Physical chemical studies on the specific interaction of an acriflavine-phosphotungstic Acid complex with double-stranded nucleic acids.

The detailed definition of the structure of DNA in chromosomes and in interphase chromatin is important for correlating the structure of the genetic material with various states of physiological activity. A general approach to developing specific reagents for a variety of such studies in solution and in tissues is to combine a chemically specific organic cation with the electron-opaque phosphotungstic acid (PTA) molecule. The reagent described in this paper was made from the interaction of acriflavine and phosphotungstic acid. The acriflavine-PTA complex (a) displays some unique absorption and fluorescence properties, (b) binds specifically to DNA and RNA by intercalation of the acriflavine moiety, and (c) is electron opaque. In addition, it binds to double-stranded synthetic polynucleotides, but not to a variety of proteins, nucleoproteins, or polysaccharides.

Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats.

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals. When thymic and splenic lymphocytes of normal or immunized animals were surface radioidinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1 percent Triton X-100 and analyzed by immunodiffusion in agarose gels both IgG and IgM were identified in thymic and splenic cells.

Cerebrospinal fluid immunoglobulin abnormalities in neurosarcoidosis.

A retrospective chart review of neurosarcoidosis at the University of Texas Medical Branch (Galveston) between 1982 and 1987 revealed 99 patients with sarcoidosis. Six patients were diagnosed with neurosarcoidosis and had electrophoresis of serum and cerebrospinal fluid performed (one patient with a ventriculoperitoneal shunt was later excluded). Cerebrospinal fluid immunoglobulins and albumin levels were determined followed by calculation of an IgG index and synthesis rate for each patient. Four (80%) of five patients had elevated IgG indexes and synthesis rates indicative of intrathecal immunoglobulin production. No patient had immunoglobulin oligoclonal bands detected. To date, results of electrophoresis of cerebrospinal fluid in neurosarcoidosis have been reported in 37 patients among four series. Of these, only nine patients (24%) have had either an elevated IgG index or synthesis rate. Our series suggests that intrathecal immunoglobulin production in neurosarcoidosis occurs more frequently than previously described. Furthermore, the elevated indexes and synthesis rates without associated oligoclonal bands suggests a polyclonal immunoglobulin response.

Statistical techniques reported in pathology journals during 1983-1985. Implications for pathology educators.

The increasingly scientific basis of medicine challenges pathology educators to incorporate quantitative skills into a very compressed curriculum. One strategy is to focus training on the statistical techniques pathologists will most commonly encounter in their literature. We identified the reporting of statistical techniques in nearly 5200 original articles published during 1983-1985 in 16 journals important to pathology. Our results suggest that a reader familiar with 12 fundamental statistical concepts can evaluate knowledgeably over 95% of the quantitative findings reported in these journals. With the exception of survival analysis and pharmacologic modeling, these techniques are typically encountered in many introductory statistical texts. In numerous articles, failure to identify the statistical methodology used made it impossible to identify the analytic procedures used and, hence, judge the scientific validity of results.

A variant of toxic shock syndrome. Clinical, microbiologic, and autopsy findings in a fatal case.

A 16-year-old boy had toxic shock syndrome (TSS); Staphylococcus aureus bacteremia developed 11 hours prior to his death, which was three days after onset of the illness. The isoelectric focusing pattern of the staphylococcal isolate differed from both non-TSS and classic TSS S aureus isolates. Anatomic findings suggest three pathogenetic mechanisms: (1) immune complex-associated pulmonary microangiitis and vasculitis in the skin and skeletal muscle; (2) parenchymal cell "microvesicular" fatty metamorphosis in the liver, myocardium, renal tubules, and pancreas, and (3) pancarditis.

Tissue culture of cells in serous effusions. Evaluation as an adjunct to cytology.

Cell pellets from peritoneal, pleural and pericardial effusions from thirty-six patients were incubated in tissue culture medium. Both the culture supernatants and the cell monolayers in the flasks were examined for malignant cells. Of the ten cases diagnosed as positive for malignant cells by the conventional cytologic smears, eight were positive on examination of the tissue culture supernatants and only six were positive by analysis of the monolayers. All the cases diagnosed as positive from the culture supernatants and from the monolayers were also positive by the conventional smear method. Hence, tissue culture of cells in serous effusions is not helpful as an adjunct to routine diagnostic cytology techniques.

Kinetics of methotrexate uptake by human tumor cells from routine pathological samples.

The kinetics of cell uptake of methotrexate were measured in vitro using primary human tumor cells from malignant pleural effusions, leukemic cells from leukopheresis samples, and normal tonsillar lymphocytes. The results were consistent with either passive diffusion or carrier mediated uptake kinetics, with the latter applicable only under conditions where the extracellular concentration of methotrexate was smaller than the drug-transport receptor binding constant. The intial rates of uptake for the same cell type differed signicantly between patients; however no differences in uptake rates were noted between cell types. All three sources of cells proved suitable for uptake studies. The results suggest that the variation in response in normally methotrexate responsive neoplasms, such as leukemia or lymphoma, may be due to between-patient differences in the cellular rates of methotrexate uptake.

Decreased anionic groups and increased permeability precedes deposition of immune complexes in the glomerular capillary wall.

To verify whether an increase in glomerular permeability precedes the deposition of immune complexes in the capillary wall, the following were studied in mice with lupus nephritis: 1) urinary proteins; 2) glomerular transfer of IgG, albumin, and anionic ferritin (AF) (isoelectric point, 4.2-4.6); and 3) anionic groups of the capillary wall as detected by binding of cationized ferritin (CF) (isoelectric point, 7.5-8.6). The glomeruli were investigated by immunofluorescence, immunoelectron, and transmission electron-microscopic studies. Urinary proteins were quantitated and characterized by electrophoresis. When mice were 5 months of age, (the time at which pathologic proteinuria was first detected), immune deposits were few and were confined to the mesangium. Although AF molecules were largely retained at the level of the lamina rara interna, focal leakage of albumin and IgG was detected across capillary walls that were not found to contain immune deposits. Focal and irregular loss of anionic groups, reflected by decreased binding of CF molecules, occurred in the laminae rarae of the basement membrane. Foot processes of glomeruli incubated with CF showed no loss of polyanion. We conclude that the increase in glomerular permeability that precedes deposition of immune complexes is due, in part, to focal loss of anionic groups of the basement membrane.

Biochemical and enzymatic characterization of thymic and splenic lymphocyte plasma membranes from inbred rats.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.

The kinetics of IgG and IgM antibody-forming cells in ACI and F344 rats immunized with poly(glu52lys33tyr15).

The cellular kinetics of antibody production in high and low responder rats immunized with poly(Glu52Lys33Tyr15) or with poly(Glu52Lys33Tyr15)/MeBSA were characterized: serum antibody and IgG and IgM antibody-forming cells in the spleen and in selected lymph nodes were assayed in male and female rats following immunization by several routes. Aggregation of the antigen with MeBSA enabled the poorly responding F344 rats to produce antibody, which was almost exclusively IgG. High responder ACI rats, under the same conditions, produced antibody of both IgG AND IgM classes. These data suggest that in low responders one defect, possibly at the T-cell level, can be overcome by aggregation but that a second defect, involving the regulation of IgM production, still exists.

Do you understand the role of software? It's the key to performance of laboratory computer systems.

Pathologists making decisions regarding computer applications in their laboratories need to understand all three aspects of computer technology: software, the user interface, and hardware. Of these, software may be the most important. This article identifies problems associated with software usage, taking into account contraints imposed by hardware and personnel, then suggests criteria for evaluating software. If pathologists are to derive maximum benefit from their investment in automated technology, they need to understand the intrinsic role of software in computer-based systems.

The effect of tumor necrosis factor binding protein and interleukin-1 receptor antagonist on the development of abdominal aortic aneurysms in a rat model.

PURPOSE: Tumor necrosis factor (TNF), interleukin 1 (IL-1), and matrix metalloproteases have been noted to be elevated in human abdominal aortic aneurysms (AAAs) as compared with normal and occlusive aortic disease. Because TNF and IL-1 have been shown to cause release of proteases that weaken the aortic matrix, it has been suggested that these cytokines may play a central role in the aortic dilatation process. To substantiate this hypothesis, we investigated the effects of TNF and IL-1 antagonists, tumor necrosis factor binding protein (TNF-BP) and interleukin-1 receptor antagonist (IL-1RA), on the development of AAAs in a well-described rat model. METHODS: Isolated segments of infrarenal aorta of 16 rats were perfused with porcine elastase. In the treated group, eight rats were given intravenous TNF-BP prior to elastase perfusion, at 48 hours and at 96 hours. In the control group, eight rats were given only intravenous vehicle at the same time intervals. Isolated segments of infrarenal aorta of an additional 16 rats were perfused with porcine elastase in a similar fashion. In the treated group, eight rats were given intraperitoneal IL-1RA prior to celiotomy and every eight hours. In the control group, eight rats were given only intraperitoneal vehicle at the same time intervals. On the sixth postoperative day, all rats underwent celiotomy and measurement of the infrarenal aortic diameter with a micrometer while the animal was alive. Aortic specimens were collected on day six for hematoxylin and eosin staining, trichrome staining, and gel polyacrylamide gel electrophoresis (PAGE) zymography. RESULTS: TNF-BP was completely able to block post elastase dilation, whereas IL-1Ra seemed to have no effect. Hematoxylin and eosin staining and trichrome staining revealed that animals treated with TNF-BP had less of an inflammatory response and preservation of the elastin and smooth muscles in the media of the aortic wall as compared with animals treated with IL-1RA or vehicle. Zymography was not able to detect significant protease activity in the aortic wall of any of the rats at six days. CONCLUSION: TNF-BP, but not IL-1RA, may inhibit the development of AAAs in this model.

Fetal intestinal transplants in syngeneic rats: a developmental model of intestinal immunity.

We conducted a longitudinal study of the development of lymphoid tissue in fetal small intestine transplanted to a subcutaneous site in adult syngeneic Fischer strain rats. Fetal jejunoileal segments obtained between 18 and 21 days of gestation were transplanted to a dorsal subcutaneous site on syngeneic adult rats. Three weeks later, intestinal segments greater than 2.5 cm in length were found in 70% of recipients. Each week for 6 wk post-transplantation, a full-thickness biopsy was obtained for histologic and immunohistologic examination. At the time of transplantation, fetal rat intestine did not display Peyer's patches, intraepithelial lymphocytes, lymphoid follicles, or IgA-containing plasma cells. These lymphoid structures reached adult levels by 4 wk after transplantation, and the sequence of development of the lymphoid structures in the transplants appeared to match the postnatal development of normal small intestine. After immunizing the in situ intestine or the transplanted fetal intestine with cholera toxin, the number of cells producing specific antibodies to the immunogen increased significantly in intestinal transplants and in situ intestine. In contrast, few if any cells synthesizing antibodies to cholera toxin developed in the transplants after i.p. immunization. This study suggests that fetal intestinal transplants behave as part of the mucosal immune system. This model may provide useful approaches to studying the development of mucosal immunity.