Tissue distribution of histo-blood group antigens.

The introduction of immunohistochemical techniques and monoclonal antibodies to specific carbohydrate epitopes has made it possible to study in detail the tissue distribution of histo-blood group antigens and related carbohydrate structures. The present paper summarizes the available data concerning the histological distribution of histo-blood group antigens and their precursor structures in normal human tissues. Studies performed have concentrated on carbohydrate antigens related to the ABO, Lewis, and TTn blood group systems, i.e. histo-blood group antigens carried by type 1, 2, and 3 chain carrier carbohydrate chains. Histo-blood group antigens are found in most epithelial tissues. Meanwhile, several factors influence the type, the amount, and the histological distribution of histoblood group antigens, i.e. the ABO, Lewis, and saliva-secretor type of the individual, and the cell- and tissue type. Oligosaccharides with blood-group specificity are synthesized by the stepwise action of specific gene-encoded glycosyltransferases. In general, this stepwise synthesis of histo-blood group antigens correlates with cellular differentiation. The H and the Se genes both encode an al-2fucosyltransferase, which is responsible for the synthesis of blood group antigen H from precursor disaccharides. A new model for the participation of the Se/H-gene-encoded glycosyl transferases in synthesis of terminal histo-blood group antigens in human tissues is proposed; the type and degree of differentiation rather than the embryologic origin determines whether it is the H or the Se gene-encoded transferases that influence expression of terminal histo-blood group antigens in tissues.

Immunohistochemical evaluation of estrogen and progesterone receptors in paraffin-embedded, formalin-fixed endometrial tissues: comparison with enzyme immunoassay and immunohistochemical analysis of frozen tissue.

Monoclonal antibody (MoAb) 1D5 with specificity to the estrogen receptor (ER), MoAb 1A6, and a polyclonal antibody (PoAb) (the latter two with specificity to the progesterone receptor [PgR]) were used to stain microwave-pretreated sections of formalin-fixed, paraffin-embedded normal and malignant endometrial tissues (n = 60). The tissues were previously evaluated for ER and PgR by enzyme immunoassay (EIA) (n = 44) and immunohistochemical analysis of frozen tissue (ICAfroz, n = 59). With results of EIA as a reference, the ER-1D5 method yielded a better agreement on receptor status, i.e., positive versus negative (74 vs. 51%) and a higher sensitivity (71 vs. 45%) but a similar high specificity (100%) than the ER-ICA method. Compared with results of PgR-EIA, the immunohistochemical assays for PgR gave similar results as to agreement (86-95%) and sensitivity (95-97%). Quantitative agreement on the fraction of cells stained for ER and PgR by immunohistochemical analysis in frozen and formalin-fixed tissue was obtained in approximately 60% of the cases. The results of semiquantitation were correlated with the results of both ICA and EIA. The MoAbs 1D5 and 1A6, as well as the anti-PgR PoAb, thus seem to be valid for evaluation of ER and PgR status in formalin-fixed endometrial tissue. Differences in the specificity of the Abs and in the sensitivity of the methods used to demonstrate ER and PgR might explain some of the discordant findings.

Loss of a novel mucin-like epithelial glycoprotein in oral and cervical squamous cell carcinomas.

Stratified squamous epithelia of oral and cervical mucosa express high levels of simple mucin-type O-linked carbohydrates, and these are known to undergo structural changes in relation to epithelial differentiation and neoplastic transformation. O-glycans in these epithelia are associated with the cell membrane, but the identity of the carrier molecule(s) remains largely unknown. We report here the identification of a membrane-bound M(r) 200,000-250,000 glycoprotein (gp230) that is expressed in stratified squamous epithelia of the oral cavity. Western blot analysis identified gp230 as a major carrier of simple-mucin type carbohydrate antigens in buccal nonkeratinized mucosal epithelium, suggesting that it may represent a mucin-like molecule. A monoclonal antibody PANH4 defining a protein epitope of gp230 was generated. The PANH4 epitope was localized by immunohistology to suprabasal cell layers of buccal epithelium and was also found in larynx, esophagus, vagina, and exocervix, but not in epidermis. Data showed that gp230 was distinct from MUC1 or CD44. It is interesting that in most cases gp230 was not expressed in squamous cell carcinomas of buccal and cervical mucosa. A few moderately differentiated carcinomas, mainly from cervix, expressed the gp230 epitope. The results suggest that a membrane-bound mucin-like molecule, gp230, is associated with the differentiated phenotype of normal mucosal stratified squamous epithelia and that expression of gp230 generally is lost in severe oral epithelial dysplasia and squamous cell carcinomas of oral and cervical mucosa.

The use of polymerase chain reaction to detect metastatic cancer cells within lymph nodes in stage I cervical carcinoma.

The aim of the study was to determine whether human papillomavirus (HPV) in lymph nodes is a useful marker for the risk of recurrence in patients with HPV-related cervical cancer. The polymerase chain reaction and DNA-DNA hybridization techniques were used to examine 149 formalin-fixed, paraffin-embedded lymph nodes that had been resected from 24 patients undergoing radical hysterectomy for stage IB cervical carcinoma. The lymph nodes were examined for the HPV type, which in each case was found in the cervical tumor. Of 18 patients with histologically negative nodes, HPV DNA was found in a lymph node in two of 10 patients who later experienced a recurrence and in three of eight patients who were alive and well for > or = 5 years after surgery. In addition, HPV was detected in the lymph nodes of two of four patients with nodal metastases at the primary surgery; four of nine histologically positive lymph nodes in these patients contained HPV. It is concluded that detection of HPV in resected lymph nodes is probably not a useful means of identifying the cervical cancer patients who might benefit from adjuvant postoperative therapy.

Simple mucin-type carbohydrates in normal and malignant human endometrium.

The simple mucin-type carbohydrate antigens, Tn, sialosyl-Tn, and T, are tumor-associated antigens of adenocarcinomas. We evaluated by immunohistochemistry the expression of Tn, sialosyl-Tn (s-Tn), T, and sialosyl-T (s-T) antigens in normal nonsecretory, early gestational, and malignant human endometrium in relation to genetic (ABO/Lewis blood-type) and hormonal factors. The blood group status had no influence on staining. Tn and s-Tn antigens were infrequently demonstrated in normal nonsecretory endometria but showed an increased expression in adenomatous hyperplasias and endometrial carcinomas, which was unrelated to estradiol (E2) levels, grade, and stage. The T disaccharide was demonstrated infrequently in both normal and malignant endometrium. Staining for T antigen showed an inverse correlation to serum E2 levels. In contrast, s-T antigen showed a pronounced but varied expression in normal and malignant endometrium, and the expression of s-T antigen was positively correlated with E2 levels in serum. Our findings suggest a hormonal influence on expression of simple mucin-type carbohydrates in human endometrium. However, the accumulation of Tn and s-Tn antigens in malignant endometrial cells seem to be unrelated to both genetic and hormonal factors. Because s-Tn is also expressed by secretory endometria, only Tn antigen can be considered a "tumor-associated" antigen of endometrial tissue.

Estrogen- and progesterone receptors in normal cycling endometrium as studied by end-point titration.

A thorough knowledge of the normal physiological fluctuations in estrogen- (ER) and progesterone receptors (PgR) is essential to characterize the changes in ER and PgR in the abnormal endometrium. We investigated the distribution of ER and PgR in frozen human cycling endometrial tissue using the commercially available ER- and PgR-ICA kits. Two-fold end-point titration (EPT) of ER and PgR antibodies was implemented to semi-quantitate more accurately ER and PgR. Semiquantitation of ER and PgR using EPT was significantly correlated to results obtained using either simple scoring or enzyme-immunoassay (EIA) methods. ER and PgR staining fluctuated in relation to the menstrual cycle. In most subphases PgR exceeded ER in both epithelial and stromal cells. Highest levels of ER and PgR were demonstrated in the glands of the functionalis in mid-to-late proliferative phases, whereas both receptors were almost undetectable by immunohistology in the glands of mid-to-late secretory phases. Endometrial stromal cells had high and nearly constant EPT values for PgR, but low values for ER throughout the menstrual cycle. EPT values for ER and PgR were generally higher in the basalis than in the functionalis but showed similar cyclic fluctuations. Our results further substantiate the view that the response to hormonal stimulation is cell-type specific, and suggest differences in steroid metabolism according to cell type and layer.

Type-1 chain histo-blood group antigens (Le(a), monosialosyl-Le(a), disialosyl-Le(a), Le(b), and H) in normal and malignant human endometrium.

Type-1 chain histo-blood group antigens such as the Lewis (Le)a, monosialosyl-Le(a), Le(b) and H antigens show an increased expression in endometrial carcinomas. However, the possibility that these antigens are expressed under genetic or hormonal influence in endometrial carcinomas has not been considered. In the present study, the expression of type-1 chain carbohydrate antigens in normal and malignant endometrium was evaluated by immunohistochemistry and related to both genetic and hormonal factors. The glands of normal, non-secretory endometria expressed, in contrast with surface epithelial cells, Le(a), Le(b), disialosyl-Le(a), and H determinants infrequently. Adenomatous hyperplasias and endometrial carcinomas showed an increased expression of type-1 chain carbohydrates that was qualitatively influenced by the erythrocyte Lewis phenotype and the secretor status. Whereas Le(a+b-) non-secretors mainly accumulated Le(a) antigen, and only limited amounts of Le(b) antigen, Le(a-b+) secretors expressed H, Le(b) and Le(a) antigens. The expression of type-1 chain antigens showed no association with the serum-oestrogen level or to the hormone-receptor status. Thus the Lewis secretor status has a qualitative influence on the increased expression of type-1 chain antigens, which, however, seem to be unrelated to hormonal factors. Our findings suggest an increased activity of the Se-gene-defined or a closely related fucosyl-transferase in neoplastic endometrial epithelial cells.

Expression of type-2 histo-blood group carbohydrate antigens (Le(x), Le(y), and H) in normal and malignant human endometrium.

Changes in expression of histo-blood group ABH and Lewis antigens are common alterations in carcinomas. Using immunohistochemistry, we have evaluated the expression of type-2 histo-blood group antigens in normal and malignant endometrial tissues in relation to genetic and hormonal factors. The Le(x), sialosyl-Le(x), and Le(y) antigens were inconstantly expressed in the normal endometrium. The expression was uninfluenced by the secretor status but was related to the ABO blood group status in Oestradiol (E2) stimulated endometria. Le(y) was expressed most frequently in proliferating endometria from blood group 0 individuals. Le(x) and Le(y) were maximally expressed in atrophic endometria, and Le(x) and Le(y) staining scores correlated inversely with serum levels of E2 in normal, non-secretory endometria. No correlation was found in adenomatous hyperplasias and endometrial carcinomas, which when compared with atrophic endometria, showed a loss of Le(x) and Le(y) and an increased H-carbohydrate expression at apical membranes. Carcinomas from non-secretors showed lower expression of Le(y) and H-antigens than carcinomas from secretors. Our findings suggest that the genetic and hormonal influence on glycosylation based on type-2 chain carbohydrates differ between normal and malignant endometrium. This difference is probably related to specific tumour-associated qualitative and quantitative changes in the fucosyltransferases.

Reproducibility of subjective immunohistochemical estrogen- and progesterone receptor determination in human endometrium.

To obtain a reliable scoring system for semi-quantitation of estrogen- (ER) and progesterone receptors (PgR) in human endometrial tissue, we investigated the reproducibility of subjective immunohistochemical ER and PgR determination. Specimens of frozen endometrial tissue were stained once (n = 129) or twice (n = 22) using the ER-ICA and PgR-ICA kits from Abbott. The semi-quantitative approach we used included subjective estimates of the overall staining intensity (I) and of the fraction of stained cells (%). Scoring was performed twice by the same observer and once by another observer (n = 87). Intra- and inter-observer agreement were evaluated using Kappa statistics. We found that more comprehensive scorings of I and % could not be agreed upon by the observers. Only simplified estimates of the fraction of cells stained and overall staining intensity were reproducible. Subjective estimates obtained by this method agreed with estimates obtained by counting (n = 38). Simplified H-scores, which were obtained by multiplication of the simplified estimates of % and I, were reproducible, too. In addition, semi-quantitation of ER and PgR by immunohistology was significantly correlated to quantitation by enzyme-immunoassay (n = 39). Thus, it was possible to reproducibly semi-quantitate ER and PgR only by employing a very simple immunohistochemical scoring of ER and PgR.

The distribution of type 1 chain ABH and related histo-blood group antigens in normal cycling human endometrium.

Cycling endometrial tissue was examined immunohistochemically for blood group antigens using a panel of monoclonal antibodies with specificity to type 1 chain carbohydrates. Staining was evaluated against the genetic background (ABO, Lewis, and saliva-secretor status) and the subphase of the menstrual cycle. Expression of type 1 chain antigens in most cases correlated with the genetic background; however, Le(a) and Le(b) antigens were in a few cases demonstrated in endometria with the erythrocyte Le(a-b-) phenotype and Le(b) antigen in erythrocyte Le(a + b-) endometria also. In addition, Le(b) antigen was preferentially expressed in endometria from blood group O individuals. Type 1 chain antigens were, in general, maximally demonstrated in the surface epithelium. Chain 1 A, H, ALe(b), Le(a), and disialosyl-Le(a) (dsLe(a)) determinants were demonstrated only sporadically in functionalis and basalis glands of cycling endometria. Staining for most type 1 chain antigens showed variations that were related to the histomorphology (layer and menstrual phase), and monosialosyl-Le(a) (msLe(a)) seemed to be a marker of secretory differentiation in the endometrium. Our results support the view that synthesis and expression of type 1 chain antigens in human endometrium is influenced by the genetic background and is modulated by the hormonal environment.

The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium.

The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le(x) were demonstrated to be uninfluenced by the genetic background. A and Ale(y) antigens were exclusively demonstrated in endometria from blood group A individuals, while Le(y) was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe(y) antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le(x), sialosyl-Le(x), Le(y)) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.

Mucin-type carbohydrates (type 3 chain antigens) in normal cycling human endometrium.

Carbohydrates related to the ABO, Tn, and T blood group systems are markers of cellular differentiation in many epithelial tissues. Using a panel of specific monoclonal antibodies, we have immunohistochemically investigated the expression of mucin-type (type 3 chain ABO-related) antigens in 64 samples of normal cycling endometria of known ABO and Lewis blood type. Tn and T antigens had a highly restricted expression in normal cycling endometrial tissue. The expression of type 3 chain H and A antigens was always compatible with the ABO blood type of the individual and seems to be regulated by the secretor genes, as secretors [Lewis (a-b+)] expressed more A and H type 3 chains than nonsecretors [Lewis (a+b-)]. The expression of type 3 chain H and A, sialyl-Tn and sialyl-T antigens showed cyclic variations in the glandular epithelium of the functionalis, but not of the basalis layer of cycling endometrium. A hormonal regulation of the enzymes involved in blood group-related carbohydrate chain elongation in human endometrium is thus possible and may participate in the specialized secretory process of human cycling endometrium.

Distribution of histo-blood group antigens in cervical and uterine endometrium.

Cell surface carbohydrates serve as differentiation and developmental markers characteristic of different cell and tissue types. Expression of these carbohydrate antigens is often significantly altered in tumors particularly in those arising from epithelial tissues. Analysis of the expression of cell surface carbohydrates in normal endometrium has shown that this glycosylation is hormonally influenced. Change in expression of carbohydrates in malignant tissue should therefore be evaluated against this normal fluctuation. In normal cervical uterine epithelium the result shows that the glycosylation of metaplastic squamous cells is different from that of original squamous cells indicating that the regulation and differentiation of the epithelium in the transformation zone is different from that of the original squamous epithelium. This variation in expression of carbohydrates seen in the metaplastic epithelium may be of importance for the development of carcinomas in this area.

Human milk-fat globule membrane antigens (Mam-3 group) in normal cycling endometrium and endometrial carcinomas--an immunohistochemical study. A preliminary report.

The expression of Mam-3 antigens in normal human endometrium and endometrial carcinomas was investigated employing the monoclonal antibodies 67D11 (anti Mam-3a), 115H10 (anti Mam-3b), and 115C2 (anti Mam-3c). Dewaxed sections of formalin-fixed, paraffin-embedded curettings of 9 proliferative-, 6 interval-, 13 secretory endometria, as well as 16 endometrial carcinomas were used. In normal cycling endometrium the Mam-3a, -3b and -3c antigens were detected at the apical membrane of surface and/or glandular epithelium only, but to a different degree. The Mam-3a antigen was only detected in the surface epithelium of 66% of proliferative and 33% of interval endometrium. The Mam-3b antigen was detected in surface and glandular epithelium of all normal endometria, but was most widespread in proliferative endometrium. The Mam-3c antigen was detected in surface epithelium of proliferative, and interval endometrium especially, and in glandular epithelium in 22% of endometria irrespective of the phase. Increased expression of Mam-3a and -3c antigens was recorded in most carcinomas. A loss of polarity of staining was seen in grade III carcinomas especially, staining of the cytoplasma was recorded in half of the carcinomas. The expression of Mam-3c antigen in carcinomas seemed to decline with increasing grade of anaplasia. All carcinomas expressed the Mam-3b antigen. The present results indicate that the Mam-3a and -3c antigens are tumor-associated antigens of endometrial carcinomas and that they might be useful as diagnostic tools in endometrial pathology.

Thomsen-Friedenreich-related antigen in human endometrium. An immunohistochemical study employing the monoclonal antibody 49H.8. A preliminary report.

The Thomsen-Friedenreich antigen (T-antigen) Gal beta 1-3Ga1NAc, is a cryptic disaccharide structure on human erythrocytes. It is masked by sialic acid and uncovered by sialidases as neuraminidase. T-antigen is a tumor-associated antigen in epithelial tissues. This study reports preliminary immunohistochemical findings on the expression of the blood group related T-antigen in human endometrium. Dewaxed sections of 10 proliferative-, 6 interval-, 14 secretory endometrium, and 16 endometrial carcinomas, either untreated or pretreated with neuraminidase, were subjected to an indirect immunoperoxidase technique using the monoclonal antibody 49H.8. The monoclonal antibody 49H.8 did not bind to normal cyclic endometrium, unless pretreated with neuraminidase. Neuraminidase treatment revealed staining of apical membranes of glandular- and surface epithelium in early or manifest secretory endometrium only. Thirteen of sixteen carcinomas stained without neuraminidase treatment, while all carcinomas stained after pretreatment with neuraminidase. Staining was more widespread than in untreated carcinomas, resembling that of secretory endometrium. Our results suggest: 1) That the sialylated 49H.8 antigen seems to be a marker of cellular differentiation in the endometrium, 2) that the antigen defined by the monoclonal antibody 49H.8 might be a tumor-associated antigen in endometrial epithelial tissue.

Independent effects of liver disease and chronic alcoholism on thyroid function and size: the possibility of a toxic effect of alcohol on the thyroid gland.

In an autopsy study we found thyroid volume significantly decreased in alcoholics with liver cirrhosis as compared to matched controls: 15 mL (range, 7 to 37 mL) v 25 mL (range, 13 to 90 mL) (P less than .01). At the same time the amount of fibrosis of the thyroid glands was higher in the alcoholics as compared to the matched controls: 20% (range, 6% to 40%) v 12% (range, 6% to 23%) (P less than .01). In order to evaluate the relative importance of alcohol consumption and liver disease on thyroid function and ultrasonically determined size, three groups of patients and matched controls (sex, age, weight, and smoking habits) were investigated: group 1, 18 patients with nonalcoholic liver cirrhosis; group 2, 21 consecutive chronic alcoholics (greater than 100 g of alcohol daily for greater than 5 years) without liver cirrhosis (all had biopsy proven fatty change or normal liver); group 3, 31 nonalcoholic patients with chronic nonhepatic, nonrenal disease. In group 1 median thyroid volume and serum FT4I, FT3I, and TSH levels were unchanged compared with the controls. In group 2 median thyroid volume was 13 mL (range, 9 to 32 mL) compared with 27 mL (range, 12 to 44 mL) in the controls (P less than .005). Serum T3 and FT3I levels were reduced, while T4, FT4I, and TSH levels were unaltered. In group 3 serum T3 and FT3I levels were reduced while serum FT4I and TSH levels and thyroid volume were unaltered compared with the controls. It is suggested that alcohol may have a toxic effect on the thyroid gland independent of the degree of liver damage.

Pattern of progression in liver injury following jejunoileal bypass for morbid obesity.

Liver biopsies from 34 patients with morbid obesity, performed before and 5-9 months after jejunoileal bypass, were studied. The patients were divided into four groups according to preoperative findings: A: no or slight steatosis (15 patients), B: moderate-severe steatosis (6), C: steatohepatitis (steatosis + lobular lymphocytic inflammation) (8), D: steatofibrosis (steatosis + pericellular fibrosis) (5). In Group A, 12 patients showed postoperative progression to either moderate/severe steatosis, steatohepatitis, or steatofibrosis. In Group B, all patients progressed to steatohepatitis or steatofibrosis, and one developed septate fibrosis. All patients in Group C progressed to steatofibrosis, and 5 developed septate fibrosis or cirrhosis. In Group D, 3 developed bridging fibrosis. Mallory bodies appeared postoperatively in 11 patients (32%), all of whom preoperatively had either severe steatosis, steatohepatitis, or steatofibrosis. Only patients with postoperative pericellular fibrosis and Mallory bodies developed deranged architecture: 6 septate/bridging fibrosis, and 3 cirrhosis. Five patients, all with deranged architecture, developed reversible liver insufficiency. Progressive liver injury after jejunoileal bypass appears to reflect aggravation of a pre-existing liver lesion. The sequence of events: increasing steatosis, lobular lymphocytic inflammation, pericellular fibrosis, Mallory bodies, and deranged architecture is similar to that of the alcoholic liver lesion, indicating common pathogenetic mechanisms.

Inverted papilloma of upper urinary tract.

Two new cases of inverted urothelial papilloma in the upper urinary tract are described and added to the 22 cases previously reported in the literature. In both cases inverted papilloma was localized beneath macroscopic normal surface, and in one of the cases the changes were found scattered widely in the upper urinary tract. The possible etiology and the symptomatology are discussed, and the need for follow-up of these patients is emphasized.