Adoptive immunotherapy with peripheral blood lymphocytes cocultured in vitro with autologous tumor cells and interleukin-2

A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×10⁶ IU m^–2 day^–1. A dose of 300 mg/m² cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3⁺ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4⁺ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230     

High-dose continuous venous infusion of interleukin-2: Influence of dose and infusion rate on tumoricidal function and lymphocyte subsets

Previous clinical studies have demonstrated a dose-response relationship between enhancement of certain immune parameters and interleukin-2 (IL-2) dose in trials with low dosages of the cytokine. This has not been demonstrated for high-dose (greater than 18×10⁶ IU/m² per day) IL-2. We completed phase II trials of sustained administration of indomethacin and ranitidine with IL-2 given as a continuous infusion over 5 days for three courses. Peripheral blood mononuclear cells, both fresh and cultured in vitro with IL-2 or IL-2 and indomethacin, were tested for tumoricidal function against K562 and Daudi targets; these results were then correlated with actual delivered dose and mean infusion rate per course. Similar correlations were calculated between delivered dose or infusion rate and absolute and proportional counts of lymphocyte subsets as determined by flow cytometry. No enhancement of in vitro tumoricidal function with either increasing delivered dose or increasing infusion rate was seen. No consistent pattern of correlation was found between the absolute counts of lymphocyte subsets after each course of IL-2 with delivered dose or infusion rate. The percent rise in absolute counts of selected T- and NK-cell subsets at the end of course 1 compared with baseline values correlated positively with infusion rate; however, a similar correlation between the infusion rate and an increase in lymphocyte tumoricidal function was lacking. Little evidence was found for improved tumoricidal function of mononuclear cells or consistent enhancement of lymphocyte subset counts in patients able to tolerate doses of IL-2 beyond 18×10⁶ IU/m² per day in a 5-day continuous infusion schedule.Presented in part at the Twenty-eighth Annual Meeting of the American Society of Clinical Oncology, May 17–19, 1992, San Diego, Calif.     

Possible role for cytotoxic lymphocytes in the pathogenesis of acute interstitial nephritis after recombinant interleukin-2 treatment for renal cell cancer

A patient with renal cell cancer developed acute renal failure due to biopsy-proven acute tubulo-interstitial nephritis (AIN) in the 6th week of continuous infusion of 9 × 10⁶ IU m^–2 day^–1 recombinant interleukin-2 (rIL-2). We investigated whether the AIN was the result of a cellular cytotoxic reaction induced by the rIL-2 treatment. The cytolytic activity of cryopreserved peripheral blood lymphocytes (PBL), isolated before and at the end of the rIL-2 treatment (at the time of AIN), was studied after 5 days of culture with or without rIL-2 or anti-CD28 and immobilized anti-CD3 antibodies. The PBL isolated before and at the end of the rIL-2 treatment showed cytolytic activity towards a number of allogeneic targets. However, only the PBL isolated at the end of the rIL-2 treatment showed, when stimulated with rIL-2 in vitro, significant cytolytic activity against an autologous renal cell line cultured from the AIN biopsy specimen and against an allogeneic renal cell cancer cell line. These PBL displayed no enhanced killing capacity towards autologous PBL and the melanoma cell line M14. These observations suggest that the AIN may be the result of a cytotoxic lymphocyte-mediated reaction induced by the rIL-2 treatment.     

Lymphokine-activated suppressor (LAS) cells in patients with gastric carcinoma

Summary T-cell-growth-factor (TCGF) activated peripheral blood lymphocytes (PBL), cultured for 14 days, showed killer cell activities against natural-killer resistant Daudi cells in a 4 h ⁵¹Cr-release assay. However, the effector cells obtained from patients with nonresectable carcinoma exhibited very much lower cytotoxicity to tumor cells. To analyze the mechanism of depression, we have attempted to examine suppressor cell activities of the TCGF-activated PBL. The assay for the suppressor cell activities was made by in vitro inhibition of cell-mediated cytotoxicity by incubating radiolabeled target tumor cells with lymphokine-activated killer (LAK) cells and TCGF-activated PBL. LAK cells were induced by cultivation with recombinant interleukin-2. TCGF-activated PBL, obtained from four out of ten patients with resectable carcinoma and nine out of ten patients with nonresectable carcinoma, significantly suppressed the LAK cell activities. However, this suppression was not observed in TCGF-activated PBL from ten normal healthy control subjects. TCGF-activated PBL with immunosuppressive reactivity were named lymphokine-activated suppressor (LAS) cells. To investigate the phenotypic characterization of TCGF-activated PBL, the cells were analyzed by two-color flow cytometry. TCGF preferentially expanded CD8⁺CD11^– cells and decreased the growth of CD8⁺CD11⁺ cells in both normal healthy control subjects and gastric cancer (resectable and nonresectable) patients. Dominantly expressed CD8⁺CD11^– cells on TCGF-activated PBL in patients — especially those with nonresectable gastric carcinoma — showed strong LAS cell activity, irrespective of the presence of killer cell activities of CD8⁺CD11^– cells in TCGF-activated PBL from normal healthy control subjects. The results suggested the generation of CD8⁺CD11^– LAS cells from cancer patients, and revealed that CD8⁺CD11^– T-cells contained killer and/or suppressor cell function. In addition, it was found that the TCGF-activated PBL from gastric cancer patients were associated with an increased proportion of CD4⁺Leu8⁺, HLA-DR⁺CD8⁺ and HLA-DR⁺CD25⁺ cells.Abbreviations LAK lymphokine-activated killer - TCGF T-cell growth factor - PBL peripheral blood lymphocytes - rIL-2 recombinant interleukin-2 - LAS lymphokine-activated suppressor This study was supported by a grant-in-aid for scientific research (no. 62570307) from the Ministry of Education, Science and Culture, Japan     

A multicellular tumor spheroid model of cellular immunity against head and neck cancer

Summary A multicellular tumor spheroid (MTS) model for head and neck cancers has been used to examine the immune function of fresh and 6-day interleukin-2(IL-2)-activated peripheral blood lymphocytes (PBL). MTS are individually cultured in the presence of effector cells, and the spheroids' growth is monitored by sizing them under an inverted microscope. Dose/response studies for IL-2 (0–100 U/ml) alone and for fresh unstimulated PBL (0–10⁵ cells/MTS) showed no effects on MTS growth. IL-2-activated PBL (0–10⁵ cells/MTS), in contrast, modulated MTS growth in a multiphasic pattern: MTS growth was unperturbed for the first 3 days and then growth inhibition occurred, followed by MTS disintegration. Histological analysis showed that intact MTS histoarchitecture correlated with unperturbed growth, and increasing cell sloughing and MTS dissolution and replacement by activated PBL correlated with growth inhibition and disintegration. Flow-cytometric sorting of lymphocyte subset populations indicated that it was the Leu19⁺CD3^– cells that produced these growth-modulatory effects. In contrast to the initial LAK cell resistance of MTS, single-cell suspensions demonstrated significant lysis in standard 4-h chromium-release assays. Differences between single cells and MTS suggest a potential for tissue-like organization as a factor in lymphokine-activated killing.Supported in part by the First Independent Investigator Award (R29 CA46 251-01) (S. P. S.) of the National Cancer Institute. The Cancer Information Systems Core Facility used in this study was funded under the National Cancer Institute grant CA 16672     

Serum levels of the low-affinity interleukin-2 receptor molecule (TAC) during IL-2 therapy reflect systemic lymphoid mass activation

Summary Expression of the low-affinity interleukin-2 (IL-2) receptor molecule (TAC) has been associated with lymphocyte activation, in vitro and in vivo [Greene WC (1987) Clin Res 35:439]. We have used an enzyme-linked immunosorbent assay (ELISA) to quantify the role of released and cell-bound IL-2 receptor following in vitro or in vivo activation of human lymphocytes with IL-2. In vitro experiments, culturing fresh peripheral blood lymphocytes in 30 U/ml IL-2 (corresponding to the steady-state IL-2 concentration achieved in patients receiving IL-2 in our clinical trials), showed that the levels of IL-2 receptor released into the culture media exceeded the levels of cell-associated receptor, with both rising in parallel to the cytotoxic activity of the peripheral blood lymphocytes (PBL) against cultured tumor cells. In 12 patients receiving high-dose IL-2 for the treatment of various malignant neoplasms, the levels of IL-2 receptor released into the serum rose dramatically during the IL-2 infusion, and then fell following cessation of the IL-2 infusion. This heightened release of IL-2 receptor into the serum occurred during the episodes of profound lymphopenia that developed within hours after patients began an IL-2 infusion. Following each 4-day infusion of IL-2, a rebound lymphocytosis was observed, as has been previously reported. Serum IL-2 receptor levels do not rebound in parallel; rather, they reach a plateau near the end of the 4-day infusion and then decrease upon cessation of IL-2. These changes in serum IL-2 receptor levels accompany changes in lytic activity of circulating PBL on Daudi target cells. These results suggest that lymphocyte populations exposed to IL-2 in vivo are activated to become cytotoxic, release TAC, and relocate in non-peripheral blood compartments. Following cessation of the IL-2 infusion these activated lymphocytes return to the peripheral circulation and do not secrete TAC as vigorously as while influenced directly by the IL-2 infusion.This research was supported by NIH contract CM-47669, NIH grants CA-32685, and RR-03186, American Cancer Society CH-237B, and a research fellowship of the Leukemia Society of America (P. Fisch), a Ewing Foundation Fellowship (S. Voss) and Lutheran Brotherhood/Life and Health Insurance Medical Research Fund M. D./Ph. D. Training Fellowship (S. Voss)     

The influence of drainage and soil phosphorus on the vegetation of Douala-Edea Forest Reserve,Cameroun

All living trees (30 cm gbh) were enumerated in 104 80×80 m plots arranged along four transects in the Douala-Edea Forest Reserve Cameroun, a system of low-lying ancient coastal sand dunes interspersed by numerous streams and swamps. The extent of permanent and seasonal swamps was recorded for each plot. Two hundred thirty taxa were recognized of which 63% were identified to species. Mean tree density was 376 ha^–1, basal area 31.0 m² ha^–1 and number of species per plot 39. The Olacaceae were the most abundant family in terms of basal area, but the Euphorbiaceae the most frequently represented. The most abundant species wasCoula edulis (Olacaceae). Twenty-two plots had most of their area permanently or seasonally swamped. Percentage sand, silt and clay ranged between 32–100, 0–64, 0–21% respectively. The ranges for other variables recorded were: pH (2.7–5.4), organic carbon (1.5–12.4%), available phosphorus (7–90 ppm) and potassium (28–188 ppm), and nitrogen (ammonium 4–40 ppm, nitrate 1–12 ppm).Classification of the plots on the basis of six soil variables provided three large distinct groups: swamp plots and non-swamp plots, the latter divided into plots of low and high available soil phosphorus. Swamp plots were distinguished by high abundances ofProtomegabaria stapfiana andLibrevillea klainei, though correspondence ordination of plots in these groups showedP. stapfiana associated with more clayey soils andLibrevillea klainei (andGluema ivorensis) on the very sandy soils. Direct gradient analysis highlighted several species associated with these lower phosphorus soils. Available soil phosphorus is not as low at Douala-Edea as in parts of Korup, and the association of these Douala-Edea soils with the Caesalpinioideae is correspondingly weaker.Nomenclature follows Aubréville (1963–1983).The field work was supported by grant numbers RR00167-14, RR00167-15 and RR0167-16 from the National Institutes of Health for the operation of the Wisconsin Regional Primate Research Center, and N.A.T.O. Scientific Affairs grant number 1748 (to PGW and JSG). It was greatly facilitated by the skill and dedication of Ferdinand Namata. R. M. Polhill and D. W. Thomas assisted considerably in the identification of plant species. Sue Gartlan collected and collated the meteorological data, besides other field support. In the field phase J.S.G., P.G.W. and D.B.McK. were researches attached to the National Office of Scientific and Technical Research (ONAREST), Yaoundé. We thank M. D. Swaine for comments on earlier drafts, R. Letouzey for checking species nomenclature, the Computer Unit at Stirling University for facilities, M. Burnett for the typing at Stirling, and the Department of Soil Science, University of Wisconsin, for undertaking the soil chemical analyses.Reprint requests to D.McC.N. at Stirling.Publication No. 23-025 of the Wisconsin Regional Primate Center.     

Interleukin-2 dose,blood monocyte and CD25+ lymphocyte counts as predictors of clinical response to interleukin-2 therapy in patients with renal cell carcinoma

Summary The purpose of this study was to determine immunological parameters in the peripheral blood that correlate with the clinical effect of interleukin-2 (IL-2) in patients with metastatic renal cell cancer. A group of 26 patients with metastatic renal cell cancer underwent IL-2 treatment using a 36-day schedule with continuous intravenous IL-2 infusion (3 × 10⁶ units m^–2 day^–1) administered from days 1 to 5 and days 12 to 16. The white blood cell count and the absolute and relative number of neutrophils, lymphocytes, eosinophils and monocytes were recorded six times in peripheral blood during the treatment. Also the blood counts of T cell and NK cell subsets and cells expressing the T cell activation markers IL-2R and VLA-1 were measured. The lymphokine-activated killer (LAK) cell cytotoxicity was measured either with or without additional in vitro stimulation by IL-2. Multivariate statistical analysis showed that the clinical responses were related to the administered dose of IL-2, to a low number of blood cells expressing IL-2 receptors and to a reduction in the blood monocyte count (P <0.05).     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

 The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3⁺PBL) and TIL (CD3⁺TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3⁺TIL and CD3⁺PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3⁺TIL produced significantly lower levels of IL-2 than CD3⁺PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3⁺TIL as compared to the CD3⁺PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3⁺TIL and CD3⁺PBL derived from patients with renal cell carcinomas. Received: 8 August 1995 / Accepted: 23 January 1996     

Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan     

An enumerative assay of purine analogue resistant lymphocytes in women heterozygous for the Lesch-Nyhan mutation

In females heterozygous for the Lesch-Nyhan (LN) mutation, there is mosaicism of peripheral blood lymphocytes (PBLs) with regard to sensitivity to 6-thioguanine (TG) inhibition of tritiated thymidine ([³H]Tdr) incorporation following phytohemagglutinin (PHA) stimulation. That there are two populations of PBLs, normal and mutant (LN-like), has been demonstrated by an autoradiographic enumerative assay. A single three-generation family containing six potentially heterozygous females was studied. Five of the six were mosaics with frequencies of TG-resistant (TG^r) PBLs ranging from 1.4×10^–3 to 4.2×10^–3 when tested at 2×10^–4 m TG. The median frequency of TG^r PBLs in 63 healthy non-LN individuals between the ages of 11 and 75 years was found to be 1.1×10^–4 (mean 1.3×10^–4) (10th and 90th percentiles—6.1×10^–5 and 2.1×10^–4) and was not age related. The sixth potentially heterozygous female in the current family had a TG^r PBL frequency of 1.9×10^–4. In the five females with elevated TG^r PBL frequencies, TG^r skin fibroblasts with frequencies ranging from 26% to 100% of the sample tested were found; in the female with the normal TG^r PBL frequency, no TG^r skin fibroblasts were found. The former group was considered to be LN heterozygous. Four of the five had been previously diagnosed as such. The latter individual is considered to be genotypically normal. Females who are heterozygotes for the LN mutation have two populations of PBLs.This work was supported by National Institutes of Health Grant No. PHS RO1 15450 and the Surgical Associates Foundation, Inc., University of Vermont College of Medicine. Elizabeth F. Allen was supported by the NIH National Research Service Award No. 5 F32 CA 05918.     

Genetic polymorphism of PIF (parotid isoelectric focusing variant) proteins with linkage to the PPP (parotid proline-rich protein) gene complex

Genetic polymorphism is found among the PIF (parotid isoelectric focusing variant) salivary proteins after separation by prolonged isoelectric focusing in pH 3.5–5.2 urea polyacrylamide slab gels subsequently stained for protein. Two PIF proteins are either present (PIF +) or absent (PIF –) from all salivas. The phenotypes are determined by autosomal inheritance of two alleles, PIF ⁺ and PIF ^–. Gene frequencies in randomly collected samples show marked racial differences: among 148 whites, PIF ⁺ is 0.66 and PIF ^– is 0.34; among 90 blacks, PIF ⁺ is 0.35 and PIF ^– is 0.65; among 78 Chinese, PIF ⁺ is 0.56 and PIF ^– is 0.44. Studies in 41 families including 129 children support the interpretation of control of PIF by a single autosomal locus. In 8 PIF+ × PIF– matings, there were 8 PIF– (6.34 expected) children. In 33 PIF+ × PIF+ matings, there were 7 PIF– (6.70 expected) children. Linkage studies indicate that PIF is closely linked to the proline-rich protein (PPP) gene complex (e.g., for six families, lod score at =0.00 of PIF/G1 is 3.58). In 107 randomly collected samples from whites, PIF is strongly associated with Db (x ₁ ² =20.02; P<0.0001) and Gl (x ₁ ² =12.58; P=0.0005) but not with Pr, Ps, Pm, and Pa proteins. These data (probably reflecting genetic disequilibrium) suggest that PIF may be closer to Db and G1 than to other identified loci of the PPP gene complex. The PPP gene complex includes at least seven genes (and probably more) that produce many acidic and basic proline-rich proteins, constituting about two-thirds of parotid salivary proteins that are thought to have important functions at the tooth surfaces.This study was supported by a grant from the National Institutes of Dental Research (DEO 3658-15). Paper No. 2435 of the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706.     

Comparative bioenergetics of permanent and temporary pond populations of the freshwater clam,Musculium partumeium (Say)

The population energetics of a temporary and a permanent pond population of Musculium partumeium in Southwest Ohio were studied. In the permanent pond (surface area = 396 m², maximum depth = 0.7 m) the population was bivoltine and iteroparous whereas in the temporary pond (surface area = 1042 m², maximum depth = 0.9 m) the population was usually univoltine and semelparous.Growth and biomass were assessed as total organic carbon and total nitrogen to provide estimates of productivity and seasonal changes in C:N for each generation. Productivity (non-respired assimilation = growth + reproduction; N-R.A. = G + R) was 6939 mgC·m^–2·a^–1 (3858 and 3353 mgC·m^–2·a^–1 for each generation) and 1661 mgC·m^–2·a^–1 for the permanent and temporary pond populations respectively. The average standing crop biomass (B) was 606.8 mgC·m^–2 (357.5 and 249.3 mgC·m^–2 for each generation) and 231.9 mgC·m^–2 with overall productivity: biomass ratios of 11.4 and 7.2 for the permanent pond and temporary pond populations respectively.Respiration rates were converted to carbon equivalents (respired assimilation = R.A.) and used to evaluate the components of total assimilation (T.A. = R.A. + N-R.A.) and the efficiency of partitioning this energy to N-R.A. for G and R. When expressed as a percentage, the production efficiencies (100 × N-R.A.:T.A.) were 50.4 and 62%, and the reproductive efficiencies (100 × R:N-R.A.) were 26.4 and 18% for the permanent and temporary pond populations respectively. The reproductive efficiencies for populations of these viviparous clams are greater than those for most oviparous molluscs.The comparative information on the energetics of these populations does not completely fit any theoretical consideration of reproductive effort or life-history strategy. These data are discussed in relation to selection for population success in temporary ponds.Funded in part by grants to Albert J. Burky from the Ohio Biological Survey and the University of Dayton Research CouncilFunded in part by grants to Albert J. Burky from the Ohio Biological Survey and the University of Dayton Research Council     

HLA class l specific t lymphocyte clones with dual alloreactive functions

Four human T lymphocyte clones exhibiting proliferative responses to class I HLA antigens were isolated from an in vitro mixed lymphocyte culture (MLC). Three clones expressed the Leu-2⁺3^– phenotype and demonstrated proliferation in response to HLA-B8, while the fourth clone expressed the Leu-2^–3⁺ phenotype and proliferated in response to HLA-A2. These clones were also cytotoxic towards cells bearing the same target antigens. Blocking studies utilizing monoclonal antibodies demonstrated that proliferation was triggered by determinants on the class I molecule itself, and these determinants appear to be spatially close to those which determine serologic allospecificity. These findings support the concept that the class I molecules themselves are the weak MLC stimulating determinants previously mapped to the HLA-A and B regions of the major histocompatibility complex.Abbreviations used in this paper B-LCL B-lymphoblastoid cell line - cpm counts per minute - FCS fetal calf serum - HS human serum - ³H-TdR tritiated thymidine - IL-2 interleukin-2 - IL2-CM interleukin-2 containing conditioned medium - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MoAb monoclonal antibody - PBL peripheral blood mononuclear leukocytes - PLT primed lymphocyte test     

Chemo-immunotherapy of murine tumors using interleukin-2 (IL-2) and cyclophosphamide

Summary The antitumor effect of interleukin-2 (IL-2), alone and in combination with cyclophosphamide was assessed in mice with established sarcoma (MCA 105, H-2^b), carcinoma (M109, H-2^d) and T lymphoma (PIR-2, H-2^b). Whereas administration of IL-2 alone (5×10⁴–10×10⁴ U, i.p. twice daily, for 4–8 consecutive days) prolonged the survival of mice with the solid neoplasms, it enhanced tumor growth and decreased survival of mice with the lymphoma. In the PIR-2 lymphoma, no IL-2 receptor (TAC) could be detected, nor could we demonstrate IL-2 tumor growth stimulation in vitro. A synergistic therapeutic effect was achieved in mice with the solid tumors, but not in mice with the lymphoma, only when IL-2 was given 1–4 days after cyclophosphamide (100–200 mg/kg). Conversely, administration of IL-2 1–4 days prior to cyclophosphamide resulted, in all three tumor systems, in enhanced tumor growth and in decreased survival as compared with mice receiving cyclophosphamide alone. Similarly, treatment with IL-2 both before and after cyclophosphamide was less efficacious than a single course of IL-2 given after-wards. It is concluded that for maximal therapeutic efficacy, IL-2 should be administered following chemotherapy, and that certain tumors may respond adversely to IL-2 treatment.     

B-Lymphocyte Proliferation during Bovine Leukemia VirusInduced Persistent Lymphocytosis Is Enhanced by T-Lymphocyte-Derived Interleukin-2

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5⁺ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-γ) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.     

Histamine in immunotherapy of advanced melanoma: a pilot study

Sixteen patients with advanced metastatic malignant melanoma were treated with a high-dose infusion of interleukin-2 (IL-2; 18×10⁶ IU/m^–2 day^–1) together with daily subcutaneous (s.c.) injections of interferon (IFN; 3×10⁶ U/m^–2 day^–1) in 5-day cycles. Nine of these patients were given histamine (1 mg s.c.) twice daily during treatment with IL-2 and IFN. In the seven patients who did not receive histamine, one partial response (that is a reduction of more than 50% in the total tumour burden) was observed in a patient with skin and lymph node melanoma. In the eight histamine-treated patients evaluable for response, four partial responses were observed. Two other patients showed regression at one site of metastasis but tumours remained unchanged at other sites. Two histamine-treated patients showed complete resolution of extensive liver metastasis. Sites of response in histamine-treated patients also included the subcutis, lymph nodes, skeleton, spleen and muscle. Lung melanoma did not respond to histamine/IL-2/IFN. Three patients with lung tumours responded with significant (more than 50%) reduction of the volume of soft-tissue tumours, suggesting that the response to histamine may be organotropic. Survival was significantly prolonged in patients receiving histamine. Our data suggest that treatment with histamine may improve the antitumour efficacy of immunotherapy in metastatic melanoma.     

The influence of topography and soil phosphorus on the vegetation of Korup Forest Reserve,Cameroun

All living trees (30 cm gbh) were enumerated in 135 80×80 m plots, each subdivided into four 40×40 m subplots, and arranged along four 5 km transect lines in the Korup Forest Reserve, Cameroun. For each plot altitude, slope and the extent of permanent and seasonal swamps were recorded.Four hundred and eleven taxa were recognized of which 66% were identified to species. Mean tree density was 471 ha^–1, basal area 27.6 m² ha^–1 and number of species per plot 75. The subfamily Caesalpinioideae (Leguminosae) was the most abundant family/subfamily in terms of basal area, but the Scytopetalaceae the most frequently represented, mainly on account ofOubanguia alata. Ten plots had at least three quarters of their area permanently swamped, and three, to a similar extent, were seasonally swamped. The ranges in sand, silt and clay content were 60–91, 0–24 and 4–20% respectively. The pH value, organic carbon content and nitrate-nitrogen concentration ranged between 4.0–5.8, 1.3–5.7% and 0–35 ppm respectively. The largest soil variations were in available phosphorus, range 2–29 ppm, and potassium, 38–375 ppm.Correspondence analysis ordination of all plots showed a major indirect floristic gradient correlated with increasing altitude, slope and soil phosphorus and potassium. Removal of the topographic effect by separate re-ordinations of four groups of plots at low, middle (2) and high altitude/slopes highlighted a strong correlation of the main floristic gradients of the middle altitude/slope groups with the concentration of available soil phosphorus.Direct gradient analysis using all plots with respect to available soil phosphorus concentration confirmed the indirect analyses. Individual species response to phosphorus were also shown by direct comparisons in the vegetation on plots of high and low available soil phosphorus concentration. Low available phosphorus soils (5 ppm) are strongly associated with species of the subfamily Caesalpinioideae, especially of the tribes Amherstieae and Detarieae. It is suggested that this result is probably due to the ability of these particular legume tribes to form associations with ectotrophic mycorrhizae.The field work was supported by grant numbers RROO167-17, RROO167-18 and RROO167-19 from the National Institutes of Health for the operation of the Wisconsin Regional Primate Center, N.A.T.O. Scientific Affairs Division Grant number 1748 (to PGW and JSG), N.E.R.C. grant number GR3/3455 (to PGW), and was greatly facilitated by the skill and dedication of Ferdinand Namata. Meteorological data were kindly provide by E. P. Cundall (Plantations du Cameroun, Lobé). JSG acknowledges the support and encouragement of Sue Gartlan. In the field phase, JSG was a researcher attached to the National Office of Scientific and Technical Research (ONAREST), Yaoundé. We are grateful to I. Alexander and M. D. Swaine for comments on earlier drafts, R. Letouzey for checking the species nomenclature, the Computer Unit of the University of Stirling for facilities, M. Burnett for the typing at Stirling and the Department of Soil Science, University of Wisconsin, for undertaking the soil analyses.Nomenclature follows Aubréville (1963–1983).Publication No. 23-024 of the Wisconsin Regional Primate Center.Reprint requests to D.McC.N. at Stirling.     

Lidocaine,Dexmedetomidine and Their Combination Reduce Isoflurane Minimum Alveolar Concentration in Dogs

The effects of intravenous (IV) lidocaine, dexmedetomidine and their combination delivered as a bolus followed by a constant rate infusion (CRI) on the minimum alveolar concentration of isoflurane (MAC_ISO) in dogs were evaluated. Seven healthy adult dogs were included. Anaesthesia was induced with propofol and maintained with isoflurane. For each dog, baseline MAC (MAC_ISO/BASAL) was determined after a 90-minute equilibration period. Thereafter, each dog received one of the following treatments (loading dose, CRI): lidocaine 2 mg kg⁻¹, 100 µg kg⁻¹ minute⁻¹; dexmedetomidine 2 µg kg⁻¹, 2 µg kg⁻¹ hour⁻¹; or their combination. MAC was then determined again after 45- minutes of treatment by CRI. At the doses administered, lidocaine, dexmedetomidine and their combination significantly reduced MAC_ISO by 27.3% (range: 12.5–39.2%), 43.4% (33.3–53.3%) and 60.9% (46.1–78.1%), respectively, when compared to MAC_ISO/_BASAL. The combination resulted in a greater MAC_ISO reduction than the two drugs alone. Their use, at the doses studied, provides a clinically important reduction in the concentration of ISO during anaesthesia in dogs.