The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier,OK-432

 Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432. Received: 29 April 1996 / Accepted: 27 July 1996     

99mTc-CD19 monoclonal antibody is not useful for imaging of B cell non-Hodgkin’s lymphoma

 In this study we investigated the applicability of ^99mTc-labeled CD19 monoclonal antibody (mAb) for tumor imaging in patients with B cell non-Hodgkin’s lymphoma. A 1-mg sample of murine CD19 mAb was labeled with approximately 550 MBq [^99mTc]pertechnetate. The labeled mAb was administered i. v. to seven patients, four without and three with pretreatment with 10 mg unlabeled CD19 mAb. The number of circulating B cells was decreased by 44±5% 1 h after injection of the radiolabeled mAb. Peripheral B cells were coated with CD19, resulting in partial modulation of CD19, most pronounced in the three pretreated patients. Whole-body images were obtained with a gamma camera and compared with results obtained by conventional imaging techniques. Initially, blood-pool activity dominated, whereas 24 h after injection the radioactivity was mainly located in the spleen, kidneys and liver. In two patients, a lesion in the spleen appeared as an unlabeled spot. In one patient, a lesion in the femur, which was detected by computed tomography (CT) and gallium-67 scans, was also seen on the CD19 scan from 1 h after administration of the radioimmunoconjugate onwards. Good imaging of bone marrow infiltration was observed in one of three patients. Lymph node involvement was not observed in any of the patients in whom affected lymph nodes were detected by CT or gallium-67 scan. In conclusion, in the present study radioimmunodetection with ^99mTc-labeled CD19 mAb was found to be inferior to CT and gallium-67 scanning in the diagnosis of patients with B cell non-Hodgkin’s lymphoma. Received: 14 March 1996 / Accepted: 14 May 1996     

The concurrent administration of OK432 augments the antitumor vaccination effect with tumor cells by sustaining locally infiltrating natural killer cells

 The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon γ in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of antitumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells. Received: 6 March 1996 / Accepted: 30 May 1996     

Analysis of anchor residues in a naturally processed HLA-DR53 ligand

 The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. Received: 29 April 1996 / Revised: 16 June 1996     

Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells

 The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC-associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug : mAb ratios of 7 : 1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 μM and 1 μM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients. Received: 22 September 1995 / Accepted: 23 November 1995     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

Sap flow rates of mangrove trees are not unusually low

 In contrast with previous reports, we observed high transpiration rates in mangrove trees. Maximum sap velocities and mean daytime sap flow rates were estimated from heat pulse velocity in entire, field grown trees of Avicennia cf. alba Blume and Rhizophora apiculata Blume. Results were within the range of values measured by identical techniques for trees in lowland dipterocarp and tropical heath forests with a similar climate in Brunei Darussalam (north Borneo). High stomatal conductance (400 mmol m^{–  2} s^{–  1}) was also measured for well insolated leaves of A. cf. alba, with midday water potentials reaching about  – 3 MPa in both species. Received: 11 September 1996 / Accepted: 27 January 1997     

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

 An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD₅₀ value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 – 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent. Received: 15 August 1995 / Accepted: 23 April 1996     

Effects of climate on vertical resin duct density and radial growth of Norway spruce [Picea abies (L.) Karst.]

 Vertical resin duct density in spruce tree-rings was used as a dendroclimatological variable and compared with radial growth. Resin duct density data were statistically stable and distributed with higher mean sensitivity and standard deviation but lower signal-to-noise rate than the corresponding growth rate. Radial growth and resin duct density relationships with climate were investigated using correlation and response function analysis. It was found that resin duct density has a significant positive response to above-normal temperature especially from June to August and a less significant negative response for above-normal precipitation from May – July during the current growing season. Ring width showed a significant negative response to above-normal precipitation from June to August but no response with temperature. Ring width and resin duct density were not related to each other. Sufficient data indicate that vertical resin duct density is a useful variable for dendroclimatology. Received: 14 March 1996 / Accepted: 24 June 1996     

Involvement of tumor necrosis factor α and very late activation antigen 4/vascular cell adhesion molecule 1 interaction in surgical-stress-enhanced experimental metastasis

 We examined the influence of surgical stress on hematogenous metastasis of malignant tumor cells. The study was performed by focusing on the involvement of inflammatory cytokines in the serum, raised acutely after surgery, and endothelial adhesion molecules in the metastatic process. Surgical stress, given to C57BL/6 mice before B16-BL6 melanoma inoculation, significantly enhanced the pulmonary metastasis. This enhancement was seen when the surgery lasted for more than 2 h. After the 2-h surgery, the enhancement of pulmonary metastasis was seen most remarkably when B16-BL6 was inoculated 24 h after surgery. The serum level of tumor necrosis factor α (TNFα) in the mice that underwent the 2-h surgery peaked 12 h after the surgery. In contrast, serum interferon γ was not detectable. Administration of an anti-TNFα mAb before the surgery inhibited the enhanced metastasis by inhibiting the increased expression of vascular cell adhesion molecule 1 (VCAM-1) on lung vascular endothelium after the surgery. Pretreatment of B16-BL6 cells with an anti-very late activation antigen 4 (anti-VLA-4) mAb completely inhibited the enhanced metastasis after surgery. Administration of an anti-VCAM-1 mAb before surgery also inhibited the enhancement. These results indicate that serum TNFα , raised by surgical stress, is critically involved in the enhanced pulmonary metastasis of mouse melanoma by inducing VCAM-1 expression on lung vascular endothelium. Received: 22 January 1996 / Accepted: 1 April 1996     

DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression

 Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH⁺ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 – 1106, S at position 1157, and VDLL at positions 1188 – 1191. A second donor typed as C4A2, A4, B1 and was also WH⁺. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 – 1106, S at position 1157, and ADLR at positions 1188 – 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene. Received: 22 November 1995 / Revised: 19 February 1996     

Degradation of radioiodinated B cell monoclonal antibodies: inhibition via a FCγ -receptor-II-mediated mechanism and by drugs

 Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37°C, 20% – 25% of initially cell-bound ¹²⁵I-CD19 mAb and ¹²⁵I-CD22 mAb was degraded in B cells, whereas almost no degradation occurred after binding of ¹²⁵I-CD20 mAb. For B cells expressing Fcγ receptor II (FcγRII), isotype-dependent degradation was noted as the CD19 IgG1 mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with ¹²⁵I-CD19 mAb or of CD20 with ¹²⁵I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcγRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant B cells and consequently result in an enhanced therapeutic effect in vivo. Received: 22 September 1995 / Accepted: 13 November 1995     

Toxicological and immunological evaluation of the MHC-non-restricted cytotoxic T cell line TALL-104

 The human MHC-non-restricted cytotoxic T cell line TALL-104 has been shown to display potent antitumor effects in several animal models with spontaneous and induced malignancies. In view of its potential future use in cancer therapy, we investigated the tolerability and target-organ toxicity of these cells in various animal species. The acute toxicity of TALL-104 cell administrations was evaluated in: (a) healthy immunocompetent mice and immunodeficient (SCID) mice bearing human tumors using multiple (up to 15) intraperitoneal (i.p.) injections, and (b) healthy dogs, tumor-bearing dogs, and healthy monkeys using multiple (up to 17) intravenous (i.v.) injections. TALL-104 cells were γ-irradiated (40 Gy) prior to administration to mice and dogs, but administered without irradiation in monkeys. Cell doses ranged from 5×10⁷/kg to 10¹⁰/kg for each injection. All regimens were well tolerated, the main clinical signs observed being transient gastrointestinal effects. Moderate and transient increases in liver transaminase levels were observed in all animal species. Discrete and transient leukocytosis with neutrophilia was also noted in dogs and monkeys after i.v injections of TALL-104 cells. Histological analysis revealed foci of hepatic necrosis with lympho-/mono-/granulocytic infiltration in immunocompetent mice injected i.p. with 5×10⁹ – 10¹⁰ cells/kg. In the same mice, the colon showed an increased number of muciparous cells and alterations in the villi structure: these alterations were completely reversed by 72 h after the last injection, while liver alterations reversed more slowly (1 week). No delayed or chronic toxicity was observed in any of the animals even when non-irradiated TALL-104 cells were administered: both immunocompetent mice and healthy dogs were found to be grossly and histopathologically normal when sacrificed (1 year and 1 month after the last TALL-104 injection respectively). TALL-104 cells did not persist in these hosts. In addition, monkeys showed no molecular signs of TALL-104-cell-induced leukemia in their blood 1 year after the last cell injection. Despite immunosuppression, most of the tumor-bearing dogs as well as the healthy dogs and monkeys developed both humoral and cellular immune responses against TALL-104 cells. The data derived from these preclinical studies suggest that administration of high doses of irradiated TALL-104 cells is well tolerated and would be unlikely to induce severe toxicity if applied in clinical trials to the treatment of patients with refractory cancer. Received: 8 September 1996 / Accepted: 28 January 1997     

Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor α and interferon γ treatment

 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E₂ values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Effects of N G-methyl-L-arginine,an inhibitor of nitric oxide synthesis,on interleukin-2-induced capillary leakage and antitumor responses in healthy and tumor-bearing mice

 We tested whether treatment with an inhibitor of nitric oxide synthesis (N ^G-methyl-L-arginine, MeArg) can ameliorate interleukin-2(IL-2)-therapy-induced capillary leak syndrome in healthy or tumor-bearing mice without compromising the antitumor effects of IL-2 therapy. Healthy or C3-L5-mammary-adenocarcinoma-bearing C3H/HeJ mice were treated with one or two rounds of various doses of IL-2 (ten injections, i. p., every 8 h) or MeArg (ten injections s. c., every 8 h) or their combination. In an additional experiment, MeArg was given chronically in the drinking water, rather than s. c. to healthy mice subjected to one round of therapy as above. Mice were killed 1 h after their last IL-2 injection to measure the water content of the lungs and pleural cavities (markers of capillary leakage), NO production (given by NO₂ ^– and NO₃ ^– levels in the serum and pleural effusion), as well as the effect of therapies on the primary tumor size and number of spontaneous lung metastatic nodules. Results revealed that all doses of IL-2 (7500 – 35 000 Cetus U/injection), as well as both rounds of IL-2 therapy, caused capillary leakage. However, no pleural effusion was seen after the second round in any of the IL-2-treated groups. MeArg therapy, given subcutaneously (5 – 20 mg kg^–1 injection^–1 in healthy and 20 mg kg^–1 injection^–1 in tumor-bearing mice), did not ameliorate IL-2-induced capillary leakage in either group of mice, and did not compromise antitumor effects of IL-2. However, subcutaneous MeArg therapy alone reduced the growth of the primary tumors, the occurrence of spontaneous lung metastases and the amount of tumor-induced pulmonary edema. When MeArg therapy was given orally (1 mg/ml drinking water), a substantial drop in NO production, as well as reduction in capillary leakage was noted in IL-2-treated healthy mice. These findings suggest that NO inhibitors could be a valuable adjunct to IL-2 therapy of cancer and infectious diseases. Received: 23 October 1995 / Accepted: 22 November 1995     

The effect of antibody protein dose on the uniformity of tumor distribution of radioantibodies: An autoradiographic study

Summary The inaccessibility of radiolabeled antibody to poorly vascularized regions of solid tumors may reduce the therapeutic efficacy of these macromolecules. Theoretical mathematical models have predicted that increasing the protein dose administered would reduce the heterogeneity of radioantibody distribution. This investigation was undertaken to evaluate this hypothesis in experimental animal models. We have utilized the technique of macroautoradiography to demonstrate an increase in tumor penetration of the lower-affinity¹²⁵I-labeled NP-4 or higher-affinity Immu-14 anti-carcinoembryonic antigen (anti-CEA) mAbs into small (60.25—0.4 g) and large (0.8–1.5 g) GW-39 and LS174T human colonic xenografts, grown subcutaneously in the nude mouse, when 400 µg unlabeled antibody is administered simultaneously with 10 µg (100 µCi) radioantibody. Further increases in protein to 800 µg result in a reduction in total tumor uptake of the antibody. These differences in mAb distribution could be visualized as early as 1 day after antibody injection. Improved mAb penetration was also achieved for the Mu-9 anti-CSAp (anti-mucin) antibody using 800 µg unlabeled antibody. An irrelevant antibody (AFP-7-31) was found to be homogeneously distributed 3 days after injection, even at a low protein dose. Attempts to improve mAb penetration by increasing the protein dose in the GS-2 colorectal tumor, a model that has low NP-4 accretion as a result physiological barriers separating antibody from antigen, were not successful. These results suggest that a more homogeneous distribution of radioantibody can be achieved by carefully selecting a dose of unlabeled antibody to coadminister. Work is currently in progress to determine the effect of improved tumor distribution of radioantibody on the therapeutic potential of a single dose of radioantibody.This work has been supported in part by USPHS grants CA-37895, CA 39841, and RR-05 903 (Division of Research Resources), National Institutes of Health, Department of Health and Human ServicesResearch fellow of the Dutch Cancer Society     

Rapid elimination of mouse/human chimeric monoclonal antibodies in nude mice

 At our laboratory we are currently evaluating the suitability of mouse/human chimeric monoclonal antibodies (cmAb) for use in radioimmunotherapy of patients with head and neck squamous cell carcinoma (HNSCC). We have developed cmAb containing the human constant IgG1 domain and the variable domains of murine mAb (mmAb) E48 and U36 respectively. We considered the tumour-bearing nude mouse to be a well-validated model for a first testing of the targeting capabilities of these cmAb in comparison with the mmAb. Therefore, 3 μg cmAb E48 (labelled with ¹²⁵I) and 3 μg mmAb E48 (labelled with ¹³¹I) were simultaneously injected into HNSCC-bearing nude mice and, at various assay times, mAb uptake in blood and other tissues was assessed. Remarkably, while in roughly 50% of the animals the biodistribution of the conjugates was similar, in the other animals cmAb E48 showed a much higher blood clearance than mmAb E48. This resulted in a lower tumour uptake of cmAb E48 in comparison with mmAb E48. To determine whether this phenomenon was related to mAb E48 or to the animal model, other cmAb-mmAb combinations were evaluated in the same way: cmAbs SF-25, 17-1A and U36 (all IgG1) were tested and all showed a rapid elimination in about 50% of the animals. Besides a decrease in blood concentration, an increase of cmAb levels in liver and spleen was observed within 24 h after injection. Isotype-specific enzyme-linked immunosorbent assays showed that mice that demonstrated a rapid elimination of cmAb from the blood had much lower endogenous IgG1, IgG2b and IgG3 titres than mice showing normal clearance. IgG2a levels were low in all mice. Biodistribution experiments with 3 μg chimeric 17-1A isoforms showed high blood clearance in a proportion of the mice for IgG1, IgG3 and IgG4, but not for IgG2. Increase of the cmAb dose to 100 μg resulted in a similar cmAb and mmAb biodistribution in all mice. Moreover, the biodistribution of the F(ab′)₂ fragment of an IgG1 cmAb was similar for all mice in contrast to that of coinjected whole IgG. On the basis of these results it can be hypothesized that, in mice with low endogenous IgG titres, cmAb with specific isotypes are rapidly removed from the blood (and ultimately from the body) by mediation of Fc-binding receptors. Apparently, in mice with high endogenous IgG titres or in mice receiving a high cmAb dose, these receptors are saturated. Furthermore, the rapid elimination of cmAb from nude mice, which may occur after injection at a low dose, is a phenomenon related to the nude mouse model. Received: 26 July 1996 / Accepted: 16 January 1997     

Seedling growth strategies and seed size effects in fourteen oak species native to different soil moisture habitats

 Seedling growth and morphology are thought to reflect evolutionary responses to habitat or influences of seed size. To test these hypotheses, we selected fourteen species of North American oaks differing in soil moisture habitat preference and seed size. Seedlings were grown for 1 – 2 years with abundant soil water and moderate soil nutrition in pots placed outdoors and in a common garden. Oak species native to xeric environments produced the smallest seedlings. Oaks from hydric soils had more shoot weight per unit of root weight and more height per unit of total plant weight than did mesic or xeric oaks. Essentially no differences in leaf area per unit of total plant weight were detected. Species with thinner and larger individual leaves tended to produce larger seedlings. Within species, seed size was generally unrelated to seedling growth, although results may have been complicated by uncontrolled genotypic variability. However, when species were compared, those with larger mean seed size produced larger seedlings. Root/shoot allometry, height growth and leaf thickness in the tested species may reflect evolutionary responses to soil moisture and flooding. Although seed size influenced seedling growth, no clear relationship between seed size and soil moisture habitat was found. Received: 26 March 1995 / Accepted: 30 November 1995     

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged. Received: 5 August 1996 /  Accepted: 23 September 1996