Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.

Expression studies of catalytic antibodies.

We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived from a number of catalytic antibodies. Expression yields of eight hybridoma- and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high density fermentations. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies.

Ultra-low concentrations of naloxone selectively antagonize excitatory effects of morphine on sensory neurons, thereby increasing its antinociceptive potency and attenuating tolerance/dependence during chronic cotreatment.

Ultra-low picomolar concentrations of the opioid antagonists naloxone (NLX) and naltrexone (NTX) have remarkably potent antagonist actions on excitatory opioid receptor functions in mouse dorsal root ganglion (DRG) neurons, whereas higher nanomolar concentrations antagonize excitatory and inhibitory opioid functions. Pretreatment of naive nociceptive types of DRG neurons with picomolar concentrations of either antagonist blocks excitatory prolongation of the Ca(2+)-dependent component of the action potential duration (APD) elicited by picomolar-nanomolar morphine and unmasks inhibitory APD shortening. The present study provides a cellular mechanism to account for previous reports that low doses of NLX and NTX paradoxically enhance, instead of attenuate, the analgesic effects of morphine and other opioid agonists. Furthermore, chronic cotreatment of DRG neurons with micromolar morphine plus picomolar NLX or NTX prevents the development of (i) tolerance to the inhibitory APD-shortening effects of high concentrations of morphine and (ii) supersensitivity to the excitatory APD-prolonging effects of nanomolar NLX as well as of ultra-low (femtomolar-picomolar) concentrations of morphine and other opioid agonists. These in vitro studies suggested that ultra-low doses of NLX or NTX that selectively block the excitatory effects of morphine may not only enhance the analgesic potency of morphine and other bimodally acting opioid agonists but also markedly attenuate their dependence liability. Subsequent correlative studies have now demonstrated that cotreatment of mice with morphine plus ultra-low-dose NTX does, in fact, enhance the antinociceptive potency of morphine in tail-flick assays and attenuate development of withdrawal symptoms in chronic, as well as acute, physical dependence assays.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Serotonin regulates mouse cranial neural crest migration.

Serotonergic agents (uptake inhibitors, receptor ligands) cause significant craniofacial malformations in cultured mouse embryos suggesting that 5-hydroxytryptamine (serotonin) (5-HT) may be an important regulator of craniofacial development. To determine whether serotonergic regulation of cell migration might underly some of these effects, cranial neural crest (NC) explants from embryonic day 9 (E9) (plug day = E1) mouse embryos or dissociated mandibular mesenchyme cells (derived from NC) from E12 embryos were placed in a modified Boyden chamber to measure effects of serotonergic agents on cell migration. A dose-dependent effect of 5-HT on the migration of highly motile cranial NC cells was demonstrated, such that low concentrations of 5-HT stimulated migration, whereas this effect was progressively lost as the dose of 5-HT was increased. In contrast, most concentrations of 5-HT inhibited migration of less motile, mandibular mesenchyme cells. To investigate the possible involvement of specific 5-HT receptors in the stimulation of NC migration, several 5-HT subtype-selective antagonists were used to block the effects of the most stimulatory dose of 5-HT (0.01 microM). Only NAN-190 (a 5-HT1A antagonist) inhibited the effect of 5-HT, suggesting involvement of this receptor. Further evidence was obtained by using immunohistochemistry with 5-HT receptor antibodies, which revealed expression of the 5-HT1A receptor but not other subtypes by migrating NC cells in both embryos and cranial NC explants. These results suggest that by activating appropriate receptors 5-HT may regulate migration of cranial NC cells and their mesenchymal derivatives in the mouse embryo.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Localization of specific erythropoietin binding sites in defined areas of the mouse brain.

The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.