Photodynamic inactivation of normal and antifungal resistant Candida species.

BACKGROUND: Susceptibility of bacterial and fungal species to the photodynamic killing effects of various photosensitizing dyes has received increasing attention. In the oral cavity oral candidiasis is primarily caused by Candida albicans. Evidence suggests that Oropharyngeal Candidiasis, found frequently in patients with immunodeficiency, present with mixed Candida organisms and are more difficult to treat than those solely due to C. albicans. In the present study we demonstrate the ability to efficiently kill antifungal resistant isolates of Candida using Photofrin induced PDT. METHODS: Candida strains from the ATCC as well as fluconazole and amphotericin B resistant and sensitive isolates from adults with AIDS were grown cultures and grown under standard conditions. Photofrin was added to appropriate cultures as dictated by experimental design. Light was delivered to assigned cultures using a 630 nm laser source at a power density of 150 mW/cm(2), for appropriate time to deliver 45-135 J/cm(2). Colony forming assays were used to determine survival. RESULTS: After illumination cultures treated with Photofrin had significant reduction in colony forming ability at all light doses examined. Isolates from AIDS patients which had demonstrated antifungal resistance showed equivalent sensitivity to photodynamic killing as did control ATCC cultures of the same strain. CONCLUSION: This study demonstrates Photofrin induced PDT can eliminate Candida species with significant efficiency as revealed by colony forming ability. Further Candida isolates from AIDS patients that had demonstrated fluconazole and amphotericin B resistance were equally susceptible to photodynamic killing.

Identifying packaging errors.

Common medical device packaging problems are identified here together with their cause and the remedy. The discussion examines the causes of poor heat seal, misleading results from the measurement of seal strength, blister flange curl, damage to sterile barrier system and failure of the ethylene oxide sterilisation process.

The DNA sequence and biological annotation of human chromosome 1.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

Apolipoprotein-D: a novel cellular marker for HGPIN and prostate cancer.

BACKGROUND: High grade prostatic intraepithelial neoplasia (HGPIN) is a putative pre-malignant lesion of the prostate. While apolipoprotein-D (Apo-D), an androgen-regulated hydrophobic transporter protein, is expressed in prostate tumors, its expression in HGPIN is unknown. METHODS: Immunoreactivity for Apo-D and another androgen-regulated protein, prostate specific antigen (PSA), was investigated in 64 radical prostatectomy tissues by video image analysis. RESULTS: Eighty two percent of prostatectomy specimens demonstrated moderate to strong Apo-D immunoreactivity in areas of HGPIN. In comparison, weak Apo-D immunoreactivity was observed in non-malignant areas in only 24% of specimens. The median (range) percentage cellular area of HGPIN immunopositive for Apo-D (9.7%, 0-42.9), and the cellular concentration of Apo-D (MIOD 3.1, 0-13.3), were intermediate between that of normal (area 0%, 0-53.5%, MIOD 0, 0-12.6) and early stage prostate cancer tissues (area 29.2%, 0-90.8%, MIOD 6.7, 0-28.1). This increase in Apo-D expression from non-malignant, through HGPIN to prostate cancer was statistically significant (P < 0.001), and contrasted with the decrease observed in PSA staining between adjacent areas of normal glands, HGPIN, and cancer (P = 0.026). CONCLUSIONS: The presence of high levels of immunoreactive Apo-D in HGPIN and prostate cancer, but not in non-malignant epithelial cells, is consistent with HGPIN being an intermediate lesion in the transition to prostate cancer, and suggests that cellular Apo-D expression is a marker of malignant transformation of the prostate.

Use of bidirectional blots in differential display analysis.

We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-beta, TNF-alpha, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA (32)P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis.

Induction of leukemia cell differentiation and apoptosis by recombinant P48, a modulin derived from Mycoplasma fermentans.

P48 is a 48-kDa monocytic differentiation/activation factor which was originally identified in the conditioned medium of the Reh and other leukemia cell lines and has recently been shown to be a Mycoplasma fermentans gene product. Previously, conditioned medium P48 has been shown to induce differentiation of HL-60 (human promyelocytic leukemia) cells. Recently our laboratory isolated cDNA clones for P48 from Reh cells and genomic clones from Mycoplasma fermentans and expressed the recombinant protein as a maltose binding protein (MBP) fusion protein in E. coli. In this report we present the initial characterization of this recombinant P48 fusion protein (rP48-MBP). We show that rP48-MBP induces differentiation of HL-60, U937 (human histiocytic lymphoma), and M1 (mouse myeloid leukemia) cell lines. Interestingly, rP48-MBP also induces apoptosis of U937 and HL-60 cells as assessed by terminal transferase (TUNEL) assays. This is the first report of induction of apoptosis by a Mycoplasma gene product. P48 is a Mycoplasma-derived immunomodulatory molecule which has differentiation and apoptosis-inducing activities and may be important in the pathophysiology of Mycoplasma infections. The recombinant protein may be useful in studying the mechanisms of differentiation, cytokine production, and apoptosis in malignant and nonmalignant hematopoietic cells.

Expression of the monocytic differentiation/activation factor P48 in Mycoplasma species.

P48 is a 48 kd monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukemia line. It induces differentiation of HL-60 promyelocytic leukemia cells along the monocytic pathway and production of IL1, TNF-alpha and IL6 in human monocytes and monocytic cell lines. Recently our laboratory isolated cDNA clones for P48 from Reh cells and genomic clones from Mycoplasma fermentans DNA and showed that P48 is a M. fermentans gene product. In this paper we report the analysis of P48 expression at the DNA, mRNA and protein levels in different Mycoplasma species. Polymerase Chain Reaction (PCR) analysis of extracted DNA using P48-specific oligonucleotide primers revealed P48 sequences in M. fermentans but not M. hominis, M. iowae, M. genitalium or M. capricolum. Southern analysis of Mycoplasma DNAs revealed hybridizing bands in M. fermentans and M. capricolum under low stringency, but only in M. fermentans under high stringency. Consistent with this, Northern blot studies revealed a single hybridizing transcript in M. fermentans but not in other Mycoplasma species tested. However, Western blot studies with anti-P48 antibodies revealed P48 antigenic material in M. fermentans, as well as M. hominis and M. iowae. These studies demonstrate that the gene for P48 is derived from M. fermentans or a closely related species and is absent in these other species tested. However, the P48 protein exhibits shared antigenic determinants among several Mycoplasma species which presently are of unknown function or significance. P48 is a Mycoplasma -derived immunomodulatory molecule which may be important in Mycoplasma pathophysiology and may be useful in understanding human haematopoietic differentiation and the control of cytokine biosynthesis.

Hematopoietic differentiation activity of a recombinant human interleukin-6 (IL-6) isoform resulting from alternatively spliced deletion of the second exon.

We have previously identified and cloned an alternatively spliced form of human interleukin-6 mRNA lacking exon II, which encodes amino acid residues known to be important in gp130-mediated signal transduction pathways. We expressed and purified the recombinant protein (rIL6-alt) resulting from this alternatively spliced mRNA and now report the initial characterization of its biologic activities with comparison to full length IL6 (rIL6-full). rIL6-alt was found to have 10(4) to 10(5) fold less activity in proliferation assays with 7TD1 murine plasmacytoma cells and did not competitively inhibit the stimulatory activity of rIL6-full. In addition, like rIL6-full, rIL6-alt had antiproliferative activity toward M1 murine myeloblast cells and was 10-200-fold less active than rIL6-full. In contrast, in assays with human HL60 promyelocytic leukemia cells, rIL6-alt had greater antiproliferative activity than rIL6-full and more strongly upregulated phagocytosis as well as surface expression of the differentiation antigen CD11b. rIL6-full and rIL6-alt upregulated the level of lysozyme mRNA in HL60 cells approximately equally. These findings suggest that IL6-alt, which lacks amino acid residues encoded by the second exon of the gene, is not a natural inhibitor of IL6-full but may be relatively tissue specific and may play a role in modulation of hematopoietic cell growth and differentiation.

Variation in hospital rates of intraaortic balloon pump use in coronary artery bypass operations.

BACKGROUND: Little is known about regional patterns of intraaortic balloon pump (IABP) use in coronary artery bypass graft (CABG) operations. Our objectives were (1) to identify clinical variables associated with IABP use, and (2) to examine risk-adjusted rates of IABP use for 12 Massachusetts hospitals performing CABG operations. METHODS: We used hospital discharge data to identify 6944 CABG surgical cases. Logistic regression was used to identify clinical variables associated with IABP use, and the resulting multivariate model was then used to risk adjust hospital rates of IABP use. RESULTS: The IABP was used in 13.4% of the CABG surgical cases. The clinical variables independently associated with IABP use were cardiogenic shock, same admission angioplasty, prior CABG operation, cardiac arrest, congestive heart failure, recent myocardial infarction, and urgent admission status. Risk-adjusted rates of IABP use varied widely across hospitals from 7.8% to 20.8% (p < 0.0001). CONCLUSIONS: Hospital rates of IABP use vary considerably in Massachusetts. This practice variation may be related to the persistent uncertainty regarding the precise clinical indications for the IABP in this patient population.

Oestrogen receptor-alpha variant mRNA expression in primary human breast tumours and matched lymph node metastases.

We have shown previously that the relative expression of a truncated oestrogen receptor-alpha variant mRNA (ER clone 4) is significantly increased in axillary node-positive primary breast tumours compared with node-negative tumours. In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-alpha variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-alpha variant mRNAs appeared conserved between any primary and its matched secondary tumour.

Identifying pre- and postoperative predictors of cost and length of stay for coronary artery bypass surgery.

Prior studies of resource use for coronary artery bypass graft (CABG) surgery have either focused on a limited number of hospitals or have used charges instead of costs. We used a large statewide database (n = 6791) to study predictors of cost and length of stay (LOS) for CABG surgery. We used linear regression to sequentially model (a) specific procedures performed, (b) preoperative patient characteristics, and (c) postoperative events to determine the relative impact of these 3 factors on resource use. We then used the resulting models to calculate adjusted mean hospital costs and LOS. These 3 factors were all significantly associated with resource use. Postoperative events were the greatest determinant of costs, while preoperative characteristics were the greatest determinant of LOS. Despite risk adjustment for these factors, resource use differed significantly across 12 hospitals (mean cost range, $22,200 to $41,900; mean LOS range, 11 to 18 days), suggesting that some institutions may need to reduce their resource use.

Prostate-specific antigen and gross cystic disease fluid protein-15 are co-expressed in androgen receptor-positive breast tumours.

Androgens regulate breast cancer cell proliferation via androgen receptor (AR)-mediated mechanisms. To investigate further the androgen-responsiveness of human breast tumours, we examined the immunohistochemical expression of the AR and two androgen-regulated proteins, prostate-specific antigen (PSA) and gross cystic disease fluid protein-15 (GCDFP-15), in 72 primary breast tumours. AR immunoreactivity was present in the nuclei of breast tumour cells and was correlated with oestrogen receptor (ER; P < 0.05) and progesterone receptor (PR; P < 0.01) status. PSA and GCDFP-15 immunoreactivity was present in the cytoplasm of tumour cells but not the adjacent stromal cells. AR-positive cells were present in 85% (61/72) of breast tumours, and 98% (43/44) of PSA-positive and 92% (44/48) of GCDFP-15-positive tumours were also positive for AR. Positive immunoreactivity for both PSA and GCDFP-15 in breast tumours was highly dependent on AR status (odds ratios of 24.0 and 4.5 respectively), but unrelated to age, ER and PR status and axillary lymph node involvement. PSA immunoreactivity was more frequently observed in moderate and well-differentiated tumours and was significantly (P < 0.001) associated with GCDFP-15 immunoreactivity. In conclusion, PSA and GCDFP-15 immunoreactivity was dependent on the presence of AR, but not ER or PR in primary breast tumours.

Evaluation of complication rates after coronary artery bypass surgery using administrative data.

Our objectives were (1) to determine if studying hospital complication rates after coronary artery bypass graft (CABG) surgery provides information not available when only mortality is studied, and (2) to reexplore the utility of ICD-9-CM administrative data for CABG outcomes assessment. Using data from Massachusetts, we identified CABG cohorts from 1990 and 1992 to respectively develop and validate multivariate risk adjustment models predicting in-hospital mortality and complications. The resulting models had good discrimination and calibration. In 1992, adjusted hospital complication rates ranged widely from 13.0% to 57.6%, while mortality rates ranged from 1.4% to 6.1%. Hospitals with high complication rates tended to have high mortality (r = 0.74, p = 0.006), but 2 of the 12 hospitals studied ranked quite differently when judged by complications rather than mortality. We conclude that (1) complications after CABG occur frequently and may provide information about hospital quality beyond that obtained from hospital mortality rates, and that (2) administrative data continue to be a promising resource for outcomes research.

Role of the androgen receptor in human breast cancer.

Although the androgen receptor (AR)3 is often co-expressed with the estrogen receptor (ER) and progesterone receptor (PR) in human breast tumors, its role in breast cancer is poorly understood. Specific growth stimulatory and inhibitory actions of androgens have been described in human breast cancer cell lines. The mechanisms by which androgens exert these contrasting growth effects are unknown. A commonly utilized second line therapy for the treatment of advanced breast cancer is high dose medroxyprogesterone acetate (MPA). Although MPA, a synthetic progestin, was thought to act exclusively through the PR, the androgenic side-effects observed in women taking MPA suggest that its action may also be mediated in part by the AR. In support of this hypothesis, the level of AR measured by radioligand binding in primary breast tumors was correlated with the duration of response to MPA treatment following failure of tamoxifen therapy. Recent data suggest that the presence of structurally altered AR in breast cancers may account for unresponsiveness to MPA in some of these cases. Further studies are warranted to determine the role of AR mediated pathways in regulating breast tumor growth. In particular, identification of androgen-regulated genes may lead to new possibilities for the hormonal treatment of breast cancer.

Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer.

The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.

Hospital cost of complications associated with coronary artery bypass graft surgery.

We identified 6,791 coronary artery bypass grafting (CABG) cases using the Massachusetts hospital discharge data to quantify the contribution of complications to the cost of a hospitalization for CABG. After adjusting for in-hospital mortality and baseline clinical severity as other contributors to cost, the additional costs associated with complications were substantial.

Treatment of dyskeratosis congenita with granulocyte colony-stimulating factor and erythropoietin.

Aplastic anaemia is both frequent and difficult to manage in patients with dyskeratosis congenita (DC). We recently treated a 23-year-old male for a year with granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Ep), with an excellent neutrophil response, and a transient effect on haemoglobin levels. G-CSF alone or combined with other cytokines may provide at least a partial effect in pancytopenic patients with DC.

Statewide quality improvement initiatives and mortality after cardiac surgery.

BACKGROUND: Recent reports from New York and northern New England claim that statewide quality improvement initiatives and outcome reporting are leading to decreased mortality following coronary artery bypass graft (CABG) surgery. OBJECTIVE: To compare trends in mortality after CABG surgery in Massachusetts (a state that has not instituted statewide outcome reporting) with the decreases reported from New York and northern New England. DESIGN: Surgical cohorts from 1990, 1992, and 1994 were used to evaluate the risk-adjusted mortality trend for Massachusetts. We present this trend along with the published trends from New York and northern New England. For comparison, we also present unadjusted Medicare mortality trends from Massachusetts, New York, northern New England, and the entire United States. SETTING: All 12 Massachusetts hospitals performing cardiac surgery (excluding a Veterans Affairs hospital). PATIENTS AND DATA SETS: Massachusetts administrative data were used to identify all patients undergoing isolated CABG surgery in 1990, 1992, and 1994. MAIN OUTCOME MEASURES: Observed and risk-adjusted in-hospital mortality. RESULTS: Observed mortality rates in Massachusetts decreased from 4.7% in 1990 to 3.5% in 1992 and to 3.3% in 1994. The corresponding risk-adjusted mortality reductions for 1992 and 1994 (relative to 1990) were 35% and 42%, respectively. The mortality reduction seen in Massachusetts is comparable to the reductions seen in New York and northern New England over similar periods. Unadjusted Medicare mortality trends were generally similar in the states under study, and in the United States as a whole. CONCLUSIONS: In-hospital mortality after CABG surgery has decreased in Massachusetts despite the absence of statewide outcome reporting. Direct program evaluations are needed to better characterize the efficacy of the ongoing statewide outcome studies in New York and northern New England.

cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product.

P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line. It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-alpha and interleukin 6 in human monocytes and monocytic cell lines. The cDNA for P48 was cloned from Reh cellular RNA using 3' reverse amplification of cDNA ends. Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells. Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated from Mycoplasma fermentans but not M. hominis, M.iowae, M.synoviae or M.lypophilum. Although initial studies using Mycoplasma culture and hybridization techniques had failed to reveal Mycoplasma infection in our Reh and Molt-4 cell lines, subsequent PCR studies using Mycoplasma genus-specific rRNA primrs revealed Mycoplasma sequences in these cell lines. Using the P48 cDNA probe, we isolated a genomic clone from M. fermentans DNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium. The 5' end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein. The P48 cDNA was expressed in pMAL in Escherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots. This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification. Consistent with this, Northern blot studies revealed a single 1 kb transcript. The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium. These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived from M. fermentans or a closely related species. P48 may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis.