Anaesthetists will continue to play a major role in the management of UK intensive care.
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Critical Care↗
Biomedical subjects
D Banerjee
Publications and source records attributed to D Banerjee.
Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity.
BACKGROUND: Anemic patients with chronic renal failure receiving recombinant human erythropoietin (rHuEPO) therapy frequently develop hypertension through an unknown mechanism. We hypothesize that EPO receptors (EPORs) on endothelial cells (ECs) in various sites of vasculature may mediate the activities of nitric oxide synthase (NOS) and/or the release of endothelin-1 (ET-1), contributing to blood pressure changes. We tested this hypothesis using primary cultures of ECs obtained from human coronary artery (HCAEC), pulmonary artery (HPAEC), dermis (HDEC), and umbilical vein (HUVEC). METHODS: EPORs were measured by 125I-EPO binding. The effect of EPO on EPOR, ET-1, and NOS mRNA levels was assessed by quantitative reverse transcription-polymerase chain reaction. Cellular NOS activity and ET-1 release into the medium was measured by the NOSdetect assay and by radioimmunoassay kits. RESULTS: Short-term (4 h) treatment with EPO (4 U/mL) did not change the number or affinity of EPOR per cell. Neither were there any changes in the amount of EPOR, ET-1, and NOS transcripts (cDNA/microg of mRNA) nor in ET-1 release and NOS activity. In HUVEC only, 24-hour exposure to EPO caused a threefold increase in NOS transcript. In other cells, EPO treatment for six days increased NOS activity by twofold to fourfold. CONCLUSIONS: We show that upon extended exposure, EPO induces NOS activity but does not affect ET-1 release. These findings indicate that the hypertensive effect of EPO is not likely to be caused by a direct effect on ECs.
Blood Pressure↗
Mastectomy follow-up by biennial mammograms: is it worthwhile?
The value of follow-up after mastectomy for breast cancer is controversial. One reason is to detect metachronous tumour in the contralateral breast, but the optimum method for achieving this is undecided. The long-term follow-up policy of our unit is annual clinical review combined with biennial mammography. We have assessed the benefit of this policy in the detection of tumours of the contralateral breast. The case notes of 216 patients undergoing mastectomy between 1978 and 1985, under the care of one consultant surgeon (DJTW) were reviewed. Follow-up was complete to December 1997, thus allowing a minimum follow-up of 12 years. The development of a metachronous tumour was recorded as was its method of detection : either clinically, by the patient or the clinician, or by routine mammography. Two-hundred and five patients were available for follow-up of the contralateral breast. Seventeen (8.3%) developed metachronous tumours. Eight were detected by the patient, 4 by the clinician and 5 by routine follow-up mammography. Biennial mammography does not appear to be beneficial in breast cancer follow-up. More work is required to determine the benefits of more frequent mammography, with or without breast self-examination and clinical review within the hospital environment or within primary care.
Journal Article↗
The treatment of respiratory pseudomonas infection in cystic fibrosis: what drug and which way?
Pseudomonas aeruginosa is a non-capsulate and non-sporing gram-negative bacillus that most commonly affects the lower respiratory system in humans. Burkholderia (previously Pseudomonas) cepacia has emerged as an important respiratory pathogen in patients with cystic fibrosis (CF). The ability of P. aeruginosa to persist and multiply in moist environments and equipment, such as humidifiers in hospital wards, bathrooms, sinks and kitchens, maybe of importance in cross-infection. P. aeruginosa infections of the lower respiratory tract can range in severity from colonisation (without an immunological response) to a severe necrotising bronchopneumonia. Infection is seen in patients with CF and other chronic lung diseases such as non-CF bronchiectasis. In patients with CF, once P. aeruginosa is established in the airways it is almost impossible to eradicate, but prior to this, aggressive treatment can delay the development of chronic infection. 30 to 40% of the present paediatric population with CF will have chronic pseudomonal infection. B. cepacia has a particular predisposition to infect patients with CF and may be distinguished from P. aeruginosa by accelerated lung disease in about one- third of patients. Overwhelming septicaemia and necrotising pneumonia are well described (cepacia syndrome); events that are rare with P. aeruginosa. With the propensity for social cross-infection, segregation policies have been accepted as means of controlling outbreaks. A number of antipseudomonal agents are available. The most commonly used are the extended-spectrum penicillins, aminoglycosides, cephalosporins, fluoroquinolones, polymixins and the monobactams. An aminoglycoside with a beta-lactam penicillin is usually considered to be the first line treatment. No trial has shown any significant clinical advantage of any particular combination regimen over another. The emergence of resistance continues to be a concern. Pipericillin, piperacillin/tazobactam and meropenem have good but equivalent antibacterial activity against P. aeruginosa. However, B. cepacia is characterised by in vitro resistance to colistin (colomycin), aminoglycosides and ciprofloxacin but better susceptibility to ceftazidime. Nebulised delivery of antipseudomonal antibiotics is thought to prevent recurrent exacerbations, reduce antibiotic usage and maintain lung function, particularly in patients with CF. Colistin, tobramycin and gentamicin are currently the most commonly prescribed nebulised antibiotics. Much effort is directed at treating chronic P. aeruginosa infection but as chronic infection is seldom if ever eradicated when first established, prevention is preferable. Early intensive treatment for P. aeruginosa infection is advocated in order to maintain pulmonary function and postpone the onset of chronic P. aeruginosa infection.
Anti-Bacterial Agents↗
AACR - 91st meeting. Bioinformatics.
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Journal Article↗
Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.
The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.
Antigens, Neoplasm↗
Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34(+) hematopoietic stem cells: implications for ex vivo tumor purging and autologous stem cell transplantation.
The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients.
Antibodies, Monoclonal↗
Expression of a novel double-mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside.
A novel fusion gene consisting of the open reading frame of a double-mutant (Phe22-Ser31) dihydrofolate reductase (dmDHFR) cDNA fused to the open reading frame of cytidine deaminase (CD) was constructed and characterized for the purpose of conferring simultaneous resistance to methotrexate (MTX) and cytosine arabinoside (ara-C). The kinetic properties of purified recombinant dmDHFR-CD fusion protein were compared with those of purified CD and dmDHFR. The fusion protein was found to retain enzymatic properties of both dmDHFR and CD, in that the Km and Kcat values of purified dmDHFR-CD protein were found to be virtually identical to those of CD and dmDHFR alone. Retrovirus-mediated expression of dmDHFR-CD in NIH 3T3 cells conferred significant resistance (10- to 12-fold) against MTX and ara-C, compared with mock- and single gene-infected cells and the level of resistance obtained was similar to that of cells expressing both CD and dmDHFR from a retroviral bicistronic vector. Infection of mouse bone marrow cells with the dmDHFR-CD construct also showed high levels of resistance to MTX and ara-C in a CFU-GM assay. This fusion protein confers resistance to two antineoplastic agents that differ in their mechanism of action, and may be useful in the design of gene transfer strategies for protection of target cells against multiple drugs. Since high-dose ara-C and MTX are used in the treatment of lymphomas, this vector may be of value in protecting human hematopoietic progenitor cells from the toxicity of these antimetabolites.
3T3 Cells↗
Transcriptional regulatory sequences within the first intron of the chicken apolipoproteinAI (apoAI) gene.
Previous studies demonstrated that the -82 to +87 nucleotides (nt) 5'-upstream region of the chicken apolipoprotein (apoAI) gene are necessary for maximum reporter chloramphenicol acetyl transferase (cat) gene activation in chicken hepatocarcinoma (LMH) cells [Bhattacharyya, N., Chattapadhyay, R., Oddoux, C., Banerjee, D., 1993. Characterisation of the chicken apolipoprotein A-I gene 5'-flanking region. DNA Cell Biol. 12, 597-604]. The -82 to +87nt contain the 5'-untranslated nt, part of the first intron, and the upstream regulatory sequences. In this study, we examined the role of the first intron in the transcriptional regulation of the chicken apoAI gene. Six different reporter cat gene constructs with or without part of the first intron were prepared and transfected into LMH, normal rat kidney (NRK) and human hepatocarcinoma (HepG2) cells. Cell extracts were prepared from each transfected cell line, and CAT activities were measured. All three cell-lines readily expressed CAT, indicating that transcriptional regulatory sequences are present within the first intron region of the chicken apoAI gene. In an enhancer assay, the first intron containing cat construct exhibited a 5.4-fold increase of reporter activity in NRK cells when compared to a SV 40 promoter containing cat plasmid, suggesting the presence of a moderate enhancer element within +29 to +87nt of the first intron. DNase I protection assays, electrophoretic mobility shift assays and binding experiments with nuclear proteins isolated from different chicken tissues and LMH cells showed interaction with +29 to +87nt. Nuclear proteins isolated from tissues like liver and intestine, that actively express apoAI gene, failed to interact with +29 to +87nt, whereas nuclear proteins isolated from tissues that are less active in apoAI gene expression readily interacted with this region. To show the binding of the LMH-specific trans-acting factors to the +50 to +68nt intron region, DNA-affinity chromatography step was performed by using 3H-labeled nuclear proteins. These studies demonstrate that the first intron region of the apoAI gene interacts with trans-acting proteins and plays an important role in transcriptional regulation of the apoAI gene.
Animals↗
Mechanisms of methotrexate resistance in acute leukemia. Decreased transport and polyglutamylation.
Drug resistance limits the effectiveness of methotrexate (MTX) for the treatment of acute leukemia. An increased understanding of the pathways involved in folate metabolism has allowed investigations of the mechanisms of resistance observed in leukemic blasts obtained from patients. Acute lymphocytic leukemia (ALL) was studied for mechanisms of acquired MTX resistance. MTX transport in 27 patients with untreated ALL and 31 patients with relapsed ALL was measured using a previously described competitive displacement assay. Only 13% of the untreated patients were considered to have impaired MTX transport whereas over 70% of the relapsed patients had evidence of impaired MTX transport. Northern analyses and quantitative RT-PCR for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of 9 samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. Acute myelocytic leukemia (AML) was studied to determine the basis of the decreased MTX polyglutamylation. Enzyme kinetics of the enzyme folylpolyglutamate synthetase (FPGS) were studied, demonstrating FPGS in the myeloid cell lines and patient samples had a higher K(m) for MTX as a substrate than lymphoid cells. Measuring gamma-glutamyl hydrolase enzyme activity allowed a more accurate prediction of steady state levels of MTX polyglutamates. A knowledge of the mechanisms of MTX resistance that occur in leukemic blasts obtained from patients may allow the development of therapeutic strategies to circumvent resistance.
Antimetabolites, Antineoplastic↗
Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours.
M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric M, 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia.
Animals↗
Purified apolipoprotein B gene regulatory factor-3 is DNA topoisomerase I.
Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. A negative cis-acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor-3 (BRF-3) interacts with this. In this paper, we report the purification and characterization of BRF-3 from rat liver nuclear extracts. BRF-3 has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonucleotide containing BRF-3 binding site in a South-Western blot. Double-stranded oligonucleotide containing mutations in the BRF-3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70-kDa and 67-kDa forms of BRF-3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.
Amino Acid Sequence↗
Angiosarcoma of the breast following radiation therapy.
Breast carcinoma is increasingly treated by conservation therapy. This includes wide local excision and axilliary node clearance followed by radiotherapy to the remaining breast. Therapeutic irradiation may be complicated by several problems, including the development of other malignant tumours. We report a case of angiosarcoma occurring following such treatment and present a review of the literature.
Breast Neoplasms↗
Writing pens are an unlikely vector of cross-infection with methicillin resistant Staphylococcus aureus (MRSA)
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Cross Infection↗
The role of quinolones in chronic obstructive pulmonary disease.
Chronic obstructive pulmonary disease is a disease state characterized by the presence of airflow obstruction due to chronic bronchitis and/or emphysema. The airflow obstruction is generally progressive. In the past asthma was often confused with chronic obstructive pulmonary disease but as the cellular inflammatory mechanisms are quite different to chronic bronchitis and emphysema it is prudent to separate this condition of airway hyper-responsiveness. Exacerbation of chronic obstructive pulmonary disease is a considerable burden on health service resources in terms of morbidity and mortality. Approximately one half of exacerbations can be attributed to bacterial pathogens, the major pathogens being Haemophilus influenzae, Streptococcus pnemoniae and Moraxella catarrhalis. Resistance to common first-line treatment antibiotics such as the beta-lactams can be variable. Newer fluoroquinolones such as grepafloxacin, levofloxacin, sparfloxacin, clinafloxacin, moxifloxacin, gatifloxacin and gemifloxacin are characterized by improved activity against Gram positive bacteria as well as their Gram negative properties. However, more randomized controlled trials need to be accomplished before the true role of quinolones in exacerbation of chronic obstructive pulmonary disease is clearly ascertained.
Journal Article↗
Overexpression of p21(waf1) decreases G2-M arrest and apoptosis induced by paclitaxel in human sarcoma cells lacking both p53 and functional Rb protein.
We examined the effect of overexpression of p21(waf1) on cytotoxicity of paclitaxel, a microtubule stabilizer, using a tetracycline-inducible expression system in human sarcoma cells (SaOs-2) that lack both functional retinoblastoma protein and p53. Under normal growth conditions, p21(waf1) is not detectable in SaOs-2 cells. Upon p21(waf1) induction by tetracycline withdrawal, we observed a reduced apoptotic response to paclitaxel with a 3- to 6-fold increase in IC50 values compared with that of cells not induced by p21(waf1). We also observed a 5-fold increase in the IC50 value when cytotoxicity to vincristine, another microtubule-disrupting agent, was assessed, whereas we observed a marked decrease in the IC50 value after p21(waf1) induction in response to etoposide, a topoisomerase II inhibitor. After treatment with paclitaxel, less accumulation of G2-M was observed in p21(waf1)-induced cells compared with non-p21(waf1)-induced cells (57% versus 74%). p21(waf1) induction also inhibited the increased cyclin B1-associated kinase activity induced by paclitaxel. Overexpression of p21(waf1) in SaOs-2 cells lacking both p53 and functional retinoblastoma protein may decrease the G2-M arrest induced by paclitaxel due to suppression of the S-G2 checkpoint, resulting in a decreased apoptotic response of cells to paclitaxel.
Antineoplastic Agents, Phytogenic↗
Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.
Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis.
Animals↗
Differential decline in Leishmania membrane antigen-specific immunoglobulin G (IgG), IgM, IgE, and IgG subclass antibodies in Indian kala-azar patients after chemotherapy.
Pathogenesis in kala-azar is associated with depressed cellular immunity and significant elevation of antileishmanial antibodies. Since these antibodies are present even after cure, analysis of the parasite-specific isotypes and immunoglobulin G (IgG) subclasses in kala-azar patients may shed new light on the immune responses during progression and resolution of infection. Using leishmanial membrane antigenic extracts, we investigated the relative levels of specific IgG, IgM, IgA, IgE, and IgG subclasses in Indian kala-azar patient sera during disease, drug resistance, and cure. Acute-phase sera showed strong stimulation of IgG, followed by IgE and IgM and lastly by IgA antibodies. IgG subclass analysis revealed expression of all of the subclasses, with a predominance of IgG1 during disease. Following sodium stibogluconate (SAG) resistance, the levels of IgG, IgM, IgE, and IgG4 remained constant, while there was a decrease in the titers of IgG2 and IgG3. In contrast, a significant (2.2-fold) increase in IgG1 was observed in these individuals. Cure, in both SAG-responsive and unresponsive patients, correlated with a decline in the levels of IgG, IgM, IgE, and all of the IgG subclasses. The stimulation of IgG1 and the persistence, most importantly, of IgE and IgG4 following drug resistance, along with a decline in IgE, IgG4, and IgG1 with cure, demonstrate the potential of these isotypes as possible markers for monitoring effective treatment in kala-azar.
Adolescent↗