Comparison of the CELLEX™ and UVAR-XTS™ closed-system extracorporeal photopheresis devices in the treatment of chronic graft-versus-host disease.

Extracorporeal Photopheresis (ECP) is a cellular immunotherapy frequently used for steroid-refractory graft-versus-host disease (GVHD). Chronic GVHD (cGVHD), response to ECP is associated with survival benefit. The UVAR-XTSTM system and the more recently developed CELLEXTM device (both TherakosTM ) are the mainstay for ECP-delivery in the UK and US. No comparison of treatment outcomes has been reported. We retrospectively compared cGVHD response and steroid reduction and withdrawal in patients treated exclusively over 12 months with either the XTS (n = 51) or CELLEX (n = 50). Our hypothesis was that there would be no difference in clinical outcome or steroid changes in the 2 matched cohorts. We also compared infection incidence, infection-related death (IRD), and treatment time. Significant clinical improvement and regular capacity to reduce or cease steroids was encountered in both cohorts; at 6 months of ECP 70% of cutaneous cGvHD patients had partial or complete responses and 85% of patients receiving steroids pre-ECP had reduced dosage. In the XTS group we unexpectedly encountered both superior steroid reduction (86% dose at least halved vs. 61% for CELLEX, P = 0.01) and withdrawal (15 vs. 5 CELLEX, P = 0.01) and a trend for superior skin disease response in the CELLEX-treated cohort at 3 months. No inter-relationship was evident. Halving or greater reduction of steroid dose by 3 or 6 months was associated with reduced risk of IRD in the XTS cohort as was withdrawal at 6 months for the combined cohorts. By 6 months, XTS-treated patients had experienced fewer antibiotic-requiring infections (mean 1.9 vs. 2.8, P = 0.025). Origins for the disparities are unclear and warrant investigation.

Assessment of the comparative agreement between chest radiographs and CT scans in intensive care units.

PURPOSE: Chest radiographs in critically ill patients can be difficult to interpret due to technical and clinical factors. We sought to determine the agreement of chest radiographs and CT scans, and the inter-observer variation of chest radiograph interpretation, in intensive care units (ICUs). METHODS: Chest radiographs and corresponding thoracic computerised tomography (CT) scans (as reference standard) were collected from 45 ICU patients. All radiographs were analysed by 20 doctors (radiology consultants, radiology trainees, ICU consultants, ICU trainees) from 4 different centres, blinded to CT results. Specificity/sensitivity were determined for pleural effusion, lobar collapse and consolidation/atelectasis. Separately, Fleiss' kappa for multiple raters was used to determine inter-observer variation for chest radiographs. RESULTS: The median sensitivity and specificity of chest radiographs for detecting abnormalities seen on CTs scans were 43.2% and 85.9% respectively. Diagnostic sensitivity for pleural effusion was significantly higher among radiology consultants but no specialty/experience distinctions were observed for specificity. Median inter-observer kappa coefficient among assessors was 0.295 ("fair"). CONCLUSIONS: Chest radiographs commonly miss important radiological features in critically ill patients. Inter-observer agreement in chest radiograph interpretation is only "fair". Consultant radiologists are least likely to miss thoracic radiological abnormalities. The consequences of misdiagnosis by chest radiographs remain to be determined.

Circulating B-cell activating factor level predicts clinical response of chronic graft-versus-host disease to extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) is an important therapeutic option in steroid-refractory chronic graft-versus-host disease (cGVHD). Few biomarkers predicting response exist. We measured serum B-cell activating factor (BAFF) in 46 cGVHD patients receiving ECP before and during treatment course. BAFF level at 1 month of ECP predicted 3- and 6-month skin disease response, with BAFF less than 4 ng/mL associated with significant skin improvement and complete resolution in 11 of 20 patients. High BAFF at 1-month ECP associated with a worsening median 6-month skin score and resolution in 1 of 10 patients. BAFF level at 3 months also predicted the likelihood of maintaining skin disease improvement at 6 months. BAFF level was not correlated directly with extracutaneous cGVHD response, although full cutaneous responders exhibited improved extracutaneous organ response rates compared with skin nonresponders (65% vs 35%). This study suggests that early BAFF measurement during ECP for cGVHD represents a potentially useful biomarker in prediction of treatment outcome.

Regulatory T Cells in Chronic Graft-Versus-Host Disease After Extracorporeal Photopheresis: Correlation With Skin and Global Organ Responses, and Ability to Taper Steroids.

BACKGROUND: Induction of immune tolerance by an increase in regulatory T (Treg) cells after extracorporeal photopheresis (ECP) is thought to contribute to how ECP exerts its therapeutic effect in patients with chronic graft-versus-host disease (cGvHD). We investigated whether percentages and absolute counts of Treg cells changed post-ECP, and examined correlation with response. METHODS: Absolute counts and % of CD4+ T cells and Treg cells (CD4 + CD25 + FOXP3 + CD127dim/-) were evaluated using flow cytometry in 32 patients with cGvHD treated by ECP for a minimum of 3 months, and up to 12 months. CD4+ or Treg cells at baseline to 12 months post-ECP were compared with changes in skin disease scores or global organ involvement, or the ability to taper steroids, at 14, 28, and 56 weeks. RESULTS: Regulatory T cells % increased significantly above any overall changes in CD4+ % at 6, 9, and 12 months post-ECP. There was no statistically significant association between Treg cells and skin or steroid response, whereas a larger increase in CD4+ count from baseline to 1 to 3 months corresponded to increased odds of being able to reduce steroid dose by 50% or greater at 14 weeks. Skin and global organ responders at 28 weeks had higher median Treg cell counts 3 months post-ECP than nonresponders, as did steroid responders at 56 weeks who were 12 months post-ECP. CONCLUSIONS: Regulatory T cell counts and % varied greatly among cGvHD patients, and the increase post-ECP was not significant until 6 months. No clear correlation was found between Treg cells and clinical improvement, suggesting that increases in Treg cell numbers and/or proportions are not driving the mechanism leading to a response after ECP.

Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease.

The thiocarbamate alcoholism drug disulfiram blocks the P-glycoprotein extrusion pump, inhibits the transcription factor nuclear factor-kappaB, sensitizes tumors to chemotherapy, reduces angiogenesis, and inhibits tumor growth in mice. Thiocarbamates react with critical thiols and also complex metal ions. Using melanoma as the paradigm, we tested whether disulfiram might inhibit growth by forming mixed disulfides with critical thiols in a mechanism facilitated by metal ions. Disulfiram given to melanoma cells in combination with Cu2+ or Zn2+ decreased expression of cyclin A and reduced proliferation in vitro at lower concentrations than disulfiram alone. In electrophoretic mobility shift assays, disulfiram decreased transcription factor binding to the cyclic AMP-responsive element in a manner potentiated by Cu2+ ions and by the presence of glutathione, suggesting that thiocarbamates might disrupt transcription factor binding by inducing S-glutathionylation of the transcription factor DNA binding region. Disulfiram inhibited growth and angiogenesis in melanomas transplanted in severe combined immunodeficient mice, and these effects were potentiated by Zn2+ supplementation. The combination of oral zinc gluconate and disulfiram at currently approved doses for alcoholism also induced >50% reduction in hepatic metastases and produced clinical remission in a patient with stage IV metastatic ocular melanoma, who has continued on oral zinc gluconate and disulfiram therapy for 53 continuous months with negligible side effects. These findings present a novel strategy for treating metastatic melanoma by employing an old drug toward a new therapeutic use.

Differential expression of melanoma-associated antigens and molecules involved in antigen processing and presentation in three cell lines established from a single patient.

Tumour cells are able to evade the immune system by using several 'escape mechanisms'. Downregulation of molecules involved in the processing and presentation of self-antigens has been reported. However, these adaptations have not been compared in metastases in different anatomical locations but derived from a single patient. We investigated three melanoma cell lines--MJT1 from the parietal lobe of the brain, MJT3 from the cerebellum and MJT5 from the left side of the neck--established from biopsies excised from a 45 year old female patient. Although human leukocyte antigen (HLA) class I was detected in all three cell lines by flow cytometry using an anti-HLA monomorphic antibody, further serological analysis demonstrated HLA B38 loss in all three cell lines, HLA B7 downregulation in MJT5 (skin metastases) and B7 loss in MJT3 and MJT1 (brain metastases) compared with the HLA type of the patient's normal autologous lymphocytes. Interferon-gamma (IFNgamma) treatment increased the expression of HLA class I and transporters associated with antigen processing 1 (TAP1) in all three cell lines. De novo HLA class II molecule expression was observed after IFNgamma treatment in MJT3 and MJT5. Western blot and reverse transcription-polymerase chain reaction results revealed heterogeneity of melanoma-associated antigen (MAA) expression in the cell lines: MJT3 cells expressed higher levels of MAAs than the other two cell lines. In conclusion, this study has demonstrated that three metastatic lesions from a single patient can have differential expression of molecules involved in antigen processing (TAP1) and presentation (HLA I and II), but that expression of these molecules is modulated by IFNgamma to a similar degree in all cell lines. In contrast, the downregulation of expression of specific MAAs between the three cell lines was unaffected by the addition of IFNgamma.

Thermally-prepared polymorphic forms of cilostazol.

Prior to this study, cilostazol, an antithrombotic drug, was thought to exist as a single crystalline phase with a melting point of approximately 159 degrees C (Form A). On cooling, melts often form a glass that, when heated, may crystallize as additional crystalline polymorphic forms. Cilostazol, when reheated, subsequently forms polymorphs that melt at approximately 136 degrees C (Form B) and 146 degrees C (Form C). Free-energy temperature diagrams estimated from calorimetry data reveal that each pair of the cilostazol polymorphs (A-B, B-C, and A-C) is monotropic. Essentially pure samples of suitable crystalline shape and size permitted single crystal structural analysis of Forms A and C. Theoretical solubility ratios calculated using calorimetry data indicate that at 37 degrees C, Form B should be more than four times more soluble and Form C should be more than two times more soluble than Form A. Forms B and C could not be crystallized from solvents. Metastable forms from super cooled melts analyzed by intrinsic dissolution and Fourier transform-Raman experiments demonstrated that Forms B and C undergo a rapid, solvent-mediated recrystallization to Form A, making dissolution rate measurements difficult.

Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs ofApterous genotypes inDrosophila melanogaster.

The aldehyde oxidase staining pattern in wing discs ofDrosophila melanogaster bearing the genotypesap blt /ap blt andap blt andap blt /ap 73n showns changes from the wild-type pattern. Extensive areas of the presumptive dorsal posterior wing blade, which are normally unstained, have enzyme activity in these mutants. In wings of these genotypes, dorsal posterior structures are replaced by dorsal anterior wing structures. A strong correlation has been found between the frequencies of various staining patterns in the discs and the extent of transformation in the cuticular structures of the wing, which is consistent with the idea that aldehyde oxidase activity can be used as an indicator in the wing disc of this transformation. Unlike the homoeotic mutationengrailed, apterous has not been interpreted as a selector gene yet the work reported here shows thatapterous alleles can cause changes resembling those of theengrailed phenotype both in aldehyde oxidase staining behaviour and in the cuticular transformation.

Polymorphic forms of cilostazol.

Two unique conformational polymorphic forms of the compound 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydroquinolin-2(1H)-one (cilostazol), C(20)H(27)N(5)O(2), have been discovered and characterized using single-crystal X-ray structural analysis. A third polymorph also exists, but acceptable crystals could not be obtained. Features of both reported polymorphic structures include a chair conformation of the cyclohexyl ring and puckering in the quinolinone ring. The major feature distinguishing the two polymorphic forms is a rotational twisting of the butoxy chain between the tetrazole and quinolinone rings. This difference in conformation influences the intermolecular forces, and hence the packing of the two molecules during crystallization.