Bone marrow-derived mesenchymal stem cell-conditioned medium ameliorates diabetic foot ulcers in rats.

OBJECTIVES: This study aimed to explore the effects of bone marrow-derived Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) treating diabetic foot ulcers in rats. METHODS: Models of T2DM rats were induced by a high-fat diet and intraperitoneal injection of STZ in SD rats. Models of Diabetic Foot Ulcers (DFUs) were made by operation on hind limbs in diabetic rats. Rats were divided into four groups (n = 6 for each group), i.e., Normal Control group (NC), Diabetes Control group (DM-C), MSC-CM group and Mesenchymal Stem Cells group (MSCs). MSC-CM group was treated with an injection of conditioned medium derived from preconditioned rats' bone marrow MSCs around ulcers. MSCs group were treated with an injection of rats' bone marrow MSCs. The other two groups were treated with an injection of PBS. After the treatment, wound closure, re-epithelialization (thickness of the stratum granulosums of the skin, by H&E staining), cell proliferation (Ki67, by IHC), angiogenesis (CD31, by IFC), autophagy (LC3B, by IFC and WB; autolysosome, by EM) and pyroptosis (IL-1ß, NLRP3, Caspase-1, GSDMD and GSDMD-N, by WB) in ulcers were evaluated. RESULTS: After the treatment wound area rate, IL-1ß by ELISA, and IL-1ß, Caspase-1, GSDMD and GSDMD-N by WB of MSC-CM group were less than those of DM group. The thickness of the stratum granulosums of the skin, proliferation index of Ki67, mean optic density of CD31 and LC3B by IFC, and LC3B by WB of MSC-CM group were more than those of DM group. The present analysis demonstrated that the injection of MSC-CM into rats with DFUs enhanced the wound-healing process by accelerating wound closure, promoting cell proliferation and angiogenesis, enhancing cell autophagy, and reducing cell pyroptosis in ulcers. CONCLUSIONS: Studies conducted indicate that MSC-CM administration could be a novel cell-free therapeutic approach to treat DFUs accelerating the wound healing process and avoiding the risk of living cells therapy.

Bone marrow-derived mesenchymal stem cell-conditioned medium ameliorates diabetic foot ulcers in rats

Abstract Objectives: This study aimed to explore the effects of bone marrow-derived Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) treating diabetic foot ulcers in rats. Methods: Models of T2DM rats were induced by a high-fat diet and intraperitoneal injection of STZ in SD rats. Models of Diabetic Foot Ulcers (DFUs) were made by operation on hind limbs in diabetic rats. Rats were divided into four groups (n = 6 for each group), i.e., Normal Control group (NC), Diabetes Control group (DM-C), MSC-CM group and Mesenchymal Stem Cells group (MSCs). MSC-CM group was treated with an injection of conditioned medium derived from preconditioned rats' bone marrow MSCs around ulcers. MSCs group were treated with an injection of rats' bone marrow MSCs. The other two groups were treated with an injection of PBS. After the treatment, wound closure, re-epithelialization (thickness of the stratum granulosums of the skin, by H&E staining), cell proliferation (Ki67, by IHC), angiogenesis (CD31, by IFC), autophagy (LC3B, by IFC and WB; autoly-sosome, by EM) and pyroptosis (IL-1β, NLRP3, Caspase-1, GSDMD and GSDMD-N, by WB) in ulcers were evaluated. Results: After the treatment wound area rate, IL-1β by ELISA, and IL-1β, Caspase-1, GSDMD and GSDMD-N by WB of MSC-CM group were less than those of DM group. The thickness of the stratum granulosums of the skin, proliferation index of Ki67, mean optic density of CD31 and LC3B by IFC, and LC3B by WB of MSC-CM group were more than those of DM group. The present analysis demonstrated that the injection of MSC-CM into rats with DFUs enhanced the wound-healing process by accelerating wound closure, promoting cell proliferation and angiogenesis, enhancing cell autophagy, and reducing cell pyroptosis in ulcers. Conclusions: Studies conducted indicate that MSC-CM administration could be a novel cell-free therapeutic approach to treat DFUs accelerating the wound healing process and avoiding the risk of living cells therapy.

Abnormal white matter within brain structural networks is associated with high-impulse behaviour in codeine-containing cough syrup dependent users.

Codeine-containing cough syrup (CCS) is considered as one of the most popular drug of dependence among adolescents because of its inexpensiveness and easy availability. However, its relationship with neurobiological effects remains sparsely explored. Herein, we examined how high-impulse behaviours relate to changes in the brain structural networks. Forty codeine-containing cough syrup dependent (CCSD) users and age-, gender-, and number of cigarettes smoked per day -matched forty healthy control (HC) subjects underwent structural brain imaging via MRI. High-impulse behaviour was assessed using the 30-item self-rated Barratt Impulsiveness Scale (BIS-11), and structural networks were constructed using diffusion tensor imaging and AAL-90 template. Between-group topological metrics were compared using nonparametric permutations. Benjamin-Hochberg false discovery rate correction was used to correct for multiple comparisons (P < 0.05). The relationships between abnormal network metrics and clinical characteristics of CCS dependent (BIS-11 total score, CCS- dependent duration and mean dose) were examined by Spearman's correlation. Structural networks of the CCSD group demonstrated lower small-world properties than those of the HC group. Abnormal changes in nodal properties among CCSD users were located mainly in the frontal gyrus, inferior parietal lobe and olfactory cortex. NBS analysis further indicated disrupted structural connections between the frontal gyrus and multiple brain regions. There were significant correlations between abnormal nodal properties of the frontal gyrus and clinical characteristics (BIS-11 total score, CCS dependent duration and mean dose) in the CCSD group. These findings suggest that the high-impulse behavioural expression in CCS addiction is associated with widespread brain regions, particularly within those in the frontal cortex. Aberrant brain regions and disrupted connectivity of structural network may be the bases of neuropathology for underlying symptoms of high-impulse behaviours in CCSD users, which may provide a novel sight to better treat and prevent codeine dependency in adolescents.

Intermittent Theta-Burst Stimulation Over the Suprahyoid Muscles Motor Cortex Facilitates Increased Degree Centrality in Healthy Subjects.

Theta-burst stimulation (TBS), a variant of repetitive transcranial magnetic stimulation (rTMS), can potentially benefit the treatment of swallowing disorders. However, the after-effects of TBS on the swallowing motor cortex remain uncertain. The newly developed graph-based analysis of the centrality approach has been increasingly used to explore brain networks. The purpose of this study was to identify degree centrality (DC) alterations in the brain network after different TBS protocols were performed over the suprahyoid muscles motor cortex in healthy subjects. A total of 40 right-handed healthy subjects (mean age: 23.73 ± 2.57 years, range: 21-30, 20 females) were included in this study and randomly assigned to two groups, including the continuous TBS (cTBS) group and the intermittent TBS (iTBS) group. All of the subjects underwent resting-state functional magnetic resonance imaging (rs-fMRI) scanning before and after TBS implementation. Compared to the baseline, cTBS resulted in increased DC values in the left inferior frontal gyrus (P < 0.01). In the iTBS group, decreased DC was observed in the left cerebellum and left medial frontal gyrus; However, increased DC was observed in several brain areas including the right superior temporal gyrus, right superior frontal gyrus, right postcentral gyri and left paracentral lobule (P < 0.01). These results indicated that cTBS mainly results in increasing DC in the ipsilateral. However, iTBS is capable of facilitating the excitability of the swallowing motor cortex and increasing the connectivity of multiple brain regions, including the bilateral sensorimotor network, and might have therapeutic potential in the treatment of swallowing disorders.

Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2.

Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.

Intermittent Theta-Burst Stimulation Reverses the After-Effects of Contralateral Virtual Lesion on the Suprahyoid Muscle Cortex: Evidence From Dynamic Functional Connectivity Analysis.

Contralateral intermittent theta burst stimulation (iTBS) can potentially improve swallowing disorders with unilateral lesion of the swallowing cortex. However, the after-effects of iTBS on brain excitability remain largely unknown. Here, we investigated the alterations of temporal dynamics of inter-regional connectivity induced by iTBS following continuous TBS (cTBS) in the contralateral suprahyoid muscle cortex. A total of 20 right-handed healthy subjects underwent cTBS over the left suprahyoid muscle motor cortex and then immediately afterward, iTBS was applied to the contralateral homologous area. All of the subjects underwent resting-state functional magnetic resonance imaging (Rs-fMRI) pre- and post-TBS implemented on a different day. We compared the static and dynamic functional connectivity (FC) between the post-TBS and the baseline. The whole-cortical time series and a sliding-window correlation approach were used to quantify the dynamic characteristics of FC. Compared with the baseline, for static FC measurement, increased FC was found in the precuneus (BA 19), left fusiform gyrus (BA 37), and right pre/post-central gyrus (BA 4/3), and decreased FC was observed in the posterior cingulate gyrus (PCC) (BA 29) and left inferior parietal lobule (BA 39). However, in the dynamic FC analysis, post-TBS showed reduced FC in the left angular and PCC in the early windows, and in the following windows, increased FC in multiple cortical areas including bilateral pre- and postcentral gyri and paracentral lobule and non-sensorimotor areas including the prefrontal, temporal and occipital gyrus, and brain stem. Our results indicate that iTBS reverses the aftereffects induced by cTBS on the contralateral suprahyoid muscle cortex. Dynamic FC analysis displayed a different pattern of alteration compared with the static FC approach in brain excitability induced by TBS. Our results provide novel evidence for us in understanding the topographical and temporal aftereffects linked to brain excitability induced by different TBS protocols and might be valuable information for their application in the rehabilitation of deglutition.

The After-Effects of Theta Burst Stimulation Over the Cortex of the Suprahyoid Muscle on Regional Homogeneity in Healthy Subjects.

Theta burst stimulation (TBS) is a powerful variant of repetitive transcranial magnetic stimulation (rTMS), making it potentially useful for the treatment of swallowing disorders. However, how dose TBS modulate human swallowing cortical excitability remains unclear. Here, we aim to measure the after-effects of spontaneous brain activity at resting-state using the regional homogeneity (ReHo) approach in healthy subjects who underwent different TBS protocols over the suprahyoid muscle cortex. Sixty healthy subjects (23.45 ± 2.73 years, 30 males) were randomized into three groups which completed different TBS protocols. The TMS coil was applied over the cortex of the suprahyoid muscles. Data of resting-state functional MRI (Rs-fMRI) of the subjects were acquired before and after TBS. The ReHo was compared across sessions [continuous TBS (cTBS), intermittent TBS (iTBS) and cTBS/iTBS] and runs (pre/post TBS). In the comparison between pre- and post-TBS, increased ReHo was observed in the right lingual gyrus and right precuneus and decreased ReHo in the left cingulate gyrus in the cTBS group. In the iTBS group, increased ReHo values were seen in the pre-/postcentral gyrus and cuneus, and decreased ReHo was observed in the left cerebellum, brainstem, bilateral temporal gyrus, insula and left inferior frontal gyrus. In the cTBS/iTBS group, increased ReHo was found in the precuneus and decreased ReHo in the right cerebellum posterior lobe, left anterior cerebellum lobe, and right inferior frontal gyrus. In the post-TBS inter-groups comparison, increased ReHo was seen in right middle occipital gyrus and decreased ReHo in right middle frontal gyrus and right postcentral gyrus (cTBS vs. cTBS/iTBS). Increased ReHo was shown in left inferior parietal lobule and left middle frontal gyrus (cTBS vs. iTBS). Increased ReHo was shown in right medial superior frontal gyrus and decreased ReHo in right cuneus (cTBS/iTBS vs. iTBS). Our findings indicate cTBS had no significant influence on ReHo in the primary sensorimotor cortex, iTBS facilitates an increased ReHo in the bilateral sensorimotor cortex and a decreased ReHo in multiple subcortical areas, and no reverse effect exhibits when iTBS followed the contralateral cTBS over the suprahyoid motor cortex. The results provide a novel insight into the neural mechanisms of TBS on swallowing cortex.

Discrimination Between Solitary Brain Metastasis and Glioblastoma Multiforme by Using ADC-Based Texture Analysis: A Comparison of Two Different ROI Placements.

RATIONALE AND OBJECTIVES: To explore the value of texture analysis based on the apparent diffusion coefficient (ADC) value and the effect of region of interest (ROI) placements in distinguishing glioblastoma multiforme (GBM) from solitary brain metastasis (sMET). MATERIALS AND METHODS: Sixty-two patients with pathologically confirmed GBM (n = 36) and sMET (n = 26) were retrospectively included. All patients underwent diffusion-weighted imaging with b values of 0 and 1000 s/mm2, and the ADC maps were generated automatically. ROIs were placed on the largest whole single-slice tumor (ROI1) and the enhanced solid portion (ROI2) of the ADC maps, respectively. The texture feature metrics of the histogram and gray-level co-occurrence matrix were then extracted by using in-house software. The parameters of the texture analysis were compared between GBM and sMET, using the Mann-Whitney U test. A receiver operating characteristic (ROC) curve analysis was performed to determine the best parameters for distinguishing between GBM from sMET. RESULTS: Homogeneity and the inverse difference moment (IDM) of GBM were significantly higher than those of sMET in both ROIs (ROI1, p = 0.014 for homogeneity and p = 0.048 for IDM; ROI2, p< 0.001 for homogeneity and p = 0.029 for IDM). According to the ROC curve analysis, the area under the ROC curve (AUC) of homogeneity in ROI1 (AUC, 0.682, sensitivity, 72.2%, specificity, 61.5%) was significantly lower than that of ROI2 (AUC, 0.886, sensitivity, 83.3%, specificity, 76.9%; p= 0.012), whereas the IDM showed no statistical significance between two ROIs (p> 0.05). CONCLUSION: The ADC-based texture analysis can help differentiate GBM from sMET, and the ROI on the solid portion would be recommended to calculate the ADC-based texture metrics.

Alterations of the amplitude of low-frequency fluctuation in healthy subjects with theta-burst stimulation of the cortex of the suprahyoid muscles.

Theta burst stimulation (TBS) has emerged as a promising tool for the treatment of swallowing disorders; however, the short-term after-effects of brain activation induced by TBS remain unknown. Here, we measured the changes in spontaneous brain activation using the amplitude of low-frequency fluctuation (ALFF) approach in subjects who underwent different TBS protocols. Sixty right-handed healthy participants (male, n=30; female, n=30; mean age=23.5y) were recruited in this study and randomly assigned to three groups that underwent three different TBS protocols. In group 1, continuous TBS (cTBS) was positioned on the left hemisphere of the suprahyoid muscle cortex. For group 2, intermittent TBS (iTBS) was placed on the left hemisphere of the suprahyoid muscle cortex. Group 3 underwent combined cTBS/iTBS protocols in which iTBS on the right hemisphere was performed immediately after completing cTBS on the left suprahyoid muscle cortex. Compared to pre-TBS, post-cTBS showed decreased ALFF in the anterior cingulate gyrus (BA 32); post-iTBS induced an increase in ALFF in the bilateral precuneus (BA 7); and post-cTBS/iTBS induced a decrease in ALFF in the brainstem, and resulted in increased ALFF in the middle cingulate gyrus (BA 24) as well as the left precentral gyrus (BA 6). Compared the effect of post-TBS protocols, increased ALFF was found in left posterior cerebellum lobe and left inferior parietal lobule (BA 40) (post-cTBS vs post-iTBS), and decreased ALFF exhibited in paracentral lobule (BA 4) (post-iTBS vs post-cTBS/iTBS). These findings indicate that multiple brain areas involved in swallowing regulation after stimulation of TBS over the suprahyoid muscles. cTBS induces decreased after-effects while iTBS results in increased after-effects on spontaneous brain activation. Moreover, iTBS can eliminate the after-effects of cTBS applied on the contralateral swallowing cortex and alter the activity of contralateral motor cortex and brainstem. Our findings provide a novel evidence for the short-term effect of TBS on spontaneous brain activation.

An antidote approach to reduce risk and broaden utility of antibody-based therapeutics.

Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.

Decreased Subcortical and Increased Cortical Degree Centrality in a Nonclinical College Student Sample with Subclinical Depressive Symptoms: A Resting-State fMRI Study.

Abnormal functional connectivity (FC) at rest has been identified in clinical depressive disorder. However, very few studies have been conducted to understand the underlying neural substrates of subclinical depression. The newly proposed centrality analysis approach has been increasingly used to explore the large-scale brain network of mental diseases. This study aimed to identify the degree centrality (DC) alteration of the brain network in subclinical depressive subjects. Thirty-seven candidates with subclinical depression and 34 well-matched healthy controls (HCs) were recruited from the same sample of college students. All subjects underwent a resting-state fMRI (rs-fMRI) scan to assess the DC of the whole brain. Compared with controls, subclinical depressive subjects displayed decreased DC in the right parahippocampal gyrus (PHG), left PHG/amygdala, and left caudate and elevated DC in the right posterior parietal lobule (PPL), left inferior frontal gyrus (IFG) and left middle frontal gyrus (MFG). In addition, by using receiver operating characteristic (ROC) analysis, we determined that the DC values in the regions with altered FC between the two groups can be used to differentiate subclinical depressive subjects from HCs. We suggest that decreased DC in subcortical and increased DC in cortical regions might be the neural substrates of subclinical depression.

Radiologic features of primary intracranial ectopic germinomas: Case reports and literature review.

RATIONALE: Germinomas are sensitive to radiation therapy and chemotherapy; therefore, correct imaging diagnosis is crucial for them. However, the imaging findings of germinomas originating from off-midline regions displayed different patterns from those originating from midline areas. PATIENT CONCERNS: The objective of this study is to describe the radiologic features of primary ectopic germinoma. We reviewed the MR and CT findings of 12 patients with histologically proven off-midline ectopic germinomas with off-midline locations. INTERVENTIONS: All of these patients underwent conventional MR images and 3 of them underwent diffusion images. Additional CT images were available in 3 patients. Analysis was focused on the shape and entity of tumors in images, signs of hemiatrophy, and the involvement of fibers in diffusion images. OUTCOMES: Well-defined (8/12) and ill-defined margin masses (4/12) were identified according to the shape of the mass. Multicystic masses were seen in 11 of the 12 patients. The solid component of the tumors had a high density (3/3) with calcifications (2/3) on CT images, iso- to hypointensity in T2WI (11/12) and restricted diffusion on apparent diffusion coefficient (ADC) maps (3/3). Hemiatrophy was observed in 5 cases and progressive hemiatrophy was observed in 1 case. Other signs included mild peritumoral edema (10/12), and hydrocephalus (7/12). Additionally, infiltration of the corticospinal tract (CST) was identified on diffusion tensor imaging (DTI) (2/2). LESSONS: The results indicate that multicysitic entities and hypointensities in solid components on T2WI and hemiatrophy are the imaging features of ectopic germinomas. DTI has potential for assessing CST involvement.

A multifunctional bispecific antibody protects against Pseudomonas aeruginosa.

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.

Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln(29)-His(53), which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu(32)-His(55). We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3(-/-) mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.

Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice.

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 -/- mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 -/- suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE -/-, LDLr -/-, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 -/- mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease.

C/EBPbeta contributes to hepatocyte growth factor-induced replication of rodent hepatocytes.

BACKGROUND/AIMS: Hepatocyte replication can be induced in vivo by hepatocyte growth factor (HGF), which might be used for gene therapy or to promote liver regeneration. However, the biochemical steps critical for this process are not clear. C/EBPbeta and C/EBPalpha are liver-enriched transcription factors that induce and inhibit hepatocyte replication, respectively. Because of their role in hepatocyte replication, this study examined the effect of HGF upon C/EBP proteins in vivo. METHODS: Rats were treated with HGF, and the effect upon C/EBPs was evaluated in liver extracts. Normal or C/EBPbeta-deficient mice were treated with HGF, and the effect upon hepatocyte replication was determined. RESULTS: HGF had no effect in rat liver upon C/EBPalpha or C/EBPbeta mRNA, nuclear protein, or nuclear DNA binding activity. However, HGF increased phosphorylated p90-RSK and ERK to 18- and 3-fold normal, respectively. These kinases phosphorylate C/EBPbeta and increase its transcriptional activity. The percentage of hepatocytes that replicated in C/EBPbeta-deficient mice after HGF administration was only 1.1%, which was lower than the value of 6.6% for hepatocytes from HGF-treated normal mice (P=0.005). CONCLUSIONS: C/EBPbeta contributes to the induction of hepatocyte replication in response to HGF in rodents, which is likely due to post-translational modifications.

Neonatal or hepatocyte growth factor-potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B.

Hemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus-based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 microg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

Transduction of hepatocytes after neonatal delivery of a Moloney murine leukemia virus based retroviral vector results in long-term expression of beta-glucuronidase in mucopolysaccharidosis VII dogs.

The use of Moloney murine leukemia virus (MLV)-based retroviral vectors (RV) can result in stable in vivo expression in the liver, but these vectors only transduce replicating hepatocytes. As newborn animals exhibit rapid growth, we evaluated the ability of MLV-based RV to transduce hepatocytes in neonatal dogs. I.v. injection of a beta-galactosidase-expressing RV at 3 days after birth resulted in transduction of 9% of hepatocytes. Prior treatment with human hepatocyte growth factor at 2.5 mg/kg did not increase transduction. Although cells from the spleen were also transduced with moderate efficiency, cells from other organs were not. Neonatal dogs with mucopolysaccharidosis VII (MPS VII) received an i.v.injection of an RV containing the canine beta-glucuronidase (cGUSB) cDNA. At several months after transduction, clusters of hepatocytes that expressed high levels of cGUSB were present in the liver, which probably derived from replication of transduced hepatocytes. At 6 months after transduction, serum GUSB levels were 73% that of homozygous normal dogs and were 34% of the peak values observed at 1 week. We conclude that neonatal delivery of an MLV-based RV results in stable transduction of hepatocytes in dogs. This approach could result in immediate correction in patients with an otherwise-lethal genetic deficiency.