DEN-3 and DEN-4 serotypes. While hepatocellular damage has been reported previously in dengue infection, acute liver failure is an extremely rare occurrence in adults. We report a patient with dengue shock syndrome who presented with acute liver failure and hepatic encephalopathy after recent travel to an endemic area.

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

Approximately one-third of the human population is latently infected withMycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintainingM. tuberculosisin the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five relatedM. tuberculosisproteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role ofM. tuberculosis rpfgenes in an established mouse model ofM. tuberculosispersistence and reactivation. After an aerosol infection with theM. tuberculosisErdman wild type (Erdman) or single-deletionrpfmutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the fiverpfdeletion mutants tested, one (ΔRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and ΔRv1009-infected mice at advanced stages of reactivation. Analysis ofrpfgene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the fiverpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting thatrpfgene expression may be regulated in a growth phase-dependent manner. To our knowledge, ΔRv1009 is the firstM. tuberculosismutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.

The granulomatous reaction is the hallmark of the host response to infection with Mycobacterium tuberculosis. Despite its apparent importance to host defence against the tubercle bacillus, the granulomatous response remains to be completely defined. The present study used histological, immunohistochemical and flow-cytometric analyses to characterize pulmonic granulomatous tissues of tuberculous mice and humans. The kinetics of recruitment of neutrophils, macrophages, dendritic cells, and T and B lymphocytes into the lungs of mice infected aerogenically with the virulent Erdman strain of M. tuberculosis was evaluated in detail in both the acute and persistent phase of infection. A hypoxia-sensing compound based on the 2-nitroimidazole structure (EF5), together with immunohistochemical studies targeting endothelial cells were used to examine the relative oxygen tension in tuberculous granulomatous tissues in mice. The results have provided evidence that: (i) the granulomatous tissues are a highly organized structure whose formation is regulated by orderly recruitment of specific immune cells exhibiting distinct spatial relationship with one another; (ii) the granulomatous reaction, at least in the mouse, may represent an exaggerated response to the tubercle bacillus that can play a role in the development of immunopathology; (iii) B lymphoid aggregates are a prominent feature in both murine and human granulomatous tissues, although the immune cells that are most prominently associated with these clusters vary among the two species; (iv) murine tuberculous granulomatous tissues are relatively aerobic, suggesting that mouse models of persistent tuberculosis may not be suitable for the study of any hypoxic response of M. tuberculosis.

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.

Introduction: Nodular regenerative hyperplasia (NRH) is a known etiology of noncirrhotic portal hypertension. Cases of biopsy-proven NRH in human immunodeficiency virus (HIV)–positive patients have been described. While these patients often have normal synthetic liver function, several reports described disease progression to liver failure. Case: We here present a 26-year-old woman with history of congenital HIV on antiretroviral therapy complicated by Pneumocystis carinii pneumonia at age 14. CD4 counts have been >300 with undetectable viral load. She was referred to our Hepatology service for evaluation of splenomegaly, elevated liver tests, and thrombocytopenia. On initial presentation, she reported easy bruising and gingival bleeding, and abdominal imaging showed evidence of portal hypertension without associated cirrhosis. Upper endoscopy was significant for large esophageal varices without bleeding stigmata. Liver biopsy showed minimal fibrosis around the portal areas without significant inflammation. The lobules showed focal zones of thin hepatocyte plates on reticulin stain with adjacent areas showing mild regenerative changes. The diagnosis of NRH was made and patient was placed on propranolol for variceal bleeding prophylaxis. Two years later, the patient presented with bleeding gastric varices warranting transjugular intrahepatic portosystemic shunt. Postprocedure course was complicated by mild encephalopathy. Subsequent magnetic resonance imaging showed a 1.7 × 1.3 cm lesion suggestive of hepatocellular carcinoma (HCC). The patient was deemed to be a candidate for liver transplantation, and she is now delisted due to ongoing pregnancy. Conclusion: This report describes the first case of HCC in an HIV patient with NRH. The possible association of NRH with HCC warrants further investigation.

Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) againstMycobacterium tuberculosisinfection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense againstM. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous,M. tuberculosisinfection of mice. Furthermore, in vitro studies have demonstrated that different strains ofM. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates ofM. tuberculosisfollowing both aerogenic and intravenous challenges. Three recently isolated and uniqueM. tuberculosisstrains were used to infect both wild-type (wt) C57BL/6 andNOS2gene-disrupted mice. Regardless of the route of infection, NOS2−/−mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain ofM. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2−/−mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2−/−mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains ofM. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused byMycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-α), we examined the effects of TNF-α on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-α in the containment of persistent tuberculosis. TNF-α neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-α-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-α-neutralized mice. In summary, results of this study suggest that TNF-α plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

Tuberculosis is a major cause of death in much of the world. Current estimates are that one-third of the world's population is infected with Mycobacterium tuberculosis. Most infected persons control the infection but in many cases may not eliminate the organism. Reactivation of this clinically latent infection is responsible for a large proportion of active tuberculosis cases. A major risk factor for reactivation of latent tuberculosis is HIV infection, suggesting a role for the CD4(+) T cell subset in maintaining the latent persistent infection. In this study, we tested the requirement for CD4(+) T cells in preventing reactivation in a murine model of latent tuberculosis. Antibody-mediated depletion of CD4(+) T cells resulted in rapid reactivation of a persistent infection, with dramatically increased bacterial numbers in the organs, increased pathology in the lungs, and decreased survival. Although CD4(+) T cells are believed to be a major source of interferon (IFN)-gamma, expression of the gene for IFN-gamma in the lungs of CD4(+) T cell-depleted mice was similar to that in control mice. In addition, inducible nitric oxide synthase production and activity was unimpaired after CD4(+) T cell depletion, indicating that macrophage activation was present even during CD4(+) T cell deficiency. These data indicate that CD4(+) T cells are necessary to prevent reactivation but may have roles in addition to IFN-gamma production and macrophage activation in controlling a persistent tuberculous infection.

A unique feature of Mycobacterium tuberculosis is its ability to establish latent infection in the human host, which can reactivate to cause disease years later. In the present study, the mechanisms involved in the control of latent tuberculous infection were examined using two murine experimental tuberculosis models. Analysis of the model involving infection of mice with a relatively low inoculum of the virulent Erdman strain of M. tuberculosis indicated that in vivo inhibition of reactive nitrogen intermediate (RNI) production by the nitric oxide synthase inhibitor aminoguanidine resulted in reactivation. This reactivation was evidenced by hepatosplenomegaly, a robust tissue granulomatous reaction, and increased bacillary load. IFN-γ, TNF-α, and inducible nitric oxide synthase were all expressed throughout the latent phase of infection. Reactivation of latent tuberculous infection by aminoguanidine treatment was confirmed using a second murine tuberculosis model based on treatment with antimycobacterial drugs. Results obtained using this drug-based model also suggested the existence of an RNI-independent antimycobacterial mechanism(s) operative in the latent phase of infection. Together, these data suggest that both RNI-dependent and -independent mechanisms contribute to the prevention of tuberculous reactivation.

Fibrous long-spacing (FLS) collagen is a distinct ultrastructural form of collagen present in normal tissue, various tumors, and tissues degraded by bacterial collagenases in vivo and in vitro. An association between FLS collagen and bacillary angiomatosis has not been previously described. Six cases of bacillary angiomatosis, including one autopsy case with disseminated disease, were examined ultrastructurally. In addition, Kaposi sarcoma (3), pyogenic granuloma (3), capillary hemangioma (3), and cavernous hemangioma (2) were examined for comparison. A vascular proliferation in a lymph node from a patient with AIDS (1) and a case of pulmonary capillary hemangiomatosis (1), also in an AIDS patient, were studied. Abundant FLS collagen was identified in 4 of 6 cases of bacillary angiomatosis, in close association with the organisms. FLS collagen was not seen beyond the immediate vicinity of the organisms. The FLS collagen in bacillary angiomatosis was seen in skin biopsies and in lung and skeletal muscle in the autopsy case; in the latter case, as well as in the two AIDS-associated, nonbacillary angiomatosis, non-Kaposi sarcoma vascular proliferations, there was a striking distribution of FLS collagen around small blood vessels. Occasional FLS collagen was observed in all three pyogenic granuloma. When present in pyogenic granuloma, FLS collagen was intermixed with subendothelial collagen. Abundant FLS collagen was identified in close association with the organisms of bacillary angiomatosis in four cases; this morphologic alteration was seen in skin as well as lung and skeletal muscle. An association between FLS collagen and endothelial cells in normal tissue (Descemet's membrane) and in certain vascular proliferations appears to exist.

(1997). Effects of Protein Calorie Malnutrition on Mice Infected with BCG. Journal of Nutritional Immunology: Vol. 5, No. 1, pp. 11-19.

Background: Immunohistochemical methods were used to study the pattern of expression of tenascin (TN) in invasive colon cancer and its relation to prognosis. Methods: Sixty patients (29 males, 31 females) with a mean age of 77 years were studied. TN expression was evaluated by immunohistochem-istry using paraffin-embedded tissue sections, TN expression levels were correlated with patient age, tumor stage, and survival. Results: TN positivity varied from trace to 4+. Staining patterns were as follows: in well-differentiated cancer, TN fibers form thick bands around invading tumor glands. In poorly differentiated cancer, TN fibers had an interstitial pattern surrounding individual tumor cells. Using Cox's proportional hazard regression method, survival was significantly related to TN score (P < 0.0001) and stage of disease (P < 0.05). No significant relationship was found between survival and age (P = 0.375). Conclusion: Patients with more TN expression had better long-term survival than patients with no or weak TN expression. Pathologic and clinical entities in colon cancer have distinct immunohistochemical TN matrix patterns that may correlate with predictive value and long-term survival.

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and -7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.

Nonalcoholic fatty liver disease (NAFLD) represents a continuum from simple hepatic steatosis alone to steatosis together with hepatitis and eventual fibrosis.1 The mechanisms underlying the development of this disease remain unclear, but NAFLD is considered the hepatic manifestation of the metabolic syndrome and therefore linked to the development of obesity and insulin resistance.2 The prevalence of the metabolic syndrome increases markedly with aging.3 In 20 to 29 year olds the prevalence is 7% but rises to 44% in individuals aged 60 to 69.3 Similarly aging has also been shown to be a risk factor for the development of human NAFLD,4,5 although some studies have suggested that this effect is limited to females.6,7 More conclusive of an effect of aging on NAFLD is evidence that the prevalence of nonalcoholic steatohepatitis (NASH) and chronic liver disease is increased in the aged. Thus, steatohepatitis8 and fibrosis9,10 are increased with aging leading to a higher mortality in aged individuals with NAFLD.11,12 Aging may promote the development of NAFLD through several mechanisms. First, aging promotes the onset of obesity and diabetes and the increase in these known risk factors for NAFLD may account for the higher prevalence of NAFLD in the aged. Supportive of this possibility is that in parallel with steep rises in obesity and insulin resistance in the pediatric population, problems that had formerly been restricted to the elderly, there has been a dramatic increase in the incidence of NAFLD in young individuals. Second, the increase in disease with age may result from the cumulative effects over many years of lifestyle or environmental factors such as the over consumption of a Western diet. Finally, physiological changes inherent to the process of aging may trigger or promote the development of components of the metabolic syndrome later in life. For example, function of the lysosomal degradative pathway of autophagy decreases with aging,13 and adequate levels of hepatic autophagy are critical to limit lipid accumulation in the liver.14,15 Another possibility is that aging is associated with increased oxidative stress,16 which has also been implicated as a mediator of NAFLD.17 Several of the known, or even currently unknown, physiological changes that occur with aging may therefore lead to NAFLD. To begin to examine the potential effects of aging on NAFLD development, we challenged mice of different ages with a defined period of high-fat diet (HFD) feeding to induce the metabolic syndrome and NAFLD. This design was intended to ask whether the aging state altered the development of steatosis and hepatitis in response to a defined period of dietary factors that induce NAFLD. Age failed to affect the development of metabolic abnormalities such as weight gain and insulin resistance. The amount of steatosis was similarly unaffected by age. However, aged mice developed more liver injury and a greater inflammatory response from a HFD. Studies in cultured hepatocytes revealed that cells from aged mice were sensitized to death from stimulation of the Fas death pathway. Aging therefore does not affect the development of simple steatosis but promotes the progression to steatohepatitis.

Gap junction-mediated communication is required for normal cellular growth and differentiation. As cancer is thought to be a manifestation of the breakdown of cell-cell communication, with the concomitant loss of growth control, it would be expected that alterations in the primary structure, processing, oligomerization or trafficking of connexin (cxn) molecules would have a profound effect on the neoplastic process. Here we a present a preliminary immunohistochemical and molecular analysis of cxn 43 expression in prostatic epithelial cells from resected human tissue. Our data indicate that benign prostatic epithelial cells express cxn 43 protein, but that this expression is diminished in more advanced, anaplastic cancer cells. These data suggest that decreased connexin expression is not involved in the initiation of prostate cancer, but rather occurs during the progression of the disease.

We have recently demonstrated that the macrophage L-arginine-dependent cytotoxic pathway effectively kills the virulent Erdman strain of Mycobacterium tuberculosis in vitro via the generation of toxic reactive nitrogen intermediates by the enzyme nitric oxide synthase. This report demonstrates that two distinct inhibitors of nitric oxide synthase (aminoguanidine and NG-monomethyl-L-arginine) render similar deleterious effects on tuberculous infection in mice, as assessed by mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of reactive nitrogen intermediates in resistance against M. tuberculosis.

Cardiac involvement is common in systemic lupus erythematosus. Classically, the term "verrucous endocarditis" was used to describe the noninfective vegetations seen on pathological inspection of heart valves. The wide use of echocardiography has led to increased frequency of detection of valve abnormalities, most commonly leaflet thickening. The vast majority of patients with systemic lupus erythematosus and valvular involvement are asymptomatic, with only a small minority progressing to hemodynamically significant pathology, generally after long disease duration. We report a patient with systemic lupus erythematosus and associated antiphospholipid syndrome, whose first presentation of disease consisted of severe, symptomatic valvular regurgitation of the mitral, aortic, and tricuspid valves requiring triple valve surgery.

The etiology of acute respiratory distress syndrome is wide and mortality is extremely high. We describe a patient dying from severe acute respiratory distress syndrome who had a tremendous recovery after receiving dexamethasone (1 g daily). This patient required positive end-expiratory pressure (up to 18 mm/Hg) and fractional inspiratory oxygen (up to 100%). Thirty-six hours after the large dose of corticosteroids, the respiratory mechanics and oxygenation were acceptable for extubation. Acute respiratory distress syndrome was proven and other etiologies of respiratory failure were ruled out by a bedside open-lung biopsy. The biopsy proven acute respiratory distress syndrome dramatically resolved with this salvage therapy. High-dose usage of corticosteroids for acute respiratory distress syndrome has tremendous potential.

Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transferring the Simian Virus 40 Large T-Antigen into primary human hepatocytes. These cells were stably transfected with a full-length HBV genome to generate a clone that expresses HBV genes and replicates HBV. The HBV-producing cells were transplanted into the livers of mice with combined immunodeficiency (Rag-2 deficient) by intrasplenic injection. Survival of the engrafted human hepatocytes was shown in several ways: fluorescent in situ hybridization (FISH) with a human-chromosome-specific DNA probe (human alpha satellite), dot-blot hybridization of the genomic DNA extracted from liver biopsy specimens with a human-specific Alu repetitive DNA probe, Blur-8, as well as with an HBV DNA probe, and secretion of human proteins into plasma. Histological examination of mouse liver up to 8 months following human cell transplant shows completely normal architecture. Determination of plasma HBV DNA levels indicated that engrafted cells secreted 3x10(7) to 3x10(8) virions per mL into the blood, and HBsAg was detected in plasma. This new murine model of HBV viremia should be useful for in vivo HBV studies.

Immunoreactivity to LN2 and LN3, monoclonal antibodies that recognize components of the class II major histocompatibility complex, was assessed in 72 cases of non-small cell lung carcinoma (NSCLC) (32 biopsy specimens, 40 resection specimens) and 64 cases of small cell carcinoma (56 biopsy specimens, 8 resections) of the lung. All cases were reviewed independently by three pathologists for histological classification. Only 1 of the 64 small cell carcinomas showed immunoreactivity for LN2, and none of the 64 cases showed reactivity for LN3. Among the non-small cell carcinomas, 25 of 48 cases were positive for LN2 and 43 of 71 were positive for LN3; the sensitivity was greater for adenocarcinoma (78.5%) than for squamous cell carcinoma (37%). A combined sensitivity of 64.7% was observed when the results of LN2 and LN3 were combined, and this sensitivity was not significantly diminished in the biopsy subset of cases (59.4%). Differentiation within histological subtypes of NSCLC (ie, well, moderate, or poorly differentiated) did not alter test sensitivity. In conclusion, LN2 and LN3, used alone or in combination, appear highly specific for non-small cell carcinoma and moderately sensitive in both biopsy and resection specimens; therefore, these antibodies may be diagnostically useful in distinguishing small cell from non-small cell carcinoma of the lung.

Infectious diseases and malnutrition represent major burdens afflicting millions of people in developing countries. Both conditions affect individuals in industrialized nations, particularly the aged, the HIV-infected, and people with chronic diseases. While malnutrition is known to induce a state of immunodeficiency, the mechanisms responsible for compromised antimicrobial resistance in malnourished hosts remain obscure. In the present study, mice fed a 2% protein diet and developing protein calorie malnutrition, in contrast to well-nourished controls receiving a 20% protein diet, rapidly succumbed to infection withMycobacterium tuberculosis. Malnourished mice exhibited a tissue-specific diminution in the expression of interferon γ, tumor necrosis factor α, and the inducible form of nitric oxide synthase in the lungs, but not the liver. The expression of these molecules critical to the production of mycobactericidal nitrogen oxides was depressed in malnourished animals in the lungs specifically at early times (in vivo, were not detectably diminished in malnourished, mycobacteria-infected mice. In contrast to the selective and early reduction of lymphokines and the inducible form of nitric oxide synthase in the lung, a marked diminution of the granulomatous reaction was observed in malnourished mice throughout the entire course of infection in all tissues examined (lungs, liver, and spleen). Remarkably, the progressively fatal course of tuberculosis observed in the malnourished mice could be reversed by restoring a full protein (20%) diet. The results indicate that protein calorie malnutrition selectively compromises several components of the cellular immune response that are important for containing and restricting tuberculous infection, and suggest that malnutrition-induced susceptibility to some infectious diseases can be reversed or ameliorated by nutritional intervention.

• L0211 HEPATOCYTE TRANSPLANTATION REPOPULATES THE LIVER AND INCREASES SURVIVAL AFTER PARTIAL HEPATECTOMY AND WHOLE LIVER IRRADIATION IN F344 RATS. C. Guha 1, B. Vikram t, S. Gupta 2, A. Sharma 1, A. Alfieri t,3, S. Gagandeep 2, R. P. Sokhi 2, G.R. Gorla l, K.E. Tanaka 4, and J. Roy Chowdhury 2. Depts. of Radiation Oncology 1, Medicine 2, and Pathology 4, Albert Einstein College of Medicine, and Dept. of Radiation Oncology 3, Beth Israel Medical Center, NY.