α1 -Adrenergic receptor-induced cytoskeletal organization and cell motility in CCL39 fibroblasts requires phospholipase D1.

The role of phospholipase D (PLD) in cytoskeletal reorganization, ERK activation, and migration is well established. Both isoforms of PLD (PLD1 and PLD2) can independently activate stress fiber formation and increase ERK phosphorylation. However, the isoform's specificity, upstream activators, and downstream targets of PLD that coordinate this process are less well understood. This study explores the role of α(1) -adrenergic receptor stimulation and its effect on PLD activity. We demonstrate that PLD1 activators, RhoA, and PKCα are critical for stress fiber formation and ERK activation, and enhance the production of phosphatidic acid (PA) upon phenylephrine addition. Ectopic expression of dominant negative PLD1 and not PLD2 blocks ERK activation, inhibits stress fiber formation, and reduces cell motility in CCL39 fibroblasts. Furthermore, we demonstrate the mechanism for PLD1 activation of ERK involves Ras. This work indicates that PLD1 plays a novel role mediating growth factor and cell motility events in α(1) -adrenergic receptor-activated cells.

Real world analysis of high-cut-off (HCO) hemodialysis with bortezomib-based backbone therapy in patients with multiple myeloma and acute kidney injury.

BACKGROUND: In patients with multiple myeloma (MM) free light chain-induced cast nephropathy is a serious complication associated with poor survival. High-cut-off (HCO) hemodialysis can reduce the amount of serum free light chains (sFLC), but data on its impact on clinical outcome is limited and contradictory. To gain further insights we collected real world data from two major myeloma and nephrology centers in Austria and the Czech Republic. METHODS: Sixty-one patients with MM and acute kidney injury, who were treated between 2011 and 2019 with HCO hemodialysis and bortezomib-based MM therapy, were analyzed. RESULTS: The median number of HCO hemodialysis sessions was 11 (range 1-42). Median glomerular filtration rate at diagnosis was 7 ± 4.2 ml/min/1.73m2. sFLC after the first HCO hemodialysis decreased by 66.5% and by 89.2% at day 18. At 3 and 6 months, 26 (42.6%) and 30 (49.2%) of patients became dialysis-independent. CONCLUSION: The widely used strategy combining HCO hemodialysis and bortezomib-based antimyeloma treatment is dissatisfactory for half of the patients undergoing it and clearly in need of improvement.

Human monocytic cells direct the robust release of CXCL10 by bronchial epithelial cells during rhinovirus infection.

BACKGROUND: Human rhinovirus (HRV) infections are a major cause of exacerbations in chronic respiratory conditions such as asthma and chronic obstructive pulmonary disease, but HRV-induced immune responses of the lower airway are poorly understood. Earlier work examining cytokine release following HRV infection has focused on epithelial cells because they serve as the principal site of viral replication, and internalization and replication of viral RNA appear necessary for epithelial cell mediator release. However, during HRV infection, only a small proportion of epithelial cells become infected. As HRV-induced cytokine levels in vivo are markedly elevated, this observation suggests that other mechanisms independent of direct viral infection may induce epithelial cell cytokine release. OBJECTIVE: Our aim was to test for the importance of interactions between human bronchial epithelial cells (HBECs) and monocytic cells in the control of mediator release during HRV exposure. METHODS: In vitro models of HRV serotype-16 (HRV16) infection of primary HBECs and human monocytic cells, in mono or co-culture, were used. We assessed HRV16-induced CXCL10 and CCL2 protein release via ELISA. RESULTS: Co-culture of human monocytic and bronchial epithelial cells promoted a synergistic augmentation of CXCL10 and CCL2 protein release following HRV16 challenge. Transfer of conditioned media from HRV16-treated monocytic cells to epithelial cultures induced a robust release of CXCL10 by the epithelial cells. This effect was greatly attenuated by type I IFN receptor blocking antibodies, and could be recapitulated by IFN-alpha addition. CONCLUSIONS: Our data indicate that epithelial CXCL10 release during HRV infection is augmented by a monocytic cell-dependent mechanism involving type I IFN(s). Our findings support a key role for monocytic cells in the amplification of epithelial cell chemokine production during HRV infection, and help to explain how an inflammatory milieu is created in the lower airways even in the absence of extensive viral replication and epithelial infection.

Variations in storm-induced bed level dynamics across intertidal flats.

Hydrodynamic forces on intertidal flats vary over a range of temporal and spatial scales. These spatiotemporal inhomogeneities have implications for intertidal flat morphodynamics and ecology. We determine whether storm events are capable of altering the long-term morphological evolution of intertidal flats, and unravel the contributions of tidal flow, wind-driven flow, waves, and water depth on inhomogeneities in bed level dynamics (bed level changes over ~days) across these areas. We complement decades of bed level measurements on eight intertidal flats in two estuaries in the Netherlands with an extensive 1-month field campaign on one of those flats. Across this intertidal flat, the hydrodynamics and morphodynamics of a storm event were captured, including the post-storm recovery. We show that individual events can persistently alter the morphological evolution of intertidal flats; magnitudes of some bed level changes are even comparable to years of continuous evolution. The morphological impacts of events are largely controlled by the relative timing of the forcing processes, and not solely by their magnitudes. Spatiotemporal variations in bed level dynamics of intertidal flats are driven by a combination of: (1) the inhomogeneous distributions of the hydrodynamic forcing processes (including the under-explored role of the wind); and (2) the linear proportionality between bed level dynamics and the local bed slope.

Levels of CEACAM6 in Peripheral Blood Are Elevated in Patients with Plasma Cell Disorders: A Potential New Diagnostic Marker and a New Therapeutic Target?

INTRODUCTION: The prognosis of multiple myeloma is still unfavorable due to inherent characteristics of the disease and the often-delayed diagnosis due to widespread and unspecific symptoms such as back pain and fatigue. Therefore, a simple diagnostic blood test would be helpful to speed up the diagnostic procedure in such patients (pts.). Here, we evaluated the diagnostic value of plasma levels of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) in the peripheral blood and bone marrow of pts. with plasma cell disorders and in healthy controls. MATERIALS AND METHODS: Immunoreactive CEACAM6 was determined in the peripheral blood and bone marrow (n = 95/100) of pts. with monoclonal gammopathy of unknown significance (MGUS: 28/37), newly diagnosed multiple myeloma (NDMM: 42/40), and relapsed/refractory multiple myeloma (RRMM: 25/23) by sandwich ELISA. RESULTS: Median CEACAM6 levels in the peripheral blood of pts. with plasma cell disorders were significantly higher than those of healthy controls (healthy controls: 15.2 pg/ml (12.1-17.1); MGUS: 19.0 pg/ml (16.4-22.5); NDMM: 18.0 pg/ml (13.4-21.2); and RRMM: 18.9 pg/ml (15.2-21.5); p < 0.001). Plasma levels of CEACAM6 discriminated healthy subjects from MGUS/NDMM pts. (AUC = 0.71, 95% CI: 0.6-0.8); i.e., a CEACAM6 level > 17.3 pg/ml has an 82% (95% CI: 70-90) predictive probability for the identification of MGUS or NDMM. Moreover, CEACAM6 levels in the bone marrow were significantly higher in RRMM pts. than in NDMM pts. (p = 0.04), suggesting a role of this molecule in disease progression. CONCLUSION: CEACAM6 plasma levels can noninvasively identify pts. with a plasma cell disorder and should be evaluated prospectively as a potential diagnostic marker. Moreover, due to high CEACAM6 levels in the bone marrow in RRMM pts., this adhesion molecule might be a therapeutic target in multiple myeloma pts.

Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci.

Gross variations in the structure of the centromere of Schizosaccharomyces pombe chromosome III (cen3) were apparent following characterization of this centromeric DNA in strain Sp223 and comparison of the structure with that of cen3 in three other commonly used laboratory strains. Further differences in centromere structure were revealed when the structure of the centromere of S. pombe chromosome II (cen2) was compared among common laboratory strains and when the structures of cen2 and cen3 from our laboratory strains were compared with those reported from other laboratories. Differences observed in cen3 structure include variations in the arrangement of the centromeric K repeats and an inverted orientation of the conserved centromeric central core. In addition, we have identified two laboratory strains that contain a minimal cen2 repeat structure that lacks the tandem copies of the cen2-specific block of K-L-B-J repeats characteristic of Sp223 cen2. We have also determined that certain centromeric DNA structural motifs are relatively conserved among the four laboratory strains and eight additional wild-type S. pombe strains isolated from various food and beverage sources. We conclude that in S. pombe, as in higher eukaryotes, the centromere of a particular chromosome is not a defined genetic locus but can contain significant variability. However, the basic DNA structural motif of a central core immediately flanked by inverted repeats is a common parameter of the S. pombe centromere.

Advances in Cardiac Biomarkers of Acute Coronary Syndrome.

Acute coronary syndrome (ACS) encompasses a pathophysiological spectrum of cardiovascular diseases, all of which have significant morbidity and mortality. ACS was once considered an acute condition; however, new treatment strategies and improvements in biomarker assays have led to ACS being an acute and chronic disease. Cardiac troponin is the preferred biomarker for the diagnosis of myocardial infarction, and there is considerable interest and efforts toward development and implementation of high-sensitivity cardiac troponin (hs-cTn) assays worldwide. Analytical and clinical performance characteristics of hs-cTn assays as well as testing limitations are important for laboratorians and clinicians to understand in order to utilize testing appropriately. Furthermore, expanding the clinical utility of hs-cTn into other cohorts such as asymptomatic community dwelling populations, heart failure, and chronic kidney disease populations supports novel opportunities for improved short- and long-term prognosis.

Cellular crossreactivity. Implications for solid organ transplantation matching.

This study evaluates the cellular crossreactivity among DR11, DR13, and DR8 molecules using TLC reagents generated in reciprocal priming combinations where the responder and stimulator cells express different microvariants of DR11. The large majority of T lymphocyte clones (TLC) derived from such stimulation detect not only the product of the specific DR11 allele expressed by the stimulator but also detect subsets of DR molecules that span serologic specificities. Thus, TLC generated in response to DR(alpha,beta1*1102) detect DR(alpha,beta1*1103) and products of specific DR13, DR8, DR2 and DR4 alleles. Whereas, TLC generated in response to DR(alpha,beta1*1104) detect DR(alpha,beta1*1101), DR(alpha,beta1*1103), and products encoded by specific DR8 and DR2 but not DR13 or DR4 alleles. Since DR11 microvariants cannot be identified serologically, this type of mismatch certainly occurs frequently between DR11 serologically matched donors and recipients. Particularly affected are populations, such as the African American population, that exhibit extensive HLA diversity and exhibit different frequencies of HLA alleles compared with those of the majority of serologically matched cadaveric donors. Rapid methods of DNA-based HLA typing now makes it feasible to utilize this methodology for allele level identificaiton of recipient and donor alleles. Based on the strength of the alloproliferative responses and on the recognition patterns of the TLC reported here, we suggest that retransplant patients might benefit by excluding subsequent donors expressing DR molecules that in vitro demonstrate strong cellular crossreactivity with DR molecules expressed by the previous donor(s) as well as those DR molecules shared with the previous donor(s). Since such a matching schema has the potential to improve retransplant allograft survival, particularly in patients from minority population groups, it should be evaluated clinically.

Hypercholesterolemia in adolescents: effectiveness of screening strategies based on selected risk factors.

This study evaluates the efficacy of the American Academy of Pediatrics' current recommendations for identifying hypercholesterolemia in teenagers. These guidelines advocate a screening strategy that is targeted only at high-risk individuals. Detailed information regarding risk factors for hypercholesterolemia was obtained from 1001 adolescents or their parents at three urban teen clinics during routine health assessments. In addition, serum cholesterol was measured in all adolescents. The population under study was 38.2% Hispanic, 33.5% white, 15.4% black, and 11.3% Asian, with a mean age of 15.6 years (range: 12 through 21 years); 52% were female. Based on the cholesterol values, the study population was divided into two groups: (1) normal cholesterol level, less than 200 mg/dL (n = 937, mean cholesterol value = 157 mg/dL) and (2) elevated cholesterol level, greater than or equal to 200 mg/dL (n = 64, mean cholesterol value = 228 mg/dL, range = 200 through 366 mg/dL). Application of the 1985 American Academy of Pediatrics criteria for selected screening identified 30 of 64 individuals with hypercholesterolemia (sensitivity = 47%). Criteria similar to 1988 recommendations identified 40 of 64 teenagers (sensitivity = 62%). A combination of 1985 and 1988 criteria yielded a sensitivity of 44 of 64, or 69%. The specificity of these criteria was 74%, 60%, and 53%, respectively. Despite the utilization of the 1985, 1988, or a combination of both American Academy of Pediatrics criteria for hypercholesterolemia screening, this study demonstrates that many adolescents with abnormal cholesterol levels would not be identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in peptide binding of DR11 and DR13 microvariants demonstrate the power of minor variation in generating DR functional diversity.

The influence of subtle HLA diversification on antigen binding was explored using murine L-cell transfectants expressing alleles in the DR11/DR13 family and a panel of peptides. The levels of binding among this family of DR microvariants were as diverse as the levels of binding among distantly related DR molecules. Even a single amino acid difference between allelic products had a profound effect on peptide binding. Specific amino acid substitutions, generated using site-directed mutagenesis to alter polymorphic residues at DR beta 32, 37, 57, 58, 67, 71, 86, demonstrated that a specific change within the context of a single DR molecule differed in its effect on the binding of specific peptides. In addition, a specific amino acid substitution had a differential effect on the binding level of a peptide to different DR molecules. Each polymorphic amino acid appeared to play a role in the binding of some peptide. Studies using the amino-terminal portion of the invariant chain CLIP peptide suggested that this peptide may offer varying degrees of competition in the binding of the cellular peptide pool in cells expressing different DR molecules. Finally, the results obtained with two strain-specific peptides from an immunodominant region of a malarial parasite show differential binding to two DR13 molecules, suggesting that immune pressure may promote parasite diversity. A dynamic interaction may exist between pathogens and the immune system shaping the HLA profile in a population. Thus even subtle diversification of the HLA molecules, possibly pathogen driven, can have a substantial effect on peptide binding and immune recognition.

DQ polymorphism: an analysis of DQw4 alpha and beta membrane proximal regions.

The complete coding sequences of cDNA clones encoding the DQw4 alpha and beta polypeptides have been determined from two individuals expressing the DRw18(3), DQw4 haplotype. Although the first domain nucleotide sequence of the DQ alpha cDNA is very similar to the DQw2 alpha sequence, the sequence of the membrane proximal region (encoding second domain, transmembrane, and cytoplasmic segments) is more similar to DQw3-like alpha-gene sequences. The nucleotide sequence of the membrane proximal region of the DQw4 beta gene is identical to the DQw8 sequence in contrast to the extensive differences in the region encoding the first domains of these polypeptides. These sequences have been used to determine the evolutionary relationships among DQ alpha and beta genes.

DR and DQ beta cDNA sequences associated with a DR2 haplotype.

Three cDNA clones encoding a DQ beta and two DR beta polypeptides have been isolated and sequenced from an American black individual expressing a DR2,DQw1 haplotype. The sequences of the cDNA clones are identical to previously described DR and DQ sequences from a DR2,Dw2 cell. The differences between DQw1-associated beta chains from DR2 and DR1 haplotypes is substantial, although a DQw1-specific sequence can be identified. The identical DQ and DR beta sequences found in unrelated individuals from different racial backgrounds suggests that class II structural polymorphism within the human population will be limited.

HLA-B alleles associated with the B15 serologically defined antigens.

Cells expressing HLA molecules in the B15 family were identified by serologic typing in routine testing of volunteer donors of various ethnic backgrounds for a bone marrow registry. DNA sequencing was used to identify HLA-B15 alleles associated with each serologic type and to examine the diversity within the B15 antigen family. Alleles which appeared predominantly in each B15 serologic cluster included: B15 with no defined serologic subdivision (B*1501), B62 (B*1501), B63 (B*1516, B*1517), B75 (B*1502, B*1521), and B76/77 (B*1513). Other B*15 alleles were also found associated with the serotypes and some of these alleles (e.g., B*1501 and B*1516) were found in two or more serologic clusters illustrating the complexity of this family. The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype. Five new B*15 alleles (B*1530, B*1531, B*1533, B*1534, B*1535) and 5 other new HLA-B alleles (B*38022, B*3910, B*4010, B*51012, and B*5108) were also identified.

The DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci.

The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population, in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a DQw4 allelic product. This study characterizes the DR subregion of that haplotype. cDNA sequence analysis has revealed a DR beta gene which differs at several positions from previously described DR3 beta 1 genes. It is postulated that a gene-conversion-like event with a DRw52 beta gene as donor has generated some of these differences. The haplotype carries a DRw52a allele as defined by oligonucleotide hybridization studies. DNA restriction fragment analysis using a family and several unrelated individuals has allowed us to identify DR alpha and beta fragments associated with the DR3(w18),DQw4 haplotype. The most striking observation is that the DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci. Several genetic mechanisms including reciprocal recombination, gene conversion, and point mutation were involved in generating the differences between these haplotypes. Once established, the DR3(w18),DQw4 haplotype appears to be relatively stable in the population.

Diversity of alleles encoding HLA-B40: relative frequencies in united states populations and description of five novel alleles.

The frequency of each B*40 allele was determined by DNA sequencing in four major United States populations: Caucasians, African Americans, Asians/Pacific Islanders, and Hispanics. Thirty-two individuals from each ethnic group, who were previously described serologically as B40, B60, or B61, were randomly selected out of a pool of 82,979 unrelated individuals for allele characterization. Out of nine different B*40 alleles identified in this study, B*4001 and B*4002 were the two most frequent B*40 alleles in all the population groups. B*4001 was the primary B*40 allele seen in Caucasians (83%) and African Americans (76%), while B*4002 was found in the majority of Hispanics (62%). The distributions of both alleles were comparable in the Asian/Pacific Islander population. These two alleles were the only B*40 alleles detected in Caucasians while four to five additional B*40 alleles were seen in the other population groups. The other B*40 alleles detected in this study included: B*4003 and B*4010 in Asian/Pacific Islanders; B*4012 and B*4016 in African Americans; and B*4004, B*4006, and B*4027 in Hispanics. Analysis revealed significant differences between Hispanics and all other groups as well as between African Americans and Asian/Pacific Islanders. This report also describes five novel B*40 alleles: B*4019, B*4020, B*4024, B*4027, and B*4028.

Effects of drugs, age and illumination on response speed of squirrel monkeys.

Four young (2 1/2 years) and four older (over 10 years) squirrel monkeys were used to study age differences in the effects of d-amphetamine, methylphenidate and illumination on response speed. Although young monkeys were faster than the old monkeys, only the older monkeys showed an illumination effect. Both d-amphetamine and methylphenidate slowed response speed but only in the older monkeys showed an illumination illumination was present. These results suggest that older, mature squirrel monkeys are more sensitive to the effects of d-amphetamine, methylphenidate and illumination than young squirrel monkeys.

Effects of d-amphetamine and methylphenidate upon auditory threshold in the squirrel monkey.

The effects of d-amphetamine sulfate and methylphenidate hydrochloride on auditory thresholds in ten squirrel monkeys were examined using a 4.2 kHz stimulus in a free field. The results indicated that d-amphetamine raised auditory thresholds but methylphenidate did not alter the thresholds. The elevation of sensory thresholds by d-amphetamine was in agreement with previous studies suggesting that the drug acts as a behavioral depressant in diurnal animals.

Methods of hydration in palliative care patients.

A strong and often polarized debate has taken place during recent years concerning the consequences of dehydration in the terminally ill patient. When a patient has a severely restricted oral intake or is found to be dehydrated, the decision to administer fluids should be individualized and made on the basis of a careful assessment that considers problems related to dehydration, potential risks and benefits of fluid replacement, and patients' and families' wishes. This review discusses the assessment of hydration status in the terminal cancer patient and the options for fluid administration in the cases where evaluation of the patients' condition has led to this indication. These include different modes of hypodermoclysis, intravenous hydration, use of the nasogastric route, and proctoclysis. Arguments for and against rehydration have been previously addressed by other authors and fall outside the scope of this review.