Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-kappaB.

The promoters of the IL-8, MCP-1, and RANTES genes contain binding sites for the redox-responsive transcription factors AP-1 and NF-kappaB, which have been shown to be important for their expression. In this overview, we present evidence from our laboratories that the stimulus-specific regulation of these chemokines by the reactive oxidant H2O2, the proinflammatory cytokine TNF-alpha, and respiratory syncytial virus (RSV) is mediated in a cell type-specific manner involving different patterns of AP-1 and NF-kappaB binding activity. Our results demonstrate that H2O2 induction of IL-8 gene expression is linked with the selective binding of AP-1 to the IL-8 promoter, whereas TNF-alpha and RSV induction of IL-8 correlates with the activation of NF-kappaB binding. We propose that the differential activation and binding of inducible transcription factors to the promoter regions of chemokine genes provides a critical regulatory mechanism by which the CXC and CC chemokines can be selectively expressed in a cell type-specific and stimulus-specific manner. Such a regulatory mechanism of differential chemokine expression could critically influence the site-specific recruitment of distinct subsets of leukocytes to sites of inflammation and injury.

Human cell line that differentiates to all myeloid lineages and expresses neutrophil secondary granule genes.

The aim of this study was to characterize a human leukemic cell line that appears capable of spontaneous differentiation to all myeloid lineages. The MPD cell line was derived using standard tissue culture techniques from the peripheral blood of a patient with an aggressive nonchronic myelogenous leukemia myeloproliferative disorder. Immunophenotyping, cytogenetic analysis, reverse transcriptase polymerase chain reaction, Northern blotting, immunoblotting, and colony assays were used to characterize the line and to assess its ability to express lineage-specific genes representative of advanced differentiation.Light microscopic morphologic analysis of the MPD cell line suggests that it has the unique property of spontaneous differentiation to mature-appearing neutrophils, macrophages, eosinophils, and basophils in proportions that approximate those found in normal bone marrow or peripheral blood. It was demonstrated that this cell line is capable of producing lineage-specific mRNA and granule proteins of at least two myeloid lineages, neutrophil and eosinophil, including neutrophil secondary granule proteins, which are not expressed in other available human cell lines. MPD cells were found to be capable of producing differentiated myeloid colonies (neutrophil, eosinophil, macrophge, mixed) in semisolid medium. The ability of MPD cells to express genetic programs associated with advanced differentiation of multiple myeloid lineages will make it a valuable tool for the study of the processes underlying lineage commitment and the regulation of expression of lineage-specific genes.

Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo.

A persistent obstacle that has hampered gene transfer experiments is the short-term nature of transgene expression in vivo. In this article we present evidence for sustained expression from primary human keratinocytes, using the retroviral vector MFG. Primary keratinocytes were transduced in culture with the MFG retroviral vector containing the coding region from factor IX cDNA. Transduced keratinocytes, which secreted on average 830 ng of factor IX/10(6) cells/24 hr in tissue culture, were used to form a bilayered skin equivalent and grafted onto nude mice under a silicone transplantation chamber. Between 0.1 and 2.75 ng of human factor IX per milliliter was found in mouse plasma for more than 1 year, suggesting that keratinocyte stem cells were both transduced and grafted. The results show, for the first time, that long-term expression is obtainable in retrovirally transduced keratinocytes after transplantation.

An ex vivo keratinocyte model for gene therapy of hemophilia B.

We are investigating whether skin-targeted gene therapy may be used to treat hemophilia B by transplanting keratinocytes transduced by factor IX-expressing retroviral vectors. No pre-clinical animal model for keratinocyte-mediated gene therapy has shown long-term efficacy in vivo. It remains unclear whether this short-term expression is due to promoter shut-off or a reduced survival of grafted genetically modified cells. The purpose of this study was to determine the fate of primary human keratinocytes superficially grafted to nude mice in a silicone transplantation chamber. In addition, vectors containing keratinocyte-specific enhancers from the human papilloma virus-16 (HPV-16) and human keratin 5 and 14 genes were used upstream of the cytomegaloviral (CMV) immediate-early promoter/enhancer to control factor IX cDNA expression to avoid promoter shut-off. Factor IX was secreted by cultured keratinocytes after transduction by each of these chimeric promoter/enhancer vectors, although the levels varied according to the particular construct used. Keratinocytes transduced by the vector containing the HPV-16 enhancer were grafted into nude mice, and human factor IX was detected in plasma at 0.02-9 ng per ml for 4-5 wk for the duration of graft survival. The HPV-16 enhancer may be a useful addition to expression vectors for keratinocyte gene therapy. The transplantation chamber can be adapted to grafting retrovirally transduced keratinocytes for gene transfer studies.

Planning and documentation. Addressing patient needs in a day surgery setting.

Day surgery has reduced the time allowed for patient contact, but it has not reduced the need for assessment, planning, and delivery of patient care. Day surgery, however, has shifted some responsibility from nurses to patients and their families. Our goals in developing a preoperative assessment plan were to ensure consistent and thorough patient assessment; enhance problem identification and guide appropriate interventions; provide evaluations based on the needs of the day surgery patient; assist patients and families in assuming increased responsibility for postoperative care; provide achievable and manageable documentation of patient information gathered in a compressed amount of time; and facilitate communication between the patient and the health care team. We feel our assessment documenting system meets these goals and allows nurses and patients to adapt to the changes in traditional roles both parties experience when day surgery procedures are performed.

Complications and range of motion following plate fixation of metacarpal and phalangeal fractures.

Eighty-two patients with 105 metacarpal and/or phalangeal fractures stabilized with plates were retrospectively reviewed to assess complications and outcomes. Despite stable fixation and early mobilization, major complications were encountered in 36% of fractures, especially with phalangeal and open fractures. Complications included stiffness, nonunion, plate prominence, infection, and tendon rupture. Forty-eight of 63 (76%) metacarpal fractures and 44 of 66 (67%) closed fractures had a final range of motion greater than 220 degrees; however, only 4 of 37 (11%) phalangeal fractures and 8 of 34 (24%) open fractures achieved this outcome. Despite technical advances in plate design and instrumentation, including lower-profile titanium plates, complications occur commonly with metacarpal and phalangeal fractures, leading to a high incidence of unsatisfactory results. We do not condemn plate fixation, and attribute many of our unsatisfactory results to the frequent use of plates in open and phalangeal fractures.

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy.

HaCaT cells, a spontaneously immortalised, nontumorigenic keratinocyte line, were used as a more amenable model than primary keratinocytes for ex vivo-mediated gene transfer. These cells were transduced with retroviral vectors containing the factor IX cDNA under the control of a cytomegaloviral (CMV) promoter/enhancer alone or as hybrids with either the human papilloma virus-16 (HPV-16), keratin 14 (hK14) or keratin 5 (hK5) regulatory elements. Unlike primary keratinocytes, HaCaT cells tolerated transduction and G418 selection well. The HPV-16 and hK5 hybrid constructs were disproportionately more active in primary keratinocytes than in the basal-like HaCaT cells. After skin grafting to athymic mice, transduced HaCaT cells differentiated to form a stratified epidermis that remained viable for at least 99 days in some mice. Factor IX in plasma of mice grafted with vectors containing the HPV-16 and hK5 elements was two- to three-fold higher than with vectors containing the CMV promoter alone. These results are consistent with the expected up-regulation in differentiated suprabasal cells by the HPV-16 and hK5 elements. Enhancers may be useful in specifically targeting the differentiated layer of the epidermis or achieving higher levels of gene expression after transplantation.

Surgical manpower, beds and output in the NHS: 1967-1977.

The availability and use of surgical manpower and beds and certain measures of surgical workload were examined in the NHS in England and Wales from 1967 to 1977 using routine health statistics. Amongst the surgical specialties, there was no consistent relationship between changes in levels of manpower and beds and operating output. For example, ENT surgery and cardiothoracic surgery had more staff and fewer beds in 1977 than in 1967, but operating output in ENT surgery decreased by 18 per cent and in cardiothoracic surgery increased by 28 per cent. Although the efficiency of bed use may have improved (average length of stay was 10.5 days in 1967 and 8.8 days in 1977), the overall use of available beds in most specialties may have decreased. The number of operations performed in each specialty per consultant surgeon was less in 1977 than 1967 except for traumatic and orthopaedic surgery. Although interpretations of routine health statistics are rarely conclusive, the results of this study suggest the possibility of a less than optimum use in 1977 compared to 1967 of surgical beds and surgeons' operating potential which might be due to lack of other resources such as usable theatre time.

Stimulation of neutrophil interleukin-8 production by eosinophil granule major basic protein.

We evaluated the ability of eosinophil granule major basic protein (MBP) to stimulate interleukin (IL)-8 production by neutrophils. MBP over the concentration range of 0.1 to 10 microM stimulated the release of up to approximately 8 ng/ml IL-8. Incubation with 2 microM MBP showed that, after a 1 h lag, the level of IL-8 release increased with time for approximately 10 h. At the 2 microM concentration, eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase did not stimulate significant levels of IL-8 production. MBP stimulated 2-fold increases in IL-8 messenger RNA (mRNA) after 1 and 3 h of incubation, which were blocked by pretreatment with actinomycin D. However, stimulation with MBP did not produce an increase in the binding activity of nuclear factor (NF)-kappaB or activator protein-1. No NF-IL-6 binding activity was detected in the same nuclear extracts. In addition, stimulation with MBP prolonged the stability of IL-8 mRNA. MBP also induced transient increases in mRNA for macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, but did not stimulate the release of either chemokine. These findings indicate that MBP is selective among the eosinophil granule proteins as a stimulus for neutrophil IL-8 release and, further, that stimulation of neutrophil IL-8 release by MBP involves both transcriptional and posttranscriptional regulation. We postulate that MBP-induced release of IL-8 by neutrophils may contribute to the pathophysiology of acute asthma and other inflammatory lung diseases.

A novel and highly divergent homolog of human eosinophil granule major basic protein.

Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.