Regulatory peptide immunocytochemistry at light- and electron microscopical levels

Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalization depends on adequate preservation of the peptide antigen and the tissue structure in which it resides; a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several 'families' of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.     

Regulatory peptide immunocytochemistry at light- and electron microscopical levels

Summary Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalisation depends on 1) adequate preservation of the peptide antigen and the tissue structure in which it resides; 2) a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several families of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.     

Immunochemical similarities among the hemolymph juvenile hormone binding proteins of moths

The hemolymph from various species of moths was analyzed for cross-reactivity with a panel of six monoclonal antibodies made against the hemolymph juvenile hormone binding protein ofManduca sexta. With the exception of one antibody, the immunoreactivity was limited to the sphingid family. One monoclonal antibody cross-reacted with a number of lepidopteran species; however, families such as Noctuidae and Pyralidae, known to have high affinity, low molecular weight juvenile hormone binding proteins, did not cross-react. Immunological cross-reactivity withManduca sexta juvenile hormone binding protein in several primitive moth families supports the current model of phylogenetic relationships in the order Lepidoptera.     

Monoclonal antibodies to L-asparaginase

Five hybridomas secreting monoclonal antibody to E. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G1 (1 clone), Ig G2 (2 clones) and Ig G3 (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (Kd) ranging between 2.5 X 10(-9) M and 6.3 X 10(-10) M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

The value of good Biozzi antibody-producing strains of mice for the production of monoclonal antibodies]

We report here that using Biozzi's high and low responder strains of Mice in the preparation of monoclonal antibodies against human T lymphocytes, we observed with the high responder strain 1) a higher number of hybrids; 2) a huge increase in the proportion of hybrids secreting on antibody directed against human lymphocytes.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

The role of glycosylation in protein antigenic properties

Glycosylation of proteins is a common event and contributes to protein antigenic properties. Most data have been obtained from model studies on glycoprotens with well-defined structure or synthetic glycopeptides and their respective monoclonal antibodies. Antibodies raised against glycoprotein antigens may be specific for their carbohydrate units which are recognized irrespective of the protein carrier (carbohydrate epitopes), or in the context of the adjacent amino acid residues (glycopeptidic epitopes). Conformation or proper exposure of peptidic epitopes of glycoproteins is also frequently modulated by glycosylation due to intramolecular carbohydrate-protein interactions. The effects of glycosylation are broad: glycosylation may 'inactivate' the peptidic epitope or may be required for its reactivity with the antibody, depending on the structure of the antigenic site and antibody fine specificity. Evidence is increasing that similar effects of glycosylation pertain to T cell-dependent cellular immune responses. Glycosylated peptides can be bound and presented by MHC class I or II molecules and elicit glycopeptide-specific T cell clones. Received 5 July 2001; received after revision 9 October 2001; accepted 11 October 2001     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Summary Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Molecular diagnosis of parasites

Summary New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene clonign and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Molecular diagnosis of parasites

New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene cloning and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Regulatory peptide receptors: visualization by autoradiography

The receptors for regulatory peptides have been extensively characterized using radioligand binding techniques. By combining these binding techniques with autoradiography it is possible to visualize at the light and electron microscopic levels the anatomical and cellular localization of these receptors. In this review we discuss the procedures used to label peptide receptors for autoradiography and the peculiarities of peptides as ligands. The utilization of autoradiography in mapping peptide receptors in brain and peripheral tissues, some of the new insights revealed by these studies particularly the problem of 'mismatch' between endogenous peptides and receptors, the existence of multiple receptors for a given peptide family and the use of peptide receptor autoradiography in human tissues are also reviewed.     

Targeted polymeric micelles for delivery of poorly soluble drugs

Polymeric micelles (micelles formed by amphiphilic block copolymers) demonstrate a series of attractive properties as drug carriers, such as high stability both in vitro and in vivo and good biocompatibility, and can be successfully used for the solubilization of various poorly soluble pharmaceuticals. These micelles can also be used as targeted drug delivery systems. The targeting can be achieved via the enhanced permeability and retention effect (into the areas with the compromised vasculature), by making micelles of stimuli-responsive amphiphilic block copolymers, or by attaching specific targeting ligand molecules to the micelle surface. Immunomicelles prepared by coupling monoclonal antibody molecules to p-nitrophenylcarbonyl groups on the water-exposed termini of the micelle corona-forming blocks demonstrate high binding specificity and targetability. Immunomicelles prepared with cancer-specific monoclonal antibody 2C5 specifically bind to different cancer cells in vitro and demonstrate increased therapeutic activity in vivo. This new family of pharmaceutical carriers can be used for the solubilization and targeted delivery of poorly soluble drugs to various pathological sites in the body.     

Recombinant anti-P protein autoantibodies isolated from a human autoimmune library: reactivity,specificity and epitope recognition

The ribosomal P proteins are specific and important autoantigens in patients affected by systemic lupus erythematosus. In this study, we describe for the first time the selection and characterization of recombinant human monoclonal anti-P protein (auto)-antibody fragments from an autoimmune patient-derived phage display antibody library. The selected recombinant anti-P antibodies specifically recognize the P proteins in immunofluorescence assays on HEp-2 cells and in immunoblotting assays, and they immunoprecipitate the P proteins under native conditions. Using both anti-P-positive patient sera and the selected recombinant anti-P antibodies, the immunodominant epitope was determined and shown to be located at the C-terminal end of the P proteins (amino acids 111-115). Inhibition of in vitro protein translation demonstrated that interaction of the monoclonal patient-derived anti-P antibodies with their native epitope functionally inhibits the activity of the P proteins on the ribosome, confirming the notion that patient autoantibodies are often directed to the functional centre of their autoantigenic target.     

Evolution of anti-Rh antibodies idiotypy in a blood donor over 9 years]

Anti-idiotypic sera were obtained in Rabbits immunized with Rh antibodies (isolated from the serum sample collected in 1974 from a Blood Donor). The sera agglutinate, at high titers, red cells coated with the immunizing antibodies and, at different titers, cells coated with antibodies from serum samples taken at other periods. Inhibition of hemagglutination of anti-idiotypic sera by different samples from the same Donor was complete with the immunizing serum and partial with other samples. These results show that idiotypes or idiotypic specificities appeared or disappeared during the period studied and represent the first observation on evolution of antibody idiotypy in Man.     

Molecular recognition in antibodies and its application

The structure and function of immunoglobulins, and the nature of the antibody — antigen interaction are described. Applications of the molecular recognition properties of antibodies are discussed in the areas of immunotherapy, immunoassay, immunotargeting and catalytic antibodies.     

Inhibition of IgM antibody-mediated aggregation ofTrypanosoma gambiense in the presence of complement

This paper deals with the immune reaction betweenTrypanosoma gambiense and monoclonal IgM mouse antibody at equivalence with or without rabbit complement. Antibody-mediated trypanosome clumps formed in the absence of complement, and were readily dissociated by complement to become free. In the presence of complement, on the other hand,T. gambiense were not aggregated by the antibody. Free parasites adhered readily to cultured peritoneal macrophages. Complement-mediated dissociation of the clumped trypanosomes in the equivalence area released a large number of previously bound surface antigens. These antigens were capable of binding again to fresh IgM antibody. Experimental results further indicated that the complement system caused a functional alteration, changing the multivalent nature of the IgM antibody in the immune complex into a univalent one. This phenomenon is of great advantage to the infected host in clearing pathogens in vivo, as it allows more antibodies to attach to trypanosomes and subsequently initiate complement activity.     

Novel cytotoxic peptides from the tropical marine cyanobacterium Hormothamnion enteromorphoides. 1. Discovery, isolation and initial chemical and biological characterization of the hormothamnins from wild and cultured material

A Caribbean cyanobacterium, Hormothamnion enteromorphoides, was found to produce a complex mixture of ichthyotoxic peptides, perhaps explaining the apparent absence of predation upon these potentially palatable life forms. Bioassay-guided fractionation was used to isolate these toxic and antimicrobial natural products, and a variety of techniques including HR FAB mass spectrometry, 2D-NMR, traditional hydrolysis-amino acid analysis, and several chemical reactions were used to define the basic structural features of the major peptide, hormothamnin A. Hormothamnin A is a cyclic undecapeptide containing six common and five uncommon or new amino acid residues. HPLC analyses indicate that the relative proportions of these peptide natural products remain relatively constant between different collection locations and years, however, they do vary seasonally. Clonal isolates of this cyanobacterium in culture produce the full spectrum of toxic peptides.     

Inhibition of IgM antibody-mediated aggregation of Trypanosoma gambiense in the presence of complement.

This paper deals with the immune reaction between Trypanosoma gambiense and monoclonal IgM mouse antibody at equivalence with or without rabbit complement. Antibody-mediated trypanosome clumps formed in the absence of complement, and were readily dissociated by complement to become free. In the presence of complement, on the other hand, T. gambiense were not aggregated by the antibody. Free parasites adhered readily to cultured peritoneal macrophages. Complement-mediated dissociation of the clumped trypanosomes in the equivalence area released a large number of previously bound surface antigens. These antigens were capable of binding again to fresh IgM antibody. Experimental results further indicated that the complement system caused a functional alteration, changing the multivalent nature of the IgM antibody in the immune complex into a univalent one. This phenomenon is of great advantage to the infected host in clearing pathogens in vivo, as it allows more antibodies to attach to trypanosomes and subsequently initiate complement activity.