Lymphangiogenesis in Crohn’s disease: an immunohistochemical study using monoclonal antibody D2-40

Crohn’s disease (CD) is a chronic inflammatory bowel disorder of unknown etiology. An involvement of the intestinal lymphatic system has been suggested. Recently, monoclonal antibodies have become available to distinguish lymphatic vessels from blood vessels. The aim of the study was to examine the distribution of lymphatic vessels in ileal and colic walls of patients affected by CD and compare it with healthy controls and other inflammatory bowel diseases. Twenty-eight cases of CD, 13 cases of other inflammatory bowel diseases, and 10 normal ileal and colic walls were studied. Immunohistochemical staining was performed using the monoclonal antibody D2-40. Quantification of lymphatic vessels was performed by identifying four fields with high density of lymphatics and then counting the number of lymphatic vessels at high resolution. Lymphatic diameter was also evaluated by using an ocular micrometer. Lymphatic vessels showed the highest density in CD specimens. The median number of lymphatics was significantly higher both in ileal and colic samples of CD than the other inflammatory diseases as well as normal controls. Moreover, in patients with CD, diffuse lymphangiectasia was also observed. The present data suggest that lymphangiogenesis and lymphangiectasia probably play a role in the pathogenesis of CD.     

Canakinumab: a human anti-IL-1β monoclonal antibody for the treatment of cryopyrin-associated periodic syndromes

The cryopyrin-associated periodic syndromes (CAPS) are a group of rare inherited inflammatory diseases associated with overproduction of IL-1β. Canakinumab, developed by Novartis AG (Basel, Switzerland), is an intravenously or subcutaneously administered, fully human monoclonal antibody that neutralizes the bioactivity of human IL-1β. Canakinumab has promising clinical safety and pharmacokinetic properties, and has demonstrated potential for the treatment of CAPS. Canakinumab was recently granted EU orphan drug status for systemic-onset juvenile idiopathic arthritis, and early clinical trials have established that administration of canakinumab every 2 weeks is both safe and effective. Subcutaneous canakinumab (approved formulation) offers some advantages over the existing IL-1β-blocking treatment, anakinra, which must be injected daily and is often not well tolerated by patients. The long-term safety of all targeted anti-IL-1 therapies in CAPS remains an unanswered question owing to the relatively short clinical experience with these agents; as canakinumab produces sustained IL-1 suppression, vigilance is necessary to diagnose the development of adverse events, especially any associated infections.     

Characterization of two distinct Cl− conductances in fused human respiratory epithelial cells

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl^– conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca²⁺, as described in the preceding publication. Cell membrane potential (V _m) and resistance (R _m) were recorded as well as their response to substitution of 90% of bath Cl^– by isethionate (V _m,ISE), by I^– (V _m,I), or by other halide anions. Fused CF cells had significantly (P<0.05) higher control V _m values (P–18.0 ±9.4 mV, ±SD, n=68) than fused non-CF cells (–12.5±6.6 mV, n=69) and responded to the Ca²⁺ ionophore A23187 with an increase in the V _m response to Cl^– substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca²⁺-stimulated Cl^– conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased V _m,ISE and V _m,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl^– conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in R _m remains unexplained. Stimulation of the Ca²⁺-regulated Cl^– conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect. In the search for the single-channel equivalent of the Ca²⁺-stimulated Cl^– conductance we injected a concentrated placental cytosol fraction containing a cytosolic inhibitor of the outwardly rectifying intermediate conductance (ORIC) Cl^– channel into fused non-CF cells stimulated either with A23187 or forskolin. However, no effect was observed. This speaks against a role of the ORIC Cl^– channel in the Ca²⁺-activated Cl^– conductance, although it cannot definitely be excluded.     

Macrophages regulate expression of α1,2-fucosyltransferase genes in human endometrial epithelial cells

The epithelial cell surface of the endometrium undergoes substantial biochemical changes to allow embryo attachment and implantation in early pregnancy. We hypothesized that tissue macrophages influence these events to promote uterine receptivity. To investigate the role of macrophages in regulating epithelial cell expression of genes linked to glycan-mediated embryo adhesion, Ishikawa, RL95-2 and HEC1A endometrial epithelial cells were cultured alone or with unactivated or lipopolysaccharide-activated monocytic U937 cells, separated using transwell inserts. Expression of mRNAs encoding two α1,2-fucosyltransferases (FUT1, FUT2) was increased in all three epithelial cell lines following co-culture with U937 cells, and was associated with increased fucosylation of cell surface glycoproteins detected using lectins from Ulex europaeus (UEA-1) and Dolichos biflorus (DBA). FUT1 induction by U937 cells also occurred in primary endometrial epithelial cells collected in luteal but not proliferative phase. Activation of the interleukin-6 (IL6)/leukemia inhibitory factor (LIF) cytokine signaling pathway with phosphorylation of STAT3 and elevated SOCS3 mRNA expression was evident in epithelial cells stimulated by U937 co-culture. Several recombinant macrophage-secreted cytokines exerted stimulatory or inhibitory effects on FUT1 and FUT2 mRNA expression, and the macrophage-derived cytokine LIF partially replicated the effects of U937 cells on both FUT1 and FUT2 expression and UEA-1 and DBA lectin reactivity in Ishikawa cells. These results suggest that macrophage-derived factors including LIF might facilitate development of an implantation-receptive endometrium by regulating surface glycan structures in epithelial cells. Abnormal phenotypes or altered abundance of uterine macrophages could contribute to the pathophysiology of primary unexplained infertility in women.     

Canakinumab: a human anti-IL-1β monoclonal antibody for the treatment of cryopyrin-associated periodic syndromes

The cryopyrin-associated periodic syndromes (CAPS) are a group of rare inherited inflammatory diseases associated with overproduction of IL-1β. Canakinumab, developed by Novartis AG (Basel, Switzerland), is an intravenously or subcutaneously administered, fully human monoclonal antibody that neutralizes the bioactivity of human IL-1β. Canakinumab has promising clinical safety and pharmacokinetic properties, and has demonstrated potential for the treatment of CAPS. Canakinumab was recently granted EU orphan drug status for systemic-onset juvenile idiopathic arthritis, and early clinical trials have established that administration of canakinumab every 2 weeks is both safe and effective. Subcutaneous canakinumab (approved formulation) offers some advantages over the existing IL-1β-blocking treatment, anakinra, which must be injected daily and is often not well tolerated by patients. The long-term safety of all targeted anti-IL-1 therapies in CAPS remains an unanswered question owing to the relatively short clinical experience with these agents; as canakinumab produces sustained IL-1 suppression, vigilance is necessary to diagnose the development of adverse events, especially any associated infections.     

Evaluation of systemic lupus erythematosus disease activity using anti-α-enolase antibody and RDW

Clinical and Experimental Medicine - The objective of the study was to investigate the value of anti-α-enolase antibody (Ab) combined with RDW in evaluating the activity of systemic lupus...     

Expression of the human β-defensin-1 induced by dehydroandrographolide in human pulmonary gland epithelial cells

In this study we observed the enhanced mRNA and protein expression of human β-defensin-1 (hBD-1) induced by dehydroandrographolide (DA) in human gland epithelial cells and explored the mechanism of DA on infection treatment. Human pulmonary gland epithelial cells were incubated with DA, then were harvested for hBD-1 expression detection. The mRNA expression of hBD-1 was detected by RT-PCR while the protein expression was detected by Western blot. It was found that DA can up-regulate mRNA and protein expression of hBD-1, the optimal concentration was 80 µM, the maximal expression of hBD-1 mRNA and protein occurred after 8 h. The DA can up-regulate mRNA and protein expression of hBD-1 in human gland epithelial cells. It suggests that β-defensin-1 may play an important role in the treatment of infective diseases with DA.     

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A₂, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart’s hydrops fetalis in which no α-globin chain is produced.     

Error-free replicative bypass of (6–4) photoproducts by DNA polymerase ζ in mouse and human cells

The ultraviolet (UV)-induced (6–4) pyrimidine–pyrimidone photoproduct [(6–4) PP] confers a large structural distortion in DNA. Here we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in promoting replication through a (6–4) TT photoproduct carried on a duplex plasmid where bidirectional replication initiates from an origin of replication. We show that TLS contributes to a large fraction of lesion bypass and that it is mostly error-free. We find that, whereas Pol η and Pol ι provide alternate pathways for mutagenic TLS, surprisingly, Pol ζ functions independently of these Pols and in a predominantly error-free manner. We verify and extend these observations in mouse cells and conclude that, in human cells, TLS during replication can be markedly error-free even opposite a highly distorting DNA lesion.     

Elimination of non-specific staining in immunohistochemistry of human liver tissues by modulation of antibody–antigen binding microenvironment

Abstract

Immunohistochemistry (IHC) is a method of routine use in histopathology for the diagnosis and differentiated diagnosis of liver diseases. IHC staining in human hepatic tissue is often obstructed by the presence of non-specific antibody interactions, the cause of high background staining. Cognizant of previous encounters with high levels of background in human hepatic tissue when using polyclonal antibodies, the potential causes were systemattically investigated by testing the impact of different antibody variants and assay conditions. Firstly, IHC was performed on hepatic tissue with immunized and non-immunized polyclonal antibodies and it was demonstrated that immunized antibodies pervaded the samples with non-specific staining, while non-immunized antibodies produced insignificant background. Following, the primary antibody was co-incubated with different concentrations of protein blocks, acid/base solutions, crystalline ionic compounds, and combinations of these individual components in order to assess their potency in removing background staining. Optimization of these assay conditions revealed that background could be significantly reduced by the addition of sodium chloride to primary antibody solution and/or protein block to sodium azide-stabilized Tris-HCl buffer, while little beneficial effect was observed from co-incubating the primary antibody with non-serum protein block/goat serum, hydrochloric acid/sodium hydroxide, or a higher concentration of peroxidase block. Together, the results suggest that hepatic background in IHC is mainly attributed to strong ionic interactions between the light chains of polyclonal antibodies and polar molecules specifically prevalent in hepatic tissue, which can be reduced by increasing the ionic strength of the antibody–antigen reaction microenvironment. Thus, the findings can enhance the specificity of hepatic tissue immunoassays and improve IHC studies in human liver tissues using polyclonal antibodies.     

Methylation of cytokines gene promoters in IL-1β-treated human intestinal epithelial cells

Objective and design

Epigenetic regulation is important in the activation of inflammatory cells. In the present study, we evaluated if DNA-methylation variations are involved in Interleukin-1β (IL-1β)-induced intestinal epithelial cells activation.

Materials and methods

Differentiated Caco-2 cells were exposed to IL-1β or to 5-azadeoxycytidine (5-azadC) for 24 or 48 h. Genome-wide methylation status was evaluated, while DNA methylation status at the promoter region of the gene encoding interleukin-6, 8 and 10 (IL-6, 8 and 10) was estimated. The levels of the corresponding gene products as well as DNA methyltransferases (DNMTs) quantity were assessed.

Results

IL-1β decreased genomic methylation of human intestinal epithelial cells and induced demethylation at cg-specific sites at the promoter of pro-inflammatory genes IL6 and IL8; conversely it did not change the methylation of the IL10 promoter. IL-1β also increased the release of IL-6 and IL-8 but did not change the IL-10 expression. Finally, cell exposure to IL-1β decreased the DNMT3b expression, increased DNMT3a and was not able to change DNMT1 expression.

Conclusions

Our results suggest a potential role of IL-1β as modulator of DNA methylation in activated differentiated Caco-2 cell line.

     

Nuclear factor kappa B plays a pivotal role in polyinosinic-polycytidylic acid-induced expression of human β-defensin 2 in intestinal epithelial cells

Intestinal epithelial cells (IECs) play an important role in protecting the intestinal surface from invading pathogens by producing effector molecules. IECs are one of the major sources of human beta-defensin 2 (hBD-2), and can produce it in response to a variety of stimuli. Although IECs express Toll-like receptor 3 (TLR-3) and can respond to its ligand, double-stranded RNA (dsRNA), hBD-2 expression in response to dsRNA has not been elucidated. In the present study, using an artificial analogue of dsRNA, polyinosinic-polycytidylic acid (poly I:C), we investigated whether the human IEC line, HT-29, can produce hBD-2 in response to poly I:C. HT-29 cells can express hBD-2 mRNA only when stimulated with poly I:C. The induction of hBD-2 mRNA expression was observed at 3 h after stimulation and peaked at 12 h of post-stimulation. Pre-incubation of the cells with nuclear factor kappa B (NF-κB)-specific inhibitor, l-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and isohelenine abolished the expression of hBD-2. Detection of the poly I:C signal by TLR-3 on the surface of HT-29 cells was revealed by pre-incubating the cells with anti-TLR-3 antibody. The 5'-regulatory region of the hBD-2 gene contains two NF-κB binding sites. A luciferase assay revealed the importance of the proximal NF-κB binding site for poly I:C-induced expression of hBD-2. Among NF-κB subunits, p65 and p50 were activated by poly I:C stimulation and accumulated in the nucleus. Activation of the p65 subunit was investigated further by determining its phosphorylation status, which revealed that poly I:C stimulation resulted in prolonged phosphorylation of p65. These results indicate clearly that NF-κB plays an indispensable role in poly I:C induced hBD-2 expression in HT-29 cells.     

Oral administration of recombinant human acid α-glucosidase reduces specific antibody formation against enzyme in mouse

Animal and human studies of enzyme replacement therapy for Pompe disease have indicated that antibodies generated against the infused recombinant human acid α-glucosidase (rhGAA) can negatively impact therapeutic outcome. In this study, we show that oral administration of rhGAA into mice can reduce the titer of anti-rhGAA antibody following immunization with rhGAA. Oral administration of rhGAA is safe and antigen specific, it offers advantages over other immunosuppressive drugs.     

Dengue virus isolation relying on antibody-dependent enhancement mechanism using FcγR-expressing BHK cells and a monoclonal antibody with infection-enhancing capacity

Background

Virus isolation is the most reliable evidence of dengue virus (DENV) infection. However, conventional virus isolation methods generally posses lower sensitivity and are time consuming as compared to other diagnostic methods such as detection of viral genome by RT-PCR, and determination of NS1 antigen and anti-DENV antibody by ELISA.

Objectives

A virus isolation method relying on the antibody-dependent enhancement mechanism was established and the assay's efficacy in DENV isolation was confirmed.

Study design

FcγR-expressing BHK cells were used for DENV isolation from patient serum samples in the presence of a flavivirus-group reactive monoclonal antibody, mAb4G2, which possesses DENV infection-enhancement activity. DENV genome copy numbers in the culture supernatant fluids of FcγR-expressing BHK cells were assessed and compared to those of parent BHK cells and C6/36 mosquito cells, a cell line commonly used for DENV isolation.

Results

The virus titer levels were higher in the culture supernatant fluid of FcγR-expressing BHK cells in the presence of enhancing antibody in comparison with other cell lines using laboratory-established strains and some clinical samples. DENV was isolated from 7 of 16 serum samples by using FcγR-expressing BHK cells in the presence of mAb4G2, but not by using cell lines commonly employed in conventional isolation assays, the FcγR-negative BHK cells and C6/36 cell lines.

Conclusions

The results demonstrate that FcγR-expressing BHK cell line in the presence of antibodies, which possess antibody dependent enhancement (ADE) activity, is a useful tool for DENV isolation.