Graft-versus-host disease after double-unit cord blood transplantation has unique features and an association with engrafting unit-to-recipient HLA match.

Manifestations of and risk factors for graft-versus-host disease (GVHD) after double-unit cord blood transplantation (DCBT) are not firmly established. We evaluated 115 DCBT recipients (median age, 37 years) who underwent transplantation for hematologic malignancies with myeloablative or nonmyeloablative conditioning and calcineurin inhibitor/mycophenolate mofetil immunosuppression. Incidence of day 180 grades II to IV and III to IV acute GVHD (aGVHD) were 53% (95% confidence interval, 44 to 62) and 23% (95% confidence interval, 15 to 31), respectively, with a median onset of 40 days (range, 14 to 169). Eighty percent of patients with grades II to IV aGVHD had gut involvement, and 79% and 85% had day 28 treatment responses to systemic corticosteroids or budesonide, respectively. Of 89 engrafted patients cancer-free at day 100, 54% subsequently had active GVHD, with 79% of those affected having persistent or recurrent aGVHD or overlap syndrome. Late GVHD in the form of classic chronic GVHD was uncommon. Notably, grades III to IV aGVHD incidence was lower if the engrafting unit human leukocyte antigen (HLA)-A, -B, -DRB1 allele match was >4/6 to the recipient (hazard ratio, 0.385; P = .031), whereas engrafting unit infused nucleated cell dose and unit-to-unit HLA match were not significant. GVHD after DCBT was common in our study, predominantly affected the gut, and had a high therapy response, and late GVHD frequently had acute features. Our findings support the consideration of HLA- A,-B,-DRB1 allele donor-recipient (but not unit-unit) HLA match in unit selection, a practice change in the field. Moreover, new prophylaxis strategies that target the gastrointestinal tract are needed.

Mutations in the BRCA1 gene in families with early-onset breast and ovarian cancer.

We analysed 50 probands with a family history of breast and/or ovarian cancer for germline mutations in the coding region of the BRCA1 candidate gene, using single-strand conformation polymorphism (SSCP) analysis on PCR-amplified genomic DNA. A total of eight putative disease-causing alterations were identified: four of these are frameshifts and two are nonsense mutations. In addition, we found two missense mutations, one of which changes the final cysteine of the BRCA1 zinc finger motif to glycine. These data are consistent with a tumour suppressor model, and support the notion that this candidate gene is in fact BRCA1. The heterogeneity of mutations, coupled with the large size of the gene, indicates that clinical application of BRCA1 mutation testing will be technically challenging.

Spatial dependency of the PPG morphology at right carotid common artery.

PhotoPlethysmoGraphy (PPG) is ubiquitously employed in wearable devices for health monitoring. Photodiode signal inversion is observed in rare occasions, most of the time when the sensor is pressed against the skin. We report in this article such observations made at the right common carotid artery site. Indeed we have systematically observed a photodiode signal inversion when the PPG sensor is placed where the pulse is the best felt at the carotid. In addition to be inverted, the pulse is steeper during the systolic phase. Such inversion has implications in terms of pulse arrival time (PAT) measurements In our experiments, this causes a difference of 20 ms in the carotid PAT when measured at the absolute maximum slope. The mechanical and optical properties of tissues must be better accounted to explain the PPG signal morphology. Clinical Relevance- Understanding the role of mechanical tissue properties seems relevant in order to obtain more reproducible results in PPG signal analysis.

Evaluation of a dual-PPG system for pulse transit time monitoring.

This work presents a new dual-photoplethysmographic (PPG) system for pulse transit time (PTT) monitoring. An experiment has been set up in order to compare the PTT measurement between carotid and radial arteries from two systems: our physiological multimodal platform (PMP) and the Complior® tonometer. This work explores the comparison between such optical and mechanical modalities. The results show that the PPG device tends to overestimate the PTT (RMSE = 16 ms). Furthermore, both mechanical and optical signals have been superposed and demonstrated that pulse morphologies are quite similar.Clinical Relevance-Carotid-radial pulse wave velocity (PWV) is compared on a small cohort of subjects and significant differences are observed between optical and mechanical-based systems.

Blood pressure measurement by coupling an external pressure and photo-plethysmographic signals.

This work presents a new technique for the noninvasive measurement of systolic blood pressure (sBP), mean blood pressure (mBP) and diastolic blood pressure (dBP) using photo-plethysmographic (PPG) sensors when an artery is exposed to an external pressure. Two sets of experiments were performed: a first study combining PPG with an oscillometric device and the second combining PPG with a force/pressure sensor. These two experiments enable the estimation of BP values. PPG results were found to be comparable to oscillometric results, with bias and precision errors of -0.81 and 5.26 for sBP, 1.12 and 5.61 for mBP and 1.67 and 9.09 for dBP (n = 28). Furthermore, amplification over the brachial-to-finger path was found to be 1.02 ± 0.08 for mBP (n = 20) confirming that mBP does not undergo any amplification along the arterial tree whereas, for sBP, an amplification of 1.20 ± 0.12 (n = 7) was found for green wavelength, 1.35 ± 0.09 (n = 6) for red wavelength and 1.36 ± 0.09 (n = 6) for infrared wavelength. Thus, amplification for sBP is bigger for red and infrared wavelengths compared to green one.

Transfer of potassium. A new measure of cell-cell coupling.

Mammalian cells of different species differ in sensitivity to ouabain. This sensitivity is expressed as reduced intracellular K+ content, reduced rates of protein synthesis, and cessation of cell multiplication. Using 86Rb+ as a measure of intracellular K+, we found higher levels of radioactivity in mixtures of ouabain-sensitive and -resistant cells cultured in the presence of ouabain than predicted from pure cultures of the two component cell types. The simplest explanation is that K+ and 86Rb+ are being transferred from ouabain-resistant to ouabain-sensitive cells, enhancing the total intracellular 86Rb+ in the culture. A function, "index of cooperation," expresses this enhancement as a number ranging from 0 to 1, and permits comparisons to be made under various culture conditions and using various cell types. An index of cooperation greater than 0 requires cell contact, since no enhancement occurs when contact between two cell types in the same culture is prevented. The index of cooperation for a number of different cell combinations agrees with other measures of cell-cell interaction associated with gap junctions, such as electrical coupling and metabolic cooperation. Coculture of ouabain-sensitive and ouabain-resistant cells in the presence of ouabain also leads to restoration of the capacity for protein synthesis. Autoradiography shows that this restoration occurs in the sensitive cell type and is dependent upon contact with ouabain-resistant cells. Furthermore, sensitive cells are able to multiply in the presence of ouabain when cocultured with resistant cells. Thus K+, presumably transferred to sensitive cells through gap junctions, is able to counteract the toxic effects of ouabain on intracellular K+ levels and protein synthesis, and to restore growth.

Impaired growth hormone secretion in the adult population: relation to age and adiposity.

Growth hormone (GH) release was studied in adults of normal stature, ages 21-86 yr. The subjects were 85-115% of ideal body weight, between the 5th and 95th percentiles in height, and free of active or progressive disease. 9 to 12 individuals in each decade from thirds to ninth were evaluated. The following criteria of GH status were measured: serum GH concentration, analyzed by radioimmunoassay at half-hour intervals for 4 h after onset of sleep, and at 1-h intervals from 8 a.m. to 4 p.m. in 52 subjects; daily retention of N, P, and K in response to 0.168 U human (h)GH/kg body wt3/4/day in 18 subjects; and plasma somatomedin C (SmC) level before and during exogenous hGH treatment in 18 subjects. All 10 individuals, 20-29 yr old, released substantial amounts of endogenous GH during both day and night (average peak serum GH obtained during day and night was 7.3 and 20.3 ng/ml, respectively); average plasma SmC was 1.43 U/ml (95% tolerance limits, 0.64-2.22 U/ml). There was no significant effect of exogenous hGH on elemental balances or on plasma SmC. In contrast, 6 of 12 individuals 60-79 yr old showed the following evidences of impaired GH release; peak waking and sleeping serum GH less than 4 ng/ml; plasma SmC less than 0.38 U/ml; a significant retention in N, P, and K; and a significant rise in plasma SmC, in response to exogenous hGH. Plasma SmC, serum GH during sleep, serum GH during the day, retentions of N, P, and K in response to exogenous hGH, and rise in plasma SmC in response to hGH were all intercorrelated (P less than 0.05). Plasma SmC less than 0.38 U/ml corresponded to peak nocturnal serum GH less than 4 ng/ml. The prevalence of plasma SmC less than 0.38 U/ml increased progressively from age 20 to 90: third decade, 0%; fourth, 11%; fifth, 20%; sixth, 22%; seventh, 42%; eight, 55%; and ninth, 55%. Within each decade, plasma SmC was inversely related to adiposity.

Prolonged inhibition of protein and glycoprotein synthesis in tumor cells treated with muconomycin A.

Several agents were compared for their ability to inhibit protein synthesis for long periods in tumor cells growing in culture. Mouse B16 melanoma cells, treated with high concentrations of cycloheximide or pactamycin for 1 hour and then washed repeatedly, recovered their ability to incorporate [3H]leucine into protein in about 4 hours, while cells treated with emetine recovered in 12 hours. After similar treatment with muconomycin A, however, incorporation of [3H]leucine remained inhibited for at least 30 hours. During this time the cells remained attached to the culture dishes, were able to exclude trypan blue dye, and retained nearly normal levels of rubidium-86 content. When another, untreated, population of cells was added to the muconomycin-treated cells, protein synthesis was not inhibited in the untreated population; action of the drug was thus shown to be confined to the treated cells. In melanoma cells treated with neuraminidase and muconomycin, measurement of glycoprotein synthesis (as determined by sialic acid analysis) showed that muconomycin also inhibited restoration of sialic acid content. Brief treatment with muconomycin, therefore, appeared to be sufficient for prolonged inhibition of protein and glycoprotein synthesis.

Increased tumor immunity in mice inoculated with muconomycin A-treated B16 melanoma cells.

B16 melanoma cells were treated in vitro with muconomycin A, a long-lasting inhibitor of protein and glycoprotein synthesis, to reduce cellular sialic acid. Two i.p. inoculations of 10(7) muconomycin-treated cells into female C57BL/6 mice, followed by challenge with homologous live cells, resulted in a significant decrease in tumor incidence when compared to the results of inoculation with untreated cells (p less than 0.01). Inoculation of mice with cells treated with neuraminidase resulted in little or no decrease in tumor incidence. Effective immunity was dependent on the number of cells injected and was found only with the i.p. route of inoculation into female mice.

Autoimmune hemolysis and immune thrombocytopenic purpura after cord blood transplantation may be life-threatening and warrants early therapy with rituximab.

Autoimmune hemolysis (AH) and immune thrombocytopenic purpura (ITP) are recognized complications after cord blood transplantation (CBT). We evaluated the incidence and characteristics of AH/ITP after double-unit CBT in a day 100 landmark analysis of 152 patients (median age 36 years, range 0.9-70 years) transplanted for hematologic malignancies with myeloablative or nonmyeloablative conditioning and calcineurin inhibitor (CNI)/mycophenolate mofetil. With a median 5.2-year (range 1.6-9.7 years) survivor follow-up, 10 patients developed autoimmune cytopenias (8 AH, 1 ITP, 1 both) at a median of 10.4 months (range 5.8-24.5) post CBT for a 7% cumulative incidence 3 years after the day 100 landmark. Six patients presented with severe disease (hemoglobin ⩽6 g/dL and/or platelets <20 × 109/L). All AH patients were direct antiglobulin test positive. All 10 cases developed during immunosuppression taper with 8 having prior acute GVHD. All 10 patients received rituximab 2-18 days after diagnosis, and corticosteroids combined with rituximab within <7 days was the most effective. No patient died of AH/ITP. AH/ITP occurs infrequently after CBT but may be life-threatening requiring emergency therapy. Rituximab combined with corticosteroids at diagnosis is warranted in patients with severe disease.

Bisphenolic compounds that enhance cell cation transport are found in commercial phenol red.

We have isolated two bisphenolic compounds (4 and 5) that have a marked effect on K+ and Na+ concentrations in human cells from commercial preparations of the pH indicator dye phenol red (phenolsulfonphthalein). We used a bioassay to identify active chromatographic fractions from the lipophilic impurities present in phenol red, and we determined the structure of two active components (4 and 5) by 1H and 13C NMR and mass spectrometry. When added to human fibroblasts in serum-free medium, the bisphenol fluorene derivative 9,9-bis(4'-hydroxyphenyl)-3-hydroxyfluorene (5) produced a rapid loss of K+ and a gain of Na+, at low concentrations, with an EC50 between 30 and 60 ng/mL (80-160 nM). The 2- and 4-hydroxy isomers of the fluorene 5 (i.e., compounds 6 and 7), prepared by synthesis, had similar activity, although compound 6 was somewhat less potent. The bisphenol xanthene derivative 9,9-bis(4'-hydroxyphenyl)xanthene (4) elicited a similar biological response but was less potent than 5-7; it also had a strong effect on cell adhesion, causing release of cells from the plastic substrate at concentrations as low as 2-5 microg/mL (5.5-14 microM). The structures of xanthene (4) and fluorene (5) bisphenols have been confirmed by synthesis from xanthone and hydroxyfluorenone, respectively, by Friedel-Crafts alkylation with phenol. In the latter case, the desired 3-hydroxyfluorene isomer was formed in situ by rearrangement of the 1-hydroxy isomer.

The subcellular distribution of nitric oxide synthase relative to the NR1 subunit of NMDA receptors in the cerebral cortex.

Results from several electrophysiological studies predict that the neuronal NO-synthesizing enzyme, nNOS, resides within spines formed by pyramid-to-pyramid axo-spinous synaptic junctions of the cortex. On the other hand, light microscopic neuroanatomical detection of nNOS within pyramidal neurons has been difficult, suggesting that these neurons contain nNOS at levels below threshold for detection. Our results obtained by electron microscopic immunocytochemistry indicate that nNOS occurs within spiny neurons, such as those of pyramidal neurons, albeit discretely within their spines. Dual electron microscopic immunocytochemistry, whereby antigenic sites to the NR1 subunit of NMDA receptors are probed simultaneously with sites immunoreactive for nNOS, reveals that some, although not all, nNOS within spines co-exist with NR1 subunits. Additionally, immunoreactivity for the NR1 subunit is detectable within nNOS-axons, indicating that NO may be generated in response to axo-axonic interactions with glutamatergic axons in the vicinity and independently of action potential propagation. Immunoreactivity for NR1 subunits within axons (with or without nNOS-immunoreactivity) may additionally serve to confer receptivity of these axons to NO generated coincidentally with activity. Analysis of the visual cortex of monocular adult animals indicates that the level of nNOS within neurites is dependent on chronic activity levels of the surrounding neuropil and independent of somatic input level. Together, these findings point to plasticity of nNOS neurons within adult brain tissue, involving regulation of subcellular nNOS distribution.

Influence of endogenous albumin binding on blood-material interactions.

A method has been developed to enhance the albumin affinity of a number of medical polymers, based on alkylation of the surface with straight-chain 16- or 18-carbon alkyl groups. This method has been demonstrated to induce the rapid binding of albumin from single and binary protein solutions, from plasma, and apparently, from whole blood. The bound albumin resists fluid shear or chemically induced desorption. Fibrinogen adsorption is inhibited in vitro and in vivo. Complement protein C3 activation from plasma is inhibited. Fibrin formation and platelet aggregation is inhibited in short-term in vivo experiments. Long-term catheter implant studies suggest that the C18 alkylation is more effective than most, if not all, currently available treatments for the retention of a clean, biocompatible, blood-contacting surface. No data have been obtained to date that conflict with the hypothesis that a renewable albumin layer, so formed, blocks the adsorption or conformational alteration of plasma proteins that otherwise might initiate or participate in various host defenses.

Preservation of ultrastructure and antigenicity for EM immunocytochemistry following intracellular recording and labeling of single cortical neurons in brain slices.

Knowledge of the distribution of neurotransmitters, neuromodulators, and transmitter receptors operating at specific synaptic sites on cortical neurons is essential for understanding the precise mechanisms that underlie the dynamic properties of cortical microcircuitry. We report on a new combination of techniques for analyzing chemically-specified synaptic input to individual cortical neurons first electrophysiologically characterized in the in vitro brain slice preparation. We tested the feasibility of this approach by performing intracellular recordings and biocytin injections in guinea pig medial prefrontal cortex slices and then by performing dual preembedding immunocytochemistry in order to localize neuronal nitric oxide synthase relative to single biocytin-filled neurons. The recorded cell and nitric oxide synthase immunoreactivity were visualized by light and electron microscopy utilizing both peroxidase and silver intensified gold stains. Single neurons were also dually visualized with fluorescence for light microscopy and with silver intensified gold for electron microscopy. Our findings indicate that both antigenicity and ultrastructure can be well preserved in tissue first used for in vitro slice experiments. This combination of methods should be widely applicable for analyzing the subcellular distribution of neuronal molecules such as receptors, channels and enzymes on physiologically characterized mammalian neurons.

Nitric oxide synthase in the visual cortex of monocular monkeys as revealed by light and electron microscopic immunocytochemistry.

Recent results indicate that nitric oxide (NO) can play an important role in neuronal excitability by modifying the strength of activated synapses and regulating local cerebral blood flow. We sought to determine whether the level of NO synthase (NOS) could, in turn, also be regulated by neural activity. Results using a polyclonal anti-NOS antibody showed that, in cortical area V1 of monocular monkeys, NOS-immunoreactivity is diminished in lamina 4C neuropil of the deprived ocular dominance columns relative to the immediately adjacent non-deprived columns. Closer examination of lamina 4C indicated that the intercolumnar difference in NOS-immunoreactivity does not reflect differences in the distribution of NOS-labeled perikarya, since relatively few neurons were immunoreactive for NOS in lamina 4C of either monocular or normal binocular monkeys. Electron microscopy revealed that the majority (> 80%) of NOS-immunoreactive profiles in lamina 4C are axon terminals. NOS-immunoreactive spines and dendritic shafts also are present but these are more prevalent in the superficial laminae. In order to determine whether the intercolumnar differences in lamina 4C neuropil correspond to altered densities of NOS cells in the superficial laminae, we performed a series of quantitative analyses. In the superficial laminae, NOS-cells occur as two distinguishable classes: a few that are large and intensely NOS-immunoreactive and many more (ca. 24-fold) that are small and lightly immunoreactive. Analysis of the distribution of 559 small and 105 large NOS-immunoreactive cells within 40-microns-thick tangential sections spanning laminae 2-3 showed that the number of cells (large and small together) associated with each blob is approximately 14 for both deprived (lighter) and non-deprived (darker) blobs. These cells are distributed evenly from the center to periphery of columns. Analysis of the distribution of NOS-cells in the infragranular laminae also did not reveal any columnar differences. These observations suggest that local neural activity may be coupled to NO release via alteration of NOS protein levels specifically within distal axonal processes of neurons. This mechanism could operate in conjunction with the more instantaneous catalytic activation of NOS. Ultrastructural analyses further suggest that NO may act as an anterograde and retrograde messenger arising from terminals in addition to its proposed role as a retrograde messenger arising from dendrites.

NMDA-R1 subunit of the cerebral cortex co-localizes with neuronal nitric oxide synthase at pre- and postsynaptic sites and in spines.

The majority of nitric oxide's (NO) physiologic and pathologic actions in the brain has been linked to NMDA receptor activation. In order to determine how the NO-synthesizing enzyme within brain, neuronal NO synthase (nNOS), and NMDA receptors are functionally linked, previous studies have used in situ hybridization techniques in combination with light microscopic immunocytochemistry to show that the two are expressed within single neurons. However, this light microscopic finding does not guarantee that NMDA receptors are distributed sufficiently close to nNOS within single neurons to allow direct interaction of the two. Thus, in this study, dual immuno-electron microscopy was performed to determine whether nNOS and NMDA receptors co-exist within fine neuronal processes. We show that nNOS and the obligatory subunit of functional NMDA receptors, i.e. the NMDA-R1, co-exist within dendritic shafts, spines and terminals of the adult rat visual cortex. Axon terminals form asymmetric synaptic junctions with the dually labeled dendrites, suggesting that the presynaptic terminals release glutamate. Axons and dendrites expressing one without the other also are detected. These results indicate that it is possible for the generation of NO to be temporally coordinated with glutamatergic synaptic transmission at axo-dendritic and axo-axonic junctions and that NO may be generated independently of glutamatergic synaptic transmission. Together, our observations point to a greater complexity than previously recognized for glutamatergic neurotransmission, based on the joint versus independent actions of NO relative to NMDA receptors at pre- versus postsynaptic sites.

Is age a risk factor for surgery?

The question of age as a risk factor for surgery is complicated. There are many factors that have an impact on mortality rates in surgical care of the elderly. The most important of these are the physiologic changes with aging, underlying disease states, the type of procedures performed, and whether the procedure is performed as an emergency. Although there are many risks in performing surgery in elderly patients, there are many patients who do well and benefit from undergoing surgical procedures. Age alone should never be used as the criterion to deny surgery indicated in an elderly patient.

Cost-effectiveness of computerized tomography and magnetic resonance imaging in dementia.

The benefits and costs of routinely using computerized tomography (CT) to diagnose surgically treatable causes of dementia compared to a more selective strategy were assessed, using a decision-analytic model, for hypothetical cohorts at 60, 70, and 80 years of age. The model was also used to project what the impact would be if magnetic resonance imaging (MRI) were to replace CT, assuming that MRI is a perfect test. Given plausible assumptions, routine CT could be expected to detect between 1 425 and 14 930 additional surgically treatable cases at an extra cost of between $0 and $49 million per 100 000 persons scanned. Replacing CT with MRI might yield an additional 70 to 150 cases of surgically treatable dementia, at an additional cost of $20-$30 million. Given current treatment limitations in dementia, it appears that, as a clinical tool, MRI will have little immediate health impact on this problem.

Effects of diet supplementation with wheat bran on serum estrogen levels in the follicular and luteal phases of the menstrual cycle.

There is both epidemiologic and experimental support for the hypothesis that a high-fiber diet can reduce breast cancer risk; this may be due, at least in part, to a reduction in circulating estrogens. This study examined the effects of three levels of wheat bran supplementation (5, 10, and 20 g/d for 2 mo) on the major serum estrogens during both the luteal and follicular phases of the menstrual cycle. The 10- and 20-g supplements, which increased the total dietary fiber intakes to approximately 20 and 32 g/d, respectively, resulted in significant decreases in the luteal serum estrone (P < 0.05 and < 0.02, respectively). The serum estradiol was significantly reduced in the 10-g wheat bran group after 2 mo (P < 0.05); the 20-g supplemented group showed a significant decrease in estradiol at 1 mo (P < 0.02), but not at 2 mo. No changes occurred in the estrone sulfate concentrations. During the follicular phase, the 10-g wheat bran group exhibited a significant reduction in the serum estrone (P < 0.02). Only the serum estrone sulfate showed any reduction with the 20-g supplement, and this just failed to achieve significance (P = 0.07). Serum sex hormone-binding globulin levels were unaffected by wheat bran. When of long duration, these effects may be sufficient to favorably influence breast cancer risk in Western women.