Vein adaptation to arterialization in an experimental model.

PURPOSE: The events preceding myointimal thickening in vein grafts after vascular reconstructions are not well characterized. Indeed, the injury response associated with vein graft arterialization may be different than that observed in the balloon angioplasty model. Therefore, we used a rat model to study the early cellular response after arterialization of vein grafts. METHODS: Epigastric veins were placed as femoral artery interposition grafts in 37 male Lewis rats (weight range, 350-400 g). Vein grafts and contralateral epigastric veins were harvested at different time points (6 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 30 days, and 70 days). Tissue specimens were processed for histology and immunohistochemistry with antibodies for the proliferating cell nuclear antigen (PCNA) and for different cell types. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used as a means of determining the presence of apoptosis. Electron microscopy was used as means of assessing the integrity of the endothelial cell surface (SEM) and confirming the presence of apoptosis (TEM). Specimens were also snap frozen in liquid nitrogen for RNA isolation and molecular analysis. RESULTS: At 1 day, endothelial denudation with platelet deposition on the surface was shown by means of SEM. Both apoptosis and necrosis of smooth muscle cells (SMCs) were present in the media, along with monocyte infiltration. Cellular proliferation and apoptosis were most intense within the first week of implantation. PCNA staining was first seen in the adventitial fibroblasts and microvessels, then in the medial SMCs at 3 days. With reverse transcriptase polymerase chain reaction, upregulation of vascular endothelial growth factor (VEGF) messenger RNA (mRNA) was noted at 1 day. Myointimal thickening progressively developed, with no apparent diminution of the luminal area as long as 70 days after implantation. By means of the analysis of the transforming growth factor beta1, mRNA showed expression during intimal thickening and accumulation of extracellular matrix. Reendothelialization was complete at 30 days. CONCLUSIONS: These observations indicate that the cellular composition in our vein graft model is similar to human stenotic explants. Endothelial denudation is observed in rat vein grafts with complete regeneration by 30 days. VEGF mRNA is upregulated at 1 day, followed by proliferation of microvessel endothelial cells in the adventitia. Cellular proliferation and apoptosis are minimal after 21 days, with progressive intimal thickening likely to be the result of matrix accumulation.

Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies.

The authors questioned whether an automated kinetic mode assay of combined cytomegalovirus (CMV) late viral message and immediate and early antigens might result in a more sensitive and timely CMV diagnosis relevant to speedy treatment in the transplant setting. Toward this end, two cohorts were studied using automated in situ hybridization (ISH) for CMV as well as immunohistochemistry (IHC). The first cohort of patients consisted of 19 cases that were histologically positive (CMV-associated cytopathic change). A second cohort consisted of 10 cases that were histologically negative, yet culture positive. From the first cohort of histologically positive cases, 100% were positive by both ISH and IHC run on separate slides. In the second cohort, CMV was detected overall in 70% of cases (50% by ISH alone and 30% by IHC alone). These results indicate that a combined assay of ISH and IHC can detect more cases than routine hematoxylin and eosin staining or either assay alone. In two illustrative cases, used to demonstrate the feasibility of combining ISH and IHC, the authors used a combined two-color assay (ISH and IHC) performed sequentially on the same slide. The combined assays resulted in colocalized single cell message and protein in some cells and demonstrated more positive cells overall (some positive by IHC alone, some by ISH alone, and some by both) than either assay alone. The combined dual color assay can be completed within 4 to 5 hours giving the prospect of a same day result, which is faster than shell vial technique with immunofluorescence (24 to 48 hours) or culture (7 to 14 days). This study demonstrates that combining CMV message and protein assays results in a more sensitive assay and, when carried out in the kinetic mode, allows a speedy result relevant to early anti-CMV therapy.

A multidisciplinary approach to the diagnosis of cutaneous T-cell lymphomas.

The cutaneous T-cell lymphomas (CTCLs) comprise a spectrum of non-Hodgkin lymphomas with a predilection for the skin. This heterogeneous group of CTCLs include the prototypic CTCL mycosis fungoides (MF) and the recently described Ki-1+ lymphomas. MF is notoriously difficult to diagnose in its early stages. The histologic appearance of early MF is indistinguishable from that of chronic dermatitis. The limitations of light microscopy in the diagnosis of the CTCLs have led to the development of other diagnostic laboratory techniques. The best approach to the diagnosis of the CTCLs is a multidisciplinary one and should include ultrastructural morphometry, immunophenotyping, immunogenotyping, and histologic evaluation whenever possible. It is the purpose of this overview to point out the strengths and weaknesses of each of these techniques and, together with clinical input, to provide a comprehensive and rational approach to patient care.

Immunobiologic factors predictive of clinical outcome in diffuse large-cell lymphoma.

The prognostic importance of immunobiologic factors in diffuse large-cell lymphoma (DLCL) is studied in 105 consecutive DLCL patients. Multivariate results using the Cox proportional hazards model clearly indicate that the Ki-67 index (P = .002), a marker of cell proliferation activity, and the presence or absence of human leukocyte antigen-DR (HLA-DR) (P = .007) are strong predictors of survival even in the presence of established clinical factors of stage (P = .015) and symptoms (P = .050). Using these four variables, prognostic groups were formed identifying patient groups with varying degrees of risk. The group of patients with three or four risk factors present at the time of diagnosis had a median survival of 4 months compared with a median survival of 59 months for the group with no risk factors. Similarly, prognostic groups for disease-free survival (DFS) were constructed based on the proportional hazards model that involved B versus T phenotype (P = .035) and HLA-DR (P = .054). Median DFS for the patient group with one or two risk factors present was 11 months compared with 43 months with no risk factors present. This study suggests immunobiologic parameters are important predictors of clinical outcome in DLCL patients and are of value in identifying subgroups of patients who have not responded to currently available therapy. The practical significance of this study is to identify parameters that may suggest specific changes in therapy of patient subgroups.

T-cell antigen-positive multiple myeloma.

We report the simultaneous expression of T-cell antigens on the myeloma cells from six patients with multiple myeloma (MM). These six patients come from a total population of 215 samples (115 direct samples, clinical incidence of 5.2%) of plasmacytic malignancies immunotyped at the University of Arizona. Four patients expressed T helper antigen (Leu 3, CD4), one expressed T-cell antigen receptor (Leu 4, CD3), and one expressed E-rosette antigen receptor (Leu 5, CD2). The presenting clinical features, histology, and plasma cell morphology showed no differences from multiple myeloma patients who did not express T-cell antigen. However, although the survival duration ranged from 5 to 93 mo overall, survival from demonstration of T antigen expression was very short, (2 to 7+ mo), with five of six (80%) patients dying less than or equal to 5 mo after study. The reason for T antigen expression is unknown. It may indicate that myeloma can arise from a normally minor subpopulation of B cells involved with immunoregulation; conversely, it could be a coincidental aberrancy associated with malignant change in the plasma cells.

Nuclear contour irregularity correlates with Leu-9-, Leu-8- cells in benign lymphoid infiltrates of skin. An ultrastructural morphometric and quantitative immunophenotypic analysis suggesting the normal T-cell counterpart to the malignant mycosis fungoides/Sézary cell.

The purpose of the present study was to determine if a nonmalignant skin-associated T-cell might exhibit nuclear irregularity and represent the normal counterpart to the malignant Sézary T-cell found in mycosis fungoides (MF) and the Sézary syndrome (SS). Punch skin biopsies from ten benign lymphoid infiltrates of skin were subjected to ultrastructural morphometric analysis and quantitative immunohistochemistry (on serial frozen sections) using a battery of monoclonal antibodies. Form factor (FF) (4 pi A/p2) was used to assess nuclear contour irregularity. The lymphoid infiltrates were morphometrically heterogeneous (range of FF values: 0.482-0.704). Immunophenotypically, there was marked variation in host response to the variety of antigens presented. Linear regression analysis was used to determine if nuclear contour irregularity showed any statistical relationship to immunophenotypically defined lymphocyte subpopulations. It was determined that nuclear contour irregularity (mean FF values) did not correlate with the presence of any surface antigen tested. This included the antigens Leu-2a, Leu-3a + b, Leu-4, Leu-6, Leu-7, Leu-8, Leu-9, Leu-14, and Ia. There was, however, significant correlation of increased nuclear contour irregularity with the presence of Leu-9- and Leu-8- cells (r = 0.7 and 0.6, respectively), lymphocyte subsets reportedly deficient in MF and the SS. This finding leads to the speculation that the Leu-9-, Leu-8- reactive T-cell with an irregular nucleus might represent the normal counterpart to the malignant clonally expanded T-cell found in MF and the SS. It was also determined that helper to suppressor ratios varied 68-fold from 0.2 to 13.5 among these ten benign lymphoid infiltrates of skin. This finding underscores the futility of using helper to suppressor ratios as a diagnostic tool in defining T-cell malignancies.

Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67.

To assess the prognostic significance of the growth fraction in diffuse large cell lymphoma (DLCL), we studied 105 DLCL patients with the monoclonal antibody Ki-67 applied to frozen tissue sections. Ki-67 detects a nuclear antigen associated with cell proliferation not found in resting cells. Ki-67 findings and other clinical prognostic factors were correlated with outcome using univariate and multivariate analyses in the proportional hazards model. High proliferative activity, defined as nuclear Ki-67 expression in greater than 60% of malignant cells (Ki-67 greater than 60), was found to be a strong predictor of poor survival among these patients (P = .003, log-rank). The 19 patients with Ki-67 greater than 60% had a median survival of 8 months compared with a median survival of 39 months for the 86 patients with Ki-67 less than or equal to 60%. Examination of pretreatment clinical variables indicated the patient groups were similar with regard to age, sex, stage, B symptoms, tumor bulk, and lactate dehydrogenase (LDH). Both patient groups received comparable curative intent therapy and showed comparable complete response rate precluding treatment differences as modifying outcome. Multivariate analysis indicated Ki-67 is an independent predictor of survival (multivariate P = .006). Further statistical analysis using only B-cell DLCL patients treated with CHOP (63 patients) indicated that Ki-67 greater than 60 retained strong prediction of poor outcome (P = .002, log-rank) among this homogeneous group. We conclude that high proliferative activity (Ki-67 greater than 60) is an independent factor allowing laboratory prediction of probable poor outcome of DLCL.

Burkitt's-like lymphoma of T-cell type.

Over an 8-yr period, we studied 29 cases of Burkitt's/Burkitt's-like lymphoma and unexpectedly found 2 Burkitt's-like cases of the T-cell type. One case presented as diffuse adenopathy in a 35-yr-old male. A second case presented as a jaw mass in a 2-yr-old girl with Down's syndrome. Histologically, each case demonstrated usual Burkitt's-like morphology (intermediate-size cells with high nuclear/cytoplasmic ratio, 1 to 3 prominent nucleoli, high mitotic rate, basophilic cytoplasm, and cytoplasmic vacuolation). Ultrastructural morphometric data corroborated the Burkitt's-like nature of these neoplasms. Immunologically, the neoplasms were of "novel" T-cell phenotype, as seen in peripheral T-cell lymphoma (PTL). The cases showed variable expression of activation antigens (e.g., Ia) and weak to moderate expression of proliferation antigens as measured by Ki-67. This modest proliferative activity (less than 25% Ki-67 expression) contrasts with Burkitt's-like lymphomas of the B-cell type which usually show greater than 80% Ki-67 expression. The jaw tumor also demonstrated positivity for human progenitor cell antigen (HPCA) as commonly found in leukemia. Both cases mimic granulocytic sarcoma by virtue of their eosinophilic/myelocytic recruitment--a phenomenon previously reported in association with PTL. The patients have survived 62 wk and 20 wk, respectively, surpassing the survival rates seen in our concurrent B-cell Burkitt's-like lymphomas (12 wk). Burkitt's-like lymphoma of the T-cell type appears to be a distinctive immunological subset of potential clinical and prognostic relevance.

A comprehensive immunotopographic map of human thymus.

We have achieved a comprehensive immunotopographic mapping of human thymus by using a large battery of monoclonal antibodies and the methodological refinement of comparative serial tissue section immunohistochemistry, allowing analysis of multiple phenotypes in the same tissue site. Previous immunohistochemical studies of thymus have concentrated on the majority T-cell and epithelial cell populations. Besides demonstrating the complexity of T-cell antigenic expression (e.g., simultaneous cortical expression of Leu 2, Leu 3, CALLA, Tdt, and Leu 6), we delineate surprisingly complex B-cell zones (e.g., septal B-follicles with DRC+C3d+ dendritic cells and zonal maturation of B-cells). Whereas septal B-follicles were found in 25% of cases, medullary B-cells were universally present as a substantial minority component. This expanded immunotopographic knowledge of the complex T-, B-, epithelial, and reticulum cell neighborhoods suggests that the thymus is an organ capable of a broad repertoire of immunological responses, not limited to T-cell development.

Signet-ring cell lymphoma of T-cell origin. An immunocytochemical and ultrastructural study relating giant vacuole formation to cytoplasmic sequestration of surface membrane.

In contrast to previous accounts of signet-ring lymphoma as a B-cell neoplasm, we report a case of signet-ring, large-cell lymphoma of T-cell lineage. Immunologic and ultrastructural studies were performed on a subcutaneous mass noted initially, as well as on an enlarged lymph node that developed later, in a 69-year-old man. Immunologic assessment indicated strong expression of T-helper antigen (Leu 3a + b), universal T-antigens (Leu 1, 5), and Ia. There was an absence of T-suppressor/cytotoxic antigen (Leu 2a), universal T-antigens (Leu 4, 9), and immunoglobulin light and heavy chains. Collectively, these findings indicate a mature T-cell lymphoma of T-helper type in an activated (Ia+) state. In contrast to previous reports of T-cell and Ia occurring solely as surface antigens, we demonstrated pools of cytoplasmic Leu 1, 3, 5 and Ia that displaced the nucleus. The ultrastructure of the giant cytoplasmic vacuoles was identical to the microvesicle-containing vacuoles reported in signet-ring cell lymphomas of B-cell lineage. In our case of T-cell lineage, we found substantial evidence of endocytosis by the neoplastic cells and numerous giant multivesicular bodies. The pools of cytoplasmic T and Ia antigens may result from abnormal internalization of surface T-antigens or the sequestration of T-antigen-containing Golgi-derived vesicles. Our combined immunologic and ultrastructural findings suggest that aberrant membrane recycling may be the common denominator of signet-ring formation in both B- and T-cell signet-ring lymphomas.

Establishment of two new myeloma cell lines from bilateral pleural effusions: evidence for sequential in vivo clonal change.

Two new human myeloma cell lines have been established from a 36-year-old woman with refractory IgG kappa multiple myeloma in whom bilateral malignant pleural effusions developed. The malignant plasma cells from each effusion were set up in a liquid culture using an L-15 medium containing catalase, glutathione, selenous acid, ascorbic acid, insulin, transferrin, additional glutamine hydrocortisone, and 2-mercaptoethanol and designated as M-3 medium. Two IgG kappa cell lines, LB -831 and LB-832, were established and proved to be Epstein-Barr virus negative using the internal repeat sequence DNA probe. Characteristic plasma cell morphology was evident by light and electron microscopy. Immunotyping revealed an IgG kappa , B1+, B2-, Ia (HLA-DR)+, CALLA+ phenotype for each cell line as well as for the original pleural fluid and bone marrow myeloma cells. The supernatants also contained IgG kappa, beta 2 microglobulin, and large amounts of osteoclast-activating factor (indicating bone-resorbing activity). Cytogenetic analysis of the LB-831 cell line revealed a nearly triploid highly abnormal karyotype with numerous clonal chromosomal abnormalities involving chromosomes 1, 3, 5, 7, 13, and 15; several structurally abnormal marker chromosomes; and a putative homogeneously staining region on chromosome 7p at band p22. Analysis of the LB-832 cell line revealed several additional clonal abnormalities. These additional cytogenetic changes suggest that in vivo sequential clonal evolution occurred in this patient. Therefore, two new but related cell lines have been established, which should prove useful for further biological studies.

Immunotopography of splenic lymphoma of small cleaved B-cell type.

Using a battery of monoclonal antibodies on snap-frozen sections, we delineated the immunoarchitecture of two splenic small cleaved cell lymphomas (SCL). Both cases had light- and heavy-chain restricted immunoglobulin (lg) expression signifying replacement of splenic white pulp by a single B-cell clone. Both the monotypia and aberrant topography of lg expression in SCL contrasted with the usual polyclonal, zonal lg expression in reactive splenic B-cell zones. Pan B antigens (B1, B4, and L14) were constant in expression as expected for B-cell neoplasms, while B-cell maturation antigens (B2, IgD, and CALLA) were variably expressed, suggesting that different SCL may derive from separate phases of B-cell ontogeny. Close association of SCL with dendritic reticulum cells suggests SCL may derive from splenic secondary follicles or home to these sites. The variable T-cell component detected by a T-cell panel (Leu 1-9) indicates the substantial range of T-cell reactivity in splenic SCL. We emphasize the immunologic aberrancy of splenic SCL when compared to normal splenic B-cell immunotopography. Further, we illustrate the utility of serial tissue section immunochemistry in revealing complex neoplastic cell phenotypes and in revealing the relationships of reactive cells to neoplastic cells.

Further delineation of the immunoarchitecture of the human spleen.

Using a battery of monoclonal antibodies directed at B and T cell antigens, we delineate the immunotopography of the human spleen. The tissue section immunohistochemical methods employed demonstrate the complexity of white pulp reactivity with both light and heavy chain immunoglobulin expression appearing polyclonal. Zonal expression of heavy chains suggests an anatomic basis for the known sequence of heavy chain switching due to immunoglobulin gene rearrangement. Localization of universal B-cell antigen, B1, delineates the entire B-cell zone whereas antibodies directed at universal T antigens (e.g. Leu 1, 4, 9) delineate the T cell zone. Leu 14 demonstrates striking B-zone staining in normal spleen and in hairy cell leukemia, suggesting that leukemic cells in this disease arise from Leu 14+ B cells. Leu 2a staining occurs not only on lymphoid cells but probably also on sinusoidal endothelial lining cells.