Establishment of Cotesia flavipes (Hymenoptera: Braconidae) in sugarcane fields of Ethiopia and origin of founding population.

Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) is used as a classical biological control agent against Chilo partellus (Swinhoe) (Lepidoptera: Crambidae), a serious exotic pest of cereal crops in eastern and southern Africa. This parasitoid has been introduced into several African countries for the control of C. partellus in maize, Zea mays L., and sorghum, Sorghum bicolor (L.), but it has never been released in Ethiopia. It is hypothesized that it spread into Ethiopia from populations released in Kenya and Somalia to become the predominant parasitoid of C. partellus in maize and sorghum fields of the country. In recent surveys conducted in Ethiopia, C. flavipes was recovered from C. partellus in sugarcane, Saccharum L. spp. hybrids, at a site >2,000 km from the nearest known release sites in Kenya and Somalia. These findings question published hypotheses that estimate the dispersal rate of C. flavipes to be 60 km per year in Africa, and they suggest that since its release in Africa this parasitoid has developed strains adapted to searching particular host plants infested by particular stem borers. The anomalies between our results and previous reports evoked the hypothesis that C. flavipes in Ethiopian sugarcane might be a different strain. To test this hypothesis, we compared partial COI gene sequences of C. flavipes collected from sugarcane in Ethiopia and those of specimens from other African countries to determine the origin of the Ethiopian population. In addition, COI sequences were obtained for C. flavipes from other continents. The C. flavipes population established in Ethiopian sugarcane is most closely related to the populations released against C. partellus in maize in other parts of Africa, which were derived from the original population imported from Pakistan. The dispersal rate of the parasitoid was estimated to be >200 km per year.

Diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon, NeuroVue Red and NeuroVue Green and their use for double and triple labeling of neuronal profile.

We describe here diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon (near infrared), NeuroVue Red and NeuroVue Green. Using pair-wise comparisons between the new dyes and existing dyes (DiI, DiA, DiD, DiO, PKH2, PKH26) applied to the left and the right side of fixed spinal cord preparations, we show that NeuroVue Maroon (excitation maximum 647 nm) surpasses all other dyes in this study in signal to noise ratio. We also present data showing the utility of these new dyes for both double labeling and triple labeling in combination with each other or existing lipophilic tracers. Using mice bearing the PLP-eGFP transgene, we demonstrate that either NeuroVue Maroon or NeuroVue Red can readily be combined with eGFP labeling. Double labeling experiments using NeuroVue Red and eGFP allowed us to demonstrate that every fiber in the neonatal ear is surrounded by developing Schwann cells.

Optimization of PKH67 labeling conditions for proliferation monitoring in daunorubicin-treated leukemic cells.

Daunorubicin (DNR) is commonly used to treat acute myeloid leukemia (AML). The aim of this study was to determine whether PKH67 dye dilution could be used for differential proliferation monitoring in chemosensitive (K562S) and chemoresistant (K562R) leukemic sublines after drug treatment. Cells were labeled with PKH67 and treated for 2 h with a sublethal dose of DNR or vincristine either immediately or after 3 h in fresh medium. Viability (TOTO-3 exclusion) and DNR uptake (total cellular DNR fluorescence) were assessed by flow cytometry, and nuclear DNR accumulation was determined by confocal laser microspectrofluorimetry. Immediate DNR treatment led to enhanced DNR uptake and decreased viability in PKH67-labeled K562S, whereas no excess toxicity was seen if DNR treatment was delayed for 3 h. Treatment with vehicle control (Diluent C) gave similar results. In contrast, PKH67 labeling had no effect on K562S viability after vincristine treatment. For K562R, DNR uptake measured at 120 min was unaltered by prior exposure to PKH67 or vehicle, but viability was again significantly reduced after immediate DNR treatment. As with K562S, delaying DNR treatment for 3 h normalized viability in K562R. The excess DNR toxicity seen for PKH67-labeled K562S appears to be drug related, since it is not seen with vincristine, and may be due to the daunosamine sugar moiety present in DNR. However, with the addition of a 3-h incubation in fresh medium prior to drug exposure, PKH67 dye dilution can be used for proliferation monitoring of both K562S and K562R cells after treatment with DNR.

A flow cytometric assay for simultaneous assessment of drug efflux, proliferation, and apoptosis.

BACKGROUND: Proliferative status and multidrug resistance play a key role in determining cell response to chemotherapy. There is a need to develop multiple labeling method that allows simultaneous assessment of multidrug resistant (MDR) phenotype, proliferative status, apoptosis related changes in mitochondrial potential, in chemosensitive and chemoresistant tumor cell populations. METHODS: A three-color labeling was performed using Hoechst 33342 (DNA), JC1 (mitochondrial potential), and a far red fluorescent membrane intercalating dye: PTIR271 (proliferation). RESULTS: Combined staining of DNA and mitochondrial potential allows identification of subpopulations expressing and MDR phenotype mediated by P-glycoprotein (Pgp), and, in Pgp negative subpopulations, identification of apoptotic cells and evaluation of cell cycle status in viable cells. Addition of a far red fluorescent membrane intercalating dye, PTIR271, allows simultaneous monitoring of cell division status by dye dilution in both drug sensitive and drug resistant populations. CONCLUSION: This triple labeling is an interesting method to study the proliferation status of drug sensitivity and drug resistance in viable tumor cells.

A novel drug delivery system using IL-2 activated NK cells and Zyn-linked doxorubicin.

Adoptively transferred IL-2 activated NK (A-NK) cells selectively accumulate within tumor metastases which recommends them as vehicles for locoregional drug delivery. Zyn-Linkers are membrane-binding lipophilic dyes which can be coupled by a variety of conjugation chemistries to therapeutic agents. We have previously demonstrated that A-NK cells labeled with PKH26 are able to accumulate within established B16 melanoma pulmonary metastases by 16 h at a concentration of over 600 cells/mm2 of tumor tissue (Basse et al. J. Exp. Med. 174: 479 1991). Zyn-205 is a prodrug in which doxorubicin is attached to a similar Zyn-Linker through an acid-sensitive bond. We have optimized the ex vivo labeling conditions and found that a 10 min incubation with 25 microM Zyn-205 results in the uptake of over 10(8) drug molecules per cell with no effect on either cell viability or cytolytic activity up to 24 h after labeling. Given these parameters, the amount of drug which may be carried to and concentrated in metastatic lesions represents a local concentration of approximately 15 microM. In addition, A-NK cells carrying Zyn-Linked doxorubicin at an equivalent dose of 25 micrograms/kg was therapeutically comparable to a systemic dose of 8 mg/kg (320x more) in the 3LL model of experimental metastasis. These data indicate that A-NK cells bearing Zyn-Linked chemotherapeutic agents represent a unique and feasible method to target chemotherapeutic agents to cancer metastases and that therapeutic doses can be attained without unwanted systemic exposure.

Usefulness of PKHs for studying cell proliferation.

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies. (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.

PKH26 probe in the study of the proliferation of chemoresistant leukemic sublines.

Proliferative status and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia. Although classical methods for proliferative assessment such as tritiated thymidine or BrdUrd incorporation, are correlated with treatment outcome, they are time consuming and difficult to standardize. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine rate and extent proliferation in drug sensitive and resistant cell lines. When cells labelled with this fluorescent membrane intercalating dye divide, each resulting daughter cell receives half of the dye. Using flow cytometric analysis, it is possible to estimate the number of cells having undergone different numbers of cell divisions. Four different questions were addressed in these studies: a) does PKH26 give stable and reproducible labelling? b) does labelling with PKH26 alter cellular proliferation characteristics? c) is PKH26 a substrate for PGP and MRP? d) does PKH26 labelling alter PGP expression and/or PGP activity? We found that PKH26 labelling is stable, reproducible and has no effect on cell proliferation. It does not modify PGP activity or expression, nor does it appear to be a substrate for PGP or MRP, since the rate of decrease in fluorescence intensity is similar for sensitive and resistant cells which are proliferating at the same rate. Using the dye dilution method, it is possible to simultaneously assess PGP, proliferative status, and level of PGP expression. We conclude that the methods developed here provide a simpler, more complete means for assessment of the effects of the drug therapy on sensitive and resistant cell populations in patients with hematologic malignancies.

Zyn-Linker delivery of antirheumatic agents.

Despite our increasing ability to manage rheumatoid arthritis through systemic medication, refractory joints require local administration of more aggressive therapy in a substantial number of patients. These studies tested whether a new class of molecules designated Zyn-Linkers could deliver and retain therapeutics in a joint. Zyn-Linkers are synthetic lipid-like molecules designed to insert into cell membranes and enhance drug delivery to cells. After intra-articular injection into the knee of NZW rabbits, Zyn-Linkers bound rapidly and homogenously to synovial lining cells. Chelating Zyn-Linkers which contained Re-186 or Y-90 were synthesized to evaluate localization and retention after intra-articular injection. Initial studies using Re-186 Zyn-Linker gave excellent localization as evaluated by whole-body imaging: counts in the knee region represented > 90% of counts present in the whole body for at least 4-6 days postinjection. Similar results were obtained using a Y-90 Zyn-Linker and this agent was used for biodistribution studies due to its greater stability and ease of preparation. Efficacy and safety of Y-90 Zyn-Linker as a potential radiation synovectomy agent were estimated by extrapolation of biodistribution data to humans. A therapeutically effective dose of 8,000 cGy to synovium was calculated to require intra-articular injection of 3.4 mCi Y-90 Zyn-Linker, a value less than or equal to doses of particulate Y-90 agents used clinically in Europe. The predicted safety profile for Y-90 Zyn-Linker was excellent, with estimated doses to nontarget organs and tissues falling well within FDA-recommended safety levels for research-only radiopharmaceuticals. In addition to exhibiting desirable localization and retention properties, Zyn-Linkers may also be synthesized to release antirheumatic drugs such as methotrexate at controlled rates. This suggests substantial potential for these drug delivery molecules as chemical synovectomy agents which may be used concurrently with systemic chemotherapy to improve management of refractory joints.

Mechanisms of adoptive immunotherapy: improved methods for in vivo tracking of tumor-infiltrating lymphocytes and lymphokine-activated killer cells.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced cancer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor activity and optimize treatment protocols. Traditional cell tracking methods such as fluorescent protein labeling and radiolabeling using 111In, 125I, or 51Cr are limited by isotope half-life, leakage or transfer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows long-term cell tracking but is laborious to perform and difficult to quantitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and 125I-PKH95) which stably partition into lipid regions of the cell membrane to track immune cells in vivo. Concentrations of each tracking compound which had no adverse effects were determined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up to 5 microM for PKH95 and 20 microM for PKH26. TIL proliferation was unaltered by labeling with up to 5 microM PKH95, 20 microM PKH26, or a combination of 15 microM PKH26 and 5 microM PKH95. In vivo cytotoxic effector function and in vivo therapeutic efficacy of lymphokine-activated killer cells and TIL were also unimpaired by labeling with 20 microM PKH26 or 1 microM 125I-PKH95. Subsequent studies in an adoptive transfer immunotherapy model used 125I-PKH95 to track the biodistribution of TIL in tumor and in non-tumor-bearing animals and PKH26 fluorescence to monitor microdistribution within tissues and distinguish TIL from host T-cells. The results suggest that differential accumulation, selective retention, or proliferation at the tumor site cannot account for the observed pattern of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeutic effect.

Manipulation of human B cells to confer immortality.

Electroporation was used to deliver genomic DNA from a lymphoid tumor to activated/stimulated human peripheral blood lymphocytes to create immortalized lymphoid cell lines. Activation of the recipient lymphocytes was essential for efficient immortalization. A panel of human B cell transfectant clones, each phenotypically representing specific stages of differentiation, resulted from the transfection. Monoclonal antibody production was measured, and the level produced depended on the phenotype of the cells, with the more mature B cell transfectants secreting up to 10 micrograms/mL of immunoglobulin. The transfectants were stable with respect to their morphological appearance, growth rate, and antibody production. Chromosome analysis indicated that the transfectants displayed a normal karyotype, devoid of abnormalities. We have shown that electroporation is an effective method of immortalizing human lymphocytes at different stages of differentiation. The transfectants provide a panel of cells that can readily be studied with respect to their phenotypic/karyotypic stability, regulation, and production of immunoglobulin, lymphokines, and growth factors. These data demonstrate the feasibility of generating immortalized human B cells to provide an important resource for the study of B cell differentiation and immortalization.

Radioactive cell membrane labelling.

Building upon earlier studies with fluorescent probes, the authors describe a new cell tracking compound, PKH95, with a radioactive signal, which has been developed specifically for high-sensitivity cell tracking and biodistribution studies.

Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations.

Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin. Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light-scattering properties.

An improved clonal excess assay using flow cytometry and B-cell gating.

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti-kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non-B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B-cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

Flow cytometric monitoring of human immunodeficiency virus-infected patients. Simultaneous enumeration of five lymphocyte subsets.

The utility of CD4 lymphocytes in monitoring disease progression and prognosis of human immunodeficiency virus (HIV)-infected patients is well established. We have modified a previously described antibody cocktail to provide complete lymphocyte subset analysis on 100-200-microL samples of whole blood. This method optimizes accuracy of CD4 lymphocyte assessments and provides simultaneous assessment of four other lymphocyte subtypes of interest in specimens with absolute lymphocyte counts as low as 300 X 10(6)/L. Lymphocytes are classified as Thelper (CD3+CD4+); Tsuppressor (CD3+CD8+); Tnull (CD3+CD4-CD8-, putative gamma delta T-cell receptor); B (CD19+CD20+); or natural killer (CD3-CD16+CD56+). The method positively discriminates against contamination of lymphocyte scatter gates by monocytes and unlysed erythrocytes and is compatible with a variety of cell preparation procedures. Increased accuracy of CD4 lymphocyte determinations and simultaneous identification of other lymphocyte subsets whose relationship to disease progression is under study make this an efficient and informative method for disease monitoring and evaluation of therapy in HIV-infected patients.

Procedural guidelines for performing immunophenotyping by flow cytometry.

Flow cytometry is a rapidly expanding technology that is moving from the research laboratory into the clinical laboratory. Recent advances in availability and reproducibility of monoclonal antibody reagents specific for a wide range of cell types coupled with lower costs for increasingly automated flow cytometers with powerful and user friendly data analysis capabilities have made flow cytometry the method of choice for immunophenotyping in the clinical laboratory. However, there is great variability in the level and type of quality assurance procedures used from laboratory to laboratory. A subcommittee established by the National Committee for Clinical Laboratory Standards (NCCLS), composed of representatives from industry, academia, professional societies, and regulatory agencies, has drafted consensus procedures which address specific problems and suggested solutions for performance of immunophenotyping by flow cytometry. This paper is based on the authors' discussions with the NCCLS Committee but does not represent an official NCCLS position. The official NCCLS document on this subject (H42) is expected to be published in 1989.

Methotrexate inhibits macrophage activation as well as vascular and cellular inflammatory events in rat adjuvant induced arthritis.

We demonstrated previously that variables of macrophage activation are associated with the development and progression of the arthritic lesion in the model of adjuvant induced arthritis. This association was investigated further by assessing the ability of antiarthritic agents to modulate variables of macrophage activation in direct comparison to effects on the arthritic lesion. Whereas indomethacin effectively reduced hindpaw edema, it had no significant effect on Ia expression or on any measurement of activation. Prednisolone inhibited hindpaw edema and the production of interleukin-1 (IL-1) by splenic macrophages. Only methotrexate inhibited hindpaw edema and all variables of macrophage activation (PGE2 and IL-1 production, cyanine dye accumulation) as well as the influx of Ia positive macrophages into synovial tissue.

Macrophage activation in rat models of inflammation and arthritis. Systemic activation precedes arthritis induction and progression.

The association between the induction and progression of adjuvant-induced arthritis (AA) and the development of synovial and systemic macrophage activation was assessed by studying the temporal development of these parameters in a rat model. Rats with AA developed significant edema of the uninjected hind leg beginning 10 days post-adjuvant injection, with progressive increases in edema continuing through day 17. Several parameters of macrophage activation, including the enhanced ability to secrete interleukin-1 and prostaglandin E2, kill tumor cells, accumulate fluorescent cyanine dyes, emigrate into the peritoneal cavity and synovium, and express Ia antigen, as well as the decreased ability to secrete superoxide anion, were associated temporally with the development of the arthritic lesion. In addition to the temporal association between macrophage activation and development of arthritis, a positive correlation between macrophage activation and arthritis induction was seen with the use of synthetic adjuvants at arthritogenic and nonarthritogenic doses. These data taken together suggest that induction and progression of AA in rats is associated with both systemic (blood, spleen, and peritoneal cavity) and local (synovium) macrophage activation.

Antiarthritic and immunoregulatory activity of spirogermanium.

Spirogermanium is a novel metal containing azaspirane compound with reported antitumor activity. The results of the present investigation demonstrate that spirogermanium also exhibits antiarthritic and immunoregulatory activities after p.o. administration to rats. Spirogermanium decreased hindleg inflammatory lesions of adjuvant arthritic rats when administered p.o. before or after the development of the arthritic lesions. After termination of spirogermanium administration, the adjuvant-injected hindleg lesions remained significantly suppressed for at least 2 weeks postdrug treatment; whereas, the uninjected, immune-mediated hindleg inflammation tended to increase postdrug treatment. In multiparameter ex vivo studies, untreated arthritic rats exhibited enhanced cyanine dye fluorescence in peripheral blood monocytes, enhanced interleukin (IL)-1 production by adherent spleen cells and depressed IL-2 and IL-3 production by splenic lymphocytes. Spirogermanium normalized these changes to various degrees, with the exception of the depressed IL-2 and IL-3 production. Spirogermanium administered to normal nonarthritic rats decreased mitogenic responses of spleen cells to Concanavalin A which was found to be caused, at least in part, by enhanced suppressor cell activity. The antiarthritic and immunoregulatory profile of spirogermanium appeared to be different from the profiles of the antiarthritic agents, auranofin and indomethacin.

Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory.

As the diagnostic utility of lymphocyte subset analysis has been recognized in the clinical research laboratory, a wide variety of reagents and cell preparation, staining and analysis methods have also been described. Methods that are perfectly suitable for analysis of smaller sample numbers in the biological or clinical research setting are not always appropriate and/or applicable in the setting of a high volume clinical reference laboratory. We describe here some of the specific considerations involved in choosing a method for flow cytometric analysis which minimizes sample preparation and data analysis time while maximizing sample stability, viability, and reproducibility. Monoclonal T- and B-cell reagents from three manufacturers were found to give equivalent results for a reference population of healthy individuals. This was true whether direct or indirect immunofluorescence staining was used and whether cells were prepared by Ficoll-Hypaque fractionation (FH) or by lysis of whole blood. When B cells were enumerated using a polyclonal anti-immunoglobulin reagent, less cytophilic immunoglobulin staining was present after lysis than after FH preparation. However, both preparation methods required additional incubation at 37 degrees C to obtain results concordant with monoclonal B-cell reagents. Standard reagents were chosen on the basis of maximum positive/negative separation and the availability of appropriate negative controls. The effects of collection medium and storage conditions on sample stability and reproducibility of subset analysis were also assessed. Specimens collected in heparin and stored at room temperature in buffered medium gave reproducible results for 3 days after specimen collection, using either FH or lysis as the preparation method. General strategies for instrument optimization, quality control, and biohazard containment are also discussed.