Hospital outbreak of vancomycin-resistant enterococci caused by a single clone of Enterococcus raffinosus and several clones of Enterococcus faecium.

A mixed outbreak caused by vancomycin-resistant Enterococcus raffinosus and Enterococcus faecium carrying the vanA gene was analysed. The outbreak occurred in a large hospital in Poland and affected 27 patients, most of whom were colonised, in three wards, including the haematology unit. The E. raffinosus isolates had a high-level multiresistant phenotype and were initially misidentified as Enterococcus avium; their unambiguous identification was provided by multilocus sequence analysis. The molecular investigation demonstrated the clonal character of the E. raffinosus outbreak and the polyclonal structure of the E. faecium isolates. All of the isolates carried the same Tn1546-like element containing an IS1251-like insertion sequence, located on a c. 50-kb conjugative plasmid. One of the E. faecium clones, found previously to be endemic in the hospital, was probably the source of the plasmid. The results of the study suggest that difficulties in identification may have led to an underestimate of the importance of E. raffinosus in vancomycin-resistant enterococci (VRE) control strategies.

Induction of immune resistance against L1210 lymphatic leukemia in mice after chemoradiotherapy of the leukemia and reconstitution with bone marrow purged from the leukemia with mafosfamide.

Lymphatic leukemia L1210-bearing semisyngeneic Balb/c x DBA/2Wf F1 (CD2F1) mice were subjected to chemoradiotherapy (2 x 100 mg/kg of cyclophosphamide i.p. and 1000 cGy of total body irradiation) and reconstitution with 10(7) syngeneic bone marrow cells i.v. The bone marrow obtained from leukemic mice was previously ex vivo purged of the leukemia cells with mafosfamide (ASTA Z7654) and stored in liquid nitrogen. Eight weeks after cytoreductive therapy and bone marrow transplantation we tried to immunize the mice against the lethal dose of the leukemia by i.p. injections of L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth). About 75% of such mice were able to reject the subsequent 10(3) L1210 leukemia cell challenge, as compared with 70% of normal immunized mice and 55% of mice reconstituted with bone marrow cells not treated with mafosfamide.

Early induction of immune resistance against leukemia in mice after lethal irradiation followed by syngeneic bone marrow transplantation and injection of syngeneic leukocytes.

BALB/cxDBA/2Wf F1 (CD2F1) mice were lethally irradiated and reconstituted with syngeneic bone marrow cells untreated or treated with 75 micrograms/ml of mafosfamide. One day after bone marrow transplantation some groups of mice were injected with syngeneic splenocytes, peripheral blood leukocytes, or thymocytes. Seven days after marrow grafting the anti-L1210 leukemia immunization of mice, consisting of four i.p. injections of 10(6) L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth in vivo), was started. Strong resistance against leukemia could be obtained only in mice receiving splenocytes or peripheral blood leukocytes, not in mice injected with thymocytes or in those not receiving any cells. In vitro elimination of various subpopulations from among the splenocytes before their injection into the mice made it possible to deduce which are necessary for early induction of antitumor resistance after bone marrow transplantation in mice. These cells are: Thy 1.2-, Ig-, AsGM 1-, Mac 1+, 1-Ad+/-, are adherent and nonsusceptible to carrageenan toxicity.

Recovery of the ability to induce immune resistance against L1210 lymphatic leukemia in semisyngeneic CD2F1 mice after lethal irradiation and reconstitution with bone marrow purged of leukemia with mafosfamide (ASTA Z 7654).

Balb/c x DBA/2 F1 (CD2F1) mice were lethally irradiated (TBI) and reconstituted with syngeneic bone marrow cells (SBMT) untreated or treated with mafosfamide (ASTA Z 7654) for ex vivo purging of semisyngeneic L1210 leukemia (TBI + SBMT or TBI + SBMT-Maf mice, respectively). At various times after irradiation and reconstitution mice were injected intraperitoneally four times at weekly intervals with 10(6) immunogenic L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth in vivo). As positive controls we immunized normal (non-irradiated) CD2F1 mice. Full resistance against L1210 leukemia (as compared to normal immunized mice) could be obtained in TBI + SBMT and TBI + SBMT-Maf mice when the immunization procedure was started from day +28 or day +56 after transplantation, respectively. Earlier immunization of TBI + SBMT mice (from day +14) or TBI + SBMT-Maf mice (from day +14 or +28) caused only partial resistance against the leukemia.

Early induction of immune resistance against leukemia in lethally total body irradiated mice reconstituted with syngeneic bone marrow cells obtained from previously immunized donor mice.

BALB/c x DBA/2 F1 (CD2F1) mice were lethally irradiated and reconstituted with syngeneic bone marrow cells (SBMC) obtained from normal or previously immunized (against L1210 lymphatic leukemia) donors. These recipient mice are called TBI + SBMT or TBI + Imm-SBMT mice, respectively. TBI + Imm-SBMT, but not TBI + SBMT mice, were able to develop strong immune resistance against L1210 leukemia, but not against MOPC 104E plasmacytoma, if the immunization procedure (four i.p. injections at weekly intervals of immunogenic L1210 cells) was started as early as 7 days posttransplantation. Incubation of Imm-SBMC with mafosfamide (ASTA Z7654) before grafting abrogated the ability of the recipient mice to develop early resistance against the leukemia. Treatment of Imm-SBMC with monoclonal or polyclonal antibodies plus complement showed that two or three subpopulations of Imm-SBMC were necessary for the transfer of immune information against leukemia: T lymphocytes with phenotype Thy 1.2+, Lyt 1+2-, I-Ad-, macrophages with phenotype Mac-1+, I-Ad-, and probably asialo-GM 1+ cells. Recipient mice immunized against L1210 leukemia before TBI + SBMT do not develop early resistance to the leukemia.

Return of immunohematopoietic impairment a long time after murine syngeneic bone marrow transplantation.

We have tested the immunologic status and hematologic parameters of mice 2 months (short-term survivors) or 18 months (long-term survivors) after lethal total body irradiation and syngeneic bone marrow transplantation (SBMT), and of normal mice of corresponding age. Long-term SBMT survivors showed significantly lowered bone marrow and spleen cellularities, decreased numbers of CFU-S in hemopoietic organs and severe impairment in the formation of CFU-F colonies compared with short-term SBMT survivors and normal mice. The peripheral blood parameters (hematocrit, erythrocytes, reticulocytes, platelets, white blood cells and granulocyte counts), however, remained unaltered. In long-term SBMT survivors we also observed a relative increase of Lyt-2+ lymphocytes (CD8+, cytotoxic/suppressor) and Mac-1+ cells among splenocytes. At the same time the L3T4+/Lyt-2+ ratio (CD4+/CD8+) was decreased. Relative contents of Ig+, Thy-1+ and L3T4+ cells were unchanged. The ability of splenocytes to generate IL-2R+ cells after in vitro stimulation with concanavalin A was greatly diminished. In summary, the immunohematopoietic status after initial normalization is again impaired in long-term SBMT survivors.

The kinetics of immunologic and hematologic recovery in mice after lethal total body irradiation and reconstitution with syngeneic bone marrow cells treated or untreated with mafosfamide (ASTA Z 7654).

The immunohematopoietic reconstitution of mice lethally irradiated (TBI) and reconstituted with syngeneic bone marrow cells untreated or treated with mafosfamide (ASTA Z 7654) [TBI + SBMT or TBI + SBMT-Maf mice, respectively] was examined. The number of CFU-S was greatly reduced in TBI + SBMT-Maf mice compared with those in TBI + SBMT mice. The recovery of blood parameters (hematocrit, reticulocytes, erythrocytes, white blood cells, granulocytes, platelets) and of bone marrow and spleen cells, but not of peritoneal exudate cells, was slightly delayed in TBI + SBMT-Maf mice compared with those in TBI + SBMT mice. The time for immune system regeneration was, however, considerably longer in TBI + SBMT-Maf than in TBI + SBMT mice, as measured by the incidence of Ig+, Thy-1.2+, L3T4+, Lyt-2+, and IL-2R+ cells in the spleens. The appearance of Mac-1+ and asialo-GM 1+ cells was only slightly prolonged or unchanged, respectively.

Susceptibility patterns of Enterococcus spp. isolated in Poland during 1996.

Susceptibility of Enterococcus spp. isolated from various clinical specimens to different antimicrobial agents was evaluated. Of the 346 enterococcal isolates obtained from four regional Polish hospitals during 6 months of 1996, 261 (75.4%) were identified as Enterococcus faecalis, 75 (21.7%) as Enterococcus faecium and ten (2.9%) as other enterococcal species. High-level resistance to gentamicin was expressed by 33.4% of E. faecalis and 86.5% of E. faecium strains and corresponding streptomycin resistance by 43.9 and 82.4%, respectively. Over 80% of E. faecium isolates were resistant to ampicillin. None of the isolates was resistant to teicoplanin, however 7.9% of E. fecalis and 1.4% of E. faecium strains were moderately susceptible to vancomycin.

Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides.

To detect HL-60 human promyelocytic leukemia cell proteins involved in the uptake of gangliosides from the culture medium we used photoreactive, 4-azidosalicylic acid (ASA) acylated and radioiodinated (200 Ci/mmole) derivatives of GM3, GD3, GM1, and FucGM1 gangliosides. Gangliosides-ASA, added to the medium at 15-20 nM concentration, followed a similar time course of uptake. After 1 min incubation cell bound gangliosides-ASA could not be removed with trypsin, but only 5-10% remained after incubation with BSA. The proportion of cell bound gangliosides-ASA resistant to BSA treatment increased with time of incubation up to 76% after 20 h. As shown on TLC, GM3- and GD3-ASA were catabolized to LacSph-ASA and ceramide-ASA, while GM1-ASA was hydrolyzed to GM2-ASA. FucGM1-ASA was converted to GM1-ASA very slowly. Upon irradiation with UV lamp, cell bound gangliosides-ASA crosslinked to and photolabeled many proteins but the distribution of radioactivity after SDS/PAGE was very uneven and did not correlate with Coomassie staining. In all experiments the 42 kDa protein bands were most intensely photolabeled. Photolabeling of 42 kDa proteins decreased with time of incubation as compared to lower molecular mass pro teins. With all gangliosides-ASA used similar but not identical protein photolabeling patterns were obtained. Photolabeling patterns with GM3- and GD3-ASA differed from those with GM1- and FucGM1-ASA.

A novel restriction endonuclease UnbI, a neoschizomer of Sau96I from an unidentified psychrofilic bacterium from Antarctica is inhibited by phosphate ions.

A novel type II restriction endonuclease UnbI was isolated from an unidentified psychrofilic bacterial strain from Antarctica. UnbI recognizes and cleaves the sequence 5'-GGNCC-3', producing 5 nucleotide long sticky ends. In this respect it differs from its neoschizomer Sau96I and all other restriction enzymes recognizing this sequence. UnbI has a relatively low temperature optimum of 15 degrees C to 20 degrees C and its activity is completely inhibited by inorganic phosphate.

Preparation and some properties of highly purified human serum kallikrein.

A 3000--6000-fold purified kallikrein was obtained from human serum in 10--25% yield by chromatography on QAE-Sephadex A-50, Molselect CM-50 and on soybean trypsin inhibitor (SBTI)-AH-Sepharose 4-B. The enzyme had a specific activity of 14--23 U, as measured by BAEE hydrolysis. Some properties of highly purified kallikrein are described.

Preparation and some properties of human serum kallikrein immobilized on ARM-Sepharose.

A highly purified human serum kallikrein immobilized on CH-Sepharose 4-B was obtained. KM values for N-benzoyl-L-arginine ethyl ester and N-tosyl-L-arginine methyl ester hydrolysis of this preparation were 1.10 x 10(-3) M and 3.6 x 10(-4) M, respectively; pH optimum of hydrolysis of these esters were found to be 8.2 and 8.5, respectively. The immobilized kallikrein possessed kininogenase activity and was capable of activating prekallikrein.

Immunogenicity of cyclophosphamide-treated tumor cells.

The immunogenicity of cyclophosphamide-treated murine tumor cells was tested on syngeneic or semisyngeneic mice. The majority of mice immunized with modified Ehrlich carcinoma cells, L1210 leukemia cells or AA sarcoma cells survived after challenge with a lethal dose of unmodified tumor cells. Plasmacytomas RPC-5 and MOPC 104E were less immunogenic and L-1 sarcoma cells were not immunogenic.

A simple method of bone marrow cryopreservation.

Mouse bone marrow cells (BMC) were frozen by the simple two step method and stored in liquid nitrogen. The reconstitutive potency of fresh and frozen BMC were compared. BMC-fresh and BMC-frozen had a similar potential of colony formation in spleen and reconstitution of lethally total body irradiated (TBI) mice.

Leukemia L 1210 cells induce depletion of Lyt 2+ thymocytes.

The thymocytes were analyzed on the 7th day after i.p. inoculation of 10(6) leukemia L 1210 cells to syngeneic DBA/2 Wf mice. A three-fold decrease of the total number of thymocytes was found as well as 1.7-fold decrease of the per cent of thymocytes with Lyt 2+ phenotype, while the per cent of cells with phenotypes Thy 1.2+ and Lyt 1+ was unchanged.

Cellular composition of spleen and peritoneal exudate in mice after injection of cyclophosphamide-treated L 1210 leukemia cells.

The composition of peritoneal exudate and spleen cells of CD2F1 mice after fourfold i.p. administration of L 1210 leukemia cells treated with cyclophosphamide (L 1210-CY cells) were examined. The number of cells in peritoneal cavity did not increase, however, the spleen weight rose after administration of L 1210-CY cells. The per cent of lymphocytes T was increased 2.5 times but the content of macrophages and lymphocytes B was normal in the peritoneal cavity after L 1210-CY cells injections. In the spleen an 1.4 times increase of the per cent of lymphocytes B, but normal level of macrophages and lymphocytes T were observed.

Immunogenic and nondividing mafosfamide-treated L 1210 cells--comparison of lines resistant and nonresistant to cyclophosphamide.

Cyclophosphamide/mafosfamide-resistant L 1210 cell line [L 1210(Cy)R] was established from a sensitive parental line. The L 1210(Cy)R line was resistant to cyclophosphamide at the dose of 100 mg/kg. Cells of L 1210(Cy)R line were more immunogenic for semisyngeneic CD2F1, mice as compared with parental line. They grew slower in immunocompetent mice compared to immunosuppressed mice. It has been shown that L 1210(Cy)R cells treated with mafosfamide at high concentration not only retained immunogenicity but were even more immunogenic than parental L 1210 cells. In conclusion, it was possible to produce immunogenic, nondividing leukemia cells even when cells were resistant to the cytostatic used for cell modification.

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.

The influence of phosphorothioate oligodeoxynucleotides on various organs in vivo.

To characterize the distribution and toxicity of phosphorothioate antisense oligodeoxynucleotides ([S]ODNs) in vivo, the mice, previously injected with BV173 leukemic cells (Philadelphia chromosome-positive chronic myeloid leukemia blast-crisis), received intravenously 26-mer BCR-ABL antisense oligodeoxynucleotides (1 mg/mouse/day) for 9 consecutive days. Our investigation revealed that [S]ODNs were distributed to almost all organs except the brain with the highest level in the liver, spleen and kidneys. They were also detected in CD10+ leukemic cells isolated from spleen and bone marrow. Intracellular distribution assay showed the presence of [S]ODNs most prominently in nuclear and cytoplasmic fractions. Our data demonstrated no significant toxicity of [S]ODNs except the increase in spleen weight.

Cyclophosphamide- or ifosfamide-treated L 1210 leukaemia cells: immunogenicity, viability and metabolism.

We have demonstrated that lymphoid leukaemia L 1210 cells treated with cyclophosphamide or ifosfamide were immunogenic for semisyngeneic CD2F1 mice and that they effectively prevented the development of the later inoculated leukaemia L 1210 cells. Non-dividing and immunogenic L 1210 cells were only obtained when an appropriate dose of cyclophosphamide was applied. These cells lost their immunogenicity after killing by repeated freezing and thawing or after fixing with glutaraldehyde. Non-dividing immunogenic L 1210 cells treated with cyclophosphamide possessed the histocompatibility and tumour-associated transplantation antigens and had the ability to synthesize proteins, the RNA and partially the DNA.