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To study the basic relationships between estrogen receptor immunocytochemical assay (ERICA) and dextran-coated charcoal (DCC) techniques, 116 women affected by breast cancer were admitted to the study from June 1985. In 56% of cases the tumor sample came from patients with operable disease and in 44% from patients with advanced inoperable disease. We found an overall agreement of 80% between the two methods characterized by a high sensitivity (95%) and a low specificity of ERICA versus DCC. ER/ICA negative results were found in 20 out of 78 ER/DCC positive cases; on the contrary, discordant results were found in only 3 out of 38 cases described by DCC as ER negative. 29 patients with advanced disease and ER/DCC positive tumors were considered for clinical analysis and reliability of each hormone receptor assay. A clinical response was reached in 69% of ER/DCC positive patients and on 64% of ER/ICA positive ones (16/25). However, it must be noted that 4/29 cases described as negative by ERICA were clinically responsive to hormone therapy.

The present study describes the estrogen receptor (ER) detection by an immunocytochemical assay kit (ER-ICA) on cryostat sections of 78 primary breast carcinomas. Results are compared with quantitatively measured ER levels, which were obtained by the dextran-coated charcoal (DCC) method. An excellent overall correlation between the logarithm of the ER levels, estimated by this technique, and the semiquantitative immunocytochemical evaluation was found, i.e. r = 0.73. Since the ER-ICA can be easily handled without radioactivity being involved and since it is more representative of the total tumor, we conclude (as other groups before us) that the ER-ICA is an easy-to-handle and reliable technique presenting many advantages over the DCC method.

Anti-Iatk monoclonal antibodies (mAbs) were found to inhibit syngeneic mixed lymphocyte reaction (SMLR) of mice with k and a haplotypes (H-2k and H-2a) of the major histocompatibility complex (MHC) by acting on responder T cells but not stimulator cells. Only the early phase of SMLR was inhibited by anti-Iat mAbs. The inhibitory effect was due to the blocking of autoreactive T cells but not induction of suppressor lymphocytes. Cross-linking of Iat epitopes induced T cell proliferation of k haplotype strain. The inhibitory pattern of SMLR by four anti-Iat mAbs varied among different strains of mice. The inhibitory pattern seemed to depend on MHC and unidentified non-MHC background genes possessed by stimulator cells, suggesting that the shape of the MHC recognition site must have different conformation depending on both MHC and non-MHC gene products recognized. However, the inhibitory pattern of SMLR by anti-Iat mAbs of strains which differ at the I-J locus: B10.A(5R) and B10.A(3R) was dependent on the genotype of stimulator cells. The same was observed in B10.S(9R) and B10.HTT strains. The inhibitory activity of anti-Iat mAbs was entirely directed against responder T cells and was not passively carried out by the stimulators. It was concluded that Iat epitopes are I region controlled determinants that are utilized by T cells as receptors for self Ia antigens. It is suggested that anti-Iat mAbs react with an idiotype on the receptor(s) for Ia or I-J, or possibly a "receptor" for these receptors.

The classical morphological criteria in the diagnosis of hepatocellular carcinoma (HCC) include: (a) the similarity of tumor cells to hepatic cord cells; (b) the trabecular nature of the growth with capillary and canaliculi formation, and (c) the intravascular growth of trabecular carcinoma. These criteria apply to the most common variants of HCC but they do not suffice in all cases. That makes additional criteria and certain refinements necessary. A promising approach to the diagnosis of HCC is that based upon consideration by the pathologist of some relevant aspects of the natural history of this tumor. A panel of tests exploring the various functions and properties of liver cells should be set up. Tests for bile and fibrinogen synthesis are most important because they reflect specific and exclusive properties of the original cell line. Bile synthesis in tumor tissue is reflected by the finding of cholecholatestasis, namely bilirubinostasis and retention of copper and copper-binding proteins. The positive immunostaining for fibrinogen may appear in the form of cytoplasmic granules occurring in 50% of HCC, or in the form of fibrinogen-ground-glass (G-G) inclusions, representing a specific feature of HCC. During neoplastic transformation oncofetal proteins may reappear, alpha-fetoprotein (AFP) being very common. Despite sensitivity of AFP, this test, similar to alpha-1-antitrypsin (AAT), has very low specificity, because of the widespread occurrence of these proteins in a variety of tumors. The selection of special 'clones' such as Mallory bodies and fibrinogen-G-G is of particular value, because of the specificity of these peculiar cytoplasmic changes. Although rare, the presence of HBV antigens in tumor tissue is virtually pathognomonic for HCC. The availability of nonneoplastic liver tissue for morphological examination is of great help, because it may carry key information or markers of the development stages of HCC: cirrhosis, liver cell dysplasia, HBV antigens, congenital metabolic disorders, such as hemochromatosis and AAT deficiency. The two latter conditions represent the link with the last working hypothesis of the present study, i.e. that during neoplastic transformation hepatocytes may 'switch' their 'phenotype' thus escaping the storage phenomena, which continue to occur in nonneoplastic hepatocytes. This study provides a guideline to a dynamic approach to the diagnosis of HCC. The rationale is listed in 5 points; among them, bile production, fibrinogen synthesis, Mallory body and fibrinogen-G-G selection, HBV antigen expression can be considered at present as confident markers for the morphological diagnosis of HCC.

Heparin-induced alterations of platelet function have repeatedly been reported over the last 30 years. The development of low-molecular-weight (LMW) heparin fractions prompted us to compare the effect of unfractionated (UF) heparin and LMW heparin on platelet function. Heparin was applied intravenously to healthy volunteers in a dose of 100 U (antifactor Xa)/kg body weight. Sequential evaluation of platelet function ex vivo confirmed that UF heparin may activate platelets in vivo. Studying the ADP-induced fibrinogen binding to platelets in vivo, we were able to show that the effect of heparin on platelets is antibody- and antithrombin-III-independent. LMW heparin fractions in general exhibit a reduced platelet activating activity although there are differences between the LMW heparin fractions available. The stimulating effect of LMW heparin on platelets disappears below a mean molecular weight of 3,000 daltons.

In a clinical, between-patient study we investigated the effects of a VLMW Heparin fragment (CY 222) versus standard heparin (SH) in plasma exchanges (n = 10) on coagulation factors (CF). Fibrinogen (FGN), II, V, VIIF + X, IX, XI, XII, VIIIc, VIIIRag, VIIIvwf and ATIII, ProtC, ProtS, Plasminogen (PGN), Activated Thromboplastin time (APTT), Prothrombin time (PT), Thrombin time (TT), anti factor Xa (Axa), DDimer, Platelet count. Heparin was administered as a bolus and by infusion during the session, CY 222 as a bolus dose only; 1 to 1.5 plasma volume was exchanged with substitution by 5% albumin. Results (mean, s.d.) at the end of the session (End) and 4 hours later (T4) were analyzed and showed no differences between groups (with CY 222 and with SH) for technical and clinical findings. Biologically, CF were similar in both groups except for Factor XII levels at the end of the session, and Factors II, V, XII at T4. Prolonged APTT in all samples appear related to low FGN. Significant differences in Axa activities were found for each treatment when compared with its own standard, suggesting different ranges of activities of both drugs. Changes in D-dimer levels differed during the session and four hours after the session with the drug tested, and could be related to their mode of administration. Clinical efficiency and tolerance were excellent both with CY 222 and SH.

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.

Ovarian cancer specimens from 45 patients were assayed for the presence of estrogen receptor (ER) utilizing highly specific monoclonal antiestrophilin antibodies and the peroxidase-antiperoxidase technique. Results were compared with the conventional ER determination of the dextran charcoal method (DCC) and were in concordance in 91% of cases. This technique was also used to analyze ER in fine needle aspiration biopsy specimen from four patients with metastatic ovarian tumor. These results suggest that antireceptor monoclonal antibody in immunohistochemical analysis is an effective tool in the evaluation of estrogen receptor content in ovarian cancer. This technique can be easily performed at community hospitals and may be extended to the evaluation of fine needle aspiration biopsies and to analyze specimens of insufficient size for biochemical assay.

Now that monoclonal antibodies against the estrogen receptor (ER) have become available, it is possible to detect the presence of ER immunohistochemically. In order to evaluate the validity of the immunohistochemical (ERICA) method in a routine detection of ER, we have tested the method in a multicenter investigation. As reference is used a conventional biochemical assay, dextran-coated-charcoal assay (DCC). The investigation analyzes the validity of the ERICA-assay, including a semiquantitative estimation of the ER-content, when the method is used in several independent departments of pathology, and the interobserver variation of the analysis. The results show that the qualitative detection of ER, obtained by the ERICA-assay and the DCC-assay has a concordance of about 90% for all the participating departments. The concordance is around 60-70%, when a semiquantitative estimation of the ER-content is used. The interobserver variation is also very low, with full agreement in 58 of 59 cases. The ERICA-assay may, therefore, be a relevant alternative to the DCC-assay. However, its clinical importance still has to be fully elucidated.

Estrogen receptor (ER) immunocytochemical assay (ER-ICA) was assessed human breast carcinomas. The patient's age, the tumor size, the histological type and SBR grade, the presence or the absence of axillary lymph node metastases and of vessel invasion in tumor borders were recorded in all the cases included in the series (n = 469) estrogen and progesterone receptors were concomitantly evaluated (DCC method) and or immunoenzymatic assays. Monoclonal H222 sp gamma and PAP procedure (Abbott kit) were applied in frozen sections, tumor imprints and fine needle aspirates. A computerized system of image analysis referred to as SAMBA (TITN), permitted to achieve a multiparametric quantitative analysis of ER positive surfaces. With this system, in each tumor, the cellularity, the percentage ER surface versus the total cell surface and versus the epithelial (keratin positive) surface, integrated optical density (IOD), mean optical density, index of the concentration of labelled objects, and IOD histograms, were obtained and correlated to histological and biochemical data. It was shown that: 1) ER antigenic sites were heterogeneously distributed in ER positive tumors, with a specific nuclear localization in epithelial cells; 2) the SAMBA 200 multiparametric analysis of the ER sites distribution in tissue was appropriate, accurate, reproductible and therefore more reliable than the semi-quantitative analysis; 3) the standardization and the complete automation of this method of immunoprecipitates evaluation on tissue section permit to daily and to routinely analyse a large number of preparation; 4) there was a correlation between ER binding sites evaluation (DCC) and ER antigenic sites immunodetection (ER-ICA and ER-EIA); 5) there was a correlation between the SAMBA evaluation of ER-ICA and other histological prognostic factors such as small tumor size, low SBR grade, the absence of axillary lymph node metastasis and vessel invasion; 6) the preliminary SAMBA analysis of ER-ICA in tissue sections, imprints and fine needle aspirates suggest that fine needle aspirates may not reflect accurately the tumor cell heterogeneity.

Transferrin saturation was determined in 11,431 men and 10,639 women aged 15 or more drawn from different areas in southern and central Finland and attending a multiphasic health screening examination in 1967-1972. All the 163 men and 66 women with transferrin saturation greater than or equal to 70% at the initial examination and still alive at the end of 1983 were invited to a re-examination. Of the invited persons, 76% attended the re-examination. Transferrin saturation and serum ferritin were the initial screening methods in the re-examination. All persons with suspected hemochromatosis were clinically examined and a laparoscopy was performed. Four men and four women were found with unequivocal hemochromatosis. Only one of these cases was diagnosed beforehand. According to these data the prevalence of hemochromatosis in Finland is about 50/100,000.

A 1,1,2-triphenylbut-1-ene with a 4-OH group at one C-1 phenyl ring and a chlorocarbamate mustard moiety at the second C-1 ring (compound 3) was synthesized in order to obtain a "cytotoxic estrogen" with a specific antitumor effect on estrogen-receptor-containing tumors. This compound was tested in comparison to the carrier (compound 1) and a compound (2) having a carbamate mustard group on both C-1 phenyl rings. The estrogen receptor affinity of compound 3 was only about one-quarter lower than that of compound 1, but much higher than that of compound 2. Compounds 2 and 3 showed only partially irreversible binding to the receptor owing to their relatively low alkylating properties. The growth inhibition of the receptor-positive MCF-7 breast cancer cell line by compound 3, but not by compound 1 or 2, was more pronounced than the inhibition of the receptor-negative line MDA. In vivo the hormone-dependent, transplantable mammary tumor MXT M3.2 of the mouse was much better inhibited by compound 3 than its hormone-resistent line MXT OVEX. Compounds 1-3 had no antiestrogenic properties in the mouse, but estrogenic activity was in the order 1 greater than 3 greater than 2. From these results and because the antitumor activity of compound 3 was superior to that of compounds 1 and 2 in the hormone-dependent tumor models, a selective, receptor-mediated cytotoxic effect of compound 3 on estrogen-receptor-positive tumors in obvious.

We developed a lymphocyte ferritin antibody-binding test (LFABT) to measure lymphocyte surface ferritin (LSF) and used it in cases of malignant and other diseases associated with elevated serum ferritin. LSF was elevated in 33 of 83 patients with a variety of malignant neoplasms in all stages of disease. LSF was also elevated in 2 of 5 patients with infectious mononucleosis, but was normal in all 15 patients with rheumatoid arthritis, bacterial infections and hemochromatosis. LSF and serum ferritin levels do not correlate. These findings suggest the usefulness of LFABT as a diagnostic tool and demonstrate the biologic significance of LSF.

There have been some reports on the risk of developing hemosiderosis in hemodialysis patients when heavily transfused and simultaneously possessing hemochromatosis alleles (HA). We evaluated 99 patients on chronic hemodialysis estimating their serum ferritin (SF) levels, transfusion rate, and prevalence of HLA A3, B7 and B14 alleles, which are considered to be more frequent in idiopathic hemochromatosis. We analyzed the patients as a whole group and also separately as low or high transfusion groups. There was no correlation between the number of HA and the mean SF levels. The presence of HA is not a risk factor for the development of hemosiderosis when excessive transfusions and parenteral iron administration are avoided.

1. GH in Australia is significantly associated with the HLA-A3 antigen, which is the only independent marker for the disease (B7 in linkage disequilibrium with A3). 2. The haplotype A3, B7, DR2 is the only one with increased prevalence in this disease, presumably due to its being the predominant haplotype among early immigrants. 3. Exceptions to HLA association in GH are rare and can be explained by: (1) incorrect HLA serotyping, (2) chromosomal recombination, or (3) rare homozygous-homozygous mating. 4. These data are consistent with GH being due to a mutant gene or genes in close proximity to HLA-A. 5. Heavy alcohol ingestion does not lead to expression of hemochromatosis in heterozygous subjects.

The authors evaluated the ability of a monoclonal antibody immunoperoxidase procedure (ERICA [Estrogen Receptor Immunocytochemical Assay], information from Regulatory Affairs Department, Abbott Laboratories, North Chicago, IL) to detect estrogen receptor in aspiration biopsy cytology (ABC) specimens from breast cancer routinely taken by fine-needle aspiration during office diagnostic evaluation. Results were correlated with biochemical values determined from dextran-coated charcoal (DCC) assay on tumor tissue obtained subsequently at operation. ERICA had positive results in 32 of 41 DCC-positive cases (sensitivity, 78%) and in 5 of 17 DCC-negative cases (specificity, 71%). The semiquantitative degree of ERICA positivity correlated with the concentration of estrogen receptor by DCC. Results of both assays correlated with the histologic grade of the tumor and patient age. Estrogen receptor can be determined by immunocytochemistry in ABC specimens in a community hospital. However, the sensitivity and specificity of this procedure compared with biochemical assay, and eventual response to hormonal therapy, require further investigation.

Our present state of knowledge regarding estrogen and progesterone receptors (ER and PgR) has led to changes in treatment strategies: patients without receptors in their tumor tissues cannot be expected to respond to endocrine therapy. Furthermore, groups of patients with specifically good or poor prognoses can be selected. Treatment of the disease now approaches being of a rational rather than of an empirical nature. However, it is imperative that we achieve a considerably higher level of understanding before we can predict, with high probabilities, which patients will benefit from endocrine therapy. Only through a coordinated effort by many centers can we hope to attain this goal. In such collaborations there are several factors that must be taken into consideration if reproducible conclusions are to be reached: a) sampling of the tumor biopsy for analysis, b) potential differences in assay procedures which may affect results, and c) the composition of the population studied. Since the traditionally used ligand binding assay (dextran-coated charcoal (DCC) method) is highly sensitive even to slight modifications in assay procedure, intra- and interlaboratory standardization of receptor analyses is challenging. Accordingly, correlations between receptor status and/or concentrations and the clinical course of the disease from different centers often demonstrate discrepant results. With the greater reproducibility and sensitivity of the newly developed immunoenzymometric assay (IEMA) methods, many of these problems might be solved in the future and inter-center clinical studies will thus be facilitated. In the national Danish Breast Cancer Cooperative Group (DBCG) project, approximately 90% of all patients with primary breast cancer are registered. Estrogen and progesterone receptor (ER and PgR) determinations have been performed on tumor tissue from approximately 30% of these patients in one single laboratory. The results of these analyses are presented here for approximately 4,000 patients in relation to age, menopausal status, tumor size, grade of anaplasia, and lymph node involvement. Biologically and clinically there appear to be three fundamental types of tumor tissues; hormone responsive (ER+PgR+ and ER-PgR+), hormone non-responsive (ER-PgR-), and tissues of a more dubious hormone responsive nature (ER+PgR-), which occur predominantly among postmenopausal patients. Several lines of evidence indicate that among the postmenopausal patients there may be an estrogen binding molecule similar to but distinct from the normal, physiologically functioning ER molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

A survey of literature data and the author's own observation of 18 patients with hemochromatosis (H) have shown that H is a polysyndrome disease. Men aged 35 to 60 suffer more frequently. Its most significant symptoms and syndromes are skin hyperpigmentation, hypersideremia, liver cirrhosis, diabetes, cardiomyopathy, and endocrinopathy. Three variants in a course of H--mild, average severe and severe (complicated)--were defined with regard to the duration of disease, a degree of morphofunctional changes and insufficiency of the affected organs. Intravital diagnosis of H was possible in clinical awareness and in the detection of hypersideremia and hemosiderin in liver and skin biopsy specimens. Multimodality therapy (blood-letting, desferal and insulin) promoted compensation of metabolic derangement, stabilization and even regression of disease.

In summary, the work reviewed in the present paper indicates that 1. Iron and the iron-binding proteins can act as regulators of immune function, and not only as a result of a nutritional dependence of lymphoid cells on transferrin and transferrin-iron. Subsets of cells of the immune system respond differently to increases in iron concentration in vitro and in vivo. 2. Macrophages and lymphocytes differ in the H and L subunit content of the ferritins synthesized in response to increases in iron concentration in vitro. 3. NK activity by adherent and nonadherent cells differ in their susceptibility to the enhancing effect of lactoferrin in vitro. 4. Responses to mitogen stimulation by PHA and Con A are diminished, while the PWM response remains unaffected by exposure to acidic ferritins or by increasing concentrations of iron in vitro and in vivo. 5. Pretreatment of effector but not target cells with iron results in diminished responses in the MLR, an effect that appears to be related to the HLA-A locus. 6. In situ hybridization studies indicate that transferrin is synthesized by a specific subset of the T lymphocytes. 7. Transient increases in serum iron concentration above the full saturation of transferrin, reproducing the clinical situation frequently seen in hereditary hemochromatosis, are followed by a series of cellular changes in the synovium that can be correlated to changes in the course of an experimental model of arthritis in the rat.

Patients with previous myocardial infarction were tested for antithrombin-III (AT-III) activity and selenium levels in their plasma and compared with sex- and age-matched healthy control individuals. Patients and controls showed a positive correlation between AT-III and selenium levels (r = 0.27, p = 0.015). After calculatory adjustment for this correlation, selenium was found to be significantly negatively correlated with disease. Multivariate analysis of differences between patients and controls indicated that triglyceride levels in serum had the greatest discriminatory ability (r2 = 0.169), followed by AT-III (r2 = 0.072) and selenium (r2 = 0.056). The increased AT-III levels were correlated with the use of warfarin and beta blockers in the patients, but these drugs could not explain the comparatively low selenium levels in the patients. Serum total cholesterol and plasma fatty acid composition had no discriminatory power in multivariate testing. The various fatty acid did not show co-variation with the selenium levels. The clinical significance of these observations is not clear, but they are consistent with the hypothesis that selenium is an important determinant in cardiovascular disease. The relation between AT-III and selenium should be further evaluated.

No abstract available.