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.

Assessment of Enhanced Thermal Effect Due to Gold Nanoparticles during MR-Guided High-Intensity Focused Ultrasound (HIFU) Procedures Using a Mouse-Tumor Model.

An in vivo study was conducted using a mouse tumor model, to assess the utility of using gold nanoparticles (gNPs) during HIFU procedures to locally enhance heating at low powers. Tumors were grown using melanoma tumor cells (B16/F10) subcutaneously on the right flanks of mice (C57Bl/6J). Physiologically relevant concentrations (0 and 0.125%) of gNPs were directly injected into the tumors. Sonications at acoustic powers of 10 and 30 W were performed for a duration of 16 s inside a magnetic-resonance system. Temperature increases and lesion volumes were calculated and compared for procedures with and without gNPs. Histopathology study was conducted using a cleaved caspase 3 antibody and hematoxylin and eosin staining after removing the tumors from the mice. For an acoustic power of 30 W, end-of-sonication temperature increases of 25.4 ± 3.8 °C (0% gNP) and 42.2 ± 4.6 °C (0.125% gNP) were measured. Using cleaved caspase 3 antibody, it was observed that more than 1% of nuclei are affected in the case of 0.125% and 30 W but only 0.01% of nuclei are affected in the 0% case. For 30 W and a gNP concentration of 0.125%, a lesion volume of 0.33 ± 0.22 mm3 was obtained, while no lesion was observed without gNP's.

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.

Sepsis Induces Adipose Tissue Browning in Nonobese Mice But Not in Obese Mice.

Severe sepsis and septic shock are the biggest cause of mortality in critically ill patients. Obesity today is one of the world's greatest health challenges. Little is known about the extent of involvement of the white adipose tissue (WAT) in sepsis and how it is being modified by obesity. We sought to explore the involvement of the WAT in sepsis. We hypothesize that sepsis induces browning of the WAT and that obesity alters the response of WAT to sepsis. Six-week-old C57BL/6 mice were randomized to a high-fat diet to induce obesity (obese group) or control diet (nonobese group). After 6 to 11 weeks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 18, and 72 h after CLP and epididymal WAT (eWAT), inguinal WAT, and brown adipose tissue (BAT) harvested. Both types of WAT were processed for light microscopy and transmission electron microscopy to assess for morphological changes in both obese and nonobese mice. Tissues were processed for immunohistochemistry, image analyses, and molecular analyses. BATs were used as a positive control. Nonobese mice have an extensive breakdown of the unilocular lipid droplet and smaller adipocytes in WAT compared with obese mice after sepsis. Neutrophil infiltration increases in eWAT in nonobese mice after sepsis but not in obese mice. Nonobese septic mice have an increase in mitochondrial density compared with obese septic mice. Furthermore, nonobese septic mice have an increase in uncoupling protein-1 expression. Although the WAT of nonobese mice have multiple changes characteristic of browning during sepsis, these changes are markedly blunted in obesity.

Proximal Femoral Growth Modification: Effect of Screw, Plate, and Drill on Asymmetric Growth of the Hip.

BACKGROUND: Guided growth has long been used in the lower extremities but has not been applied to varus or valgus deformity in the hip, as may occur in children with cerebral palsy or developmental dysplasia of the hip. The purpose of this study was to determine if screw, plate, or drilling techniques decreased the femoral neck-shaft angle (NSA) and articular trochanteric disease (ATD), as well as describe growth plate structural changes with each method. METHODS: Twelve 8-week-old lambs underwent proximal femoral hemiepiphysiodesis (IACUC approved) using either a screw (n=4), plate (n=4), or drilling procedure (n=4). Postoperative time was 6 months. Radiographs taken after limb harvest were used to measure NSA and ATD. Differences between treated and control sides were determined by 1-tailed paired t tests and Bonferroni (α=0.05/3). Histology was obtained for 1 limb pair per group. Proximal femurs were cut in midcoronal plane and the longitudinal growth plates were examined for structural changes. RESULTS: The mean NSA measured 7 degrees less than controls in this model using the screw technique, and this difference was statistically significant. Differences between the control and the treated groups did not reach statistical significance for either the plate or the drill group. Differences in ATD were not statistically significant, although there was a trend for larger ATD measurements using the screw technique. Histologically, physeal changes were observed on the operative sides in screw and plate specimens, but not drill specimens, compared with contralateral sham control. The screw specimen exhibited the most severe changes, with growth plate closure over half the section. The plate specimen showed focal loss of the physis across the section, but with no evidence of closure. CONCLUSIONS: This study builds on previous work that indicates screw hemiepiphysiodesis can effectively alter the shape of the proximal femur, and result in a lower neck-shaft ankle (or lesser valgus). This study suggests that implantation of a screw is likely to be more effective than a plate or drilling procedure in decreasing the NSA in skeletally immature hips. CLINICAL SIGNIFICANCE: If further preclinical, and later clinical, studies demonstrate reproducible efficacy, guided growth of the proximal femur may eventually become a viable option for treatment or prevention of hip deformity in select patients.

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.

IgG1 protects against renal disease in a mouse model of cryoglobulinaemia.

Immunoglobulins protect against disease to a considerable extent by activating complement and stimulatory immunoglobulin crystallizable fragment receptors (Ig FcRs), and aggregating microbial pathogens. Yet IgG1, the predominant murine serum Ig isotype, cannot activate complement by the classical pathway, binds more avidly to an inhibitory than to stimulatory FcRs, and has limited ability to aggregate pathogens. In these regards, it resembles human IgG4 (ref. 4). We hypothesized that limited ability to activate effector mechanisms might protect against immune complex immunopathology. Here we show that IgG1-deficient (γ1(-)) mice, immunized with a potent antigen, develop lethal renal disease soon after they begin to produce antigen-specific antibody, whereas similarly immunized wild-type mice remain healthy. Surprisingly, renal disease in this model is complement and FcR independent and results from immune complex precipitation in glomerular capillaries, as in some cryoglobulinaemic humans. IgG3, which self-associates to form large immune complexes, accounts for more than 97% of the mouse Ig in this cryoglobulin; furthermore, glomerular disease develops when mice are injected with IgG3 anti-trinitrophenyl (TNP) monoclonal antibody followed by a TNP-labelled protein. Renal disease is prevented in both active and passive immunization models by antigen-specific IgG1; other isotypes are less potent at preventing disease. These observations demonstrate the adaptive significance of Ig isotypes that poorly activate effector mechanisms, reveal an immune-complex-dependent, complement- and FcR-independent nephrotoxic mechanism, and suggest that isotypes that poorly activate effector mechanisms may be useful for inhibiting immune complex immunopathology.

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.

Extended healing validation of an artificial tendon to connect the quadriceps muscle to the Tibia: 180-day study.

Whenever a tendon or its bone insertion is disrupted or removed, existing surgical techniques provide a temporary connection or scaffolding to promote healing, but the interface of living to non-living materials soon breaks down under the stress of these applications, if it must bear the load more than acutely. Patients are thus disabled whose prostheses, defect size, or mere anatomy limit the availability or outcomes of such treatments. Our group developed the OrthoCoupler™ device to join skeletal muscle to prosthetic or natural structures without this interface breakdown. In this study, the goat knee extensor mechanism (quadriceps tendon, patella, and patellar tendon) was removed from the right hind limb in 16 goats. The device connected the quadriceps muscle to a stainless steel bone plate on the tibia. Mechanical testing and histology specimens were collected from each operated leg and contralateral unoperated control legs at 180 days. Maximum forces in the operated leg (vs. unoperated) were 1,400 ± 93 N (vs. 1,179 ± 61 N), linear stiffnesses were 33 ± 3 N/mm (vs. 37 ± 4 N/mm), and elongations at failure were 92.1 ± 5.3 mm (vs. 68.4 ± 3.8 mm; mean ± SEM). Higher maximum forces (p = 0.02) and elongations at failure (p=0.008) of legs with the device versus unoperated controls were significant; linear stiffnesses were not (p=0.3). We believe this technology will yield improved procedures for clinical challenges in orthopedic oncology, revision arthroplasty, tendon transfer, and tendon injury reconstruction.

MHC class I-specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice.

Transfusion-related acute lung injury (TRALI), a form of noncardiogenic pulmonary edema that develops during or within 6 h after a blood transfusion, is the most frequent cause of transfusion-associated death in the United States. Because development of TRALI is associated with donor antibodies (Abs) reactive with recipient major histocompatibility complex (MHC), a mouse model has been studied in which TRALI-like disease is caused by injecting mice with anti-MHC class I monoclonal Ab (mAb). Previous publications with this model have concluded that disease is caused by FcR-dependent activation of neutrophils and platelets, with production of reactive oxygen species that damage pulmonary vascular endothelium. In this study, we confirm the role of reactive oxygen species in the pathogenesis of this mouse model of TRALI and show ultrastructural evidence of pulmonary vascular injury within 5 min of anti-MHC class I mAb injection. However, we demonstrate that disease induction in this model involves macrophages rather than neutrophils or platelets, activation of complement and production of C5a rather than activation of FcγRI, FcγRIII, or FcγRIV, and binding of anti-MHC class I mAb to non-BM-derived cells such as pulmonary vascular endothelium. These observations have important implications for the prevention and treatment of TRALI.

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.

An artificial tendon to connect the quadriceps muscle to the tibia.

No permanent, reliable artificial tendon exists clinically. Our group developed the OrthoCoupler™ device as a versatile connector, fixed at one end to a muscle, and adaptable at the other end to inert implants such as prosthetic bones or to bone anchors. The objective of this study was to evaluate four configurations of the device to replace the extensor mechanism of the knee in goats. Within muscle, the four groups had: (A) needle-drawn uncoated bundles, (B) needle-drawn coated bundles, (C) barbed uncoated bundles, and (D) barbed coated bundles. The quadriceps tendon, patella, and patellar tendon were removed from the right hind limb in 24 goats. The four groups (n = 6 for each) were randomly assigned to connect the quadriceps muscle to the tibia (with a bone plate). Specimens were collected from each operated leg and contralateral unoperated controls both for mechanical testing and histology at 90 days post-surgery. In strength testing, maximum forces in the operated leg (vs. unoperated control) were 1,288 ± 123 N (vs. 1,387 ± 118 N) for group A, 1,323 ± 144 N (vs. 1,396 ± 779 N) for group B, 930 ± 125 N (vs. 1,337 ± 126 N) for group C, and 968 ± 109 N (vs. 1,528 ± 146 N) for group D (mean ± SEM). The strengths of the OrthoCoupler™ legs in the needled device groups were equivalent to unoperated controls (p = 0.6), while both barbed device groups had maximum forces significantly lower than their controls (p = 0.001). We believe this technology will yield improved procedures for clinical challenges in orthopaedic oncology, revision arthroplasty, tendon transfer, and tendon injury reconstruction.

Global gene expression profile progression in Gaucher disease mouse models.

BACKGROUND: Gaucher disease is caused by defective glucocerebrosidase activity and the consequent accumulation of glucosylceramide. The pathogenic pathways resulting from lipid laden macrophages (Gaucher cells) in visceral organs and their abnormal functions are obscure. RESULTS: To elucidate this pathogenic pathway, developmental global gene expression analyses were conducted in distinct Gba1 point-mutated mice (V394L/V394L and D409 V/null). About 0.9 to 3% of genes had altered expression patterns (≥ ± 1.8 fold change), representing several categories, but particularly macrophage activation and immune response genes. Time course analyses (12 to 28 wk) of INFγ-regulated pro-inflammatory (13) and IL-4-regulated anti-inflammatory (11) cytokine/mediator networks showed tissue differential profiles in the lung and liver of the Gba1 mutant mice, implying that the lipid-storage macrophages were not functionally inert. The time course alterations of the INFγ and IL-4 pathways were similar, but varied in degree in these tissues and with the Gba1 mutation. CONCLUSIONS: Biochemical and pathological analyses demonstrated direct relationships between the degree of tissue glucosylceramides and the gene expression profile alterations. These analyses implicate IFNγ-regulated pro-inflammatory and IL-4-regulated anti-inflammatory networks in differential disease progression with implications for understanding the Gaucher disease course and pathophysiology.