The military gear microbiome: risk factors surrounding the warfighter.

Combat extremity wounds are highly susceptible to contamination from surrounding environmental material. This bioburden could be partially transferred from materials in immediate proximity to the wound, including fragments of the uniform and gear. However, the assessment of the microbial bioburden present on military gear during operational conditions of deployment or training is relatively unexplored. Opportunistic pathogens that can survive on gear represent risk factors for infection following injury, especially following combat blasts, where fibers and other materials are embedded in wounded tissue. We utilized 16S rRNA sequencing to assess the microbiome composition of different military gear types (boot, trouser, coat, and canteen) from two operational environments (training in Hawai'i and deployed in Indonesia) across time (days 0 and 14). We found that microbiome diversity, stability, and composition were dependent on gear type, training location, and sampling timepoint. At day 14, species diversity was significantly higher in Hawai'i samples compared to Indonesia samples for boot, coat, and trouser swabs. In addition, we observed the presence of potential microbial risk factors, as opportunistic pathogenic species, such as Acinetobacter, Pseudomonas, and Staphylococcus, were found to be present in all sample types and in both study sites. These study outcomes will be used to guide the design of antimicrobial materials and uniforms and for infection control efforts following combat blasts and other injuries, thereby improving treatment guidance during military training and deployment.IMPORTANCECombat extremity wounds are vulnerable to contamination from environments of proximity to the warfighter, leading to potential detrimental outcomes such as infection and delayed wound healing. Therefore, microbial surveillance of such environments is necessary to aid the advancement of military safety and preparedness through clinical diagnostics, treatment protocols, and uniform material design.

Prevalence and Epidemiology of Mycoplasma genitalium in a Pacific-Region Military Population.

BACKGROUND: Mycoplasma genitalium is an important emerging sexually transmitted pathogen commonly causing urethritis in men, cervicitis, and pelvic inflammatory disease in women with potential of infertility. Accumulating evidence identifies the prevalence of M. genitalium similar to long recognized pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae. The purpose of this study was to establish the prevalence and epidemiology of M. genitalium in a mid-Pacific military population. METHODS: A prospective analysis was conducted from routine specimens collected as standard of care for sexually transmitted infection (STI) testing at Tripler Army Medical Center on Oahu, HI. The prevalence of M. genitalium was determined using the Aptima M. genitalium assay, a transcription-mediated amplification test. A multivariate analysis was performed to assess the associations for this infection with other STIs and demographic factors. RESULTS: A total of 1876 specimens were tested in a 6-month period including 6 sample types from 1158 females and 718 males. Subject ages ranged from 18 to 76 years, with a median of 24 years (interquartile range, 21-29 years). The prevalence of M. genitalium was 8.8% overall (n = 165), 7.1% in females and 11.6% in males. Coinfection with M. genitalium occurred with another sexually-transmitted pathogen in 43 patients (18.3%), with C. trachomatis as the most common organism (n = 38). CONCLUSIONS: These data contribute to the evidence base for M. genitalium and STI screening in an active-duty military.

Midline craniofacial malformations with a lipomatous cephalocele are associated with insufficient closure of the neural tube in the tuft mouse.

BACKGROUND: Genetic variations affecting neural tube closure along the head result in malformations to the face and brain, posing a significant impact on health care costs and the quality of life. METHODS: We have established a mouse line from a mutation that arose spontaneously in our wild-type colony that we called tuft. Tuft mice have heritable midline craniofacial defects featuring an anterior lipomatous cephalocele. RESULTS: Whole-mount skeletal stains indicated that affected newborns had a broader interfrontal suture where the cephalocele emerged between the frontal bones. Mice with a cephalocele positioned near the rostrum also presented craniofacial malformations such as ocular hypertelorism and midfacial cleft of the nose. Gross and histological examination revealed that the lipomatous cephalocele originated as a fluid filled cyst no earlier than E14.5 while embryos with a midfacial cleft was evident during craniofacial development at E11.5. Histological sections of embryos with a midfacial cleft revealed the cephalic neuroectoderm remained proximal or fused to the frontonasal ectoderm about the closure site of the anterior neuropore, indicating a defect to neural tube closure. We found the neural folds along the rostrum of E9 to E10.5 embryos curled inward and failed to close as well as embryos with exencephaly and anencephaly at later stages. Whole-mount in situ hybridization of anterior markers Fgf8 and Sonic hedgehog indicated closure of the rostral site was compromised in severe cases. CONCLUSION: We present a model demonstrating how anterior cranial cephaloceles are generated following a defect to neural tube closure and relevance to subsequent craniofacial morphogenesis in the tuft mouse.

Craniofacial features resembling frontonasal dysplasia with a tubulonodular interhemispheric lipoma in the adult 3H1 tuft mouse.

Intracranial lipomas are rare, but 45% of them occur along the midline cisterns between the hemispheres and are often associated with corpus callosum hypoplasia and craniofacial defects. They are difficult to detect as they are generally asymptomatic and visible by MRI or by postmortem examination. The exact cause of these interhemispheric lipomas is not known, but they arise from a developmental defect resulting in the maldifferentiation of mesenchymal cells into mesodermal derivatives that are not normally present. We have identified a new mouse mutant called tuft, exhibiting a forebrain, intracranial lipoma with midline craniofacial defects resembling frontonasal dysplasia (FND) that arose spontaneously in our wild-type 3H1 colony. The tuft trait seems to be transmitted in recessive fashion, but approximately 80% less frequent than the expected Mendelian 25%, due to either incomplete penetrance or prenatal lethality. MRI and histologic analysis revealed that the intracranial lipoma occurred between the hemispheres and often protruded through the sagittal suture. We also observed a lesion at the lamina terminalis (LT) that may indicate improper closure of the anterior neuropore. We have mapped the tuft trait to within an 18 cM region on mouse chromosome 10 by microsatellite linkage analysis and identified several candidate genes involved with craniofacial development and cellular differentiation of adipose tissue. Tuft is the only known mouse model for midline craniofacial defects with an intracranial lipoma. Identifying the gene(s) and mutation(s) causing this early developmental defect will help us understand the pathogenesis of FND and related craniofacial disorders.

A Longitudinal Evaluation of the Bacterial Pathogens Colonizing Chronic Non-Healing Wound Sites at a United States Military Treatment Facility in the Pacific Region.

PURPOSE: The biology of chronic wounds is complex and many factors act concurrently to impede healing progress. In this study, the dynamics of microflora changes and their antibiotic susceptibility patterns were evaluated longitudinally over 30 days using data from 28 patients with a total of 47 chronic lower extremity wounds. MATERIALS AND METHODS: In this study, colonized wound isolates were characterized using cultural, biochemical, and VITEK 2 methods. Antibiotic susceptibility patterns of the wound isolates were analyzed using various phenotypic assays. Furthermore, antimicrobial resistance patterns and the presence of mutations were evaluated by a genotypic assay, whole-genome sequencing (WGS). RESULTS: Staphylococcus aureus and Pseudomonas aeruginosa were found to be the most common strains at early time points, while members of Enterobacteriaceae were prevalent at later stages of infection. Antimicrobial resistance testing and whole-genome sequencing revealed that the molecular and phenotypic characteristics of the identified wound pathogens remained relatively stable throughout the study period. It was also noted that Enterobacter and Klebsiella species may serve as reservoirs for quinolone resistance in the Pacific region. CONCLUSION: Our observations showed that wounds were colonized with diverse bacteria and interestingly their numbers and/or types were changed over the course of infection. The rapid genetic changes that accompanied the first 4 weeks after presentation did not directly contribute to the development of antibiotic resistance. In addition, standard wound care procedures did not appear to select for resistant bacterial strains. Future efforts should focus on defining those genetic changes associated with the wound colonizing microorganisms that occur beyond 4 weeks.

Rapid qualitative antibiotic resistance characterization using VITEK MS.

Development and standardization of simple, timely, and cost-effective antibiotic susceptibility assays are much needed to address the emergence of global resistance. The use of matrix-assisted laser desorption/ionization time of flight mass spectrometry is routine for bacterial identification. This study evaluated 2 assays using the VITEK MS for rapid detection and accurate differentiation of bacterial antibiotic susceptibility. We describe an extraction method and direct-on-target microdroplet growth assay (DOT-MGA). Non-susceptible and susceptible strains of Staphylococcus aureus, Enterococcus species, Escherichia coli, and Klebsiella pneumoniae were tested. The liquid extraction method and DOT-MGA proved to be reliable assays providing consistent differentiation between non-susceptible and susceptible strains. Results from this study support VITEK MS and these assays as rapid and accurate tools to augment traditional susceptibility testing. If implemented clinically, these assays can reduce the cost of patient care and the time to deliver critically needed treatment.

An evaluation of the impact of clinical bacterial isolates on epithelial cell monolayer integrity by the electric Cell-Substrate Impedance Sensing (ECIS) method.

Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (µFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa, ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli, MRSA, and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure. Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1ß production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence, which may have broader implications governing the course of treatment compared to traditional methods alone.

Lysyl oxidase-like 2 promotes migration in noninvasive breast cancer cells but not in normal breast epithelial cells.

A growing number of studies indicate the importance of the lysyl oxidase family in the promotion of epithelial neoplasms towards their more aggressive forms. However, the role of individual family members in carcinoma progression has yet to be ascertained. In this study, we analyzed LOXL2 expression in malignantly transformed MCF-7 and normal MCF-10A mammary epithelial cell line clones stably transduced with LOXL2 in vitro, and in normal and cancerous breast tissue samples in vivo. We found LOXL2 to be catalytically active in both MCF-7 and MCF-10 clones. LOXL2 overexpression promoted a more mesenchymal morphology in both cell types, but LOXL2-induced increase in migratory ability could only be established in MCF-7 clones. We demonstrated altered localization of the LOXL2 protein in breast cancer tissue compared to normal mammary tissue, and altered localization and processing of LOXL2 protein in breast cancer cell lines compared to normal cell lines, which may allow LOXL2 to interact with different intra and extracellular components during tumor progression. Results support the role of LOXL2 in selectively promoting a metastatic phenotype in breast tumor cells. Additional data suggest epigenetic molecular mechanisms in tumor specific regulation of LOXL2 expression that could be explored as a molecular target in the prevention of breast cancer progression.

Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis.

The epithelial to mesenchymal transition has recently been implicated as a source of fibrogenic myofibroblasts in organ fibrosis, particularly in the kidney. There is as yet minimal evidence for the epithelial to mesenchymal transition in the liver. We hypothesized that this process in biliary epithelial cells plays an important role in biliary fibrosis and might be found in patients with especially rapid forms, such as is seen in biliary atresia. We therefore obtained liver tissue from patients with biliary atresia as well as a variety of other pediatric and adult liver diseases. Tissues were immunostained with antibodies against the biliary epithelial cell marker CK19 as well as with antibodies against proteins characteristically expressed by cells undergoing the epithelial to mesenchymal transition, including fibroblast-specific protein 1, the collagen chaperone heat shock protein 47, the intermediate filament protein vimentin, and the transcription factor Snail. The degree of colocalization was quantified using a multispectral imaging system. We observed significant colocalization between CK19 and other markers of the epithelial to mesenchymal transition in biliary atresia as well as other liver diseases associated with significant bile ductular proliferation, including primary biliary cirrhosis. There was minimal colocalization seen in healthy adult and pediatric livers, or in livers not also demonstrating bile ductular proliferation. Multispectral imaging confirmed significant colocalization of the different markers in biliary atresia. In conclusion, we present significant histologic evidence suggesting that the epithelial to mesenchymal transition occurs in human liver fibrosis, particularly in diseases such as biliary atresia and primary biliary cirrhosis with prominent bile ductular proliferation.

A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure.

Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse.

Induction of heat shock proteins by hyperglycemic cerebral ischemia.

Hyperglycemia worsens the neuronal death induced by cerebral ischemia. A previous study demonstrated that diabetic hyperglycemia suppressed the expression of heat shock protein 70 (HSP70) in the liver. The objective of this study is to determine whether hyperglycemia exacerbates ischemic brain damage by suppressing the expression of heat shock proteins (HSPs) in the brain. Both normoglycemic and hyperglycemic rats were subjected to a transient global cerebral ischemia of 15 min and followed by 0.5, 1 and 3 h of reperfusion. The expression of stress-related genes and levels of HSP proteins were determined by DNA microarray, immunocytochemistry and Western blot analyses. The results showed that hyperglycemic ischemia upregulated the expressions of hsp70, hsp90A, hsp90B, heat shock cognate 71 kD protein (hsc70) and mthsp70. Protein levels of HSP70 and HSP60 were enhanced by hyperglycemia compared with normoglycemia. The results suggested that hyperglycemia-exacerbated ischemic brain damage is not mediated by the suppression of the HSPs. The increased levels of HSPs and mthsp70 suggest that the cell and the mitochondrion had strong stress responses to hyperglycemic ischemia.

Up-regulation and altered distribution of lysyl oxidase in the central nervous system of mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis.

Mutations of the copper-zinc superoxide dismutase (SOD1) gene can result in the development of amyotrophic lateral sclerosis (ALS). The exact cellular mechanisms causing ALS are not known, but oxidative stress is thought to play a prominent role. Lysyl oxidase (LOX) is one of the genes that are known to be up-regulated in ALS patients. In this study, we examined LOX localization in wild type rat and mouse brain sections using immunohistochemistry coupled with laser-scanning confocal microscope. The results showed that LOX, an extracellular matrix protein, was expressed in the choroid plexus, blood vessel walls, brain matrix, and neurons of normal rat and mice. In neurons, LOX was localized within the cytoplasm. LOX immunoreactivity increased in neurons of the spinal cord, brain stem and cortex, and the Purkinje cells of the cerebellum in transgenic G93A SOD1 (mSOD1) mouse model of ALS. In situ hybridization indicated that LOX gene expression was enhanced in the neurons of the spinal cord, brain stem, cortex, caudoputamen and cerebellum in mSOD1 mice compared with wild type controls. LOX enzyme activity was increased in mSOD1 mice. An increase in the amount of LOX mRNA, protein and enzyme activity was coincidental with late stage ALS, indicating that LOX may be associated with the progression of the neurodegenerative process in the mSOD1 model of ALS.

Comparative immunocytochemical localization of lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) proteins: changes in the expression of LOXL during development and growth of mouse tissues.

Lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are extracellular enzymes that deaminate peptidyl lysyl residues involved in the cross-linking of fibrillar collagens and elastin. While LOX is required for the survival of newborn mice, the role of LOXL during development remains unclear. Studies have shown that the same cell types express LOX and LOXL in the same tissues, but no functional differences have been established. We have compared the immunohistochemical localization of LOX and LOXL in various tissues from normal, young adult mice. LOX and LOXL were co-localized in the skin, aorta, heart, lung, liver and cartilage, but were localized to different areas in the kidney, stomach, small intestine, colon, retina, ovary, testis and brain. LOXL expression was further examined in tissues from different developmental stages. In embryonic mice (10.5-14.5 dpc), LOXL immunostaining was abundant in the heart, liver, intestine, and neural tube. LOXL was present in most major organs in late fetal (16.5 dpc) and newborn mice, but generally diminished as animals aged. Immunoreactivity was significantly reduced in the heart, lung, kidney and liver of 2 year-old mice, but remained prevalent in the skin and tongue. LOX and LOXL were also found in the nuclei of cells in a number of tissues. These results indicate that LOXL has a role during mouse development and in the maintenance of adult tissues.

Life span extension and reduced neuronal death after weekly intraventricular cyclosporin injections in the G93A transgenic mouse model of amyotrophic lateral sclerosis.

OBJECT: The authors investigated whether cyclosporin A (CsA), a cyclophilin ligand with mitochondrial permeability transition pore-blocking and calcineurin-inhibiting properties, affects motor function, neuronal death, and life span in the G93A transgenic mouse model of familial amyotrophic lateral sclerosis (FALS). METHODS: The G93A mice received weekly intracerebroventricular injections of CsA (20 microg/mouse/week) starting at the age of 65 days, and physical performance on an exercise wheel was monitored beginning at 84 days of age. Mice were allowed to survive for clinical observation of body weight, hindlimb weakness, and life span or until a defined end stage or were killed at 110 days of age for histological analysis. CONCLUSIONS: Treatment with CsA significantly delayed the onset of hindlimb weakness and also extended the time from its onset to paralysis. The overall life span of CsA-treated G93A mice was significantly extended, by 12% compared with vehicle-treated transgenic littermates. The CsA also prolonged physical performance on the exercise wheel and delayed weight loss. Histologically, there was significant preservation of both cervical and lumbar spine motor neurons and also tyrosine hydroxylase-positive dopaminergic substantia nigra neurons in 110-day-old CsA-treated mice compared with their transgenic littermates. The local administration of CsA directly into the brain ventricles is an effective means of central nervous system drug delivery (because CsA does not readily cross the blood-brain barrier), which in this study ameliorated clinical and neuropathological features of FALS in G93A mice. The remarkably low intrathecal CsA dose required for neuroprotection reduces potential adverse effects of systemic immunosuppression or nephrotoxicity seen with chronic systemic delivery of the drug.

Transcriptome analysis of PPARγ target genes reveals the involvement of lysyl oxidase in human placental cytotrophoblast invasion.

Human placental development is characterized by invasion of extravillous cytotrophoblasts (EVCTs) into the uterine wall during the first trimester of pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in placental development, and activation of PPARγ by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARγ target genes, microarray analysis was performed using GeneChip technology on EVCT primary cultures obtained from first-trimester human placentas. Gene expression was compared in EVCTs treated with the PPARγ agonist rosiglitazone versus control. A total of 139 differentially regulated genes were identified, and changes in the expression of the following 8 genes were confirmed by reverse transcription-quantitative polymerase chain reaction: a disintegrin and metalloproteinase domain12 (ADAM12), connexin 43 (CX43), deleted in liver cancer 1 (DLC1), dipeptidyl peptidase 4 (DPP4), heme oxygenase 1 (HMOX-1), lysyl oxidase (LOX), plasminogen activator inhibitor 1 (PAI-1) and PPARγ. Among the upregulated genes, lysyl oxidase (LOX) was further analyzed. In the LOX family, only LOX, LOXL1 and LOXL2 mRNA expression was significantly upregulated in rosiglitazone-treated EVCTs. RNA and protein expression of the subfamily members LOX, LOXL1 and LOXL2 were analyzed by absolute RT-qPCR and western blotting, and localized by immunohistochemistry and immunofluorescence-confocal microscopy. LOX protein was immunodetected in the EVCT cytoplasm, while LOXL1 was found in the nucleus and nucleolus. No signal was detected for LOXL2 protein. Specific inhibition of LOX activity by ß-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARγ targets and that LOX activity is a negative regulator of trophoblastic cell invasion.

Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity.

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

Lysyl oxidase-like 2 expression is increased in colon and esophageal tumors and associated with less differentiated colon tumors.

Lysyl oxidase-like 2 (LOXL2) belongs to an amine oxidase family whose members have been implicated in crosslink formation in stromal collagens and elastin, cell motility, and tumor development and progression. We previously demonstrated the association between increased LOXL2 expression and invasive/metastatic behavior in human breast cancer cells and mouse squamous and spindle cell carcinomas, interaction between LOXL2 and SNAIL in epithelial-mesenchymal transition, and localization of the LOXL2 gene to 8p21.2-21.3, within a minimally deleted region in several cancers, including colon and esophagus. In the present study, we analyzed LOXL2 expression in colon and esophageal tumors, and explored methylation as a regulator of LOXL2 expression. Immunohistochemistry using normal tissues demonstrated intracellular localization of LOXL2 in colonic enteroendocrine cells and esophageal squamous cells at the luminal surface, but not in mitotically active cells. Tissue array analysis of 52 colon adenocarcinomas and 50 esophageal squamous cell carcinomas revealed presence of LOXL2 expression in 83 and 92% of the samples, respectively, and a significant association between increased number of LOXL2-expressing cells and less-differentiated colon carcinomas. We determined that the methylation status of the 1150 bp 5' CpG island may contribute to the regulation of the gene. Loss of heterozygosity studies, using a microsatellite within intron 4 of the LOXL2 gene, revealed that loss of LOXL2 was unlikely to play a major role in either colon or esophageal tumors. These results suggest that increased LOXL2 expression in colon and esophageal cancer may contribute to tumor progression.

Dysregulation of pulmonary elastin synthesis and assembly in preterm lambs with chronic lung disease.

Failed alveolar formation and excess, disordered elastin are key features of neonatal chronic lung disease (CLD). We previously found fewer alveoli and more elastin in lungs of preterm compared with term lambs that had mechanical ventilation (MV) with O(2)-rich gas for 3 wk (MV-3 wk). We hypothesized that, in preterm more than in term lambs, MV-3 wk would reduce lung expression of growth factors that regulate alveolarization (VEGF, PDGF-A) and increase lung expression of growth factors [transforming growth factor (TGF)-alpha, TGF-beta(1)] and matrix molecules (tropoelastin, fibrillin-1, fibulin-5, lysyl oxidases) that regulate elastin synthesis and assembly. We measured lung expression of these genes in preterm and term lambs after MV for 1 day, 3 days, or 3 wk, and in fetal controls. Lung mRNA for VEGF, PDGF-A, and their receptors (VEGF-R2, PDGF-Ralpha) decreased in preterm and term lambs after MV-3 wk, with reduced lung content of the relevant proteins in preterm lambs with CLD. TGF-alpha and TGF-beta(1) expression increased only in lungs of preterm lambs. Tropoelastin mRNA increased more with MV of preterm than term lambs, and expression levels remained high in lambs with CLD. In contrast, fibrillin-1 and lysyl oxidase-like-1 mRNA increased transiently, and lung abundance of other elastin-assembly genes/proteins was unchanged (fibulin-5) or reduced (lysyl oxidase) in preterm lambs with CLD. Thus MV-3 wk reduces lung expression of growth factors that regulate alveolarization and differentially alters expression of growth factors and matrix proteins that regulate elastin assembly. These changes, coupled with increased lung elastase activity measured in preterm lambs after MV for 1-3 days, likely contribute to CLD.

Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation.

Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (K(d)) of 2.5 nm. This was comparable with our measured K(d) of LOX binding to tropoelastin (1.9 nm) and type I collagen (5.2 nm), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity.

A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progression.

The transcription factor Snail controls epithelial-mesenchymal transitions (EMT) by repressing E-cadherin expression and other epithelial genes. However, the mechanisms involved in the regulation of Snail function are not fully understood. Here we show that lysyl-oxidase-like 2 and 3 (LOXL2 and LOXL3), two members of the lysyl-oxidase gene family, interact and cooperate with Snail to downregulate E-cadherin expression. Snail's lysine residues 98 and 137 are essential for Snail stability, functional cooperation with LOXL2/3 and induction of EMT. Overexpression of LOXL2 or LOXL3 in epithelial cells induces an EMT process, supporting their implication in tumor progression. The biological importance of LOXL2 is further supported by RNA interference of LOXL2 in Snail-expressing metastatic carcinoma cells, which led to a strong decrease of tumor growth associated to increased apoptosis and reduced expression of mesenchymal and invasive/angiogenic markers. Taken together, these results establish a direct link between LOXL2 and Snail in carcinoma progression.