Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength.

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1-3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20,000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 x 10(4) cells injected i.p.) consisted of i.p. injections of 5000 or 20,000 U IL-2/mouse given on days 10-14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity.

Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov.

A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S. thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of priority, S. thermonitrificans is a later subjective synonym of S. thermovulgaris. Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S. thermovulgaris. The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S. thermodiastaticus.

Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline derivatives: DNA topoisomerase II inhibitors.

A series of new 5H-indolo[2,3-b]quinoline derivatives bearing methoxy and methyl groups at C-2 and C-9 was synthesized (according to the modified Graebe-Ullmann reaction). These compounds were evaluated for their antimicrobial and cytotoxic activity and tested as inhibitors of DNA topoisomerase II. Lipophilic and calf thymus DNA binding properties of these compounds were also established. In the SAR studies we used quantum-mechanical methodology to analyze the molecular properties of the drugs. All of the 5H-indolo[2,3-b]quinolines tested were found to inhibit the growth of gram-positive bacteria and pathogenic fungi at MIC ranging between 2.0 and 6.0 microM. They showed also cytotoxic activity in vitro against several human cancer cell lines of different origin (ID50 varied from 0.6 to 1.4 microM), and stimulated the formation of topoisomerase-II-mediated pSP65 DNA cleavage at concentration between 0.2 and 0.5 microM. The most active indolo[2,3-b]quinolines which had the greatest contribution to the increase in the Tm of DNA displayed also the highest DNA binding constants and the highest cytotoxic activity. The differences in DNA binding properties and cytotoxic activity seem to be more related to steric than electrostatic effects.

Therapeutic potential of biological response modifiers against transplantable mouse tumors of spontaneous origin. II. Local interleukin 2 treatment of tumors of different immunogenic strength.

This report describes tumor susceptibility to interleukin 2 (IL-2) therapy in relation to tumor immunogenicity. The following lines recently established from spontaneous tumors were used: X5, X6, and X9 mammary tumors of DBA/2, BALB/c and CBA origin, respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumors of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and M109 Madison lung carcinoma (M109) of BALB/c origin, were used as control systems. Mice were transplanted with different inocula of tumor cells at day 0; IL-2 treatment was initiated on day 1-3 or 10 and consisted of daily injections of 5000 or 20,000 IU/mouse given 5 times a week for 3 weeks. SL2 (i.p. - 2 x 10(4) cells) treatment consisted of i.p. injections of 5000 or 20,000 IU IL-2 given on days 10-14. IL-2 therapy of SL2 bearing DBA/2JIco mice resulted in the significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2 bearing DBA/2Crliw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the 5 recently established tumors resulted, in general, in transient growth inhibition of the i.m. transplants of line X5, X6, and X7 provided IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumor cells, and the best results being achieved at cell numbers close to the dose inducing tumor growth in 50% of animals (TD50). We found that the tumors responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and as such might express foreign antigens.

[Diagnostic molecular microbiology--identification of Staphylococcus epidermidis]. / Molekularna diagnostyka mikroorganizmów--identyfikacja Staphylococcus epidermidis.

The species Staphylococcus epidermidis is the predominant coagulase-negative staphylococci (CNS) isolated from clinical sources. S. epidermidis is now recognized as an important nosocomial pathogen. Identification of CNS is often performed using diagnostic kits based on biochemical or immunological reactions. However, these kits are often unreliable for the identification of CNS species including S. epidermidis. Currently, ribosomal RNA (rRNA) analyses are the most powerful methods for determining phylogenetic relationships among microorganisms and also for identification of species. Several aspects of construction of ribosomal probes for identification of CNS species are presented and discussed. Additionally, the application of restriction fragment length polymorphisms (RFLP) of rRNA genes for differentiation of clinical isolates of S. epidermidis is shown.

Organization and nucleotide sequence analysis of the ribosomal gene set (rrnB) from Streptomyces lividans.

Streptomyces lividans (Sl) contains six ribosomal RNA (rRNA) gene sets, rrnA-F (Suzuki, Y., Ono, Y., Nagata, A. and Yamada, T. (1988) Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J. Bacteriol. 170, 1631-1636). We have cloned the rrnB gene cluster. Physical mapping revealed that rrnB gene set is located on a 290 kb Asel fragment in the 11 to 12 o'clock region of the S. coelicolor A3(2) chromosome. The complete nucleotide (nt) sequence of Sl 23S rRNA has been determined. The structural gene of the Sl 23S rRNA codes for the 3108 nt RNA chain. The G+C content of the 23S rRNA is 57.3 mol%. The length of the spacer region between the 23S and 5S genes is 99 bp. Analysis of the sequences between the 16S and 23S genes and downstream of the 5S rRNA gene failed to identify any tRNA-like sequences. A secondary structure model of Sl 23 rRNA is proposed, based on the earlier published model of Gutell and Fox (Nucleic Acids Res. 16 (1988) 175-269).

Rapid detection of Staphylococcus saprophyticus using primer specific PCR.

Staphylococcus saprophyticus is one of the most frequently encountered clinically significant members of the coagulase-negative staphylococci. A set of species-specific PCR primers was defined for the detection of Staphylococcus saprophyticus. These primers target variable regions (V3 and V6) of the 16S rRNA gene. Primer-specific PCR has potential applications in epidemiological studies and diagnosis of Staphylococcus saprophyticus.

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex.

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.

Staphylococcus pulvereri sp. nov., isolated from human and animal specimens.

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus pulvereri, was isolated from human and animal specimens. The complete 16S rRNA sequence of the type strain of S. pulvereri, NT215, was determined and compared with the sequences of 16S rRNAs from the other staphylococci. Strains of S. pulvereri were differentiated from other novobiocin-resistant Staphylococcus species by their biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is NT215 (= PCM 2443T).

Distribution of oxoacyl synthase homology sequences within Streptomyces DNA.

A genomic DNA sequence of Streptomyces strain ISP 5485 was cloned, sequenced and compared with corresponding information from nuclei acid data banks. The DNA sequence was unique, but showed homology to DNA coding for the condensing enzyme, 2-oxoacyl synthase, of the deoxyerythronolide B synthase complex (DEBS) from Saccharopolyspora erythraea NRRL 2338. A subfragment of the sequenced DNA was used to construct a gene-specific probe that formed part of the putative 2-oxoacyl synthase gene. The PCR-amplified and labelled probe was used in hybridization experiments involving 33 streptomycete strains that produced different classes of antibiotics. The probe showed widespread homology with DNA considered to be part of analogous genes within genomes of different polyketide producers. The implications of the probe homology to bacterial chromosomal DNA are discussed.

Therapeutic potential of biological response modifiers against transplantable mouse tumors of spontaneous origin. I. Characterization of biological properties of tumor lines and preliminary data on response to cytostatic drugs and biomodulators.

Several transplantable lines were derived from spontaneous tumors of mouse strains of low and high cancer incidence (CBA, BALB/c and DBA/2), and eight of them were used for an evaluation of the therapeutic potential of BRM. The lowest number of cells inducing tumors was in most cases around 10(4) cells. In transplantation tests, tumors of CBA and DBA/2 mice were non-immunogenic, while tumors of BALB/c mice showed different level of immunogenicity. Transplantation of a small number of tumor cells in admixture with BCG resulted in no tumor growth of the immunogenic line X6 and no effect on take rate of non-immunogenic line X1. Sensitivity of studied tumor lines to BCNU, CY and ADR was moderate. Therapy of the tumors with postulan and CMA, two biomodulators already shown to be active against various experimental tumors, failed. The significant therapeutic response was observed in DBA/2 mice bearing i.p. transplant of lymphosarcoma X19 and treated with i.p. injections of CMA.

Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe.

Staphylococcus epidermidis is the most medically significant of the coagulase-negative staphylococci. An oligonucleotide probe (pSe) for identification of S. epidermidis was defined by comparing the sequences of the 16S rRNA variable region V6 from numerous coagulase-negative staphylococci. In order to increase the sensitivity of the detection, polymerase chain reaction amplification of the variable region with primers based on the conserved flanking sequences was applied. The detection limit of the polymerase chain reaction assay combined with pSe probe was shown to be 1 fg which corresponds to about one single bacterium. Additionally, a sensitive, non-radioisotopic system with chemiluminescence detection was tested.

A carboline derivative as a novel mammalian DNA topoisomerase II targeting agent.

The DNA intercalating, ellipticine analog drug, 5,11-dimethyl-5H-indol[2,3-b]quinoline, is able to stabilize in vitro the topoisomerase II-DNA cleavable complex and to induce DNA breaks in BPV I episome in rat fibroblasts. Cytotoxicity studies with DC3F cells resistant to ellipticine strongly suggest that topoisomerase II is a cellular target involved in the mechanism of cytotoxic action of this carboline derivative.