Jonathan Alexander Green

Inhibition of glial scarring in the injured rat brain by a recombinant human monoclonal antibody to transforming growth factor-beta2.

The transforming growth factor‐βs (TGF‐βs) are potent fibrogenic factors implicated in numerous central nervous system (CNS) pathologies in which fibrosis and neural dysfunction are causally associated. In this study, we aim to limit the fibrogenic process in a model of CNS scarring using a recombinant human monoclonal antibody, derived from phage display libraries and specific to the active form of the TGF‐β2 isoform. The implicit inference of the work was that, as such antibodies are potential pharmacological agents for the treatment of human CNS fibrotic diseases, validation of efficacy in a mammalian animal model is a first step towards this end. Treatment of cerebral wounds with the anti‐TGF‐β2 antibody led to a marked attenuation of all aspects of CNS scarring, including matrix deposition, formation of an accessory glial‐limiting membrane, inflammation and angiogenesis. For example, in the wound, levels of: (i) the connective tissue components fibronectin, laminin and chondroitin sulphate proteoglycan; and (ii) wound‐responsive cells including astrocytes and macrophages/microglia, were markedly reduced. Our findings suggest that such synthetic anti‐fibrotic TGF‐β antibodies are potentially applicable to a number of human CNS fibrotic diseases to arrest the deposition of excessive extracellular matrix components, and maintain and/or restore functional integrity.

A fully human antibody neutralising biologically active human TGFβ2 for use in therapy

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.

Production of Polyclonal Antisera

All immunochemical procedures require a suitable antiserum or monoclonal antibody raised against the antigen of interest Polyclonal antibodies are raised by injecting an immunogen into an animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest. In producing antibodies, several parameters must be considered with respect to the final use to which the antibody will be put. These include (1) the specificity of the antibody, i.e., the ability to distinguish between different antigens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the titer of the antibody, which determines the optimal dilution of the antibody in the assay system. A highly specific antibody with high avidity may be suitable for immunohistochemistry, where it is essential that the antibody remains attached during the extensive washing procedures, but may be less useful for immunoaffinity chromatography, as it may prove impossible to elute the antigen from the column without extensive denaturation.All immunochemical procedures require a suitable antiserum or monoclonal antibody raised against the antigen of interest Polyclonal antibodies are raised by injecting an immunogen into an animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest. In producing antibodies, several parameters must be considered with respect to the final use to which the antibody will be put. These include (1) the specificity of the antibody, i.e., the ability to distinguish between different antigens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the titer of the antibody, which determines the optimal dilution of the antibody in the assay system. A highly specific antibody with high avidity may be suitable for immunohistochemistry, where it is essential that the antibody remains attached during the extensive washing procedures, but may be less useful for immunoaffinity chromatography, as it may prove impossible to elute the antigen from the column without extensive denaturation.

The ultrastructural immunolocalization ofγ-glutamyltranspeptidase in rat lung: Correlation with the histochemical demonstration of enzyme activity

Summaryγ-Glutamyltranspeptidase (γ-GT) was localized in slices of rat lung, at the ultrastructural level, by pre-embedding immunogold labelling. Antiserum was raised against the protein purified from rat kidney. The enzyme was found to be concentrated on the lumenal surface of the non-ciliated (‘Clara’) cells of the bronchiolar epithelium and, to a lesser degree, on the surface of type II alveolar pneumocytes. This immunological localization was consistent with the distribution of reaction product, in both slices and resin sections incubated to demonstrate γ-GT activity. γ-GT is probably involved in the utilization of reduced gluthathione (GSH) present in the fluid lining the airway epithelium.

Sex-linked hepatic uroporphyria and the induction of cytochromes P450IA in rats caused by hexachlorobenzene and polyhalogenated biphenyls

A marked sex difference in the development of uroporphyria occurred after administration of polychlorinated and polybrominated biphenyls (PCBs and PBBs), as well as hexachlorobenzene (HCB), to F344 rats for 15 weeks. Thus the propensity of female rats to develop uroporphyria appears to be a general response to this class of halogenated chemicals. A heat-stable inhibitor(s) of liver uroporphyrinogen decarboxylase was extractable from uroporphyric livers. Although oxidation of uroporphyrinogen I to uroporphyrin I by hepatic microsomes from rats pretreated with porphyrogenic regimes of HCB and PCBs was induced, there was no correlation with the in vivo sex difference in porphyria development. Levels of total cytochrome P450 and pentoxyresorufin and benzyloxyresorufin dealkylase activities (associated with cytochrome P450IIB1) were greater in microsomes from control, HCB, PCB and PBB treated male rats than females. In contrast, ethoxyresorufin deethylase activity (associated with cytochrome P450IA1) was always significantly greater in females. These findings were confirmed by immunoblotting with polyclonal antibodies to cytochromes P450IA1, IA2 and IIB1. Immunocytochemical studies showed that, even after 30 weeks of HCB exposure, cytochromes P450IA1 and P450IA2 were still more highly induced in female liver, especially in the centrilobular region. The results are consistent with the association of cytochrome P450IA isoenzymes with uroporphyria development, although the sex difference in P450IA levels alone may not be marked enough to provide the complete explanation for the pronounced susceptibility of females to HCB.

Degree of ethoxyquin-induced nephrotoxicity in rat is dependent on age and sex.

The toxicity of ethoxyquin (EQ) to rat kidney was examined in males which were either weanling or adult at the beginning of the experiment, and also in adult females. Female rats were much less susceptible to the toxic effects of EQ than males of the same age. In males damage to the cortex, mainly as an acceleration of the normal ageing process, was similar in both age groups, but rats exposed to EQ as weanlings also suffered from extensive papillary necrosis. Male rats were more prone than females to proteinuria, which was greatly exacerbated by EQ in both age groups. Thus there is very little evidence of nephrotoxicity in adult female rats on exposure to EQ at 0.5% in the diet for 26 weeks. In males, the initial age of the animal, as well as the length of treatment, influences the extent of damage.

Immunohistochemical detection of BromodeoxyurIDine-Labeled Nuclei for In Vivo Cell Kinetic Studies

The classical technique for identifying cells engaged in DNA synthesis is by their uptake of [(3)H]-thymidine, detected using autoradiography. However, this method can be inconvenient, as specialized darkroom and radioisotope facilities are required, with the potential health hazard that handling isotopes entails. Bromodeoxyuridine (BrdU), the halogenated 5-substituted derivative of deoxyuridine, is a thymidine analog specifically incorporated into the DNA of proliferating cells during S phase. This is now a well-established alternative to (3)H thymidine, since it has been shown that labeling indices for the two molecules are the same (1,2). The development of a monoclonal antibody (3) that recognizes BrdU incorporated into single-stranded DNA has resulted in several techniques using immunocytochemical staining to detect incorporated BrdU in frozen, paraffin- and plastic-embedded sections of tissue by light microscopy. It has also proved extremely valuable for studies in conjunction with flow cytometry and even, for in vivo studies of human tumor cell kinetics (see this vol., Chapter 43 ). We describe here a method to detect DNA synthesis by in vivo labeling of nuclei with BrdU, followed by indirect immunological detection in paraffin-embedded tissue (4).The classical technique for identifying cells engaged in DNA synthesis is by their uptake of [(3)H]-thymidine, detected using autoradiography. However, this method can be inconvenient, as specialized darkroom and radioisotope facilities are required, with the potential health hazard that handling isotopes entails. Bromodeoxyuridine (BrdU), the halogenated 5-substituted derivative of deoxyuridine, is a thymidine analog specifically incorporated into the DNA of proliferating cells during S phase. This is now a well-established alternative to (3)H thymidine, since it has been shown that labeling indices for the two molecules are the same (1,2). The development of a monoclonal antibody (3) that recognizes BrdU incorporated into single-stranded DNA has resulted in several techniques using immunocytochemical staining to detect incorporated BrdU in frozen, paraffin- and plastic-embedded sections of tissue by light microscopy. It has also proved extremely valuable for studies in conjunction with flow cytometry and even, for in vivo studies of human tumor cell kinetics (see this vol., Chapter 43 ). We describe here a method to detect DNA synthesis by in vivo labeling of nuclei with BrdU, followed by indirect immunological detection in paraffin-embedded tissue (4).

double Label Immunohistochemistry on Tissue Sections Using Alkaline Phosphatase and PeroxIDase Conjugates

One of the most common methods in immunohistochemistry involves the use of an antibody to the antigen of interest detected indirectly with an enzyme-labeled antispecies secondary antibody. The enzyme catalyzes the formation of a colored insoluble reaction product at the antigen site. It is possible, with careful choice of reagents, to label two antigens simultaneously, resulting in two different colored reaction products (1). Cells or tissue sections can also be double-labeled with two antispecies secondary antibodies carrying different fluorochromes (see this vol., Chapter 42 ), or by using suitable antibodies conjugated to different sizes of colloidal gold (see this vol., Chapter 19 ).One of the most common methods in immunohistochemistry involves the use of an antibody to the antigen of interest detected indirectly with an enzyme-labeled antispecies secondary antibody. The enzyme catalyzes the formation of a colored insoluble reaction product at the antigen site. It is possible, with careful choice of reagents, to label two antigens simultaneously, resulting in two different colored reaction products (1). Cells or tissue sections can also be double-labeled with two antispecies secondary antibodies carrying different fluorochromes (see this vol., Chapter 42 ), or by using suitable antibodies conjugated to different sizes of colloidal gold (see this vol., Chapter 19 ).