Expression of Salmonella typhimurium virulence plasmid genes in cultured macrophages.
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Animals↗
Biomedical subjects
P Riikonen
Publications and source records attributed to P Riikonen.
A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.
A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.
Amino Acid Sequence↗
Recombinant human granulocyte-macrophage colony-stimulating factor in children with chemotherapy-induced neutropenia.
Twenty children 1-17 (median, 5.5) years of age received GM-CSF during chemotherapy-induced neutropenia at the dose of 5 micrograms/kg/day, continued until the absolute neutrophil count (ANC) exceeded 500 x 10(6)/liter. Twelve children with solid tumors received GM-CSF after courses of conventional chemotherapy (VP-16 + ifosfamide or "6 in 1"). One course followed by GM-CSF was compared to identical courses without GM-CSF in the same patients. Eight children with recurrent/poor risk malignancies received GM-CSF after marrow-ablative therapy and autologous bone marrow transplantation (ABMT). Their engraftment data were compared to matched historical controls. In both groups GM-CSF accelerated myeloid recovery, which was preceded by the appearance of immature myeloid elements in bone marrow. The ANC levels of 200, 500, and 1,000 x 10(6)/liter were exceeded 2, 3 (P < 0.05), and 6 (P < 0.005) days earlier with GM-CSF in the conventional chemotherapy group, and 6, 10 (P < 0.05), and 9 days earlier in the ABMT group, as compared to the controls. All adverse effects observed were mild, including skin rashes, nasal stuffiness, general achiness, nausea, and fever. We conclude that GM-CSF is well tolerated in children and accelerates myeloid recovery in chemotherapy-induced neutropenia.
Adolescent↗
The virulence plasmid does not contribute to growth of Salmonella in cultured murine macrophages.
The virulence plasmid, characteristic of many serovars of Salmonella sp., and specifically its spv genes, promote intracellular growth of the bacteria in the liver and spleen and are essential for the virulence of these Salmonella serovars in the mouse. In an attempt to establish an in vitro model for studying its function, we evaluated its effect on the intracellular growth of the bacteria in macrophages in culture. We used a number of different macrophage-like cell lines (J774-A.1, IC-21 and PU5-1.8), as well as peritoneal or splenic macrophages from genetically Salmonella-sensitive (Itys, BALB/c) or resistant (Ityr, C3H/HeN) mice, and at different states of activation, stimulated in vivo or in vitro with lipopolysaccharide and/or recombinant gamma interferon. These were found to differ in their ability to suppress or sustain intracellular growth of several Salmonella serovars, but in all cases the growth was independent of the spv genes.
Animals↗
Plasmid-mediated serum resistance in Salmonella enterica.
The ability of Salmonella enterica to survive an incubation of 2 h in fresh, pooled guinea-pig serum was determined comparing strain sets of serovariants Dublin and Typhimurium, harbouring or lacking the virulence plasmid. All strains showed marked serum resistance, which was slightly decreased among cured strains of Typhimurium. However, when introduced into a rough Escherichia coli strain, all Typhimurium plasmids substantially increased the resistance of the host strain to guinea-pig serum, whereas the Dublin plasmid did not. The traT gene, previously shown to affect serum resistance, was identified on all Typhimurium plasmids, but not on the Dublin plasmid. Mutational inactivation of the traT gene on the Typhimurium plasmids eliminated the serum resistance mediated by the plasmids.
Animals↗
Cytokine and acute-phase reactant levels in serum of children with cancer admitted for fever and neutropenia.
Serum concentrations of tumor necrosis factor-alpha (TNF alpha), interleukin (IL)-1 beta, IL-6, and the acute-phase reactants C-reactive protein (CRP) and serum amyloid A (SAA) were measured on admission in 46 neutropenic children with cancer in 81 episodes of fever. The aim was to find out whether any of these variables would differentiate true bacteremia from fever due to other causes. In most episodes serum concentrations of TNF alpha and IL-1 beta were elevated. IL-6 was detectable in 68%, but the serum concentration was elevated in only 15%. SAA proved to be more sensitive than CRP for the early detection of bacteremia. However, not even SAA was sufficiently accurate at the individual level. We conclude that the cytokine and acute-phase protein levels found were related to the febrile response but did not correlate with documented bacterial etiology.
Acute-Phase Proteins↗
Amino-terminal sequence analysis of four plasmid-encoded virulence-associated proteins of Salmonella typhimurium.
We have expressed four virulence-associated proteins encoded by the Salmonella typhimurium plasmid pEX102 in Escherichia coli. The genes coding for the proteins MkaA, MkaB, MkaC and MkaD subcloned in the vectors pUC19 or Bluescript KS+ directed substantial production of these proteins in E. coli. The same host harbouring pEX102 did not produce these proteins in detectable amounts. The proteins were exclusively found in the particulate fraction. The amino-terminal sequence analysis showed that the N-termini of these proteins corresponded to the ones predicted from the open reading frames found previously in the DNA sequence of the virulence determinant and that no N-terminal signal sequence processing of the proteins occurred.
Amino Acid Sequence↗
The mkaC virulence gene of the Salmonella serovar typhimurium 96 kb plasmid encodes a transcriptional activator.
The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four MKa proteins indicated that mkaC enhances the expression of beta-galactosidase from the mkaA-, mkaB- and mkaC-lacZ gene fusions in trans. An mkaD-lacZ fusion or mkaA-lacZ fusion that did not contain DNA proximal to mkaB was not inducible with MkaC, indicating that at least mkaB and mkaA are induced together as an operon. MkaC is thus the first virulence protein whose function has been resolved.
Escherichia coli↗
Imipenem compared with ceftazidime plus vancomycin as initial therapy for fever in neutropenic children with cancer.
Two antibiotic regimens were assessed, imipenem as monotherapy and ceftazidime plus vancomycin as combination therapy, for initial empiric therapy in febrile neutropenic children with cancer. In a prospective randomized trial of 89 evaluable consecutive episodes, 45 were treated with imipenem and 44 with ceftazidime-vancomycin. In 87% of the episodes the neutropenia was severe. Of the 89 episodes 20% were bacteremias, 10% were clinically defined focal infections and 70% were considered fevers of unknown origin. The initial treatment was successful in 82% of the imipenem group and 59% of the ceftazidime plus vancomycin group. Both regimens were well-tolerated. There was no mortality, probably owing to the prompt admission and institution of antimicrobial therapy. All of the patients were treated until neutrophil recovery; no recurrent infections were seen. In conclusion imipenem monotherapy was well-tolerated and effective as initial therapy for fever in neutropenia in children.
Adolescent↗
High-dose thiotepa with autologous bone marrow rescue in pediatric solid tumors.
High-dose thiotepa was given as a single agent at a total dose of 1125 mg/m2 with autologous bone marrow rescue to nine patients with recurrent/refractory/poor risk pediatric malignancies (primitive neuroepithelial tumor (PNET), two; neuroblastoma, one; Wilms' tumor, one; osteosarcoma, one; Ewing's sarcoma one, Hodgkin's disease one, high-grade glioma, two). The response rate in these heavily pretreated patients was 71% (five out of seven evaluable patients) including two complete responses (Wilms', glioma), three partial responses (osteosarcoma, Ewing's sarcoma, Hodgkin's disease), and two with stable disease (PNET, glioma). The median duration of response was 2.5 months. The extramedullary toxicity was acceptable with symptoms mainly of skin and gastrointestinal tract. The data indicate that high-dose thiotepa is effective in several types of recurrent pediatric solid tumors, and merits further evaluation in combination regimens.
Adolescent↗
Simultaneous hepatoblastoma in identical male twins.
Identical male twin infants who in 1987 presented with hepatoblastoma at the age of 7 months are reported. Twin B was admitted for investigation due to enlarged liver and spleen. He was found to have an inoperable hepatoblastoma of fetal type and was treated with chemotherapy and local irradiation. However, 7 months after the chemotherapy the tumor recurred. The asymptomatic twin A was examined because of the known familial cases, and he proved to have an identical tumor. He was successfully radically operated on and 2 years after the chemotherapy is still doing well with no evidence of tumor. As the mother and the maternal grandmother had polyps in the colon, quite probably the hepatoblastomas of the twins were associated with familial adenomatous polyposis.
Carcinoma, Hepatocellular↗
Initial P-glycoprotein expression in childhood acute lymphoblastic leukemia: no evidence of prognostic impact in follow-up.
Treatment results in childhood acute lymphoblastic leukemia (ALL) have improved remarkably during the past 20 years, but still 25% of children cannot be permanently cured. Drug resistance is a major cause of poor outcome. One of the most investigated resistance mechanisms is the P-glycoprotein (P-gp)-mediated multiple-drug resistance (MDR). The authors prospectively analyzed P-gp using flow cytometry with monoclonal antibody JSB1 in a population-based series of 103 children with ALL treated according to intensive Nordic ALL protocols. Increased P-gp expression was detected in 55 patients (53%). With a cutoff value of 1% P-gp-positive blasts in bone marrow, no difference was found in event-free survival (EFS) or overall survival between children with low vs. increased P-gp expression. The 4-year EFS in the whole series was 77%. Patients with T-ALL had higher P-gp levels than the others, 3.6% vs. 1.0% (p = .002). P-gp expression did not correlate with the white blood cell count, age, sex, or cytogenetics. The authors conclude that the level of P-gp expression cannot be used as a tool for treatment stratification in childhood ALL.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Reduced bone mineral density in long-term survivors of childhood acute lymphoblastic leukemia.
PURPOSE: Osteoporosis and pathologic fractures are occasionally found in patients with childhood acute lymphoblastic leukemia (ALL). This study was performed to determine the degree of possible osteopenia in long-term survivors of childhood ALL. PATIENTS AND METHODS: Lumbar spine (L2-L4) and femoral neck bone mineral densities (BMDs) (g/cm2) were measured in 29 survivors (aged 12 to 30 years, median 17) of childhood ALL 2 to 20 (median 8) years after discontinuation of chemotherapy. These results were compared with those from 273 healthy controls and expressed as a percentage of the age- and sex-matched control values (mean +/- standard deviation). RESULTS: Lumbar and femoral BMDs were significantly reduced in survivors of childhood ALL. Particularly, male gender (lumbar: 91.7 +/- 10.4%, p = 0.008; femoral: 91.9 +/- 11.3%, p = 0.005) and a history of cranial irradiation (lumbar: 93.0 +/- 8.9%, p = 0.005; femoral: 94.4 +/- 13.3%, p = 0.03) were associated with low lumbar and femoral BMDs. CONCLUSIONS: The detected deficit in bone density in survivors of childhood ALL may predispose these patients to osteoporotic fractures later in adulthood. A follow-up of BMD in survivors of childhood ALL should facilitate the identification of patients who would require specific therapeutic interventions to prevent further decrease of their skeletal mass and preserve their BMD.
Absorptiometry, Photon↗
Changing pattern of treatment policies invalidates the use of C-reactive protein level and hyponatremia as indicators of sepsis in children with malignancies.
We evaluated serum C-reactive protein (CRP) level and serum sodium concentration as early indicators of bacteremia in neutropenic children in two different series in 1983-1984 (49 bacteremias) and 1989-1990 (29 bacteremias). During the earlier period, the goal was to avoid unnecessary antimicrobial therapy. Currently a neutropenic patient is placed on antimicrobial therapy at the first sign of fever. In 1983-1984 the serum CRP concentration was elevated in every case, whereas in 1989-1990 it was normal in 34% cases (P = .0001). Hyponatremia was detected on admission in 84% and 52% cases (P = .0001). The urinary sodium concentration was elevated in most cases. The mortality in bacteremia was 22% in 1983-1984 compared to 3% (P = .025) in 1989-1990. Prompt initiation of empirical antimicrobial therapy in children with fever and neutropenia invalidates the use of hyponatremia and an elevated CRP level as early indicators of sepsis.
Adolescent↗
Multiple drug resistance mediated by P-glycoprotein is not a major factor in a slow response to therapy in childhood ALL.
In childhood acute lymphoblastic leukemia (ALL), early response to treatment is an important prognostic factor and drug resistance is a major cause of poor outcome. One of the most investigated resistance mechanisms is P-glycoprotein (P-gp)-mediated multiple drug resistance (MDR). We analyzed P-gp using flow cytometry with monoclonal antibody JSB1 in a series of 118 children with ALL, 103 at diagnosis and 15 at relapse. Increased P-gp expression was found in 55 (53%) patients at diagnosis and in 11 (73%) at relapse. We also analyzed the bone marrow aspirate slides for early response to treatment in a central review. No correlation was found between P-gp and early response. Patients with T-ALL had higher P-gp levels than the others, 5.3% versus 1.0% (P = .002). We conclude that P-gp-mediated multiple drug resistance is not a factor in a slow response to ALL induction therapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Infections occurring during the courses of anticancer chemotherapy in children with ALL: a retrospective analysis of 59 patients.
In a retrospective analysis we evaluated the occurrence of infections in 59 children with acute lymphoblastic leukemia (ALL) during the entire duration of their anticancer chemotherapy. We recorded a total of 245 infection episodes, 118 (50%) being during neutropenia and 119 (50%) during nonneutropenia. The infections most commonly detected during neutropenia were fevers of undetermined origin (36%), clinically or microbiologically defined focal infections (33%), and bacteremias (28%). During nonneutropenia, upper respiratory tract infections (55%) were the most common. Patients needed hospitalization for infections for a total of 1951 days (i.e., a mean of 33 days per patient) and the mean number of infection episodes was 4.2 per patient. Recurrent fever developed in 21% of the children with bacteremia. Mortality caused by bacteremias was 10%. Infections during the chemotherapy of ALL were a significant cause of morbidity in children, but mortality was low.
Adolescent↗