Pregnancy outcomes in patients with systemic lupus erythematosus with or without a history of lupus nephritis.

BACKGROUND: Pregnancy is an important issue for many women with systemic lupus erythematosus (SLE). This study examined maternal and fetal outcomes among SLE women with or without a history of lupus nephritis (LN). METHODS: We retrospectively analyzed 98 pregnancies in 57 women previously diagnosed with SLE who gave birth at our hospital. RESULTS: There were 44 pregnancies in women with a history of LN and 54 pregnancies in those without. Fetal loss was observed in 16.1% of SLE pregnancies when excluding induced abortion, and preeclampsia and SLE flare were observed in 12.2 and 6.1% of SLE pregnancies, respectively. No significant differences were evident between women with or without LN in rate of fetal loss, preeclampsia or SLE flare. Women with a history of LN exhibited a significantly shorter duration of gestation (37.0 weeks vs. 38.4 weeks, P = 0.006) and lower birth weight (2484 g vs. 2746 g, P = 0.007) than those without LN. Multivariate analysis revealed glucocorticoid dose but not history of LN, as an independent risk factor for preterm delivery and low birth weight. CONCLUSION: This study was unable to conclude that a history of LN predicted pregnancy outcomes among SLE women. Instead, a higher dose of glucocorticoid at conception was unexpectedly associated with preterm delivery and low birth weight. Further studies are awaited to verify the relationship.

Analysis of the expression of candidate genes for type 1 diabetes susceptibility in T cells.

Type 1 diabetes is characterized by T-cell-mediated autoimmune destruction of pancreatic ß-cells. Currently, approximately 50 type 1 diabetes susceptibility genes or chromosomal regions have been identified. However, the functions of type 1 diabetes susceptibility genes in T cells are elusive. In this study, we evaluated the correlation between type 1 diabetes susceptibility genes and T-cell signaling. The expression levels of 22 candidate type 1 diabetes susceptibility genes in T cells from nonobese diabetic (NOD), control C57BL/6 (B6), and NOD-control F1 hybrid mice were analyzed in response to 2 key immunoregulatory cytokines: interleukin-2 (IL-2) and transforming growth factor ß (TGF-ß). Exogenous gene expression studies were also performed in EL4 and Jurkat E6.1 T-cell lines. Significant differences in the expression of Clec16a, Dlk1, Il2, Ptpn22, Rnls, and Zac1 (also known as Plagl1) were observed in T cells derived from the 3 strains of mice, and TGF-ß differentially influenced the expression of Ctla4, Foxp3, Il2, Ptpn22, Sh2b3, and Zac1. We found that TGF-ß induced Zac1 expression in both primary T cells and EL4 cells and that exogenous expression of Zac1 and ZAC1 in T-cell lines altered the expression of Il2 and DLK1, respectively. The results of our study indicate the possibility that additional genetic pathways underlying type 1 diabetes susceptibility, including those involving Clec16a, Dlk1, Rnls, Sh2b3, and Zac1 under IL-2 and TGF-ß signaling in T cells, may be shared between human and NOD mice.

Vitronectin regulates the axon specification of mouse cerebellar granule cell precursors via αvß5 integrin in the differentiation stage.

Vitronectin, an extracellular matrix protein, controls the differentiation of cerebellar granule cell precursors (CGCPs) via αvß5 integrin, particularly in the initial stage of differentiation to granule cells. In this study, we determined whether vitronectin regulates axon specification in this initial differentiation stage of CGCPs. First, we analyzed whether vitronectin deficiency, ß5 integrin knockdown (KD), and ß5 integrin overexpression affect axon specification of primary cultured CGCPs. Vitronectin deficiency and ß5 integrin KD inhibited axon formation, while vitronectin administrated- and ß5 integrin overexpressed-neurons formed multiple axons. Moreover, KD of ß5 integrin suppressed vitronectin-induced multiple axon formation. These findings indicate that vitronectin contributes to regulating axon specification via αvß5 integrin in CGCPs. Next, we determined the signaling pathway involved in regulating vitronectin-induced axon specification. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited vitronectin-induced multiple axon specification, and lithium chloride, an inhibitor of glyocogen synthase kinase 3 beta (GSK3ß), attenuated the inhibitory effect of vitronectin-KO and ß5 integrin KD on the specification of CGCPs. In addition, vitronectin induced the phosphorylation of protein kinase B (Akt) and GSK3ß in neuroblastoma Neuro2a cells. Taken together, our results indicate that vitronectin plays an important factor in axon formation process in CGCPs via a ß5 integrin/PI3K/GSK3ß pathway.

A case of a patient with neurofibromatosis type I who developed pneumothorax and eosinophilic pleural effusion after suffering from COVID-19 pneumonia.

Coronavirus disease 2019 (COVID-19) has become a global pandemic since its discovery in December 2019, and as the disease continues to evolve, varying complications associated with it continue to arise. In this regard, computed tomography has played an extremely important role in the diagnosis and evaluation of COVID-19 pneumonia and its complications. We encountered a case of a male patient with neurofibromatosis (type I) who developed concurrent pneumothorax and pleural effusion during his recovery period from severe COVID-19 pneumonia. Pulmonary fibrosis and emphysema were also confirmed. Furthermore, an eosinophil pleural effusion appeared and was prolonged during the healing process of COVID-19. This clinical presentation suggests that fibrosis and emphysema formation due to neurofibromatosis may have caused pneumothorax and pleural effusion.

First-in-human randomised trial and follow-up study of Plasmodium falciparum blood-stage malaria vaccine BK-SE36 with CpG-ODN(K3).

BACKGROUND: BK-SE36 is blood-stage malaria vaccine candidate that is undergoing clinical trials. Here, the safety and immunogenicity of BK-SE36 with a novel adjuvant, CpG-ODN(K3) (thus, BK-SE36/CpG) was assessed in a phase 1a trial in Japan. METHODS: An investigator-initiated, randomised, single-blind, placebo-controlled, dose-escalation study was conducted at Osaka University Hospital with 26 healthy malaria naïve Japanese male adults. The trial was conducted in two stages: Stage/Group 1, half-dose (n = 7 for BK-SE36/CpG and n = 3 for control) and Stage/Group 2, full-dose (n = 11 for BK-SE36/CpG and n = 5 for control). There were two intramuscular vaccinations 21 days apart for both half-dose (0.5 ml: 50 µg SE36 + 500 µg aluminum + 500 µg K3) and full-dose (1.0 ml: 100 µg SE36 + 1000 µg aluminum + 1000 µg K3). A one-year follow-up was done to monitor changes in autoimmune markers and vaccine-induced antibody response. RESULTS: BK-SE36/CpG was well tolerated. Vaccination site reactions were similar to those observed with BK-SE36. During the trial and follow-up period, no subject had clinical evidence of autoimmune disease. The full-dose group had significantly higher titres than the half-dose group (Student's t-test, p = 0.002) at 21 days post-second vaccination. Antibody titres remained above baseline values during 12 months of follow-up. The vaccine induced antibody was mostly composed of IgG1 and IgM, and recognised epitopes close to the polyserine region located in the middle of SE36. CONCLUSIONS: BK-SE36/CpG has an acceptable safety profile. Use of CpG-ODN(K3) greatly enhanced immunogenicity in malaria naïve Japanese adults when compared to BK-SE36 alone. The utility of BK-SE36/CpG is currently under evaluation in a malaria endemic setting in West Africa. TRIAL REGISTRATION: JMACCT Clinical Trial Registry JMA-IIA00109.

Promotion of differentiation in developing mouse cerebellar granule cells by a cell adhesion molecule BT-IgSF.

Brain- and testis-specific immunoglobulin superfamily (BT-IgSF) (also known as IgSF11), one of the immunoglobulin superfamily proteins, is a cell adhesion molecule, expressed in the developing cerebellum. We hypothesized that BT-IgSF might have some function in the development of cerebellum, although the physiological roles of BT-IgSF in the cerebellum remain unclear. To investigate the role of BT-IgSF in the development of mouse cerebellum, we first determined the presence of BT-IgSF in the newborn mouse cerebellum; its expression level was found to be much higher than that in the adults. BT-IgSF was abundantly expressed in the molecular layer, where cerebellar granule cell precursors (CGCPs) are in the differentiation stage during migration. We subsequently analyzed the effects of BT-IgSF-knockdown and -overexpression on the proliferation and differentiation of primary cultured CGCPs. BT-IgSF suppressed the proliferation of CGCPs, and promoted their differentiation into cerebellar granule cells. Taken together, our results suggested that BT-IgSF is one of the important cell adhesion molecules that regulate the developmentof mouse cerebellum.

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins.

In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.

Crystallization and preliminary X-ray crystallographic analysis of two vascular apoptosis-inducing proteins (VAPs) from Crotalus atrox venom.

VAPs are haemorrhagic snake-venom toxins belonging to the reprolysin family of zinc metalloproteinases. In vitro, VAPs induce apoptosis specifically in cultured vascular endothelial cells. VAPs have a modular structure that bears structural homology to mammalian ADAMs (a disintegrin and metalloproteinases). VAP1 is a homodimer with a MW of 110 kDa in which the monomers are connected by a single disulfide bridge. VAP2 is homologous to VAP1 and exists as a monomer with a MW of 55 kDa. In the current study, several crystal forms of VAP1 and VAP2 were obtained using the vapour-diffusion method and diffraction data sets were collected using SPring-8 beamlines. The best crystals of VAP1 and VAP2 generated data sets to 2.5 and 2.15 angstroms resolution, respectively.

In vitro selection of restriction endonucleases by in vitro compartmentalization.

Restriction endonucleases are widely used in laboratory applications from recombinant DNA technology to diagnostics, but engineering of restriction enzymes by structure-guided design and in vivo directed evolution is at an early stage. Here, we report the use of an in vitro compartmentalization system for completely in vitro selection of restriction enzymes. Compartmentalization of a single gene in a rabbit reticulocyte in vitro transcription/translation system serves to isolate individually synthesized enzymes from each other. In each compartment, an active enzyme cleaves only its own encoding gene, whereas genes encoding inactive enzymes remain intact. Affinity selection of the cleaved DNA encoding active restriction endonucleases was accomplished by the use of streptavidin-immobilized beads and dUTP-biotin, which was efficiently incorporated into the cohesive end of the cleaved DNA using a DNA polymerase. We confirmed that genes encoding active restriction endonuclease FokI could be selected from a randomized library. This method overcomes the limitations of current in vivo technologies and should prove useful for rapid screening and evolution of novel restriction enzymes from diverse mutant libraries, as well as for studies of catalytic and evolutionary mechanisms of restriction enzymes.

Antibody titres and boosting after natural malaria infection in BK-SE36 vaccine responders during a follow-up study in Uganda.

The malaria vaccine BK-SE36 is a recombinant protein (SE36) based on the Honduras 1 serine repeat antigen-5 of Plasmodium falciparum, adsorbed to aluminium hydroxide gel. The phase Ib trial in Uganda demonstrated the safety and immunogenicity of BK-SE36. Ancillary analysis in the follow-up study of 6-20 year-old volunteers suggest significant differences in time to first episodes of clinical malaria in vaccinees compared to placebo/control group. Here, we aimed to get further insights into the association of anti-SE36 antibody titres and natural P. falciparum infection. Children who received BK-SE36 and whose antibody titres against SE36 increased by ≥1.92-fold after vaccination were categorised as responders. Most responders did not have or only had a single episode of natural P. falciparum infection. Notably, responders who did not experience infection had relatively high anti-SE36 antibody titres post-second vaccination compared to those who were infected. The anti-SE36 antibody titres of the responders who experienced malaria were boosted after infection and they had lower risk of reinfection. These findings show that anti-SE36 antibody titres induced by BK-SE36 vaccination offered protection against malaria. The vaccine is now being evaluated in a phase Ib trial in children less than 5 years old.

High solubility of random-sequence proteins consisting of five kinds of primitive amino acids.

Searching for functional proteins among random-sequence libraries is a major challenge of protein engineering; the difficulties include the poor solubility of many random-sequence proteins. A library in which most of the polypeptides are soluble and stable would therefore be of great benefit. Although modern proteins consist of 20 amino acids, it has been suggested that early proteins evolved from a reduced alphabet. Here, we have constructed a library of random-sequence proteins consisting of only five amino acids, Ala, Gly, Val, Asp and Glu, which are believed to have been the most abundant in the prebiotic environment. Expression and characterization of arbitrarily chosen proteins in the library indicated that five-alphabet random-sequence proteins have higher solubility than do 20-alphabet random-sequence proteins with a similar level of hydrophobicity. The results support the reduced-alphabet hypothesis of the primordial genetic code and should also be helpful in constructing optimized protein libraries for evolutionary protein engineering.

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins.

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.