Category On the fate of primordial germ cells injected into early mouse embryos.

A novel isolator-based system promotes viability of human embryos during laboratory processing., On the fate of primordial germ cells injected into early mouse embryos.

Downregulated RRS1 inhibits invasion and metastasis of BT549 through RPL11‑c‑Myc‑SNAIL axis

by Yasmin
Regulator of ribosome synthesis 1 (RRS1) is a key factor in ribosome biosynthesis and other cellular functions. High level of RRS1 in breast cancer cell lines is associated with increased cell proliferation, invasion and migration. RRS1 controls the assembly of the 60s subunit and maturation of 25S rRNA during ribosome biosynthesis.
In this study, lentiviral transfection of sh‑RNA was used to knock down the level of RRS1, to detect the effect of RRS1 on cell function and to explore the specific mechanism of RRS1 affecting cell invasion and metastasis by COIP and dual‑luciferase reporter gene assays.
The present study found that RRS1 knockdown reduced the accumulation of ribosome protein L11 (RPL11) in the nucleolus, which then migrated to the nucleoplasm and bound to c‑Myc. This inhibited trans‑activation of SNAIL by c‑Myc and eventually decreased the invasion and metastasis capacity of the human breast cancer cell line BT549.
Taken together, RRS1 regulates invasion and metastasis of human breast cancer cells through the RPL11‑c‑Myc‑SNAIL axis. The findings are of great significance for exploring the mechanism of breast cancer invasion and metastasis and the corresponding regulatory factors.

Jumonji-C domain-containing protein 5 suppresses proliferation and aerobic glycolysis in pancreatic cancer cells in a c–Myc-dependent manner

Despite the importance of metabolic reprogramming in cancer cells, the molecular mechanism regulating the tumor metabolic shift is still poorly understood. Deregulation of Jumonji-C domain-containing protein 5 (JMJD5) has been associated with multiple facets of biological processes in cancer cells.
However, the role of JMJD5 in pancreatic cancer cells has seldom been discussed and requires further investigation. In the present study, by silencing or overexpressing JMJD5 in pancreatic cancer cells, we examined the impact of JMJD5 on cell proliferation and glucose metabolism. Using a dual luciferase assay, we assessed the effect of JMJD5 on the transcriptional activity of the c-Myc target gene.
Analyzing The Cancer Genome Atlas and the Gene Expression Omnibus datasets revealed that low JMJD5 expression was associated with poor prognosis in patients with pancreatic cancer.
JMJD5 loss promoted pancreatic cancer cell proliferation and induced a cellular metabolic shift from oxidative phosphorylation to glycolysis. In addition, in vivo experiments confirmed that ectopic JMJD5 expression inhibited cancer cell growth and the expression of glycolytic enzymes, such as lactate dehydrogenase and phosphoglycerate kinase 1.
Moreover, JMJD5 negatively regulated c-Myc expression, the main regulator of cancer metabolism, leading to decreased c-Myc-targeted gene expression. Overall, the present study indicated that decreased JMJD5 expression promoted cell proliferation and glycolytic metabolism in pancreatic cancer cells in a c-Myc-dependent manner.

Imaging-Based Screening of Deubiquitinating Proteases Identifies Otubain-1 as a Stabilizer of c–MYC

The ubiquitin-proteasome pathway precisely controls the turnover of transcription factors in the nucleus, playing an important role in maintaining appropriate quantities of these regulatory proteins. The transcription factor c-MYC is essential for normal development and is a critical cancer driver. Despite being highly expressed in several tissues and malignancies, the c-MYC protein is also continuously targeted by the ubiquitin-proteasome pathway, which can either facilitate or inhibit c-MYC degradation. Deubiquitinating proteases can remove ubiquitin chains from target proteins and rescue them from proteasomal digestion.
This study sought to determine novel elements of the ubiquitin-proteasome pathway that regulate c-MYC levels. We performed an overexpression screen with 41 human proteases to identify which deubiquitinases stabilize c-MYC. We discovered that the highly expressed Otubain-1 (OTUB1) protease increases c-MYC protein levels.
Confirming its role in enhancing c-MYC activity, we found that elevated OTUB1 correlates with inferior clinical outcomes in the c-MYC-dependent cancer multiple myeloma, and overexpression of OTUB1 accelerates the growth of myeloma cells. In summary, our study identifies OTUB1 as a novel amplifier of the proto-oncogene c-MYC.

Rational design of small-molecules to recognize G-quadruplexes of c–MYC promoter and telomere and the evaluation of their in vivo antitumor activity against breast cancer

DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging.
We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity.
The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures.
The ligand-G4 interaction studied with 1H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures.
Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.

c–Myc Targets HDAC3 to Suppress NKG2DL Expression and Innate Immune Response in N-Type SCLC through Histone Deacetylation

SCLC is an aggressive malignancy with a very poor prognosis and limited effective therapeutic options. Despite the high tumor mutational burden, responses to immunotherapy are rare in SCLC patients, which may be due to the lack of immune surveillance.
Here, we aimed to examine the role and mechanism of oncogene MYC in the regulation of NKG2DL, the most relevant NK-activating ligand in SCLC-N.
Western Blotting, Immunofluorescence, flow cytometry, quantitative real-time PCR (qRT-PCR), Co-Immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and Cytotoxicity assay were used on H2227 cells, H446 cells, and other SCLC cell lines, and we found that c-Myc negatively regulated NKG2DL expression in SCLC-N cells. Mechanistically, c-Myc recruited HDAC3 to deacetylate H3K9ac at the promoter regions of MICA and MICB, suppressing the MICA/B expression of SCLC-N cells and the cytotoxicity of NK cells.
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Treatment with selective HDAC3 inhibitor up-regulated the expression of NKG2DL on SCLC-N cells and increased the cytotoxicity of NK cells. Furthermore, analysis of the CCLE and Kaplan-Meier plotter data performed the negative correlation between MYC and NKG2DL in SCLC-N cells and the correlation with the prognosis of lung cancer patients.
Collectively, the results provided the new insight into the role and mechanism of c-Myc/HDAC3 axis in NKG2DL expression and innate immune escape of SCLC-N, suggesting the potential target for SCLC-N immunotherapy.
A novel isolator-based system promotes viability of human embryos during laboratory processing., in women undergoing in vitro fertilisation., On the fate of primordial germ cells injected into early mouse embryos.

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide

by Yasmin
DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8).
The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in the immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin.
We fused a 5′ terminal cDNA fragments for the Fab region of 2H8 mAb with 3′ terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flow cytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb.
Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.

Thoracic Society of Australia and New Zealand Position Statement on Acute Oxygen Use in Adults: ‘Swimming between the flags‘

Oxygen is a life-saving therapy but, when given inappropriately, may also be hazardous. Therefore, in the acute medical setting, oxygen should only be given as treatment for hypoxaemia and requires appropriate prescription, monitoring and review.
This update to the Thoracic Society of Australia and New Zealand (TSANZ) guidance on acute oxygen therapy is a brief and practical resource for all healthcare workers involved with administering oxygen therapy to adults in the acute medical setting.
It does not apply to intubated or paediatric patients. Recommendations are made in the following six clinical areas: assessment of hypoxaemia (including use of arterial blood gases); prescription of oxygen; peripheral oxygen saturation targets; delivery, including non-invasive ventilation and humidified high-flow nasal cannulae; the significance of high oxygen requirements; and acute hypercapnic respiratory failure.
There are three sections which provide (1) a brief summary, (2) recommendations in detail with practice points and (3) a detailed explanation of the reasoning and evidence behind the recommendations. It is anticipated that these recommendations will be disseminated widely in structured programmes across Australia and New Zealand.

Decitabine and Vorinostat with FLAG Chemotherapy in Pediatric Relapsed/Refractory AML: Report from the Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) Consortium

Survival outcomes for relapsed/refractory pediatric acute myeloid leukemia (R/R AML) remain dismal. Epigenetic changes can result in gene expression alterations which are thought to contribute to both leukemogenesis and chemotherapy resistance.
We report results from a phase I trial with a dose expansion cohort investigating decitabine and vorinostat in combination with fludarabine, cytarabine, and G-CSF (FLAG) in pediatric patients with R/R AML [NCT02412475]. Thirty-seven patients enrolled with a median age at enrollment of 8.4 (range, 1-20) years. There were no dose limiting toxicities among the enrolled patients, including two patients with Down syndrome. The recommended phase 2 dose of decitabine in combination with vorinostat and FLAG was 10 mg/m2 .
The expanded cohort design allowed for an efficacy evaluation and the overall response rate among 35 evaluable patients was 54% (16 complete response (CR) and 3 complete response with incomplete hematologic recovery (CRi)). Ninety percent of responders achieved minimal residual disease (MRD) negativity (<0.1%) by centralized flow cytometry and 84% (n=16) successfully proceeded to hematopoietic stem cell transplant.
Two-year overall survival was 75.6% [95%CI: 47.3%, 90.1%] for MRD-negative patients vs. 17.9% [95%CI: 4.4%, 38.8%] for those with residual disease (p<0.001). Twelve subjects (34%) had known epigenetic alterations with 8 (67%) achieving a CR, 7 (88%) of whom were MRD negative.
Correlative pharmacodynamics demonstrated biologic activity of decitabine and vorinostat and identified specific gene enrichment signatures in non-responding patients.
Overall, this therapy was well-tolerated, biologically active, and effective in pediatric patients with R/R AML, particularly those with epigenetic alterations. This article is protected by copyright. All rights reserved.

Dento-facial infections in children – A potential red flag for child neglect?

Background: Healthcare professionals are often confronted with children presenting to the emergency department with dento-facial infections. These infections may be associated with dental neglect and as such could be a marker for general neglect. The aim of this retrospective study was to ascertain whether dento-facial infections can be used as an indicator for general neglect.
Method: All children aged 16 years and under, who were admitted for surgical incision and drainage of dento-facial infection between January 2017 and January 2019 at King’s College Hospital were examined retrospectively. All patients were discussed with the local safeguarding team/local authority to establish whether they were previously known to social services.
Results: This study showed that in our cohort, 48% of children admitted with dento-facial infection were already known to social services and one (2%) had been recently referred. The most commonly affected age group were 5-8-year-olds (50%) indicating that these children have an increased risk of neglect. An average of 5.6 teeth were extracted and four (10%) patients required extra-oral drainage. The average hospital stay was 2.26 days.
Conclusion: Our retrospective study revealed that social services were already aware of 48% of patients under the age of 16, who were admitted to hospital with a dento-facial infection.
This suggests a relationship between dental neglect and generalised neglect. Families of children presenting with dento-facial infection should be supported in accessing appropriate dental services for their children and clinicians should consider dento-facial infection a potential ‘red flag’ for generalised neglect.
Keywords: Child neglect; Dental neglect; Dento-facial infections; Maxillofacial.

Reduced Expression of Emotion: A Red Flag Signalling Conversion to Psychosis in Clinical High Risk for Psychosis (CHR-P) Populations

Objective: In this hypothesis-testing study, which is based on findings from a previous atheoretical machine-learning study, we test the predictive power of baseline “reduced expression of emotion” for psychosis.Method: Study participants (N = 96, mean age 16.55 years) were recruited from the Prevention of Psychosis Study in Rogaland, Norway. The Structured Interview for Prodromal Syndromes (SIPS) was conducted 13 times over two years. Reduced expression of emotion was added to positive symptoms at baseline (P1-P5) as a predictor of psychosis onset over a two-year period using logistic regression.
Results: Participants with a score above zero on expression of emotion had over eight times the odds of conversion (OR = 8.69, p < .001).
Data indicated a significant dose-response association. A model including reduced expression of emotion at baseline together with the positive symptoms of the SIPS rendered the latter statistically insignificant.
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Conclusions: The study findings confirm findings from the previous machine-learning study, indicating that observing reduced expression of emotion may serve two purposes: first, it may add predictive value to psychosis conversion, and second, it is readily observable.
This may facilitate detection of those most at risk within the clinical high risk of psychosis population, as well as those at clinical high risk. A next step could be including this symptom within current high-risk criteria. Future research should consolidate these findings.
A novel isolator-based system promotes viability of human embryos during laboratory processing., in women undergoing in vitro fertilisation., On the fate of primordial germ cells injected into early mouse embryos.

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA

by Yasmin
Background: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA.
Methods: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels.
Results: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs.
Conclusions: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.
Keywords: Hexahistidine tag; Isothermal DNA amplification; Recombinase polymerase amplification (RPA); uvsY.

A Strategy to Fight against Triple-Negative Breast Cancer: pH-Responsive Hexahistidine-Metal Assemblies with High-Payload Drugs

Triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer, is difficult to be targeted therapeutically due to negative expression of the bioreceptor, which leads to the poorest overall four-year survival rate among all cancer subtypes.
We proposed that the nanomedicine featuring high payload and pH-responsive release of the loaded drugs could assist the TNBC treatment. In the present study, the His6-metal assemblies (HmA) were employed to encapsulate the doxorubicin (Dox), and the effect of HmA loaded with Dox (HmA@Dox) on treating TNBC was evaluated in vitro and in vivo.
We found that the participation of Dox in the formation of HmA leads to high loading efficiency (99.4% for concentration ≤ 1 mg/mL) and the loading capacity (50.7% for concentration ≥ 10 mg/mL) of Dox encapsulated into HmA. HmA@Dox exhibited a narrow size distribution on the nanoscale, a pH-responsive release of loaded Dox, a quick endocytosis process, and fast lysosome escape. Most importantly, the HmA@Dox showed high efficacy in killing various breast cancer cells (MCF-7, MDA-MB-231, and MDA-MB-453) in vitro and depressing the development of TNBC in vivo.
Our results demonstrated that such a strategy for designing a nanomedicine with high payload and responsive release of drugs to the environment around the tumor was of great importance to treat TNBC.

Efficient Delivery of Antibodies Intracellularly by Co-Assembly with Hexahistidine-Metal Assemblies (HmA)

Purpose: There has been a substantial global market for antibodies, which are based on extracellular targets. Binding intracellular targets by antibodies will bring new chances in antibody therapeutics and a huge market increase. We aim to evaluate the efficiency of a novel delivery system of His6-metal assembly (HmA) in delivering intracellular antibodies and biofunctions of delivered antibodies.
Methods: In this study, the physicochemical properties of HmA@Antibodies generated through co-assembling with antibodies and HmA were well characterized by dynamic light scatter. The cytotoxicity of HmA@Antibodies was investigated by Cell Counting Kit-8 (CCK-8). The endocytic kinetics and lysosome escape process of HmA@Antibodies were studied by flow cytometry and fluorescent staining imaging, respectively. Compared to the commercialized positive control, the intracellular delivery efficiency by HmA@Antibodies and biofunctions of delivered antibodies were evaluated by fluorescent imaging and CCK-8.
Results: Various antibodies (IgG, anti-β-tubulin and anti-NPC) could co-assemble with HmA under a gentle condition, producing nano-sized (~150 nm) and positively charged (~+30 eV) HmA@Antibodies particles with narrow size distribution (PDI ~ 0.15). HmA displayed very low cytotoxicity to divers cells (DCs, HeLa, HCECs, and HRPE) even after 96 h for the feeding concentration ≤100 μg mL-1, and fast escape from endosomes. In the case of delivery IgG, the delivery efficiency into alive cells of HmA was better than a commercial protein delivery reagent (PULSin).
For cases of the anti-β-tubulin and anti-NPC, HmA showed comparable delivery efficiency to their positive controls, but HmA with ability to deliver these antibodies into alive cells was still superior to positive controls delivering antibodies into dead cells through punching holes.
Conclusion: Our results indicate that this strategy is a feasible way to deliver various antibodies intracellularly while preserving their functions, which has great potential in various applications and treating many refractory diseases by intracellular antibody delivery.
Keywords: antibody; coordination polymer; intracellular delivery; nanocarrier; peptide assembly.

Efficient delivery of cytosolic proteins by protein-hexahistidine-metal co-assemblies

Proteins play key roles in most biological processes, and protein dysfunction can cause various diseases. Over the past few decades, tremendous development has occurred in the protein therapeutic market due to the high specificity, low side effects, and low risk of proteins.
Currently, all protein drugs on the market are based on extracellular targeting; more than 70% of intracellular targets remain un-druggable. Efficient delivery of cytosolic proteins is of significance for protein drugs, advanced biotechnology and molecular cell biology. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for intracellular protein delivery.
Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged protein encapsulation particles(Protein@HmA) through a co-assembly process with a high loading capacity and loading efficiency.
Protein@HmA was able to deliver proteins with diverse physicochemical properties through multiple endocytosis pathways, and the protein could quickly escape from endosomes.
In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells. Our results demonstrate that this strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics,
 STATEMENT OF SIGNIFICANCE: : Intracellular targets with protein drugs may provide a new way for the treatment of many refractory disease. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for efficient intracellular protein delivery.
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Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged particles (Protein@HmA) with a high loading efficiency.
Protein@HmA was able to deliver different proteins through multiple endocytosis pathways, and the protein could quickly escape from endosomes. In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells.
This strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics.
A novel isolator-based system promotes viability of human embryos during laboratory processing., On the fate of primordial germ cells injected into early mouse embryos.

Complement Inhibitors Vitronectin and Clusterin Are Recruited from Human Serum to the Surface of Coronavirus OC43-Infected Lung Cells through Antibody-Dependent Mechanisms

by Yasmin
Little is known about the role of complement (C’) in infections with highly prevalent circulating human coronaviruses such as OC43, a group of viruses of major public health concern. Treatment of OC43-infected human lung cells with human serum resulted in C3 deposition on their surfaces and generation of C5a, indicating robust C’ activation.
Real-time cell viability assays showed that in vitro C’-mediated lysis of OC43 infected cells requires C3, C5 and C6 but not C7, and was substantially delayed as compared to rapid C’-mediated killing of parainfluenza virus type 5 (PIV5)-infected cells.
In cells co-infected with OC43 and PIV5, C’-mediated lysis was delayed, similar to OC43 infected cells alone, suggesting that OC43 infection induced dominant inhibitory signals.
When OC43-infected cells were treated with human serum, their cell surfaces contained both Vitronectin (VN) and Clusterin (CLU), two host cell C’ inhibitors that can alter membrane attack complex (MAC) formation and C’-mediated killing.
VN and CLU were not bound to OC43-infected cells after treatment with antibody-depleted serum. Reconstitution experiments with purified IgG and VN showed that human antibodies are both necessary and sufficient for VN recruitment to OC43-infected lung cells-novel findings with implications for CoV pathogenesis.

Additivity in effects of vitronectin and monoclonal antibodies against alpha-helix F of plasminogen activator inhibitor-1 on its reactions with target proteinases.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators.
Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5.
Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2.
Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases.
The same step in the reaction mechanism remains to limit for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki).
These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.

Activated vitronectin as a target for anticancer therapy with human antibodies.

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites.
Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs).
In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin.
One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/kappa monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues.
Iodine 123-radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.

New insights into heparin binding to vitronectin: studies with monoclonal antibodies.

Vitronectin is a plasma glycoprotein that binds to a variety of ligands. There is considerable debate regarding the dependency of these binding interactions upon the conformational status of vitronectin, the role of multimerization and how the binding of different ligands can change vitronectin’s conformational state.
We have developed a method of capturing vitronectin directly from fresh plasma using solid-phase monoclonal antibodies. Various biotin-labelled secondary monoclonal antibodies were used to quantify the bound vitronectin and to measure its degree of denaturation.
Using these tools we demonstrated that one monoclonal antibody partially denatured vitronectin without direct multimerization.
Treatment of vitronectin in plasma with soluble heparin produced a similar degree of denaturation. These results led to a proposed adaptation of the unfolding/refolding pathways for chemically denatured vitronectin originally presented by Zhuang and co-workers in 1996 [Zhuang, Blackburn and Peterson (1996) J. Biol. Chem. 271, 14323-14332 and Zhuang, Li, Williams, Wagner, Seiffert and Peterson (1996) J. Biol. Chem. 271, 14333-14343]. The adapted version allows for the production of a more stable partially unfolded intermediate, resulting from the binding of particular ligands.
We also demonstrated that the avidity of heparin binding to vitronectin is governed by both the conformational state of the monomer and multimerization of the molecule.

Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding.

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central beta-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including alpha-helix F.
In order to elucidate this mechanism,
we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against alpha-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1).
One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1.
When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in alpha-helix F and the loop connecting alpha-helix F and beta-strand 3A as being important for the inhibitory function of PAI-1.
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Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators.
These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.
A novel isolator-based system promotes viability of human embryos during laboratory processing., On the fate of primordial germ cells injected into early mouse embryos.

Short-Term Therapy with Anti-ICAM-1 Monoclonal Antibody Induced Long-Term Liver Allograft Survival in Non-Human Primates

by Yasmin
Tolerance induction remains challenging following liver transplantation and the long-term use of immunosuppressants, especially calcineurin inhibitors, leads to serious complications.
We aimed to test an alternative immunosuppressant, a chimeric anti-ICAM-1 monoclonal antibody, MD-3, for improving outcomes of liver transplantation. We used a rhesus macaques liver transplantation model and monkeys were divided into three groups: no immunosuppression (n=2), conventional immunosuppression (n=4), and MD-3 (n=5).
Without immunosuppression, liver allografts failed within a week by acute rejection. Sixteen-week-long conventional immunosuppression that consisted of prednisolone, tacrolimus, and an mTOR inhibitor, prolonged liver allograft survival; however, recipients died of acute T cell-mediated rejection (day 52), chronic rejection (day 62, 66) or adverse effects of mTOR inhibitor (day 32).
In contrast, 12 weeks-long MD-3 therapy with transient conventional immunosuppression in the MD-3 group significantly prolonged the survival of liver allograft recipients (5, 96, 216, 412, 730 days; P = 0.0483). MD-3 effectively suppressed intragraft inflammatory cell infiltration, anti-donor T cell responses and donor-specific antibody with intact anti-cytomegalovirus antibody responses.
However, this regimen ended in chronic rejection. In conclusion, short-term therapy with MD-3 markedly improved liver allograft survival to 2 years without maintenance of immunosuppressant. MD-3 is therefore a promising immune-modulating agent for liver transplantation.

Fc-engineering significantly improves the recruitment of immune effector cells by anti-ICAM-1 antibody MSH-TP15 for myeloma therapy

Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet.
In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody’s modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2.
Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models – reflecting the importance of Fc-dependent mechanisms of action also in vivo.
The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.

Effective targeted therapy for drug-resistant infection by ICAM-1 antibody-conjugated TPGS modified β-Ga2O3:Cr3+ nanoparticles.

The prevalence of antibiotic resistance and lack of alternative drugs have posed an increasing threat to public health. Here, we prepared β-Ga2O3:Cr3+ nanoparticles modified with ICAM1-antibody-conjugated TPGS (I-TPGS/Ga2O3) as a novel antibiotic carrier for the treatment of drug-resistant infections.
 Methods: I-TPGS/Ga2O3 were firstly characterized by measuring particle size, morphology, crystal structure, drug loading capacity, and in vitro drug release behaviors. The in vitro antibacterial activities of I-TPGS/Ga2O3/TIG were evaluated using standard and drug-resistant bacteria. The internalization of I-TPGS/Ga2O3 was observed by fluorescence confocal imaging, and the expression levels of the efflux pump genes of TRKP were analyzed by real-time RT-PCR.
In vitro cellular uptake and in vivo biodistribution study were performed to investigate the targeting specificity of I-TPGS/Ga2O3 using HUEVC and acute pneumonia mice, respectively. The in vivo anti-infective efficacy and biosafety of I-TPGS/Ga2O3/TIG were finally evaluated using acute pneumonia mice.
 Results: It was found that TPGS could down-regulate the over-expression of the efflux pump genes, thus decreasing the efflux pump activity of bacteria. I-TPGS/Ga2O3 with small particle size and uniform distribution facilitated their internalization in bacteria, and the TPGS modification resulted in a significant reduction in the efflux of loaded antibiotics.
These properties rendered the encapsulated tigecycline to exert a stronger antibacterial activity both in vitro and in vivo. Additionally, targeted delivery of I-TPGS/Ga2O3 mediated by ICAM1 antibodies contributed to a safe and effective therapy.
 Conclusion: It is of great value to apply I-TPGS/Ga2O3 as a novel and effective antibiotic delivery system for the treatment of drug-resistant infections.
A novel isolator-based system promotes viability of human embryos during laboratory processing., On the fate of primordial germ cells injected into early mouse embryos.

Polyclonal and monoclonal antibodies against chicken gizzard 5‘-nucleotidase inhibit the spreading process of chicken embryonic fibroblasts on laminin substratum.

by Yasmin
Polyclonal and monoclonal antibodies raised against chicken gizzard 5′-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5′-nucleotidase.
However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard.
In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5′-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.

Clinical and immunological heterogeneity of canine subepidermal blistering dermatoses with anti-<em>laminin</em>-332 (<em>laminin</em>-<em>5</em>) auto-<em>antibodies</em>.

Laminin-332 (laminin-5) is a basement membrane heterotrimeric protein composed of alpha-3, beta-3 and gamma-2 laminin chains. Laminin-332 polypeptides are targeted by auto-antibodies in human patients with mucous membrane (cicatricial) pemphigoid or, more rarely, subepidermal vesicular diseases that resemble epidermolysis bullosa acquisita (EBA) or bullous pemphigoid (BP).
The objectives of this report were to characterize the clinical, histopathological and immunological characteristics of nine dogs with auto-antibodies targeting laminin-332. Immunological investigations consisted of direct immunofluorescence (IF), indirect IF with intact and salt-split canine gingival, and salt-split normal or laminin-332-deficient human skin, immunoblotting with purified human laminin-332 and immunoblotting with recombinant NC1 domain of human collagen VII.
All dogs exhibited varying degrees of skin blistering and ulceration associated with microscopic subepidermal vesiculation with or without inflammatory cells. Indirect IF established that circulating IgG auto-antibodies bound the dermal side of salt-split canine lip and human skin. In five dogs, IgG variably recognized the basement membrane of laminin-332-deficient human skin (three dogs negative, two dogs positive).
In all nine dogs, IgG auto-antibodies detected purified human laminin-332 by immunoblotting. In two dogs, additional targeting of collagen VII-NC1 was present.
These observations establish laminin-332 as a novel basement membrane antigen in dogs with autoimmune blistering diseases with variable clinical phenotypes. The names ‘acquired junctional epidermolysis bullosa’, ‘anti-laminin-332 mucous membrane pemphigoid (MMP)’ and ‘mixed auto-immune subepidermal blistering dermatosis’ are proposed for dogs with clinical signs reminiscent of EBA, MMP or BP respectively.

Bullous pemphigoid positive for anti-BP180 and anti-<em>laminin</em> <em>5</em> <em>antibodies</em> in a patient with graft-vs-host disease.

We report the case of a 55-year-old female with bullous pemphigoid (BP) who was positive for anti-BP180 and anti-laminin 5 antibodies after development of graft-vs-host disease (GVHD) caused by a bone marrow transplant. She had tense blisters on her trunk and extremities.
Histologic examination showed a subepidermal blister and marked lymphocytic infiltration, especially eosinophils. Direct immunofluorescence revealed a linear deposition of IgG on the base membrane zone. Indirect immunofluorescence on 1M NaCl split skin revealed a linear IgG deposition to both sides of the epidermal and the dermal layers.
Immunoblot assays using human epidermal extracts and BP180 NC16a domain recombinant protein confirmed the presence of IgG antibodies against BP180 and recombinant BP180 NC16a domain protein. Furthermore, immunoblotting using laminin 5 purified from human keratinocyte extract as the substrate demonstrated reactivity against the gamma2 and beta3 subunits but not the alpha3 subunit of laminin 5.
We diagnosed BP and treated her with prednisolone (40 mg/day). Both skin and oral lesions resolved without leaving scars on the bulla. Immune disturbance as well as destruction of basal epidermal cells and base membrane by GVHD may result in the induction of autoimmune blistering diseases with unusual clinical and laboratory manifestations.

Ocular ‘non-scarring’ mucous membrane pemphigoid associated with anti-<em>laminin</em>-<em>5</em> <em>antibodies</em>.

Mucous membrane pemphigoid is a rare, chronic autoimmune disease characterized by subepidermal blistering and scarring, predominantly affecting mucous membranes. Ocular involvement frequently occurs and often represents the only manifestation of the disease.
We describe a 62-year-old woman with a bilateral 18-month duration of conjunctival hyperaemia, associated with erythema and oedema of the eyelids, lacking any typical ocular signs of mucous membrane pemphigoid such as sub-conjuctival fibrosis and scarring. Histology was not significant.
Direct immunofluorescence of the conjunctiva showed IgG, IgA and complement deposition along the basement membrane zone. Immunoprecipitation analysis of affinity purified laminin-5 revealed a band consistent with the beta3 chain of laminin-5. This represents the first case of pure ocular mucous membrane pemphigoid associated with anti-laminin-5 antibodies.

Detection of <em>laminin</em> <em>5</em>-specific auto-<em>antibodies</em> in mucous membrane and bullous pemphigoid sera by ELISA.

Mucous membrane pemphigoid (MMP) is an autoimmune bullous disease that primarily affects mucous membranes leading to a scarring phenotype. MMP patients produce auto-antibodies (auto-ab) that preferentially recognize two components of the dermoepidermal basement membrane zone (BMZ): bullous pemphigoid (BP)180 and laminin 5 (LN5). Since detection of disease-specific auto-ab may be critical for the diagnosis of MMP, we developed an ELISA with affinity-purified native human LN5. A total of 24 MMP, 72 BP, and 51 control sera were analyzed for LN5-specific auto-ab: 18/24 (75.0%) MMP and 29/72 (40.3%) BP sera were LN5 reactive.
Sensitivity and specificity of the LN5 ELISA for MMP were 75% and 84.3%, respectively, and 40.3% and 88.2% for BP, respectively.
The LN5 ELISA was more sensitive than a dot blot assay with native LN5, which detected LN5-reactive IgG in 14/24 (58.3%) MMP and 16/72 (22.2%) BP sera. In MMP, but not BP, levels of LN5-reactive IgG correlated with disease severity. Furthermore, IgG reactivity to LN5 of the MMP and BP sera was not significantly associated with IgG reactivity against other autoantigens of the BMZ, such as BP180 or BP230. Thus, the established LN5 ELISA holds great promise as a novel diagnostic and prognostic parameter for MMP.

<em>Antibody</em>-induced activation of beta1 integrin receptors stimulates cAMP-dependent migration of breast cells on <em>laminin</em>-<em>5</em>.

The beta1 integrin-stimulating antibody TS2/16 induces cAMP-dependent migration of MCF-10A breast cells on the extracellular matrix protein laminin-5. TS2/16 stimulates a rise in intracellular cAMP within 20 min after plating. Pertussis toxin, which inhibits both antibody-induced migration and cAMP accumulation, targets the Galphai3 subunit of heterotrimeric G proteins in these cells, suggesting that Galphai3 may link integrin activation and migration via a cAMP signaling pathway.

Anti-epiligrin cicatricial pemphigoid with <em>antibodies</em> against the gamma2 subunit of <em>laminin</em> <em>5</em>.

BACKGROUND
Cicatricial pemphigoid (CP) is a scarring subepithelial mucocutaneous blistering disease characterized by anti-basement membrane zone autoantibodies. Anti-epiligrin CP is an uncommon variant that has been recently characterized. Severe laryngeal involvement is infrequently observed in all forms of CP and has been documented in only 2 patients with anti-epiligrin CP.
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METHODS
We report a case of CP exhibiting extensive laryngeal and ocular involvement. Histological, immunofluorescence, and immunoprecipitation studies confirmed the diagnosis of anti-epiligrin CP. Immunoblotting studies demonstrated the presence of antibodies against the alpha3 and the gamma2 subunit of laminin 5.
CONCLUSIONS
This article expands the diversity of the clinical and immunopathologic features of this newly characterized variant of CP.
On the fate of primordial germ cells injected into early mouse embryos.
On the fate of primordial germ cells injected into early mouse embryos.

On the fate of primordial germ cells injected into early mouse embryos.

by Yasmin

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a brand new embryo in the subsequent technology. PGCs are additionally the cell of origin of multilineage teratocarcinomas.

In vitro, mouse PGCs can provide rise to embryonic germ (EG) cells – pluripotent stem cells that may contribute to main chimaeras when launched into pre-implantation embryos. Thus, PGCs can provide rise to pluripotent cells in the course of the developmental cycle, throughout teratocarcinogenesis and by in vitro tradition. However, there isn’t any proof that PGCs can differentiate instantly into somatic cell varieties.

Furthermore, it’s typically assumed that PGCs don’t contribute to chimaeras following injection into the early mouse embryo. However, these knowledge have by no means been formally revealed. Here, we current the main knowledge from the unique PGC-injection experiments carried out 40 years in the past, alongside outcomes from newer research in three separate laboratories.

These outcomes have knowledgeable and influenced present fashions of the relationship between pluripotency and the germline cycle. Current applied sciences enable additional experiments to substantiate and develop upon these findings and permit definitive conclusions as to the developmental efficiency of PGCs.

On the fate of primordial germ cells injected into early mouse embryos.

On the fate of primordial germ cells injected into early mouse embryos.

MC Chang–Reproductive biologist of distinction 1908-1991.

This article evaluations the outstanding life and main scientific achievements of the reproductive biologist M.C. Chang. His scholarly profession progressed from college in Peking, through Edinburgh, Scotland, and Cambridge, England, to the newly based Worcester Foundation for Experimental Biology in Massachusetts. At every stage, the hand of fate is famous as are the help and encouragement of key professors.

Chang’s personal contributions on capacitation of spermatozoa, in vitro fertilisation of mammalian eggs, and transplantation of oocytes and embryos are all introduced out, as is his important enter to the creation and improvement of a steroid contraceptive capsule.

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He strongly inspired younger reproductive biologists who labored in his laboratory, and applauded the world-wide distinction of his scholar and affiliate, R. Yanagimachi, as a specialist in mammalian fertilisation. Finally, Chang’s continued emotions in the direction of his homeland are contrasted with the actuality of his American life after 1945, itself a examine in poignancy.