Immune cell-derived C3a and C5a costimulate human T cell alloimmunity.

Emerging evidence indicates that complement provides costimulatory signals for murine T cells but whether complement impacts human T cells remains unclear. We observed production of complement activation products C3a and C5a during in vitro cultures of human T cells responding to allogeneic dendritic cells (DC). Both partners expressed the receptors for C3a (C3aR) and C5a (C5aR) and C3aR- and C5aR-antagonists inhibited T cell proliferation. Recombinant C3a/C5a promoted CD4(+) T cell expansion, bypassed the inhibitory effects of CTLA4-Ig, and induced AKT phosphorylation, the latter biochemically linking C3aR/C5aR to known T cell signaling pathways. Lowering DC C3a/C5a production by siRNA knockdown of DC C3 reduced T cell alloresponses. Conversely downregulating DC expression of the complement regulatory protein decay-accelerating factor increased immune cell C3a/C5a and augmented T cell proliferation, identifying antigen presenting cells as the dominant complement source. Pharmacological C5aR blockade reduced graft versus host disease (GVHD) scores, prolonged survival, and inhibited T cell responses in NOD scid γc(null) mouse recipients of human peripheral blood mononuclear cells, verifying that the mechanisms apply in vivo. Together our findings unequivocally document that immune cell-derived complement impacts human T cell immunity and provide the foundation for future studies targeting C3aR/C5aR as treatments of GVHD and organ transplant rejection in humans.

Fibroblast growth factor 23 (FGF23) and alpha-klotho stimulate osteoblastic MC3T3.E1 cell proliferation and inhibit mineralization.

Elevated serum levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) are found in patients with phosphate wasting diseases and chronic kidney disease-mineral and bone disorder (CKD-MBD). These diseases are associated with rickets and renal osteodystrophy, respectively. FGF23 is secreted from osteoblastic cells and signals through FGFRs, membrane coreceptor alpha-Klotho (Klotho), and, possibly, a circulating form of Klotho. Despite the absence of detectable Klotho on osteoblastic cells, studies have suggested that forced FGF23 expression in osteoblasts inhibited mineralization. Thus, we examined the effects of exogenously applied FGF23 on osteoblastic MC3T3.E1 cell proliferation and differentiation, with and without soluble Klotho. MC3T3.E1 cells were cultured in osteoblast differentiation medium, supplemented with FGF23 (0.1-1,000 ng/mL), Klotho (50 ng/mL), the combination FGF23 + Klotho, and FGF2 (100 ng/mL) as a control. Neither FGF23 nor Klotho exposure affected proliferation of day 4 growth phase cells or mineralization of day 14 cultures. In contrast, FGF23 + Klotho resulted in inhibition of mineralization and osteoblast activity markers at day 14, and a slight, reproducible induction of proliferation. Inhibition of FGFR1, but not FGFR2 or FGFR3, completely restored FGF23 + Klotho-induced inhibition of alkaline phosphatase (ALP) activity at day 7. ALP activity was partially restored by the MAPK inhibitor U0126 but not inhibitors p38 and P13K. Thus, soluble Klotho enables FGF23 signaling in MC3T3.E1 cells, likely through FGFR 1(IIIc). Elevated FGF23 actions, in part, appear to parallel FGF2 with lower potency. In addition to affecting bone via indirect phosphate wasting pathways, supraphysiological FGF23 and soluble Klotho may directly impact bone in diseases with elevated FGF23 levels.

Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol.

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10(-11)-10(-7) M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10(-8) M) + DM or paricalcitol (10(-8) M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required.

Guidelines and the adoption of 'lipid rescue' therapy for local anaesthetic toxicity.

Gathering evidence from animal experiments, an editorial in this journal and published human case reports culminated in the Association of Anaesthetists of Great Britain and Ireland recommending in August 2007 that lipid emulsion be immediately available to all patients given potentially cardiotoxic doses of local anaesthetic drugs. This development offered an opportunity to track the adoption of an innovation by anaesthetists in the UK and to gauge the effects of guidelines. Two surveys, each of 66 NHS hospitals delivering acute care within London and its penumbra, examined the adoption of lipid emulsion therapy. After the publication of the editorial in autumn 2006, the spread of 'lipid rescue' was rapid. The timing of the adoption and the impetus for innovation varied substantially between the sampled hospitals. When the formal guidelines were published, approximately half of the hospitals surveyed did not have lipid rescue. Of those that subsequently adopted it, half attributed their decision to the guidelines. At the end of 2007, there remained a small number of hospitals that had yet to adopt lipid rescue. Lipid rescue's adoption by anaesthetists in the UK offers a rare example of swift uptake of an innovation. National guidelines accelerated the adoption of innovation by some hospitals.

False-positive presentation of Battle's sign during hepatic encephalopathy.

INTRODUCTION: Battle's sign is a classical clinical sign that has long been held to be synonymous with fracture of the basal skull. As such the presence of Battle's sign is a strong indicator that a basal skull fracture could be present in the head injured patient, as exemplified by its inclusion as a major risk factor in scoring systems designed to assess the likelihood of basal skull fracture. DISCUSSION: We present a case that describes the occurrence of this classic clinical sign in an unlikely setting and, for the first time since it was described more than 120 years ago, re-examine the pathologic basis for its appearance.

Development of bioelectrical impedance-derived indices of fat and fat-free mass for assessment of nutritional status in childhood.

OBJECTIVES: (1) To develop a method of manipulating bioelectrical impedance (BIA) that gives indices of lean and fat adjusted for body size, using a large normative cohort of children. (2) To assess the discriminant validity of the method in a group of children likely to have abnormal body composition. DESIGN: Two prospective cohort studies. SETTING: Normative data: Avon Longitudinal Study of Parents and Children (ALSPAC), population based cohort; proof of concept study: tertiary feeding clinic and special needs schools. SUBJECTS: Normative data: 7576 children measured aged between 7.25 and 8.25 (mean 7.5) (s.d.=0.2) years; proof of concept study: 29 children with either major neurodisability or receiving artificial feeding, or both, mean age 7.6 (s.d.=2) years. MEASURES: Leg-to-leg (Z (T)) and arm-to-leg (Z (B)) BIA, weight and height. Total body water (TBW) was estimated from the resistance index (RI=height(2)/Z), and fat-free mass was linearly related to TBW. Fat mass was obtained by subtracting fat-free mass from total weight. Fat-free mass was log-transformed and the reciprocal transform was taken for fat mass to satisfy parametric model assumptions. Lean and fat mass were then adjusted for height and age using multiple linear regression models. The resulting standardized residuals gave the lean index and fat index, respectively. RESULTS: In the normative cohort, the lean index was higher and fat index lower in boys. The lean index rose steeply to the middle of the normal range of body mass index (BMI) and then slowly for higher BMI values, whereas the fat index rose linearly through and above the normal range. In the proof of concept study, the children as a group had low lean indices (mean (s.d.) -1.5 (1.7)) with average fat indices (+0.21 (2.0)) despite relatively low BMI standard deviation scores (-0.60 (2.3)), but for any given BMI, individual children had extremely wide ranges of fat indices. The lean index proved more stable and repeatable than BMI. CONCLUSIONS: This clinical method of handling BIA reveals important variations in nutritional status that would not be detected using anthropometry alone. BIA used in this way would allow more accurate assessment of energy sufficiency in children with neurodisability and may provide a more valid identification of children at risk of underweight or obesity in field and clinical settings.

Checkpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential.

The spindle checkpoint detects errors in kinetochore attachment to microtubules and delays anaphase if attachment is improper. The checkpoint is activated by attachment-sensitive components including Mad2 and certain phosphorylated proteins detected by the 3F3/2 antibody. We have studied Mad2 and 3F3/2 immunofluorescence in grasshopper spermatocytes. As in other cells, unattached kinetochores are loaded with Mad2 and are highly phosphorylated, whereas after proper attachment, Mad2 is lost and kinetochores are dephosphorylated. What is it about proper attachment that produces these changes--is it microtubule attachment itself or is it the tension from mitotic forces that follows proper attachment? Using micromanipulation, we created an intermediate state, weak attachment, that provides an answer. Weakly attached kinetochores are not under tension and have few kinetochore microtubules. Despite the absence of tension, many weakly attached kinetochores lose their Mad2 and become dephosphorylated. Therefore we conclude that microtubule attachment determines both Mad2 binding and phosphorylation. Nevertheless, tension plays an absolutely essential role. Tension elevates the number of kinetochore microtubules to the level necessary for the complete loss of Mad2 and dephosphorylation from all kinetochores. This gives a reliable 'all clear' signal to the checkpoint, allowing the cell to progress to anaphase.

In vitro activities of linezolid against multiple Nocardia species.

Linezolid was tested by broth microdilution against 140 clinical Nocardia isolates belonging to seven species. The MIC at which 50% of the strains are inhibited (MIC50) and MIC90 for all species other than Nocardia farcinica were 2 and 4 microg/ml. Linezolid is the first antimicrobial agent demonstrated to be active against all Nocardia species.

Activities of linezolid against rapidly growing mycobacteria.

Linezolid is an oxazolidinone available as an oral drug which has activity against most gram-positive bacteria. However, few species of the genus Mycobacterium have been studied. We tested 249 clinical isolates and 10 reference strains of rapidly growing mycobacteria for susceptibility to linezolid by broth microdilution. Clinical species included the Mycobacterium fortuitum group (n = 74), M. abscessus (n = 98), M. chelonae (n = 50), M. mucogenicum (n = 10), and M. fortuitum third biovariant complex (10). The modal MIC for M. mucogenicum was 1.0 microg/ml, and the MIC at which 90% of the isolates tested are inhibited (MIC(90)) was 4 microg/ml; the modal MIC for the M. fortuitum group was 4 microg/ml, and the MIC(90) was 16 microg/ml; the modal MIC for the M. fortuitum third biovariant complex was 4 microg/ml, and the MIC(90) was 8 microg/ml; the modal MIC for M. chelonae was 8 microg/ml, and the MIC(90) was 16 microg/ml; and the modal MIC for M. abscessus was 32 microg/ml, and the MIC(90) was 64 microg/ml. Based on peak levels of linezolid in serum of 15 to 20 microg/ml, we propose the following broth MIC breakpoints for these species: susceptible, < or = 8 microg/ml; moderately susceptible, 16 microg/ml; and resistant, > or =32 microg/ml). These studies demonstrate the excellent potential of linezolid for therapy of rapidly growing mycobacteria.

Squamous cell carcinoma of the hallux.

This article reports on a case of malignant degeneration of a hallux nail bed ulcer of 30 years' duration. Histologically, this lesion was determined to be a squamous cell carcinoma, a type of lesion that is also known as Marjolin's ulcer. The diagnosis, histologic findings, and treatment of patients with cutaneous squamous cell carcinoma are discussed.

Developmental sex differences in amino acid neurotransmitter levels in hypothalamic and limbic areas of rat brain.

GABA, glutamate and aspartate are the predominant amino acid neurotransmitters in the mammalian brain. We have previously reported a developmental sex difference in messenger RNA levels of glutamate decarboxylase, the rate-limiting enzyme in GABA synthesis [Davis A. M. et al. (1996) Horm. Behav. 30, 538-552]. Males were found to have significantly higher levels of messenger RNA in many steroid-concentrating regions of the hypothalamus and limbic system on day 1 of life. Therefore, in this study, we have examined levels of amino acid neurotransmitters during early postnatal development in many of the same or related brain areas. We found that levels of all three transmitters change as animals age. While both GABA and aspartate concentrations increase, glutamate levels decrease. In addition, there are sex differences in neurotransmitter levels in several areas examined, including the ventromedial and arcuate nuclei of the hypothalamus, and the CA1 region of the hippocampus. Sex differences for GABA occur only on postnatal days 1 and 5. However, sex differences in aspartate occur later in development (postnatal day 20). The CA1 region of males has a significantly greater concentration of GABA, glutamate and aspartate than females on postnatal day 1. In addition, treatment of females with testosterone propionate on the day of birth results in increased GABA levels, suggesting that these sex differences may be the result of hormone exposure during development. We hypothesize that these hormonally mediated sex differences in amino acid transmitters early in development contribute to the establishment of sexually dimorphic neuronal architecture in the adult.

Preoperative magnetic resonance staging of rectal cancer with an endorectal coil and dynamic gadolinium enhancement.

BACKGROUND: The aim of the study was to assess the value of endorectal coil magnetic resonance imaging (MRI) with gadolinium enhancement in the preoperative staging of rectal cancer. METHODS: In addition to standard evaluation, patients with rectal lesions were assessed by MRI obtained with a pelvic phased-array coil in combination with an endorectal coil. RESULTS: The study group comprised 29 patients with rectal cancer staged with an endorectal coil who had surgery without preoperative adjuvant therapy. In addition to standard T1- and T2-weighted images, dynamic contrast-enhanced images were acquired in all patients. Considerable interobserver variation was noted, particularly for pathological tumour stage pT1 or pT2 (kappa = 0.36). Compared with pathological findings, endorectal MRI correctly staged nine patients, overstaged 16 and understaged four. Whilst lymph node metastases were accurately detected in 70 per cent of patients, the positive predictive value was only 58 per cent. CONCLUSION: MR staging of rectal cancer with an endorectal coil and gadolinium enhancement is inaccurate for early tumours (stage T1 or T2) and is associated with a considerable degree of interobserver variation for individual scan sequences.

Tension-sensitive kinetochore phosphorylation in vitro.

Many cells have a checkpoint that detects a single misattached chromosome and delays anaphase, allowing time for error correction. Detection probably depends on tension-sensitive kinetochore protein phosphorylation. Somehow, mechanical tension, or some consequence of tension, produces a chemical change, dephosphorylation. The mechanism of tension-mediated dephosphorylation can be approached using an in vitro system. Earlier work showed that the kinetochores of washed chromosomes from a mammalian cell line can be phosphorylated in vitro simply by incubation with ATP and a phosphatase inhibitor. We confirm this for chromosomes from insect meiotic cells. Thus, kinetochores of washed chromosomes from diverse sources contain a complete phosphorylation system: a kinase, a phosphatase and the substrate protein(s). We show that phosphorylation in vitro is sensitive to tension, as it is in living cells. This makes the conditions required for phosphorylation in vitro relevant to the process in living cells. The phosphatase is ruled out as the tension-sensitive component in vitro, leaving either the kinase or the substrate as the sensitive component. We show that a kinase extracted from mammalian cells in mitosis phosphorylates the kinetochores of insect meiotic chromosomes very effectively. The mammalian kinase under-phosphorylates the kinetochore of the insect's X-chromosome, just as the native insect kinase does. This provides a clue to the evolution of a chromosome that is not detected by the checkpoint. The mammalian kinase is not tightly bound to the chromosome and thus functions primarily in solution. This suggests that the substrate's phosphorylatable groups are freely available to outside constituents, e.g. regulators, as well as to the kinetochore's own kinase and phosphatase.

Subnasoalveolar anatomy and hominoid phylogeny: evidence from comparative ontogeny.

The present analysis evaluated extant hominoid subnasal morphological variation from an ontogenetic perspective, documenting both qualitative and allometric details of subnasal maturation in Hylobates, great apes and modern humans. With respect to intraspecific variation, results of log-linear modeling procedures indicate that qualitative features of the subnasal region shown previously to discriminate extant taxa (Ward and Kimbel, 1983; McCollum et al., 1993) do not vary appreciably with either age or sex. In terms of quantitative variation, aside from observed changes in the position of the anterior attachment of the nasal septal cartilage relative to the lateral margins of the nasal cavity, the morphology of the subnasal region does not vary appreciably with age. Furthermore, it was found that sexual dimorphism in subnasal form is present only in Pongo and Gorilla and is the result of sexual bimaturism rather than sexual variation in canine size. In considering interspecific variation in subnasal form, there is a propensity among hominoid taxa for the nasal cavity floor to be free of substantial topographic relief. The smoothly continuous nasal floor topography identified in the majority of hominoid taxa appears to be produced by extensive resorption of the anterior nasal cavity floor that accompanies an upward rotation of the anterior maxilla during craniofacial ontogeny. Comparisons of ontogenetic allometric trajectories indicate that relatively little of the variation in hominoid subnasal form can early be attributed to variation in body/cranial size. Instead, variation in craniofacial orientation, vascular anatomy and incisor size and inclination were identified as potential mediators of hominoid subnasoalveolar anatomy. Although results of this analysis confirm that many detail of the orangutan subnasal morphology are derived for this taxon, there is only little conclusive evidence to support recent reports that the morphology displayed by Gorilla is primitive for great apes.

Hepatic angiomyelolipoma and tuberous sclerosis.

A patient with angiomyelolipoma of the liver, together with radiological evidence of pancreatic, renal and bony lesions characteristic of tuberous sclerosis, is described. Although the patient had no other clinical features of tuberous sclerosis, her daughter was found to suffer from the classical triad of this syndrome and also has had hepatic lipomatous lesions and bony involvement. This is the first histologically proven case of hepatic angiomyelolipoma associated with tuberous sclerosis.

A prospective study of upper gastrointestinal hemorrhage in patients with hepatocellular carcinoma.

Our purpose was to determine, in a prospective study, the causes of gastrointestinal hemorrhage in patients with hepatocellular carcinoma, and the relationship of portal vein invasion with variceal hemorrhage in these patients. During an 11-month period, 55 patients presented with hepatocellular carcinoma presented with signs and/or symptoms of upper gastrointestinal hemorrhage. Forty-seven percent had bleeding from varices, whereas the majority, 53%, had a nonvariceal bleeding source. Among those with nonvariceal bleeding, duodenal ulceration was the commonest cause. Direct tumor invasion into the gastrointestinal tract was found in three patients. Tumor invasion of the portal venous system was detected by ultrasound examination in 76% of the variceal bleeders, compared to only 45% of the nonvariceal bleeders. Despite the very high frequency of cirrhosis among patients with hepatocellular carcinoma, the source of bleeding was variceal in less than half of the patients. Portal vein invasion is a risk factor for subsequent variceal bleed.

Technical note: an evaluation of the routine use of rectal air in computed tomography of the pelvis.

We have performed computed tomographic examinations of the pelvis in 50 patients, using rectally administered air to outline the rectosigmoid colon. Bowel cleansing was not performed and smooth muscle relaxants were not used. The procedure was well tolerated by all patients, and diagnostic images were obtained in all cases. There were no complications. This is a safe, low cost and easily administered form of bowel contrast. We recommend it as the technique of choice in routine pelvic computed tomography.

Radiation pneumonitis after selective internal radiation treatment with intraarterial 90yttrium-microspheres for inoperable hepatic tumors.

PURPOSE: To investigate the clinical, histopathological, and radiological features of radiation pneumonitis arising as a complication of selective internal radiation treatment for liver tumors. To correlate the development of radiation pneumonitis with the degree of lung shunting as assessed by 99mTechnetium-labeled macroaggregated albumin (Tc-MAA) scan. METHODS AND MATERIALS: Five out of 80 patients who had inoperable hepatic tumors and underwent treatment with intraarterial 90Yttrium- (90Y)-microspheres, developed progressive restrictive ventilatory dysfunction without an infective or cardiovascular cause. Histopathological evidence of a pneumonitis and the presence of microspheres in the lung tissue suggested a diagnosis of radiation pneumonitis. The clinical course, radiological and histopathological findings, percentage tumor shunting to the lungs (lung shunting, as predicted by gamma camera scanning after intraarterial Tc-MAA), and the estimated radiation dose to the lungs were analyzed. In an attempt to reduce pulmonary shunting of the microspheres, three patients received partial hepatic embolization with inert particles before selective internal radiation therapy. RESULTS: In the five patients who developed radiation pneumonitis, lung shunting percentages (as predicted by Tc-MAA scan) ranged from 13.1 to 45.6% (median 23.7%). The estimated whole lung radiation dose ranged from 10.43 Gy to 36.44 Gy (median 25.04 Gy). Among 75 patients who did not develop radiation pneumonitis, the percentage lung shunting ranged from less than 1% to 15% (median 6%). Nine patients had lung shunting greater than 13% and five of them developed radiation pneumonitis, whereas this developed in none of those in whom shunting was below 13%. The onset of radiation pneumonitis ranged from 1 to 6 months after internal radiation treatment. All five patients exhibited characteristic plain radiographic and computerized tomographic changes comprising extensive consolidation with well-defined lateral margins. Clinical improvement after corticosteroid treatment was seen in two patients. Three patients died from respiratory failure and two from other causes. Partial hepatic arterial embolization reduced the degree of lung shunting to less than 13%, but did not prevent the development of radiation pneumonitis. CONCLUSION: Radiation pneumonitis may become a complication after intraarterial 90Y-microspheres treatment when lung shunting, as assessed by Tc-MAA scan, is high (above 13%). Prescribed activity of 90Y and lung shunting of Tc-MAA should be considered together before giving selective internal radiation (SIR) therapy for hepatic tumors, and preferably avoided if the lung shunting is above 13%.

Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint.

Some cells have a quality control checkpoint that can detect a single misattached chromosome and delay the onset of anaphase, thus allowing time for error correction. The mechanical error in attachment must somehow be linked to the chemical regulation of cell cycle progression. The 3F3 antibody detects phosphorylated kinetochore proteins that might serve as the required link (Gorbsky, G. J., and W. A. Ricketts. 1993. J. Cell Biol. 122:1311-1321). We show by direct micromanipulation experiments that tension alters the phosphorylation of kinetochore proteins. Tension, whether from a micromanipulation needle or from normal mitotic forces, causes dephosphorylation of the kinetochore proteins recognized by 3F3. If tension is absent, either naturally or as a result of chromosome detachment by micromanipulation, the proteins are phosphorylated. Equally direct experiments identify tension as the checkpoint signal: tension from a microneedle on a misattached chromosome leads to anaphase (Li, X., and R. B. Nicklas. 1995. Nature (Lond.). 373:630-632), and we show here that the absence of tension caused by detaching chromosomes from the spindle delays anaphase indefinitely. Thus, the absence of tension is linked to both kinetochore phosphorylation and delayed anaphase onset. We propose that the kinetochore protein dephosphorylation caused by tension is the all clear signal to the checkpoint. The evidence is circumstantial but rich. In any event, tension alters kinetochore chemistry. Very likely, tension affects chemistry directly, by altering the conformation of a tension-sensitive protein, which leads directly to dephosphorylation.