Clinical Pathology - Research Publications
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ItemNo Preview AvailablePlasmodium Falciparum: Cytoadherence occurring in the absence of knobs uses the thrombospondin receptor (CD36)(Elsevier BV, 1990)P. falciparum is the cause of the lethal form of malaria which results in thousands of deaths each year. The primary cause of death, cerebral malaria, is associated with the sequestration of erythrocytes infected with the mature stages of P. falciparum (trophozoites and schizonts) in the post capillary venules of the brain. The identification of the parasite protein(s) involved in this process will provide important vaccine candidate molecules and knowledge about the pathological processes involved in cell-cell adhesion in general. The mechanism of cytoadherence is studied in vitro using cultured lines of P. falciparum which bind to umbilical vein endothelial cells and C32 amelanotic melanoma cells. Mature stages of the parasite may induce knob-like protrusions in the erythrocyte membrane, and it was previously thought that ‘knobs’ were necessary although not sufficient for cytoadherence to occur both in vitro and during natural infection. We have derived a clone of the Brazilian isolate of P. falciparum, ITG2F6, and selected for cytoadherence by repeated passage over amelanotic melanoma cells. Chromosome analysis using pulsed-field gradient electrophoresis and DNA amplification using the polymerase chain reaction reveal that this clone has deleted the gene coding for knobs. Furthermore, cytoadherence which is independent of knobs occurs via the receptor for the platelet protein, thrombospondin.
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ItemThe role of genetic factors in predisposition to squamous cell cancer of the head and neck.(Springer Science and Business Media LLC, 1999-02)
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ItemA phase III study of radiotherapy with and without continuous-infusion fluorouracil as palliation for non-small-cell lung cancer(NATURE PUBLISHING GROUP, 1997)This study assesses the effect of adding continuous-infusion fluorouracil to palliative thoracic radiation therapy (RT) on the rate and duration of symptom relief in patients with advanced non-small-cell lung cancer (NSCLC). Two hundred eligible patients with NSCLC were randomized to receive either 20 Gy in five daily fractions as palliation for intrathoracic disease or the same RT with concurrent continuous infusion of 1 g m(-2) day(-1) fluorouracil for 5 days. Survival, response and rates of symptom relief in the two groups were compared according to treatment intent, and toxicities were compared according to treatment received. The overall response rate was higher in patients randomized to the combination (29%) than in patients randomized to RT alone (16%) (P = 0.035). However, there were no significant differences between the treatment arms in terms of overall or progression-free survival or in palliation of symptoms. Patients treated with RT plus fluorouracil had significantly more acute toxicity, including nausea and vomiting (P = 0.01), oesophagitis (P = 0.0003), stomatitis (P = 0.0005) and skin reaction (P = 0.003). This study suggests for the first time an interaction between RT and infusional fluorouracil in NSCLC. Although RT plus fluorouracil resulted in a significantly higher response rate than achieved with RT alone, this did not translate into more effective palliation. Because the combination produced significantly more toxicity than RT alone, it is not recommended for the palliative treatment of NSCLC. Nevertheless, these results suggest that opportunities may exist for exploitation of the observed enhancement of antitumour effect in the setting of high-dose radical RT for NSCLC.
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ItemClonal anergy of B cells: a flexible, reversible, and quantitative concept.(Rockefeller University Press, 1996-05-01)
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ItemAntigen-driven B cell differentiation in vivo.(Rockefeller University Press, 1993-07-01)The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.
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Itemc-Ki-ras gene mutations in dysplasia and carcinomas complicating ulcerative colitis.(Springer Science and Business Media LLC, 1991-07)One hundred and nine samples comprising carcinomas, adenomas, dysplastic, inflamed and normal mucosa from patients with sporadic colon cancer and ulcerative colitis (UC) were analysed for c-Ki-ras mutations. DNA was extracted from archival paraffin-embedded material, amplified using the polymerase chain reaction (PCR) and the PCR products analysed using restriction enzyme digestion. Forty-two per cent (14/33) of the sporadic carcinoma controls contained Ki-ras codon 12 mutations in contrast to 24% (8/33) of ulcerative colitis carcinomas. A significantly higher c-Ki-ras mutation rate was observed in rectal carcinomas (72%) in comparison to colonic carcinomas (28%) in control patients (P less than 0.04), while the opposite was observed in UC patients. The difference between the incidence of c-Ki-ras mutations in rectal carcinomas in UC (9%) and in sporadic rectal carcinomas (72%) was also significant (P less than 0.01). This lower prevalence rate and different site distribution of c-Ki-ras mutations in UC carcinomas compared to sporadic carcinomas suggests that specific genetic differences may underlie the causation of carcinomas arising in these situations.
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ItemRapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.(Springer Science and Business Media LLC, 1993-06)In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).
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ItemRECOMBINANT SOLUBLE HUMAN FC-GAMMA-RII - PRODUCTION, CHARACTERIZATION, AND INHIBITION OF THE ARTHUS REACTION(ROCKEFELLER UNIV PRESS, 1993-11-01)A recombinant soluble form of human Fc gamma RII (rsFc gamma RII) was genetically engineered by the insertion of a termination codon 5' of sequences encoding the transmembrane domain of a human Fc gamma RII cDNA. Chinese hamster ovary cells were transfected with the modified cDNA and the secreted rsFc gamma RII purified from the tissue culture supernatant (to > 95%, assessed by SDS-PAGE) using heat aggregated human immunoglobulin G (IgG) immunoaffinity chromatography. The IgG-purified rsFc gamma RII was relatively homogeneous (approximately 31,000 M(r)) whereas the total unpurified rsFc gamma RII secreted into the tissue culture supernatant was heterogeneous relating to N-linked glycosylation differences. Functional in vitro activity of the rsFc gamma RII was demonstrated by: (a) ability to bind via the Fc portion of human IgG and mouse IgG (IgG2a > IgG1 > > IgG2b); (b) complete inhibition of binding of erythrocytes sensitized with rabbit IgG to membrane-bound Fc gamma RII on K562 cells; and (c) inhibition of the anti-Leu4-induced T cell proliferation assay. Blood clearance and biodistribution studies show the rsFc gamma RII was excreted predominantly through the kidney in a biphasic manner, with an alpha-phase (t1/2 approximately 25 min) and a beta-phase (t1/2 approximately 4.6 h); the kidneys were the only organs noted with tissue-specific accumulation. In vivo, the administration of rsFc gamma RII significantly inhibited the immune complex-mediated inflammatory response induced by the reversed passive Arthus reaction model in rats. There was a specific and dose-dependent relationship between the amount of rsFc gamma RII administered, and the reduction in the size and severity of the macroscopic inflammatory lesion. Histological analysis of the skin showed a diffuse neutrophil infiltrate in both control and rsFc gamma RII-treated rats, however the perivascular infiltrate and the red cell extravasation was less intense in the rsFc gamma RII-treated group. It is likely that complement activation leads to neutrophil chemotaxis, but neutrophil activation via Fc gamma RII, which results in inflammatory mediator release, is inhibited. The data indicate that rsFc gamma RII is a potential therapeutic agent for the treatment of antibody or immune complex-mediated tissue damage.
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ItemCells capable of bone production engraft from whole bone marrow transplants in nonablated mice(ROCKEFELLER UNIV PRESS, 1999-02-15)Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.
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ItemDELAYED HEMATOPOIETIC DEVELOPMENT IN OSTEOPETROTIC (OP/OP) MICE(ROCKEFELLER UNIV PRESS, 1993-01-01)Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.