An immunohistochemical study of the clearance of apoptotic cellular fragments.

We investigated the distribution and fate of apoptotic bodies during human development and in the adult, using an antibody (M30) that recognizes a neo-epitope formed early in the apoptotic cascade by caspase cleavage of cytokeratin 18. In the fetus, we found extensive accumulation of M30-positive, non-phagocytosed fragments in the red pulp of the spleen, subcutaneous and submucosal vessels, the interstitium of the lung, and the glomerular mesangium of the kidneys. In the liver, M30-immunoreactive fragments were found inside macrophages in the sinusoids. The number of these fragments and the intensity of the immunostaining increased with the gestational age of the fetus. In the adult, M30-positive fragments were barely detectable in normal tissues. However, many pathological situations, including both chronic degenerative processes and metastatic cancer, were associated with accumulation of M30-positive fragments in the red pulp of the spleen. In the liver and kidney, no fragments could be detected. Remarkably, 13 of the 16 patients with metastasized cancer showed pronounced accumulation of M30-positive fragments containing hematoxylin-reactive material in the red pulp of the spleen. In the non-cancerous cases, such DNA-containing fragments were only seen in 9 of 94 cases. The results show that when apoptotic activity is high, as during development in the fetus or during metastasis and other pathological processes in the adult, the phagocytic clearance of apoptotic bodies can be overloaded. These apoptotic fragments then accumulate in the spleen. The visual detection of apoptotic fragments is concluded to reflect increased cell turnover.

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30-positive cells appear in the 'apoptotic' subG1 peak. Tests with synthetic peptides define positions 387-396 of CK18, with a liberated C-terminus at the caspase cleavage site DALD-S, as the ten-residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo-epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin-fixed material.

Family caregiving in cases of hip fracture.

The growing incidence and cost and the serious nature of hip fracture in the elderly require a closer examination of the family's role in the rehabilitation process and how it can be assisted in that role. In this prospective study, 57 family caregivers provided information before the hospital discharge of the patient with hip fracture and at 2, 8, and 14 weeks postdischarge. They were asked about caregiving demands and problems, caregiver mood, expectations about recovery, and advice to future caregivers. A brief follow-up was conducted at 6 months. The care recipients were all women and had been living at home before injury. The postdischarge location of the patient (e.g., in a residence shared with the caregiver, in a different residence, or in a nursing home) was a major factor in the types of caregiving activities but not in total demand. Nonspouses cited the most problems. Mood distress did not change over time. Caregivers appeared to have unrealistic expectations about the length of the recovery period; by 14 weeks, 35% judged the care recipient's mobility to be worse than expected, and 20% felt that the patient had more pain than expected. The most frequent advice to future caregivers was to have patience and to give encouragement.

Notable relationship between the level of tumor marker TPS in serum and survival in breast cancer.

Tissue Polypeptide Specific antigen (TPS) in serum was measured once during the follow-up of 200 breast cancer patients and compared with survival. Within 12 months, patients with normal TPS (< 80 U/L) exhibited a 3% death rate (3/96), which was undistinguishable from the mortality of normal females of corresponding age. Patients with moderate TPS (80-400 U/L) suffered 19% death (14/72), and patients with high TPS (> 400 U/L) 72% death (23/32). The relative risk (RR) of death within 6 months was 1 with normal TPS, 8 with moderate TPS, and 48 with high TPS. RR for 12 months was 1, 6, and 23, respectively. Serum TPS at admission had a significant predictive value with regard to survival up to 12 months.

Posthospital convalescence in older women with hip fracture.

Women who had lived at home before hip fracture repair (N = 120, M age = 79.9) were interviewed before hospital discharge and at 2, 8, and 14 weeks postdischarge to determine (a) early recovery patterns in function and mood, (b) factors predictive of assistance needed in mobility and perceived mobility compared to prefracture status, (c) problems faced, and (d) advice to others. The mobility pattern was that of a relatively rapid gain until 8 weeks, with a smaller gain from 8 to 14 weeks. Affective mood distress was low except in those going to nursing homes. Somatic mood distress was high, decreasing only gradually. Factors predictive of needed assistance in mobility and of perceived mobility included both those without potential for nursing intervention (age, prefracture mobility, how fell, and type of surgical procedure), and those with the potential for intervention (affective distress, fatigue, and urinary problems). Persistent problems related to limitations in mobility, especially in dressing. Overwhelmingly, subjects advised the need for maintaining a good mental attitude.

Early outcomes after hip fracture among women discharged home and to nursing homes.

In a prospective design, early outcomes after hip fracture were compared in three groups of formerly community-living women: those discharged home from the hospital (n = 58), those discharged to a nursing home (NH) and staying there < or = 1 month (n = 23), and those staying > 1 month (n = 39). Data were collected on mobility and mood states prior to hospital discharge and at 2, 8, and 14 weeks. Overall recovery ratings were obtained at the latter three times; readiness for discharge from hospital and nursing home also was examined. The short-stay group did as well in regaining mobility as the home-discharge group and both rated their overall recovery similarly. Affective mood distress was associated with discharge destination site. The short-stay NH group had a greater proportionate lack of designated caregivers than either of the other two groups. Research is needed to identify features of nursing homes as well as characteristics of patients that contribute to positive outcomes in the large number of hip fracture patients currently discharged to these institutions.

Immunoradiometrical measurement of tissue polypeptide specific antigen (TPS) in normal, healthy, nonpregnant and pregnant Japanese women.

Using a monoclonal immunoradiometrical assay, we measured the concentrations of the specific epitope M3 of tissue polypeptide antigen (TPA), namely, tissue polypeptide specific antigen (TPS), in serum and amniotic fluid obtained from normal, healthy, nonpregnant and pregnant Japanese women (NHNJW and NHPJW). The cut-off value of the serum TPS level was set at 130 U/l, based on the mean +2 standard deviations in the NHNJW. The serial measurement of serum TPS levels demonstrated the significant temporal elevation of TPS level near the time of ovulation (+/- 3 days) in the ovulatory women. In the NHPJW, the mean serum TPS level and the positivity rate (> 130 U/l) increased with the advance of gestation, reaching 183.7 U/l and 66.7%, respectively, in the third trimester. Maternal serum levels were much elevated just before (mean: 982.3 U/l) and after (mean: 824.6 U/l) delivery, and were reduced to about one-fourth by 3 days. In addition, markedly high TPS concentrations were found in the retroplacental blood (one case: 9,076 U/l) and the amniotic fluid (mean: 11,650 U/l). The serum TPS level correlated well (r = 0.871) with the serum TPA level in the TPS range of 2-2,000 U/l, while a poor correlation (r = 0.273) was found in the TPS range of 2-500 U/l. The present study thus obtained fundamental data on TPS in Japanese women. The fluctuation and change of serum TPS levels during the menstrual cycle and in pregnancy should be taken into consideration when we apply TPS as a tumor marker for women.

Distribution of tissue polypeptide antigen (TPA) in normal mucosa, in colitis ulcerosa, in adenomas and in carcinomas of the human colorectum. An immunohistochemical study.

65 carcinomas with their normal resection margins, 30 adenomas of the colorectum, and also ulcerative colitis biopsies from 10 cases were analysed immunohistochemically for pattern and intensity of expression of Tissue Polypeptide Antigen (TPA). In normal colon, and in well- and moderately-differentiated carcinomas, a cell membrane type staining pattern was predominant. In ulcerative colitis, in carcinoma cell groups within mucus of mucinous carcinomas or in single cells at the invasion front of all grades of carcinomas, a strong cytoplasmic type staining pattern was found. The cytoplasmic pattern was also found in poorly differentiated carcinomas, but with weaker staining intensity. The relationship between staining intensity and pattern and carcinoma grade was significant, whereas a similar relationship with the Dukes stages was not significant.

Detection of triple antibody-binding lymphocytes in standard single laser flow cytometry using colloidal gold, fluorescein and phycoerythrin as labels.

Goat anti-mouse antibodies conjugated with colloidal 40 nm gold particles (G40) were used as secondary layers to stain human T lymphocytes in an attempt to extend the polarized light epi-illumination microscopic technique to flow cytometric multiparameter analysis. G40-labelled T cells were further stained with phycoerythrin (PE, red)- and fluorescein (FITC, green)-conjugated antibodies with specificity for the same cells. The cell samples were then analysed on a standard flow cytometer equipped with one laser operating at 488 nm. G40-labelled cells were detected in the side scatter (90 degrees) channel and fluorescent cells in the red and green fluorescence channels, respectively. Single labelling of the same T cell subsets with either G40-, FITC- or PE-conjugated antibodies yielded similar results, and cell mixture experiments did not show interference between gold and fluorescence labels. Triple staining experiments with three differently conjugated antibodies showed that subpopulations of cells were labelled in an independent manner with one, two or three antibodies in proportions expected from fluorescence controls. It was, therefore, possible to detect subset, sub-subset and activation markers on individual T cells. Our experiments show that immunogold staining of cell populations can be detected in routine one-laser flow cytometry. Further, the gold label can be combined to give two-color staining with ordinary red and green fluorochrome-conjugated antibodies thereby permitting rapid and precise triple antibody staining of individual cells. This methodology provides a powerful tool for the analysis of cellular antigenic diversity among, e.g., immunocompetent cells.

Clinical results of immunoscintigraphy in a variety of malignant tumors with special reference to immunohistochemistry.

Radioimmunoscintigraphy was performed in 52 patients with a variety of malignant tumors (colorectal, melanoma, lung, testicular, ovarian, bladder, carcinoid). Respective antibodies or their F(ab')2 fragments against CEA (n = 23), melanoma antigen 225.28 S (n = 18), TPA (n = 4), beta HCG (n = 5) and HMFG2 (n = 2) were selected by immunohistochemistry of the primary tumor. Most patients were suspected of recurrence or of hitherto unknown distant or local metastases. Overall accuracy was 61% (32/52). False negatives amounted to 33% (17/52). Useful additional clinical information-not available by CT, ultrasonics or serum levels of tumor markers-was obtained in 17 out of 52 patients (= 33%). From these results it seems obvious that antibodies used for radioimmunoscintigraphy should be selected on the basis of immunohistochemistry.

Distribution of TPA and cytokeratins in gastrointestinal carcinomas as revealed by immunohistochemistry.

The presence and distribution of tissue polypeptide antigen (TPA) were assessed in gastrointestinal carcinomas of different origin, morphology and degree of differentiation. Immunocytochemistry was employed, using the PAP technique on formalin-fixed, paraffin-embedded material and compared with the results obtained with antibodies to cytokeratins. Like cytokeratins, TPA was a reliable marker of epithelial differentiation and showed tissue distribution patterns similar to cytokeratins, as revealed by antibodies with broad-range cytokeratin immunoreactivity. In most carcinomas, TPA-specific immunostaining was less intense than in non-neoplastic tissue. No direct relationship between intensity of TPA staining and morphological degree of differentiation and proliferation was found. TPA staining was most pronounced at the periphery of the cells. In stratified epithelium, i.e. oesophageal mucosa, basally located cells exceeded superficial cells in TPA immunoreactivity in contrast to the cytokeratin antibodies which decorated the more superficially placed cell layers. TPA and cytokeratin staining patterns were similar in neoplastic and non-neoplastic gastric, intestinal mucosa, as well as in biliary tract epithelium. Antral and cardial mucoid glands of the stomach as well as gastric carcinomas of the pylorocardial type remained unstained with both types of antibodies. Similar staining with TPA and cytokeratin antibodies was also observed in pancreatic and liver tissue. In this study, hepatocytes were, although weakly, stained by TPA antibodies and an identical staining was found with benign and malignant hepatocellular neoplasms. Ductal and ductular TPA-staining was most conspicuous and so was the immunoreactivity of cholangiocellular carcinomas. A comparison between TPA and cytokeratins was also made by immunoblotting which revealed immunoreactivity of antibodies to TPA with cytokeratin polypeptides of different species (man, mouse) and organs (epidermis, liver), particularly with the cytokeratin component 8 of human liver and the related component A of mouse liver. The significance of this finding is uncertain until the pertinent epitopes have been revealed by monoclonal mapping of the components which exhibit similar molecular weights by SDS polyacrylamide gel electrophoresis.

Distribution of tissue polypeptide antigen (TPA) in normal human tissues: Immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissues.

The distribution of tissue polypeptide antigen (TPA) was studied in unfixed, methanol-, 95% ethanol-1% acetic acid (EA)-, and formalin-fixed paraffin-embedded sections of all adult human tissues using an indirect immunoperoxidase method. The specific staining patterns were virtually identical in unfixed and alcohol-fixed tissues, but in formalin-fixed tissues this similarity was found only after fixation for up to 24 hr and pretreatment with protease for 15 min. Although prolongation of formalin fixation beyond 48 hr increasingly diminished the TPA reactivity, TPA could still be demonstrated in tissues fixed in formalin for up to 6 months. TPA was found to be a cytoplasmic constituent of almost all adult human duct and cavity lining, simple, and stratified epithelia. TPA was not demonstrated in epidermis, renal proximal convoluted and testicular tubules, basket-like myoepithelial cells, nor in most glandular acini, including hepatocytes and pancreatic acinar cells. The TPA staining was also negative in all non-epithelial tissues, including lymph nodes and bone marrow. The well-defined epithelial distribution and the comparable demonstrability in differently preserved tissues make TPA a useful tool for the identification of cells of epithelial character.

Detection of tissue polypeptide antigen in salivary glands and salivary gland tumors. An immunohistochemical study.

Tissue polypeptide antigen (TPA) was analyzed immunohistochemically in parotid gland tissue. This antigen, which is generally regarded as a proliferative antigen, was detected in the ductal system of the normal parotid gland. Parotid gland tumors were analyzed as well: pleomorphic adenomas; cystadenolymphomas; adenoid cystic carcinomas, and mucoepidermoid tumors. TPA could be found in distinct parts of every kind of tumor. However, apart from the TPA-positive cells, negative cells could be observed. The implications of these investigations are discussed.

Differential immunocytochemical localization of tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA) in breast cancer.

The presence of tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA) in serial sections of 44 breast carcinomas, one case of neurofibrosarcoma, and one case of fibroadenoma was demonstrated by immunocytochemistry using the indirect peroxidase technique. The neurofibrosarcoma was negative for TPA and CEA. All 44 specimens of breast cancer were positive for TPA and/or CEA. In 18 cases the staining was strongly positive for TPA and CEA. The overall concordance of staining was 43%. TPA was more prominent than CEA in 52% of the tumors while CEA was more prominent in 7%. This is in relative agreement with the values for TPA and CEA in serum from patients with breast cancer, where TPA was higher than CEA in 41% and CEA higher than TPA in 9%. The finding of a difference between and within tumors indicated that TPA and CEA may be synthesized during partly different phases of the proliferative cycle. Assay of TPA and CEA in cytological specimens and in serum should be of clinical interest.

Specificity and basis of the tissue polypeptide antigen.

TPA is an antigenic molecule which is generally present in human carcinoma tumors. Proliferating cells of tumor or normal origin produce and release TPA. Chemical characterization has shown TPA to be an unbranched peptide chain with an apparent Mr of 4.3 X 10(4) and a frictional ratio of 2.7. The electrophoretic mobility is close to that of beta 2-globulin, the IP is 4.5, and the sedimentation constant is 4.5S. The amino acid sequence of the specific determinant is known and has been synthetically produced. Antibodies to TPA obtained by affinity chromatography with BrCN-activated Sepharose, charged with isolated TPA, are responsible for reactivity with TPA by hemagglutination, radioimmunoassay, immunoelectrophoresis, immunodiffusion in gel, and immunocytochemistry. TPA is nonspecies specific and can be found down to fishes. In cell culture (HeLa) perinuclear formation of TPA is seen early in the S phase. After cell division TPA is externalized leaving the cell free of visible TPA. At this stage a rise of TPA in the cell culture medium is observed. These findings together with extensive clinical studies and use of TPA have led to the hypothesis that TPA is related to proliferative activity in general. Since cancer is based on proliferation of cells, and the genes of cancer cells are not different from those of other cells, it could be helpful to use TPA as a monitor of cell proliferation in the further search for local and general causes of proliferation. This can be done by looking for TPA in body fluids by radioimmunoassay and at the cellular level by immunocytochemistry.

Immuno-histochemical localization of tissue polypeptide antigen (TPA) and carcino-embryonic antigen (CEA) in breast cancer. A comparative study.

The presence of Tissue Polypeptide Antigen (TPA) and Carcino-embryonic Antigen (CEA) in serial sections of 32 breast carcinomas and one case of neurofibrosarcoma was demonstrated by immunocytochemistry using the indirect peroxidase technique. The neurofibrosarcoma was negative for TPA and CEA. All 32 specimens of breast cancer were positive for TPA and/or CEA. In 12 cases the staining was strongly positive for both TPA and CEA. In other cases either TPA or CEA was more abundant in the tumor. The overall concordance of staining was 58%, which is in relative agreement with the correlation between values for TPA and CEA in serum from patients with breast cancer. The finding that in addition to the difference between tumors, there was a difference within tumors was interesting. This indicated that TPA and CEA may be synthesized during partly different phases of the proliferative cycle.

The clinical significance of tissue polypeptide antigen (TPA) in the urine of bladder cancer patients.

A 2-stage study has been carried out to evaluate the usefulness of urinary tissue polypeptide antigen (TPA) levels in patients with bladder cancer as an adjunct to the routine procedures for detection of bladder tumours. Two-hour urine samples were collected from 83 bladder cancer patients and normal individuals for the first part of the study, 24-h samples from 54 patients and normal individuals for the second part. Urinary TPA was determined using radioimmunoassay. In 2-h samples there was no significant difference in the amounts of TPA/l in any of the groups. In contrast, the TPA results of 24-h urine samples (n = 54) were markedly different from 2-h samples and the former correlated very well with the presence or absence of bladder cancer. A reason for this difference may be circadian rhythm effects.