Biomedical subjects
Publications and source records attributed to C T Pham.
Evidence on topical negative pressure from randomised controlled trials, non-randomised comparative studies and case studies is considered. This is the first systematic review on this therapy to consider results by wound type.
OBJECTIVE: To determine if and to what extent postnatal women's preferences for birth outcomes differ from those of midwives and medical staff, and whether any variations in utility scores are associated with demographic variables. DESIGN: Cross sectional cohort study. SETTING: The Women's and Children's Hospital, Adelaide. POPULATION: A total of 180 participants which included 90 postnatal women, 59 midwives and 31 medical staff. METHODS: Preferences (utility scores) were measured by direct interviews using utility techniques: the visual analogue scale and the standard gamble. MAIN OUTCOMES MEASURES: Preferences (utility scores) for eight birth outcomes. RESULTS: Women assigned higher utility scores for the five birth outcomes of jaundice requiring phototherapy, admission to neonatal nursery, shoulder dystocia, nerve palsy and transient neurological symptoms than midwives, which suggested that women regarded these outcomes as less severe (P < 0.01). Utility scores for the women and medical staff were similar. The majority of postnatal women, midwives and medical staff preferred permanent neurological sequelae to perinatal death. Eighty-nine percent of postnatal women preferred permanent neurological sequelae to perinatal death compared with 71% of midwives (P < 0.01), and 68% of medical staff (P < 0.01). CONCLUSION: Utility values for important birth outcomes varied between women who had recently given birth and health professionals. Clinical practice should recognise and respect the preferences of women, with appropriate balance between their preferences, those of health professionals and the known benefits of care.
Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI -/- mice. We find that DPPI -/- mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI -/- mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI -/- BMMCs is 25% that of DPPI +/- BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.
Cathepsin G is a neutral serine protease that is highly expressed at the promyelocyte stage of myeloid development. We have developed a homologous recombination strategy to create a loss-of-function mutation for murine cathepsin G. Bone marrow derived from mice homozygous for this mutation had no detectable cathepsin G protein or activity, indicating that no other protease in bone marrow cells has the same specificity. Hematopoiesis in cathepsin G-/- mice is normal, and the mice have no overt abnormalities in blood clotting. Neutrophils derived from cathepsin G-/- mice have normal morphology and azurophil granule composition; these neutrophils also display normal phagocytosis and superoxide production and have normal chemotactic responses to C5a, fMLP, and interleukin-8. Although cathepsin G has previously shown to have broad spectrum antibiotic properties, challenges of mice with Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli yielded survivals that were not different from those of wild-type animals. In sum, cathepsin G-/- neutrophils have no obvious defects in function; either cathepsin G is not required for any of these normal neutrophil functions or related azurophil granule proteases with different specificities (ie, neutrophil elastase, proteinase 3, azurocidin, and/or others) can substitute for it in vivo.
To date, the normal transcriptional regulation of the human beta-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human beta-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human beta-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F(1) mice showed that 2 lines express human beta globin at levels approximately equivalent to the endogenous mouse beta-major genes. One line expresses no human beta globin, while the remaining 6 lines show intermediate expression levels. Complete gamma-->beta-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic-->adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.
Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that has been implicated in the processing of granzymes, which are neutral serine proteases exclusively expressed in the granules of activated cytotoxic lymphocytes. In this report, we show that cytotoxic lymphocytes derived from DPPI-/- mice contain normal amounts of granzymes A and B, but these molecules retain their prodipeptide domains and are inactive. Cytotoxic assays with DPPI-/- effector cells reveal severe defects in the induction of target cell apoptosis (as measured by [(125)I]UdR release) at both early and late time points; this defect is comparable to that detected in perforin-/- or granzyme A-/- x B-/- cytotoxic lymphocytes. DPPI therefore plays an essential role in the in vivo processing and activation of granzymes A and B, which are required for cytotoxic lymphocyte granule-mediated apoptosis.
Human granzyme H is a neutral serine protease that is expressed predominantly in the lymphokine-activated killer (LAK)/natural killer (NK) compartment of the immune system. The gene that encodes this granzyme is located between the granzyme B and cathepsin G genes on human chromosome 14q11.2. Although the murine orthologue of human granzyme H has not yet been identified, murine granzymes C, D, E, F, and G also lie between the murine granzyme B and cathepsin G genes on murine chromosome 14; murine granzymes C, D, and F are also highly expressed in LAK cells, but minimally in cytotoxic T lymphocytes (CTL). We therefore tested whether the 5' flanking region of human granzyme H contains the cis-acting DNA sequences necessary to target a reporter gene to the LAK/NK compartment of transgenic mice. A 1.2-kb fragment of 5' flanking human granzyme H sequence was linked to an SV40 large T-antigen (TAg) reporter gene and used to create six transgenic founder lines. SV40 TAg was specifically expressed in the LAK cells of these mice, but not in resting T or NK cells, in CTL, or in any other tissues. Most mice eventually developed a fatal illness characterized by massive hepatosplenomegaly and disseminated organ infiltration by large malignant lymphocytes. Cell lines derived from splenic tumors were TAg+ and NK1.1(+) large granular lymphocytes and displayed variable expression of CD3, CD8, and CD16. Although these cell lines contained perforin and expressed granzymes A, B, C, D, and F, they did not exhibit direct cytotoxicity. Collectively, these results suggest that the 5' flanking sequences of the human granzyme H gene target expression to an NK/T progenitor compartment and to activated NK (LAK) cells. Mice and humans may therefore share a regulatory "program" for the transcription of NK/LAK specific granzyme genes.
Human CR2 (CD21) is a B lymphocyte protein whose surface expression is restricted primarily to the mature cell stage during development. To study the transcriptional mechanisms that govern cell- and stage-restricted CR2 expression, we first performed transient transfection analysis using constructs extending from -5 kb to +75 bp (-5 kb/+75) in the CR2 promoter. The promoter was found to be broadly active, with no evidence of cell- or stage-specific reporter gene expression. However, the addition of a 2.5-kb intronic gene segment (containing a DNase I hypersensitive site) to the (-5-kb/+75) construct resulted in appropriate reporter gene expression, defined as the silencing of the (-5-kb/+75) promoter activity only in non-CR2-expressing cells. Interestingly, appropriate reporter gene expression required stable transfection of the constructs in cell lines, suggesting nuclear matrix or chromatin interactions may be important for appropriate CR2 gene expression. Importantly, transgenic mice also required the intronic silencer to generate lymphoid tissue-specific reporter gene expression. Some transgenic founder lines did not demonstrate reporter gene expression, however, indicating that additional transcriptional regulatory elements are present in other regions of the CR2 gene. In summary, these data support the hypothesis that human CR2 expression is regulated primarily by an intronic silencer with lineage- and B cell stage-specific activity.
Granzyme B (GzmB) is a neutral serine protease found in cytotoxic lymphocytes; this enzyme is critically involved in delivering the rapid apoptotic signal to susceptible target cells. GzmB has been difficult to study and has not yet been produced in non-mammalian systems because of the complex processing events that are thought to be required for its activation. In this report, we have successfully produced fully active, soluble recombinant GzmB (rGzmB) in a yeast-based system by fusing GzmB cDNA in frame with yeast alpha-factor cDNA, using the yeast KEX2 signal peptidase to release the processed enzyme into the supernatant of yeast cultures. We expressed the proenzyme form of GzmB as well and determined that pro-GzmB is efficiently converted to its active form by the cysteine proteinase dipeptidyl peptidase I. The fully processed enzyme was able to hydrolyze the synthetic substrate N-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 17 s-1 and catalytic efficiency kcat/Km of 181,237 M-1 S-1; the recombinant enzyme is therefore at least twice as active as purified native GzmB. In addition, the recombinant enzyme hydrolyzes Boc-Ala-Ala-Met thiobenzyl ester with a kcat of 3.2 S-1 and a catalytic efficiency kcat/Km of 65,306 M-1 S-1. Purified rGzmB can also cleave the putative substrate caspase-3 into its signature p20/p10 forms. Unlike caspases, rGzmB is not sensitive to inhibition by several peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK, as well as Zn2+ (a known inhibitor of caspase-3). Structural studies of rGzmB may allow us to better understand the substrate specificity of this enzyme and to design better inhibitors.
CD8+ cytotoxic lymphocytes, natural killer cells and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4+ T cells utilize Fas and other mechanisms to induce cell death. The molecular mechanisms used by these pathways to induce target cell apoptosis may converge on common death substrates.
Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that catalyzes the sequential removal of dipeptides from the amino termini of various protein substrates. We have isolated a cDNA coding for murine DPPI from mouse thymus and spleen cDNA libraries. The deduced amino acid sequence codes for a protein of 462 amino acid residues; comparison of this deduced sequence with that of rat and human DPPI revealed 90.1% and 77.8% identity, respectively. Using DPPI cDNA, we obtained two BAC (Bacterial Artificial Chromosome) clones that contained the murine DPPI locus. The DPPI gene consists of seven exons and 6 introns, and spans approximately 20 kilobases. Using fluorescence in situ chromosome hybridization, we localized murine DPPI to chromosome 7D3-E1.1. We determined that DPPI protein is widely distributed in mouse tissues, although its relative abundance varies from tissue to tissue. In contrast to previous reports, we show here that DPPI mRNA and protein levels and enzymatic activity are unchanged during in vitro T cell activation, implying that this enzyme is not rate-limiting for granzyme processing.
Granzymes are neutral serine proteases that are stored in the specialized lytic granules of cytotoxic lymphocytes. A mutation introduced into the granzyme B locus leads to a severe defect in the ability of cytotoxic lymphocytes to induce apoptosis in susceptible target cells, and reduces the severity of class I-dependent acute graft-versus-host disease (GvHD). However, granzyme B-independent cytotoxicity also exists: in CD8+ cells, most of it is perforin-dependent, but in CD4+ cells, the Fas system and an additional pathway are involved. The identification of these pathways and their physiological relevance may lead to new approaches for inhibiting cytotoxic lymphocyte functions.
BACKGROUND: Rheumatoid neutrophilic dermatitis (RND) is a recently recognized, rare cutaneous manifestation of rheumatoid arthritis. It occurs in patients with severe rheumatoid arthritis and is typically asymptomatic. Rheumatoid neutrophilic dermatitis was originally described by Ackerman in 1978. Since that time, 8 patients with this disease have been described in the literature. OBSERVATIONS: We report 2 cases of RND. Findings of skin biopsy specimens from both patients revealed characteristic signs of dermal leukocytosis and leukocytoclasia without vasculitis. The pathogenesis of the neutrophilic infiltrate is unclear. Processes that may play a role in the pathogenesis of RND include immune complex activations, cell adhesion and migration, and cytokine release. CONCLUSIONS: Rheumatoid neutrophilic dermatitis falls into the spectrum of neutrophilic vascular reactions described by Jorizzo and Daniels. Although early reports suggest that prominent leukocytoclasia is not a feature of RND, our findings confirm the observations of Lowe et al that leukocytoclasia can be seen in RND and may be striking. It is important for dermatologists to be aware of this rare manifestation of rheumatoid arthritis.
Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in "knockout" mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the beta-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5' gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the beta-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3' to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these-long distance effects are unknown, the expression of PGK-Neo can be "captured" by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.
BACKGROUND: Topical PUVA therapy has become a useful alternative for patients who cannot tolerate the systemic side effects of nausea and headache or are concerned about the ophthalmologic risk associated with oral PUVA therapy. However, there is no study to date on the systemic absorption of psoralen after the localized application of topical paint PUVA. OBJECTIVE: This study was designed to assess the plasma level of 8-methoxypsoralen (8-MOP) after paint PUVA therapy for patients with palmoplantar psoriasis or eczema. METHODS: Reverse-phase high-pressure liquid chromatography was used to determine 8-MOP plasma levels in eight patients with palmoplantar psoriasis and two with eczema. Three patients receiving oral PUVA therapy served as the control group. RESULTS: Plasma levels of 8-MOP taken 1, 6, and 24 hours after topical PUVA treatments of patients with palmoplantar psoriasis were undetectable. One patient with hand eczema consistently had detectable 8-MOP levels 1 hour after topical PUVA treatments. CONCLUSION: This report indicates that there is minimal, if any, systemic absorption of 8-MOP after topical PUVA treatment of patients with palmoplantar psoriasis.
In order to gain further insight into the mechanisms of calcium (Ca) homeostasis in hypercalciuria, we studied 32 lithiasic patients who were divided into three groups: normocalciuric patients (NC; n = 11), patients with absorptive hypercalciuria (AH; n = 12) and patients with renal hypercalciuria (RH; n = 9). The patients were investigated on 3 occasions: during a random diet, after a Ca-restricted diet and during a Ca tolerance test. The following determinations were made: Ca intake, Ca tubular reabsorption (Ca TR), plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] and parathyroid hormone (PTH) levels, natriuresis and urinary protein-bound Gla. The latter was measured as a marker of urinary nephrocalcin excretion. Ca TR was decreased in hypercalciuric patients (HC). AH but not RH patients normalized their Ca TR during fasting. Plasma PTH and 1,25(OH)2D levels were similar in all the groups on the 3 occasions. Natriuresis was elevated in RH during the fasting period (p < 0.02 vs. AH). Compared with NC, protein-bound Gla urinary excretion rates (UER) were enhanced in AH after the Ca-restricted period (p < 0.02) and in RH during fasting (p < 0.02). In AH, a strong positive correlation was found between Ca TR and protein-bound Gla UER (r = 0.79, p = 0.002) following a Ca-restricted diet. In the HC group as a whole, fasting protein-bound Gla UER were correlated to plasma 1,25(OH)2D levels (r = 0.68, p < 0.001). In conclusion, the results suggest that PTH directly or indirectly through 1,25(OH)2D increases nephrocalcin synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
BACKGROUND: The importance of emotional factors in dermatologic disorders has been recognized for several decades. However, there have been few reports on the management of psychodermatologic disorders. Meanwhile, there has been tremendous progress in the pharmacologic treatment of these conditions. This article describes major categories of psychodermatologic disorders and provides practical management guidelines, emphasizing the use of selected psychopharmacologic agents. OBSERVATIONS: Psychodermatologic conditions can be divided into three broad subgroups: psychophysiologic disorders in which the severity of primary cutaneous disease is influenced by the patients' emotions; primary psychiatric disorders in which the skin conditions are self-induced and reflect underlying psychopathologic conditions; and secondary psychiatric disorders in which the patients experience psychologic problems as a result of disfigurement associated with the skin conditions. There are many ways to manage these psychodermatologic conditions, including psychotherapy, behavioral therapy, and psychopharmacotherapy. To determine which pharmacologic agent is most appropriate, the nature of the underlying psychopathologic condition such as anxiety, depression, psychosis, and obsession/compulsion should be identified. Furthermore, there are nonpsychiatric disorders encountered by dermatologists in which psychopharmacologic agents may be more efficacious than conventional therapeutics. CONCLUSION: We conclude that psychopharmacologic agents provide a useful option to the management of psychodermatologic disorders.
The Vietnamese are the fastest-growing Asian/Pacific Islander ethnic group in California. Data from Vietnam and elsewhere suggest that cervical cancer and breast cancer are major contributors to cancer morbidity and mortality among Vietnamese women. However, little is known about the cancer knowledge and screening practices of Vietnamese women. Using a structured 57-item written questionnaire, we conducted a mailed survey of 400 randomly selected Vietnamese adult women in San Francisco. Overall, 107 women responded (31%). Of the respondents, 54 (52%) indicated that there was little one could do to prevent cancer. More than a third (39%) thought that breast or cervical cancer could be caused by poor hygiene, and about one-third (29%) thought that these cancers could be contagious. Although virtually all (97%) had heard of cancer, one-third did not know that a breast lump could be a sign of breast cancer (37%) or that abnormal vaginal bleeding could be a sign of cervical cancer (39%). Many (55%) did not know that family history was a risk factor for breast cancer and three-fourths (74%) did not know that having multiple sexual partners was a risk factor for cervical cancer. Fourteen (13%) had not heard of breast self-examination. Of 31 women aged > or = 40, 10 (34%) had never had a mammogram, and of 92 women aged > or = 18, 50 (54%) had never had a Papanicolaou test.(ABSTRACT TRUNCATED AT 250 WORDS)