In situ expression of interleukin-4 (IL-4) receptors in human brain tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary glioblastoma cell cultures.

We have reported that human malignant glioma cell lines express high levels of plasma membrane interleukin-4 receptors (IL-4R). We have also reported that biopsy/surgical samples or primary explant cell cultures from brain tumors express mRNA and protein for the IL-4Ralpha chain, a primary IL-4-binding protein. However, whether IL-4R are expressed in brain tumors in situ has not been resolved. In addition, expression of IL-4R on the cell surface of various normal brain tissues is not known. We examined the expression of IL-4R by using a monoclonal antibody to the IL-4Ralpha chain (also known as IL-4R beta) in surgical/biopsy samples of brain tumor tissues by immunohistochemistry. Our data indicate that 15 of 18 glioblastoma multiforme (GBMs) tumors obtained from two different institutions and 12 other brain tumor samples are moderately to intensely positive for IL-4Ralpha. In contrast, although IL-4Ralpha mRNA was expressed, no IL-4R protein was detectable in two adult and one pediatric brain tissue specimens. In addition, a commercially available human neural tissue grid containing fixed tissues from various areas of brain showed no positive staining for the IL-4Ralpha chain. IL-4Ralpha expression was also demonstrated on astrocytoma grades I, II, and III. Because IL-4 cytotoxin comprised of a circularly permutated IL-4 and a mutated form of Pseudomonas exotoxin [IL4(38-37)-PE38KDEL] is cytotoxic to IL-4R-expressing cells, we tested whether primary GBM explant cell cultures are sensitive to IL-4 cytotoxin. Our data indicate that 13 of 15 GBM cell cultures were 25-74 times more sensitive to IL-4 cytotoxin compared with normal human astrocytes or the NT2 neuronal cell line. These observations indicate that human brain tumors in situ overexpress IL-4R compared with normal brain tissues, thus confirming our previous conclusions that IL-4R in brain tumors may serve as an attractive target for anticancer therapy.

Hamster mitochondrial transfer RNA lacks T and the universal GUUCG sequence.

Earlier inferences (based on incorporation of radio-activity from methyl-labeled methionine) that mammalian mitochondrial tRNA lacks ribothymidine ('T') have been confirmed using [14C]uridine as precursor. Moreover, oligonucleotide fingerprint analysis of 32P-labeled samples have shown that hamster mitochondrial tRNA lacks the 'universal' pentanucleotide of conventional tRNA in which T normally occurs, CTpsiCG, in modified or unmodified form.

Intratumoral administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma.

Human glioblastoma but not normal brain cells express numerous receptors for the cytokine interleukin (IL)-4. To target these receptors, we have investigated the safety and activity of directly infusing IL-4(38-37)-PE38KDEL, a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE), into recurrent malignant high-grade gliomas. IL-4(38-37)-PE38KDEL (IL-4-toxin) was infused over a 4-8-day period into gliomas of nine patients by one to three stereotactically placed catheters. No apparent systemic toxicity occurred in any patient. The infusion of IL-4-toxin in six of nine patients showed glioma necrosis as evidenced by diminished gadolinium enhancement on magnetic resonance imaging. Seven of nine patients underwent craniotomy because of increased intracranial pressure at 16-101 days after the beginning of infusion. In six of these seven patients, partial-to-extensive tumor necrosis with edema was confirmed pathologically. No histological evidence of neurotoxicity to normal brain was identified in any patient. Two patients were not operated on; by magnetic resonance imaging, one showed mottled gadolinium enhancement, and the other showed extensive necrosis of tumor leading to complete remission; this patient remains disease-free > 18 months after the procedure. We conclude that direct glioma injection of IL-4(38-37)-PE38KDEL is safe without systemic toxicity. Local toxicity seemed attributable mainly to tumor necrosis or occasionally to the volume of infusion. Histological evidence of toxicity to normal brain was not observed and in many patients, could be pathologically excluded. Additional patients are being treated to determine the maximal tolerated concentration and volume of IL-4(38-37)-PE38KDEL.

Detection of human herpesvirus 6 sequences in lymphoma tissues by immunohistochemistry and polymerase chain reactions.

We have analyzed one Hodgkin's and six non-Hodgkin's lymphomas for the presence of human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR) and antigens by immunohistochemistry. A large cell B-cell lymphoma and a T-cell lymphoma were positive for HHV-6 antigens by immunohistochemistry. Using PCR primers from the HHV-6 ZVH14 transforming DNA segment, all seven tumors were positive. EBV DNA was not detected by PCR using sequences from EBV internal repeat-3 region as primers. The data suggest an etiologic involvement of HHV-6 in some human lymphomas. Although all seven of the lymphoma tissues examined contained HHV-6 DNA, expression of HHV-6 antigens was observed in only two cases, suggesting that expression of proteins was not required for transformation.

Interleukin-4 receptor expression in vivo on human AIDS-related Kaposi's sarcoma.

We have investigated the expression of interleukin-4 receptors (IL-4R) in acquired immunodeficiency syndrome (AIDS)-related Kaposi's Sarcoma (KS) in situ by immunohistochemistry. Frozen and fixed sections from five patch stage and two nodular stage KS lesions were stained with anti-IL-4R monoclonal antibody with similar results. Skin biopsies from the clinically apparent lesions and adjacent clinically uninvolved skin were also examined. We observed that individual KS cells lining the irregular vascular spaces were stained with anti-IL-4R antibody, although the degree of staining was variable. The epithelioid and oval cells appear to stain more than the spindle cells in plaque stages or nodular lesions. The sections from nonclinically involved skin also contained a few cells with features of KS, singly or in clusters that also stained for IL-4R. Skin sections from four normal donors did not stain with IL-4R antibody except for hair follicles, sweat glands, and faint staining of blood vessels. KS sections were also stained with antibodies to basic fibroblast growth factor (FGF), S100, fibronectin, and von Willebrand factor. KS lesions from clinically involved and uninvolved skin sections were positive for all four antibodies. Thus, the differences between KS lesion and clinically uninvolved skin adjacent to a KS lesion may be more quantitative than qualitative. The IL-4 receptors on KS cells were functional as IL-4 modulated intercellular adhesion molecule 1 (ICAM-1) on these cells. Taken together, our results suggest that AIDS-KS cells express elevated levels of IL-4R compared to normal endothelial and skin cells and, thus, the receptors for IL-4 on KS may serve as an attractive target for anticancer therapy.

Head and neck cancers, but not benign lesions, express interleukin-4 receptors in situ.

We have previously reported on the expression of interleukin-4 receptor (IL-4R) on many solid cancer cell lines. In the present study, we have examined the expression of IL-4R on head and neck cancers in situ by immunohistochemistry. Seven primary squamous cell carcinomas of the head and neck region were stained by a monoclonal antibody to human IL-4Rp140 protein (M-57). We report that all squamous cell carcinoma samples were positively stained although at variable intensity with anti-IL-4R antibody. Tumors stained with IgG control did not show any staining. On the other hand, six benign lesions from the same anatomical area showed faint or no staining at all. Three uterine endometrium samples were also negative for the IL-4R expression. In contrast to published contrary results, we did not observe any effect of IL-4 on the proliferation of four squamous cell carcinoma of head and neck (SCCHN) cell lines examined. These results demonstrate that human squamous carcinoma of the head and neck express IL-4R, which could be targeted for diagnosis and therapy by anti-IL-4 receptor antibody fused to toxins or radionuclides or alternatively by IL-4 toxins.

An albumin-like protein is the major secretory protein of ovarian epithelial cells in vivo and in vitro.

Contemporary experimental techniques were used to evaluate the protein secretion of ovarian epithelium. The protein composition of 14 ovarian cyst fluids (OCF) from either cystadenomas or cystadenocarcinomas, and conditioned media (CM) from seven ovarian carcinoma lines in culture, were analyzed by SDS-PAGE under reducing conditions, and Western immunoblots. The major protein common to all the above samples was a 65 kDa protein that, by densitometry, constituted between 43% and 77% of OCF protein and 19% and 38% of CM protein. By Western blot analysis, this band was immunologically related to human albumin. Moreover, immunoreactivity to albumin was demonstrated in ovarian epithelium in vivo. Ovarian epithelium synthesizes and releases an albumin-like protein that constitutes the major secretory protein. This may suggest an osmotic mechanism for cyst enlargement in ovarian cystadenomas.

"Compartmentalization" of Escherichia coli ribosomes and ribonucleic acid.

Escherichia coli spheroplasts lysed by Brij 58 and deoxycholate were separated into supernatant (S) and membrane fractions by low-speed centrifugation. The membrane fraction was further divided into that which was releasable by deoxyribonuclease (fraction D) and that which was not (M). In the presence of 10(-2)m Mg(2+), the S, D, and M fractions contained, respectively, 60, 20, and 20% of the total cellular ribonucleic acid (RNA). Ribosomal and transfer RNA (rRNA, tRNA) were found in each fraction. The M + D fraction RNA was labeled more by a pulse label. Incorporation of uracil into the D fraction continued only as long as the uptake of exogenous uracil, suggesting that this was a major primary site of RNA synthesis. From pulse-labeled cells, each fraction contained precursor rRNA, and there was a 10S RNA in the M fraction. Ninety per cent of the ribosomal subunits and the ribosomal precursor particles, 26 and 43S, were in the S fraction. Precursor RNA (17S) was found in the 26S precursor particles. The D fraction contained 38% of the polysomes (this does not consider polysomes, if any, of the M fraction) which were labeled four times as much as the supernatant polysomes by a 1-min pulse of uracil. These results are interpreted to mean that new RNA is associated with a cytoplasmic membrane-RNA polymerase-DNA complex.

Comparison of supernatant-and ribosome-bound rat liver tRNA.

Transfer RNAs were separated into a ribosome bound fraction and a supernatant (cytoplasmic) fraction. The nucleoside composition, 2-dimensional PAGE pattern and in vivo labeling were compared. 12 minor nucleosides were identified by HPLC. In general, the minor nucleosides, especially N2-methyl-guanidine and ribothymidine, were higher in the ribosome-bound fraction. The PAGE patterns were similar but there were quantitative and qualitative differences among the smaller spots. In vivo labeling by 32P showed that new tRNA goes preferentially to the ribosome but mixing does occur. The results suggest the existence of two compartments of tRNA.

Ribosome patterns in Escherichia coli growing at various rates.

The distribution of ribosomes, 30 and 50S subunits and polysomes, at three different growth rates of Escherichia coli strains B and K-12 has been studied. The usual percentage of subunits is about 20%. However, at the lowest growth rate (mu = generations/hour), mu = 0.45 at 30C, the proportion of subunits is about 30%. An exceptional situation exists in K-12 strains growing at maximum growth rate, mu = 1.35, where the percentage of subunits is 45%. Several points of control over ribosome production are thus indicated. It is suggested that "subunit pool" is essentially a reserve. Furthermore, the polysome content when related to deoxyribonucleic acid content varies directly with the growth rate, which indicates the average efficiency of polysomes in protein synthesis does not vary over the range of growth rates tested.

Thermal denaturation of mesophilic and thermophilic 5S ribonucleic acids.

The Tm of Bacillus stearothermophilus 5S ribonucleic acid (RNA) is 1.5 +/- 0.5 C higher than that of 5S RNAs from B. subtilis and Escherichia coli. Melting in 50% methanol and in formaldehyde indicate that both base stacking and helical regions are involved in the slightly increased thermal stability of B. stearothermophilus 5S RNA. It is probable that the 5S RNA makes only a minor contribution to the thermostability of B. stearothermophilus 50S ribosomal subunits.

Transfer RNA of a collagen producing tissue, chick-embryo calvaria.

Transfer RNA was isolated from calvaria prepared from chick embryos incubated for 15-17 days. The chargeability of the unfractionated tRNA with ten amino acids tested was very similar to that of unfractionated tRNA from adult chicken liver when data were expressed on the basis of pmoles of amino acceptance per A260 unit of tRNA. However, the relative amount of tRNA in calvaria is only about one-fourth the amount in liver. Analysis of individual species of tRNA by two-dimensional electrophoresis on polyacrylamide gels shows that there are fewer isoaccepting species of tRNA in calvaria than in liver.