An efficient genotyper and star-allele caller for pharmacogenomics.

High-throughput sequencing provides sufficient means for determining genotypes of clinically important pharmacogenes that can be used to tailor medical decisions to individual patients. However, pharmacogene genotyping, also known as star-allele calling, is a challenging problem that requires accurate copy number calling, structural variation identification, variant calling, and phasing within each pharmacogene copy present in the sample. Here we introduce Aldy 4, a fast and efficient tool for genotyping pharmacogenes that uses combinatorial optimization for accurate star-allele calling across different sequencing technologies. Aldy 4 adds support for long reads and uses a novel phasing model and improved copy number and variant calling models. We compare Aldy 4 against the current state-of-the-art star-allele callers on a large and diverse set of samples and genes sequenced by various sequencing technologies, such as whole-genome and targeted Illumina sequencing, barcoded 10x Genomics, and Pacific Biosciences (PacBio) HiFi. We show that Aldy 4 is the most accurate star-allele caller with near-perfect accuracy in all evaluated contexts, and hope that Aldy remains an invaluable tool in the clinical toolbox even with the advent of long-read sequencing technologies.

The genetic assimilation in language borrowing inferred from Jing People.

OBJECTIVES: The Jing people are a recognized ethnic group in Guangxi, southwest China, who are the immigrants from Vietnam during the 16th century. They speak Vietnamese but with lots of language borrowings from Cantonese, Zhuang, and Mandarin. However, it's unclear if there is large-scale gene flow from surrounding populations into Jing people during their language change due to the very limited genetic information of this population. MATERIALS AND METHODS: We collected blood samples from 37 Jing and 3 Han Chinese individuals from Wanwei, Shanxin, and Wutou islands in Guangxi and genotyped about 600,000 genome-wide single nucleotide polymorphisms (SNPs). We used Principal Component Analysis (PCA), ADMIXTURE analysis, f statistics, qpWave and qpAdm to infer the population genetic structure and admixture. RESULTS: Our data revealed that the Jing people are genetically similar to the populations in southwest China and mainland Southeast Asia. But compared with Vietnamese, they show significant evidence of gene flow from surrounding East Asians. The admixture proportion is estimated to be around 35-42% in different Jing groups using southern Han Chinese as a proxy. The majority of the paternal lineages of Jing people are most likely from surrounding East Asians. DISCUSSION: We conclude that the formation and language change of present-day Jing people have involved genetic assimilation of surrounding East Asian populations. The language borrowing, in this case, is not only a cultural phenomenon but has involved demic diffusion.

Wrist-ankle Acupuncture Increases Pain Thresholds in Healthy Adults.

Context • Wrist-ankle acupuncture (WAA) has been used to relieve both chronic and acute pain in China. Some research has shown that WAA can increase the pain thresholds in pain patients, but the ability of WAA to affect the pain thresholds in healthy adults is unknown. Objective • The study intended to assess the influence of WAA on the pain thresholds of healthy adults. Design • This is an observational study. Setting • This study was conducted in the School of Traditional Chinese Medicine at the Second Military Medical University (Shanghai, China). Participants • Participants were 50 healthy university students aged 19-23 y. Intervention • In the theory of WAA, each side of the body and each limb are longitudinally divided into 6 regions, with 1 needling point defined for each region at the wrist or ankle. The theory indicates that needling a point should relieve pain in a point's corresponding region. For the study, a needle was inserted and retained for 30 min in the Upper 2 point of the left wrist of each participant. Outcome Measures • The pressure pain threshold was measured by a handheld algometer at a position in the left Upper 2 region corresponding to the site of the needling and at positions in the right Upper 2 region as well as the left and right Upper 3 regions not corresponding to the site of the needling. The measurements were taken at 40 min before needling, 5 min after needling, 30 min after needling when the needles were removed, and 70 min after needling. Results • The immediate influence of the WAA on the pain threshold was not significant at 5 min after needling (P > .05). However, at 30 min after needling when the needles were removed, the increases in the pain thresholds were statistically significant when compared to those at 40 min before needling, which were the measurements at baseline (P ≤ .01). At 70 min after needling, the pain thresholds remained higher than those at 40 min before needling (P < .05). From 40 min before needling to 70 min after needling, the pain thresholds in the different positions showed a continuous increase. Conclusions • The WAA had an analgesic effect on pressure-induced pain not only in the corresponding but also in the noncorresponding regions of the needling point in healthy adults. The immediate analgesic effect of the WAA at 5 min after needling was not obvious, but the effects at 30 min and 70 min after needling were statistically significant.

FGF-2 Gene Polymorphism in Osteoporosis among Guangxi's Zhuang Chinese.

Osteoporosis is a complex multifactorial disorder of gradual bone loss and increased fracture risk. While previous studies have shown the importance of many genetic factors in determining peak bone mass and fragility fractures and in suggesting involvement of fibroblast growth factor-2 (FGF-2) in bone metabolism and bone mass, the relationship of FGF-2 genetic diversity with bone mass/osteoporosis has not yet been revealed. The current study investigated the potential relevance of FGF-2 gene polymorphism in osteoporosis among a Zhuang ethnic Chinese cohort of 623, including 237 normal bone mass controls, 227 osteopenia, and 159 osteoporosis of different ages. Bone density was examined by calcaneus ultrasound attenuation measurement, and single nucleotide polymorphisms (SNPs) and linkage disequilibrium analyses were performed on five SNP loci of FGF-2 gene. Significant differences were found in bone mass in males between the 45-year-old and ≥70-year-old groups (p < 0.01), and in females among 55, 60, 65 and 70-year-old groups (p < 0.05). Males had higher bone mass values than females in the same age (over 55-year-old) (p < 0.05). The proportions of individuals with normal bone mass decreased with age (65.2% to 40% in males, and 50% to 0% in females), whereas prevalence of osteoporosis increased with age (15.4% to 30% in men, and 7.7% to 82% in women). Out of five FGF-2 SNP loci, the TA genotype of rs308442 in the osteoporosis group (40.2%) was higher than in the control group (29.5%) (p < 0.05). The TA genotype was significantly correlated with the risk of osteoporosis (odds ratio OR = 1.653), 95% confidence interval (CI): 1.968-1.441). Strong linkage disequilibrium in FGF-2 gene was also detected between rs12644427 and rs3747676, between rs12644427 and rs3789138, and between rs3747676 and rs3789138 (D' > 0.8, and r² > 0.33). Thus, the rs308442 locus of FGF-2 gene is closely correlated to osteoporosis in this Zhuang ethnic Chinese cohort, and the TA may be the risk genotype of osteoporosis.

An Intelligent Channel Estimation Algorithm Based on Extended Model for 5G-V2X.

Car networking systems based on 5G-V2X (vehicle-to-everything) have high requirements for reliability and low-latency communication to further improve communication performance. In the V2X scenario, this article establishes an extended model (basic expansion model) suitable for high-speed mobile scenarios based on the sparsity of the channel impulse response. And propose a channel estimation algorithm based on deep learning, the method designed a multilayer convolutional neural network to complete frequency domain interpolation. A two-way control cycle gating unit (bidirectional gated recurrent unit) is designed to predict the state in the time domain. And introduce speed parameters and multipath parameters to accurately train channel data under different moving speed environments. System simulation shows that the proposed algorithm can accurately train the number of channels. Compared with the traditional car networking channel estimation algorithm, the proposed algorithm improves the accuracy of channel estimation and effectively reduces the bit error rate.

Wrist-ankle acupuncture for primary dysmenorrhea: a randomized controlled trial evaluating the efficacy of an analgesic strap.

Background: Drawing on the principles of wrist-ankle acupuncture (WAA), our research team has developed a portable device for WAA point compression, termed the acupressure wrist-ankle strap (AWA). The current study aims to evaluate the efficacy of the AWA in alleviating pain associated with primary dysmenorrhea. Methods: A single-blind, randomized clinical trial was conducted from April 1, 2019, to December 31, 2019. 78 participants with primary dysmenorrhea were recruited from Shanghai University of Traditional Chinese Medicine. All participants were treated on the first day of menstruation for 30 min. Participants in the AWA group used the AWA, the internal side of which is equipped with a tip compression component, while participants in the non-acupressure wrist-ankle acupuncture(NAWA)group used the NAWA, with the inside tip pressing parts removed. The main outcome was the difference in visual analogue scale (VAS) score between baseline and 30 minutes after randomization. Results: A total of 78 participants aged 18 to 30 years were included in the intention-to-treat analyses. The VAS scores (mean [standard deviation]) in the AWA group were significantly lower than those in the NAWA group at each time point of intervention (5 minutes: 95% CI, [-1.27 to -0.68], p < 0.001; 10 minutes: 95% CI, [-2.34 to -1.51], p < 0.001; 30 minutes: 95% CI, [-3.74 to -2.72], p < 0.001). In the AWA group, 16 participants reported "obvious relief" of dysmenorrhea pain while 23 did not; the average onset time of analgesia they reported were (21.50 ± 3.65) min, while no subjects in NAWA group reported obvious pain relief. The pain threshold (mean [standard deviation]) at SP9 of both sides in AWA group decreased significantly after intervention that in NAWA group (Left: 95% CI, [-5.02 to -1.81], p < 0.001; Right: 95% CI, [-7.67 to -4.24], p < 0.001). There was no significant change in the temperature at CV4 in either group (95% CI, [-0.63 to -0.66], p = 0.970). Conclusion: This trial substantiates our hypothesis that the AWA provides immediate analgesic effects. The AWA represents an effective and safe non-invasive physical therapy option, which patients can self-administer to alleviate abdominal pain.

Geny: A Genotyping Tool for Allelic Decomposition of Killer Cell Immunoglobulin-Like Receptor Genes.

Accurate genotyping of Killer cell Immunoglobulin-like Receptor (KIR) genes plays a pivotal role in enhancing our understanding of innate immune responses, disease correlations, and the advancement of personalized medicine. However, due to the high variability of the KIR region and high level of sequence similarity among different KIR genes, the currently available genotyping methods are unable to accurately infer copy numbers, genotypes and haplotypes of individual KIR genes from next-generation sequencing data. Here we introduce Geny, a new computational tool for precise genotyping of KIR genes. Geny utilizes available KIR haplotype databases and proposes a novel combination of expectation-maximization filtering schemes and integer linear programming-based combinatorial optimization models to resolve ambiguous reads, provide accurate copy number estimation and estimate the haplotype of each copy for the genes within the KIR region. We evaluated Geny on a large set of simulated short-read datasets covering the known validated KIR region assemblies and a set of Illumina short-read samples sequenced from 25 validated samples from the Human Pangenome Reference Consortium collection and showed that it outperforms the existing genotyping tools in terms of accuracy, precision and recall. We envision Geny becoming a valuable resource for understanding immune system response and consequently advancing the field of patient-centric medicine.

Pharmacokinetics and brain uptake of an IgG-TNF decoy receptor fusion protein following intravenous, intraperitoneal, and subcutaneous administration in mice.

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine active in the brain. Etanercept, the TNF decoy receptor (TNFR), does not cross the blood-brain barrier (BBB). The TNFR was re-engineered for BBB penetration as a fusion protein with a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse and mediates transport via the endogenous BBB TfR. To support future chronic treatment of mouse models of neural disease with daily administration of the cTfRMAb-TNFR fusion protein, a series of pharmacokinetics and brain uptake studies in the mouse was performed. The cTfRMAb-TNFR fusion protein was radiolabeled and injected into mice via the intravenous, intraperitoneal (IP), or subcutaneous (SQ) routes of administration at doses ranging from 0.35 to 10 mg/kg. The distribution of the fusion protein into plasma following the IP or SQ routes was enhanced by increasing the injection dose from 3 to 10 mg/kg. The fusion protein demonstrated long circulation times with high metabolic stability following the IP or SQ routes of injection. The IP or SQ routes produced concentrations of the cTfRMAb-TNFR fusion protein in the brain that exceed by 20- to 50-fold the concentration of TNFα in pathologic conditions of the brain. The SQ injection is the preferred route of administration, as the level of cTfRMAb fusion protein produced in the brain is comparable to that generated with intravenous injection, and at a much lower plasma area under the concentration curve of the fusion protein as compared to IP administration.

Comparison of registered and published primary outcomes in randomized controlled trials of gastroenterology and hepatology.

OBJECTIVES. The need for trial registration as well as the benefits it has brought for the transparency of medical research has been recognized for years. Trial registration has turned from an exception to a mandatory guideline in recent years. The present study aimed to examine the characteristics of registered randomized controlled trials (RCTs) in a sample of recently published gastroenterology RCTs, and to assess the consistency of registered and published primary outcome (PO) in RCTs. METHODS. Articles published in the top five "general and internal journals" and top five "gastroenterology and hepatology journals" categories between 2009 and 2012 were searched in PubMed. Basic characteristics and the registration information were identified and extracted from the included RCTs. PO consistency analysis was conducted to compare between the registered and published format. RESULTS. A total of 305 RCTs were included; among them 252 could be identified with a registration number. Nearly half of these RCTs were funded solely by industry (141/305, 46.3%). ClinicalTrials.gov was the most popular registry for these RCTs (214/252, 84.9%). A total of 155 RCTs were included in the PO consistency analysis. Among them, 22 (14.2%) RCTs had discrepancies between POs registered in the trial registry compared to the published article. CONCLUSIONS. Based on the results of the present study, selective outcome reporting of gastroenterology RCTs published in leading medical journals has been much improved over the past years. However, there might be a sampling bias to say that consistency of registered and published POs of gastroenterology RCTs has been better than before.

Deep asymmetric extraction and aggregation for infrared small target detection.

Infrared small target detection is widely applied in military and civilian fields. Due to the small size of infrared targets, textural detail is missing. Common target detection methods extract semantic feature by narrowing down the feature map several times, which may lead to the small targets lost in deep layers and are not effective for infrared small target detection. To solve this problem, we propose a novel network called deep asymmetric extraction and aggregation. The network mainly consists of two processes - the vertical feature extraction and the horizontal feature aggregation, both of which are enhanced by an asymmetric attention mechanism. In the vertical process, the use of asymmetric attention mechanism combined with the reduction of down-sampling makes the small target better retained in the deep layers. Then through the horizontal process, shallow spatial feature and deep semantic feature are aggregated to further highlight the small targets while suppressing background noise. Experiments on the public datasets NUAA-SISRT, NUDT-SISRT and MDvsFA-cGan show that our proposed network outperforms the state-of-the-art methods in terms of detection accuracy and parameter efficiency.

Intelligent Device for Harvesting the Vibration Energy of the Automobile Exhaust with a Piezoelectric Generator.

With increasing consumption of energy and increasing environmental pollution, research on capturing the vibration energy lost during transportation and vehicle driving is growing rapidly. There is a large amount of vibration energy in the automobile exhaust system that can be recycled. This paper proposes a self-powered intelligent device (SPID) using a piezoelectric energy generator. The SPID includes a piezoelectric generator and sensor unit, and the generator is installed at the end of the automobile exhaust system. The generator adopts a parallel structure of four piezoelectric power generation units, and the sensing unit comprises light-emitting diode warning lights or low-power sensors. A simulated excitation experiment verifies the working state and peak power of the piezoelectric generator unit, which can achieve 23.4 µW peak power. The self-power supply and signal monitoring functions of the intelligent device are verified in experiments conducted for driving light-emitting diode lights and low-power sensors. The device is expected to play a crucial role in the field of intelligent driving and automobile intelligence.

Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Frequent tumor recurrence after surgery is related to its poor prognosis. Although gene expression signatures have been associated with outcome, the molecular basis of HCC recurrence is not fully understood, and there is no method to predict recurrence using peripheral blood mononuclear cells (PBMCs), which can be easily obtained for recurrence prediction in the clinical setting. METHODS: According to the microarray analysis results, we constructed a co-expression network using the k-core algorithm to determine which genes play pivotal roles in the recurrence of HCC associated with the hepatitis B virus (HBV) infection. Furthermore, we evaluated the mRNA and protein expressions in the PBMCs from 80 patients with or without recurrence and 30 healthy subjects. The stability of the signatures was determined in HCC tissues from the same 80 patients. Data analysis included ROC analysis, correlation analysis, log-lank tests, and Cox modeling to identify independent predictors of tumor recurrence. RESULTS: The tumor-associated proteins cyclin B1, Sec62, and Birc3 were highly expressed in a subset of samples of recurrent HCC; cyclin B1, Sec62, and Birc3 positivity was observed in 80%, 65.7%, and 54.2% of the samples, respectively. The Kaplan-Meier analysis revealed that high expression levels of these proteins was associated with significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed that cyclin B1 (hazard ratio [HR], 4.762; p = 0.002) and Sec62 (HR, 2.674; p = 0.018) were independent predictors of HCC recurrence. CONCLUSION: These results revealed that cyclin B1 and Sec62 may be candidate biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

Brain-penetrating IgG-iduronate 2-sulfatase fusion protein for the mouse.

Mucopolysaccharidosis (MPS) type II (Hunter's syndrome) is caused by mutations in the iduronate 2-sulfatase (IDS) fusion protein. MPS-II affects the brain, and enzyme replacement therapy is not effective in the brain, because the enzyme does not cross the blood-brain barrier. To treat mouse models of MPS-II with brain-penetrating IDS, the lysosomal enzyme was reengineered as an IgG-IDS fusion protein. The mature human IDS was fused to the carboxyl terminus of both heavy chains of the chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and the fusion protein is designated cTfRMAb-IDS. The purity and identity of the fusion protein was confirmed by electrophoresis and Western blotting with antibodies to mouse IgG and human IDS. The EC50 of binding of the cTfRMAb-IDS fusion protein to the mouse TfR (0.85 ± 0.15 nM) was comparable to the EC50 of binding of the cTfRMAb (0.78 ± 0.05 nM). The IDS enzyme activity of the cTfRMAb-IDS fusion protein was 126 ± 1 nmol · h⁻¹ · µg⁻¹ protein. After intravenous injection in the mouse, the cTfRMAb-IDS fusion protein was rapidly removed from plasma and distributed to tissues, including brain and spinal cord. The uptake of the fusion protein by brain or spinal cord was 1.3 ± 0.1 and 2.2 ± 0.2% injected dose/g, respectively, which is 100-fold greater than the brain uptake of IDS alone. This work shows that a lysosomal sulfatase can be reengineered as an IgG-enzyme fusion protein that rapidly penetrates the brain after intravenous administration.

Synthesis of bisethylnorspermine lipid prodrug as gene delivery vector targeting polyamine metabolism in breast cancer.

Progress in the development of nonviral gene delivery vectors continues to be hampered by low transfection activity and toxicity. Here we proposed to develop a lipid prodrug based on a polyamine analogue bisethylnorspermine (BSP) that can function dually as gene delivery vector and, after intracellular degradation, as active anticancer agent targeting dysregulated polyamine metabolism. We synthesized a prodrug of BSP (LS-BSP) capable of intracellular release of BSP using thiolytically sensitive dithiobenzyl carbamate linker. Biodegradability of LS-BSP contributed to decreased toxicity compared with nondegradable control L-BSP. BSP showed a strong synergistic enhancement of cytotoxic activity of TNF-related apoptosis-inducing ligand (TRAIL) in human breast cancer cells. Decreased enhancement of TRAIL activity was observed for LS-BSP when compared with BSP. LS-BSP formed complexes with plasmid DNA and mediated transfection activity comparable to DOTAP and L-BSP. Our results show that BSP-based vectors are promising candidates for combination drug/gene delivery.

[Association of MMP14 gene polymorphisms and osteoporosis in Zhuang men from Baise region of Guangxi].

OBJECTIVE: To assess the association between membrane type 1 matrix metalloproteinase gene (MT1-MMP, MMP14) polymorphisms and osteoporosis in Zhuang men from Baise region of Guangxi. METHODS: Genotypes of 5 loci (rs1003349, rs3751488, rs2269213, rs2236303 and rs743257) of MMP14 gene in 301 Zhuang men were determined with single base extension methods, and bone mineral density (BMD) at left calcaneus was evaluated with quantitative ultrasound with measured values of broadband ultrasonic attenuation (BUA). The subjects were divided according to BMD into osteoporosis group, osteopenia group and normal bone density group. RESULTS: All selected loci were in Hardy-Weinberg equilibrium (P> 0.05). By multiple linear stepwise regression analysis, polymorphisms of the five loci were not associated with BUA. But a significant higher risk of osteoporosis was found in individuals with MMP14 rs1003349 GT genotype (vs. GG genotype; P<0.05) and rs2236303 CC and CT genotypes (vs. TT genotype; P<0.05). Genetic linkage between rs1003349 and rs2236303 was also discovered (D'= 0.839, r(2) = 0.458, P<0.01). Compared with the normal bone density group, the frequency of a G-T haplotype of rs1003349 and rs2236303 was significantly lower in the osteoporosis group (P<0.05). And the risk of osteoporosis for individuals with G-C and T-C haplotypes was 2.556 (95% CI: 1.029-6.349, P = 0.038) and 5.111 (95% CI: 1.341-19.485, P = 0.011) compared with G-T haplotype. CONCLUSION: Polymorphisms of rs1003349 and rs2236303 loci of MMP14 gene are associated with the susceptibility of osteoporosis in Zhuang men in Guangxi. G-C and T-C haplotypes for loci rs1003349 and rs2236303 may increase the disease risk.

A traditional Chinese herbal medicine compound preparation versus interventional therapy after resection of small hepatocellular carcinoma: 22-year follow-up.

OBJECTIVE: To compare the long-term effectiveness of compound Ruanjianhugan(RJH)tablets and interventional therapy (IT) in patients after resection of small hepatocellular carcinoma (HCC). in 399 patients after resection of small HCC who were admitted between January 1987 and December 2008 in the Department of Hepatobiliary Surgery and Center of Minimally Invasive Surgery, First Affiliated Hospital of Guangxi Medical University. Four groups were based on different therapy modes: a TCM-only (TCMO) group, a TCM combined with interventional therapy (TCM-IT) group, an interventional therapy-only (ITO) group, and a simple operation (SO) group. Prognostic factors were correlated with overall survival (OS) and OS rates were calculated with the Kaplan-Meier method, and multivariate analyses for factors affecting survival were evaluated by the Cox proportional hazard model. RESULTS: The median OS was 151.20 months in the TCM-IT group, 43.87 months in the ITO group, and 20.77 months in the SO group. All survival rates of the TCMO group were higher than those of the other three groups (>50%). The 5-, 10-, and 15-year OS in the TCMO and ITO patients were 83.94%, 45.50%, and 71.22% and 33.34%, 55.58%, and 9.26%, respectively (risk ratio, 0.209; 95% confidence interval, 0.126-0.347; P = 0.000). Multivariate analysis revealed that the independent risk factors were therapy mode (P = 0.000), sex (P = 0.005), family history (P = 0.011), TNM classification of malignant tumor staging (P = 0.000), medical care-seeking behavior (P = 0.021), and maximum diameter (P = 0.030). CONCLUSION: Long-term oral use of compound RJH tablets may improve OS for small HCC after resection compared with IT.

[Assessment of research papers in medical university staff evaluation].

Medical university staff evaluation is a substantial branch of education administration for medical university. Output number of research papers as a direct index reflecting the achievements in academic research, plays an important role in academic research evaluation. Another index, influence of the research paper, is an indirect index for academic research evaluation. This paper mainly introduced some commonly used indexes in evaluation of academic research papers currently, and analyzed the applicability and limitation of each index. The author regards that academic research evaluation in education administration, which is mainly based on evaluation of academic research papers, should combine the evaluation of journals where the papers are published with peer review of the papers, and integrate qualitative evaluation with quantitative evaluation, for the purpose of setting up an objective academic research evaluation system for medical university staff.

Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor.

Biologic tumor necrosis factor (TNF)-α inhibitors do not cross the blood-brain barrier (BBB). A BBB-penetrating TNF-α inhibitor was engineered by fusion of the extracellular domain of the type II human TNF receptor (TNFR) to the carboxyl terminus of the heavy chain of a mouse/rat chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb-TNFR fusion protein and etanercept bound human TNF-α with high affinity and K(D) values of 374 ± 77 and 280 ± 80 pM, respectively. Neuroprotection in brain in vivo after intravenous administration of the fusion protein was examined in a mouse model of Parkinson's disease. Mice were also treated with saline or a non-BBB-penetrating TNF decoy receptor, etanercept. After intracerebral injection of the nigral-striatal toxin, 6-hydroxydopamine, mice were treated every other day for 3 weeks. Treatment with the cTfRMAb-TNFR fusion protein caused an 83% decrease in apomorphine-induced rotation, a 67% decrease in amphetamine-induced rotation, a 82% increase in vibrissae-elicited forelimb placing, and a 130% increase in striatal tyrosine hydroxylase (TH) enzyme activity. In contrast, chronic treatment with etanercept, which does not cross the BBB, had no effect on neurobehavior or striatal TH enzyme activity. A bridging enzyme-linked immunosorbent assay specific for the cTfRMAb-TNFR fusion protein showed that the immune response generated in the mice was low titer. In conclusion, a biologic TNF inhibitor is neuroprotective after intravenous administration in a mouse model of neurodegeneration, providing that the TNF decoy receptor is reengineered to cross the BBB.

Delivery of a peptide radiopharmaceutical to brain with an IgG-avidin fusion protein.

The genetic engineering, host cell expression, purity, identity, and in vivo brain drug targeting properties are described for a new IgG-fusion protein, designated the cTfRMAb-AV fusion protein. Avidin (AV) is fused to the carboxyl terminus of the heavy chain of the genetically engineered chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR). The TfRMAb binds the endogenous TfR on the blood-brain barrier (BBB), which triggers transport into brain from blood. The cTfRMAb-AV fusion protein is produced in stably transfected Chinese hamster ovary cells, which are grown in serum free medium under conditions of biotin starvation. Following affinity purification, the purity and identity of the cTfRMAb-AV fusion protein were verified by electrophoresis and Western blotting. The affinity of the cTfRMAb for the murine TfR is high, K(I) = 4.6 ± 0.5 nM, despite fusion of avidin to the antibody heavy chain. The model peptide radiopharmaceutical used in this study is the Aß(1-40) amyloid peptide of Alzheimer's disease (AD), which in a brain-penetrating form could be used to image the amyloid plaque in brain in AD. The BBB transport and brain uptake of the [(125)I]-Aß(1-40) peptide was measured in mice injected intravenously (IV) with the peptide either free or conjugated to the cTfRMAb-AV fusion protein. The brain uptake of the free Aß(1-40) peptide was very low, 0.1% of injected dose (ID)/gram brain following i.v. injection, and is comparable to the brain uptake of a brain blood volume marker. However, the brain uptake of the Aß(1-40) peptide was high, 2.1 ± 0.2% ID/gram brain, following attachment of the biotinylated peptide to the cTfRMAb-AV fusion protein. Capillary depletion analysis showed the peptide penetrated the brain parenchyma from blood. The cTfRMAb-AV fusion protein is a new drug delivery system that can target to mouse brain monobiotinylated peptide or antisense radiopharmaceuticals.

Brain-penetrating tumor necrosis factor decoy receptor in the mouse.

Biologic tumor necrosis factor inhibitors (TNFIs) include TNF decoy receptors (TNFRs). TNFα plays a pathologic role in both acute and chronic brain disease. However, biologic TNFIs cannot be developed as brain therapeutics because these large molecule drugs do not cross the blood-brain barrier (BBB). To enable penetration of the brain via receptor-mediated transport, the human TNFR type II was re-engineered as an IgG fusion protein, where the IgG part is a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb part of the fusion protein acts as a molecular Trojan horse to ferry the TNFR across the BBB via transport on the endogenous BBB TfR. cTfRMAb-TNFR was expressed by stably transfected Chinese hamster ovary cells and purified by affinity chromatography to homogeneity on electrophoretic gels. The fusion protein reacted with antibodies to both mouse IgG and the human TNFR and bound TNFα with high affinity (K(d) = 96 ± 34 pM). cTfRMAb-TNFR was rapidly transported into mouse brain in vivo after intravenous administration, and the brain uptake of the fusion protein was 2.8 ± 0.5% of injected dose per gram of brain, which is >45-fold higher than the brain uptake of an IgG that does not recognize the mouse TfR. This new IgG-TNFR fusion protein can be tested in mouse models of brain diseases in which TNFα plays a pathologic role.