Assessment of G Protein-Coupled Oestrogen Receptor Expression in Normal and Neoplastic Human Tissues Using a Novel Rabbit Monoclonal Antibody

In addition to the classical oestrogen receptors, ERα and ERβ, a G protein-coupled oestrogen receptor (GPER) has been identified that primarily mediates the rapid, non-genomic signalling of oestrogens. Data on GPER expression at the protein level are contradictory; therefore, the present study was conducted to re-evaluate GPER expression by immunohistochemistry to obtain broad GPER expression profiles in human non-neoplastic and neoplastic tissues, especially those not investigated in this respect so far. We developed and thoroughly characterised a novel rabbit monoclonal anti-human GPER antibody, 20H15L21, using Western blot analyses and immunocytochemistry. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded human tissue samples. In normal tissue, GPER was identified in distinct cell populations of the cortex and the anterior pituitary; islets and pancreatic ducts; fundic glands of the stomach; the epithelium of the duodenum and gallbladder; hepatocytes; proximal tubules of the kidney; the adrenal medulla; and syncytiotrophoblasts and decidua cells of the placenta. GPER was also expressed in hepatocellular, pancreatic, renal, and endometrial cancers, pancreatic neuroendocrine tumours, and pheochromocytomas. The novel antibody 20H15L21 will serve as a valuable tool for basic research and the identification of GPER-expressing tumours during histopathological examinations.     

Immunohistochemical Evaluation of Adaptor Protein FAM159B Expression in Normal and Neoplastic Human Tissues

FAM159B is a so-called adaptor protein. These proteins are essential components in numerous cell signalling pathways. However, little is known regarding FAM159B expression in normal and neoplastic human tissues. The commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 was initially characterised for its specificity using Western blot analyses and immunocytochemistry and then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Confirmation of FAM159B’s predicted size and antibody specificity was achieved in BON-1 cells, a neuroendocrine tumour cell line endogenously expressing FAM159B, using targeted siRNA. Immunocytochemical experiments additionally revealed cytoplasmic expression of the adaptor protein. Immunohistochemical staining detected FAM159B expression in neuronal and neuroendocrine tissues such as the cortex, the trigeminal ganglia, dorsal root and intestinal ganglia, the pancreatic islets and the neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, but also in the syncytiotrophoblasts of the placenta. FAM159B was also expressed in many of the 28 tumour entities investigated, with high levels in medullary and anaplastic thyroid carcinomas, parathyroid adenomas, lung and ovarian carcinomas, lymphomas and neuroendocrine tumours of different origins. The antibody HPA011778 can act as a useful tool for basic research and identifying FAM159B expression in tissue samples.     

Recombinant Human Thymosin β4 (rhTβ4) Modulates the Anti-Inflammatory Responses to Alleviate Benzalkonium Chloride (BAC)-Induced Dry Eye Disease

Dry eye disease (DED) is a multifactorial ocular disorder that interferes with daily living and reduces quality of life. However, there is no most ideal therapeutic treatment to address all the deleterious defects of DED. The purpose of this study was to investigate the ability of recombinant human thymosin β4 (rhTβ4) to promote healing in a benzalkonium chloride (BAC)-induced mice DED model and the anti-inflammatory effects involved in that process. Eye drops consisting of 0.05% and 0.1% rhTβ4 were used for treatment of DED. Tear volume and corneal staining scores were measured after 7 days. Periodic acid-Schiff staining for gobleT cells in conjunctiva, immunohistochemical staining for CD4⁺ T cells, TUNEL assay for apoptotic positive cells in cornea and conjunctiva, qRT-PCR and ELISA assays for multiple cytokines were performed. All clinical parameters showed improvement in both the 0.05% and 0.1% rhTβ4 groups. Specifically, topical application of rhTβ4 significantly increased conjunctival gobleT cells and reduced apoptotic cells in conjunctiva. Mechanically, the rhTβ4 groups showed significantly reduced inflammatory cytokine levels and CD4⁺ T cells in conjunctiva by blocking NF-κB (nuclear factor kappa B) activation, suggesting that 0.05–0.1% rhTβ4 eye drops may be used as a potential therapeutic treatment for DED.     

Interleukin-4 Aggravates LPS-Induced Striatal Neurodegeneration In Vivo via Oxidative Stress and Polarization of Microglia/Macrophages

The present study investigated the effects of interleukin (IL)-4 on striatal neurons in lipopolysaccharide (LPS)-injected rat striatum in vivo. Either LPS or PBS as a control was unilaterally injected into the striatum, and brain tissues were processed for immunohistochemical and Nissl staining or for hydroethidine histochemistry at the indicated time points after LPS injection. Analysis by NeuN and Nissl immunohistochemical staining showed a significant loss of striatal neurons at 1, 3, and 7 days post LPS. In parallel, IL-4 immunoreactivity was upregulated as early as 1 day, reached a peak at 3 days, and was sustained up to 7 days post LPS. Increased levels of IL-4 immunoreactivity were exclusively detected in microglia/macrophages, but not in neurons nor astrocytes. The neutralizing antibody (NA) for IL-4 significantly protects striatal neurons against LPS-induced neurotoxicity in vivo. Accompanying neuroprotection, IL-4NA inhibited activation of microglia/macrophages, production of reactive oxygen species (ROS), ROS-derived oxidative damage and nitrosative stress, and produced polarization of microglia/macrophages shifted from M1 to M2. These results suggest that endogenous IL-4 expressed in LPS-activated microglia/macrophages contributes to striatal neurodegeneration in which oxidative/nitrosative stress and M1/M2 polarization are implicated.     

Role of Somatostatin Signalling in Neuroendocrine Tumours

Somatostatin (SST) is a small peptide that exerts inhibitory effects on a wide range of neuroendocrine cells. Due to the fact that somatostatin regulates cell growth and hormone secretion, somatostatin receptors (SSTRs) have become valuable targets for the treatment of different types of neuroendocrine tumours (NETs). NETs are a heterogeneous group of tumours that can develop in various parts of the body, including the digestive system, lungs, and pituitary. NETs are usually slow growing, but they are often diagnosed in advanced stages and can display aggressive behaviour. The mortality rate of NETs is not outstandingly increased compared to other malignant tumours, even in the metastatic setting. One of the intrinsic properties of NETs is the expression of SSTRs that serve as drug targets for SST analogues (SSAs), which can delay tumour progression and downregulate hormone overproduction. Additionally, in many NETs, it has been demonstrated that the SSTR expression level provides a prognostic value in predicting a therapeutic response. Furthermore, higher a SSTR expression correlates with a better survival rate in NET patients. In recent studies, other epigenetic regulators affecting SST signalling or SSA–mTOR inhibitor combination therapy in NETs have been considered as novel strategies for tumour control. In conclusion, SST signalling is a relevant regulator of NET functionality. Alongside classical SSA treatment regimens, future advanced therapies and treatment modalities are expected to improve the disease outcomes and overall health of NET patients.     

Somatostatin Receptor Splicing Variant sst5TMD4 Overexpression in Glioblastoma Is Associated with Poor Survival,Increased Aggressiveness Features,and Somatostatin Analogs Resistance

Glioblastoma (GBM) is the most malignant and lethal brain tumor. Current standard treatment consists of surgery followed by radiotherapy/chemotherapy; however, this is only a palliative approach with a mean post-operative survival of scarcely ~12–15 months. Thus, the identification of novel therapeutic targets to treat this devastating pathology is urgently needed. In this context, the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4), which is produced by aberrant alternative splicing, has been demonstrated to be overexpressed and associated with increased aggressiveness features in several tumors. However, the presence, functional role, and associated molecular mechanisms of sst5TMD4 in GBM have not been yet explored. Therefore, we performed a comprehensive analysis to characterize the expression and pathophysiological role of sst5TMD4 in human GBM. sst5TMD4 was significantly overexpressed (at mRNA and protein levels) in human GBM tissue compared to non-tumor (control) brain tissue. Remarkably, sst5TMD4 expression was significantly associated with poor overall survival and recurrent tumors in GBM patients. Moreover, in vitro sst5TMD4 overexpression (by specific plasmid) increased, whereas sst5TMD4 silencing (by specific siRNA) decreased, key malignant features (i.e., proliferation and migration capacity) of GBM cells (U-87 MG/U-118 MG models). Furthermore, sst5TMD4 overexpression in GBM cells altered the activity of multiple key signaling pathways associated with tumor aggressiveness/progression (AKT/JAK-STAT/NF-κB/TGF-β), and its silencing sensitized GBM cells to the antitumor effect of pasireotide (a somatostatin analog). Altogether, these results demonstrate that sst5TMD4 is overexpressed and associated with enhanced malignancy features in human GBMs and reveal its potential utility as a novel diagnostic/prognostic biomarker and putative therapeutic target in GBMs.     

Human monoclonal antibodies to domain C of tenascin-C selectively target solid tumors in vivo

We had previously reported that splice isoforms of tenascin-C containing the extra-domain C are virtually absent in normal adult tissues but are highly abundant in high-grade astrocytomas, with a prominent peri-vascular pattern of expression. We now report that the extra-domain C of tenascin-C is strongly expressed in the majority of lung cancers, with a vascular and stromal pattern of expression. Using antibody phage technology, we have generated a human monoclonal antibody (G11), with a dissociation constant K(D) = 4.2 nM for the human domain C. The G11 antibody, expressed in scFv and in mini-antibody (SIP) format, as well as a scFv-interleukin-2 fusion protein, was then characterized in quantitative biodistribution studies using mice grafted subcutaneously with U87 gliomas, revealing a selective tumor uptake, with tumor/blood ratios up to 11.8:1 at 24 h. A radioiodinated preparation of SIP(G11) was also investigated in a double tracer study using an orthotopic rat glioma model, confirming the antibody's ability to preferentially localize at the tumor site, with tumor/brain ratios superior to the ones observed with (18)F-fluorodeoxyglucose. These tumor-targeting properties, together with the strong immunohistochemical staining of human tumor sections, indicate that the G11 antibody may be used as a portable targeting moiety for the selective delivery of imaging and therapeutic agents to gliomas and lung tumors.     

Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.     

Diagnostic Utility of Genetic and Immunohistochemical H3-3A Mutation Analysis in Giant Cell Tumour of Bone

To validate the reliability and implementation of an objective diagnostic method for giant cell tumour of bone (GCTB). H3-3A gene mutation testing was performed using two different methods, Sanger sequencing and immunohistochemical (IHC) assays. A total of 214 patients, including 120 with GCTB and 94 with other giant cell-rich bone lesions, participated in the study. Sanger sequencing and IHC with anti-histone H3.3 G34W and G34V antibodies were performed on formalin-fixed, paraffin-embedded tissues, which were previously decalcified in EDTA if needed. The sensitivity and specificity of the molecular method was 100% (95% CI: 96.97–100%) and 100% (95% CI: 96.15–100%), respectively. The sensitivity and specificity of IHC was 94.32% (95% CI: 87.24–98.13%) and 100% (95% CI: 93.94–100.0%), respectively. P.G35 mutations were discovered in 2/9 (22.2%) secondary malignant GCTBs and 9/13 (69.2%) GCTB after denosumab treatment. We confirmed in a large series of patients that evaluation of H3-3A mutational status using direct sequencing is a reliable tool for diagnosing GCTB, and it should be incorporated into the diagnostic algorithm. Additionally, we discovered IHC can be used as a screening tool. Proper tissue processing and decalcification are necessary. The presence of the H3-3A mutation did not exclude malignant GCTB. Denosumab did not eradicate the neoplastic cell population of GCTB.     

Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.     

PTP61F Mediates Cell Competition and Mitigates Tumorigenesis

Tissue homeostasis via the elimination of aberrant cells is fundamental for organism survival. Cell competition is a key homeostatic mechanism, contributing to the recognition and elimination of aberrant cells, preventing their malignant progression and the development of tumors. Here, using Drosophila as a model organism, we have defined a role for protein tyrosine phosphatase 61F (PTP61F) (orthologue of mammalian PTP1B and TCPTP) in the initiation and progression of epithelial cancers. We demonstrate that a Ptp61F null mutation confers cells with a competitive advantage relative to neighbouring wild-type cells, while elevating PTP61F levels has the opposite effect. Furthermore, we show that knockdown of Ptp61F affects the survival of clones with impaired cell polarity, and that this occurs through regulation of the JAK–STAT signalling pathway. Importantly, PTP61F plays a robust non-cell-autonomous role in influencing the elimination of adjacent polarity-impaired mutant cells. Moreover, in a neoplastic RAS-driven polarity-impaired tumor model, we show that PTP61F levels determine the aggressiveness of tumors, with Ptp61F knockdown or overexpression, respectively, increasing or reducing tumor size. These effects correlate with the regulation of the RAS–MAPK and JAK–STAT signalling by PTP61F. Thus, PTP61F acts as a tumor suppressor that can function in an autonomous and non-cell-autonomous manner to ensure cellular fitness and attenuate tumorigenesis.     

Claudin 4 Is Differentially Expressed between Ovarian Cancer Subtypes and Plays a Role in Spheroid Formation

Claudin 4 is a cellular adhesion molecule that is frequently overexpressed in ovarian cancer and other epithelial cancers. In this study, we sought to determine whether the expression of claudin 4 is associated with outcome in ovarian cancer patients and may be involved in tumor progression. We examined claudin 4 expression in ovarian cancer tissues and cell lines, as well as by immunohistochemical staining of tissue microarrays (TMAs; n = 500), spheroids present in patients' ascites, and spheroids formed in vitro. Claudin 4 was expressed in nearly 70% of the ovarian cancer tissues examined and was differentially expressed across ovarian cancer subtypes, with the lowest expression in clear cell subtype. No association was found between claudin 4 expression and disease-specific survival in any subtype. Claudin 4 expression was also observed in multicellular spheroids obtained from patients' ascites. Using an in vitro spheroid formation assay, we found that NIH:OVCAR5 cells treated with shRNA against claudin 4 required a longer time to form compact spheroids compared to control NIH:OVCAR5 cells that expressed high levels of claudin 4. The inability of the NIH:OVCAR5 cells treated with claudin 4 shRNA to form compact spheroids was verified by FITC-dextran exclusion. These results demonstrate a role for claudin 4 and tight junctions in spheroid formation and integrity.     

Regulation of Epigenetic Modifications in the Placenta during Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.     

高表达抗人TNF-α人-鼠嵌合抗体细胞株的建立及其表达产物的分析

目的建立高表达抗人TNF-α人-鼠嵌合抗体的工程细胞株。方法将构建的抗人TNF-α人-鼠嵌合抗体基因真核表达载体转染CHO细胞,并采用DHFR/MTX基因扩增策略,经反复加压和克隆筛选,提高抗体的表达量。用ELISA、Westernblot和体外中和试验分析比较抗人TNF-α人-鼠嵌合抗体与鼠亲本单抗的特性。结果获得的表达抗人TNF-α嵌合抗体的转染细胞系2F5,在静止培养4d后工程抗体的表达量约80mg/L。所表达的工程抗体优于鼠亲本单抗F7/2,并具有良好的特异性和中和活性,其相对亲和力约为2.1×10-10mol/L。结论已成功建立了高表达的嵌合抗体工程细胞株,其抗体具有潜在的临床应用前景。     

Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells.     

Anti-Semaphorin 4D Rescues Motor,Cognitive, and Respiratory Phenotypes in a Rett Syndrome Mouse Model

Rett syndrome is a neurodevelopmental disorder caused by mutations of the methyl-CpG binding protein 2 gene. Abnormal physiological functions of glial cells contribute to pathogenesis of Rett syndrome. Semaphorin 4D (SEMA4D) regulates processes central to neuroinflammation and neurodegeneration including cytoskeletal structures required for process extension, communication, and migration of glial cells. Blocking SEMA4D-induced gliosis may preserve normal glial and neuronal function and rescue neurological dysfunction in Rett syndrome. We evaluated the pre-clinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody in the Rett syndrome Mecp2T158A transgenic mouse model and investigated the contribution of glial cells as a proposed mechanism of action in treated mice and in primary glial cultures isolated from Mecp2T158A/y mutant mice. SEMA4D is upregulated in neurons while glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1-positive cells are upregulated in Mecp2T158A/y mice. Anti-SEMA4D treatment ameliorates Rett syndrome-specific symptoms and improves behavioural functions in both pre-symptomatic and symptomatic cohorts of hemizygous Mecp2T158A/y male mice. Anti-SEMA4D also reduces astrocyte and microglia activation in vivo. In vitro experiments demonstrate an abnormal cytoskeletal structure in mutant astrocytes in the presence of SEMA4D, while anti-SEMA4D antibody treatment blocks SEMA4D–Plexin B1 signaling and mitigates these abnormalities. These results suggest that anti-SEMA4D immunotherapy may be an effective treatment option to alleviate symptoms and improve cognitive and motor function in Rett syndrome.     

Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.     

A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.     

Biochemical Characterisation of Human Transglutaminase 4

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.