Lipocalin-2 is an essential component of the innate immune response to Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A. baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A. baumannii. Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A. baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A. baumannii infection is required. Here, we investigate the innate immune response to A. baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A. baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 (Lcn2), a siderophore sequestering protein, was the most highly upregulated during A. baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2-/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9, suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro, LCN2 inhibits the iron-dependent growth of A. baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2-/- mice were infected with A. baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2-/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A. baumannii. The control of A. baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.

Mitochondrial Calcium Uniporter Affects Neutrophil Bactericidal Activity during Staphylococcus aureus Infection.

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

The Innate Immune Protein S100A9 Protects from T-Helper Cell Type 2-mediated Allergic Airway Inflammation.

Calprotectin is a heterodimer of the proteins S100A8 and S100A9, and it is an abundant innate immune protein associated with inflammation. In humans, calprotectin transcription and protein abundance are associated with asthma and disease severity. However, mechanistic studies in experimental asthma models have been inconclusive, identifying both protective and pathogenic effects of calprotectin. To clarify the role of calprotectin in asthma, calprotectin-deficient S100A9-/- and wild-type (WT) C57BL/6 mice were compared in a murine model of allergic airway inflammation. Mice were intranasally challenged with extracts of the clinically relevant allergen, Alternaria alternata (Alt Ext), or PBS every third day over 9 days. On Day 10, BAL fluid and lung tissue homogenates were harvested and allergic airway inflammation was assessed. Alt Ext challenge induced release of S100A8/S100A9 to the alveolar space and increased protein expression in the alveolar epithelium of WT mice. Compared with WT mice, S100A9-/- mice displayed significantly enhanced allergic airway inflammation, including production of IL-13, CCL11, CCL24, serum IgE, eosinophil recruitment, and airway resistance and elastance. In response to Alt Ext, S100A9-/- mice accumulated significantly more IL-13+IL-5+CD4+ T-helper type 2 cells. S100A9-/- mice also accumulated a significantly lower proportion of CD4+ T regulatory (Treg) cells in the lung that had significantly lower expression of CD25. Calprotectin enhanced WT Treg cell suppressive activity in vitro. Therefore, this study identifies a role for the innate immune protein, S100A9, in protection from CD4+ T-helper type 2 cell hyperinflammation in response to Alt Ext. This protection is mediated, at least in part, by CD4+ Treg cell function.

Clinical and immunological characteristics of 150 systemic lupus erythematosus patients in Jamaica: a comparative analysis.

Background Epidemiological studies in systemic lupus erythematosus have been reported in the literature in many countries and ethnic groups. Although systemic lupus erythematosus in Jamaica has been described in the past, there has not been a detailed evaluation of systemic lupus erythematosus patients in urban Jamaica, a largely Afro-Caribbean population. The goal of this study was to describe the clinical features, particularly disease activity, damage index and immunological features, of 150 systemic lupus erythematosus subjects. Methods 150 adult patients (≥18 years) followed in rheumatology clinic at a tertiary rheumatology hospital centre (one of two of the major public referral centres in Jamaica) and the private rheumatology offices in urban Jamaica who fulfilled Systemic Lupus International Collaborating Clinics (SLICC) criteria were included. Data were collected by detailed clinical interview and examination and laboratory investigations. Hence demographics, SLICC criteria, immunological profile, systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) and SLICC/American College of Rheumatology (ACR) damage index (SDI) were documented. Results Of the 150 patients, 145 (96.7%) were female and five (3.3%) were male. The mean age at systemic lupus erythematosus onset was 33.2 ± 10.9. Mean disease duration was 11.3 ± 8.6 years. The most prevalent clinical SLICC criteria were musculoskeletal, with 141 (94%) of subjects experiencing arthralgia/arthritis, followed by mucocutaneous manifestations of alopecia 103 (68.7%) and malar rash 46 (30.7%), discoid rash 45 (30%) and photosensitivity 40 (26.7%). Lupus nephritis (biopsy proven) occurred in 42 (28%) subjects and 25 (16.7%) met SLICC diagnostic criteria with only positive antinuclear antibodies/dsDNA antibodies and lupus nephritis on renal biopsy. The most common laboratory SLICC criteria were positive antinuclear antibodies 136 (90.7%) followed by anti-dsDNA antibodies 95 (63.3%) and low complement (C3) levels 38 (25.3%). Twenty-seven (18%) met SLICC diagnostic criteria with only positive antinuclear antibodies/anti-dsDNA antibodies and lupus nephritis on renal biopsy. Mean SLEDAI score was 6.9 ± 5.1 with a range of 0-32. Organ damage occurred in 129 (86%) patients; mean SDI was 2.4 ± 1.8, with a range of 0-9. Conclusion These results are similar to the clinical manifestations reported in other Afro-Caribbean populations; however, distinct differences exist with respect to organ involvement and damage, particularly with respect to renal involvement, which appears to be reduced in our participants.

Enhanced Risk Stratification for Children and Young Adults with B-Cell Acute Lymphoblastic Leukemia: A Children's Oncology Group Report.

Current strategies to treat pediatric acute lymphoblastic leukemia rely on risk stratification algorithms using categorical data. We investigated whether using continuous variables assigned different weights would improve risk stratification. We developed and validated a multivariable Cox model for relapse-free survival (RFS) using information from 21199 patients. We constructed risk groups by identifying cutoffs of the COG Prognostic Index (PICOG) that maximized discrimination of the predictive model. Patients with higher PICOG have higher predicted relapse risk. The PICOG reliably discriminates patients with low vs. high relapse risk. For those with moderate relapse risk using current COG risk classification, the PICOG identifies subgroups with varying 5-year RFS. Among current COG standard-risk average patients, PICOG identifies low and intermediate risk groups with 96% and 90% RFS, respectively. Similarly, amongst current COG high-risk patients, PICOG identifies four groups ranging from 96% to 66% RFS, providing additional discrimination for future treatment stratification. When coupled with traditional algorithms, the novel PICOG can more accurately risk stratify patients, identifying groups with better outcomes who may benefit from less intensive therapy, and those who have high relapse risk needing innovative approaches for cure.

Abnormal prefrontal activity subserving attentional control of emotion in remitted depressed patients during a working memory task with emotional distracters.

BACKGROUND: Patients with major depressive disorder (MDD) show deficits in processing of facial emotions that persist beyond recovery and cessation of treatment. Abnormalities in neural areas supporting attentional control and emotion processing in remitted depressed (rMDD) patients suggests that there may be enduring, trait-like abnormalities in key neural circuits at the interface of cognition and emotion, but this issue has not been studied systematically. METHOD: Nineteen euthymic, medication-free rMDD patients (mean age 33.6 years; mean duration of illness 34 months) and 20 age- and gender-matched healthy controls (HC; mean age 35.8 years) performed the Emotional Face N-Back (EFNBACK) task, a working memory task with emotional distracter stimuli. We used blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to measure neural activity in the dorsolateral (DLPFC) and ventrolateral prefrontal cortex (VLPFC), orbitofrontal cortex (OFC), ventral striatum and amygdala, using a region of interest (ROI) approach in SPM2. RESULTS: rMDD patients exhibited significantly greater activity relative to HC in the left DLPFC [Brodmann area (BA) 9/46] in response to negative emotional distracters during high working memory load. By contrast, rMDD patients exhibited significantly lower activity in the right DLPFC and left VLPFC compared to HC in response to positive emotional distracters during high working memory load. These effects occurred during accurate task performance. CONCLUSIONS: Remitted depressed patients may continue to exhibit attentional biases toward negative emotional information, reflected by greater recruitment of prefrontal regions implicated in attentional control in the context of negative emotional information.

Establishing the Jamaica lupus registry: report of patients with systemic lupus erythematosus attending a major referral hospital in Jamaica.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by multisystem microvascular inflammation with the generation of autoantibodies. There are reports on demographic data and clinical manifestation of lupus in the United States of America and some other developed countries. There is a single study that has reported on the clinical and immunological features of SLE patients in Jamaica and another that reported that the prevalence of SLE in Jamaica was 5-17/100,000 in 1979. METHOD: A Jamaican lupus registry was established in 2008 at the Department of Medicine, The University of the West Indies. Data were collected using patient records and interview of patients fulfilling the American College of Rheumatology revised diagnostic criteria for SLE. Information on demographics, presence of diagnostic criteria for SLE, presence of complications and other clinical parameters were collected. RESULTS: There were a total of 107 patients that met the criteria for diagnosis of SLE at the referral centre, 96.3% of them female. Positive ANA (90.7%), arthritis (70.0%), malar rash (53.5%) and a positive dsDNA (40.1%) were the more frequent manifestations and diagnostic indices of the disease. Up to 41.7% of the SLE population suffered some form of complication. CONCLUSIONS: The initiation of a lupus registry has allowed for reporting ofpreliminary demographic, clinical and serological data and identifying of disease burden.

Complement-mediated antiserum cytotoxic reactions to human chromosome 7 coded antigen(s): immunoselection of rearranged human chromosome 7 in human-mouse somatic cell hybrids.

Immunoselection via complement-dependent lysis of human-mouse somatic cell hybrids containing chromosome 7, with antisera reactive to cell surface antigen(s) coded for by chromosome 7, has resulted in growth of somatic cell hybrids containing rearranged human chromosome 7s. Investigation of these hybrids has localized the gene(s) coding for the relevant cell surface antigen(s) to the short arm of human chromosome 7. The simian virus 40 integration site and the gene coding for human beta-glucuronidase appear to be localized to the long arm of chromosome 7 in this hybrid clone.

Endocrine stress response of Eastern Fence Lizards in fire-disturbed landscapes.

Landscape disturbances can alter habitat structure and resource availability, often inducing physiological responses by organisms to cope with the changing conditions. Quantifying the endocrine stress response through measurement of glucocorticoids has become an increasingly common method for determining how organisms physiologically respond to challenges imposed by their environment. We tested the hypothesis that Eastern Fence Lizards cope with fire disturbance effects by modulating their secretion of corticosterone (CORT). We measured the baseline and stress-induced plasma CORT of male Eastern Fence Lizards in a chronosequence of fire-altered habitats (recently burned, recovering from burn, and unburned). Although habitat use by lizards differed among burn treatments, including differences in use of canopy cover, leaf litter, and vegetation composition, we did not detect a significant effect of fire-induced habitat alteration on plasma CORT concentration or on body condition. In addition, we found no effect of blood draw treatment (baseline or stress-induced), body temperature, body condition, or time taken to collect blood samples on concentration of plasma CORT. Low intensity burns, which are typical of prescribed fire, may not be a sufficient stressor to alter CORT secretion in Eastern Fence Lizards (at least during the breeding season). Instead, lizards may avoid allostatic overload using behavioral responses and by selecting microsites within their environment that permit thermoregulatory opportunities necessary for optimal performance and energy assimilation.

Zinc intoxication induces ferroptosis in A549 human lung cells.

Zinc (Zn) is an essential trace metal required for all forms of life, but is toxic at high concentrations. While the toxic effects of high levels of Zn are well documented, the mechanism of cell death appears to vary based on the study and concentration of Zn. Zn has been proposed as an anti-cancer treatment against non-small cell lung cancer (NSCLC). The goal of this analysis was to determine the effects of Zn on metabolism and cell death in A549 cells. Here, high throughput multi-omics analysis identified the molecular effects of Zn intoxication on the proteome, metabolome, and transcriptome of A549 human NSCLC cells after 5 min to 24 h of Zn exposure. Multi-omics analysis combined with additional experimental evidence suggests Zn intoxication induces ferroptosis, an iron and lipid peroxidation-dependent programmed cell death, demonstrating the utility of multi-omics analysis to identify cellular response to intoxicants.

Globin gene expression in erythroid human fetal liver cells.

We measured steady-state levels of the human globin mRNAs in liver samples from several mid-gestational fetuses. RNA from the epsilon, gamma, beta, zeta, theta, and alpha globin genes were present in fetal liver samples isolated from 10-25-wk embryos. The abundance of all human globin mRNAs declined in older fetuses, presumably because of a gradual reduction in the proportion of erythroid precursors in the liver as development proceeds. The gamma:beta globin mRNA ratio in 10-18-wk fetal erythroblasts was 6-7:1, and in adult erythroid bone marrow the ratio was 0.02:1. In fetal liver samples, the relative abundance of epsilon transcripts was less than 1% that of gamma, and zeta transcripts less than 5% that of alpha. Embryonic transcripts declined in abundance during late fetal development and were not detected in newborn liver or adult erythroid bone marrow. theta globin mRNA also represented a minor species (less than 1% that of alpha) in fetal liver samples, but in contrast to the embryonic mRNAs, was most abundant in adult marrow samples obtained from patients with erythroid hyperplasia. These results support the hypothesis that globin protein levels are regulated by the relative amounts of each globin mRNA at various stages of erythropoietic development.

Chronic disease in the Mojave desert tortoise: Host physiology and recrudescence obscure patterns of pathogen transmission.

A seminatural, factorial-design experiment was used to quantify dynamics of the pathogen Mycoplasma agassizii and upper respiratory tract disease in the Mojave desert tortoise (Gopherus agassizii) over 2 years. Groups of initially healthy animals were separated into serologically positive (seropositive), seronegative, and artificially infected groups and paired into 23 pens. We found no evidence of long-term immune protection to M. agassizii or of immunological memory. Initially seronegative, healthy tortoises experienced an equal amount of disease when paired with other seronegative groups as when paired with seropositive and artificially infected groups-suggesting that recrudescence is as significant as transmission in introducing disease in individuals in this host-pathogen system. Artificially infected groups of tortoises showed reduced levels of morbidity when paired with initially seronegative animals-suggesting either a dilution effect or a strong effect of pathogen load in this system. Physiological dynamics within the host appear to be instrumental in producing morbidity, recrudescence, and infectiousness, and thus of population-level dynamics. We suggest new avenues for studying diseases in long-lived ectothermic vertebrates and a shift in modeling such diseases.

COMPARISON OF CURRENT METHODS FOR THE DETECTION OF CHRONIC MYCOPLASMAL URTD IN WILD POPULATIONS OF THE MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII).

Pathogens that cause subclinical diseases or exhibit low infection intensities are difficult to quantify in wild populations. Mojave desert tortoises ( Gopherus agassizii ) have been the focus of much research aimed at measuring the presence of upper respiratory disease (URTD) and URTD-associated pathogens, and techniques used to quantify disease in Gopherus species have also been used for disease surveillance in other species of turtles and tortoises of conservation concern. Published surveys of G. agassizii populations have found a relatively low prevalence of URTD, with most URTD-positive animals exhibiting moderate, intermittent signs of morbidity. Therefore, multiple tests have been developed to quantify URTD including genetic detection of the pathogens Mycoplasma agassizii and Mycoplasma testudineum , detection of M. agassizii -specific antibodies, and standardized quantification of clinical signs of URTD and body condition. These diagnostic tests have only been compared in diseased or moribund, semicaptive animals. We compared diagnostic techniques (TaqMan® and SYBR™ Green qPCR, serology, and visible examination) to detect M. agassizii -associated URTD in 126 wild desert tortoises sampled in Nevada and California, US in 2010. All had healthy body condition indices and none exhibited more than mild-to-moderate visual signs of URTD. Pairwise comparisons of diagnostic techniques indicated poor performance in diagnosing disease in individual animals. We found stronger, but inconsistent, statistical associations among diagnostic techniques at the population level. Our findings have implications for quantifying subclinical respiratory disease in tortoises.

Prostatic carcinosarcoma 15 years after combined external beam radiation and brachytherapy for prostatic adenocarcinoma: a case report.

A 65-year-old man with a history of combined pelvic external beam radiation therapy (EBRT) and brachytherapy for prostatic adenocarcinoma 15 years prior underwent total pelvic exenteration for presumed rectal sarcoma with prostatic invasion. Pathology revealed carcinosarcoma of prostatic origin. This patient exhibited the longest reported interval between initial presentation with prostatic adenocarcinoma and development of carcinosarcoma. This case is also the first reported case of prostatic carcinosarcoma occurring after combined EBRT and brachytherapy. The increasing use of such combination high-dose radiation therapy may potentially lead to an increased incidence of secondary malignancies such as prostatic carcinosarcoma in the future.

Plasma hormone profiles of young women at risk for familial breast cancer.

The plasma hormone concentrations of 30 young women, who were judged by genetic analysis to be at high risk for familial breast cancer, were compared with those of 30 matched controls identified as at low risk for the disease. The hormone measurements were obtained every second day throughout the menstrual cycle, and the results were analyzed in terms of follicular, luteal, and full-cycle mean concentrations. Comparison was carried out in a paired fashion with each high-risk and low-risk pair matched closely for height, weight, age, and reproductive history. No statistically significant differences were found in prolactin, gonadotropin, estrone, estradiol, or estriol plasma concentrations although the high-risk group displayed consistently lower values in all of the above except estriol.

Educational innovations in clinical pharmacogenomics.

Genetic and genomic discovery is revolutionizing medicine at an extraordinary pace, leading to a better understanding of disease and improved treatments for patients. This advanced pace of discovery presents an urgency to expand medical school curricula to include genetic and genomic testing (including pharmacogenomics), and integration of genomic medicine into clinical practice. Consequently, organizations and healthcare authorities have charged medical schools with training future physicians to be competent in their knowledge of genomic implementation.

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.

Fluorescence signaling of ligand binding and assembly in metal-chelating lipid membranes.

BACKGROUND: Chemical information is sometimes transmitted across cell membranes by ligand-induced assembly of receptors. We have previously designed a series of lipids with metal-chelating headgroups that can serve as general receptors for proteins containing accessible histidines. Such lipids can also be derivatized with pyrene, a fluorescent probe that has a different emission maximum when it is aggregated (excimer fluorescence) from that seen for the monomer. We set out to examine whether lipids of this kind would produce a signal in response to ligand binding. RESULTS: A model ligand, poly-L-histidine (poly(His)), bound specifically to pyrene-labeled Cu(II)-iminodiacetate lipid (Cu-PSIDA) within a membrane matrix. Binding of poly(His) induces the redistribution of Cu-PSIDA, so that it forms pyrene-rich domains that are detectable by the increased ratio of excimer to monomer fluorescence. Using rhodamine-labeled poly(His), we have shown that the receptor lipid domains correspond to poly(His)-rich domains below the lipid interface. CONCLUSIONS: The Cu-PSIDA receptor signals binding of the macromolecular ligand through its excimer fluorescence and allows the resulting domains formed by ligand assembly to be imaged. Fluorescent Cu-PSIDA can thus serve as an optical reporter of ligand-induced lipid reorganization.

Different patterns of homozygous p16INK4A and p15INK4B deletions in childhood acute lymphoblastic leukemias containing distinct E2A translocations.

The p16INK4A (p16) and p15INK4B (p15) tumor suppressor genes are inactivated by homozygous gene deletion and p15 promoter hypermethylation in a significant proportion of childhood acute lymphoblastic leukemias (ALLs). However, little is known about the potential association between p16/p15 gene alterations and specific genetic abnormalities implicated in leukemogenesis. The t(1;19)(q23;p13) and t(17;19)(q21-22;p13) are non-random translocations observed in childhood ALL that create distinct E2A fusion proteins: E2A-PBX1 and E2A-HLF, respectively. Previously, a negative association was found between the t(1;19) and homozygous p16/p15 deletions. In this study we determined p16 and p15 gene status in additional t(1;19)+ ALLs and compared this incidence to that observed in t(17;19)+ ALLs. No homozygous p16 or p15 deletions were observed among 13 t(1;19)+ ALLs analyzed. In contrast, homozygous deletions of both p16 and p15 were present in two of four t(17;19)+ ALLs. None of 10 t(1;19)+ ALLs contained p15 promoter hypermethylation. In contrast, one of the two t(17;19)+ ALLs that lacked p15/p16 homozygous deletions showed probable hemizygous p15 hypermethylation. We conclude that homozygous p16 and/or p15 deletions and p15 hypermethylation rarely accompany E2A-PBX1 fusion, but occur in concert with E2A-HLF fusion in a subset of t(17;19)+ ALLs. These findings suggest that there may be different modes of cooperative leukemogenesis in ALLs associated with different E2A fusion proteins.