Identification of alternative protein targets of glutamate-ureido-lysine associated with PSMA tracer uptake in prostate cancer cells.

Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.

Juvenile OLFM4-null mice are protected from sepsis.

Pediatric sepsis is a leading cause of morbidity and mortality in children. One of the most common and devastating morbidities is sepsis-related acute kidney injury (AKI). AKI was traditionally thought to be related to low perfusion and acute tubular necrosis. However, little acute tubular necrosis can be found following septic AKI, and little is known about the mechanism of septic AKI. Olfactomedin-4 (OLFM4) is a secreted glycoprotein that marks a subset of neutrophils. Increased expression of OLFM4 in the blood is associated with worse outcomes in sepsis. Here, we investigated a pediatric model of murine sepsis using murine pups to investigate the mechanisms of OLFM4 in sepsis. When sepsis was induced in murine pups, survival was significantly increased in OLFM4-null pups. Immunohistochemistry at 24 h after the induction of sepsis demonstrated increased expression of OLFM4 in the kidney, which was localized to the loop of Henle. Renal cell apoptosis and plasma creatinine were significantly increased in wild-type versus OLFM4-null pups. Finally, bone marrow transplant suggested that increased OLFM4 in the kidney reflects local production rather than filtered from the plasma. These results demonstrate renal expression of OLFM4 for the first time and suggest that a kidney-specific mechanism may contribute to survival differences in OLFM4-null animals.

Differential Expression of Glucose Transporters and Hexokinases in Prostate Cancer with a Neuroendocrine Gene Signature: A Mechanistic Perspective for 18F-FDG Imaging of PSMA-Suppressed Tumors.

Although the incidence of de novo neuroendocrine prostate cancer (PC) is rare, recent data suggest that low expression of prostate-specific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine hallmarks and androgen receptor (AR) suppression in PC. Previous clinical reports indicate that PCs with a phenotype similar to neuroendocrine tumors can be more amenable to imaging by 18F-FDG than by PSMA-targeting radioligands. In this study, we evaluated the association between neuroendocrine gene signature and 18F-FDG uptake-associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported 18F-FDG avidity of PSMA-suppressed tumors. Methods: Data-mining approaches, cell lines, and patient-derived xenograft models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes HK1-HK3 and GCK), and PSMA (FOLH1 gene) after AR inhibition and in correlation with neuroendocrine hallmarks. Also, we characterize a neuroendocrine-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no neuroendocrine histopathology. We measured glucose uptake in a neuroendocrine-induced in vitro model and a zebrafish model by nonradioactive imaging of glucose uptake using a fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrated that a neuroendocrine gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR inhibitors, high expression of GCK and low expression of SLC2A12 correlated with neuroendocrine histopathology and PSMA gene suppression. GLUT12 suppression and upregulation of glucokinase were observed in neuroendocrine-induced PC cell lines and patient-derived xenograft models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: A neuroendocrine gene signature in neuroendocrine PC and NELPC associates with a distinct transcriptional profile of GLUTs and hexokinases. PSMA suppression correlates with GLUT12 suppression and glucokinase upregulation. Alteration of 18F-FDG uptake-associated genes correlated positively with higher glucose uptake in AR- and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient preclinical method for monitoring nonradioactive glucose uptake.

Cyclin-like proteins tip regenerative balance in the liver to favour cancer formation.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. A variety of factors can contribute to the onset of this disease, including viral infection, obesity, alcohol abuse and non-alcoholic fatty liver disease (NAFLD). These stressors predominantly introduce chronic inflammation leading to liver cirrhosis and finally the onset of HCC; however, approximately 20% of HCC cases arise in the absence of cirrhosis via a poorly defined mechanism. The atypical cyclin-like protein Spy1 is capable of overriding cell cycle checkpoints, promoting proliferation and has been implicated in HCC. We hypothesize that Spy1 promotes sustained proliferation making the liver more susceptible to accumulation of deleterious mutations, leading to the development of non-cirrhotic HCC. We report for the first time that elevation of Spy1 within the liver of a transgenic mouse model leads to enhanced spontaneous liver tumourigenesis. We show that the abundance of Spy1 enhanced fat deposition within the liver and decreased the inflammatory response. Interestingly, Spy1 transgenic mice have a significant reduction in fibrosis and sustained rates of hepatocyte proliferation, and endogenous levels of Spy1 are downregulated during the normal fibrotic response. Our results provide support that abnormal regulation of Spy1 protein drives liver tumorigenesis in the absence of elevated fibrosis and, hence, may represent a potential mechanism behind non-cirrhotic HCC. This work may implicate Spy1 as a prognostic indicator and/or potential target in the treatment of diseases of the liver, such as HCC. The cyclin-like protein Spy1 enhances lipid deposition and reduces fibrosis in the liver. Spy1 also promotes increased hepatocyte proliferation and onset of non-cirrhotic hepatocellular carcinoma (HCC). Thus, Spy1 may be used as a potential target in the treatment of HCC.

Neuroendocrine differentiation of prostate cancer leads to PSMA suppression.

Prostate-specific membrane antigen (PSMA) is overexpressed in most prostate adenocarcinoma (AdPC) cells and acts as a target for molecular imaging. However, some case reports indicate that PSMA-targeted imaging could be ineffectual for delineation of neuroendocrine (NE) prostate cancer (NEPC) lesions due to the suppression of the PSMA gene (FOLH1). These same reports suggest that targeting somatostatin receptor type 2 (SSTR2) could be an alternative diagnostic target for NEPC patients. This study evaluates the correlation between expression of FOLH1, NEPC marker genes and SSTR2. We evaluated the transcript abundance for FOLH1 and SSTR2 genes as well as NE markers across 909 tumors. A significant suppression of FOLH1 in NEPC patient samples and AdPC samples with high expression of NE marker genes was observed. We also investigated protein alterations of PSMA and SSTR2 in an NE-induced cell line derived by hormone depletion and lineage plasticity by loss of p53. PSMA is suppressed following NE induction and cellular plasticity in p53-deficient NEPC model. The PSMA-suppressed cells have more colony formation ability and resistance to enzalutamide treatment. Conversely, SSTR2 was only elevated following hormone depletion. In 18 NEPC patient-derived xenograft (PDX) models we find a significant suppression of FOLH1 and amplification of SSTR2 expression. Due to the observed FOLH1-supressed signature of NEPC, this study cautions on the reliability of using PMSA as a target for molecular imaging of NEPC. The observed elevation of SSTR2 in NEPC supports the possible ability of SSTR2-targeted imaging for follow-up imaging of low PSMA patients and monitoring for NEPC development.

Repetitive Stresses Generate Osteochondral Lesions in Skeletally Immature Rabbits.

BACKGROUND: The origin of juvenile osteochondritis dissecans (OCD) is unknown. Existing experimental animal models of OCD most frequently involve surgically created lesions but do not examine repetitive stress as a possible cause of OCD. HYPOTHESIS: Repetitive stresses can cause OCD-like lesions in immature animals. STUDY DESIGN: Controlled laboratory study. METHODS: Six juvenile rabbits were subjected to repetitive loading forces of approximately 160% body weight to the right hindlimb during five 45-minute sessions per week for 5 weeks. The contralateral limb was the unloaded control. After 5 weeks, rabbits were euthanized and examined with radiographs, micro-computed tomography, and gross and histopathologic analysis. RESULTS: All 6 rabbits developed osteochondral lesions in loaded limbs on the medial and lateral femoral condyles, while contralateral unloaded limbs did not demonstrate lesions. Loaded limbs developed relative osteopenia in the femoral epiphysis and tibial metaphysis with associated decreased trabecular density. Loaded limbs also demonstrated increased femoral subchondral bone thickness near the lesions. Lesions did not have grossly apparent extensive articular cartilage damage; however, cartilage thickness increased on histology with reduced ossification. Loaded knees demonstrated abundant chondrocyte cloning, limited cartilage fissuring, and a focal loss of cellularity at the articular surface. CONCLUSION: Low-grade lesions in human OCD have little gross articular cartilage involvement despite substantial changes to the subchondral bone as shown on magnetic resonance imaging and radiographs. Histopathology findings in this study included cartilage thickening and chondrocyte cloning resembling those of recently published human OCD biopsy studies. Our animal model supports the hypothesis that repetitive stress to immature knees may contribute to the development of human OCD. This model may be useful in understanding the pathophysiology and healing of human OCD. CLINICAL RELEVANCE: Repetitive physiologic stress generated changes to the subchondral bone in immature animals without causing extensive articular damage. The similarities of these lesions in gross and histologic appearance with human OCD support repetitive stress as the likely the cause for human OCD.

Juvenile Osteochondritis Dissecans: Correlation Between Histopathology and MRI.

OBJECTIVE: The objective of our study was to correlate specimens of juvenile osteochondritis dissecans (OCD) lesions of the knee to MRI examinations to elucidate the histopathologic basis of characteristic imaging features. MATERIALS AND METHODS: Five children (three boys and two girls; age range, 12-13 years old) who underwent transarticular biopsy of juvenile OCD lesions of the knee were retrospectively included in this study. Two radiologists reviewed the MRI examinations and a pathologist reviewed the histopathologic specimens and recorded characteristic features. Digital specimen photographs were calibrated to the size of the respective MR image with the use of a reference scale. Photographs were rendered semitransparent and over-laid onto the MR image with the location chosen on the basis of the site of the prior biopsy. RESULTS: A total of seven biopsy specimens were included. On MRI, all lesions showed cystlike foci in the subchondral bone, bone marrow edema pattern on proton density-or T2-weighted images, and relatively thick unossified epiphyseal cartilage. In four patients, a laminar signal intensity pattern was seen, and two patients had multiple breaks in the subchondral bone plate. Fibrovascular tissue was found at histopathology in all patients. Cleft spaces near the cartilage-bone interface and were seen in all patients while chondrocyte cloning was present in most cases. Focal bone necrosis and inflammation were infrequent MRI findings. Precise correlation of the MRI appearance to the histopathologic overlays consistently was found. CONCLUSION: A direct correlation exists between the histopathologic findings and the MRI features in patients with juvenile OCD. Additional studies are needed to correlate these MRI features with juvenile OCD healing success rates.

Effect of higher frequency components and duration of vibration on bone tissue alterations in the rat-tail model.

To formulate more accurate guidelines for musculoskeletal disorders (MSD) linked to Hand-Arm Vibration Syndrome (HAVS), delineation of the response of bone tissue under different frequencies and duration of vibration needs elucidation. Rat-tails were vibrated at 125 Hz (9 rats) and 250 Hz (9 rats), at 49 m/s(2), for 1D (6 rats), 5D (6 rats) and 20D (6 rats); D=days (4 h/d). Rats in the control group (6 rats for the vibration groups; 2 each for 1D, 5D, and 20D) were left in their cages, without being subjected to any vibration. Structural and biochemical damages were quantified using empty lacunae count and nitrotyrosine signal-intensity, respectively. One-way repeated-measure mixed-model ANOVA at p<0.05 level of significance was used for analysis. In the cortical bone, structural damage quantified through empty lacunae count was significant (p<0.05) at 250 Hz (10.82 ± 0.66) in comparison to the control group (7.41 ± 0.76). The biochemical damage was significant (p<0.05) at both the 125 Hz and 250 Hz vibration frequencies. The structural damage was significant (p<0.05) at 5D for cortical bone while the trabecular bone showed significant (p<0.05) damage at 20D time point. Further, the biochemical damage increased with increase in the duration of vibration with a significant (p<0.05) damage observed at 20D time point and a near significant change (p=0.08) observed at 5D time point. Structural and biochemical changes in bone tissue are dependent upon higher vibration frequencies of 125 Hz, 250 Hz and the duration of vibration (5D, 20D).

ETS1 induces human trophoblast differentiation.

A possible role for the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) in human trophoblast cell differentiation was examined using a highly enriched fraction of human mononuclear cytotrophoblast cells (CTBs) that differentiate spontaneously in vitro to a multinucleated syncytiotrophoblast cell (STB) phenotype. ETS1 mRNA and protein levels were abundant in freshly isolated CTBs and decreased as the cells differentiated. Silencing of ETS1 expression in freshly prepared CTBs markedly attenuated syncytialization, as demonstrated by desmoplakin staining, and blocked the induction of syncytin, the transcription factor activator protein-2α, placental lactogen, and other STB-specific genes. Conversely, overexpression of ETS1 in primary trophoblast cells induced STB marker gene mRNAs and transactivated each of the gene proximal promoters. Taken together, these findings strongly suggest a critical role for ETS1 in the induction of human villus CTB differentiation. The effect of ETS1 on syncytialization likely results, at least in part, from inhibition of syncytin expression, whereas the induction of STB marker genes likely results in part from transactivation by activator protein-2α.

Dependence of vascular damage on higher frequency components in the rat-tail model.

Hand-Arm Vibration Syndrome (HAVS) is caused by hand-transmitted vibration in industrial workers. Current ISO guidelines (ISO 5349) might underestimate vascular injury associated with range of vibration frequencies near resonance. A rat-tail model was used to investigate the effects of higher frequencies >100 Hz on early vascular damage. 13 Male Sprague-Dawley rats (250 ± 15 gm) were used. Rat-tails were vibrated at 125 Hz and 250 Hz (49 m/s(2)) for 1D, 5D and 10D; D=days (4 h/day). Structural damage of the ventral artery was quantified by vacuole count using Toluidine blue staining whereas biochemical changes were assessed by nitrotyrosine (NT) staining. The results were analyzed using one-way repeated measures mixed-model ANOVA at p<0.05 level of significance. The structural damage increased at 125 Hz causing significant number of vacuoles (40.62 ± 9.8) compared to control group (8.36 ± 2.49) and reduced at 250 Hz (12.33 ± 2.98) compared to control group (8.36 ± 2.49). However, the biochemical alterations (NT-signal) increased significantly for 125 Hz (143.35 ± 5.8 gray scale value, GSV) and for 250 Hz (155.8 ± 7.35 GSV) compared to the control group (101.7 ± 4.18 GSV). Our results demonstrate that vascular damage in the form of structural and bio chemical disruption is significant at 125 Hz and 250 Hz. Hence the current ISO guidelines might underestimate vascular damage at frequencies>100 Hz.

Downregulation of glutathione S-transferase pi in asthma contributes to enhanced oxidative stress.

BACKGROUND: Glutathione S-transferase pi (GSTPi) is the predominant redox regulator in the lung. Although evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. OBJECTIVES: We sought to determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. METHODS: We elucidated the regulation of GSTPi in children with asthma and used murine models of asthma to determine the role of GSTPi in redox homeostasis. RESULTS: Our findings demonstrate that GSTPi transcript levels are markedly downregulated in allergen- and IL-13-treated murine models of asthma through signal transducer and activator of transcription 6-dependent and independent pathways. Nuclear factor erythroid 2-related factor 2 was also downregulated in these models. The decrease in GSTPi expression was associated with decreased total glutathione S-transferase activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating cysteine oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in percentage cystine) compared with wild-type mice after allergen challenge. GSTPi expression was similarly downregulated in children with asthma. CONCLUSIONS: These data collectively suggest that downregulation of GSTPi after allergen challenge might contribute to the asthma phenotype because of disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi might be an important therapeutic target for asthma, and evaluation of GSTPi expression might prove beneficial in identifying patients who would benefit from therapy targeting this pathway.

A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma.

BACKGROUND: Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined. OBJECTIVE: To evaluate the role of Serpin3a, the murine homolog of SERPINB3 and SERPINB4, in asthma. METHODS: We studied wild-type Balb/c and Serpinb3a-null mice in house dust mite or IL-13-induced asthma models and evaluated airway hyperresponsiveness, inflammation, and goblet cell hyperplasia. RESULTS: Airway hyperresponsiveness and goblet cell hyperplasia were markedly attenuated in the Serpinb3a-null mice compared with the wild-type mice after allergen challenge, with minimal effects on inflammation. Expression of sterile alpha motif pointed domain containing v-ets avian erythroblastosis virus E26 oncogene homolog transcription factor (SPDEF), a transcription factor that mediates goblet cell hyperplasia, was decreased in the absence of Serpinb3a. IL-13-treated Serpinb3a-null mice showed attenuated airway hyperresponsiveness, inflammation, and mucus production. CONCLUSION: Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production are not well understood. Our data reveal a novel nonredundant role for Serpinb3a in mediating mucus production through regulation of SPDEF expression. This pathway may be used to target mucus hypersecretion effectively.

Oral benzo[a]pyrene-induced cancer: two distinct types in different target organs depend on the mouse Cyp1 genotype.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1 and CYP1B1) can both detoxify PAHs and activate them to cancer-causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within ∼28 days, whereas Cyp1(+/+) wild-type mice remain healthy for >12 months on this regimen. In this study, male Cyp1(+/+) wild-type, Cyp1a1(-/-) and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) double-knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs-including proximal small intestine (PSI), liver and preputial gland duct (PGD)-were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(-/-) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(-/-) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(-/-) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking upregulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y-chromosome; the Rab30 oncogene was upregulated; the Nr0b2 tumor suppressor gene was downregulated; paradoxical overexpression of numerous immunoglobulin kappa- and heavy-chain variable genes was found-although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of "gene-environment interactions" in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just 1 or 2 globally absent genes.

Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein.

PURPOSE: Saposin C is a multifunctional protein known to activate lysosomal enzymes and induce membrane fusion in an acidic environment. Excessive accumulation of lipid-coupled saposin C in lysosomes is cytotoxic. Because neoplasms generate an acidic microenvironment, caused by leakage of lysosomal enzymes and hypoxia, we hypothesized that saposin C may be an effective anticancer agent. We investigated the antitumor efficacy and systemic biodistribution of nanovesicles comprised of saposin C coupled with dioleoylphosphatidylserine in preclinical cancer models. EXPERIMENTAL DESIGN: Neuroblastoma, malignant peripheral nerve sheath tumor and, breast cancer cells were treated with saposin C-dioleoylphosphatidylserine nanovesicles and assessed for cell viability, ceramide elevation, caspase activation, and apoptosis. Fluorescently labeled saposin C-dioleoylphosphatidylserine was i.v. injected to determine in vivo tumor-targeting specificity. Antitumor activity and toxicity profile of saposin C-dioleoylphosphatidylserine were evaluated in xenograft models. RESULTS: Saposin C-dioleoylphosphatidylserine nanovesicles, with a mean diameter of approximately 190 nm, showed specific tumor-targeting activity shown through in vivo imaging. Following i.v. administration, saposin C-dioleoylphosphatidylserine nanovesicles preferentially accumulated in tumor vessels and cells in tumor-bearing mice. Saposin C-dioleoylphosphatidylserine induced apoptosis in multiple cancer cell types while sparing normal cells and tissues. The mechanism of saposin C-dioleoylphosphatidylserine induction of apoptosis was determined to be in part through elevation of intracellular ceramides, followed by caspase activation. In in vivo models, saposin C-dioleoylphosphatidylserine nanovesicles significantly inhibited growth of preclinical xenografts of neuroblastoma and malignant peripheral nerve sheath tumor. I.v. dosing of saposin C-dioleoylphosphatidylserine showed no toxic effects in nontumor tissues. CONCLUSIONS: Saposin C-dioleoylphosphatidylserine nanovesicles offer promise as a novel, nontoxic, cancer-targeted, antitumor agent for treating a broad range of cancers.

Clinical, pathologic, and molecular characterization of familial eosinophilic esophagitis compared with sporadic cases.

BACKGROUND & AIMS: Eosinophilic esophagitis (EE) occurs in families. METHODS: Record review confirmed patient kinship and provided clinical information. Slide review confirmed the diagnosis (threshold peak number > or = 24 eosinophils/high-power field). RESULTS: Fifty-nine members (41 males, 18 females) of 26 families were 3 months to 47 years of age (mean age, 10.3 y) at diagnosis. The only recorded race was Caucasian. In 4 families a parent of an affected male had EE. The most common complaint at diagnosis was dysphagia (68% of patients). Endoscopy showed esophageal mucosal furrows (93% of patients) and exudates (44%). Fifty-one percent had asthma. Skin prick tests to food and aeroallergens were positive in 76% and 71%, respectively. Familial EE characteristics (clinical, endoscopic, pathologic, and global esophageal transcript expression profile analysis) were similar to sporadic EE, except among patients with mucosal furrows: familial patients had lower peak eosinophil counts in the distal esophagus (P = .03) compared with sporadic patients. The basic characteristics of EE (eg, eosinophil levels, rate of atopy) did not vary with patient age. By using genome-wide microarray analysis, no significant differences (P < .05, false-discovery rate) were observed between familial and sporadic EE. Among all patients, chest pain was more common in females (P = .02), and thickened mucosa was more common in males (P = .006). CONCLUSIONS: These data support a familial pattern of inheritance of EE and a pathogenesis shared with sporadic EE. EE should be considered in symptomatic family members of patients who have EE.

Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure.

UNLABELLED: Oxidative stress is considered to be a critical mediator in liver injury of various etiologies. Depletion of glutathione (GSH), the major antioxidant in liver, has been associated with numerous liver diseases. To explore the specific role of hepatic GSH in vivo, we targeted Gclc, a gene essential for GSH synthesis, so that it was flanked by loxP sites and used the albumin-cyclization recombination (Alb-Cre) transgene to disrupt the Gclc gene specifically in hepatocytes. Deletion within the Gclc gene neared completion by postnatal day (PND)14, and loss of GCLC protein was complete by PND21. Cellular GSH was progressively depleted between PND14 and PND28-although loss of mitochondrial GSH was less severe. Nevertheless, ultrastructural examination of liver revealed dramatic changes in mitochondrial morphology; these alterations were accompanied by striking decreases in mitochondrial function in vitro, cellular ATP, and a marked increase in lipid peroxidation. Plasma liver biochemistry tests from these mice were consistent with progressive severe parenchymal damage. Starting at PND21, livers from hepatocyte-specific Gclc knockout [Gclc(h/h)] mice showed histological features of hepatic steatosis; this included inflammation and hepatocyte death, which progressed in severity such that mice died at approximately 1 month of age due to complications from liver failure. CONCLUSION: GSH is essential for hepatic function and loss of hepatocyte GSH synthesis leads to steatosis with mitochondrial injury and hepatic failure.

Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line.

Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the 129/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The approximately 2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney.

Cationic amino acid transporter 2 regulates inflammatory homeostasis in the lung.

Arginine is an amino acid that serves as a substrate for nitric oxide synthase and arginase. As such, arginine has the potential to influence diverse fundamental processes in the lung. Here we report that the arginine transport protein, cationic amino acid transporter (CAT)2, has a critical role in regulating lung inflammatory responses. Analysis of CAT2-deficient mice revealed spontaneous inflammation in the lung. Marked eosinophilia, associated with up-regulation of eotaxin-1, was present in the bronchoalveolar lavage fluid of 3-week-old CAT2-deficient mice. The eosinophilia was gradually replaced by neutrophilia in adult mice, while eotaxin-1 levels decreased and GRO-alpha levels increased. Despite the presence of activated alveolar macrophages in CAT2-deficient mice, NO production was compromised in these cells. Examination of dendritic cell activation, which can be affected by NO release, indicated increased dendritic cell activation in the lungs of CAT2-deficient mice. This process was accompanied by an increase in the number of memory T cells. Thus, our data suggest that CAT2 regulates anti-inflammatory processes in the lungs via regulation of dendritic cell activation and subsequent T cell responses.

Developmental characteristics of adapting mouse small intestine crypt cells.

BACKGROUND & AIMS: Following massive small bowel resection (SBR), the remnant intestine undergoes an adaptive process characterized by increases in a number of physiologic and morphologic parameters. These changes are the result of a stimulus that increases crypt cell mitosis and augments cellular progression along the villus axis. To better define this process, we identified patterns of gene expression specifically within adapting intestinal crypt cells following SBR. METHODS: Laser capture microdissection was used to isolate mouse intestinal crypt cells following SBR or sham operation. Multiple biological and technical complementary DNA microarray replicates allowed rigorous statistical analyses for identification of important expression profiles. Major groups of genes were classified as to site of action, functional pathway, and possible regulatory groups. RESULTS: A total of 300 genes differentially expressed at significant levels within adapting crypt enterocytes were analyzed. Comparison of this list of differentially expressed adapting crypt cell genes with a generalized mouse gene expression database (from 82 developing and adult mouse tissues) showed the greatest overlap with developing and immature intestinal tissues. We identified prominent groups of genes involved with cell growth, signal transduction, and nucleic acid binding. Genes not previously shown to be involved with adaptation or development and maturation were identified. CONCLUSIONS: Identification of similar genes coordinately regulated during both adaptation and development, processes that share key morphologic features, provides a basis for new mechanistic insights into these shared characteristics.

Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.

Eosinophilic esophagitis (EE) is an emerging disorder with a poorly understood pathogenesis. In order to define disease mechanisms, we took an empirical approach analyzing esophageal tissue by a genome-wide microarray expression analysis. EE patients had a striking transcript signature involving 1% of the human genome that was remarkably conserved across sex, age, and allergic status and was distinct from that associated with non-EE chronic esophagitis. Notably, the gene encoding the eosinophil-specific chemoattractant eotaxin-3 (also known as CCL26) was the most highly induced gene in EE patients compared with its expression level in healthy individuals. Esophageal eotaxin-3 mRNA and protein levels strongly correlated with tissue eosinophilia and mastocytosis. Furthermore, a single-nucleotide polymorphism in the human eotaxin-3 gene was associated with disease susceptibility. Finally, mice deficient in the eotaxin receptor (also known as CCR3) were protected from experimental EE. These results implicate eotaxin-3 as a critical effector molecule for EE and provide insight into disease pathogenesis.