An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis.

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis is described. For this study expression of MHC locus II coded Ia antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein was analyzed using monoclonal antibodies in avidin-biotin-peroxidase complex staining combined with visualization of [3H]thymidine incorporation by autoradiography. Compared to spontaneous [3H]thymidine incorporation assay information is obtained at single cell level. In contrast to blast indexes calculated from MGG stained preparates, information on the expression of various functional cell surface structures as well as DNA synthesis is also obtained. Double-assay for lymphocyte phenotype and DNA synthesis by flow cytometry might be preferred to light microscopy, but the widespread use of immunoperoxidase staining and autoradiography may make this new kind of approach more easily available. Other advantages worth considering are the possibility of transporting, staining and counterstaining as microscope slides and the permanent nature of the documentation and morphological information obtained. In our experience, this method seems to be useful for studying resting peripheral blood lymphocytes as well as mitogen and antigen induced changes in the lymphocyte activation state.

Lymphocyte activation in oral lichen planus in situ.

The current study evaluated the activation of T-cell mediated cellular immune response in the oral lesions in lichen planus (OLP). Analysis was made of the ultramorphology, lymphocyte activation marker expression, DNA synthesis, and gamma-interferon production of the inflammatory cells in OLP lesions. According to these four different aspects of lymphocyte activation, only a minor fraction, 5% at the most, of all T-cells in situ were activated. This minor fraction, however, not the resting T-cells without these signs of activation, may decide the outcome of the local immune-inflammatory process in OLP. Of the inflammatory cells in situ, however, 81 +/- 5% were Ia positive. This finding may be the result of an Ia inducing capacity of the gamma-interferon produced locally by the activated T-cells.

Oral parameters for the diagnosis of Sjögren's syndrome.

Agreement is probably world wide in the medical profession that patient history is the most relevant part of the patient examination, but at the same time the most demanding part because there is no substitute to lack of knowledge and experience. These form the basis for an often subconscious process of selecting the most important pieces of information to help in decision making. We describe a stepwise forward discriminant analysis with a jackknife-validation in analysis of questionnaires as to diagnosis of Sjögren's syndrome. The second topic of current interest, also due to the need of generally acceptable criteria for diagnosis and classification, is the place of labial salivary gland (LSG) biopsy in such sets of criteria. In the histopathological part of our work we describe findings of a postmortem LSG study from the coroner's office.

Demonstration of esophageal varices postmortem by gastroesophageal phlebography.

The study presents a method for the demonstration of esophageal varices at autopsy by gastroesophageal phlebography. The veins visible on the radiographs were controlled and evaluated by studying the gross appearance of sections of the esophagus at the level of the varicose veins and by histologic examination of the sections. The phlebography was found to be useful in estimating the varicose and normal veins of the esophagus. The study indicates further that the degree of varicosity is not always proportional to the severity of cirrhosis of the liver, and there may be varicose esophageal veins in patients with liver or heart disorders without hepatic cirrhosis.

Determination of cause and mode of death before and after medicolegal autopsy: a comparative study.

Two hundred and twenty-eight consecutive medicolegal autopsies were studied as to the cause of death established by a physician, the mode of death as suggested in the police report, and findings of medical interest often discovered following autopsy. Corrections had to be made even in some cases which seemed to be obvious. With a decrease of the number of autopsies, the records of various registration bureaus would be affected. Autopsies are still necessary for control and correction of clinical causes of death.

Suicide by the use of a chain saw.

The paper describes a case of suicide by the use of a chain saw. Visceral and tissue damage caused by vibrations are documented in occupational medicine, but fatal damage is not often seen at autopsy. This paper gives an account of the damage to viscera and tissues that has been noted when strong vibrations of low frequency have been directed to the body for a very short time with fatal result after a few minutes survival time.

Fatal intoxication by Remoxipride.

The case history and toxicologic findings of a 23-year-old woman who committed suicide with Remoxipride are described. Remoxipride is a recently developed neuroleptic drug of the benzamide type. Remoxipride was detected in the liver, stomach content, blood, and urine. The concentration of Remoxipride in the blood was 230 mg/L. The recommended therapeutic level for Remoxipride should not exceed 7 to 8 mg/L. The victim had no blood alcohol, but an ethanol concentration of 0.048 g/100 L was detected in the urine. The mechanism of death from Remoxipride intoxication is not known. In clinical studies, sinus bradycardia and infrequent supraventricular and ventricular ectopic beats have been noted.

Maternal death due to unnoticed ectopic pregnancy during prostaglandin-induced abortion.

The use of prostaglandin E2 (PGE2) for the termination of pregnancy is quite common and generally is considered safe. Among the complications reported are uterine rupture and dramatic pyrexic and cardiovascular response (6). A case is reported in which a woman with an ectopic pregnancy that had not been noted at ultrasonography was admitted for abortion with PGE2 with fatal outcome. At autopsy an insignificant uterine rupture was noted in addition to the main findings.

Focal adenitis in lacrimal and salivary glands. A post-mortem study.

In the present study the submandibular, labial, and lacrimal glands of 102 postmortem subjects were studied as to the presence of inflammatory foci, fibrosis, atrophy and fatty change. Focal lymphocytic infiltration of these glands with focus scores exceeding 1, one of the findings on which the diagnosis of Sjögren's syndrome is based, was found in 19 cases (18.6%). Fibrosis, atrophy, and fatty change occurred most often in the labial salivary glands of patients over 50 years of age with or without high focus scores. Fibrosis, atrophy and fat infiltration hardly ever occurred in the lacrimal glands although this was the type of gland in which high focus scores occurred most often. The findings suggest that a wider than hitherto recognized spectrum of systemic inflammatory diseases may contribute to lymphocytic adenitis and degeneration of the exocrine glands. The diagnosis of Sjögren's syndrome should be confirmed by additional diagnostic tests.

[Malignant diseases as secondary findings in forensic autopsies]. / Maligna sjukdomar som bifynd vid rättsmedicinska obduktioner.

In a study of 228 consecutive medico-legal autopsies, malignant disease was discovered in 19 cases. The malignant diseases are discussed as to their primary sites and histologic types, age groups in which they occurred, conditions with which they may have been associated, symptoms and signs which in some cases may have warned of malignant disease, and supposed reasons why the malignant diseases were not recognized while the deceased were still alive. It was found that undetected malignant diseases occur mainly in the elderly, that chronic renal disease with scarred kidneys and cirrhosis of the liver may be predisposing conditions in cancer development, and that malignant disease in the aged may be undetected because of confusion with general weakness or because of small size and obviously slow growth of the tumor with lack of symptoms.

A postmortem study of focal adenitis in salivary and lacrimal glands.

Investigations were carried out on 102 consecutive medicolegal post-mortem subjects to identify signs of focal adenitis in labial, submandibular, and lacrimal glands. There were 19 subjects (18.6%) with focus scores exceeding 1 in one or two of the types of glands. Among the 19, five had had a disease which is generally associated with Sjögren's syndrome or is autoimmune in nature. In another 63 subjects occasional lymphocytic foci were found in one, two, or all three types of gland. Fibrosis, atrophy, and fatty change had occurred most often in the labial salivary glands in those over 50 years of age with or without high focus scores. Fibrosis, atrophy, and fat infiltration had hardly ever occurred in the lacrimal glands, although this is the type of gland in which high focus scores occur most often. The results suggest that there may be a common autoimmune basis for Sjögren's syndrome and other diseases, the mechanisms of which are still incompletely understood. A focus score exceeding 1 may be an indicator of Sjögren's syndrome, but the diagnosis should not be established on the biopsy findings alone.

Focus score in the diagnosis of Sjögren's syndrome.

According to current agreements, the verification of SS should consider all three components of diagnostic relevance: focal sialadenitis, keratoconjunctivitis sicca and an associated disease. Focus score (the number of mononuclear cell infiltrates containing at least 50 inflammatory cells in a 4 mm2 glandular section) of greater than 1 is probably the single most important test result in the diagnosis of the oral component in SS. Without such quantitation mild, nonspecific inflammation is often falsely classified as focal sialadenitis suggesting an underlying SS. This was confirmed by a retrospective study of a slide library, which showed that most cases descriptively diagnosed as focal sialadenitis actually had focus scores of less than 1. Furthermore, conditions other than SS can result in focus scores of greater than 1. Firstly, in 8/17 patients, sialolithiasis of the submandibular glands was associated with focus scores of greater than 1, usually with little or no histological signs of obstruction and/or infection. Secondly, focus score values of greater than 1 were found in the labial salivary glands in 6/40 coroner's autopsies although the subjects had no clinical history or findings suggesting SS; simultaneous specific involvement of both salivary and lacrimal glands was, however, observed in 0/40 of the subjects. Based on our observations we suggest that a postmortem diagnosis of SS should be made on the demonstration of focus score of greater than 1 in both the labial salivary glands and the lacrimal glands.

Distribution of lactoferrin in human salivary glands.

An immunoperoxidase technique was used to study the distribution of lactoferrin (LF) in human salivary glands from autopsy tissues fixed in Carnoy's fluid for the optimal preservation of LF antigenicity. Specific LF staining was seen in the intralobular ducts of all salivary glands but never in the interlobular ducts; acinar LF was detected in most serous demilunes of the mixed glands and in some but not all acinar cells of the pure serous glands. No LF was detected in the acinar cells of the pure mucous glands. In the oral tissues studied the only additional cells containing LF were polymorphonuclear leucocytes. Whether LF in the salivary glands is synthesized locally and/or ultrafiltrated from the blood is at present unclear. Present evidence indicates that the biological role of salivary LF is antibacterial.

Phenotypic in situ characterization of lymphocytes in mild alcoholic liver disease.

The subsets and expression of activation markers of inflammatory mononuclear cells in mild alcoholic liver disease were studied using monoclonal antibodies and an avidin-biotin-peroxidase complex (ABC) method. There were few B cells and monocytes in situ. Most of the mononuclear cells were of T cell origin, T4 and T8 positive cells being evenly distributed in a ratio of 2 to 1. This contrasts with the present and earlier findings on autoimmune liver diseases. The frequency of Ia (HLA-DR) positive cells varied between 15 and 30% of all mononuclear cells, which indicates activation of the local T cells. This implies that T cells in alcoholic fatty liver are not innocent bystanders but actively participate in the local inflammatory process. Staining for Tac, 4F2 and T9 was completely negative, however. These findings suggest that the local T cell activation may be incomplete and that it is not as extensive as in autoimmune liver diseases.

Expression of MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein during different phases of a normal PHA-driven lymphocyte activation in vitro.

This study characterizes the temporal profile of MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein lymphocyte activation markers in relation to each other during different phases of PHA-dependent cellular activation in vitro. Binding of these monoclonal lymphocyte activation-probes was visualized by the avidin-biotin-peroxidase complex method. Maximum PHA-dependent MHC class II antigen expression of 27 +/- 3% was observed on culture day 1, but later no significant differences were observed in MHC class II antigen expression between PHA-driven or culture media-containing control cultures. On the contrary, interleukin-2 receptor (78 +/- 6%) and transferrin receptor (75 +/- 5%) expression reached a maximum on culture day 3, coinciding with a maximum proliferative response. On culture day 5, when 3H-thymidine incorporation was already on the decline, gp 40/80 glycoprotein reached a maximum PHA-dependent expression of 78 +/- 2%, which differed significantly from interleukin-2 receptor (60 +/- 8%, p less than 0.05) and transferrin receptor (51 +/- 8%, p less than 0.01) expression. This study suggests that MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein, although all of them are lymphocyte-activation markers, differ as to the chronological sequence of their appearance and disappearance. Their combined use in lymphocyte-activation marker profile assay therefore gives valuable information about the lymphocyte activation state.

T cell subsets in the blast cell population in phytohemagglutinin-stimulated peripheral blood in vitro and in rheumatoid arthritis synovial fluid in vivo.

The immunomodulatory T4/T8 ratio was studied in the total and activated lymphocyte populations by a method combining visualisation of 3H-thymidine incorporating blasts with autoradiography (AR) with simultaneous identification of the respective lymphocyte subsets using monoclonal antibodies in avidin-biotin-peroxidase complex (ABC) staining. In rheumatoid arthritis synovial fluid the activated T4/T8 ratio (calculated from T cells in the S phase of the cell cycle) was significantly different from the total T4/T8 ratio (calculated for all the T cells) (0.45 +/- 0.05 versus 0.69 +/- 0.05, P less than 0.01). Similarly, the activated and total T4/T8 ratios were also significantly different in the phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cell cultures at days 3 and 5.

Lymphocyte activation in vivo in the intestinal mucosa of patients with Crohn's disease.

We analyzed lymphocyte activation state in vivo in lamina propria by applying a panel of monoclonal antibodies for cellular activation markers and by identifying different subsets of 3H-thymidine incorporating lymphoblasts by combining autoradiography with immunoperoxidase staining. In focal lymphoblastoid infiltrates 75 +/- 8% of all inflammatory cells displayed Ia, but lymphoid cells expressing 4F2 (40 +/- 6%) and T9 (14 +/- 3%) activation markers were less frequent. The expression of receptors for interleukin-2 (Tac) was particularly low (4 +/- 1%). There were very few 3H-thymidine incorporating cells (labeling index approx. 1%) and most of them belonged to the T cell series. T4+ blasts outnumbered T8+ blasts, and in this numerically minor subpopulation the activated T4/T8 ratio was 2.9 +/- 0.7. Our results suggest that, although a big part of the local inflammatory lymphocytes in Crohn's disease may be activated according to the expression of Ia and 4F2 activation markers, only a fraction of them possess interleukin-2 receptor and have been pushed to the S-phase of the cell cycle.

T lymphocytes and mononuclear phagocytes in the skin infiltrate of systemic and discoid lupus erythematosus and Jessner's lymphocytic infiltrate.

T Lymphocytes and mononuclear phagocytes were measured quantitatively with the histochemical acid alpha-naphthyl acetate esterase (ANAE) method from paraffin sections of skin affected by systemic and discoid lupus erythematosus and by Jessner's lymphocytic infiltrate. The composition of the patchy cutaneous mononuclear cell infiltrates was similar in these three disorders.

Surgical technique and operative mortality in coronary artery bypass. A postmortem analysis with castangiography.

Post-mortem analysis with castangiography was performed on 54 patients who died within 30 days of coronary artery bypass surgery. Myocardial failure was the cause of 85% of the deaths. There were 215 coronary anastomoses (4.0 +/- 1.1/patient), 24% of which were non-functioning. Most of the occlusions were due to various technical failures. The most striking features were 1) high occlusion rate (25%) in sequential vein grafts and 2) disastrous complications of coronary endarterectomies. Compared with preoperative angiographic data, only 15 (28%) of the 54 patients were found to have 'complete' revascularization, with patent grafts and all stenosed coronary arteries bypassed. The need for recognition and avoidance of technical complications is stressed: Failures of surgical technique constitute a major risk factor in coronary artery surgery.

Mineral fiber concentration in lung tissue of mesothelioma patients in Finland.

The mineral fibers in lung tissue samples of 19 mesothelioma patients and 15 randomly selected autopsy cases were analyzed using low-temperature ashing, scanning electron microscopy (SEM) and x-ray microanalysis. The fiber concentration ranged from 0.5 to 370 million fibers per gram of dry tissue in the mesothelioma group and from less than 0.01 to 3.2 million fibers per gram of dry tissue in the autopsy group. In 80% of the mesothelioma patients and in 20% of the autopsy cases, the fiber concentration exceeded 1 million fibers per gram of dry tissue. Amphibole asbestos fibers predominated in both groups, and only a few chrysotile fibers were found. In the lungs of six mesothelioma patients, anthophyllite was the main fiber type. The overall analytical precision of sample preparation and fiber counting with SEM was 22%.