Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

Hunner-Type (Classic) Interstitial Cystitis: A Distinct Inflammatory Disorder Characterized by Pancystitis,with Frequent Expansion of Clonal B-Cells and Epithelial Denudation

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm²) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.     

IMD-4690, a Novel Specific Inhibitor for Plasminogen Activator Inhibitor-1, Reduces Allergic Airway Remodeling in a Mouse Model of Chronic Asthma via Regulating Angiogenesis and Remodeling-Related Mediators

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.     

The Association of Elastin Gene Variants with Two Angiographic Subtypes of Polypoidal Choroidal Vasculopathy

Objective

To compare the association of elastin (ELN) gene variants between two different angiographic phenotypes of polypoidal choroidal vasculopathy (PCV).

Methods

We included 411 treatment-naïve PCV patients and 350 controls in the present study. PCV was classified into two phenotypes (152 Type 1 and 259 Type 2) according to the presence or absence of feeding vessels found in indocyanine-green angiography. Single nucleotide polymorphisms (SNPs) in the ELN region including rs868005, rs884843, rs2301995, rs13239907 and rs2856728 were genotyped using TaqMan Genotyping Assays.

Results

In the allelic association analyses, rs868005 showed the strongest association with Type 2 PCV (allelic odds ratio 1.56; p = 7.4x10^-6), while no SNP was significantly associated with Type 1 PCV. Genotype association analyses revealed the significant association of rs868005 with Type 2 PCV in log additive model and predominant model (odds ratio 1.75; p = 1.5x10^-6 and odds ratio 1.60; p = 0.0044, respectively), but not with Type 1 PCV. These findings were further corroborated by another control group in the literature.

Conclusions

There may be significantly different associations in genetic variants of elastin between two angiographic phenotypes of PCV.     

A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXβ, and ATXγ) and Novel Autotaxins (ATXδ and ATXε)

Background

Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXβ, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated.

Methods

Anti-ATXβ and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXβ and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of “classical ATX” (ATXα, ATXβ, and ATXγ) and “novel ATX” (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples.

Results

The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups.

Conclusions

We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.     

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle

Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.     

Spatially Extensive Standardized Surveys Reveal Widespread,Multi-Decadal Increase in East Antarctic Adélie Penguin Populations

Seabirds are considered to be useful and practical indicators of the state of marine ecosystems because they integrate across changes in the lower trophic levels and the physical environment. Signals from this key group of species can indicate broad scale impacts or response to environmental change. Recent studies of penguin populations, the most commonly abundant Antarctic seabirds in the west Antarctic Peninsula and western Ross Sea, have demonstrated that physical changes in Antarctic marine environments have profound effects on biota at high trophic levels. Large populations of the circumpolar-breeding Adélie penguin occur in East Antarctica, but direct, standardized population data across much of this vast coastline have been more limited than in other Antarctic regions. We combine extensive new population survey data, new population estimation methods, and re-interpreted historical survey data to assess decadal-scale change in East Antarctic Adélie penguin breeding populations. We show that, in contrast to the west Antarctic Peninsula and western Ross Sea where breeding populations have decreased or shown variable trends over the last 30 years, East Antarctic regional populations have almost doubled in abundance since the 1980’s and have been increasing since the earliest counts in the 1960’s. The population changes are associated with five-year lagged changes in the physical environment, suggesting that the changing environment impacts primarily on the pre-breeding age classes. East Antarctic marine ecosystems have been subject to a number of changes over the last 50 years which may have influenced Adélie penguin population growth, including decadal-scale climate variation, an inferred mid-20th century sea-ice contraction, and early-to-mid 20^th century exploitation of fish and whale populations.     

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.