Non-intercepted dose errors in prescribing anti-neoplastic treatment: a prospective, comparative cohort study.

BACKGROUND: The incidence of non-intercepted prescription errors and the risk factors involved, including the impact of computerised order entry (CPOE) systems on such errors, are unknown. Our objective was to determine the incidence, type, severity, and related risk factors of non-intercepted prescription dose errors. PATIENTS AND METHODS: A prospective, comparative cohort study in two clinical oncology units. One institution used a CPOE system with no connection to the electronic patient record system, while the other used paper-based prescription forms. All standard prescriptions were included and reviewed. Doses were recalculated according to the guidelines of each institution, using the patient data as documented in the patient record, the paper-based prescription form, or the CPOE system. A non-intercepted prescription dose error was defined as ≥10% difference between the administered and the recalculated dose. RESULTS: Data were collected from 1 November 2012 to 15 January 2013. A total of 5767 prescriptions were evaluated, 2677 from the institution using CPOE and 3090 from the institution with paper-based prescription. Crude analysis showed an overall risk of a prescription dose error of 1.73 per 100 prescriptions. CPOE resulted in 1.60 and paper-based prescription forms in 1.84 errors per 100 prescriptions, i.e. odds ratio (OR) = 0.87 [95% confidence interval (CI) 0.59-1.29, P = 0.49]. Fifteen different types of errors and four potential risk factors were identified. None of the dose errors resulted in the death of the patient. CONCLUSIONS: Non-intercepted prescribing dose errors occurred in <2% of the prescriptions. The parallel CPOE system did not significantly reduce the overall risk of dose errors, and although it reduced the risk of calculation errors, it introduced other errors. Strategies to prevent future prescription errors could usefully focus on integrated computerised systems that can aid dose calculations and reduce transcription errors between databases.

Effect of enhanced ultraviolet germicidal irradiation in the heating ventilation and air conditioning system on ventilator-associated pneumonia in a neonatal intensive care unit.

OBJECTIVE: The objective of this study was to test the hypothesis that enhanced ultraviolet germicidal irradiation (eUVGI) installed in our neonatal intensive care unit (NICU) heating ventilation and air conditioning system (HVAC) would decrease HVAC and NICU environment microbes, tracheal colonization and ventilator-associated pneumonia (VAP). STUDY DESIGN: The study was designed as a prospective interventional pre- and post-single-center study. University-affiliated Regional Perinatal Center NICU. Intubated patients in the NICU were evaluated for colonization, and a high-risk sub-population of infants <30 weeks gestation ventilated for ≥ 14 days was studied for VAP. eUVGI was installed in the NICU's remote HVACs. The HVACs, NICU environment and intubated patients' tracheas were cultured pre- and post-eUVGI for 12 months. The high-risk patients were studied for VAP (positive bacterial tracheal culture, increased ventilator support, worsening chest radiograph and ≥ 7 days of antibiotics). RESULT: Pseudomonas, Klebsiella, Serratia, Acinetobacter, Staphylococcus aureus and Coagulase-negative Staphylococcus species were cultured from all sites. eUVGI significantly decreased HVAC organisms (baseline 500,000 CFU cm(-2); P=0.015) and NICU environmental microbes (P<0.0001). Tracheal microbial loads decreased 45% (P=0.004), and fewer patients became colonized. VAP in the high-risk cohort fell from 74% (n=31) to 39% (n=18), P=0.04. VAP episodes per patient decreased (Control: 1.2 to eUVGI: 0.4; P=0.004), and antibiotic usage was 62% less (P=0.013). CONCLUSION: eUVGI decreased HVAC microbial colonization and was associated with reduced NICU environment and tracheal microbial colonization. Significant reductions in VAP and antibiotic use were also associated with eUVGI in this single-center study. Large randomized multicenter trials are needed.

Treatment of severe hypodontia-oligodontia--an interdisciplinary concept.

The authors' experience with oral rehabilitation of patients suffering from oligodontia (i.e. six or more congenitally missing permanent teeth, third molars excluded) is reported. The concept is based on an interdisciplinary team approach involving pedodontists, orthodontists, maxillofacial surgeons and prosthodontists. A series of 112 consecutive patients suffering from oligodontia were referred from 1997 to 2001. Ten of the patients (8.9%) suffered from ectodermal dysplasia. The total number of missing teeth was 1126, with an average of 10 per patient. Ninety-two patients had either finished treatment or were on an active treatment schedule. Of these, 97% underwent some kind of orthodontic treatment. Of the 112 patients, 51 had finished treatment at the end of the follow-up period (mean 28 months, range 1-68). Of these, fixed implant-supported prosthetic restoration was used in 90% to replace missing teeth, often combined with alveolar ridge augmentations (73%), sinus floor augmentation (43%), inferior alveolar nerve transposition (18%) and orthognathic surgery (27%). Early diagnosis, and comprehensive treatment planning with good coordination and timing of the individual treatment phases are decisive for a successful treatment outcome. The therapeutic concept is presented with special emphasis on surgical aspects.

Epidermal growth factor receptor gene copy number and protein level are not associated with outcome of non-small-cell lung cancer patients treated with chemotherapy.

BACKGROUND: Survival benefit of non-small-cell lung cancer (NSCLC) patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors is predicted by high EGFR gene copy number and by strong EGFR protein expression. Clinical relevance of these features in patients treated with chemotherapy has not been reported. PATIENTS AND METHODS: This study included 82 NSCLC patients treated with chemotherapy. There were 45% of females, 6% of never smokers and 45% of patients diagnosed with adenocarcinoma. EGFR gene copy number was evaluated by fluorescence in situ hybridization and EGFR protein level by immunohistochemistry. RESULTS: High EGFR gene copy number and protein level were found in 33% and 71% of patients, respectively. Both markers were significantly associated (P = 0.01). For objective response and disease control, there was no difference between patients defined as negative or positive for both EGFR gene copy number (P = 0.39 and P = 1.00, respectively) and for EGFR protein (P = 1.00 and P = 0.80, respectively). There were no differences in progression-free and overall survival according to EGFR gene copy number (P = 0.76 and P = 0.82, respectively) and protein level (P = 0.67 and P = 0.62, respectively). CONCLUSION: In chemotherapy-treated NSCLC patients, EGFR gene copy number was positively associated with protein level but none of the features were predictive for either treatment response or survival.

Evidence for immunological priming and increased frequency of CD4+ CD25+ cord blood T cells in children born to mothers with type 1 diabetes.

Maternal transmission of islet autoantibodies to children born to mothers with type 1 diabetes (T1D) has been shown to protect from autoantibodies and diabetes development later in life. However, the factors conferring disease protection are poorly understood. The aim of this study was to evaluate comparatively proinflammatory cytokines, autoantibodies and lymphocyte subsets in cord blood (CB) of children born to mothers with either T1D (n = 13), gestational diabetes (GDM) (n = 32) or healthy mothers (n = 81) in relation to transplacental passage of autoantibodies. The results are consistent with early priming of the fetal immune system only in children born to mothers with T1D. Levels of interleukin (IL)-1beta (P = 0.022), tumour necrosis factor (TNF)-alpha (P = 0.002) and IL-8 (P = 0.0012), as well as the frequency of CD4(+) CD25(+) T cells (P < 0.01) were significantly increased, and the increased levels correlated positively with anti-GAD65 autoantibody (GADA) levels. Moreover, CD4(+) CD25(+) T cells of children born to T1D mothers exhibited a more pronounced memory phenotype with increased CCR4 expression and down-regulation of CD62L. These data suggest that early activation of the fetal immune system as a consequence of maternal autoimmunity and transplacental passage of GADA may influence the generation and expansion of fetal regulatory T cells. This might induce an early antigen-specific immunological tolerance that could protect against T1D later in life.

Adjuvant oil induces waves of arthritogenic lymph node cells prior to arthritis onset.

A single intradermal injection of the adjuvant-oil squalene induces T cell mediated arthritis in DA rats. The chain of events leading from nonspecific provocation of the immune system to arthritis is largely unknown. Previous studies have demonstrated that lymph node (LN) cells are of pathogenic importance, i.e. cells from LNs draining the injection site can transfer arthritis to naïve DA rats. Recently we have demonstrated cellular uptake of adjuvant oil in draining lymph nodes but also that nondraining LNs become hyperplastic and harbour arthritogenic cells. Here, we aimed to determine from which time-point prior to arthritis onset arthritogenic cells appear in draining inguinal and nondraining axillary/brachial LNs, respectively. We demonstrated that the ability to transfer arthritis was strongly dependent on the time-point after adjuvant-injection with clear-cut differences between draining and nondraining LN cells. Cells harvested at day 5 postinjection (p.i) were not able to transfer arthritis, while at day 8 p.i, a first wave of arthritogenic cells appeared in draining LNs. The ability to transfer arthritis was associated with a pro-inflammatory cytokine profile as indicated by the IL-1beta and IFNgamma expression in cells from draining LNs. Subsequently, at day 11 p.i., just before arthritis onset, arthritogenic cells appeared also in nondraining LNs. These results shed new light on the induction of arthritic diseases, implicating a two step mechanism for the development of pathogenic cells. Firstly, a pro-inflammatory burst in responding lymphoid organs leading to a local pool of arthritogenic cells and, secondly, a transmission of arthritogenecity to other LNs and precipitation of disease in peripheral joints.

Brimonidine formulation in polyacrylic acid nanoparticles for ophthalmic delivery.

In ocular drug delivery, a major problem is providing an adequate concentration of a therapeutic agent in the precorneal area. Mucoadhesive carriers such as polyacrylic acid in sub-colloidal, nanoparticulate form, have a strong potential for ophthalmic drug delivery. A formulation of brimonidine loaded in polyacrylic acid nanoparticles has been prepared for potential delivery in ophthalmic therapy. The particles were prepared by a reverse microemulsion polymerization technique and their sizes were in the range of 50 nm. In a preliminary biocompatibility test, Caco-2 cells (human primary colonic tumour adenocarcinoma) and human corneal epithelial cells incubated with polyacrylic acid nanoparticles were found to retain their viability over varying times. The loading efficiency of the drug brimonidine in the particles was shown to be between 80-85% and pH dependent. The bioadhesive polyacrylic hydrogel nanoparticles, used in the present study, exhibited superior loading properties for brimonidine, and the formulation was stable for more than 5 weeks. When the drug-loaded nanoparticles were dispersed in a phosphate buffer saline (pH = 7.4), the drug was slowly released over several hours. Two-photon laser scanning microscopic studies of dye-conjugated polyacrylic acid nanoparticles demonstrated the accumulation of the particles on the surface and intercellular spaces of Caco-2 cells.

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.

Myeloperoxidase activity as a lung injury marker in the lamb model of congenital diaphragmatic hernia.

PURPOSE: The antioxidant system is the primary intracellular defense system of the lung against oxygen toxicity (neutrophil sequestration). The CDH lamb model antioxidant system is deficient. It is hypothesized that pulmonary neutrophil sequestration may play a part in the acute lung injury of CDH patients. Myeloperoxidase (MPO) is a major constituent of neutrophil cytoplasmic granules and its activity therefore is a direct measure of neutrophil presence and an indirect indicator of lung injury. METHODS: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days' gestation and were delivered by cesarean section at 140 to 145 days. Eight littermate lambs served as controls. Lambs were either killed before ventilation or were ventilated conventionally for 4 hours with 100% O(2) and then killed. The lungs were dissected en bloc and snap frozen. The samples were homogenized, sonicated, freeze-thawed, and separated by density centrifugation. Supernatants were analyzed for myeloperoxidase (MPO) activity by spectrophotometry with o-dianisidine dihydrochloride and hydrogen peroxide at 460 nm. The MPO activity was normalized to the protein content of the supernatant and expressed as units of MPO activity per milligram of protein. RESULTS: There was significantly more MPO activity in the CDH-ventilated lungs than controls similarly ventilated (3,203 +/- 665 versus 1,220 +/- 194, P =.001). There was no difference in MPO activity between the CDH and control lungs (318 +/- 57 v 348 +/- 61; P =.5). There was no difference between right and left lungs in any group. CONCLUSION: Ventilation and hyperoxia leads to neutrophil accumulation in lung tissue, which is most pronounced in the CDH lung tissue. This is a further clue to the pathophysiology of iatrogenic lung injury in CDH. The myeloperoxidase assay may now be used to evaluate antenatal or postnatal antioxidant therapies for iatrogenic lung injury in CDH.

Upregulation of keratinocyte growth factor in the tracheal ligation lamb model of congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) carries a high mortality rate of 60% because of associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. Prenatal tracheal ligation has been shown to improve lung growth in experimental models. This could be caused by a direct effect of increased endothoracic pressure in utero, secondary to the induction of specific growth factors, or both. Keratinocyte growth factor (KGF) is involved in normal lung organogenesis and is a potent mitogen of alveolar type II cells. The authors have therefore investigated the protein and mRNA levels of keratinocyte growth factor in the lung tissue of control, CDH, and CDH tracheal ligation lambs. METHODS: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days' gestation. Tracheal ligation was performed at 110 days in 4 lambs, and they were delivered by cesarean section at 140 to 145 days. Twin littermates served as controls. The lungs were dissected en bloc and snap frozen. KGF protein levels were determined by ELISA. Total RNA was isolated, and a RNase protection assay was performed using an ovine cDNA probe for KGF, and a human cDNA probe for GAPDH (house keeping control). Densitometric analysis was used to quantify the relative amounts of mRNA in each sample. RESULTS: There was a significant decrease in the KGF protein levels of the CDH samples (110 v 73.2 pg/mg protein, P =.02). This decrease was mirrored by a significant fall in the level of mRNA expression for KGF (0.694 v 0.235, P = .02). Tracheal ligation normalized the KGF protein levels (96.1 pg/mg protein). This elevation of KGF protein was accompanied by an upregulation of KGF gene expression to control levels (0.56). CONCLUSIONS: Tracheal ligation clearly is accompanied by an upregulation of keratinocyte growth factor protein and gene expression. It is not yet clear whether keratinocyte growth factor is solely responsible for the growth observed in these tracheal ligation preparations. Further growth factor blocking experiments are required.

Responses of the rat immune system to arthritogenic adjuvant oil.

T-cell mediated inflammatory joint diseases with similarities to rheumatoid arthritis (RA) can be triggered in arthritis-prone rat strains by intradermal injection of adjuvant oils. The pathogenesis of oil-induced arthritis (OIA) remains elusive, and a largely unresolved question is how the rat immune system responds to arthritogenic oils such as incomplete Freund's adjuvant (IFA). Here we report that IFA already induces increased plasma levels of the acute-phase reactants (APR) fibrinogen and alpha1-acid glycoprotein at day 4 postinjection (p.i). In contrast, no early responses were detected in the joints before infiltration of the T cells, which coincided with arthritis onset at 11-14 days post injection (d.p.i.) The infiltrating cells were possibly derived from draining lymph nodes (LN), which were hyperplastic and contained increased cell numbers from 4 days p.i. and onwards. The magnitude of the early increase in cell numbers and APR was regulated by non-major histocompatibility complex (MHC) genes, as determined by comparison between arthritis-susceptible DA rats and arthritis-resistant but MHC-identical LEW.lAV1 and PVG.1AV1 rats. Arthritisprone DA rats developed a weak acute-phase response, suggesting that this systemic response may be counteracting disease. The DA rats also had the largest early increase in LN-cell numbers, suggesting that the LN hyperplasia is part of a disease pathway. The analysis of hyperplastic LN after in vivo labelling with bromodeoxyuridine (BrdU) revealed increased numbers and proportions of proliferating lymphocytes, including T cells. Furthermore, polymerase chain reaction (PCR)-analysis of LN cytokine mRNA revealed upregulation of interleukin (IL)-1beta at 4 d.p.i. We conclude that adjuvant oil exposure triggers both systemic acute phase reactions and local activation of the peripheral lymphoid system. These responses are genetically regulated and may determine arthritis development and susceptibility.

Depressed immunity and impaired proliferation of hematopoietic progenitor cells in patients with complete spinal cord injury.

The bone marrow is supplied with both sensory and autonomic neurons, but their roles in regulating hematopoietic and immunocompetent cells are unknown. Leukocyte growth and activity in patients with stable and complete spinal cord injuries were studied. The innervation of the bone marrow below the injury level lacked normal supraspinal activity, that is, a decentralized bone marrow. Lymphocyte functions were markedly decreased in injured patients. Long-term colony formation of all hematopoietic cell lineages, including dendritic cells, by decentralized bone marrow cells was substantially reduced. It was concluded that nonspecific and adaptive lymphocyte-mediated immunity and growth of early hematopoietic progenitor cells are impaired in patients with spinal cord injuries. Possibly, this reflects cellular defects caused by the malfunctioning neuronal regulation of immune and bone marrow function.

IL-13 can substitute for IL-4 in the generation of dendritic cells for the induction of cytotoxic T lymphocytes and gene therapy.

Immunization with tumor-associated antigen pulsed dendritic cells (DC) has been shown to elicit both protective and therapeutic antitumor immunity in a variety of animal models and is currently being investigated for the treatment of cancer patients in clinical trials. In this study we show that DC can be generated from peripheral blood mononuclear cells of healthy donors as well as breast and melanoma cancer patients using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13) and that these DC have many of the same characteristics as DC differentiated using GM-CSF and IL-4. The DC generated in GM-CSF and IL-13 are CD14- and express high levels of the cell surface markers CD86, HLA-DR, and CD58, as do DC generated in GM-CSF and IL-4. The purity and yield of both DC populations are not significantly different. Furthermore, both populations of DC are effective at presentation of alloantigen as determined in a mixed lymphocyte response, and both are able to process and present soluble tetanus toxoid antigen to CD4+ T cells. Because we are interested in the generation of DC for antigen-specific cytotoxic T lymphocyte (CTL) generation, we compared the ability of peptide-pulsed DC differentiated in GM-CSF and IL-4 versus GM-CSF and IL-13 for the generation of influenza and MART-1 specific CTL. Both populations of DC induced CD3+ CD8+ CD4- and CD56- CTL, which could lyse the appropriate targets in an antigen-specific manner. Finally, both GM-CSF and IL-4 DC and GM-CSF and IL-13 DC yielded similar beta galactosidase expression levels after transduction with recombinant adenovirus containing the LacZ gene. These results suggest that DC generated in GM-CSF and IL-13 may be useful for immunotherapy and gene therapy protocols.

Tracheal ligation and mechanical ventilation do not improve the antioxidant enzyme status in the lamb model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: The antioxidant enzyme (AOE) system is the primary intracellular defense system of the lung against oxygen toxicity. The authors recently demonstrated depressed levels of catalase, glutathione peroxidase, and superoxide dismutase in congenital diaphragmatic hernia (CDH) lambs compared with controls. The aim of this study was to determine whether tracheal ligation (TL) or mechanical ventilation (recently shown to stimulate growth and surfactant metabolism, respectively) could induce an elevation in AOE activity. METHODS: Four nonventilated lambs with surgically created CDH and TL and five CDH lambs ventilated for 4 hours were studied. Lung tissue was analyzed for AOE and the results compared with untreated CDH lambs. RESULTS: Both ventilation and TL failed to elevate AOE activity above that of untreated CDH lambs. CONCLUSIONS: The data provide further evidence that TL does not improve lung metabolism or maturation. Mechanical ventilation, which often involves high oxygen delivery, is a necessary and often beneficial therapeutic modality. In the CDH neonate compromised not only by low baseline levels of the AOE, but also by an inability to induce enzyme synthesis in response to hyperoxia, mechanical ventilation may, by causing lung injury, be contributing to the high morbidity and mortality rate associated with CDH.

Lung physiological and metabolic changes in lambs with congenital diaphragmatic hernia after administration of prenatal maternal corticosteroids.

BACKGROUND/PURPOSE: Improved outcomes of preterm infants born to mothers treated prenatally with corticosteroids have been documented. The authors investigated the role of prenatal maternal corticosteroid therapy in congenital diaphragmatic hernia (CDH). METHODS: Five CDH lambs of ewes given 0.5 mg/kg betamethasone intravenously 24 hours before delivery (single-dose), four CDH lambs of ewes similarly dosed at 48 and 24 hours before delivery (double-dose), five untreated CDH lambs and five control lambs were studied. After 2 hours of ventilation, compliance, arterial oxygen (PO2) and carbon dioxide (pCO2) concentrations were recorded. Lavage protein and phospholipid levels were measured, and lung tissue was analyzed for antioxidant enzyme activity (AOE). RESULTS: No improvement in gas exchange was noted in either treatment group. Significant increases in compliance (P = .02) were noted in the double-dose steroid group, which were different from that of untreated CDH lambs or controls. Minimal changes in AOE activities were seen with steroid administration. CONCLUSIONS: Although the metabolic changes were not significant, the marked improvement in compliance seen in the double-dosed steroid group suggests a potential role for prenatal maternal corticosteroids in CDH. Further timing and dosage studies are warranted in this model.

Contractile properties of intralobar pulmonary arteries and veins in the fetal lamb model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Pulmonary hypertension plays a significant role in the pathophysiology of congenital diaphragmatic hernia (CDH). Although there has been an intensive research effort directed at mediators that may cause pulmonary vasoconstriction, no single agent has been identified. The authors hypothesize that there may be an alteration in the cGMP-nitric oxide (NO) pathway of vasodilatation contributing to the pulmonary hypertension observed in CDH. The purpose of these studies is to begin to elucidate vasoactive properties of pulmonary vessels with particular attention to the cGMP-NO pathway of vasodilatation in fetal lambs with CDH. METHODS: Fourth-generation pulmonary arteries and pulmonary veins were dissected from both right and left lungs of eight, 139-day gestational fetuses with surgically created CDH. Vessels were studied with standard isolated tissue bath techniques. Experiments examined basal release of NO in endothelium-intact PVs and PAs of both right and left lungs by measuring the contractile force of vessels constricted with norepinephrine (NE) in the presence and absence of the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA). Concentration-response curves to the vasodilating agents zaprinast and A23187 were also obtained in vessels contracted by NE. RESULTS: Left and right pulmonary artery responses to NE are enhanced over those of historic controls. Pretreatment of left pulmonary arteries with L-NA enhances the vasoconstrictor response to NE, whereas right PAs show no increased response. Relaxation responses to A23187 and zaprinast, in both left and right pulmonary arteries were not different from control lambs. Relaxation responses of both left and right pulmonary veins to A23187 and zaprinast are blunted compared with controls. This blunting is significantly more in left pulmonary veins than right. Further, right but not left pulmonary veins display enhanced vasoconstrictive response to NE after L-NA pretreatment. CONCLUSIONS: The NO-cGMP pathway of vasodilatation is abnormal in the near term, fetal lamb with CDH. These abnormalities were most apparent in pulmonary veins and may reflect abnormal NOS activity or content between left and right lungs of the fetal lamb with CDH. Pulmonary arteries from CDH lambs have basal and stimulated NO release equal to that of historic controls but appear to be hypersensitive to exogenous vasoconstrictors.

Interleukin-1alpha, interleukin-6 and interleukin-8 in cervico/vaginal secretion for screening of preterm birth in twin gestation.

BACKGROUND: The purpose was to determine the prognostic value of interleukin (IL) 1-alpha, IL-6 and IL-8 in cervico/vaginal secretion for preterm birth (<37 weeks of gestation) in twin pregnancies. METHODS: The study included screening of 121 women with twin pregnancies with sampling at 24, 26, 28, 30, 32 and 34 weeks of gestation. IL-1alpha, IL-6 and IL-8 was analyzed with ELISA immunoassays. The detection limit was 30 pg/mL for IL-1 and IL-8 and 40 pg/mL for IL-6. Vaginal fluid was smeared and dried for later evaluation of bacterial vaginosis (presence of clue cells). RESULTS: Spontaneous preterm birth occurred in 36 women and 65 women were delivered at term. IL-8 was significantly higher (p=0.03) in samples from women delivered preterm (median 3.72 ng/g mucus, range <0.07-220.00) compared with samples from women delivered at term (median 3.03 ng/g mucus, range <0.08-378.60). At 28 weeks of gestation, IL-8 (cut off 1.75 ng/g mucus) was associated with preterm delivery (relative risk 2.2, CI 95% 1.1-4.5) with a sensitivity, specificity, positive and negative predictive value of 78.8, 45.8, 44.8 and 79.4%, respectively. The levels of IL-1alpha and IL-6 were not significantly associated with preterm birth. Bacterial vaginosis was found in 47/541 (8.7%) samples analyzed. The levels of IL-1alpha and IL-8 were significantly higher in samples positive for bacterial vaginosis than in negative samples (p<0.0001 and p<0.01, respectively). There was no significant association between the level of IL-6 and bacterial vaginosis. CONCLUSIONS: IL-8, but not IL-1alpha and IL-6, was associated with preterm delivery but the relationship was too weak to be of predictive value for preterm birth in twin pregnancies. IL-1alpha and IL-8, but not IL-6, were associated with bacterial vaginosis.

Improved targeting of brain tumors using dexrazoxane rescue of topoisomerase II combined with supralethal doses of etoposide and teniposide.

Dexrazoxane (ICRF-187) is a catalytic inhibitor of the nuclear enzyme DNA topoisomerase II (topo II). It protects cells against topo II poisons, such as etoposide and teniposide, by hindering the DNA cleavage reaction of the target enzyme. We have previously shown that this antagonism also extends to an in vivo model. Thus, ICRF-187 protected mice against supralethal doses of etoposide and amsacrine, and the etoposide LD10 dose increased as much as 3.6-fold when combined with ICRF-187 (B. Holm, Cancer Chemother. Pharmacol., 38: 203-209, 1996). We describe here how scheduling of this drug combination can be optimized and used. Interestingly, ICRF-187 can protect when it is given after etoposide. Although timing is very critical here, ICRF-187 was able to completely protect when given 10 min after etoposide. This rescue principle resembles methotrexate rescue by folinic acid. We also found scheduling to be crucial because ICRF-187 did not protect when etoposide was given once a day for five days, whereas effective protection was seen when etoposide was used three times, once every four days. Similar investigations were performed with teniposide in combination with ICRF-187. The combination with ICRF-187 allowed a 3.4-fold teniposide dose escalation. Such dose escalations could be advantageous in specific situations. One such case is when the tumor is situated in a pharmacological sanctuary, e.g., in the brain. ICRF-187 is hydrophilic and does not cross the blood-brain barrier, whereas the lipophilic etoposide and teniposide do. Therefore, ICRF-187 would protect normal tissues and allow a cytotoxic dose of etoposide to reach the central nervous system (CNS). We therefore studied the combinations using L1210 or EHR2 cells inoculated into the CNS of mice. L1210 presented a leukemic CNS model with leptomeningeal spread and infiltration of liver, spleen, and lymph nodes, whereas EHR2 cells acted as a solid tumor with no evidence of extracerebral disease. In all experiments, the combination of high-dose etoposide and ICRF-187 was significantly superior to an equitoxic dose of etoposide alone. Such superiority was also seen when treatment was given on days 4, 8, and 12 after tumor inoculation. Here etoposide alone resulted in a mean increased life span of 12.3%, whereas the rescue regimen yielded an increase of 47% (P < 0.0001). In conclusion, DNA topo II rescue by catalytic inhibitors is a new strategy enabling significant epipodophyllotoxin dose escalations; in this study, we have demonstrated the superiority of this strategy in two in vivo CNS tumor models. This concept is now being tested in a clinical trial.

Chinese hamster ovary cells resistant to the topoisomerase II catalytic inhibitor ICRF-159: a Tyr49Phe mutation confers high-level resistance to bisdioxopiperazines.

Anticancer drugs targeted to the nuclear enzyme DNA topoisomerase II are classified as poisons that lead to DNA breaks or catalytic inhibitors that appear to completely block enzyme activity. To examine the effects of the bisdioxopiperazine class of catalytic inhibitors to topoisomerase II, we investigated a Chinese hamster ovary (CHO) subline selected for resistance to ICRF-159 (CHO/159-1). Topoisomerase IIalpha content in CHO/159-1 cells was reduced by 40-50%, compared to wild-type CHO cells, whereas the beta isoform was increased by 10-20% in CHO/159-1 cells. However, the catalytic activity of topoisomerase II in nuclear extracts from CHO/159-1 cells was unchanged, as was its inhibition by the topoisomerase II poison etoposide (VP-16). No inhibition of topoisomerase II catalytic activity by ICRF-187 was seen in CHO/159-1 cells up to 500 microM, whereas inhibition was evident at 50 microM in wild-type CHO cells. VP-16-mediated DNA single-strand breaks and cytotoxicity were similar in the two sublines. ICRF-187 could abrogate these VP-16 effects in the wild-type line but had no effect in CHO/159-1 cells. Western blots of topoisomerase IIalpha after incubation of CHO cells with ICRF-187 demonstrated a marked band depletion, whereas this effect was completely lacking in CHO/159-1 cells, and an equal effect of VP-16 was observed in both lines. These data imply that the CHO/159-1 topoisomerase IIalpha lacks sensitivity to bisdioxopiperazines and that the mechanism of resistance in this cell line does not confer cross-resistance to topoisomerase II poisons, suggesting that mutations conferring resistance to bisdioxopiperazines can occur at sites distinct from those responsible for resistance to complex stabilizing agents. Accordingly, CHO/159-1 cDNA showed two heterozygous mutations in the proximal NH2-terminal part of topoisomerase IIalpha (Tyr49Phe and delta 309Gln-Gln-Ile-Ser-Phe313), which is in contrast to those induced by topoisomerase II poisons, which cluster further downstream. Site-directed mutagenesis and transformation of the homologous Tyr50Phe coding mutation in human topoisomerase IIalpha in a temperature-conditional yeast system demonstrated a high-level resistance to ICRF-193, compared to cells expressing wild-type cDNA, but none toward the poisons VP-16 or amsacrine, thus confirming that the Tyr50Phe mutation confers specific resistance to bisdioxopiperazines. Thus, these results indicate that the region of the protein involved in ATP-binding also plays a critical role in sensitivity to bisdioxopiperazines, a result consistent with the known requirement for the formation of an ATP-bound closed clamp for bisdioxopiperazine activity. These results may enable a more precise understanding of the interaction of topoisomerase II-directed drugs with their target enzyme.