Specificity and Function of Monoclonal Antibodies Reactive with Discrete Structural Elements of Bacterial Lipopolysaccharide

Myofibroblasts, characterised by high expression of α-smooth muscle actin (α-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via αv and β1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in α-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins αv, β1, αvβ3 and αvβ5 induced the expression of α-SMA and its organization into stress fibers. Expression of α-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of α-SMA. The expression and organization of α-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of α-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of α-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

Download Full-text

Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Antibodies ◽

10.3390/antib9030048 ◽

2020 ◽

Vol 9 (3) ◽

pp. 48

Author(s):

Jessica Ramadhin ◽

Vanessa Silva-Moraes ◽

Thomas Norberg ◽

Donald Harn

Keyword(s):

Drug Delivery ◽

Flow Cytometry ◽

Monoclonal Antibodies ◽

Structure And Function ◽

Enzyme Linked Immunosorbent Assay ◽

Incomplete Freund’S Adjuvant ◽

Delivery Platform ◽

Igg1 Mabs ◽

Human Milk Oligosaccharide ◽

And Function

Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

Download Full-text

Combination of Anti-CD4 with Anti-LFA-1 and Anti-CD154 Monoclonal Antibodies Promotes Long-Term Survival and Function of Neonatal Porcine Islet Xenografts in Spontaneously Diabetic NOD Mice

Cell Transplantation ◽

10.3727/000000007783465244 ◽

2007 ◽

Vol 16 (8) ◽

pp. 787-798 ◽

Cited By ~ 15

Author(s):

Hossein Arefanian ◽

Eric B. Tredget ◽

Ray V. Rajotte ◽

Gregory S. Korbutt ◽

Ron G. Gill ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Nod Mice ◽

Β Cells ◽

Short Term ◽

Term Survival ◽

Long Term Survival ◽

Porcine Islet ◽

Term Administration ◽

And Function

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet β-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for T1DM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing β-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.

Download Full-text

Epithelial Tonofilaments: Investigating Their Form and Function Using Monoclonal Antibodies

Cold Spring Harbor Symposia on Quantitative Biology ◽

10.1101/sqb.1982.046.01.038 ◽

1982 ◽

Vol 46 (0) ◽

pp. 387-402 ◽

Cited By ~ 35

Author(s):

E. B. Lane ◽

M. W. Klymkowsky

Keyword(s):

Monoclonal Antibodies ◽

Form And Function ◽

And Function

Download Full-text

Abstract 1832: Anti-nicastrin monoclonal antibodies as therapeutics for invasive breast cancer: Characterization and function

10.1158/1538-7445.am2012-1832 ◽

2012 ◽

Author(s):

Aleksandra Filipovic ◽

Ylenia Lombardo ◽

Deonarain Mahendra ◽

Catherine Tralau-Stewart ◽

Cordingley Hayley ◽

...

Keyword(s):

Breast Cancer ◽

Monoclonal Antibodies ◽

Invasive Breast Cancer ◽

And Function

Download Full-text

The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

mBio ◽

10.1128/mbio.00448-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Rogério F. Lourenço ◽

Saumya Saurabh ◽

Jonathan Herrmann ◽

Soichi Wakatsuki ◽

Lucy Shapiro

Keyword(s):

Dna Binding ◽

Caulobacter Crescentus ◽

Genetic Material ◽

Bacterial Cells ◽

Structural Elements ◽

Content Type ◽

Time Dynamic ◽

Associated Proteins ◽

And Function

ABSTRACT Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA. IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

Download Full-text

Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation

Blood ◽

10.1182/blood.v95.1.164 ◽

2000 ◽

Vol 95 (1) ◽

pp. 164-172 ◽

Cited By ~ 49

Author(s):

Mariagrazia De Luca ◽

David A. Facey ◽

Emmanuel J. Favaloro ◽

Mark S. Hertzberg ◽

James C. Whisstock ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Platelet Membrane ◽

Membrane Glycoprotein ◽

Platelet Membrane Glycoprotein ◽

And Function ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481–Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf–dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700–Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf–induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494–Leu-512; CR2, Phe-536–Ala-554; CR3, Arg-578–Glu-596; CR11 and CR15, Ala-564–Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700–Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

Download Full-text

Identification and characterization of an endothelial, cell-specific antigen with a monoclonal antibody

Blood ◽

10.1182/blood.v66.4.816.816 ◽

1985 ◽

Vol 66 (4) ◽

pp. 816-823 ◽

Cited By ~ 12

Author(s):

WM Parks ◽

RD Gingrich ◽

CE Dahle ◽

JC Hoak

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Vascular Endothelium ◽

Umbilical Vein ◽

Polyacrylamide Gel Electrophoresis ◽

Primary Cultures ◽

Specific Antigen ◽

Myeloma Cell ◽

Membrane Components ◽

And Function

Abstract The purpose of these studies was to use monoclonal antibodies to identify and characterize plasma membrane components unique to the vascular endothelium. Our assumption is that such components may perform some of the specialized functions of the endothelium and, by their identification with antibody probes, we may be able to study further their function and structure. Thus, primary cultures of human umbilical vein endothelium were used to immunize mice whose spleen cells were fused with the mouse myeloma cell NS-1. HEC-1 is a monoclonal antibody derived from such a fusion that appears to react with an antigen located only on endothelial cells. The antigen has been characterized by immunoprecipitation and polyacrylamide gel electrophoresis as a glycoprotein with a mol wt of 180,000 daltons under nonreducing conditions and 90,000 daltons under reducing conditions. Despite a close resemblance to a membrane component shown by others to be a receptor for transferrin, several lines of evidence reported in this paper indicate that this is not the function of the HEC-1 antigen. These data show that monoclonal antibodies can be used to identify and characterize membrane components of the vascular endothelium. Moreover, these probes can be used to inquire about the structure and function of the antigen with which they react.

Download Full-text

Production of Monoclonal Antibodies Against ADAMTS13 Determining Its Expression in Different Human Tissues.

Blood ◽

10.1182/blood.v104.11.3949.3949 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3949-3949

Author(s):

Weiqiang Gao ◽

Jiang Su ◽

Xia Bai ◽

Fei Shen ◽

Changgeng Ruan

Keyword(s):

Monoclonal Antibodies ◽

Recombinant Protein ◽

Von Willebrand Factor ◽

Positive Staining ◽

Human Tissues ◽

Cdna Sequences ◽

Normal Tissues ◽

And Function ◽

Von Willebrand ◽

Willebrand Factor

Abstract von Willebrand factor-cleaving protease/ADAMTS13 is a plasma metalloproteinase that degrades unusually large von Willebrand factor multimers (UL-vWF) derived from endothelial cells (ECs) and megakaryocytes (MCs) into small peptides circulating in blood. It is documented that transcript mRNAs of the protease are present in many human tissues; however, the protein expression of ADAMTS13 remains to be elucidated. In the present work, the gene of metalloproteinase domain of human ADAMTS13 was cloned into the multiclone site of pET28a(+). After induced by IPTG, the recombinant protein was purified using a Ni-NTA column and the Bal b/c mice were immunized with the protein. Screened with ELISA, three monoclonal antibodies against the metalloproteinase domain of ADAMTS13 were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belonged to IgG1 subclass. The quantity of them in ascites were 4 mg/ml, and their titers were as high as 1×10−5. The data of competitive ELISA showed that SZ-112 and SZ-113 recognized different epitopes of the recombinant protein. Western blot results demonstrated that SZ-112 not only reacted against the recombinant protein, but also recognized the full-length recombinant ADAMTS13 protein that expressed in CHO cell line (the vectors containing the ADAMTS13 cDNA sequences were provided by Prof. Sadler JE). The immunoprecipitation results showed that the two antibodies could react to an approximately 200 KDa protein in platelet lysate. Then, the expression panels of ADAMTS13 in human normal tissues were investigated using immunohistochemistry with the monoclonal antibodies. And the protease was found to be present in many kinds of tissues such as liver, spleen, ovary, prostate, bladder, small intestine, thyroid and thymus with significantly positive staining. The protease was also present in lung, uterus, large intestine and heart but stained weakly. We did not found the protease in brain. In most of these organs, the protease was expressed in epithelium of the tissues. While in liver, spleen and thymus, it was mainly presented in a subgroup of the solid tissue cells. Moreover, the preliminary results showed that the expression of ADAMTS13 slightly decreased in liver tissues of patients suffering form hepatitis type B and cirrhosis. In conclusion, our data indicated that two novel monoclonal antibodies against the metalloproteinase domain of human ADAMTS13 were successfully prepared, and the expression of ADAMTS13 in different tissue and specific locality might be associated with the regulation and function of the protease, which would contribute to the further research of the deficiency mechanism of the proteases in some disorders.

Download Full-text