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We report on the operation of a 25.8 Gbit/s DML TOSA over a wide temperature range (WTR) with extremely high eye-mask margin for Ethernet applications. The TOSA is comprised of a co-axial TO-CAN package with an LC-type receptacle for the optical connector and an FPC for the electrical interface. By combining a wide bandwidth InGaAlAs WTR-DFB chip and a novel TOSA design, extremely high Ethernet mask margins exceeding 30% at 25.8 Gbit/s over a case temperature of -- 5 degC to 75 degC is achieved. This TOSA enables small form factor modules, such as SFP+, to operate at up to 70 degC.

A survey of five newly developed microsatellite DNA markers in skipjack tuna Katsuwonus pelamis revealed high levels of polymorphism in two samples off the west coast of India (Minicoy Island and Kochi coast) and one sample off the Japan coast. Although significant differentiation (P < 0·01) in the number of specific alleles was observed between Minicoy and Kochi samples, the F-statistics values among the samples were very low (average = 0·0014) and not significant (P = 0·284).

The federal government is critically examining its responsibilities and opportunities for bringing the new field of gene therapy to fruition and for assuring public confidence in this new area of biomedicine. The evolving mechanisms for review and regulation in human gene transfer studies in the United States are being enhanced by increasingly effective interactions between the Food and Drug Administration and the National Institutes of Health.

We have recently reported that IL-13R may share a component with IL-4R. Here we report that both IL-4 and IL-13 share signaling events in human colon carcinoma cell lines (HT-29 and WiDr). IL-13 caused rapid phosphorylation of the three out of four members of the known Janus family of kinases (JAKs). We show that JAK2 kinase is rapidly phosphorylated and activated in response to IL-13. Within 1 min of activation, JAK2 was phosphorylated, and peaked in 10 min. In addition, IL-13 phosphorylated insulin response substrate-1, IL-4R p140, JAK1, and Tyk2, but not JAK3 kinase. IL-4 also stimulated all three kinases and substrates, but unlike in immune cells, IL-4 did not involve JAK3 activation for its signaling in colon cancer cell lines. Furthermore, JAK2 associated with the IL-4R p140 before and after stimulation with IL-13. Both IL-13 and IL-4 induced phosphorylation of IL-4 STAT (STAT6) but not STAT1, STAT3, or STAT5. 125I-IL-13 did not bind to colon cancer cell lines, but unlabeled IL-13 competed for the binding of 125I-IL-4. Our data suggest that IL-13 utilizes IL-4R and its signaling pathway, and JAK2 may play an important role in the function of IL-4R and IL-13R in colon cancer cells.

The ongoing shortage of human organs and tissues for transplantation, coupled with scientific and biotechnological advances, has catalyzed new attempts to use animal tissues in humans — a field kno...

Scientific advances in the past decade have made the clinical testing of somatic-cell therapy and gene therapy a reality. Early trials in humans suggest that important new diagnostic and therapeutic tools are on the horizon. The objectives of this article are to examine the regulation of somatic-cell and gene therapy by the Food and Drug Administration (FDA) in the context of the agency's traditional role in the development of biologic products and to stimulate discussion in areas in which policy is still being formulated. The technology of somatic-cell and gene therapy has moved from the bench to clinical evaluation with . . .

An antisense oligonucleotide phosphorothioate, previously shown to inhibit HIV-1 viral expression in chronically infected H9 cells, was fluorescently labeled to study oligonucleotide fluxes and localization within living cells. Observations based on flow cytometry and fluorescence microscopy show the following: within around 0.5-2 h, an apparent steady-state distribution of the oligonucleotide is achieved in which the intracellular oligonucleotide concentration is less than that present in the external medium; following oligonucleotide uptake and resuspension of the cells in oligonucleotide-free medium, an oligonucleotide efflux, with a time constant similar to that for uptake, is observed (although a significant fraction of the phosphorothioate remains within the cell); cellular uptake as a function of the external oligonucleotide concentration is nonlinear, being more efficient at lower concentrations (less than 2 microM); and a predominant oligonucleotide localization within the cell nucleus and perinuclear organelles is observed.

We investigated the long-term effects of continuous gamma interferon (gamma-IFN) treatment on A431, a human squamous carcinoma cell line. Cells were grown in an in vitro culture system, which over time produces cohesive cell masses ("tumoroids") exhibiting three-dimensional, histotypically differentiated structures, e.g., keratin "pearls", intercellular bridges (desmosomes), elongated flattened cells (squames) and stratification. The effects of gamma-IFN on cell growth, morphology and stage of differentiation were assessed at different treatment times by light and electron microscopy and by immunohistochemical staining using antibodies to keratins 1 and 14 and to filaggrin, markers of specific stages of keratinocyte differentiation. Our results show that A431 cells have the capacity for spontaneous differentiation, that this capacity is significantly enhanced and accelerated by gamma-IFN treatment leading to terminal differentiation and extensive cell death by 2 wk. Despite continuous exposure to IFN, a small number of viable, undifferentiated cells remain. Their proliferation, evident by 3 wk, reconstitutes the tumoroid which once again contains the full range of differentiating cell types.

With this research we present a system that suggests valuable complementary information relevant to a user's topic of interest, in the form of keywords. For this purpose we have automatically constructed a Web search directory called TORISHIKI-KAI from a large collection of Web documents, using state of the art knowledge acquisition methods. TORISHIKI-KAI maps out the use context of the terms input by the user, and classifies topically related search terms according to semantic categories such as potential troubles, methods or tools in order to help the user find potentially valuable "unknown unknowns".

Background Among all hematologic malignancies, B-cell chronic lymphocytic leukemia (BCLL) has the highest familial clustering (three- to sevenfold increase), strongly suggesting a genetic component to its etiology. Familial BCLL can be used as a model to study the early pathogenesis of this disease. Methods We examined nine kindreds from the National Cancer Institute's Familial BCLL Registry, consisting of 19 affected members with BCLL and 33 clinically unaffected first-degree relatives. Flow cytometric immunophenotyping to detect a B-cell monoclonal lymphocytosis (BCML) was performed. Monoclonality was confirmed by polymerase chain reaction analysis of whole blood DNA. Cell cycle analysis for aneuploidy was conducted. Results In all affected individuals, we observed the classic BCLL CD5/CD19/CD20/CD23 immunophenotypic patterns. Six of the 33 unaffected individuals (18%) had evidence of BCML. Additional individuals (13/33, 39%) showed some other abnormality, whereas 14 individuals (42%) were normal. Based on an estimated prevalence of 0.7% for BCML in the general population, the finding of six subjects (18%) with clonal abnormalities in this relatively modest sample was significantly greater than expected (i.e., 18% vs. 0.7%, P < 5.7 × 10−9). Conclusions Individual components of BCML and other B-cell abnormalities were observed in almost half of the apparently unaffected individuals. Our findings suggested that BCML may be an early detectable abnormality in BCLL. The spectrum of some of these observed abnormalities suggested the involvement of different B-cell subpopulations or different pathways in clonal evolution. Population-based, longitudinal studies will be required to determine the incidence of BCML and other B-cell abnormalities and their relation to disease progression in BCLL and other closely related B-cell lymphoproliferative disorders. Cytometry Part B (Clin. Cytometry) 52B:1–12, 2003. Published 2003 Wiley-Liss, Inc.

Updated information on this topic from the Food and Drug Administration is available at http://www.fda.gov/bbs/topics/ANSWERS/2003/ANS01190.html. Although most of today's gene-therapy trials are ta...

Several reports have suggested that the prevalence of asthma in adults is currently increasing. However, recent prevalence of asthma has not reported in Japan, especially in rural-mountain areas. To investigate the prevalence of asthma in adults in Japan, we conducted clinical epidemiological research on 5066 inhabitants of Menda town, in a rural-mountain area of Japan. The study population comprised 98.7% of adults in the town, including senior high school students whose age were more than 15 years old. The prevalence of asthma among adults was 3.6%. The ratio of prevalence in males to prevalence in females was 1.44. Peaks prevalences were observed in the age ranges of 15-19 and70 years old in males, and 15-19, 40-49 and70 years old in females.

We have previously reported on the expression of interleukin-4 receptors (IL-4R) on many human epithelial cancer cells; however, the binding characteristics, structure, function, and signal transduction through the IL-4R in cancer cells is not known. IL-4 binding characteristics were determined in human colon carcinoma cell lines by a 125I-IL-4 binding assay, which demonstrated that the HT-29 and WiDr colon cancer cell lines expressed high affinity IL-4R (Kd = 200 pM). Cross-linking experiments revealed a major band of 140 kDa and a broad band at 70 kDa. While the common gamma chain of IL-2R is associated with IL-4R in immune cells and is similar in size to the 70-kDa protein, this chain was not expressed in these colon cancer cells. Interestingly, IL-13, which has many functions similar to IL-4, inhibited 125I-IL-4 binding to both the 140- and 70-kDa molecules. Next, we investigated the mechanism of IL-4-induced signal transduction in colon cancer cells. After stimulation with IL-4, a 170-kDa band was primarily phosphorylated within 1 min of exposure and was identified as insulin receptor substrate-1. In addition, by immunoprecipitation assay, three other phosphorylated bands were identified as JAK1, JAK2, and Tyk2 tyrosine kinases. The phosphorylation of JAK1 and JAK2 was induced by IL-4 stimulation; however, Tyk2 was constitutively phosphorylated, and IL-4 treatment further augmented this phosphorylation. The kinetics and in vitro kinase assays demonstrated that JAK1, JAK2, and Tyk2 were phosphorylated within minutes and that JAK1 and JAK2 were activated after IL-4 exposure. Contrary to observations in immune cells. JAK3 mRNA was neither detected in colon cancer cells nor did IL-4 treatment cause phosphorylation of JAK3. These data indicate that in colon carcinoma cells JAK1, JAK2, Tyk2, and insulin receptor substrate-1 are phosphorylated after IL-4 stimulation. In addition, as is the case in lymphoid cells, IL-4 activated and phosphorylated signal transducers and activators of transcription (IL-4-STAT or STAT-6) protein in both colon cancer cell lines. These results indicate that the IL-4R complex is composed of different subunits in different tissues and shares a component with the IL-13R complex. In addition, we demonstrate for the first time that like its family members (e.g. IL-3 and GM-CSF), IL-4 can phosphorylate and activate JAK-2 kinase.

A flow cytometric analysis of five B cell chronic lymphocytic leukemic (B-CLL) cell lines was undertaken using 129 unknown reagents from the blind panel (BP) and 72 reagents from the known CD panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B cell section (Vienna, 1989). The five cell lines examined were: SeD (PNAS 75, 5706, 1978), B-CLL-LCL (BLOOD 71, 9, 1988), JVM-HH and JVM-2(INT J CAN 38, 531, 1986), and WR#1 (TH and BD). The reagents were #1-129 (blinded panel) and reagents 1-44 and 53-84 (CD panel with CD23 reagents missing). Positivity was defined as greater than 30% of the cells having a three fold increase or more in mean channel fluorescence. Fourty-three reagents of the blinded panel were negative by these criteria while all remaining reagents were positive on all five lines. SeD showed the lowest reactivity; B-CLL-LCL and JVM-2 showed the most reactivity; JVM-HH and WR#1 were intermediate. The known CD panel confirmed the reactivity of the blinded panel. An average immunophenotype was constructed and compared to published normal EBV lymphoblastoid cell lines and several differences were noted. There was an absence or significant decrease in the expression of CD19, CD21, CD22 and CD37 while there was an increased expression of CD38, CD54, CD74 and CD76. The heterogeneity observed between the B-CLL lines may in part be due to polymorphisms but is more likely to represent the underlying heterogeneity seen in common and familial B-CLL.2+öff

Two independent P-element enhancer detection lines were obtained that express lacZ in a pattern of longitudinal stripes early in germband elongation. In this paper, molecular and genetic characterization of a gene located near these transposons is presented. Sequence analysis of a cDNA clone from the region reveals that this gene has a high degree of similarity with the Drosophila snail gene ( Boulay et al., 1987 ). The sequence similarity extends over 400 nucleotides, and includes a region encoding five tandem zinc finger motifs (72% nucleotide identity; 76% amino acid identity). This region is also conserved in the snail homologue from Xenopus laevis (76% nucleotide identity; 83% amino acid identity) ( Sargent and Bennett, 1990 ). We have named the Drosophila snail -related gene escargot (esg) , and the region of sequence conservation common to all three genes the ‘snailbox’. A number of Drosophila genomic DNA fragments cross-hybridize to a probe from the snailbox region suggesting that snail and escargot are members of a multigene family. The expression pattern of escargot is dynamic and complex. Early in germband elongation, escargot RNA is expressed in a pattern of longitudinal stripes identical to the one observed in the two enhancer detection lines. Later in development, escargot is expressed in cells that will form the larval imaginal tissues. escargot is allelic with l(2)35Ce , an essential gene located near snail in the genome.

We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WB) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lower values. Autofluorescence values were comparable by all methods except Immuno-lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45-stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q-Prep uniformly gave the highest purity of CD45-positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45-stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.

Tumorigenicity testing has been an integral part of cell substrate characterization. With the combination of recombinant technology and traditional cell culture, we have entered an era in which many of the principles developed with vaccine production may need to be modified and new ones developed. Models of tumorigenicity are reviewed in the light of current knowledge; the applicability and necessity of such testing in new areas such as somatic cell therapy will also be presented.

The advent of hybridoma technology has provided an unlimited supply of monoclonal, ‘monospecific’ antibodies (Mab) that has fueled an unprecedented surge in the study of tumor immunology and biology [1]. For the first time, investigators have at their disposal a continuous source of pure antibody that makes possible the identification and extensive characterization of a wide variety of tumor antigens expressed by human carcinomas, melanomas, leukemias, and lymphomas. Subsequent studies have also led to the development of novel assays using Mabs for tumor diagnosis [2–5], as well as a variety of clinical protocols demonstrating radioimmunolocalization of an antibody to occult lesions in a variety of cancer patients [6–15]. In addition to these clinical studies, the use of immunohistochemical techniques, radioimmunoassays (RIA), Western blotting, and other experimental analyses have led to a better understanding of some of the basic principles that characterize tumor antigen expression.

Heterogeneity in the expression of tumor-associated antigens, as defined by the binding of monoclonal antibodies, is a characteristic common to most, if not all, human carcinoma cell populations. Antigen-negative cells within the population can escape detection and therapy by their failure to bind the appropriate antibody. Therefore, the extent of antigenic heterogeneity is an important consideration when designing protocols for the management of cancer by administration of monoclonal antibodies. One approach to counteracting the effect of antigenic heterogeneity is the use of clone A of recombinant human leukocyte interferon (Hu-IFN-alpha A). Administration of Hu-IFN-alpha A in vivo effectively increased the amount of tumor antigen expressed by a human colon xenograft in situ and augmented the localization of a radiolabeled monoclonal antibody to the tumor site. Concomitant administration of Hu-IFN-alpha A and monoclonal antibody may thus be effective in overcoming the antigenic heterogeneity of carcinoma cell populations and in enhancing the efficacy of monoclonal antibodies in the detection and treatment of carcinoma lesions.

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.

An epithelial cell line, LS123, was established in 1974 from the second in a series of three primary colonic tumors resected from a Caucasian female. The cell line is aneuploid, releases low concentrations of carcinoembryonic antigen (CEA), fails to grow progressively in nude mice, and forms colonies only in enriched semisolid medium developed for tumor stem cells. LS123 cells grow on confluent cell monolayers and in either low serum or serum-free medium. In the chick embryonic skin assay, LS123 cells grew as a well-differentiated abnormal colonic epithelium with little mitotic activity but with some indication of invasion. On floating collagen gels LS123 cells formed a one to three-cell-layer-thick undifferentiated epithelial sheet. The apparent low invasiveness of the cells of this line is supported by the patient's history of three primary colon tumors without systemic metastases during the past 30 yr. Therefore, although LS123 cells possess several properties associated with neoplasia, they have little invasive potential. Thus, LS123 cells may represent an important model for the study of human colon cancer.

Stimulation of transformed T cells leads to both lymphokine secretion and inhibition of spontaneous growth. Studies performed with an Ag-specific T cell hybridoma demonstrated that growth inhibition is an early (within 1 h) manifestation of activation. Experiment in which extracellular Ca2+ was chelated or in which cyclosporine A was included indicated that activation-associated growth inhibition is a two-step process. The first phase is the establishment of a G1/S cell cycle block; it does not require extracellular Ca2+ and is not prevented by the addition of cyclosporine A. The second phase is cell lysis. It can be detected 4 to 6 h after activation, requires the presence of extracellular Ca2+, and is prevented when stimulation occurs in the presence of cyclosporine A. The observation that both Ca2+ depletion and cyclosporine A prevented IL-2 secretion at all time points indicates that the pathways leading to lymphokine secretion and the G1/S block diverge early in the course of the cellular response, and establish the cell cycle block as a distinct activation event with unique characteristics.

An organ culture method has been developed in which human tumor cells and chick embryonic tissues are cocultured on agar platforms surrounded by replaceable liquid medium. This simple system permits such cultures to remain viable for up to 60 d; the extended culture period allows the tumor cells to invade, organize, and differentiate and to form microscopic tumors in embryonic tissues similar to tumors formed by the tumor cells in vivo in nude mice. These microtumors can also be examined for tumor-associated antigen expression by conventional immunoperoxidase techniques.

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.

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.

The present study advances our knowledge of the newly described immunodeficient RIIIAnN mouse. This strain was found to have markedly reduced spleen size and low serum levels of IgM, IgA, IgG1, IgG2α, and IgG2β. Studies of F2 and backcross mice demonstrated that the defective immunoglobulin production was due to multiple genes: a dominant gene for low IgA and recessive genes for the others. RIII mice, like xid-bearing mice, had a markedly reduced splenic plaque-forming cell (PFC) response to Type III pneumococcal polysaccharide; however, unlike xid-bearing mice, the RIIIAnN mice produced substantial serum antibody responses to pneumococcal polysaccharide, polyriboinosinic · polyribocytidylic acid, and trinitrophenyl-Ficoll. In addition, spleen cells from RIIIAnN mice proliferated normally when stimulated with anti-μ, whereas those from xid mice did not. Thus, RIIIAnN mice represent a model of hypogamma-globulinemia and restricted immunodeficiency. They differ from other immunodeficient mice, and should assist in the analysis of immunodeficiency, autoimmunity, and regulation of class-specific immunoglobulin production.

Cells were stained for DNA content with Hoechst 33258 combined with propidium iodide and analyzed on an epi-fluorescence flow cytometer. Scatterplots of red versus blue fluorescence of control cells showed a straight line indicating that both dyes were measuring proportional amounts of cellular DNA. Scatterplots of cells incubated with the thymidine analog bromodeoxyuridine, however, showed a displacement from the control straight line that increased with longer exposure time to bromodeoxyuridine. These results suggest that bromodeoxyuridine incorporation in DNA can be detected by flow cytometry.

Previous studies have shown that normal as well as neoplastic B-cell lines vary substantially in their response to the antiproliferative effects of human interferon (HIF). In this study we took advantage of a recent method to generate long-term continuous normal T-cell cultures (CTC) to investigate the effects of HIF on proliferating lymphoid cells. Normal CTC proved to be resistant to inhibition of proliferation; up to 1000 u HIF had little effect on [3H] TdR uptake, and up to 2000 u HIF had little effect on cell-cycle progression, measured by flow cytometry. Proliferating normal B cells were also resistant to the antiproliferative effect. Nor did up to 500 m HIF inhibit RNA synthesis or immunoglobulin biosynthesis of normal B cells. In contrast, a neoplastic myeloma B cell, a Burkitt's lymphoma cell and a neoplastic leukaemic T cell showed marked inhibition of [3H] TdR uptake and cell cycle progression with as little as 5 u HIF. These results suggest that amounts of HIF sufficient to inhibit proliferation of some neoplastic lymphoid cells have little effect on T- and B-cell proliferation and differentiation of normal B lymphocytes.

Anti-Tac monoclonal antibody identifies the receptor for interleukin 2 (IL 2, or T cell growth factor) present on activated human T lymphocytes. By using tritiated anti-Tac, we now report a sensitive and specific binding assay to evaluate cell surface IL 2 receptor expression. IL 2 receptors on human peripheral blood lymphocytes can be detected within 6 hr after PHA stimulation. PHA-induced receptor expression is inhibited by actinomycin D and cycloheximide, but not by mitomycin C, suggesting a requirement for de novo RNA and protein synthesis, but not DNA synthesis. Scatchard analysis of [3H]-anti-Tac binding to lymphocytes stimulated with PHA for 3 days revealed from 20,000 to 60,000 molecules of antibody bound per cell, and a Kd of 1 to 3 x 10(-10) mol/l. Sequential binding studies of activated human lymphocytes maintained in long-term culture with IL 2 demonstrated a progressive decline in receptor number correlating with diminished growth rate. Restimulation with lectin or antigen increased the number of IL 2 receptors, suggesting that IL 2 dependent immune responses may be regulated, at least in part, by IL 2 receptor expression. Receptor number was also increased by PMA. Moreover, similar effects were produced by incubation with phospholipase C but not interleukin 1. Because both PMA and phospholipase C result in activation of protein kinase C, these data suggest the possibility that activation of protein kinase C may induce IL 2 receptor expression.

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.

Human interferon decreased DNA but not RNA synthesis in a human colon carcinoma cell line, WiDr; in addition, there was a two- to three-fold increase in the expression of a tumor-associated antigen, carcinoembryonic antigen. In contrast, interferon had no effect on a normal human diploid cell line, WI-38. Thus, in addition to its anti-cellular effect against tumor cells, interferon can also modulate tumor antigenicity.

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.

The present study advances our knowledge of the newly described immunodeficient RIII/AnN mouse. This strain was found to have markedly reduced spleen size and low serum levels of IgM, IgA, IgG1, IgG2 alpha, and IgG2 beta. Studies of F2 and backcross mice demonstrated that the defective immunoglobulin production was due to multiple genes: a dominant gene for low IgA and recessive genes for the others. RIII mice, like xid-bearing mice, had a markedly reduced splenic plaque-forming cell (PFC) response to Type III pneumococcal polysaccharide; however, unlike xid-bearing mice, the RIII/AnN mice produced substantial serum antibody responses to pneumococcal polysaccharide, polyriboinosinic . polyribocytidylic acid, and trinitrophenyl--Ficoll. In addition, spleen cells from RIII/AnN mice proliferated normally when stimulated with anti-mu, whereas those from xid mice did not. Thus, RIII/AnN mice represent a model of hypogammaglobulinemia and restricted immunodeficiency. They differ from other immunodeficient mice, and should assist in the analysis of immunodeficiency, autoimmunity, and regulation of class-specific immunoglobulin production.

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.

The original report that certain poly(ADP-ribose) synthetase inhibitors block the repair of DNA chain breaks [1] has been confirmed by a number of laboratories [2–4]. In spite of this, a systematic approach to answering the fundamental question of whether synthetase plays an obligatory role in the repair process has not been undertaken. We have attempted to evaluate this possibility by comparing the potency of six synthetase inhibitors for inhibiting synthetase and for blocking the repair of γ-ray-induced DNA chain breaks. The results of this analysis were decidely mixed — some inhibitors being more portent in blocking repair than predicted on the basis of their Ki for synthetase and some being less effective than anticipated. Consequently, assays were made of the potency of the inhibitors for reducing cell viability or for affecting RNA synthesis. Cell viability was reduced by the synthetase inhibitors in direct relationship to their potency as synthetase inhibitors, with one exception. The effects of the inhibitors on the uptake of RNA precursors were, however, not related to their Ki’s for synthetase.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.