Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpß-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpß-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity.

Therapeutic monoclonal antibodies are the fastest growing class of biological therapeutics for the treatment of various cancers and inflammatory disorders. In cancer immunotherapy, some IgG1 antibodies rely on the Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells. It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody. In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity. As such, various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. This review discusses the relevance of antibody core fucosylation to ADCC, different strategies to produce afucosylated antibodies, and an update of afucosylated antibody drugs currently undergoing clinical trials as well as those that have been approved.

Acute changes in arterial stiffness following exercise in people with metabolic syndrome.

BACKGROUND: This study aims to examine the changes in arterial stiffness immediately following sub-maximal exercise in people with metabolic syndrome. METHODS: Ninety-four adult participants (19-80 years) with metabolic syndrome gave written consent and were measured for arterial stiffness using a SphygmoCor (SCOR-PVx, Version 8.0, Atcor Medical Private Ltd, USA) immediately before and within 5-10min after an incremental shuttle walk test. The arterial stiffness measures used were pulse wave velocity (PWV), aortic pulse pressure (PP), augmentation pressure, augmentation index (AI), subendocardial viability ratio (SEVR) and ejection duration (ED). RESULTS: There was a significant increase (p<0.05) in most of the arterial stiffness variables following exercise. Exercise capacity had a strong inverse correlation with arterial stiffness and age (p<0.01). CONCLUSION: Age influences arterial stiffness. Exercise capacity is inversely related to arterial stiffness and age in people with metabolic syndrome. Exercise induced changes in arterial stiffness measured using pulse wave analysis is an important tool that provides further evidence in studying cardiovascular risk in metabolic syndrome.

Bruton's tyrosine kinase phosphorylates DDX41 and activates its binding of dsDNA and STING to initiate type 1 interferon response.

The innate immune system senses cytosolic dsDNA and bacterial cyclic dinucleotides and initiates signaling via the adaptor STING to induce type 1 interferon (IFN) response. We demonstrate here that BTK-deficient cells have impaired IFN-ß production and TBK1/IRF3 activation when stimulated with agonists or infected with pathogens that activate STING signaling. BTK interacts with STING and DDX41 helicase. The kinase and SH3/SH2 interaction domains of BTK bind, respectively, the DEAD-box domain of DDX41 and transmembrane region of STING. BTK phosphorylates DDX41, and its kinase activities are critical for STING-mediated IFN-ß production. We show that Tyr364 and Tyr414 of DDX41 are critical for its recognition of AT-rich DNA and binding to STING, and tandem mass spectrometry identifies Tyr414 as the BTK phosphorylation site. Modeling studies further indicate that phospho-Tyr414 strengthens DDX41's interaction with STING. Hence, BTK plays a critical role in the activation of DDX41 helicase and STING signaling.

Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

DOK3 is required for IFN-ß production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-ß production. Macrophages lacking DOK3 were impaired in IFN-ß synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-ß gene expression. Interestingly, polyinosinic-polyribocytidylic acid-induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 complex was compromised in dok3(-/-) macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-ß promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton's tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-ß production.

Effect of an IT-supported home-based exercise programme on metabolic syndrome in India.

Summary We studied the effectiveness of a home-based exercise programme with information technology (IT) support in people with metabolic syndrome in India. Ninety-four participants with metabolic syndrome (mean age 50 years) were randomized into two groups. Both groups received a 12-week home exercise programme and Group 2 received additional IT support for health education. Before and after the exercise programme, participants were measured for arterial stiffness using applanation tonometry, exercise capacity using an incremental shuttle walk test and quality of life (QoL) using the SF-36 questionnaire. Sixty-one participants completed the post intervention tests. There was a significant reduction in systolic blood pressure, mean pressure and aortic systolic pressure in both groups. Pulse wave velocity, aortic pulse pressure and aortic diastolic pressure showed significant reductions only in Group 2. There were no significant changes in QoL measures, except vitality in Group 2. There was significant improvement in fasting blood glucose in Group 2, cholesterol in Group 1 and triglycerides in both groups. The participants' exercise capacity did not change significantly, although the mean duration of regular exercise was 7.2 weeks for Group 1 and 10.0 weeks for Group 2 (P = 0.019). Metabolic syndrome was reversed in 16% of the participants in both groups. IT support, through mobile text messages and phone calls, may be helpful in metabolic syndrome. Longer-term studies are now required.

Golgi phosphoprotein 3 mediates the Golgi localization and function of protein O-linked mannose ß-1,2-N-acetlyglucosaminyltransferase 1.

GOLPH3 is a highly conserved protein found across the eukaryotic lineage. The yeast homolog, Vps74p, interacts with and maintains the Golgi localization of several mannosyltransferases, which is subsequently critical for N- and O-glycosylation in yeast. Through the use of a T7 phage display, we discovered a novel interaction between GOLPH3 and a mammalian glycosyltransferase, POMGnT1, which is involved in the O-mannosylation of α-dystroglycan. The cytoplasmic tail of POMGnT1 was found to be critical for mediating its interaction with GOLPH3. Loss of this interaction resulted in the inability of POMGnT1 to localize to the Golgi and reduced the functional glycosylation of α-dystroglycan. In addition, we showed that three clinically relevant mutations present in the stem domain of POMGnT1 mislocalized to the endoplasmic reticulum, highlighting the importance of identifying the molecular mechanisms responsible for Golgi localization of glycosyltransferases. Our findings reveal a novel role for GOLPH3 in mediating the Golgi localization of POMGnT1.

Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant.

The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.

Some commonly used caspase substrates and inhibitors lack the specificity required to monitor individual caspase activity.

Many designated substrates and inhibitors have been widely used to investigate the roles of caspases in apoptotic death during mammalian cell culture. However, the specificities of these substrates and inhibitors have not been systematically evaluated. As a result, conclusions on the roles of specific caspases in apoptotic cells have been published inaccurately. In this study, the interaction between seven commercially available human caspases and their designated substrates and inhibitors was studied. Ac-YVAD-pNA, the designated substrate for caspase-1, is found to be the most specific substrate. All other substrates tested demonstrate cross-reactivity with several caspases. In relation to the enzyme, Caspase-2 is the most specific caspase, followed by caspase-9 and -6. Caspase-3 and -7 cleave three substrates efficiently. The designated substrates for capsase-1 and -8 are not even their best substrates. Fluoromethylketone (fmk) inhibitors exhibit no specificity towards different caspases even at low concentrations. In contrast, aldehyde inhibitor potency shows a distinct relationship to pNA substrate cleavage. These results show that some commonly used caspase substrates and inhibitors lack the specificity required to monitor individual caspase activity.

Mitochondrially targeted Bcl-2 and Bcl-X(L) chimeras elicit different apoptotic responses.

The Bcl-2 family of proteins interacts at the mitochondria to regulate apoptosis. However, the anti-apoptotic Bcl-2 and Bcl-X(L) are not completely localized to the mitochondria. In an attempt to generate Bcl-2 and Bcl-X(L) chimeras that are constitutively localized to the mitochondria, we substituted their C-terminal transmembrane tail or both the C-terminal transmembrane tail and the adjacent loop with the equivalent regions from Bak or Bax mutant (BaxS184V) as these regions determine the mitochondrial localization of Bak and Bax. The effects of these substitutions on subcellular localization and their activities were assessed following expression in HeLa and CHO K1 cells. The substitution of the C-terminal tail or the C-terminal tail and the adjacent loop of Bcl-2 with the equivalent regions from Bak or the Bax mutant resulted in its association with the mitochondria. This change in subcellular localization of Bcl-2 chimeras triggered cells to undergo apoptotic-like cell death. The localization of this Bcl-2 chimera to the mitochondria may be associated with the disruption of mitochondrial membrane potential. Unlike Bcl-2, the loop structure adjacent to the C-terminal tail in Bcl-X(L) is crucial for its localization. To localize the Bcl-X(L) chimeras to the mitochondria, the loop structure next to the C-terminal tail in Bcl-X(L) protein must remain intact and cannot be substituted by the loop from Bax or Bak. The chimeric Bcl-X(L) with both its C-terminal tail and the loop structure replaced by the equivalent regions of Bak or Bax mutant localized throughout the entire cytosol. The Bcl-X(L) chimeras that are targeted to the mitochondria and the wild type Bcl-X(L) provided same protection against cell death under several death inducing conditions.

Laparoscopic adjustable gastric banding versus laparoscopic gastric bypass for morbid obesity: a single-institution comparison study of early results.

Laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic adjustable gastric banding (LAGB) are common surgical procedures for morbid obesity, but few studies have compared LRYGB and LAGB. All patients who underwent LRYGB and LAGB by a single surgeon at Legacy Health System were identified from a prospectively maintained database. Preoperatively, most patients were allowed to choose between LRYGB and LAGB. Age, sex, body mass index (BMI), complications, mortality, and weight loss were examined. From October 2000 to November 2003, 219 patients underwent LRYGB and 154 patients underwent LAGB. Mean preoperative BMI was 49.5+/-6.6 and 50.9+/-9.4 kg/m2, respectively (P=0.10). Mean age was 42+/-9 and 47+/-11 years (P<0.001). The LAGB group had a higher proportion of male patients (21% versus 7%, P<0.001). Patients undergoing LRYGB had longer operative times (134 versus 76 minutes, P<0.001), more blood loss (43 versus 28 ml, P<0.01), and longer hospital stays (2.6 versus 1.3 days, P<0.001). Excess weight loss was 35% for LRYGB versus 19% for LAGB at 3-month follow-up (P<0.001), 49% versus 25% at 6 months (P<0.001), 64% versus 36% at 12 months (P<0.001), 70% versus 45% at 24 months (P<0.001), and 60% versus 57% at 36 months (P=0.85). Major complications occurred in 7% and 6% (P=0.58) and minor complications occurred in 18% and 20% (P=0.65) of patients, respectively. Reoperation occurred in 21 patients (10%) after LRYGB and 31 (20%) patients after LAGB (P<0.01). Of patients undergoing reoperation, eight (38%) LRYGB patients and one (3%) LAGB patient required open laparotomy. One death occurred in each group. Patients undergoing laparoscopic adjustable gastric banding have shorter operative times, less blood loss, and shorter hospital stays compared with laparoscopic gastric bypass patients. The incidence of major and minor complications is similar; however, morbidity after LRYGB is potentially greater and the reoperation rate is higher in the LAGB group. Early weight loss is greater with gastric bypass, but the difference appears to diminish over time.

Postoperative objective outcomes for upright, supine, and bipositional reflux disease following laparoscopic nissen fundoplication.

HYPOTHESIS: Traditionally, patients with gastroesophageal reflux disease fall into 3 categories based on 24-hour pH testing and the clinical occurrence of their acid exposure. Patients with upright reflux are believed to do worse following surgery compared with supine or bipositional reflux patients. We assessed objective postoperative outcomes for patients with upright, supine, and bipositional reflux following laparoscopic Nissen fundoplication to determine if there is a category of refluxing patient who should be counseled against antireflux surgery. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Esophageal physiology laboratory at a tertiary care teaching hospital. PATIENTS: A total of 225 patients (supine, 45; upright, 92; bipositional, 88) with preoperative and postoperative 24-hour pH measurements, manometry results, and standardized symptom assessment forms were included in the study. INTERVENTIONS: A Nissen fundoplication was performed based on 24-hour pH and manometry result. Esophageal manometry was performed with a water-perfused system, and 24-hour pH was measured with a digital capture device. MAIN OUTCOME MEASURES: Preoperative and postoperative symptom correlation, 24-hour pH, and manometric variables. RESULTS: There was a significant difference in preoperative symptom correlation between groups. Patients with bipositional reflux disease have significantly worst reflux disease (percentage of time with a pH <4, total number of reflux episodes, longest reflux episode, and Johnson-DeMeester score) and the weakest preoperative lower esophageal sphincter pressure. Postoperative symptom correlation was low among all 3 groups. There was no significant difference in postoperative 24-hour pH or manometry among groups. Success following surgery was achieved in 73.3% with supine reflux, 80.4% with upright reflux, and 75.0% with bipositional reflux. CONCLUSIONS: Patients with bipositional reflux have the most severe disease. Supine, upright, and bipositional reflux patients perform equally well following laparoscopic fundoplication as defined by objective outcome criteria.

Manometric abnormalities and gastroesophageal reflux disease in the morbidly obese.

BACKGROUND: Obesity is an epidemic in the USA. Many disorders are associated with obesity including gastroesophageal reflux disease (GERD). However, the prevalence of GERD and esophageal motility disorders in the morbidly obese population is unclear. METHODS: During evaluation for bariatric surgery, 61 morbidly obese patients underwent preoperative 24-hr pH and esophageal manometry. A single reviewer evaluated all 24-hr pH and manometric tracings. Johnson-DeMeester score >14.7 was considered diagnostic of GERD. Manometric criteria for motility disorders were from published values. All values are given as mean +/- SD. RESULTS: Mean age was 44.4 + 10.3 years. 55 of the patients (90%) were female. Mean BMI was 50.1 +/- 7.2 kg/m(2). 23 patients (38%) complained of GERD symptoms (reflux and/or heartburn). 1 patient (2%) complained of noncardiac chest pain. Mean Johnson-DeMeester score was 19.6 +/- 17.8. Mean intragastric and intrabolus pressures were both elevated (8.3 +/- 1.6 mmHg and 15 +/- 9 mmHg). 33 patients (54%) had abnormal manometric findings: 10 had a mechanically defective LES, 11 had a hypertensive LES, 2 had diffuse esophageal spasm, 3 had nutcracker esophagus,1 had ineffective esophageal disorder and 14 had nonspecific esophageal motility disorder. Some patients had more than one disorder. 20 patients (33%) had significantly elevated (>180 mmHg) contraction amplitudes at the most distal channel (210.0 +/- 28.7 mmHg). CONCLUSIONS: Prevalence of manometric abnormalities in the morbidly obese is high. Presence of a nut cracker-like distal esophagus in the morbidly obese is significant and warrants further evaluation.