Early intervention and disease modification in atopic dermatitis-the current state of the field and barriers to progress.

Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease representing a major source of global disability burden. Disease-modifying therapies are showing promise in chronic inflammatory disorders such as rheumatoid arthritis and Crohn's disease with method and timing of initial treatment impacting long-term disease outcomes. Whether disease-modifying therapies, specifically those used as an early interventional approach, impacts disease course and comorbidity development in AD is not well-understood. We reviewed the progress in disease modification strategies, emphasizing early intervention approaches in common (or proto-typical) inflammatory diseases. Although more common in other fields, disease modification approaches are becoming increasingly investigated in dermatology, though studies in AD are lacking. Despite significant limitations in ongoing and completed studies, early data are promising and suggest that both the choice and timing of early intervention approach can affect long-term disease course and comorbidity development. To best improve AD patient outcomes, more research is needed to further explore the impact of early disease-modifying therapies. Future studies should focus on identifying the most effective approaches and extend the early results to a more inclusive set of comorbidities and longer-term outcomes.

A pilot randomized study comparing extralevator with conventional abdominoperineal excision for low rectal cancer after neoadjuvant chemoradiation.

AIM: The aims of this study were to assess the feasibility of performing an extralevator abdominoperineal excision (ELAPE) after neoadjuvant chemoradiation (NCRT), to compare the rates of circumferential resection margin (CRM) involvement and intra-operative perforation (IOP) of the specimen, and to assess the amount of tissue removed around the muscularis propria (MP)/internal sphincter (IS) of the lower rectum in patients with low rectal cancer undergoing ELAPE compared with conventional abdominoperineal excision (CAPE) after NCRT. METHOD: This was an open-label, parallel-arm pilot randomized trial conducted in India. Twenty patients were randomized to one of the study arms. The surgical specimens were fixed, serially cross-sectioned and photographed. Using specialized morphometry software, the amount of tissue resected with each operation was measured. RESULTS: There was a nonsignificant trend towards more IOPs (30% vs 0%, P = 0.06) and a higher CRM involvement rate (40% vs 20%, P = 0.32) in the CAPE arm. ELAPE removed a significantly greater amount of tissue around the IS/MP when compared with CAPE (mean ± SD: 1911.39 ± 382 mm2 vs 1132.03 ± 371 mm2 , P < 0.001). The mean distance from the IS/MP to the CRM was significantly greater in the ELAPE arm both in the posterior (mean ± SD: 28.28 ± 3 mm vs 9.63 ± 3 mm, P < 0.001) and lateral (mean ± SD: 13.69 ± 3 mm vs 9.72 ± 3 mm, P = 0.009) parts of the rectum but not in the anterior part (mean ± SD: 6.74 ± 2 mm vs 6.10 ± 4 mm, P = 0.64). The short-term morbidity was not significantly different between the two procedures. CONCLUSION: ELAPE removed more tissue in the lower rectum and resulted in a lower rate of IOP and CRM involvement when compared with CAPE, even after NCRT.

Insulin resistance is associated with increased circulating lipocalin-2 levels in polycystic ovary syndrome: a systematic review and meta-analysis.

INTRODUCTION: This meta-analysis was conducted to compare the circulating lipocalin-2 levels in polycystic ovary syndrome (PCOS). EVIDENCE ACQUISITION: Relevant studies were retrieved by online database and manual searching. The standardized mean differences (SMDs) with 95% confidence intervals (CIs) were obtained by a random-effects meta-analysis. The subgroup analysis based on the Body Mass Index (BMI) and the homeostasis model assessment-estimated insulin resistance (HOMA-IR) was conducted. Meta-analysis of correlations and meta-regression were performed for the associations of lipocalin-2 with the metabolic and hormonal covariates. The funnel plot analysis was used for publication bias. EVIDENCE SYNTHESIS: The combined effect size including a total of 13 studies showed no significant difference in lipocalin-2 levels between PCOS and control groups. However, the subgroup meta-analysis based on insulin resistance showed a significant difference in the circulatory lipocalin-2 levels in PCOS as compared to controls in both HOMA-IR<3 (SMD=-1.15, Z=2.42, P=0.02) and HOMA-IR>3 subgroups (SMD=0.91, Z=2.43, P=0.02). CONCLUSIONS: There were significant associations of lipocalin-2 with age, BMI, estrogen and hyperandrogenism in PCOS. Lipocalin-2 level alterations in PCOS are associated to insulin resistance. More is the insulin resistance, higher is the lipocalin-2 level in PCOS as compared to controls.

Pharmacological effects of D-Pinitol - A comprehensive review.

In recent years, the application of phytochemicals to prevent or treat diseases has received greater attention. These phytochemicals have little or no toxicity against healthy tissues and are thus considered as ideal compounds. An impressive number of modern drugs are obtained from natural sources based on their traditional value. D-Pinitol is a natural compound that is derived from soy and soy products. It is a potentially active molecule that belongs to the class of inositols. D-pinitol has been pharmacologically evaluated for its potent antioxidant, anti-diabetic, anti-inflammatory, anti-cancer, hepatoprotective, cardioprotective, renoprotective, neuroprotective, immunosuppressive, and anti-osteoporotic efficacies. This review is an attempt to validate the plausible pharmacological effects of D-pinitol using various in vivo and in vitro studies. PRACTICAL IMPLICATIONS: The consumption of plant-based products has been significantly increased all over the world. The active phytochemicals that are found in plants are stated to have numerous health promoting functions for the treatment of diabetes, cancer, inflammation, cardiac diseases, liver dysfunction, and many other. D-Pinitol is abundantly present in soybeans that possess notable therapeutic activities. Understanding the effects of D-Pinitol would potentially help in applying this compound in clinical research for the treatment of different disorders.

A Systematic Review and Meta-analysis of Efficacy and Safety of Dexmedetomidine Combined With Intrathecal Bupivacaine Compared to Placebo.

BACKGROUND:  Dexmedetomidine has been approved as a sedative agent in critical patients. It is also frequently used as an adjuvant with local anesthetic in spinal anesthesia. However, its use as an adjuvant has not been approved due to the paucity of data. The present systematic review and meta-analysis were undertaken to synthesize evidence for efficacy and safety when dexmedetomidine is combined with bupivacaine in spinal anesthesia. METHODS: A literature search was done using PubMed, Google Scholar, Embase, and Cochrane Library. Search results were screened and eligible studies were included to perform a systematic review and meta-analysis using the software 'Review Manager (RevMan) version 5.4.1' using a random effect model. Cochrane's' Risk of Bias tool (RoB2)' was used for quality assessment. Mean and standard deviation was used to calculate the standardized mean difference and its forest plot for efficacy measures. For the adverse event, a number of events were used to determine the risk ratio and its forest plot using RevMan software. Publication bias is visualized using a funnel plot. RESULTS:  A total of 21 randomized control trials evaluating the efficacy and safety of intrathecal dexmedetomidine were included in the meta-analysis. A total of 1382 participants was included in this meta-analysis. The effect estimates for efficacy parameters, i.e. duration of the sensory block having SMD 2.33; CI, 1.83-2.83, motor block with SMD 1.83, CI 1.21, 2.46, and analgesia SMD 2.81; CI, 2.11-3.51. The risk ratio for adverse effects, i.e. nausea/vomiting, bradycardia, hypotension was not significant whereas it was significant for the incidence of shivering with RR 0.38; CI 0.23-0.97. The overall risk of bias among included studies was either of 'some concern' or 'high risk.' CONCLUSIONS:  Intrathecal dexmedetomidine when combined with bupivacaine was found to significantly increase the three efficacy parameters, i.e. duration of sensory block, motor block, and analgesia. It also appears to be safe with no increased risk of bradycardia or hypotension. It is also associated with decreased postoperative shivering.

Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil.

Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this "uncultivated majority" remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages.IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.

Complete and draft genome sequences of six members of the Aquificales.

The Aquificales are widespread in marine and terrestrial hydrothermal environments. Here, we report the complete and draft genome sequences of six new members of the Aquificales: two marine species, Persephonella marina strain EX-H1 and Hydrogenivirga strain 128-5-R1 (from the East Pacific Rise, 9 degrees 50.3'N, 104 degrees 17.5'W, and the Eastern Lau Spreading Center, 176 degrees 11.5'W, 20 degrees 45.8'S, respectively), and four terrestrial isolates, Sulfurihydrogenibium azorense strain Az-Fu1, Sulfurihydrogenibium yellowstonense strain SS-5, and Sulfurihydrogenibium strain Y03AOP1 (from Furnas, Azores, Portugal, and Calcite Springs and Obsidian Pool in Yellowstone National Park, United States, respectively), and the only thermoacidophilic isolate, Hydrogenobaculum strain Y04AAS1 (from a stream adjacent to Obsidian Pool). Significant differences among the different species exist that include nitrogen metabolism, hydrogen utilization, chemotaxis, and signal transduction, providing insights into their ecological niche adaptations.

Bioimpedance Spectroscopy-Guided Ultrafiltration Normalizes Hydration and Reduces Intradialytic Adverse Events in Hemodialysis Patients.

Chronic fluid overload as well as excessive fluid removal are associated with increased morbidity and mortality in hemodialysis (HD) patients. The clinical method to probe the dry weight is often inaccurate and the bioimpedance spectroscopy (BIS) is shown to improve the accuracy. We compared the impact of BIS and clinical methods to guide ultrafiltration (UF) in a randomized controlled study on the intradialytic complications and blood pressure control in prevalent HD patients. Fifty patients on maintenance HD were randomized to BIS method (BIS-group) and clinical method (CL-group) to guide UF. The body composition monitor (BCM) was done post-HD in all patients at baseline and 2-weeks interval during the study period of 6 months to determine the hydration status, but the result was revealed only to the nephrologist managing the patients in BIS-group to probe the dry weight. The endpoints of the study were blood pressure control, intradialytic complications and anti-hypertensive drug burden. The mean age was 56.0 ± 12.0 years and 70% were male. There was significant increase in patients with normal hydration in BIS-group (20% vs. 88%, p = 0.0001), but remained similar to baseline in CL-group (40% vs. 48%, p = 0.3) at 6 months. The incidence of intradialytic hypotension was significantly reduced in BIS-group (4.84 ± 3.0 vs. 2.8 ± 3.13 events/patient/6 months, p = 0.003). There was 35% reduction in hypertensive pill burden in BIS-group with similar blood pressure, compared to CL-group. Post-dialysis underhydration was more common than over or normal hydration at baseline in our population, indicating that clinical method to probe dry weight often resulted in hypovolemia. BIS method to determine dry weight resulted in normalization in volume status and consequently resulted in significant reduction in intradialytic hypotension and anti-hypertensive pill burden over 6-month period.

Seasonal variation in the presentation of abdominal pain.

BACKGROUND: Anxiety and depression, conditions frequently associated with childhood chronic abdominal pain (AP), are characterized by seasonal exacerbations. A seasonal pattern characterized by a higher frequency of consultations for AP during winter has been suspected but has never, to our knowledge, been demonstrated. We hypothesize the presence of a seasonal variation in AP consultations with a winter predominance. AIMS: To determine the seasonal distribution of AP consultations among centers across time and geographic latitude. PATIENTS AND METHODS: This was a retrospective cohort study. The number of outpatient consultations from primary care clinics and every pediatric specialty clinic that resulted in a diagnosis of AP and the total number of outpatient consultations (2001-2004) from 6 tertiary care institutions (Chicago, Pittsburgh, Wilmington, Pensacola, Orlando, Jacksonville) was determined. Rates of consultations were compared across time and between cities. Four time periods of interest, with 2- and 3-month definitions, were arbitrarily selected. Seasonal rates across time were compared separately for each of the 2-month (January-February vs June-July) and 3-month periods (January-March vs June-August). Logistic regression models for each city were used to determine variations in the rate of outpatient AP cases by season or by year. RESULTS: A total of 3,929,522 outpatient consultations and 73,591 AP consultations were analyzed. The rates of AP consultations were consistently higher in the winter months at all of the sites. The results differed between sites at northern and southern latitudes. CONCLUSIONS: There seems to be a seasonal variation in consultation patterns for AP at the tertiary care level. A possible role of daylight hours, climate, latitude, and stress is proposed.

Impact of Malnutrition, Inflammation, and Atherosclerosis on the Outcome in Hemodialysis Patients.

Malnutrition, inflammation, and atherosclerosis are significant problems in patients on hemodialysis. A prospective, observational study in 100 hemodialysis patients for 2 years was conducted. The primary outcomes were hospitalizations and mortality at the end of 2 years. The mean age was 61 ± 11.3 years and 69% were male. Seven patients did not complete the study (five underwent transplant and two were shifted to other units). Serum albumin was significantly lower in malnourished patients at 6 months from the beginning of the study period (3.58 vs. 3.79 g/dl, P = 0.001). Malnutrition based on subjective global assessment (SGA) was seen in 30 (32%) patients: mild to moderate in 27 (29%) and severe in 3 (3%). Inflammation was seen in 73 (78.5%) patients and intimal-medial thickness of >1.1 mm indicating significant atherosclerosis was seen in 73 (78.5%) patients. Modified SGA score and malnutrition-inflammation score (MIS) were significantly more in the malnourished group. Statistically significant association was seen between hospitalization and mortality in the malnourished population, and the odds ratio of death in malnourished patients was 9.83 (95% confidence interval: 2.8-34.3, P < 0.001). There was a moderate correlation between malnutrition assessed by modified SGA and MIS score (r = 0.54, P < 0.001). Mortality rate was 37% in patients with mild to moderate and 67% in severe malnutrition. Hospital admission was seen in 43 (46%) patients and was significantly more common in malnourished compared to well-nourished patients (77% vs. 32%, P < 0.001). Multiple logistic regression analysis showed that malnutrition by Modified SGA was the only significant variable associated with mortality at 2 years, and addition of MIS score did not improve the predictive ability of the model to modified SGA. We recommend the use of modified SGA and serial serum albumin to monitor nutrition in hemodialysis patients.

Weight loss and reduced body mass index: a critical issue in children with multiorgan chronic graft-versus-host disease.

Weight loss and malnutrition are major problems in patients with chronic graft-versus-host disease (GVHD). In adults, low body mass index (BMI) is a predictor for mortality; however, weight loss and BMI have not been studied in pediatric chronic GVHD. A retrospective study on 18 children with extensive chronic GVHD was completed. Median age at SCT was 12.3 (range 0.6-23) years; age at chronic GVHD diagnosis was 12.5 (1-23) years. Patients with multiorgan involvement had a mean maximal decrease in BMI of 20.9% and most dropped below 10th percentile in expected weight-for-age. This change in BMI not only indicates a significant decrease in weight but often a plateau in stature. In contrast, patients with one organ system involved had a mean maximal decrease in BMI of 5% and did not fall below 10th percentile. All patients with multiorgan involvement required salvage therapy beyond steroids and CSA. Three patients died due to complications of chronic GVHD. Weight loss and malnutrition (as reflected by a decrease in BMI) are clinically significant issues in children with multisystem chronic GVHD. Weight loss is likely another systemic manifestation of the disease and may contribute, along with other factors such as increased immunosuppression and infection, to increased mortality in this group.

HRAS1 rare minisatellite alleles and breast cancer in Australian women under age forty years.

BACKGROUND: A recent meta-analysis of 23 studies supported the empirically derived hypothesis that women who lack one of the four common minisatellite alleles at the HRAS1 locus are at increased risk of breast cancer. These studies relied on visual sizing of alleles on electrophoretic gels and may have underreported rare alleles. We determined whether this hypothesis applied to early-onset breast cancer by using a new method to size minisatellite alleles. METHODS: We conducted a population-based, case-control-family study of 249 Australian women under 40 years old at diagnosis of a first primary breast cancer and 234 randomly selected women, frequency matched for age. We sized HRAS1 minisatellite alleles with an Applied Biosystems model 373 automated DNA sequencer and GENESCAN(TM) software. All P values are two-sided. RESULTS: We found no association of rare alleles with breast cancer, before or after adjustment for risk factors and irrespective of how their effects were modeled (crude odds ratio = 1.04; 95% confidence interval [CI] = 0.071-1.53; P =.8). The rare allele frequency was 0. 173 (95% CI = 0.149-0.197), three times the pooled estimate of 0.058 (95% CI = 0.050-0.066) from previous studies (P<.001), and was similar for case subjects, 0.177 (95% CI = 0.143-0.221), and control subjects, 0.169 (95% CI = 0.135-0.203) (P =.7). CONCLUSION: There was no support for an association between rare HRAS1 alleles and the risk of early-onset breast cancer, despite 80% power to detect effects of the magnitude of those associations (1.7-fold) previously suggested. IMPLICATIONS: The question of whether cancer risk is associated with rare minisatellite HRAS1 alleles needs to be revisited with the use of new methods that have a greater ability to distinguish rare alleles from similarly sized common alleles.

Increased mutation frequency following treatment with cancer chemotherapy.

The relationship between somatic mutation and cancer was studied by measuring in vivo mutation frequency and in vitro mutability using lymphocytes from 28 untreated adult patients with solid tumors, 14 untreated patients with lymphoma, and 27 patients with solid tumors or lymphoma who had been treated with chemotherapy and/or radiotherapy. In vivo mutation frequency in untreated patients did not differ from that of controls, except perhaps in patients with lymphoma, who showed a slight increase. Lymphocytes from untreated patients with solid tumors or lymphoma did not show a greater increase in mutations induced after X-radiation or UV radiation than did lymphocytes from controls. For all the untreated patients, the geometric mean mutation frequency was 6.72 X 10(-6), and it was significantly increased to 19.57 X 10(-6) following chemotherapy and 34.40 X 10(-6) following chemotherapy and radiotherapy. The results suggest that excessive systemic exposure to mutagens or inherent susceptibility to mutagenesis are not important etiological factors in at least the majority of patients with cancer. The mutations produced by treatment may be related to the late side effects of therapy such as second neoplasms.

Mutation rate of normal and malignant human lymphocytes.

The genetic stability of normal and neoplastic lymphocytes was compared by using base-line mutation frequency and mutation rate/cell generation. Mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were studied by enumerating thioguanine-resistant cells in a clonogenic assay. The base-line ("spontaneous") mutation frequency was 1.52 X 10(-6), 6.38 X 10(-6), and 1.06 X 10(-6) for normal cells from three individuals and was 1.16 X 10(-3), 6.08 X 10(-5), and 3.06 X 10(-5) for the three malignant cell lines, Jurkat (JM), HRIK, FMC-Hu1B, respectively. The mutation cell/generation rate was 24.6 X 10(-8), 15 X 10(-8), and 5.5 X 10(-8) for lymphocytes from the three normal individuals, and 666.4 X 10(-8), 52.8 X 10(-8), and 131 X 10(-8) for the three malignant cell lines. The results suggest that neoplastic lymphocytes are more genetically unstable than normal lymphocytes.

Effects of new N-alkyl analogues of adriamycin on in vitro survival and cell cycle progression of L1210 cells.

The effects of N-benzyladriamycin-14-valerate (AD198) and N,N-dimethyladriamycin-14-valerate (AD199), two novel lipophilic N-alkyl derivatives of Adriamycin (ADR), on cell growth and cell cycle distribution were investigated in L1210 cells grown in suspension. Following a 1-h exposure to the drug levels selected, growth inhibition was noticeable in all cultures for most or all of the observation period of 96 h. With flow cytometry, an asynchronous cell population was measured with respect to cellular DNA, RNA, and light scatter (size) properties following a 1-h incubation with the various ADR analogues. In addition, flow cytometric techniques were utilized to determine whether drug treatment altered the sensitivity of DNA in situ to acid-induced denaturation or to binding by small DNA-intercalating dyes. Unlike the parent compound ADR or its DNA-nonbinding derivative N-trifluoroacetyladriamycin-14-O-hemiadipate (AD143), the N-alkyl derivatives AD198 and AD199 only slightly affected L1210 cell cycle traverse over the first 5 h posttreatment. However, by 24 h, AD199 (0.62 micrograms/ml) caused an S- and G2 + M-phase accumulation which became more dramatic at 48 and 72 h. AD198 (3.27 micrograms/ml) also caused an accumulation of cells predominantly in G2 + M phase at longer culture times (48 to 96 h). The two half-substituted congeners N-benzyladriamycin (AD288) and N,N-dimethyladriamycin (AD280) affected L1210 cell cycle traverse over a similar time scale at concentrations of 12.3 and 4.17 micrograms/ml, respectively. AD280 blocked cells in G1 and G2 + M whereas AD288 caused predominantly a G2 + M accumulation. While neither ADR nor AD143 interfered appreciably with binding and fluorescence of the intercalating dye acridine orange, all of the N-alkyl analogues tested reduced the fluorescence signal of acridine orange-stained L1210 cells by 26 to 60%. This effect lasted, with decreasing intensity, for at least 48 h following a 1-h exposure to the drugs. In addition, while ADR appeared to stabilize DNA in situ against acid-induced denaturation, all N-alkyl derivatives, to varying degrees, tended to increase DNA denaturability. Thus alkylation at the glycoside amine combined with the lipophilic 14-valerate side chain function accounts for several new biochemical and biological properties of AD198 and AD199, relative to ADR.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA topoisomerase II-mediated interaction of doxorubicin and daunorubicin congeners with DNA.

Three groups of doxorubicin and daunorubicin analogues, differing by their substituents on the chromophore and sugar moieties, were used in this study. The 3'-N-unsubstituted (Group 1), 3'-N-acyl (Group 2), and 3'-N-alkyl (Group 3) analogues were tested for: (a) in vivo antitumor activity and in vitro cytotoxicity; (b) cellular or tissue uptake and metabolic conversion; (c) strength of DNA intercalation; and (d) interaction with DNA topoisomerase II (topo-II). Compounds of Group 1 were cytotoxic, were strongly intercalative, and, except for those with C-14 side chain substitution, induced the formation of topo-II-DNA cleavable complexes. As shown previously, esterolysis of C-14-acyl substituents was required to yield a metabolite which can interact with topo-II in the purified system. The C-14-substituted compounds of Group 2 and their C-14-unsubstituted metabolites were cytotoxic. These drugs were weak intercalators, and the C-14-unsubstituted cogeners induced cleavable complex formation in the purified system, but with reduced potency relative to doxorubicin. The type of the 3'-N-position substituent determined whether Group 3 analogues were cytotoxic and strong intercalators, or less active and nonintercalating. Although C-14-unsubstituted intercalators of Group 3 did not form cleavable complexes in the purified system, they were cytotoxic. The study shows that DNA intercalation is required but not sufficient for the activity by topo-II-targeted anthracyclines. In addition to the planar chromophore which is involved in intercalation, two other domains of the anthracycline molecule are important for the interaction with topo-II: (a) substitution of the C-14 position totally inhibits drug activity in the purified system, but enhances cytotoxicity by aiding drug uptake and presumably acting on other cellular targets; and (b) substitutions on the 3'-N position of the sugar ring can, depending on the nature of the substituent, inhibit intercalation and/or topo-II-targeting activity. These findings may provide guidance for the synthesis and development of new active analogues.

Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells.

Direct reversal of O6 adducts caused by chemotherapy agents is accomplished in mammalian cells by the protein O6-methylguanine DNA methyltransferase (MGMT). Some tumors overexpress MGMT and are resistant to alkylator therapy. One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O6-benzylguanine (6-BG) and protection of sensitive tissues, such as hematopoietic stem and progenitor cells, using genetic modification with 6-BG-resistant MGMT mutants. We have used retroviral-mediated gene transfer to transduce murine hematopoietic bone marrow cells with MGMT point mutants showing resistance to 6-BG depletion in vitro. These mutants include proline to alanine and proline to lysine substitutions at the 140 position (P140A and P140K, respectively), which show 40- and 1000-fold resistance to 6-BG compared with wild-type (WT) MGMT. Lethally irradiated mice were reconstituted with murine stem cells transduced with murine stem cell virus retrovirus expressing each mutant, WT MGMT, or mock-infected cells and then treated with a combination of 30 mg/kg 6-BG and 10 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU alone. Compared with mice treated with BCNU alone, significant myeloid toxicity and death occurred in mice reconstituted with mock-infected or WT MGMT (<0.1 probability of survival) or the P140A mutant (0.13 probability of survival) MGMT cDNAs. In contrast, after an initial period of mild cytopenia, mice reconstituted with the P140K mutant (0.83 probability of survival) recovered nearly normal blood counts, even during continued treatment. Comparison of peripheral blood neutrophils after completion of 5 weekly treatments in these animals showed a direct correlation between the treatment and in vivo selection for progeny of transduced cells (pretreatment, approximately 8-12% transduced cells; no treatment, approximately 6% transduced cells; BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced cells). To determine whether this selection occurred at the stem cell level, bone marrow from each treatment group was infused into secondary recipients. Whereas animals that received bone marrow from untreated animals reconstituted with 2% transduced cells, animals receiving marrow from 6-BG/BCNU-treated animals reconstituted with 94% transduced cells, demonstrating nearly complete selection for stem cells in the primary animals. Mice reconstituted with marrow from animals treated with BCNU only demonstrated 23% transduced cells, consistent with partial selection of stem cells in the primary mice. The levels of transduced cells also correlated with survival during a second round of intensive combination chemotherapy (probability of survival: 6-BG/BCNU, 1.0; BCNU alone, >0.70; no treatment, <0.1). These data demonstrate that mutant MGMT expressed in the bone marrow can protect mice from time- and dose-intensive chemotherapy and that the combination of 6-BG and BCNU leads to uniform selection of transduced stem cells in vivo in mice.

Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Clinical utility of urine neutrophil gelatinase-associated lipocalin measured at admission to predict outcomes in heterogeneous population of critically ill patients.

Urine neutrophil gelatinase-associated lipocalin (uNGAL) is a reliable early biomarker of acute kidney injury (AKI) in a homogeneous patient population. However, its utility in a heterogeneous population of critically ill, in whom the time of onset of renal insult is often unclear, is not clearly established. We evaluated the ability of a single measurement of uNGAL in a heterogeneous adult population, on admission to intensive care unit (ICU), to predict the occurrence of AKI and hospital mortality. One hundred and two consecutive adult patients had uNGAL measured within 8 h of admission to ICU. The demographic and laboratory data were collected at admission. The diagnosis of AKI was based on AKI Network (AKIN) criteria. The primary outcome was the development of AKI, and the secondary outcome was hospital mortality. The mean age was 54 ± 16.4 years and 65% were males. Urine NGAL (ng/ml) was 69 ± 42 in patients with AKI (n = 42) and 30.4 ± 41.7 in those without AKI (P < 0.001). The area under the receiver operating characteristic (ROC) curve for prediction of AKI was 0.79 and for serum creatinine (SCr) was 0.88. The sensitivity and specificity for a cut-off value of uNGAL of 75 ng/ml to predict AKI were 0.5 and 0.85 respectively. uNGAL > 75 ng/ml was a strong (odd ratio = 5.17, 95% confidence interval: 1.39-19.3) and independent predictor of hospital mortality. A single measurement of uNGAL at admission to ICU exhibited good predictive ability for AKI though the sensitivity was low. The predictive ability of uNGAL was inferior to simultaneously measured SCr at admission, hence limited its clinical utility to predict AKI. However, admission uNGAL was a strong, independent predictor of hospital mortality.