Changes in plasma TIMP-1 levels after resection for primary colorectal cancer.

BACKGROUND: Increased plasma levels of tissue inhibitor of metalloproteinases (TIMP-1) are associated with poor outcome in colorectal cancer (CRC), however postoperative changes in plasma TIMP-1 levels after resections for CRC have not been thoroughly evaluated. MATERIALS AND METHODS: Plasma samples were collected from 45 patients with primary CRC, preoperatively, 2 hours after surgery, and at days 1, 2, 7, 28, 45, 60, 75 and 90 after surgery. TIMP-1 and CEA levels were determined using the ARCHITECT Immunoanalyzer. RESULTS: Postoperatively, the mean (geometric) TIMP-1 level increased and had a maximum level at day 1 (p < 0.0001). The mean TIMP-1 level then declined to a level at day 90 similar to the mean preoperative level. CONCLUSION: A mean decline in plasma TIMP-1 levels was not observed within 90 days. However, individual significant reductions of plasma TIMP-1 levels did occur within 28-60 days postoperatively.

Assessment of the biological variation of plasma tissue inhibitor of metalloproteinases-1.

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) measurements in plasma may be useful for the early detection and prognosis of colorectal cancer (CRC). Data on analytical performance and normal intra- and interindividual biological variation are required in order to interpret the utility of TIMP-1 in CRC. The aim of this study was to establish the biological and analytical variation of plasma TIMP-1 in volunteers. MATERIAL AND METHODS: Three separate studies were undertaken. 1: Plasma was collected from 23 volunteers 6 times within a 3-week period, first in September 2004 (round [R] 1), then repeated in May 2005 (R2) and May 2006 (R3) in the same group of individuals. TIMP-1 levels were determined by the MAC15 ELISA assay and with the Abbott ARCHITECT i2000 Immunoanalyzer. 2: Circadian variation was evaluated in plasma collected 7 times within a 24-hour period (n=16). 3: Effects of physical exercise were evaluated in plasma collected before and after bicycling (n=14). In studies 2 and 3 TIMP-1 levels were determined with the MAC15 ELISA assay only. RESULTS: A significant correlation between TIMP-1 MAC15 and ARCHITECT i2000 was shown (rs=0.78, p<0.002), with consistently higher levels being detected by the ARCHITECT i2000. Median levels of TIMP-1 (ARCHITECT) at 8 a.m. in each round were 74.9 ng/mL (range 65.7-89.9) (R1), 87.3 ng/mL (range 72.7-127.9) (R2), and 81.9 ng/mL (range 66.8-113.6) (R3). The within-subject variation was 10.7%, the variation between rounds was 7.4%, and the intraclass correlation was 46.2%. Comparison between the 3 rounds and time of collection showed that TIMP-1 values decreased by 11% after storage for more than 16 months (p=0.0002). A systematic circadian variation in plasma TIMP-1 levels was not observed (p=0.17). No significant variation of plasma TIMP-1 was found in relation to physical exercise (p=0.92 [global test]). CONCLUSION: Levels of plasma TIMP-1 in volunteers show limited circadian, day-to-day, week-to-week and season-to-season variation. In addition, physical exercise has no impact on plasma TIMP-1 levels. Possible storage-dependent decreases in plasma TIMP-1 levels warrant further investigation.

Characterization of monoclonal antibodies directed against squamous cell carcinoma antigens: report of the TD-10 Workshop.

Thirteen monoclonal antibodies directed against squamous cell carcinoma antigens (SCCA1 and SCCA2) were obtained from five international collaborating laboratories participating in the ISOBM TD-10 Workshop. Native and recombinant forms of SCCA were used in a wide variety of approaches to determine the reactivity and specificity of these antibodies. Based on reactivity, the antibodies could be divided into three groups: the SCCA1-reactive group containing those that reacted only with recombinant SCCA1 (rSCCA1) and native SCCA1 (nSCCA1) antigens, the SCCA2-reactive group containing those that reacted only with recombinant SCCA2 (rSCCA2), and the pan-reactive group containing those antibodies that reacted with rSCCA1, nSCCA1, and rSCCA2. Binding to radioiodinated rSCCA1 showed that all reactive antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Binding to labelled rSCCA2 demonstrated that five antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Antibody reactivity on Western blots was tested with nonreduced and reduced native and recombinant SCCA1 and SCCA2. In general, these findings showed that reduction had little effect on binding to SCCA1, but often a strong effect on the binding to SCCA2. Binding of antibodies to rSCCA1 and rSCCA2 in complexes with cathepsin L and G, respectively, was used to assist in the localization of epitope regions in enzyme-complexed SCCA. Cross-inhibition experiments showed that SCCA1-reactive antibodies represent two different epitope groups, and this is supported by their ability to make SCCA1-specific assays by combining antibodies from the two epitope groups. The SCCA2-reactive group represents two related antibodies and one unique as seen in cross-inhibition, but they do not form successful assay combinations. Classification of the pan-reactive antibodies is more difficult, as some epitope groups differ when results from rSCCA1 are compared with rSCCA2 as the target. However, two antibodies are outstanding, SCC107 and SCC113, as they are high-affinity antibodies which react equally well with free and protease complexes of SCCA1 and SCCA2. The precise location of epitopes was further studied using sequential overlapping peptides and homology modelling. The findings from this workshop strongly indicate that the recombinant antigens (rSCCA1 and rSCCA2) are very similar in epitope structure to the native counterparts in saliva, and squamous epithelium from normal and cancer tissues. Therefore, it is reasonable to conclude that the specificities found are reliable and have application for antibody measurement of all forms of squamous cell carcinoma in serum except SCCA2 in complex with its protease.

Determination of the "reflex range" and appropriate cutpoints for percent free prostate-specific antigen in 413 men referred for prostatic evaluation using the AxSYM system.

OBJECTIVES: Prostate-specific antigen (PSA) exists in the serum in two clinically important molecular forms: free PSA and PSA complexed to alpha 1-antichymotrypsin. Total PSA approximates the sum of the free and complexed forms. Preliminary investigations have illustrated the potential benefits of using percent free PSA to enhance the clinical utility of PSA in distinguishing benign prostate disease from prostate cancer. The current study defines the optimal range of total PSA for measuring percent free PSA (reflex range) and generates appropriate cutpoints for percent free PSA within this range. METHODS: A total of 413 patients, 225 (54%) with benign prostate disease (mean age, 67 years) and 188 (46%) with prostate cancer (mean age, 66 years), who had PSA values between 2.0 and 20.0 ng/mL participated in the investigation. All patients underwent a sextant biopsy to establish the diagnosis. The serum specimens were assayed with the AxSYM PSA assay (total PSA) and AxSYM Free PSA assay (Abbott Laboratories; Abbott Park, IL). Percent free PSA was calculated for all patients. Receiver operating characteristic (ROC) curves were generated for various ranges of total PSA to determine the reflex range that maximized the increase in sensitivity and specificity of percent free PSA over total PSA. Within the optimal range, the ROC curves were utilized to generate cutpoints for percent free PSA to be used in clinical practice. RESULTS: The appropriate reflex range for the utility of percent free PSA was 3.0 to 10.0 ng/mL. The appropriate cutpoint for percent free PSA when the total PSA value was 3.0 to 4.0 ng/mL to achieve 90% sensitivity for the detection of prostate cancer was 0.19. This approach resulted in a biopsy rate of 73% and a cancer detection rate of 44% in men with a total PSA value between 3.0 and 4.0 ng/mL. The appropriate cutpoint for percent free PSA when the total PSA value was 4.1 to 10.0 ng/mL to ensure 95% sensitivity for detection of prostate cancer was 0.24. Within the range of 4.1 to 10.0 ng/mL, this approach resulted in 13% fewer negative biopsies and failure to detect 5% of the cancers. CONCLUSIONS: Percent free PSA should be utilized in patients with a total serum PSA value between 3.0 and 10.0 ng/mL. In patients with a total PSA value between 3.0 and 4.0 ng/mL, percent free PSA enhanced the detection of prostate cancer (improving sensitivity). In patients with a total PSA concentration ranging from 4.1 to 10.0 ng/mL, negative biopsies were eliminated (improving specificity).

Structural comparison of prostate-specific antigen and human glandular kallikrein using molecular modeling.

OBJECTIVES: Prostate-specific antigen (PSA), the most useful tumor marker for prostate cancer, is one of three members of the human kallikrein family of serine proteases. PSA and human glandular kallikrein (hK2, previously called hGK-1) share extensive homology and are both produced in the prostate under androgen control. Our goals were to use molecular modeling techniques to generate models of the tertiary structure of PSA and hK2 and to compare their molecular features and areas of homology using these models. METHODS: Models of PSA and hK2 were generated by extrapolating from available crystallographic coordinates and amino acid sequences of homologous members of the serine protease family using standard comparative methods. RESULTS: Porcine kallikrein (57% homology) and rat tonin (53% homology) were used as templates for PSA. Porcine kallikrein (67% homology) was used as a template for hK2. The models were superimposed to define regions of nonhomology between PSA and hK2. CONCLUSIONS: Three-dimensional protein models of PSA and hK2 were generated. These models have potential uses in analyzing antigen-antibody interactions, modeling of inhibitor complexes of both PSA and hK2, and furthering our understanding of the molecular interactions involved in the clinical detection of PSA and hK2.

Infection-associated hemophagocytic syndrome. Evidence for Epstein-Barr virus gene expression.

Autopsy specimens from a patient with infection-associated hemophagocytic syndrome (IAHS) were evaluated for the presence of Epstein-Barr virus DNA and RNA using in situ hybridization. Frozen sections of liver, lymph node, and spleen were probed with EBV Bam HI-H & W, gamma interferon, and SP-65 plasmid DNA as a negative control probe. Hybridization patterns before and after treatment with ribonuclease A were examined. Both EBV probes produced diffuse hybridization throughout the tissues; in addition, there were some foci of extremely heavy concentrations of silver granules. A gamma interferon probe showed evidence of hybridization, but the overall intensity was not as great as with EBV probes. Pretreatment with ribonuclease A dramatically decreased hybridization in all tissues to EBV probes, but hybridization with SP-65 was unaffected. The elimination of EBV hybridization with ribonuclease A pretreatment provides the first evidence of EBV gene expression in an IAHS patient.

Prognostic factors in childhood T-cell acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Two hundred fifty-three children with newly diagnosed T-cell acute lymphoblastic leukemia (ALL), who were treated uniformly with modified LSA2L2 therapy, were evaluated using univariate and recursive partition analyses to define clinical or biologic features associated with risk of treatment failure. Overall event-free survival (EFS) at 4 years was 43% (SE = 4%). Factors examined included white blood cell (WBC) level, age, gender, race (black v other), presence of a mediastinal mass, hepatomegaly, splenomegaly, marked lymphadenopathy, hemoglobin level, platelet count, blast cell expression of antigens such as the common acute lymphoblastic leukemia antigen (CALLA, CD10), HLA-DR, and T-cell-associated antigens (CD3, CD4, CD8, CD7, CD5, and THY). Univariate analysis showed that age less than or equal to 5 or less than or equal to 7 years, WBC level less than 10, less than 25, less than 50 or less than 100 x 10(3)/microL, and blast cell expression of CD4, CD8, or CALLA were associated with significantly better EFS, while hepatomegaly and splenomegaly were associated with worse EFS. Recursive partitioning analysis showed that the most important single favorable prognostic factor was a WBC level less than 50 x 10(3)/microL and, for patients with WBC counts below this level, the most important predictor of EFS was blast cell expression of the pan-T antigen defined by the monoclonal antibody (MoAb), L17F12 (CD5). For patients with higher WBC levels, the most important predictor of EFS was blast cell expression of THY antigen. The recursive partitioning analysis defined three groups of patients with widely varied prognoses identified as follows: (1) those with a WBC count less than 50 x 10(3)/microL who lacked massive splenomegaly and had blasts expressing CD5 had the best prognosis (66%, SE = 7%, EFS 4 years, n = 84); (2) those with (b1) WBC counts less than 50 x 10(3)/microL with either massive splenomegaly or who had blasts lacking CD5 expression, or (b2) WBC counts greater than 50 x 10(3)/microL with expression of the THY antigen had an intermediate prognosis (39%, SE = 7% EFS at 4 years, n = 94); (3) those with WBC counts greater than 50 x 10(3)/microL and whose blasts lacked expression of THY antigen had the poorest outcome (EFS = 19% at 4 years, SE = 8%, n = 63). A three-way comparison of EFS according to these groupings showed significant differences among the three patient groups (P less than .001). The recursive partitioning was able to classify 241 (95%) of the patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunologic and clinicopathologic features of common acute lymphoblastic leukemia antigen-positive childhood T-cell leukemia. A Pediatric Oncology Group Study.

The immunologic and clinicopathologic features of common acute lymphoblastic leukemia antigen (CALLA)-positive and CALLA-negative T-acute lymphoblastic leukemia (ALL) and of CALLA-positive non-T, non-B ALL (common ALL) of childhood were compared. Twenty-seven percent of children with T-ALL had blasts that expressed CALLA. This expression was not associated with a significantly different incidence of expression of sheep erythrocyte-rosette receptors, glucocorticoid receptors, peanut agglutinin receptors, or T-cell antigens. CALLA-positive T-cell blasts were more likely to express a p24 leukemia-associated antigen (CD9, 50% versus 8%) and Ia antigens (39% versus 8%) than were CALLA-negative blasts. Patients with CALLA-positive and CALLA-negative T-ALL had similar clinicopathologic features at diagnosis. In contrast, compared to patients with common ALL, patients with CALLA-positive T-ALL were older, had higher leukocyte counts, and an increased incidence of splenomegaly, lymphadenopathy and mediastinal mass, similar to patients with CALLA-negative T-ALL. Patients with CALLA-positive T-ALL were more likely to achieve a complete remission (95% versus 83%, P = 0.055) and tended to have an increased duration of event-free survival (P = 0.07) than did patients with CALLA-negative T-ALL. The expression of T-cell antigens is more important than the expression of CALLA in defining biologically similar subgroups of childhood ALL. Preliminary evidence suggests that within T-ALL the expression of CALLA may be prognostically important.

Effect of differentiation on the expression of a nucleolar antigen with a molecular weight of 145,000 in HL-60 cells.

Mr 145,000 nucleolar protein antigen (p145) is associated with growing cells (R. L. Ochs et al., J. Cell Biol., 101: 211a, 1985) and has been found in a broad range of human cancers (J. W. Freeman et al., Cancer Res., 46: 3593-3598, 1986). In this study the presence of nucleolar antigen p145 was examined in the human promyelocytic tumor cell line HL-60 which was induced to differentiate by retinoic acid. Differentiation was monitored by morphological changes, [3H]thymidine accumulation, the ability of cells to reduce nitroblue tetrazolium, and cell number. The monoclonal antibody to nucleolar antigen p145 produced bright immunofluorescence in all cycling interphase HL-60 cells; during mitosis only diffuse staining was detected. Nucleolar antigen p145 in HL-60 cells was undetectable after 132 h of treatment with retinoic acid. The absence of nucleolar antigen p145 was associated with an 81% decline in thymidine accumulation and apparent inactivation of ribosomal and nonribosomal DNA transcription as observed by electron microscopy. The loss in expression of the antigen also correlated with increased nitroblue tetrazolium-positive cells, appearance of morphologically distinct myeloid cells, and termination of cell proliferation. These data indicate that the expression of nucleolar antigen p145 occurred in cycling HL-60 cells but not in terminally differentiated noncycling HL-60 cells.

Clinicopathologic aspects of E rosette negative T cell acute lymphocytic leukemia: a Pediatric Oncology Group study.

The Pediatric Oncology Group has studied 1,367 patients with non-B cell acute lymphocytic leukemia (ALL) of whom 186 (14%) had blasts that reacted with previously well-characterized heteroantisera, recognizing a T-lymphocyte specific surface membrane antigen (PT+). In 87 of these T cell cases, the leukemic cells failed to form at least 20% of sheep erythrocytic rosettes at 4 degrees C. Comparison of clinicopathologic features among PT-, E-PT+, and E+ groups of patients revealed significant differences among them. E-PT+ patients were older than PT- patients, had higher white blood cell counts (WBCs) and were more likely to have a mediastinal mass, and thus contained a higher proportion of poor-risk patients. However, the E-PT+ patients were also significantly different from the more traditionally-defined E+ patients in that they had lower WBCs and hemoglobin levels, and less frequent lymphadenopathy or mediastinal mass. In many respects, then, E-PT+ patients were intermediate in character between PT- and E+ patients. Our findings support the notion that further subclassification of ALL using antibodies recognizing lineage-specific surface determinants will permit recognition of groups of patients with distinct clinicopathologic features that may differ in prognosis or response to therapy. Such classification also focuses future biological studies on the pathogenesis of leukemias to immunologically well-defined subgroups of lymphoid neoplasms.

Monoclonal antibody definition of T cell acute leukemia: a Pediatric Oncology Group study.

Leukemic blasts from 774 children with newly diagnosed acute lymphocytic leukemia (ALL) have been phenotyped by microcytotoxicity testing with a panel of monoclonal antibodies and heteroantisera as part of a Pediatric Oncology Group classification study of acute leukemia. One hundred twenty-two cases, or 16% were designated as T cell leukemia based on the reactivity of blast cells with previously well-characterized antisera (PT) against a T lymphocyte-associated antigen. Using this antisera-based definition as a standard, we looked for a monoclonal antibody combination that would be a suitable substitute. An algorithm calling for reactivity with either monoclonal antibody 3A1 or Leu-1 was a 92% sensitive and 97% specific predictor of PT reactivity. Only 27 of 755 cases of leukemia were incorrectly classified using this algorithm. Subsequently, Ficoll-Hypaque-separated bone marrow cells from 118 additional patients with ALL (21 of whom had T cell ALL) were stained by immunofluorescence using a combination of directly fluoresceinated 3A1 and Leu-1. Reactivity of 20% or more of the cells with this antibody combination was a 100% sensitive and 94% specific indicator of T cell ALL defined by PT positivity; with a higher cutoff value for positive values, or the use of supplemental tests, even this small number of false-positives could be eliminated. We conclude that this monoclonal antibody combination is a satisfactory replacement for our heteroantisera definition of T cell ALL.

Phylogenetic distribution of a 24,000 dalton human leukemia-associated antigen on platelets and kidney cells.

The distribution of a 24,000-dalton human leukemia-associated antigen, p24, was examined using the BA-2 and DU-ALL-1 monoclonal antibodies. BA-2 and DU-ALL-1 bound to human, gorilla, orangutan, macaque, and rabbit platelets but did not bind to mouse, rat, guinea pig, dog, horse, sheep, or goat platelets. Orangutan platelets demonstrated a decreased level of binding with BA-2 and DU-ALL-1. In addition, BA-2, but not DU-ALL-1, bound to chimpanzee platelets suggesting that the chimpanzee has lost the epitope of p24 detected by DU-ALL-1. Immunoperoxidase analysis of kidney tissue with BA-2 and DU-ALL-1 revealed staining of distal tubules and glomeruli, which occurred in a similar phylogenetic distribution to that of p24 on platelets. A monoclonal antibody to the high molecular weight common ALL antigen, J-5, reacted with glomeruli and proximal tubules from human, chimpanzee, orangutan, mangaby , rhesus, and rabbit kidneys but failed to react with rat or mouse kidney.

A monoclonal antibody reactive with a second epitope of the 67,000-dalton human T cell antigen.

Hybridomas were produced against the T-cell CLL derived-cell line, SKW3, by the fusion of hyperimmune spleen cells with P3 myeloma cells. One clone, designated DU-SKW3-1, was shown to produce a murine IgG2b antibody reactive with an antigen expressed on normal thymocytes and peripheral blood T cells. This antigen was not detected on human B cells, erythrocytes, monocytes, granulocytes, or platelets. D-SKW3-1 also reacted with T-ALL, T-CLL, and B-CLL cells, but did not react with common ALL or acute myelocytic or monocytic leukemias. Immunoprecipitation of lactoperoxidase-iodinated, detergent-solubilized PBL demonstrated that DU-SKW3-1 reacted with a protein with an apparent mass of 67,000 daltons (p67), which had identical mobility to the antigen precipitated by L17F12, Cocapping experiments suggested that DU-SKW3-1 and L17F12 detected the same molecule: however, DU-SKW3-1 was unable to block the binding of L17F12. In addition, DU-SKW3-1 reacted with the T lymphocytes of both the great apes and old world monkeys, in contrast to L17F12 and two other p67 monoclonals, T101 and 10.2, which reacted only with the cells of the great apes. This data suggests that DU-SKW3-1 may react with a second, less phylogenetically restricted epitope on the p67 T cell-/CLL-associated molecule.

Classification of human leukemia by membrane antigen analysis with xenoantisera.

Rabbit and monkey antisera after appropriate absorption were rendered specific for normal or leukemic lymphoid- and myeloid-associated antigens. Antisera defining a common peripheral blood T-cell antigen, a thymus leukemia antigen, HLA-DR or Ia-like antigen, common acute lymphoblastic leukemia antigen (CALLA), and a myeloid-monocyte (M) antigen were used in a microcytotoxicity assay to classify leukemic cells from 30 patients in a double blind study. The antisera to the M antigen reacted with adherent peripheral blood cells and polymorphonuclear leukocytes and failed to react with nonadherent mononuclear cells and enriched T-cells and chronic lymphocytic leukemia cells. The M antisera also reacted with U937, a monocytic-type cell line, and with HL60, a promyelocytic-type cell line, but failed to react with T and B lymphoblastoid cell lines. The specificities of the other antisera have been described in previous reports. Cells from three of the patients could not be phenotyped by microcytotoxicity testing. Cells from 25 patients had a consensus morphological or histochemical diagnosis of either acute lymphoblastic leukemia or acute nonlymphocytic leukemia. The serological classification of these patients using the five types of antisera listed above were consistent with the consensus diagnosis. In addition, the lymphoid cancers were further subclassified as to T-, B-, or thymus antigen types. There was no consensus lymphoid versus myeloid diagnosis on cells from two patient. The serological classification in both cases favored a diagnosis of myeloid rather than lymphoid leukemia.

Distribution of common acute lymphoblastic leukemia antigen in nonhematopoietic tissues.

The common acute lymphoblastic leukemia antigen (CALLA), as defined by J-5 murine monoclonal antibodies, was detected on renal tubular and glomerular cells from fetal and adult donors by an indirect immunoperoxidase technique. CALLA could also be detected on epithelial cells of the fetal small intestine and on myoepithelial cells of adult breast but not on myoepithelial cells of the salivary gland. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated 125I-labeled membrane antigens from dissociated renal cells demonstrated that the antigen migrated as a 90,000 mol wt antigen rather than the 98,000-100,000 mol wt antigen noted on CALLA-positive tissue culture cell lines. The data suggest that the determinant defined by the J-5 monoclonal antibody is neither a lymphoid cell-specific differentiation antigen nor a leukemia-specific antigen.

Relationship between adenosine deaminase and a human thymus leukemia antigen isolated from MOLT 4 cells.

The relationship between adenosine deaminase (ADA) and a human membrane thymus leukemia (HTL) antigen-detected by an antithymocyte serum was explored. A freeze-thaw extract of the T-cell-derived cell line, MOLT 4, was applied to an immunoabsorbant column of a rabbit antiserum to calf ADA. The bound MOLT 4 ADA was eluted with 6 M urea. The recovered ADA had a specific activity of 490 mumol of adenosine deaminated per min per mg protein, and the yield was 32%. No HTL antigenic activity was detected in the purified ADA. In addition, no ADA activity was detected in the unbound fraction containing the HTL antigenic activity, supporting the conclusion that ADA and HTL antigen are independent molecules. Affinity-purified anti-calf ADA was not cytotoxic for several HTL antigen-positive cells, including thymocytes, MOLT 4, and thymus-derived acute leukemia lymphoblasts.

Detection and partial characterization of human thymus-leukemia antigens.

Two monkey antisera against human thymocytes after absorption with human erythrocytes and peripheral blood leukocytes were shown to detect human thymus-leukemia (HTL)-like antigens. These sera were cytotoxic for thymocytes (> 90% lysis at a 1:10 dilution) but were nonreactive with enriched peripheral blood T- and B-lymphocytes or with cells from myeloid or B-cell lymphoid leukemias. Most (16/17) sheep erythrocyte rosette-forming acute lymphoblastic leukemia (ALL) cells reacted with these sera. Cells from patients with T-cell chronic lymphocytic leukemia, lymphoblastic lymphoma (LBL), and thymoma were also positive. Three of 4 T-cell lymphoblastoid lines derived from ALL patients reacted with these sera. Absorption of the sera with MOLT-4F cells, thymocytes, or LBL cells removed the reactivity against all types of cells tested. However, sera absorbed with the T-cell line HSB remained cytotoxic for thymocytes, MOLT-4F, and most (6/9) T-cell cancers tested. The peripheral blood cell-absorbed sera precipitated a molecule with an apparent molecular weight of 48,000 from lactoperoxidase-labeled thymocytes but not from similarly labeled peripheral blood lymphocytes. The ability of the sera to precipitate this antigen was decreased by absorption with thymocytes, MOLT-4, or LBL cells but not by absorption with HSB, SB, or non-T, non-B ALL cells. Sequential precipitation studies suggested that the HTL antigen was not associated with beta 2 microglobulin.

Cultural, morphological, cell membrane, enzymatic, and neoplastic properties of cell lines derived from a Hodgkin's disease lymph node.

A neoplastic cell line (designated HuT11) has been established in continuous culture from an involved lymph node of a patient with Stage IIA Hodgkin's disease of the mixed cellularity type. The HuT11 line has been morphologically heterogeneous, consisting of mononucleate lymphoid-like cells, polygonal epithelioid cells, and mono-, bi-, and multinucleate giant cells. Four clones initiated from isolated binucleate giant cells of the HuT11 line also have been successfully established as continuous cell lines. The cloned lines have been morphologically distinct and more homogeneous, although typical giant cells have consistently appeared throughout the long-term culture of each. The HuT11 lines have grown as monolayers in McCoy's Medium 5A supplemented with 10% fetal calf serum, with generation times of 12 to 14 hr and high saturation densities. Cytogenetic studies showed that early and later passages of HuT11 cells were aneuploid, and all cell lines were successfully heterotransplanted in the hamster cheek pouch. Repeated indirect immunofluorescence examinations have shown each cell line to be negative for Epstein-Barr virus nuclear antigen. Indirect immunofluorescence tests in which monospecific immunoglobulins were used revealed positive membrane reactions for the gamma (heavy)-chain and kappa (light)-chain of human immunoglobulin G in approximately 20% of viable cells in each line; however, direct immunofluorescence with anti-human immunoglobulin G F(ab')2 reagent failed to confirm these reactions. Rosette tests for B- and T-lymphocyte and macrophage membrane receptors yielded negative results. All cell lines were strongly phagocytic for latex particles and neutral red dye. Cytochemical stains of the monolayers revealed abundant esterase, fluoride-resistant nonspecific esterase, acid phosphatase, and leucine aminopeptidase activities, while lysozyme assays were negative. Although some properties of the HuT11 lines have suggested a macrophage derivation, an undifferentiated lymphoid cell origin of the Hodgkin's neoplastic cell remains a possibility.