Downregulation of SPARC Expression Enhances the Fusion of BeWo Choriocarcinoma Cells.

Syncytiotrophoblasts, which are formed by the fusion of villous cytotrophoblasts, play an essential role in maintaining a successful pregnancy. Secreted protein acidic and rich in cysteine (SPARC) is a non-structural Ca2+-binding extracellular matrix glycoprotein involved in tissue remodeling and cell proliferation, differentiation, and migration. Previous studies have revealed that SPARC is expressed in villous and extravillous cytotrophoblasts in the first trimester and that RNA interference targeted at SPARC significantly inhibited invasion of human extravillous trophoblast HTR8/SVneo cells. However, the involvement of SPARC in cytotrophoblast fusion remains unknown. This study aimed to investigate the role of SPARC in cytotrophoblast fusion, using the BeWo choriocarcinoma cell line as a model of villous cytotrophoblasts. Immunohistochemical analysis was conducted to assess SPARC expression in normal human placentas using placental tissues obtained during the first and third trimesters of pregnancy. We investigated the effects of SPARC knockdown on trophoblast differentiation markers and cell fusion in BeWo cells using small interfering RNA. Immunohistochemical analysis revealed that SPARC expression was high in the early gestational chorionic villi and low in the late gestational chorionic villi. SPARC knockdown increased the expressions of human chorionic gonadotropin and Ovo-like transcriptional repressor 1; however, glial cells missing transcription factor 1, syncytin-1, and syncytin-2 showed no significant changes. The assessment revealed that SPARC knockdown significantly enhanced cell fusion compared to the non-silencing control. Our data suggest that SPARC plays a vital role in regulating trophoblast fusion and differentiation during placental development.

Immunomics-Guided Antigen Discovery for Praziquantel-Induced Vaccination in Urogenital Human Schistosomiasis.

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P<0.0001) and intestinal egg burdens (50-54%, P<0.0003) were achieved in mice vaccinated with cystatin in two independent trials. This study has revealed numerous antigens that are targets of DIV antibodies in urogenital schistosomiasis and offer promise as subunit vaccine targets for a drug-linked vaccination approach to controlling schistosomiasis.

Factors Associated with Medication Non-Adherence among Patients with Lifestyle-Related Non-Communicable Diseases.

This cross-sectional study explored the association between medication non-adherence and its factors in patients with non-communicable diseases (NCDs) using an online structured questionnaire emailed to 30,000 people (aged over 20 years who lived in Japan at the time of the survey). The questions concerned respondents' characteristics, medication non-adherence, health beliefs, lifestyles, and trouble taking medication. Factors related to non-adherence were analyzed among patients with lifestyle-related NCDs categorized into two age groups: 20-59, and >60 years. Unintentional (p < 0.001) and intentional (p < 0.001) non-adherence were more common among patients aged 20-59 than in older adults. NCD patients aged 20-59 experienced significantly more trouble taking medication than older adults. Multiple regression analysis showed that for patients aged 20-59 with NCDs, unintentional non-adherence was significantly and positively associated with current smoking habits (ß = 0.280, p < 0.001), while intentional non-adherence was significantly and positively associated with alcohol consumption (ß = 0.147, p = 0.020) and current smoking habits (ß = 0.172, p = 0.007). In patients aged 20-59, unhealthy eating habits (ß = -0.136, p = 0.034) and lack of exercise (ß = -0.151, p = 0.020) were negatively associated with intentional non-adherence. In conclusion, factors affecting medication non-adherence in patients with lifestyle-related diseases are related to health awareness, lifestyle, and medication barriers.

Distinct SARS-CoV-2 antibody reactivity patterns in coronavirus convalescent plasma revealed by a coronavirus antigen microarray.

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.

A novel translation inhibitor, mersicarpine, inhibits S-phase progression and induces apoptosis in HL60 cells.

Mersicarpine is an aspidosperma alkaloid isolated from the Kopsia genus of plants. Its intriguing structural features have attracted much attention in synthetic organic chemistry, but no biological activity has been reported. Here, we report the effects of mersicarpine on human leukemia cell line HL60. At concentrations above 30 µm, mersicarpine reversibly arrested cell cycle progression in S-phase. At higher concentrations, it induced not only production of reactive oxygen species, but also apoptosis. Macromolecular synthesis assay revealed that mersicarpine specifically inhibits protein synthesis. These results suggest that mersicarpine is a novel translation inhibitor that induces apoptosis.

What ATP binding does to the Ca2+ pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2.

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+ transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+ binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

A Case of Uterine Cervical Adenocarcinoma in Which Initial Total Laparoscopic Hysterectomy Was Performed for Suspected Atypical Endometrial Hyperplasia.

The patient was a 69-year-old multiparous female (gravida/para, 3/3) who had hypertension and arrhythmia. Her history included cerebral infarction treated with conservative therapy. She visited our hospital for atypical genital bleeding. She was diagnosed with atypical glandular cells (AGC) based on cervical cytology, atypical cells in endometrial cytology, and atypical endometrial hyperplasia on preoperative endometrial biopsy, and underwent total laparoscopic hysterectomy. However, in a postoperative pathologic examination, she was diagnosed with stage IB1 cervical adenocarcinoma without endometrial abnormality. AGC appeared in cervical cytology before surgery, but a surgical plan was not made with consideration of cervical adenocarcinoma.

The Influence of B Cell Depletion Therapy on Naturally Acquired Immunity to Streptococcus pneumoniae.

The anti-CD20 antibody Rituximab to deplete CD20+ B cells is an effective treatment for rheumatoid arthritis and B cell malignancies, but is associated with an increased incidence of respiratory infections. Using mouse models we have investigated the consequences of B cell depletion on natural and acquired humoral immunity to Streptococcus pneumoniae. B cell depletion of naïve C57Bl/6 mice reduced natural IgM recognition of S. pneumoniae, but did not increase susceptibility to S. pneumoniae pneumonia. ELISA and flow cytometry assays demonstrated significantly reduced IgG and IgM recognition of S. pneumoniae in sera from mice treated with B cell depletion prior to S. pneumoniae nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted S. pneumoniae colonized mice were still partially protected against both lung infection and septicemia when challenged with S. pneumoniae after reconstitution of their B cells. These data indicate that although B cell depletion markedly impairs antibody recognition of S. pneumoniae in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity.

Acquired Factor XI Deficiency with Lupus Anticoagulant in a Pregnant Woman Diagnosed by the Eruptions and Pain in Fingers.

We report a case of acquired factor XI deficiency with lupus anticoagulant (LA) in a 28-year-old primigravida who presented with finger pain and eruptions on her palms and fingers during the 3rd trimester of pregnancy. The patient complained of pain and reddening of the fingers at 30 weeks of gestation. She was referred to our tertiary center with a diagnosis of preeclampsia and suspected collagen disease at 35 weeks of gestation. Erythema was seen on the fingers and palms, and she presented with pain and cryesthesia on the fingers. Laboratory investigations revealed an activated partial thromboplastin time of 51 s (normal, 23-40 s), although it was normal during the 30th and 34th gestational weeks, LA with an anticardiolipin-beta2-glycoprotein I complex antibody, and low level of clotting XI activity (25 U/mL). On week 37 day 0 of gestation, the patient presented with severe hypertension. An urgent Cesarean section was performed after transfusion of two units of fresh frozen plasma. There was no excessive bleeding during the surgery or the postpartum period. The symptoms on her fingers and palms gradually improved after surgery. Our case indicates that dermatoses of pregnancy may become a starting point for the diagnosis of autoimmune diseases and coagulation abnormalities. When a patient presents with an atypical symptom, as in our case, the possibility of various diseases should be considered.

A Case of Recurrent Peritoneal Cancer in Which an Antitumor Effect Was Obtained Using a Combination of Etoposide and a Chinese Herbal Medicine with Maintenance of Daily Life.

The patient was a 50-year-old multiparous female (gravida/para 4/2) who had divorced. She was followed up for 1 year and 5 months after completion of initial treatment for peritoneal cancer (preoperative chemotherapy + optimal surgery + chemotherapy). A gradual increase in the tumor marker CA125 occurred, and computed tomography and ultrasonography showed bilateral neck, left supraclavicular and right axillary lymphadenopathy. The patient wanted to continue her job. Therefore, she was treated with etoposide (25 mg) daily for 3 weeks and TJ-48 (juzen-taihoto, 7.5 g) daily for 4 weeks, and then followed up. After two weeks, swelling of lymph nodes had been reduced or eliminated and tumor marker CA125 was negative. The only adverse reaction was slight numbness and the patient continued to work while receiving the same drugs orally for 2 years and 8 months without any symptoms or recurrence. This case shows that a combination of etoposide and TJ-48 has an antitumor effect on recurrent progressive peritoneal cancer while allowing a patient to work and have a normal daily life.

Recurrent Cervical Cancer with Intestinal Perforation That Was Related to Bevacizumab after Long-term NSAIDs Administration and Was Treated with Laparoscopy-assisted Anastomosis.

Bevacizumab is an effective drug for recurrent/advanced cervical cancer. A 59-year-old patient diagnosed with FIGO stage I B2 squamous cell carcinoma of the cervix at our hospital was treated with concurrent chemoradiotherapy as initial treatment. The outcome was judged as close to CR. Local recurrence in the irradiation field and paraaortic lymph node metastasis were noted 2 months after completion of this treatment. Chemotherapy of bevacizumab combined with paclitaxel plus carboplatin (TC) was initiated for recurrent cervical cancer. At 17 days after the 4th cycle, abdominal pain suddenly developed, and a close examination detected free air on abdominal CT, based on which intestinal perforation was diagnosed. Laparoscopic surgery performed to investigate the intraabdominal cavity showed that the small intestine was perforated at 2 sites. These were treated with laparoscopy-assisted partial resection of the small intestine and functional end-to-end anastomosis. Drug therapy for the recurrent cervical cancer was considered, but the primary disease rapidly aggravated and the patient died of the primary disease 11 months after completion of the initial treatment.

Serologic responses to the PfEMP1 DBL-CIDR head structure may be a better indicator of malaria exposure than those to the DBL-α tag.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). METHODS: A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. RESULTS: Malian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. CONCLUSIONS: Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

Antibodies to Peptides in Semiconserved Domains of RIFINs and STEVORs Correlate with Malaria Exposure.

The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria.IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Transient Cannabinoid Receptor 2 Blockade during Immunization Heightens Intensity and Breadth of Antigen-specific Antibody Responses in Young and Aged mice.

The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.

Effect of genistein and daidzein on the proliferation and differentiation of human preadipocyte cell line.

Isoflavones are known to have several biological activities, including a hypolipidemic effect. However, the mechanism of the lipid lowering effect of genistein remains to be elucidated. There is conflicting evidence on the effect of genistein for the deposition of adipocyte tissues. We examined the effect of the isoflavones on the growth and differentiation of human preadipocyte cells, AML-I. Growth arrest accompanied by the appearance of characteristics of apoptosis was observed by genistein or daidzein treatment under the adipogenic stimulation. The expressions of apoptosis-related proteins, Bad, Akt, and p-Akt, were modulated in the genistein-treated cells by Western blot analysis. On the other hand, exposure of AML-I to the isoflavones increased accumulation of cytoplasmic lipid droplets. Actually, the cytoplasmic expressions of fatty acid synthase (FAS) and peroxisome proliferator-activated receptor (PPAR)-gamma were increased in the genistein-treated cells. Glycosylated forms of the isoflavones genistein and puerarin did not have such activities. These results suggested that only aglycon forms of isoflavones induced not only apoptosis but also lipogenesis in the preadipocyte cell line AML-I. The possible mechanism of these phenomena has been discussed in the text.

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity.

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.

Role of regucalcin as an activator of sarcoplasmic reticulum Ca2+-ATPase activity in rat heart muscle.

The expression of regucalcin, a regulatory protein of Ca(2+) signaling, and its effect on Ca(2+) pump activity in the microsomes (sarcoplasmic reticulum) of rat heart muscle was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in heart muscle using rat regucalcin-specific primers. Results with Western blot analysis showed that regucalcin protein was present in the cytoplasm, although it was not detected in the microsomes. Microsomal Ca(2+)-ATPase activity was significantly increased in the presence of regucalcin (10(-10)-10(-8) M) in the enzyme reaction mixture. This increase was not seen in the presence of thapsigargin (TP) (10(-5) M), a specific inhibitor of the microsomal Ca(2+) pump enzyme. Regucalcin (10(-10)-10(-8) M) significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. The effect of regucalcin (10(-8) M) in increasing microsomal Ca(2+)-ATPase activity was completely prevented in the presence of digitonin (10(-3) or 10(-2)%), which has a solubilizing effect on membranous lipid, or N-ethylmaleimide (NEM), a modifying reagent of sulfhydryl (SH) groups. Dithiothreitol (DTT; 5 mM), a protecting reagent of SH groups, increased markedly Ca(2+)-ATPase activity. In the presence of DTT (5 mM), regucalcin could not significantly enhance the enzyme activity. Also, the effect of regucalcin in increasing Ca(2+)-ATPase activity was completely inhibited by the addition of vanadate (1 mM), an inhibitor of phosphorylation of enzyme. In addition, the effect of regucalcin on Ca(2+)-ATPase activity was not significantly modulated in the presence of dibutyryl cyclic AMP (10(-4) M), inositol 1,4,5-trisphosphate (10(-3) M), or calmodulin (5 microg/ml) which is an intracellular signaling factor. The present study demonstrates that regucalcin can activate Ca(2+) pump activity in rat heart microsomes, and that the protein may act the SH groups of Ca(2+)-ATPase by binding to microsomal membranes.