Comparison of immunocytochemical and steroid-binding assays for estrogen receptor in human breast tumors.

An estrogen receptor immunocytochemical assay which uses monoclonal antibodies to the estrogen receptor protein [Nature (Lond.), 307: 745-747, 1984] was applied to several human tissues, including human breast tumors, and the results were compared to those of steroid-binding assays performed on cytosol extracts of the same tissues. Specific immunoperoxidase staining in fixed, frozen sections was confined to the nucleus of selected cell populations within each tissue examined. In 117 human breast cancers, the presence or absence of nuclear staining was significantly associated with the concentration of cytosolic estrogen receptor. Thirty-eight estrogen receptor immunocytochemical assay-positive tumors were further assessed for several quantifiable features of the staining, including intensity, cellularity, and the proportion of tumor cells stained. Of these, epithelial cellularity showed the highest degree of correlation with the results of steroid-binding assays.

Prognostic usefulness of estrogen receptor immunocytochemical assays for human breast cancer.

Breast cancers of postmenopausal patients at high risk for recurrence participating in an adjuvant therapy protocol were independently assayed for estrogen receptor by conventional dextran-coated charcoal steroid binding assays and by immunocytochemistry (ER-ICA) to compare the two assays and to assess the prognostic usefulness of ER-ICA. The ER-ICA was based on a monoclonal antibody to the estrogen receptor and was applied to lightly fixed, frozen sections of the cancers. Excellent agreement was found between the two estrogen receptor methods. It was found that a combination of the distribution of ER-ICA stained cells and the overall staining intensity gave a statistically significant correlation with the quantitative estrogen receptor dextran-coated charcoal steroid binding assay value. In addition, the overall appraisal of the lesion as ER-ICA positive or negative as well as the ER-ICA staining intensity and proportion of ER-ICA stained cancer cells related to patient disease-free interval and survival, independent of patient lymph node involvement. This relationship of ER-ICA status to prognosis appeared not to relate only to responses to adjuvant tamoxifen treatment since it also was observed with patients who did not receive the antiestrogen.

Differential responses of prelactating and lactating mammary gland to similar tissue concentrations of progesterone.

Serum and mammary tissue concentrations of progesterone and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OHP) were measured by competitive protein-binding assays and gas-liquid chromatography, respectively, in pregnant and lactating rats. The concentration of progesterone in mammary tissue of pregnant rats closely paralleled the serum concentration, particularly when tissue concentration was expressed as ng/mg DNA. The variability in tissue progesterone on the last day of pregnancy was relatively great, but there was a good inverse relation between the appearance of lactose and the progesterone concentration. Serum progesterone levels declined to their lowest values at 1-3 days of lactation (10 plus or minus 1 ng/ml); the tissue concentration declined even more rapidly after parturition. The tissue 20alpha-OHP concentration, which was more closely related to serum progesterone among animals than to serum 20alpha-OHP, remained high after parturition suggesting that the presence of 20alpha-OHP has no effect on lactogenesis and that progesterone is decreased in the tissue by 20alpha-reduction. Following postpartum ovulation, serum progesterone increased to 74 plus or minus 6 ng/ml at 6-9 days of lactation; tissue progesterone also increased to levels found in rats pregnant 14-19 days, yet no change in lactose content of the glands of suckled rats occurred, and the biosynthetic capacity in terms of the RNA/DNA ratio increased. Serum 20alpha-OHP also rose, but the tissue concentration was unchanged, suggesting that saturating levels were present throughout pregnancy and lactation. Since no rapid increase in DNA was associated with lactogenesis, differentiation of nonsecretory parenchymal cells into daughter cells with the secretory capacity must occur earlier in pregnancy. Progesterone, therefore, must inhibit lactogenesis by preventing expression of the genetic potential of daughter cells. Once differentiation has been completed, however, the presence of progesterone in the tissue has no effect on the biosynthesis of milk constituents.

Functional human thyroid cells and their insulin-like growth factor-binding proteins: regulation by thyrotropin, cyclic 3',5' adenosine monophosphate, and growth factors.

The regulation of de novo synthesis of thyroid hormones in primary cultures of human thyroid cells has been examined and correlated with the regulation of the synthesis of the insulin-like growth factor-binding proteins (IGFBPs). In the serum-free culture medium, insulin and TSH (0.01-0.3U/L)were found to be obligatory additives for iodide uptake and organification. In the presence of TSH, cells reorganized into 3D follicles, which stored thyroglobulin. High concentrations of TSH ( > 1U/L), epidermal growth factor, protein kinase C activation with phorbol esters, and transforming growth factor beta 1 all were strongly inhibitory to iodide metabolism and thyroid hormone synthesis. Conditioned medium from the thyroid cell cultures contained at least 5 125I-IGF-labeled bands IGFBPs, including the two glycosylation variants of IGFBP-3. TSH, at concentrations optimal for iodide uptake, inhibited the secretion of all these binding proteins. These effects were mimicked by forskolin and the cell-permeable analog of cAMP, dibutyryl cAMP. The changes in IGFBP proteins were reflected by marked reductions in the steady-state levels of the messenger RNAs of IGFBP-3 and IGFBP-5. This reduction was less pronounced for IGFBP-4. In contrast, protein kinase C activation with phorbol esters and transforming growth factor beta, and high TSH concentrations enhanced IGFBP secretion. Steady-state levels of IGFBP-3 and IGFBP-5 messenger RNAs were elevated after treatment with transforming growth factor-beta and high TSH concentrations. This Study shows that enhanced production of IGFBPs is correlated with inhibition of thyroid function and that TSH, through cAMP, is one factor capable of inhibiting IGFBP production.

The prognostic value of immunohistochemical estrogen receptor analysis in paraffin-embedded and frozen sections versus that of steroid-binding assays.

Estrogen receptors (ER) were independently analyzed using dextran-coated charcoal assays (ER-DCC) and immunohistochemical assays in frozen (ER-ICA) and paraffin-embedded tissue (ER-PAR) from 130 human breast cancer specimens drawn from postmenopausal high-risk patients registered in the Danish Breast Cancer Cooperative Group. ER was best detected with the ER-DCC assay followed by the ER-ICA (relative sensitivity 87%) and the ER-PAR assays (relative sensitivity 71%). The semiquantified staining features of the immunohistochemical assays were statistically significantly correlated with each other and with ER-DCC. Analysis of disease-free interval (DFI) and overall survival (OS) showed that all assays allowed statistically significant discrimination between a high risk and a low risk group, although the sensitivity differences tended to be reflected as small differences in clinical discriminatory power. The patient groups were then stratified according to adjuvant treatment [radiotherapy (RT) versus radiotherapy and tamoxifen (RT + TAM)]. The survival advantage was tied primarily to the receptor status itself in the steroid-binding assays, but was linked to both the receptor status and the adjuvant treatment in the immunohistochemical assays. Thus, the relative risks in terms of DFI and OS were of the same relative magnitude in the RT and RT + TAM groups for ER-DCC assays using a cut-off level of 10 fmol/mg cytosol protein, while there were large differences in the relative risks between RT and RT + TAM groups for ER-ICA and ER-PAR assays. We conclude that an ER assay in fresh tissue should be given first priority, but if there is no fresh tissue, an ER assay in paraffin-embedded tissue offers a reasonably good alternative as a prognosticator and an equivalent alternative as a predictor of the response to endocrine treatment.

Cytokine and chemokine expression kinetics after corneal transplantation.

BACKGROUND: Allogeneic rejection is the most common cause of corneal graft failure. The aim of this work was to establish the kinetics of cytokine and chemokine mRNA expression before and after onset of corneal graft rejection. METHODS: Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway-->Lewis inbred rat model in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis-->Lewis) corneal transplants were used as controls. Donor and recipient cornea was examined by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxyanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1beta, IL-1RA, IL2, IL-6, IL-10, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), transforming growth factor (TGF)-beta1, and macrophage inflammatory protein (MIP)-II and by nonquantitative RT-PCR for IL4, IL-5, IL-12 p40, IL-13, TGF-beta2, monocyte chemotactic protein-1 (MCP-1), MIP-1alpha, MIP-1beta, and RANTES (for regulated upon activation normal T cell expressed and secreted). RESULTS: A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3-9), there was an elevation of the majority of the cytokines examined, including IL-1beta, IL-6, IL-10, IL-12 p40, and MIP-II. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after observed rejection onset in the allografts, including TGF-beta1, TGF-beta2, and IL-1RA. The T cell-derived cytokines IL-4, IL-13, and IFN-gamma were detected only during the rejection phase in allogeneic recipients. CONCLUSIONS: There is an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that probably drives angiogenesis, leukocyte recruitment, and affects leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines (IL-2, IL-12 p40, IFN-gamma), Th2 cytokines (IL-4, IL-6, IL-10, and IL-13), and antiinflammatory/Th3 cytokines (TGF-beta1/2 and IL-1RA).

Transferrin receptor-mediated gene transfer to the corneal endothelium.

BACKGROUND: The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. METHODS: Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. RESULTS: Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eight-fold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. CONCLUSIONS: Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.

The use of monoclonal antibodies to estrogen receptors (ER) for immunoperoxidase detection of ER in paraffin sections of human breast cancer tissue.

Two monoclonal antibodies against MCF-7 human estrogen receptors were used for immunoperoxidase staining of paraffin sections of human breast cancer tissue. The staining was predominantly located in the nucleus of epithelial cells. Variation in the staining intensity was observed among individual cells. A significant positive correlation between the number of positively stained cells and cytosol estrogen receptor content (fmol of bound estrogen/mg of protein) was observed. The potential and the limitations of the present techniques are discussed.

Age-related immunogenicity of meningococcal polysaccharide vaccine in aboriginal children and adolescents living in a Northern Manitoba reserve community.

OBJECTIVE: To determine the total and functional serogroup C antibody response to a quadrivalent meningococcal polysaccharide vaccine in a group of aboriginal infants, children and adolescents. A secondary objective was to determine their prevalence of meningococcal carriage. DESIGN: Open prospective, before and after intervention study. SUBJECTS: Aboriginal children ages 0.5 to 19.9 years, living in a single Northern community and eligible for a public health immunization campaign conducted in all Manitoba native reserve communities to control a meningococcal serogroup C, electrophoretic type (ET) 15 outbreak. No outbreak cases had occurred in the community at the time of the study. METHODS: Total serogroup C capsular polysaccharide antibody (CPA) and functional bactericidal antibody (BA) responses were measured by enzyme-linked immunosorbent assay and bactericidal assay, respectively. RESULTS: Neisseria meningitidis was recovered from the oropharynx of 13 (5.2%) of 249 aboriginal children including 4 (1.6%) serogroup C isolates, all with the designation C:2a:P1.2,5 ET15. Paired sera from 152 children were available for assay. For CPA the geometric mean concentrations and proportions with > or =2 microg/ml before and after immunization were 0.69, 18% and 12.3, 96%, respectively. A significant increase in serum CPA was achieved by children of all ages, with the greatest response occurring after age 11 years. Among infants < lyear old 89% achieved concentrations of > or =2 microg/ml. For BA the pre- and post-vaccine geometric mean titers were 1.02 and 45.9. The response was significantly associated with age. BA titers > or =1:8 were present, before and after immunization, respectively, in 0 and 0% of infants <1 year old, 0 and 20% of 1- to 1.4-year-olds, 0 and 50% of 1.5- to 1.9-year-olds and 1 and 100% of > or =2-year-olds. CONCLUSION: The age-related total and functional group C meningococcal antibody response after quadrivalent polysaccharide vaccine among aboriginals is similar to that reported for Caucasian children. After age 2 all children made excellent CPA and BA responses. In the younger age groups the BA response was blunted but 82 to 95% achieved CPA titers of > or =2 microg/ml.

Neuronal apoptosis inhibitory protein expression after traumatic brain injury in the mouse.

Apoptosis of brain cells is triggered by traumatic brain injury (TBI) and is blocked by caspase inhibitors. The neuronal apoptosis inhibitor protein (NAIP), which has been shown to inhibit apoptosis by both caspase-dependant and caspase-independent mechanisms, is neuroprotective in rat models of cerebral ischemia and axotomy. In order to gain a better appreciation of CNS apoptosis following head injury in general and the possible involvement of NAIP specifically, we have configured a mouse model of TBI. In addition to demonstrating apoptosis, the spatiotemporal expression or levels of a number of proteins with apoptosis modulating effects have been determined. Apoptosis of neurons and oligodendrocytes following TBI was observed in brain sections which were triple-stained with in situ end labeling, bisbenzimide and immunofluorescent stain for neuron specific nuclear protein and myelin-associated glycoprotein, respectively. Further evidence for apoptosis following TBI in this model was obtained in brain samples using ligation-mediated PCR amplification of DNA fragments and gel electrophoresis. The temporal profile of apoptosis was similar to the temporal profile of microglial activation determined by CD11b staining and TNFa expression induced by TBI. NAIP staining in sections of cerebral cortex and subcortical white matter increased at 6 h and decreased towards control levels at 24 h post-TBI. Temporal changes in the expression of NAIP were also observed using Western blot analysis of brain samples removed from injured cortex and sub-cortical white matter. At the time that NAIP expression decreased markedly (24 h post-TBI), procaspase-3 levels also decreased, PARP cleavage increased, and the highest levels of apoptosis were observed. These findings have implications in our understanding of traumatically induced programmed cell death and may be useful in the configuration of therapies for this common injury state.

The effect of Kawasaki disease on cognition and behavior.

OBJECTIVE: To determine whether there are associated long-term deficits in the cognitive, academic, or behavioral outcomes of children with a previous episode of Kawasaki disease. DESIGN: Cohort analytic study. SETTING: A tertiary care pediatric hospital in Ottawa, Ontario. PARTICIPANTS: Thirty-two patients with a past diagnosis of Kawasaki disease. Siblings of the patients with Kawasaki disease were eligible to be controls. MEASURES: A blinded psychometrist (Y.K.) assessed cognition by the appropriate Wechsler Intelligence scale, academic achievement by the Wechsler Individual Achievement Test, and behavior by the Achenbach Child Behavior Checklist. RESULTS: No differences were found in cognitive or academic measures and the mean scores corresponded closely to national norms. Parents rated their children who had Kawasaki disease as having significantly more internalizing (P<.03) and attentional (P<.02) behavior problems than controls; the risk of a clinically significant behavioral score was 3.3 times greater (P<.03; 95% confidence interval, 1.1-9.9) than for sibling controls. CONCLUSIONS: While no effect on cognitive development or academic performance was demonstrated, these results provide preliminary indication of a post-Kawasaki disease deficit in internalizing and attentional behavior.

Antibody-recognized [125I]estradiol-receptor complex in ovarian epithelial carcinoma.

Monoclonal antibody against human breast cancer estrogen receptor was used to demonstrate binding of a gamma- and Auger electron-emitting estrogen to the estrogen receptor in ovarian epithelial carcinomas. When cytosols of estrogen receptor-rich ovarian adenocarcinomas were analyzed on sucrose gradients containing 0.4 M KCl, the presence of monoclonal antibody against breast cancer estrogen receptor caused a binding peak for [125I]- and [3H]estradiol to shift from the 4S to the 8-9S region, indicating antibody complex formation with ovarian adenocarcinoma estrogen receptor. The antibody-shifted peak of 8-9S [125I]- and [3H]estradiol binding was totally inhibited by a 100-fold molar excess of diethylstilbestrol (DES), but not by testosterone or progesterone, indicating a preference for estrogen binding by the antibody-shifted estrogen receptor. When estrogen receptor from the nuclear fraction of ovarian adenocarcinomas was incubated with [125I]estradiol at 1C, in the presence of 0.4 M NaSCN to facilitate exchange with endogenous ligand, binding occurred that was inhibitable by DES and restricted to the 4S region. Under these conditions the nuclear estrogen receptor was also shifted to the 8-9S region by the presence of the monoclonal antibody against estrogen receptor.

Studies on the formation of electrostatic complexes between benzethonium chloride and anionic polymers.

The chemistry of a mixture of benzethonium chloride and a copolymer of methoxyethylene-maleic anhydride was investigated. This mixture was of interest because it was effective in reducing dental plaque, calculus, and gingival inflammation in vivo. Evidence from dialysis, pH measurements, and stoichiometry demonstrated that the benzethonium cation and the anion of the hydrolyzed copolymer formed an electrostatic complex. An emulsion was produced when a stoichiometric excess of either component was present, but this mixture coacervated at stoichiometric quantities. The stability of the complex was pH dependent, and it did not form in 50% acetone. The complex was decomposed by simulated saliva, mainly due to calcium and magnesium ions, but was unaffected by salivary proteins. Other anionic polymers also formed this type of complex.

Toothbrushing with hydrogen peroxide-sodium bicarbonate compared to toothpowder and water in reducing periodontal pocket suppuration and darkfield bacterial counts.

This study attempted to isolate and test the therapeutic effectiveness of toothbrushing with hydrogen peroxide-sodium bicarbonate supplemented with scaling and systemic antibiotics. Forty-two subjects selected for pocket suppuration were divided into two groups. Group I was treated sequentially with brushing, scaling and systemic antibiotics. Group II was treated with brushing and scaling performed concurrently. Half of each group were control subjects using an inert toothpowder. Subjects were monitored by darkfield microscopy for spirochetes and motile rods. In Group I, toothbrushing alone showed approximately a 1/3 reduction in the number of suppuration sites, no difference between experimental and control subgroups and no significant changes in the darkfield counts. Scaling, whether subsequent to the toothbrushing (Group I) or concurrent with the toothbrushing (Group II), showed a statistically significant reduction (about 70%) in the number of suppuration sites in all subgroups. Darkfield counts after scaling were reduced significantly in some subgroups but not in others. The addition of systemic antibiotics in Group I resulted in an almost total elimination of suppuration sites and spirochetes in 15 subjects, but there were no significant differences between the test and control subgroups. In both Groups I and II, neither experimental peroxide-bicarbonate subgroup could be differentiated statistically from its toothpowder-water control at any time.