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α-Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds monomeric α-subunits of human hemoglobin A (HbA) and modulates heme iron oxidation and subunit folding states. Although AHSP·αHb complexes autoxidize more rapidly than HbA, the redox mechanisms appear to be similar. Both metHbA and isolated met-β-subunits undergo further oxidation in the presence of hydrogen peroxide (H(2)O(2)) to form ferryl heme species. Surprisingly, much lower levels of H(2)O(2)-induced ferryl heme are produced by free met-α-subunits as compared with met-β-subunits, and no ferryl heme is detected in H(2)O(2)-treated AHSP·met-α-complex at pH values from 5.0 to 9.0 at 23 °C. Ferryl heme species were similarly not detected in AHSP·met-α Pro-30 mutants known to exhibit different rates of autoxidation and hemin loss. EPR data suggest that protein-based radicals associated with the ferryl oxidation state exist within HbA α- and β-subunits. In contrast, treatment of free α-subunits with H(2)O(2) yields much smaller radical signals, and no radicals are detected when H(2)O(2) is added to AHSP·α-complexes. AHSP binding also dramatically reduces the redox potential of α-subunits, from +40 to -78 mV in 1 m glycine buffer, pH 6.0, at 8 °C, demonstrating independently that AHSP has a much higher affinity for Fe(III) versus Fe(II) α-subunits. Hexacoordination in the AHSP·met-α complex markedly decreases the rate of the initial H(2)O(2) reaction with iron and thus provides α-subunits protection against damaging oxidative reactions.

Purpose of Review: The aim is to report on recent observations related to the natural history of paroxysmal nocturnal hemoglobinuria (PNH) and to review new therapeutic strategies for controlling the hemolysis of PNH., Recent Findings: This review focuses on studies designed to characterize the long-term outcome of patients with PNH treated with eculizumab and to define the relationship between PNH and bone marrow failure syndromes. New therapeutic strategies aimed at controlling extravascular as well as intravascular hemolysis are also examined., Summary: Long-term safety and efficacy of eculizumab was observed in a large group of patients. Survival for the group was not different from that of a sex-matched and age-matched control group from the general population. Thrombotic complications were rare and deaths due to PNH or complications of therapy were not observed. These studies suggest that patients with clinical PNH who are treated with eculizumab have a benign clinical course. Patients with bone marrow failure who have PNH cells detected by high-sensitivity flow cytometry have aplastic anemia or low-risk myelodysplastic syndrome. For patients with a percentage of PNH cells that is below the threshold for producing laboratory evidence of hemolysis (subclinical PNH), expansion of the clone to a size sufficient to produce clinical PNH is not observed. Approximately 50% of patients with bone marrow failure who have clinical evidence of PNH at presentation will require PNH-specific therapy. Novel reagents that target the alternative pathway of complement C3 convertase are being developed with a goal of inhibiting both the extravascular and the intravascular hemolysis of PNH.

Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

Background: Gram negative bacteria require iron for growth and virulence. It has been shown that certain pathogenic bacteria such as Neisseria gonorrhoeae possess a periplasmic protein called ferric binding protein (FbpA), which is a node in the transport of iron from the cell exterior to the cytosol., Scope of Review: The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane., Major Conclusions: Here we establish that FbpA may be considered a bacterial transferrin on structural and functional grounds. Data are presented which suggest a continuum whereby FbpA may be considered as a naked iron carrier, as well as a Fe-chelate carrier, and finally a member of the larger family of periplasmic binding proteins., General Significance: An investigation of the molecular mechanisms of action of FbpA as a member of the transferrin super family enhances our understanding of bacterial mechanisms for acquisition of the essential nutrient iron, as well as the modes of action of human transferrin, and may provide approaches to the control of pathogenic diseases. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: Constitutive activation of the Janus kinase 2 (JAK2) due to a somatic mutation (JAK2(V617F)) arising in hematopoietic stem cells plays a central role in the pathophysiology of myeloproliferative neoplasms (MPNs). To investigate the hypothesis that drugs that inhibit JAK2 have therapeutic potential, we developed a small molecule inhibitor, SGI-1252, that targets the adenosine triphosphate-binding and solvent pocket of the protein., Materials and Methods: Established cells lines each expressing different JAK2(V617F) copy numbers, a cell line transfected with wild-type and mutant JAK2, ex vivo expanded erythroid progenitor cells from patients with MPNs, and a murine xenograft model were used to characterize the activity of SGI-1252., Results: In vitro studies showed that SGI-1252 potently inhibits the kinase activity of wild-type JAK2, JAK2(V617F) and JAK1, but not JAK3. SGI-1252 blocked phosphorylation of signal transducers and activators of transcription 5, a downstream target of JAK2 and inhibited expression of the JAK2-dependent antiapoptotic gene BCL-X(L). Additional studies confirmed induction of apoptosis in JAK2(V617F)-positive cell lines by SGI-1252. Moreover, cell lines transfected with either wild-type JAK2 or JAK2(V617F) were equally susceptible to the antiproliferative effects of SGI-1252 and the antiproliferative activity of SGI-1252 toward ex vivo--expanded erythroid progenitors from patients with polycythemia vera and primary myelofibrosis appeared independent of the JAK2(V617F) allele burden. Pharmacodynamic studies in a murine xenograft model demonstrated both anti-tumor activity and inhibition of signal transducers and activators of transcription 5 phosphorylation by SGI-1252, and the drug was active and well-tolerated whether delivered intraperitoneally or orally., Conclusions: Together, these studies support further development of SGI-1252 for clinical use., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Despite the availability of safe, effective targeted therapy that controls intravascular hemolysis, the management of paroxysmal nocturnal hemoglobinuria (PNH) remains complicated because of disease heterogeneity and close association with BM failure syndromes. The purpose of this review is to provide a framework for individualizing treatment based on disease classification. According to the recommendations of the International PNH Interest Group, patients can be placed into one of the following 3 categories: (1) classic PNH, (2) PNH in the setting of another BM failure syndrome, or (3) subclinical PNH. The PNH clone in patients with subclinical disease is insufficiently large to produce even biochemical evidence of hemolysis, and consequently, patients who fit into this category require no PNH-specific therapy. Patients with PNH in the setting of another BM failure syndrome (usually aplastic anemia or low-risk myelodysplastic syndrome) have at least biochemical evidence of hemolysis, but typically the PNH clone is small (< 10%) so that hemolysis does not contribute significantly to the underlying anemia. In these cases, the focus of treatment is on the BM failure component of the disease. Intravascular hemolysis is the dominant feature of classic PNH, and this process is blocked by the complement inhibitor eculizumab. The thrombophilia of PNH also appears to be ameliorated by eculizumab, but the drug has no effect on the BM failure component of the disease. Low-grade extravascular hemolysis due to complement C3 opsonization develops in most patients treated with eculizumab, and in some cases is a cause for suboptimal response to treatment. Allogeneic BM transplantation can cure classic PNH, but treatment-related toxicity suggests caution for this approach to management.

Transferrin, the human iron transport protein, binds Ti(IV) even more tightly than it binds Fe(III). However, the fate of titanium bound to transferrin is not well understood. Here we present results which address the fate of titanium once bound to transferrin. We have determined the redox potentials for a series of Ti(IV) complexes and have used these data to develop a linear free energy relationship (LFER) correlating Ti(IV) <==> Ti(III) redox processes with Fe(III) <==> Fe(II) redox processes. This LFER enables us to compare the redox potentials of Fe(III) complexes and Ti(IV) complexes that mimic the active site of transferrin and allows us to predict the redox potential of titanium-transferrin. Using cyclic voltammetry and discontinuous metalloprotein spectroelectrochemistry (dSEC) in conjunction with the LFER, we report that the redox potential of titanium-transferrin is lower than -600 mV (lower than that of iron-transferrin) and is predicted to be ca. -900 mV vs. NHE (normal hydrogen electrode). We conclude that Ti(IV)/Ti(III) reduction in titanium-transferrin is not accessible by biological reducing agents. This observation is discussed in the context of current hypotheses concerning the role of reduction in transferrin mediated iron transport., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Regulatory T cells (Tregs) are an immunosuppressive T cell subset that functions to prevent autoimmunity and to regulate physiologic immune reactions. Tregs are also present in the tumor microenvironment and appear to play an important role in the pathophysiology of malignant processes. Available data suggests that this role is context-dependent, as a higher density of tumor infiltrating Tregs at diagnosis may be associated with either a positive or a negative clinical outcome. Negative prognostic associations are found primarily in solid tumors such as ovarian carcinoma, while positive associations have been reported in various lymphomas, most prominently in those of germinal center (GC) B cell derivation. Most of these observations are correlative, however, as mechanistic studies have lagged behind descriptive observations because of a lack of informative animal models. Nonetheless, the available data are intriguing and provide compelling support for the hypothesis that Tregs are pathobiologically relevant. This review focuses on studies of the role of CD4 (+)CD25 (+)FOXP3 (+) Tregs in hematopoietic malignancies and clonal myeloid neoplasms.

The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway-mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.

This article discusses the etiology of paroxysmal nocturnal hemoglobinuria (PNH) and its relationship to marrow hyperplasia. The author posits that the defining clinical pathology of PNH (ie, complementmediated intravascular hemolysis) is an epiphenomenon that is a consequence of an orchestrated response (ie, natural selection of PIGA-mutant stem cells) to a specific type of bone marrow injury (ie, immune mediated). Management of PNH is discussed also.

Background and Objective: In 2005, we developed and implemented the Emergency Anaesthetic Simulated Experience course at the Cheshire and Merseyside Simulation Centre.Emergency Anaesthetic Simulated Experience aims to teach clinical and team resource management skills to junior trainees in anaesthesia. Here we present 'proof-of-concept' in terms of long-term retention and transferability of acquired skills into subsequent clinical practice., Methods: An electronic questionnaire sent to 73 trainees, 9-20 months after the course, invited open-ended responses, addressing four areas; namely, real-life encounters with the same scenario as on the course, approach to real-life anaesthetic emergencies in general, approach to real-life routine anaesthesia and need to attend similar courses in the future, with their underlying reasons., Results: Qualitative analysis of the descriptive responses showed that the lessons learnt in the context of simulated emergencies were applied by candidates themselves to real-life emergencies in general and to routine practice., Conclusion: Team resource management skills learnt in a single educational intervention, based on simulated anaesthetic emergencies, are retained over the long term, translated into clinical practice and are transferable across the breadth of clinical activities.

The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is episodic hemoglobinuria, and it was this feature that captured the attention of European physicians in the latter half of the 19th century, resulting in careful observational studies that established PNH as an entity distinct from paroxysmal cold hemoglobinuria and march hemoglobinuria. Curiosity about the etiology of the nocturnal aspects of the hemoglobinuria led the German physician Paul Strübing to develop the prescient hypothesis that the erythrocytes of PNH are abnormally sensitive to hemolysis when the plasma is acidified during sleep because of accumulation of carbon dioxide and lactic acid as a result of slowing of the circulation. Investigation of the intricate pathophysiology that underlies the abnormal sensitivity of PNH erythrocytes to hemolysis in acidified serum produced a number of remarkable scientific achievements that involved discovery of the alternative pathway of complement, identification of the membrane proteins that regulate complement, discovery of a novel mechanism for attachment of proteins to the cell surface, and identification of the genetic basis of the disease. These discoveries were made steadily over a period of more than 100 years, and each generation of physicians and scientists made important contributions to the field. The mysteries of PNH have been solved in a particularly satisfying way because the precision and orderliness of the solutions made clearly understandable what had seemed at the times prior to resolution to be problems of nearly insurmountable complexity. The history of PNH is an inspirational reminder of the elegant complexity of nature, the rewards of curiosity and the power and beauty of science.

The molecular basis of PNH is known. Somatic mutation of the X-chromosome gene PIGA accounts for deficiency of glycosyl phosphatidylinositol-anchored proteins (GPI-AP) on affected hematopoietic stem cells and their progeny. However, neither mutant PIGA nor the consequent deficiency of GPI-AP provides a direct explanation for the clonal outgrowth of the mutant stem cells. Therefore, PNH differs from malignant myelopathies in which clonal expansion is directly attributable to a specific, monogenetic event (e.g., t(9;22) in CML) that bestows a growth/survival advantage upon the affected cell. Multiple, discrete PIGA mutant clones are present in many patients, suggesting that a selection pressure that favors the PNH phenotype (i.e., GPI-AP deficiency) was applied to the bone marrow. The nature of this putative selection pressure, however, is speculative, as is the basis of clonal expansion. In many patients, the majority of hematopoiesis is derived from PIGA mutant stem cells. Yet clonal expansion is limited (nonmalignant), and the contribution of the mutant clones to hematopoiesis may remain stable for decades. Understanding the basis of clonal selection and expansion will not only delineate further the pathophysiology of PNH but also provide new insights into stem cell biology and suggest novel therapeutic strategies for enhancing marrow function.

Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol-anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGA-mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3' untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow.

The developmental gene PAX 3 is expressed in the early embryo in developing muscle and elements of the nervous system, including the brain. Since no one has investigated the expression of the isoforms of PAX 3 in the neuroectodermal tumors melanoma and small cell lung cancer (SCLC), we have carried out a comprehensive screening for the expression of the isoforms PAX 3a-e using RT-PCR in human melanoma cell lines, primary human ocular and secondary cutaneous melanomas. We have identified 2 new isoforms of PAX 3, g and h, which we have isolated, cloned and sequenced. Sets of primers for each isoform were designed and their specificity was confirmed by sequence analysis of the products. The isoforms PAX 3a-e were detected in all human cutaneous melanoma cell lines (8/8), but only PAX 3c (1/2) and PAX 3d (2/2) in ocular melanoma cell lines. The same PAX 3 isoforms were detected in more than 80% of human cutaneous melanomas: PAX 3a and b (15/17), PAX 3c (14/17), PAX 3d (16/17) and PAX 3e (15/17). In contrast the results for 7 SCLC cell lines were PAX 3a (0/7), PAX 3b (1/7), PAX 3c (3/7), PAX 3d (6/7), PAX 3e (2/7); 8/8 cutaneous melanoma cell lines and 8/8 ocular melanoma tissues, together with 14/17 cutaneous melanoma tissues screened, expressed the new isoform PAX 3g. All 8 cutaneous melanoma cell lines expressed PAX 3h, but it was not detectable in any of the tumor tissues (0/20). Neither of the 2 ocular melanoma cell lines expressed the 2 new isoforms. Comparison of the different amplicon staining intensities on a gel suggests that PAX 3c and PAX 3d are the predominant transcripts expressed, with relatively low expression of PAX 3e and PAX 3h. We propose that these and the 2 new isoforms we have discovered may be important in oncogenesis and differential diagnosis of melanomas or SCLC., (Copyright 2003 Wiley-Liss, Inc.)

Tinea of the skin and nails is a common problem in remote Aboriginal communities of the Top End of Australia. A retrospective study was performed on data collected from 104 patients from several indigenous communities. Worksheets were filled in by district medical officers and rural general practitioners, detailing the extent of the tinea. Patients were prescribed between 4 and 12 weeks of 250 mg daily oral terbinafine. Fifty-two patients were followed up, with 45 having a good response to treatment (87%) and with 22 of these patients having full clearance of tinea (42%). A prospective study with 44 subjects was performed. The extent of the tinea was documented and fungal scrapings/clippings were taken. Forty subjects were recruited and given oral terbinafine (2-12 weeks depending on skin/nail involvement) or topical terbinafine if oral treatment was contraindicated. Twenty-five of the 40 (63%) subjects were reviewed. Twenty-three (92%) subjects that were followed up improved clinically, with 8/25 (32%) clearing completely. Twenty (80%) of followed-up patients complied fully with treatment. Terbinafine was found to be a well-tolerated and effective treatment of tinea of the skin and nails.

Paroxysmal nocturnal hemoglobinuria (PNH) arises in the setting of bone marrow injury. Thus, management decisions must take into account whether symptoms are a consequence of the underlying marrow failure or of the expansion of the clone of the PIG-A mutant hematopoietic cells. The primary clinical manifestations of PNH are intravascular hemolysis and thrombophilia. Currently available options for treatment of the hemolysis of PNH are unsatisfactory, but the recent development of specific inhibitors of complement for use in treating human disease should make possible effective management of this pathology. The fundamental basis of the thrombophilia of PNH has not been elucidated. Currently, empiric anticoagulant therapy is the foundation for treating the thromboembolic complications of PNH. The role of warfarin prophylaxis, however, remains an area of active debate. Pregnancy in a patient with PNH presents special concerns about fetal/maternal well-being because of the high potential for thromboembolic complications. Bone marrow transplantation can be considered curative, but the decision to recommend this treatment must take into account factors related both to PNH and to comorbid conditions. Refining the technology for both gene therapy (by transducing stem cells with a functional PIG-A gene) and autotransplantation (by using stem cells selected for the expression of glycosyl phosphatidylinositol-anchored proteins) remain challenges for the future.

Objectives: To reduce the length of the Nottingham Leisure Questionnaire (NLQ) in order to make it more suitable for postal use, and to evaluate its test-retest reliability, sensitivity, stability and validity in relation to other measures of activities of daily living (ADL), mood and handicap., Method: The NLQ was shortened and the response categories collapsed. Results from a previous trial which had used the NLQ were reanalysed to establish if significant group differences were maintained. The new version of the NLQ was subsequently tested for test-retest reliability on a new group of patients from the Nottingham stroke register who were asked to complete it twice. The new NLQ and other measures were sent to patients in a multicentre rehabilitation trial (TOTAL) six and twelve months after recruitment for postal completion., Subjects: One hundred and thirty-seven consecutive patients from the Nottingham stroke register and 466 patients with a stroke in a multicentre rehabilitation trial., Results: The original NLQ was reduced from 37 to 30 items and from five to three response categories. Data from an earlier study were reanalysed and differences between treatment groups remained. The results of a test-retest analysis using kappa showed that six items had excellent agreement, 15 good and nine fair, suggesting acceptable test-retest reliability. Results from the rehabilitation trial showed that the subjects performed all items and few additional activities were suggested. Higher NLQ scores were associated with higher subscores on the Nottingham Extended Activities of Daily Living Scale (NEADL) and lower NLQ scores with living alone and worse emotional health., Conclusion: The NLQ has been successfully modified for postal self-administration but there is potential for further development.

We studied the relationship between the neuroendocrine and inflammatory responses to hip arthroplasty and functional recovery in 102 patients undergoing elective arthroplasty for osteoarthritis. Blood samples were collected for up to 7 days after surgery and analysed for concentrations of norepinephrine, epinephrine, cortisol, interleukin-6 and C-reactive protein. The primary outcome measures were milestones in hospital, times to walk 10 and 25 m, pain on discharge from hospital, and function 1 and 6 months after surgery. Walking distances in hospital were significantly delayed in patients with greater interleukin 6 and C-reactive protein concentrations, but few neuroendocrine measures had significant correlations with functional recovery in hospital. Multivariate analysis showed that the interleukin 6 concentration on day 1 was the unique predictor of time to walk 10 and 25 m, and that the day 2 concentration of C-reactive protein was the unique predictor of pain on discharge from hospital. No significant correlations were found between the inflammatory and neuroendocrine variables and recovery at 1 and 6 months. We conclude that the inflammatory response affects immediate functional recovery after hip arthroplasty.

The complement system is the primary effector of humoral immunity. Because of its enormous destructive capacity, mechanisms for confining the activity of the system to the desired target and elaborate safeguards for protecting self against complement-mediated injury have evolved. Human cells, particularly those found at sites of inflammation (e.g., hematopoietic and endothelial cells), express highly specialized membrane constituents that act independently or in concert with plasma regulatory proteins to inhibit the functional activity of complement. Decay-accelerating factor (DAF), or CD55, directly inhibits the formation and stability of the amplification C3 and C5 convertases of both the classical and the alternative pathways. Failure of a cell to regulate the amplification C3 and C5 convertases allows the generation of the potentially cytolytic membrane attack complex (MAC), or C5b-9 (consisting of the complement components C5b, C6, C7, C8, and C9). The primary cellular regulator of the MAC is the membrane inhibitor of reactive lysis (MIRL), or CD59, which restricts complement-mediated lysis by blocking assembly of the MAC (primarily at the stage of C9 binding and polymerization). This unit provides a basic protocol for isolating CD55 and CD59, along with two support protocols describing separate functional assays for CD59 and CD55.

Objective: To evaluate the effects of leisure therapy and conventional occupational therapy (OT) on the mood, leisure participation and independence in activities of daily living (ADL) of stroke patients 6 and 12 months after hospital discharge., Design: Multicentre randomized controlled trial., Setting and Participants: Four hundred and sixty-six stroke patients from five UK centres., Main Outcome Measures: The General Health Questionnaire (12 item), the Nottingham Extended ADL Scale and the Nottingham Leisure Questionnaire, assessed by post, with telephone clarification., Results: Four hundred and forty (94%) and 426 (91%) subjects were alive at 6 and 12 months, respectively. Three hundred and seventy-four (85% of survivors) and 311 (78% of survivors) responded at 6 and 12 month follow-up respectively. At six months and compared to the control group, those allocated to leisure therapy had nonsignificantly better GHQ scores (-1.2: 95% CI -2.9, +0.5), leisure scores (+0.7, 95% CI -1.1, +2.5) and Extended ADL scores (+0.4: 95% CI -3.8, +4.5): the ADL group had nonsignificantly better GHQ scores (-0.1: 95% CI -1.8, +1.7) and Extended ADL scores (+1.4: 95% CI -2.9, +5.6) and nonsignificantly worse leisure scores (-0.3: 95% CI -2.1, +1.6). The results at 12 months were similar., Conclusion: In contrast to the findings of previous smaller trials, neither of the additional OT treatments showed a clear beneficial effect on mood, leisure activity or independence in ADL measured at 6 or 12 months.

Changes in circulating levels of catecholamines, cortisol, glucose, interleukin-6 and C-reactive protein and in the leucocyte count were investigated for 7 days after surgery in 158 patients undergoing hip or knee arthroplasty. We compared the responses to the two operations, and also examined the effects of pathology (osteoarthritis and rheumatoid arthritis) on the changes associated with knee arthroplasty. Exploratory factor analysis was applied to the data to identify the variables and sampling times that could be used in future to provide a concise description of the response. Patients undergoing knee arthroplasty showed significantly greater changes in noradrenaline, adrenaline and glucose levels, but not in cortisol levels, compared with those undergoing hip arthroplasty. Interleukin-6 and C-reactive protein concentrations were also significantly greater in knee patients than hip patients; however, when corrected for pathology, many of these differences were not significant. Minimal effects of pathology (chronic inflammation with rheumatoid arthritis) were found on the hormonal changes in knee patients. In particular, there was little evidence to support the inference from animal data that the hypothalamic-pituitary-adrenal axis is impaired. The expected increases in interleukin-6 and C-reactive protein concentrations were found in the rheumatoid arthritis patients. Exploratory factor analysis showed that the response could be separated into six components, accounting for 60% of the total variance, and identified the variables and sampling times indicative of each. In conclusion, there are differences in the hormonal, but not inflammatory, responses to hip and knee arthroplasty. Little evidence was found for an important effect of pathology on the changes associated with knee surgery. Factor analysis provided a useful summary of the data.

Compelling evidence indicates that mutations in PIG-A are necessary for the development of paroxysmal nocturnal hemaglobinuria (PNH), however, it is unclear why mutant PIG-A stem cells have a selective advantage. Further, multiple, discrete PIG-A mutations have been detected in the peripheral blood and bone marrow of patients with PNH, but the contribution of the different mutant clones to hematopoiesis is variable. This observation implies that factors in addition to mutant PIG-A influence the proliferative properties of the abnormal cells. To investigate the etiology of the selective advantage and the clonal dominance in PNH, gene expression in cells with mutant PIG-A was analyzed. Representational difference analysis was used to compare the pattern of cDNA expression between a human lymphoblastoid cell line with mutant PIG-A and its wild-type counterpart. These experiments demonstrated that the pattern of gene expression was different between the two cells lines in that the PIG-A mutant cells failed to express antiquitin mRNA. Transfection of the mutant cells with normal PIG-A restored expression of glycosyl phosphatidylinositol anchored proteins but not antiquitin. These experiments demonstrate that differences in the pattern of gene expression can occur independent of the PIG-A mutation. Depending upon the functional properties of the involved genes, these differences could influence the proliferative properties of PIG-A mutant cells and contribute to the selective advantage and clonal dominance that characterize PNH., (Copyright 1999 Wiley-Liss, Inc.)

Study Objective: This study was conducted as part of the MRC cognitive function and ageing study. It aimed to estimate the lifetime prevalence of self reported physical illnesses and other health related events, and the prevalence of limiting disability in people over 65 in six areas of England and Wales., Design: Screening phase of a two stage prevalence study., Setting: Geographically delimited areas in four urban and two rural areas including institutions., Participants: Random population samples of people in their 65th year and above on the sample definition date, interviewed between 1989 and 1994. In Newcastle, Nottingham, and Oxford (urban) and in Cambridgeshire and Gwynedd(rural), the sample was stratified to provide equal numbers in the 65-74 and 75 years and over age groups. In Liverpool (urban), equal numbers in the five year age groups were taken., Main Results: Age standardised prevalences were calculated for each geographical area, sex, and age group (65-74, 75+). Many conditions were more prevalent in the older age group including stroke, Parkinson's disease, arthritis, diabetes, and shingles but hypertension was more common in the younger age group. Conditions that were more prevalent in men included angina, heart attack, stroke, head injury, and peptic ulcers while hypertension, shingles, pernicious anaemia, and thyroid disease were more common in women. There was a complex pattern of area differences for individual conditions. Cambridgeshire had generally low prevalences for many diseases, including vascular problems, Gwynedd and Newcastle had less healthy elderly populations, and Nottingham and Newcastle had the highest percentages of housebound., Conclusions: This study provides the most robust available estimates for life-time prevalence of a variety of health conditions on a regional and national basis. It shows the greatly increased prevalence of disability in the very old population, particularly women.

We have compared the pharmacokinetics of cisatracurium with atracurium when given by bolus dose followed by continuous infusion. Twenty healthy patients were anaesthetised with thiopentone, midazolam, fentanyl and 70% nitrous oxide in oxygen. Ten patients (Group C) were randomly allocated to receive cisatracurium 0.1 mg.kg-1 and 10 patients (Group A) were given atracurium 0.5 mg.kg-1. Neuromuscular block was monitored using a mechanomyograph. When the first twitch of the train-of-four had recovered to 5% of control, an infusion of cisatracurium 3 micrograms.kg-1.min-1 was started in Group C and an infusion of atracurium 10 micrograms.kg-1.min-1 was started in Group A. The infusion rates were adjusted to maintain the first twitch of the train-of-four at 5% of control. The times to 90% and maximum depression of the first twitch of the train-of-four were significantly longer after cisatracurium than atracurium (2.2 and 3.4 min compared with 1.3 and 1.8 min, respectively; p < 0.01 in each instance). No significant differences were found in recovery parameters between the two groups. Blood samples were taken at regular intervals following the bolus, during the infusion and for 8 h thereafter. The plasma samples were analysed using high-performance liquid chromatography for cisatracurium and atracurium (using a method which distinguishes between the three geometric isomer groups), laudanosine and monoquaternary alcohol. The results were analysed using the Non-linear Mixed Effects Model program. A two-compartment model was fitted to the data. The different isomer groups of atracurium have different pharmacokinetics, the trans-trans group having the highest clearance (1440 ml.min-1) and the cis-cis group the lowest (499 ml.min-1). The clearance of cisatracurium (425 ml.min-1) is less than that of cis-cis atracurium and its elimination half-life is longer (34.9 min and 21.9 min, respectively). The plasma concentration of laudanosine after cisatracurium was one-fifth of that after atracurium.

Aims: To determine the effects of hepatic cirrhosis on the pharmacodynamics and pharmacokinetics of rocuronium bromide., Methods: We studied 21 healthy patients and 17 patients with mild or moderate cirrhosis (Child-Pugh Class A and B). Patients were premedicated with diazepam orally; anaesthesia was induced with fentanyl and thiopentone, and maintained with isoflurane 0.6% (end-tidal) and nitrous oxide 66% in oxygen. The compound action potential of the adductor pollicis muscle in response to supramaximal stimulation of the ulnar nerve was recorded using the train-of-four (TOF) twitch technique. A bolus dose of rocuronium 0.6 mg kg(-1) was then given. Venous blood samples were taken for up to 8 h, and plasma rocuronium concentrations determined by h.p.l.c., Results: The time to onset of neuromuscular block and maximal block achieved did not differ between the two groups. The mean (s.d.) recovery times were prolonged in the cirrhotic compared with the healthy group: 25% recovery T1:T0, 53.7 (18.1) vs 42.3 (14.2) min; 50% recovery T1:T0, 73.9 (33.9) vs 52.6 (19.8) min; 75% recovery T1:T0, 84.2 (24.5) vs 66.8 (27.2) min (all P<0.05); recovery of T4:T1 to 70%, 114.9 (31.7) vs 76.1 (28.8) min (P<0.01). A pharmacokinetic and pharmacodynamic model was fitted to the data for each patient. Three compartments were used to model the pharmacokinetic data; an effect compartment was added to model the pharmacodynamic data. Plasma clearance was significantly reduced in the cirrhotic group (2.66 (0.60) vs 3.70 (1.03) ml kg(-1) min (-1); P<0.005). The central (V1) and steady state volumes of distribution (V(ss)) did not differ significantly between the groups. The slow redistribution (t1/2,lambda1) and elimination (t1/2,z) half-lives were both significantly prolonged in cirrhosis (28.3 (12.1) vs 16.8 (4.6) min, P < 0.005; and 143 (80) vs 92 (40) min, P < 0.05 respectively). The exit rate constant for the effect compartment k(eo) was significantly increased in the cirrhotic group (0.25 (0.18) vs 0.16 (0.06) min(-1); P < 0.05), but cirrhosis had no significant effect on the parameters of the concentration-effect relationship Cp(ss)(50) and gamma., Conclusions: Hepatic elimination is an important pathway in the clearance of rocuronium, and delayed disposition causes the effect to be prolonged.

Human immunodeficiency virus type 1 (HIV-1) can be either resistant or sensitive to complement-mediated destruction depending on the host cells. Incorporation of different levels of host cell CD46, CD55 and CD59 may account for this differential sensitivity to complement. However, it has not been determined whether CD46, CD55 and CD59 can all be incorporated at levels which protect virions. To determine whether each of these proteins can protect HIV-1, virions were derived from CHO cells expressing either human CD46, CD55 or CD59. Virions were shown to incorporate both glycosyl phosphatidylinositol (GPI)-anchored CD55 and CD59 as well as transmembrane CD46. Importantly, all three virus preparations were significantly more resistant to complement lysis than control virus. This study demonstrates that HIV-1 incorporates both transmembrane and GPI-anchored complement control proteins from host cells and that both types of protein increase complement resistance of virus.

Congenic C5-deficient and C5-sufficient mice were infected with group B streptococci (GBS) to determine if the polymorphonuclear leukocyte (PMNL) chemoattractant C5a contributes to PMNL recruitment in GBS infection and if GBS C5a-ase reduces C5a-induced PMNL recruitment in vivo. PMNL accumulation was greater in the peritoneum and air spaces of C5-sufficient mice than in C5-deficient mice. Administration of human C5 to C5-deficient mice caused a significant increase in PMNL recruitment following infection with C5a-ase-negative GBS. GBS C5a-ase did not reduce PMNL accumulation in C5-sufficient mice but reduced PMNL recruitment in C5-deficient mice reconstituted with human C5. These data indicate that C5a is important for rapid PMNL recruitment to sites of GBS infection and that GBS C5a-ase inactivates human, but not murine, C5a in vivo. Reduction of the acute inflammatory response by C5a-ase likely contributes to GBS virulence in human neonates.

Procoagulant albumin (Pro-Alb) is an anionic form of albumin isolated from normal human plasma that regulates vascular endothelial cell hemostatic properties, including induction of tissue factor activity. We investigated the biochemical modification of Pro-Alb that was associated with procoagulant-inducing activity. Tryptic digestion of Pro-Alb identified greatest bioactivity in the carboxy-terminus of the molecule, a region associated with lipid binding sites. Activated charcoal treatment and phopholipase C digestion reduced the procoagulant-inducing activity of Pro-Alb, and Pro-Alb contained 2.3-fold more phosphorus than inactive albumin. We conclude that modification of albumin by phospholipid imparts tissue factor-inducing activity to Pro-Alb.

Stroke patients often fail to resume full lives, even if they make a good physical recovery, and social and leisure pursuits show a particular decline. The usual goals of rehabilitation are mobility and independence in self-care, but recovery in a broader sense may be impeded if health professionals concentrate exclusively on these. Leisure has been shown to be closely associated with life satisfaction and would be a worthwhile, and now measurable, goal of rehabilitation. Elderly people show a decline in leisure activity which has been studied extensively and may provide a useful model for the more rapid decline seen in stroke patients. Further research is needed to confirm the finding that specialized occupational therapy can be effective in raising leisure activity, and to show whether this will translate into improved psychological well-being.

Membrane inhibitor of reactive lysis (MIRL, CD59) is an 18-kDa glycosylphosphatidylinositol-anchored protein that regulates formation of the membrane attack complex of complement. The purpose of these studies was to characterize the gene that encodes CD59. Our experiments redefined the structural organization of the gene by identifying a previously unrecognized alternatively spliced exon. Analysis by PCR of cDNA derived from a variety of cultured human cell lines and from PBMC showed that transcripts containing the alternatively spliced exon sequence were expressed concordantly with transcripts lacking that sequence. Primer extension studies demonstrated that the transcriptional start site of alternatively spliced CD59 mRNA is the same as that of transcripts without the alternatively spliced exon sequence, suggesting that expression of both forms of CD59 mRNA is regulated similarly. Analysis of the promoter region showed that the first 70 nucleotides immediately 5' of the transcriptional start site of the CD59 gene are essential for both constitutive and PMA-responsive transcription; however, responsiveness to PMA is cell line specific. Together, these studies have redefined the organization of the CD59 gene and identified regions of the promoter involved in constitutive and PMA-inducible transcription.

A 10-year-old girl with paroxysmal nocturnal hemoglobinuria (PNH) received an infusion of syngeneic bone marrow without preparative marrow ablation or immunosuppression. Following transplant, the patient became asymptomatic in concordance with an increase in the percentage of peripheral blood cells with normal expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). However, molecular analysis suggested engraftment of a relatively small number of donor stem cells and persistence of an abnormal stem cell with mutant PIG-A. During 17 months of observation, the percentage of cells with normal GPI-AP expression gradually decreased, while intravascular hemolysis progressively increased. Approximately 16.5 months post-transplant, the patient once again became symptomatic. Together, these results indicate that syngeneic marrow infusion provided a clinical benefit by increasing the proportion of erythrocytes with normal expression of GPI-anchored complement regulatory proteins without supplanting the abnormal stem cells. However, evidence of insidious disease progression following the marrow infusion implies that the abnormal stem cells have a survival advantage relative to the transplanted stem cells. Thus, these studies contribute in vivo data in support of the hypothesis that PNH arises as a consequence of a pathological process that selects for hematopoietic stem cells that are GPI-AP-deficient.

The purpose of this review is to summarize recent studies that have led to a more complete understanding of the molecular basis of paroxysmal nocturnal hemoglobinuria (PNH). Somatic mutations of PIG-A arising in pluripotent hematopoietic stem cells are necessary for the development of PNH. PIG-A is an X-linked gene that is essential for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that serves as a membrane anchor for a functionally diverse group of cell surface proteins. Consequently, the progeny of stem cells with mutant PIG-A are deficient in all GPI-anchored proteins (GPI-AP). Among the GPI-AP that are expressed on hematopoietic cells are two important regulators of the complement system, decay-accelerating factor, (CD55) and membrane inhibitor of reactive lysis, (CD59). It is the deficiency of erythrocyte CD55 and CD59 that accounts for the intravascular hemolysis and hemoglobinuria that are the clinical hallmarks of PNH. A remarkable feature of PNH is that the peripheral blood is a mosaic composed of variable proportions of GPI-AP+ and GPI-AP- cells and that, in an individual patient, the GPI-AP- cells can be derived from multiple mutant stem cells. Currently, however, there is no evidence that the PIG-A mutation per se provides a proliferative advantage. Thus, PNH is not a monoclonal disease with a malignant phenotype. Rather, the mutant stem cells appear to dominate hematopoiesis because under some pathological conditions, GPI-AP deficiency is advantageous. The close association of PNH with aplastic anemia suggests that the selection pressure arises as a consequence of a specific type of bone marrow injury.

Analysis of the X-linked gene PIG-A from haemopoietic cells of a female PNH patient showed a homozygous C-55-T substitution that caused replacement of arginine with tryptophan at codon 19. Aval restriction analysis of PIG-A cDNA demonstrated that the patient was homozygous for this mutation, whereas her mother was heterozygous and her father was hemizygous. Flow cytometry, however, showed normal expression of glycosyl phosphatidylinositol anchored proteins on blood cells of the patient's mother and father. Therefore the C-55-T mutation is an inherited sequence variant that does not account for the PNH phenotype of this patient.

The hypertriglyceridemia commonly observed in uremia has been attributed to an abnormally high inhibitor activity in plasma for lipoprotein lipase (LPL) and hepatic lipase (HL), both of which have a key role in lipoprotein metabolism. The purpose of this investigation was to establish a relationship between plasma lipase inhibitor activity and hypertriglyceridemia, identify the main plasma lipase inhibitor, and determine the basis for the greater inhibitor activity in uremia. In a mixed population of normal (N = 8) and uremic subjects (N = 12), log-transformed plasma triglycerides correlated with both inhibitor activity and uremic status. However, inhibitor activity was the only retained predictor variable for triglycerides in a multiple linear regression model (r = 0.91; P < 0.0001). An inhibitor isolated from normal plasma was identified as a particle containing apolipoprotein A-I (apo A-I) and 3% phospholipid. This particle, which has pre-beta electrophoretic mobility and a Stokes' radius of 54 A, therefore corresponds to a form of the previously described pre-beta-HDL (free apo A-I) in the non-lipoprotein fraction of plasma. Comparison of normal and uremic plasma indicated that the greater lipase inhibitor activity in the latter could be attributed to an increased concentration of apo A-I in the non-lipoprotein fraction of plasma (pre-beta-HDL), as well as to increased inhibition by the uremic lipoproteins. The increased plasma lipase inhibitor activity may be important in the pathogenesis of hypertriglyceridemia in chronic renal failure.

The purpose of these studies was to determine the molecular basis of the phenotypic mosaicism that is a defining feature of paroxysmal nocturnal hemoglobinuria (PNH). Analysis of T cell clones from a female patient revealed four distinct phenotypes based on surface expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). When PIG-A (the gene that is mutant in PNH) from these clones was analyzed, four discrete somatic mutations were identified. Analysis of X chromosomal inactivation among the abnormal T cell clones was consistent with polyclonality. Together, these studies demonstrate that the phenotypic mosaicism that is characteristic of PNH is a consequence of genotypic mosaicism and that, at least in this case, PNH is a polyclonal rather than a monoclonal disease. That four distinct somatic mutations were present in a single patient suggests that in conditions that predispose to PNH PIG-A may be hypermutable.

We have studied 12 critically ill, sedated patients who required a neuromuscular blocking drug to assist mechanical ventilation in an intensive care unit. Patients were randomized to receive an infusion of cis-atracurium 0.18 mg kg-1 h-1 (group 1, n = 6) or atracurium 0.6 mg kg-1 h-1 (group 2, n = 6) preceded, if necessary, by a bolus dose of 2 x ED95 of the same drug (cis-atracurium 0.1 mg kg-1 or atracurium 0.5 mg kg-1). Neuromuscular block was monitored using an accelerograph and the infusion rate adjusted regularly so that it was possible to detect the first response to train-of-four (TOF) stimulation of the ulnar nerve at the wrist. Blood samples were obtained for estimation of plasma cis-atracurium and laudanosine concentrations (group 1) or the three groups of atracurium isomers and laudanosine (group 2). There was no apparent haemodynamic or allergic response to either drug. The mean infusion time in group 1 was 37.6 h and in group 2, 27.5 h. On termination of the infusion, the time for the TOF ratio to reach 0.7 was similar in the two groups (group 1 = 60 min; group 2 = 62 min). The mean infusion rate of cis-atracurium was 0.19 mg kg-1 h-1 and of atracurium 0.47 mg kg-1 h-1 (expressed as mg of bis-cation): cis-atracurium was 2.5 times more potent than atracurium. Using the NONMEM program, a single compartment pharmacokinetic model was fitted to the plasma concentrations of cis-atracurium and the cis-cis, cis-trans and trans-trans isomers of atracurium. The mean population pharmacokinetic values for cis-atracurium were: volume of distribution (V) = 21,900 (SEM 416) ml; clearance (Cl) = 549 (79) ml min-1; half-life (T1/2) = 27.6 (3.6) min; and for the three groups of atracurium isomers were: cis-cis, V = 15,100 (720) ml, Cl = 449 (42) ml min-1, T1/2 = 23.4 (1.2) min; cis-trans, V = 18,000 (667) ml, Cl = 1070 (43) ml min-1, T1/2 = 11.7 (0.1); trans-trans, V = 13,100 (1280) ml, Cl = 1560 (55) ml min-1, T1/2 = 5.8 (0.4) min. Plasma laudanosine concentrations were lower in the cis-atracurium (peak value 1.3 micrograms ml-1) than in the atracurium (maximum 4.4 micrograms ml-1) group.

Vascular endothelium regulates multiple aspects of platelet function through secretion of a variety of substances, including von Willebrand factor, nitric oxide, and prostacyclin (PGI2). The objective of this study was to determine whether procoagulant albumin (P-A1), a modified form of albumin present in normal human plasma could modulate endothelial cell secretion of these substances. P-A1 did not affect constitutive secretion of von Willebrand factor or nitric oxide, but did increase PGI2 secretion in a time- and concentration-dependent manner. Pre-treatment of endothelial cells with aspirin, or use of suramin, a broad-specificity inhibitor, prevented the response to P-A1. Prostaglandin H synthase-2 contributed to the P-A1-induced PGI2 secretion. These results indicate that in addition to inducing tissue factor activity and reducing protein C activation and fibrinolysis, P-A1 also modulates vascular endothelial cell PGI2 secretion, and potentially, platelet function.

The current study was undertaken to determine whether the human T cell leukemia/lymphoma oncovirus type I (HTLV-I) and the herpesvirus human cytomegalovirus (HCM) incorporate host cell-derived C regulatory proteins. Our experiments showed that both CD59 and CD55 were associated with the external membrane of HTLV-I derived from MT2 cells, since virus could be captured by mAbs to these proteins, and antisera to CD55 and CD59 induced C-mediated lysis of HTLV-I virions. Additionally, both CD55 and CD59 were detected by immunoblot analysis of purified HTLV-I. Purified HCMV produced in human foreskin fibroblasts (HFF) also contained both CD55 and CD59, as detected by immunoblot analysis. However, treatment with anti-CD55, but not anti-CD59, reduced the HCMV infectious titer in the presence of C. Additional studies determined whether HTLV-I-associated CD55 and CD59 participated in the resistance of the virus to C-mediated lysis. Treatment of virus with phosphatidylinositol-specific phospholipase C (PI-PLC), which removes glycosylphosphatidylinositol-anchored CD55 and CD59, increased the sensitivity of HTLV-I to C-mediated destruction in the presence of anti-HTLV-I Abs. Reconstitution of PI-PLC-treated virus with purified CD55 and CD59 restored resistance to C. These experiments show that HTLV-I and HCMV acquire C control proteins from host cells. Together with our previous experiments showing that both CD55 and CD59 are present on HIV-1, these studies demonstrate a mechanism by which a variety of enveloped viruses may acquire resistance to C-mediated destruction.

To ascertain the effects of chronic renal failure on the pharmacokinetics of 1R-cis 1'R-cis atracurium besylate (a stereoisomer, designated 51W89), we gave a bolus dose of 0.1 mg kg-1 (2 x ED95) to 17 patients with end-stage renal failure and to 15 patients with normal renal function undergoing elective surgery. All patients received thiopentone, fentanyl and midazolam i.v. and 70% nitrous oxide in oxygen. Blood samples were obtained over 8 h and plasma analysed for 51W89 and laudanosine concentration, using high pressure liquid chromatography. A two-compartment model was fitted to the 51W89 plasma concentration data using the NONMEM program, to estimate pharmacokinetic variables and to determine the influence of renal failure, age, weight and sex. Clearance of 51W89 was found to be reduced by 13% in renal failure. The typical value of T1/2 beta was 4.2 min longer in renal failure than in the healthy patients (34.2 vs 30.0 min, P < 0.005). In the healthy patients, clearance of 51W89 was greater in males, but it decreased with increasing age by approximately 1.5 ml min-1 yr-1. Mean plasma laudanosine concentrations were significantly higher in the renal failure group; nevertheless, they were approximately one-tenth of those reported after atracurium.