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Biomedical subjects

C T Wittwer

Publications and source records attributed to C T Wittwer.

At least 19 recordsLinked to original sources
PubMed1992

Cytomegalovirus infection, fetal liver disease, and neonatal hemochromatosis.

Neonatal hemochromatosis is an uncommon disorder, clinicopathologically defined by severe and generally fatal liver disease of intrauterine onset associated with extrahepatic siderosis that spares reticuloendothelial elements (hemochromatotic siderosis). The agent or agents of liver disease in neonatal hemochromatosis are not known. It also is not known if intrauterine liver disease of defined infective etiology can lead to hemochromatotic siderosis. We present two patients with fetal liver disease and hemochromatotic siderosis whose cases help address these points. In the first patient rare hepatobiliary and numerous renal tubular cytomegalovirus (CMV) inclusions were found; CMV infection was confirmed by the polymerase chain reaction. Studies of the mother of the second patient 1, 5, and 9 weeks post-partum showed recent seroconversion against CMV; seroconversion against other infectious agents (toxoplasma, rubella, herpes, parvovirus B19, hepatitis A/B/C) was not present. Histologic, immunohistochemical, in situ hybridization, or polymerase chain reaction evidence of CMV infection was not present in infant tissues, even though peripartum maternal seroconversion against CMV was observed. We conclude that hemochromatotic siderosis may accompany chronic fetal liver disease of defined infective etiology (patient no. 1) and that recent maternal seroconversion against CMV in the presence of severe fetal liver disease does not necessarily mean that transplacentally acquired CMV infection caused the fetal liver disease (patient no. 2). Polymerase chain reaction documentation of infective-agent genomic sequences in fetal or infant tissues permits more accurate interpretation of maternal serologic data.

Adolescent↗
PubMed1992

Digoxigenin-labeled probes amplified from genomic DNA detect T-cell gene rearrangements.

The detection of clonal rearrangements of the human T-cell receptor by Southern hybridization is a useful tool to diagnose morphologically difficult lymphoid proliferations. Widespread application of this method has been facilitated by the advent of sensitive nonradiolabeled probes. Although a limited number of nonradiolabeled DNA probes are commercially available, other probes must be obtained through a time-consuming and technically difficult procedure of amplification, isolation, and labeling of plasmid-cloned DNA sequences. A simple and time-saving procedure to simultaneously amplify and nonradioactively label DNA probes for use in gene rearrangement studies is described. Specifically, a method using the polymerase chain reaction to amplify and label with digoxigenin large quantities of probe to the constant region of the T-cell receptor directly from genomic DNA is described. The resultant probes are specific for the T-cell receptor-constant region, detect the appropriate germline configuration in placental DNA, and identify rearranged clonal T-cell proliferations. The polymerase chain reaction digoxigenin-labeled probes are suitable for detection by conventional colorimetric methods or by chemiluminescent detection schemes.

Amino Acid Sequence↗
PubMed1992

The distribution of immunoreactive topoisomerase II Protein in human tissues and neoplasms.

Western blotting experiments with topoisomerase II antiserum were performed to measure type II topoisomerase content in human tissues. In this study, antiserum produced against the C-terminus of recombinant HeLa topoisomerase II recognized both p170 and p180 forms of the enzyme. Antisera specifically made against p180 selectively recognized that enzyme form. By using these antisera, a remarkable difference in topoisomerase content between the tissues studied was observed. Tissues with a high proportion of cycling cells contained predominantly the p170 form of the enzyme, but also smaller amounts of p180. In terminally differentiated organs or in neoplasms with a low percentage of cycling cells, topoisomerase II immunoprotein was not detected in whole tissue extracts. In term human placenta, this was because this tissue contained predominantly the p180 enzyme form, yet the concentration of this form of the enzyme was apparently in too low a concentration to be detected by the antibody in crude extracts. It was readily observed after concentration by chromatography on hydroxylapatite.

Blotting, Western↗
PubMed1992

Identification of monoclonal B-cell populations by rapid cycle polymerase chain reaction. A practical screening method for the detection of immunoglobulin gene rearrangements.

Alternatives to Southern blot hybridization for gene rearrangement analysis are being studied because of the time, labor, cost, and radioisotopes required for this technique. We have utilized a rapid, hot air, thermocycling polymerase chain reaction (PCR) system to examine various lymphoproliferative disorders for immunoglobulin heavy chain (IgH) gene rearrangements. This unique system amplifies DNA from 10 microliters samples placed in glass capillary tubes, over a total cycle time of about 30 minutes. Amplified bands are easily visualized on ethidium bromide-stained agarose gels. Forty-one monoclonal B-cell proliferations, 27 reactive lymphoid hyperplasias, 17 T-cell lymphomas and 3 cases of Hodgkin's disease were studied. All 88 cases were fully characterized by morphologic, immunophenotypic, and genotypic (Southern blot) analyses. Each case was separately evaluated by PCR with two primer pairs: 1) IgH variable region (VH) and IgH joining region (JH) and 2) bcl-2 and JH. Thirty-four of 41 monoclonal B-cell proliferations revealed a distinct band (within an expected base pair range) with 1 or both primer combinations supporting B-cell monoclonality; the other 7 cases were considered false negatives. The 47 entities without IgH gene rearrangements detectable by Southern analysis demonstrated no amplified product or a smear of amplified DNA with no distinct band. The overall specificity of PCR was 100%, and the sensitivity was 83% when directly compared with Southern blot analysis. Although its sensitivity is currently less than optimal, PCR is a rapid and practical screening method for the detection of IgH gene rearrangements. If a positive result is obtained no further analysis is required; however, if there is a negative result, standard Southern blot analysis should be performed to definitively exclude the presence of a monoclonal B-cell population in the sample.

B-Lymphocytes↗
PubMed1991

Rapid cycle DNA amplification: time and temperature optimization.

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.

Globins↗
PubMed1990

Minimizing the time required for DNA amplification by efficient heat transfer to small samples.

Hot-air temperature cycling of 1- to 10-microliters samples in glass capillary tubes can amplify DNA by the polymerase chain reaction in 15 min or less. A rapid temperature cycler of low thermal mass was constructed to change sample temperatures among denaturation, annealing, and elongation segments in a few seconds. After 30 cycles of 30 s each, a 536-bp beta-globin fragment of human genomic DNA was easily visualized with ethidium bromide on agarose gels. With rapid cycling, amplification yield depended on polymerase concentration. The time required for DNA amplification can be markedly reduced from prevailing protocols if appropriate equipment and sample containers are used for rapid heat transfer to the sample.

DNA↗
PubMed1990

Human DNA topoisomerase II: evaluation of enzyme activity in normal and neoplastic tissues.

We have used both a quantitative filter binding assay and a decatenation assay to measure DNA topoisomerase II activity. The filter binding assay, which measures catenating activity, is able to detect topoisomerase II activity at 50-100-fold lower protein concentrations than the decatenation assay. Because of this remarkable sensitivity, we have been able to quantitate topoisomerase II activity in a variety of normal and neoplastic human tissues. The highest level of enzyme activity in normal tissues was found in the spleen and thymus. The highest level of enzyme activity in neoplasms was found in those that clinically behave in an aggressive manner and had a high proliferative status by flow cytometry. Surprisingly, these high topoisomerase II values in the neoplastic specimens are in the same range of values found in normal nonproliferating tissue. Since much previous data indicate that the enzyme is apparently a property of only proliferating cells, this finding might suggest that human tissues contain more than one form of the enzyme. The finding that 35-65% of the topoisomerase II activity in human tissues is resistant to teniposide suggests that more than one enzyme form exists.

Adolescent↗
PubMed1990

Flow cytometric DNA content analysis of a case of pilomatrix carcinoma showing multiple recurrences and invasion of the cranial vault.

Pilomatrix carcinomas are rare neoplasms of the skin that may be locally aggressive or metastatic. The differentiation of these tumors from benign pilomatrixomas depends on a constellation of microscopic features, some of which may be equivocal or absent in individual biopsy specimens. We encountered a tumor with distinct pilomatrix differentiation (lobulated nests of basaloid cells, ghost cells, focal calcification) that recurred multiple times and ultimately invaded the cranial vault. Despite this aggressive behavior, the tumor was difficult to separate from benign pilomatrixoma on morphologic grounds. Because DNA content flow cytometry has proved useful in the prediction of aggressive behavior in various solid tumors, we analyzed this neoplasm by flow cytometry. Neither aneuploid peaks nor a high proliferative fraction were seen in this example of pilomatrix carcinoma.

Carcinoma↗
PubMed1990

Mild pantothenate deficiency in rats elevates serum triglyceride and free fatty acid levels.

Pantothenate-derived coenzymes are extensively involved in intermediary metabolism, particularly in the metabolism of lipids. Using a rat model of diet-induced pantothenate deficiency, we hoped in this study to relate pantothenate status, as indicated by serum pantothenate and hepatic coenzyme A, to levels of circulating triglycerides and free fatty acids. Although commercial "vitamin-free" casein contained approximately 3 mg total pantothenate per kilogram, marked changes in serum pantothenate and hepatic CoA still occurred during weeks when rats were fed purified diets supplemented with 0 to 1600 mg pantothenate per kilogram of diet. Conditions in different experiments included ad libitum or pair-feeding of 3- or 6-wk-old rats and blood sampling from 3 to 24 h after feeding. Under most conditions of mild pantothenate deficiency in which weight differences between groups were not statistically significant, serum triglyceride and free fatty acid levels were significantly elevated in pantothenate-deficient groups. Mild pantothenate deficiency appears to have observable in vivo effects on triglyceride metabolism before severe deficiency causes significant weight loss.

Animals↗
PubMed1990

Relationship of OKT3 sensitization and vascular rejection in cardiac transplant patients receiving OKT3 rejection prophylaxis.

We prospectively and serially monitored plasma levels of OKT3 in 20 patients who were receiving 14- or 21-day rejection prophylaxis with OKT3. We retrospectively compared plasma OKT3 levels with biopsy scores assessed by light microscopy and immunofluorescence, clinical findings, human antimouse antibody (HAMA) production assessed by a blocking assay and by ELISA, and circulating immune complex levels assessed by a flow cytometric Raji cell assay. Using these methods, we evaluated the relationship of OKT3 sensitization, a humorally mediated immune response, to the development of vascular rejection in these patients. We found that 6 of 20 patients had declines in plasma OKT3 levels to less than 50% of their steady-state value before the conclusion of therapy (OKT3 consumption). This fall in plasma OKT3 preceded a significant rise in the CD 3 lymphocyte level by up to 3 days. All 6 patients showed HAMA production by either blocking or ELISA assay (P = less than 0.02) and developed vascular rather than cellular rejection (P = less than 0.01). OKT3 sensitization was significantly more common in patients treated with 21-day rejection prophylaxis (4 of 6 patients, P = less than 0.01). Only 4 of 14 other patients showed vascular rejection; 2 of these 4 also developed HAMA without OKT3 consumption and both had been treated with 21-day rejection prophylaxis with OKT3. None of the 20 patients showed significant levels of circulating immune complexes. This study demonstrates that OKT3 sensitization is strongly associated with vascular rejection. Vascular rejection was usually demonstrated 7 days after OKT3 consumption was seen and was coincident with HAMA production. By contrast, 4 patients without OKT3 sensitization had vascular rejection demonstrable in the early posttransplant period; in such patients, prospective immunofluorescence of biopsies was the only reliable indicator of this rejection type. The higher incidence of vascular rejection in these 20 patients was definitely related to the use of 21-day OKT3 rejection prophylaxis. Overall, 7 of the 12 patients treated with this regimen developed vascular rejection. Allograft and patient survival among patients with vascular rejection was significantly worse than in patients with cellular rejection (P = less than 0.01). Prospective monitoring of patients treated with OKT3 by serial plasma levels and by biopsy immunofluorescence will identify patients at risk for these types of humoral rejection.

Antibodies, Anti-Idiotypic↗
PubMed1990

A comparison study of two methods of peanut agglutinin staining with S100 immunostaining in 29 cases of histiocytosis X (Langerhans' cell histiocytosis)

Staining with peanut agglutinin (PNA) and with polyclonal antibody to S100 protein have both been recommended as aids in the diagnosis of histiocytosis X (Langerhans' cell histiocytosis). Although a large body of literature attests to the utility of S100 staining in this condition, the few published studies that involve PNA staining have employed varying methods of radically different results. We studied formaldehyde solution-fixed, paraffin-embedded tissue from 29 cases of histiocytosis X by using standard avidin-biotin immunostaining for S100 protein, as well as two published methods of PNA staining. All cases stained positively for S100 protein. By utilizing prior trypsinization and a three-step procedure, all cases also stained with PNA. With a two-step PNA-staining procedure, however, 9 cases failed to stain with PNA, and 3 cases showed a diffuse staining pattern that was not considered characteristic of Langerhans' and histiocytosis X cells. We concluded that both the S100 and the three-step PNA procedures are sensitive methods for the diagnosis of histiocytosis X.

Adolescent↗
PubMed1989

Automated polymerase chain reaction in capillary tubes with hot air.

We describe a simple, compact, inexpensive thermal cycler that can be used for the polymerase chain reaction. Based on heat transfer with air to samples in sealed capillary tubes, the apparatus resembles a recirculating hair dryer. The temperature is regulated via thermocouple input to a programmable set-point process controller that provides proportional output to a solid state relay controlling a heating coil. For efficient cooling after the denaturation step, the controller activates a solenoid that opens a door to vent hot air and allows cool air to enter. Temperature-time profiles and amplification results approximate those obtained using water baths and microfuge tubes.

Air↗
PubMed1989

Enzymes for liberation of pantothenic acid in blood: use of plasma pantetheinase.

Reported normal concentrations for human whole-blood total pantothenic acid vary from 1.1 to 12 mumol/L. This wide range may partly arise from the various enzymes used for liberation of pantothenic acid from coenzyme A, particularly the source of pantetheinase. A purified pantetheinase from pig kidney had greater than 100 times the specific activity and less than 0.01 times the pantothenate content of other commonly used extracts. Endogenous pantetheinase activity in human plasma was identified (11.2 +/- 2.0 mumol pantothenate .min-1.L-1, n = 29) and found comparable to the activity usually added from exogenous sources for liberation of pantothenate from whole blood (1-13 mumol.min-1.L-1). Alkaline phosphatase alone liberated as much pantothenate from hemolyzed whole blood as did alkaline phosphatase with pantetheinase. Previous reports of total blood pantothenate may be elevated by pantothenate in the pantetheinase extracts, an unnecessary source of error. Whole-blood total pantothenate concentrations less than 4.6 mumol/L are normal and do not indicate deficiency, as is often currently quoted.

Alkaline Phosphatase↗
PubMed1988

Clinical, ultrastructural immunohistochemical and DNA content analysis of lymphomas having features of interdigitating reticulum cells.

Interdigitating reticulum cells (IRC) are dendritic, nonphagocytic histiocytes found in thymus-dependent areas of lymphoid tissues. We report two cases of large cell lymphoma having features of IRC in patients 13 and 17 years old. In each case the diagnosis was suggested by light and electron microscopic features, positive immunoperoxidase staining for S-100 protein, and additional immunoperoxidase findings. Both patients presented with aggressive lymphomas and were treated with intensive combination chemotherapy. Each patient achieved a complete clinical remission, then rapidly relapsed and died with disseminated disease 3 and 9 months after presentation. Novel findings included strong staining of specimens with an antibody to epithelial membrane antigen, staining of one specimen with peanut agglutinin in the pattern previously described in normal IRC, and a DNA content analysis that showed aneuploid stem cell lines in both patients.

Adolescent↗