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Biomedical subjects

R E Cunningham

Publications and source records attributed to R E Cunningham.

At least 19 recordsLinked to original sources
PubMed1995

Lamellar-phase polymorphism in interdigitated bilayer assemblies.

Bilayers composed of 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine (C(18)C(10)PC) adopt a mixed-interdigitated gel-phase packing where the short chains of the C(18)C(10)PC molecules pack end-to-end while their long chains span the entire hydrocarbon width of the bilayer. Calorimetric cooling scans of freshly prepared hand-shaken bilayer suspensions of C(18)C(10)PC exhibit a single exothermic phase transition at 14.6 degrees C, whereas suspensions incubated at temperatures below 2 degrees C for several days exhibit an additional phase-transition exotherm at 17.9 degrees C. Calorimetric and electron microscopic evidence is presented that low-temperature incubation of C(18)C(10)PC bilayer suspensions composed of liposomes of heterogeneous size leads to the conversion of those liposomes in the suspension below about 0.2 microns in diameter into planar lamellar sheets. These lamellar sheets are the origin of the phase-transition exotherm at 17.9 degrees C, whereas the phase-transition exotherm at 14.6 degrees C arises from the liposomes in the suspension. We also show that phosphatidylcholine bilayer suspensions, induced to interdigitate by ethanol, exhibit a similar thermotropic behavior. The implication of these findings for the reversibility of interdigitated gel to liquid-crystalline phase transitions and the role of phospholipid molecular geometry in the formation of interdigitated bilayers are addressed.

Calorimetry, Differential Scanning↗
PubMed1994

Proliferating-cell nuclear antigen immunocytochemistry in the evaluation of gastrointestinal smooth-muscle tumors.

Current methods do not provide a way always to distinguish benign from malignant gastrointestinal smooth-muscle tumors. We compared immunocytochemical assessment of proliferating-cell nuclear antigen expression with flow cytometry and mitotic figure-counting as a prognostic marker in 85 gastrointestinal smooth-muscle tumors. Although proliferating-cell nuclear antigen expression was associated with poor prognosis in univariate statistical analysis, it was not significant in multivariate proportional hazards models that included either the mitotic index or aneuploidy of the flow-cytometric G2M peak. We conclude that proliferating cell nuclear antigen assessment is not warranted in the routine evaluation of gastrointestinal smooth-muscle tumors.

Biomarkers, Tumor↗
PubMed1993

Predicting prognosis of gastrointestinal smooth muscle tumors. Role of clinical and histologic evaluation, flow cytometry, and image cytometry.

Both flow cytometry (FCM) and morphometry have been proposed as techniques for predicting the prognosis of gastrointestinal (GI) smooth muscle tumors (SMTs). In particular, DNA aneuploidy by FCM has been associated with high histologic grade and shortened survival, whereas the DNA index determined by image cytometry has been proposed as a criterion for the diagnosis of malignancy. To further define the potential roles of these two techniques, we performed a variety of morphometric and FCM measurements on paraffin blocks from 122 patients with GI SMTs, with a median follow-up period of 6 years, together with assessments of tumor size and mitotic activity. None of the morphometric measurements (nuclear perimeter, area, form factor, longest diameter, average ferret diameter, equivalent diameter, and DNA index) was a significant prognostic factor when analyzed using a univariate Cox model. In contrast, the flow cytometric mean channel number, the fraction of cells in G2M, aneuploidy of the G0/G1 peak, aneuploidy of the G2M peak, tumor size, and mitotic activity index were statistically significant in univariate models, together with the patient age and sex, and whether or not the patient presented with metastases. In a multivariate model, > 10 mitotic figures per 50 high-power fields and metastases indicated a poor prognosis. If metastasis was not allowed to enter the model, the mitotic index and aneuploidy of the G2M peak portended a poor prognosis.

Female↗
PubMed1993

bcl-2 rearrangement in Hodgkin's disease. Results of polymerase chain reaction, flow cytometry, and sequencing on formalin-fixed, paraffin-embedded tissue.

We examined 81 cases of Hodgkin's disease for evidence of the t(14;18) translocation, using the polymerase chain reaction assay on lysates of formalin-fixed, paraffin-embedded tissue. Seven of 74 amplifiable cases (9%) were positive for the translocation, which involves the bcl-2 oncogene and the immunoglobulin heavy chain gene. Two of these cases were sequenced and the breakpoints had the same pattern found in follicular lymphoma. The nuclei from one of the cases were sorted into large and small subpopulations. The t(14;18) signal was more intense in the large nucleus subpopulation, which contained a greater proportion of Reed-Sternberg-like nuclei. These results are consistent with the hypothesis that Reed-Sternberg cells carry the translocation, but they do not exclude the possibility that the translocation is found in cells representing the reactive component of Hodgkin's disease. The results also demonstrate that routinely processed material is suitable for polymerase chain reaction-based analysis of translocations, although the sensitivity is reduced 10- to 100-fold, compared with fresh tissue.

Base Sequence↗
PubMed1989

Prognostic significance of DNA ploidy in carcinoma of prostate.

Flow cytometry was used to measure the DNA content in archived paraffin-embedded human prostatic cancer tissue for 69 patients with known outcomes that presented between 1975 and 1982. Of these, 51 patients had clinically localized lesions and were surgically staged prior to radical prostatectomy, while 18 patients presented with advanced Stage D2 disease. Thirty-six of 37 (97.3%) pathologic Stage B lesions were diploid. In contrast, the majority (72.2%) of patients with metastatic disease had aneuploid tumors. The average Gleason grade for aneuploid tumors was 8.2 +/- 1.98 versus 5.5 +/- 1.89 for diploid tumors (p less than 0.01). For 51 patients with clinically localized tumors, 13.9 percent of diploid tumors with a low Gleason sum (2 to 6) had extracapsular spread of tumor or regional lymph node involvement compared with 83.3 percent of aneuploid tumors with high Gleason scores (7 to 10). The addition of DNA ploidy to degree of glandular differentiation may enhance the prognostic evaluation of prostatic tumors and eventually improve our ability to select patients who are likely to benefit from radical prostatectomy.

Aged↗
PubMed1988

Expression of genes for platelet-derived growth factor in adult human venous endothelium. A possible non-platelet-dependent cause of intimal hyperplasia in vein grafts and perianastomotic areas of vascular prostheses.

Neointimal fibromuscular hyperplasia (NFH) in vein grafts and perianastomotic zones of vascular prostheses has been attributed to the effects of platelet-derived growth factor (PDGF) released by platelets interacting with bypass conduits. But inhibition of platelet aggregation often fails to prevent NFH, and recurrent growth of intact, platelet-free endothelium over perianastomotic areas where NFH occurs is inconsistent with the concept of sustained PDGF release from platelets causing NFH progression at late times after surgical procedures. Cultured bovine aortic endothelial cells (ECs) and human umbilical vein ECs have been shown to release a PDGF-like molecule. We report that confluent cultured fourth passage adult human saphenous vein ECs (AHSVECs) grown in the presence of heparin (100 micrograms/ml) and retina-derived growth factor (RDGF) studied by Northern blotting transcribed a messenger ribonucleic acid (mRNA) of 3.9 kb, strongly hybridizing to PDGF B chain probes, and two species of 2.0 and 2.6 kb hybridizing to PDGF A chain probes. Withdrawal of RDGF and heparin from these cultures for 48 hours before mRNA extraction amplified the scanning densitometric mRNA signal per cell by 8.0 +/- 7.6 fold (mean +/- SD) (N = 4 cultures) for B chain mRNA and 5.2 +/- 3.6 fold (N = 3 cultures) for A chain mRNA. In addition, AHSVEC cultures released a PDGF-like substance, because 50% vol/vol AHSVEC-conditioned serum-free medium increased tritiated thymidine uptake elevenfold in PDGF receptor-bearing 3T3 cells whereas an excess (50 micrograms/ml) of nonspecific goat anti-human-PDGF antibody significantly reduced this increase by a mean of 30% to 7.0 +/- 3.4 fold (N = 6 trials, p less than 0.001). Flow cytometry determined AHSVEC cultures to be proliferating with a mean of 6.2% +/- 1.9% (N = 3 culture lines) of ECs in S phase even at confluence when deprived of EC mitogens for 48 hours. Adult human ECs, which proliferate on bypass conduits and host vessels after perioperative injury, may play a role in causing NFH by stimulating proliferation of adjacent smooth muscle cells. Prevention of NFH may require not only antiplatelet agents but also ways to prevent EC release of smooth muscle cell mitogens in response to perioperative EC injury.

Animals↗
PubMed1987

Cell growth cycle block of T cell hybridomas upon activation with antigen.

Stimulation of antigen-specific T cell hybridomas with the appropriate antigen/MHC combination, at concentrations that resulted in the secretion of the lymphokine interleukin 2, resulted in a dose-dependent decrease in both [3H]thymidine incorporation and cell growth. Flow cytometric studies demonstrated that stimulation with antigen resulted in a cell cycle block that was most evident at the G1/S border, and mixing studies revealed that bystander T cells of different antigen specificities were not affected. For at least the large majority of T cells, the G1/S cell cycle block appeared to be irreversible after 24 h of exposure to antigen. This cell cycle block may be useful as a rapid and quantitative measure of T cell hybridoma activation, as a means of selecting T cell hybridomas that have functional alterations in the reception of stimulatory signals, and may serve as a model of the induction of tolerance in immature T cells.

Animals↗
PubMed1987

Heterogeneous expression of a murine B16 melanoma-associated antigen correlates with cell cycle.

A murine monoclonal antibody (MM2-3C6) that reacts with a B16 murine melanoma-associated membrane antigen was used to study the relationship of antigen expression to the cell cycle. Dual-parameter flow cytometric measurements of membrane antigen and DNA revealed that antigen-positive cells were present throughout the cell cycle. Peak antigenic expression was noted during the late log phase of the cell growth curve with negligible antigen-negative population. The emergence of a distinct antigen-negative population (30%-40%) was noted in the late stationary phase. Cell cycle analysis indicated that the negative subpopulation was restricted to the G0/G1 phase, thus, demonstrating antigenic heterogeneity within the tumor cell population. Cell sorting was performed to analyze the origin of such heterogeneity. Following two sequential sortings, the antigen-negative cells became antigenic upon reculture. Again, at the late stationary phase, a distinct antigen-negative population (30%-40%) emerged. The sorted antigen-positive cells showed flow cytometric profiles identical to the sorted antigen-negative population upon reculture. Therefore, in this murine model, it appears that antigen expression is cell cycle dependent and such expression seems to be associated with proliferation.

Animals↗
PubMed1985

The use of flow cytometry and cell sorting in a human colon carcinoma model.

We investigated the growth characteristics of a human colon carcinoma cell line, WiDr, grown in culture flasks and on chick embryonic skin (CES). WiDr cells labeled in vitro with bromodeoxyuridine (BrdU) and analyzed by combined propidium iodide/Hoechst 33258 fluorescence showed evidence of more BrdU incorporation in early S phase as compared to late S phase. When inoculated on the CES, WiDr cells multiplied and invaded the underlying skin. Morphologic examination showed that with extended culture WiDr cells on the CES undergo progressive structural organization with the development of acini and basal lamina, structures similar to those in in vivo tumors. WiDr cells were labeled with monoclonal antibody to carcinoembryonic antigen (CEA) and the brightest 2% of the population was sorted. When subsequently grown on the CES, the sorted cells formed significantly more acinar structures at 3 and 6 days of culture than an unsorted population grown for a comparable time.

Animals↗
PubMed1985

Expression of a murine B16 melanoma-associated antigen analyzed by flow cytometry.

Understanding factors regulating expression of tumor antigens is vital to the design of rational immunotherapeutic strategies. A murine monoclonal antibody (MM2-3C6) that recognizes a B16 murine melanoma-associated membrane antigen was used to study antigen expression in relation to cell cycle, clonal heterogeneity, and growth density. Dual-parameter flow cytometric measurements of DNA content and membrane antigen demonstrated that antigen-positive cells were found throughout the cell cycle. However, some antigen-negative cells were observed and were restricted to the G0/G1 phase demonstrating the antigenic heterogeneity of this tumor line. Three sublines of B16, F1, F10, and F1r with variable metastatic potential, and 50 B16 F1 clones all expressed the antigen with a mean antigen density above background of 8.84 +/- 2.52 [( mean cell fluorescence/cell size] X 10(-3] ranging from 3.68 to 16.57. Further studies of three sublines and five clones showed antigen density fluctuated over an 8-day growth period in culture with daily changes of medium. During log growth from Day 1 (1.2 +/- 0.08 X 10(4) cells/cm2) to Day 4 (20.4 +/- 3.01 X 10(4) cells/cm2), antigen density increased from 4.09 +/- 0.29 to 6.86 +/- 0.29. By Day 8 (26.5 +/- 2.86 X 10(4) cells/cm2) when the cells had been confluent for 3 days, antigen density decreased to 2.54 +/- 0.26 which was significantly lower than other days measured (P less than 0.05). It is concluded that tumor cell proliferation and cell density can modulate antigen expression in this system and, therefore, this may be a useful model to study tumor cell escape from immunotherapy.

Animals↗