Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Phase I trial of intravenous infusion of ex-vivo-activated autologous blood-derived macrophages in patients with non-small-cell lung cancer: Toxicity and immunomodulatory effects

Summary The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon (rhuIFN) [monokine-activated killer (MAK) cells] and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 × 10⁷ to 5 × 10⁸ on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate [primarly fever (75%) and chills (32%)], non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 × 10⁸ cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood postinfusion and in vitro by secretory products (IL-1. TNF, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with¹¹¹In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (<24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.This work was supported in part by a grant 6911 from the Association pour la Recherche contre le Cancer (ARC), grants from the Ligue Nationale contre le cancer and the Ligues Regionales (Bas-Rhin, Haut-Rhin) contre le cancer, and contract 891013 from the Institut National pour la Santé et la Recherche Médicale (INSERM), France     

Brain injury: New insights into neurotransmitter and receptor mechanisms

The studies reviewed here represent a continuing search for mechanisms which play a role in neurological disturbances resulting from brain injury. Focal cortical freezing lesions in rats were shown to cause a widespread decrease in local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere and this was interpreted as reflecting a depression of cortical activity. Such an interpretation was supported by the finding that in lesioned brain reduction of cerebral metabolism by pentobarbital and isoflurane was limited by the metabolic depression that has already occurred as a result of injury and by the demonstration that the energy status and substrate (glucose) supply in the cortical areas in the injured brain have not been compromised at the time when LCGU was decreased. Both the serotonergic and the noradrenergic neurotransmitter systems were implicated in functional alterations associated with injury. Cortical serotonin (5-HT) metabolism was increased throughout the lesioned hemisphere and complete inhibition of 5-HT synthesis withp-chlorophenylalanine ameliorated the decrease in cortical LCGU, interpreted as reflecting cortical functional depression. Cortical norepinephrine metabolism was bilaterally increased in focally injured brain, while prazosin, a selective ₁-noradrenergic receptor blocker, normalized cortical LCGU in the lesioned hemisphere. Low-affinity in vivo binding of [¹²⁵I]HEAT, another selective ₁-receptor ligand, was specifically increased in cortical areas of the lesioned hemisphere at the time of the greatest depression in LCGU, suggesting that ₁-adrenoreceptors may be of functional importance in injured brain. The general conclusion from this series of studies on mechanisms underlying functional disturbances in injured brain is that both the serotonergic and the noradrenergic neurotransmitter systems are involved in the widespread cortical depression which develops with time as a consequence of a focal lesion. The data are compatible with the inhibitory effects of NE and 5-HT in the cortex and with the hypothesis that these two transmitter systems affect cortical information processing.     

Phylogenetic and evolutionary implications of nuclear ribosomal DNA variation in dwarf dandelions (Krigia,Lactuceae, Asteraceae)

Restriction site and length variations of nrDNA were examined for 51 populations of seven species ofKrigia. The nrDNA repeat ranged in size from 8.7 to 9.6 kilobase (kb). The transcribed region, including the two ITSs, was 5.35 kb long in all examinedKrigia populations. In contrast, the size of the nontranscribed IGS varied from 3.35 to 4.25 kb. Eight different types of length-variations were identified among the 51 populations, including distinct nrDNA lengths in the tetraploid and diploid populations of bothK. biflora andK. virginica. However, a few variations were detected among populations of the same species or within a cytotype. All populations ofKrigia sect.Cymbia share a 600 bp insertion in IGS near the 18 S gene, and this feature suggests monophyly of the section. AllKrigia spp. had a conjugated type of subrepeat composed of approximately 75 basepairs (bp) and 125 bp. Base modifications in the gene coding regions were highly conserved among species. Forty-five restriction sites from 15 enzymes were mapped, 24 of which were variable among populations. Only four of the variable sites occurred in the rRNA coding region while 20 variable sites were detected in the noncoding regions. Collectively, 25 enzymes generated about 66 restriction sites in each nrDNA; this amounts to about 4.3% of the nrDNA repeat. A total of 50 restriction sites was variable, 28 of which were phylogenetically informative. Phylogenetic analyses of site mutations indicated that two sections ofKrigia, sect.Cymbia and sect.Krigia, are monophyletic. In addition, relationships among several species were congruent with other sources of data, such as cpDNA restriction site variation and morphology. Both length and restriction site variation supported an allopolyploid origin of the hexaploidK. montana. The average sequence divergence value inKrigia nrDNA was 40 times greater than that of the chloroplast DNA. The rapid evolution of nrDNA sequences was primarily due to changes of the IGS sequences.     

On the relationship between the Bosmina taxa coregoni and thersites (Cladocera), as indicated by subfossil remains

A succession of morphs from Bosmina coregoni f. coregoni to B. c. f. thersites, which vary in shell and antennule characters, occurred in an undated 176 cm long sediment core from the Neversdorfer See (N. Germany).     

Restriction endonuclease mapping of ribosomal RNA genes: Sequence divergence and the origin of the tetraploid treefrog Hyla versicolor

Hyla chrysoscelis (2n=24) and H. versicolor (2n=48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the western diploid population and about 50% resembling the eastern population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5 terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.This research was supported, in part, by NSF Grants CDP-8002341 and PRM-8106947 and by faculty research grants from Miami University to J.C.V.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.