Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: a biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia.

A novel whole cell bacterial biosensor, which emits light in response to the active metabolite of cytosine arabinoside (ara-C, cytarabine), ara-CTP, has been investigated and verified. The biosensor has been formulated as an ex vivo assay, designed for peripheral blood or bone marrow cells, which can produce a clinical result within a working day. The nucleoside analogue ara-C is a key agent for treatment of acute myeloid leukaemia (AML); treatment decisions are made rapidly with AML, patients often receiving same-day commencement of chemotherapy. Currently no rapid predictive test is available to select appropriate therapy for patients prior to treatment. Experiments were designed to determine optimal assay conditions using leukaemic cell lines. We observed a significant increase (~15 fold) in bioluminescence signal compared to control after 8-h incubation of the biosensor with ara-C. This corresponded to a >2-log increase in light output per bacterial cell. Interestingly, bioluminescence conferred a survival advantage to the bacteria following ara-C treatment. The assay is sensitive (lower limit of quantitation of 0.05 µM), selective, accurate (≤ 15% RE) and precise (≤ 15% coefficient of variation) over a linear concentration range of ara-CTP (0.05-0.5 µM), and detection is independent of reaction volume. Recovery of added standard was tested using ex vivo patient leukaemic cells (n=5). Stability studies on lyophilized bacterial biosensor were performed to ensure maintenance of performance over 12 months. The biosensor assay could be invaluable to the clinician, assisting with treatment selection, and potentially mitigating the risks of resistance and toxicity observed with this drug.

A novel bioluminescent bacterial biosensor for measurement of Ara-CTP and cytarabine potentiation by fludarabine in seven leukaemic cell lines.

This study evaluates an in vitro biosensor assay capable of detecting the intracellular levels of the tri-phosphorylated form of cytarabine (Ara-CTP) within one working day. The biosensor predicted the response of seven leukaemic cell lines with varying known sensitivities to cytarabine alone and in combination with fludarabine. High-performance liquid chromatography (HPLC), 3-day assessment of cellular viable mass, and flow cytometric assessment of apoptosis were used to validate biosensor performance. A correlation between the biosensor results and Ara-CTP quantitation by HPLC was confirmed (R=0.972). The biosensor was also capable of detecting enhanced accumulation of Ara-CTP following sequential pre-treatment of leukaemic cells with cytarabine ± fludarabine.

Subcorneal pustular dermatosis an immnohisto-pathological perspective.

UNLABELLED: Subcorneal pustular dermatosis (SPD) represents a chronic, relapsing sterile pustular eruption, involving the trunk and flexoral proximal extremities. A 54-year-old female presented with recurrent, flaccid pustules measuring several millimeters in diameter, on normal and mildly erythematous skin of the groin and submammary areas. Biopsies for hematoxylin and eosin (H&E) examination, direct immunofluorescence (DIF) and immunohistochemistry (IHC) analysis were performed. The H&E staining demonstrated typical features of SPD, including some damage within dermal pilosebaceous units subjacent to the subcorneal blistering process. DIF revealed strong deposits of immunoreactants IgG, IgM, fibrinogen and complement/C3, present in a shaggy pattern within the subcorneal disease areas; in focal, areas of the basement membrane junction and in focal pericytoplasmic areas of epidermal keratinocytes. IHC revealed strong positivity to HLA-DPDQDR, mast cell tryptase, CD68, and ZAP-70 in the subcorneal inflammatory infiltrate, and surrounding dermal blood vessels. Myeloperoxidase was also positive. Positive staining with the anti-ribosomal protein S6-pS240 at the edges of hair follicles and sebaceous glands subjacent to the subcorneal blisters was also noted. CONCLUSIONS: We conclude that this disorder may have several components in its etiopathology, including a possible restricted immune response and a possible genetic component; these possibilities warrant further investigation.

A bioluminescent microbial biosensor for in vitro pretreatment assessment of cytarabine efficacy in leukemia.

BACKGROUND: The nucleoside analog cytarabine (Ara-C [cytosine arabinoside]) is the key agent for treating acute myeloid leukemia (AML); however, up to 30% of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. This project aimed to construct and evaluate a self-bioluminescent reporter strain of Escherichia coli for use as an Ara-C biosensor and to design an in vitro assay to predict Ara-C response in clinical samples. METHODS: We used transposition mutagenesis to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to Ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine Ara-C uptake and phosphorylation by leukemic cells. RESULTS: Intracellular concentrations of 0.025 µmol/L phosphorylated Ara-C were detected by significantly increased light output (P < 0.05) from the bacterial biosensor. Results using AML cell lines with known response to Ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for Ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to Ara-C within 8 h. CONCLUSIONS: The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to Ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced Ara-C dose levels. The 8-h assay monitors intracellular Ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings.

IgG/IgE bullous pemphigoid with CD45 lymphocytic reactivity to dermal blood vessels, nerves and eccrine sweat glands.

CONTEXT: Bullous pemphigoid (BP), the most common autoimmune blistering disease, is mediated by autoantibodies. BP primarily affects the elderly and is characterized by the development of urticarial plaques surmounted by subepidermal blisters, and the deposition of immunoglobulins and complement at the basement membrane zone (BMZ) of the skin. BP is immunologically characterized by the development of autoantibodies targeting two structural proteins of the hemidesmosomes, BP180 (collagen XVII) and BP230 (BPAG1). CASE REPORT: A 63 -year-old Caucasian female patient was evaluated for a 4 day history of several itching, erythematous blisters on her extremities. Biopsies for hematoxylin and eosin (H&E) examination, as well as Periodic acid-Schiff (PAS), immunohistochemistry (IHC) and direct immunofluorescence (DIF) analysis were performed. RESULTS: H&E demonstrated a subepidermal blister, with partial re-epithelialization of the blister floor. Within the blister lumen, numerous neutrophils were present, with occasional eosinophils and lymphocytes also noted. Within the dermis, a mild, superficial, perivascular and periadnexal infiltrate of lymphocytes, histiocytes and occasional eosinophils was identified, with mild perivascular leukocytoclastic debris. The PAS stain was positive at the BMZ, and around selected blood vessels, nerves and sweat glands. DIF revealed linear deposits of IgG and Complement/C3 and fibrinogen at the BMZ, and around selected dermal nerves, blood vessels and sweat glands. Strong granular deposits of IgE were also observed, colocalizing with monoclonal antibodies to Collagen IV (CIV). By IHC, positive CD45 staining of lymphocytes was seen surrounding selected dermal blood vessels, eccrine sweat glands, and nerves. CONCLUSION: The patient displayed IgG, IgE, and fibrinogen autoantibodies against the BMZ, as well as around some dermal nerves and sweat glands; their binding in the skin could trigger complement activation. In addition, the role of the dermal CD45 positive lymphocytes warrants further investigation.

Presence of neutrophil extracellular traps and antineutrophil cytoplasmic antibodies associated with vasculitides.

CONTEXT: Anti-neutrophil p-ANCA antibodies are directed against antigens in the peripheral cytoplasm of both neutrophilic granulocytes and monocytes. They are detected in several autoimmune disorders and are particularly associated with systemic vasculitis. CASE REPORT: We report a case of a 54-year-old female presenting with a pruritic rash, including purpura and diffuse erythema. A biopsy with hematoxylin and eosin (H & E) analysis, direct immunofluorescence (DIF), immunohistochemistry (IHC) and enzyme-linked immunosorbent assays for ANCAs were performed. The H & E staining demonstrated leukocytoclastic vasculitis, with focal vascular fibrinoid necrosis. The DIF revealed evidence of vasculitis, the presence of p-ANCAs and neutrophil extracellular traps (NETs). The IHC displayed autoreactivity to myeloperoxidase within the vessels. The IHC aided in ruling out any intrinsic autofluorescence of the vessels. CONCLUSIONS: By observing the deposition of neutrophil extracellular traps and myeloperoxidase in inflamed skin vessels, biopsy analysis may alert physicians for rapid therapeutic intervention in patients presenting with possible vasculitides.

DNA microarray screening of differential gene expression in bone marrow samples from AML, non-AML patients and AML cell lines.

This study used cDNA microarray technology to compare gene expression profiles in acute myeloblastic leukaemia (AML) with cDNA dot-blot and real time PCR analysis of cDNA transcripts to confirm array data. Patient AML marrow samples and AML cell lines were compared with normal/non-AML samples. Screening revealed five particular genes to be significantly differentially expressed across the sample groups. The migration-inhibitory factor-related-proteins 8 and 14 (MRP-8 and MRP-14) genes, the products of which inhibit cell migration and differentiation were the most highly expressed in non-malignant cells. The high-mobility-group-protein gene (HMG-1) was up regulated in leukaemic samples and cell lines, which may be associated with aggressive disease. Also upregulated in malignant samples were genes encoding c-myc and glutathione-S-transferase pi (GSTP), the latter implicated in chemotherapy resistance. Faulty expression of such genes may contribute to the pathogenesis of AML and resistance to treatment.

New communication between dermatologists in the age of the Internet.

Dermatologists may benefit from a free Internet resource, RxDerm-L, which is an e-mall discussion group for our specialty. Initiated in November 1993, enrollment has finally passed the 1,000 mark; its daily communication reaches a significant percentage of practitioners. Proceedings of this group were monitored for 4 weeks, with special attention to authors, content, and participants. Discussions focused on diagnostic problems and treatment Issues. Members most often providing content were more than likely published and/or associated with a dermatology teaching program. In addition to questions and opinions, the daily exchange included patient images, citations, and abstracts. The average number of letters per day was 46. The ability to share images, access to collective expertise, immediacy of the discussion, and group camaraderie appear to have contributed to the success of this forum. Physicians able to endure the large quantity of e-mall may find RxDerm-L a valuable practice resource.

A novel predictive model of outcome in de novo AML based on S-phase activity and proliferative response of blast cells to haemopoietic growth factors.

This study assesses whether the kinetic response of AML cells to HGFs might help to predict initial clinical outcome of treatment in de novo AML in association with age, FAB type and karyotype. Best subset regression analysis indicated optimal variables to develop models to predict prognosis. High S-phase in surviving cells following 7 days incubation in SFM, resistance to stimulation by G+GM-CSF and poor karyotype taken in combination correctly predicted outcome in 83% of patients. The importance of high SFM S-phase may be to indicate autonomous proliferation therefore a leukemic clone more likely to regenerate following therapy at the expense of normal haemopoiesis. Kinetic studies of AML cells may be a useful predictor of outcome in addition to other more established prognostic factors.