Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Intra-operative neurophysiological mapping to identify distorted functional anatomy of the 4th ventricle in a 5-month-old infant.

BACKGROUND: Neurophysiological brainstem mapping techniques facilitate the intra-operative localisation of cranial nerve nuclei amidst distorted anatomy. Neurophysiological recording in young infants can be limited due to immature myelination and synaptogenesis, as well as an increased sensitivity to anaesthetic agents. CASE REPORT: A 5-month-old boy was diagnosed with a cystic brainstem lesion located dorsally within the pons and upper medulla. An open surgical biopsy was undertaken via a posterior fossa craniotomy, revealing a grossly distorted fourth ventricular floor. Intra-operative neurophysiological mapping produced oculomotor, facial, glossopharyngeal and vagal muscle responses allowing a deviated functional midline to be identified. Direct stimulation was used to identify an area in the floor of the fourth ventricle eliciting no cranial nerve responses and allow safe entry into the tumour cavity and biopsy. Transcranial motor evoked responses (TcMEPs), short-latency somatosensory evoked potentials (SSEPs) and brainstem auditory evoked potentials (BAEPs) were all successfully recorded throughout the procedure, despite the use of halogenated gaseous anaesthesia. CONCLUSIONS: We describe the use of brainstem mapping techniques for identification of a distorted midline on the floor of the 4th ventricle in an infant, with reproducible recordings of intra-operative TcMEPs, SSEPs and BAEPs.

Myosin S2 origins track evolution of strong binding on actin by azimuthal rolling of motor domain.

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin's α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.

Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3.

BACKGROUND: The pro-inflammatory cytokine interleukin-6 (IL6) promotes colorectal cancer (CRC) development. It is also known to regulate cytochrome P450 (CYP450) enzymes, which are involved in CRC tumour initiation and promotion via activation of chemical carcinogens. Here, IL6 regulation of CYP450 expression was investigated in CRC. METHODS: The effect of IL6 on CYP 1A1, 1B1 and 2E1 expression was determined in vitro using CRC cell lines HCT116 and SW480, and CYP450 expression was determined by immunohistochemistry in CRC tissues previously shown to have increased levels of IL6. RESULTS: In mechanistic studies, IL6 treatment significantly induced CYP1B1 and CYP2E1, but not CYP1A1, gene expression in HCT116 and SW480 cells. CYP2E1 expression regulation occurred via a transcriptional mechanism involving STAT3. For CYP1B1 regulation, IL6 downregulated the CYP1B1-targeting microRNA miR27b through a mechanism involving DNA methylation. In clinical samples, the expression of CYP1B1 and CYP2E1, but not CYP1A1, was significantly increased in malignant tissue overexpressing IL6 compared with matched adjacent normal tissue. CONCLUSIONS: Colonic inflammation with the presence of IL6 associated with neoplastic tissue can alter metabolic competency of epithelial cells by manipulating CYP2E1 and CYP1B1 expression through transcriptional and epigenetic mechanisms. This can lead to increased activation of dietary carcinogens and DNA damage, thus promoting colorectal carcinogenesis.

A HIV-1 Gp41 Peptide-Liposome Vaccine Elicits Neutralizing Epitope-Targeted Antibody Responses in Healthy Individuals.

Background: HIV-1 vaccine development is a global health priority. Broadly neutralizing antibodies (bnAbs) which target the HIV-1 gp41 membrane-proximal external region (MPER) have some of the highest neutralization breadth. An MPER peptide-liposome vaccine has been found to expand bnAb precursors in monkeys. Methods: The HVTN133 phase 1 clinical trial (NCT03934541) studied the MPER-peptide liposome immunogen in 24 HIV-1 seronegative individuals. Participants were recruited between 15 July 2019 and 18 October 2019 and were randomized in a dose-escalation design to either 500 mcg or 2000 mcg of the MPER-peptide liposome or placebo. Four intramuscular injections were planned at months 0, 2, 6, and 12. Results: The trial was stopped prematurely due to an anaphylaxis reaction in one participant ultimately attributed to vaccine-associated polyethylene glycol. The immunogen induced robust immune responses, including MPER+ serum and blood CD4+ T-cell responses in 95% and 100% of vaccinees, respectively, and 35% (7/20) of vaccine recipients had blood IgG memory B cells with MPER-bnAb binding phenotype. Affinity purification of plasma MPER+ IgG demonstrated tier 2 HIV-1 neutralizing activity in two of five participants after 3 immunizations. Conclusions: MPER-peptide liposomes induced gp41 serum neutralizing epitope-targeted antibodies and memory B-cell responses in humans despite the early termination of the study. These results suggest that the MPER region is a promising target for a candidate HIV vaccine.

Microsecond dynamics control the HIV-1 envelope conformation.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although significant progress has been made in understanding the structures of various Env conformations and transition intermediates that occur within the millisecond timescale, faster transitions in the microsecond timescale have not yet been observed. In this study, we employed time-resolved, temperature-jump small angle X-ray scattering to monitor structural rearrangements in an HIV-1 Env ectodomain construct with microsecond precision. We detected a transition correlated with Env opening that occurs in the hundreds of microseconds range and another more rapid transition that preceded this opening. Model fitting indicated that the early rapid transition involved an order-to-disorder transition in the trimer apex loop contacts, suggesting that conventional conformation-locking design strategies that target the allosteric machinery may be ineffective in preventing this movement. Utilizing this information, we engineered an envelope that locks the apex loop contacts to the adjacent protomer. This modification resulted in significant angle-of-approach shifts in the interaction of a neutralizing antibody. Our findings imply that blocking the intermediate state could be crucial for inducing antibodies with the appropriate bound state orientation through vaccination.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins.

The conserved PIK-related kinase Rad3 is required for all DNA-integrity-checkpoint responses in fission yeast. Here we report a stable association between Rad3 and Rad26 in soluble protein extracts. Rad26 shows Rad3-dependent phosphorylation after DNA damage. Unlike phosphorylation of Hus1, Crb2/Rhp9, Cds1 and Chk1, phosphorylation of Rad26 does not require other known checkpoint proteins. Rad26 phosphorylation is the first biochemical marker of Rad3 function, indicating that Rad3-related checkpoint kinases may have a direct role in DNA-damage recognition.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin.

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements.

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single-molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single-particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.

Searching for novel biomarkers of centrally and peripehrally-acting neurotoxicants, using surface-enhanced laser desorption/ionisation-time-of-flight mass spectrometry (SELDI-TOF MS).

The neurotoxicity of chemicals to humans is difficult to monitor as there are no suitable methods of detecting early neuronal dysfunction. Here, a proof of principle study was designed to assess the potential of identifying protein biomarkers in accessible biofluids for this purpose. Groups of rats were treated with a range of doses of the model neurotoxicants, acrylamide (0, 2, 10, 50mg/kg) and methylmercury (0, 0.2, 1, 5mg/kg) for up to 3 weeks and samples of serum, urine, and cerebral spinal fluid analysed by surface-enhanced laser desorption/ionisation-time-of-flight mass spectrometry. There was no neuropathology up to the highest dose tested. Protein profiles were obtained from all samples and changes in the levels of many proteins were detected in both serum and urine, although not cerebral spinal fluid. In serum, the combination of three protein ion levels with m/z values of 4968, 9402 and 12,948 was able to correctly classify the treatment groups thus: 88% control, 100% acrylamide, 92% methylmercury. In urine, three protein ions with m/z values of 4944, 12,966 and 21,992 classified correctly the groups: 67% control, 94% acrylamide, 97% methylmercury. Similar classifications using other serum and urinary protein ions were also possible. This indicates the potential of serum and urine protein biomarkers for the assessment of sub-clinical neurotoxicity.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a potent mutagen in the mouse small intestine.

Mutations in long lived stem cells are critical events in carcinogenesis. The Dlb-1 assay detects intestinal stem cell mutation at the Dlb-1 locus in Dlb-1a/b heterozygous mice by visualizing mutated clones of epithelial cells in situ which do not bind the lectin Dolichos biflorus agglutinin. We have used this assay to show that the food-derived heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent intestinal mutagen when administered either i.p. or p.o. This contrasts with the inactivity of the structurally related mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the assay which we have described previously. Immunocytochemical localization of the P-450 enzyme CYP1A2, which is responsible for the primary activation of these mutagens, shows that in untreated mice it is present in liver hepatocytes and in occasional villus epithelial cells but is absent from the target intestinal stem cell population. In addition, liver microsomes, unlike intestinal microsomes, were able to convert PhIP to the proximate mutagen N-hydroxy-PhIP. CYP1A2 immunoreactivity in beta-napthoflavone-induced animals was elevated in liver hepatocytes and increased to a lesser extent in duodenal villus epithelial cells. Treatment with beta-napthoflavone produced an unexpected 46% decrease in the number of Dlb-1 mutations in response to PhIP. Following treatment with PhIP, there was no difference in the number of Dlb-1 locus mutations between the proximal and distal ends of the small intestine in uninduced animals, indicating that the bile duct is unlikely to be responsible for transport of mutation inducing metabolites of PhIP to the small intestine. Our results demonstrate that metabolic activation of an indirect acting genotoxic agent can occur at a site other than the target tissue, and absence of the enzymes required for activation of a mutagen does not necessarily protect that tissue from its genotoxic effects.

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey (Callithrix jacchus).

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo-p-dioxin or tetrabromodibenzo-p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYP1A1, CYP1A2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYP1A2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYP1A1 is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYP1A1 and CYP1A2 in liver, but only CYP1A1 in lung. No induction of CYP1A1 or CYP1A2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYP1A2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYP1A2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a Ki of 10 microM. In the lung the induction of CYP1A1 was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYP1A1 does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

Identification of CYP2E1 in marmoset monkey.

CYP2E1 is the main enzyme responsible for chlorzoxazone 6-hydroxylase activity in human liver. Here, it is shown that marmoset monkey liver microsomal fraction catalyses this reaction at a similar rate and with a similar Km to human liver and that the activity is increased 4-fold in marmosets treated with isoniazid, a known inducer of CYP2E1. This indicates that CYP2E1 is present in marmoset liver. However conversely, an anti-peptide antibody targeted against the C-terminus of human and cynomolgus monkey CYP2E1 (Val-Ile-Pro-Arg-Ser) failed to bind to marmoset monkey hepatic microsomal fraction. To investigate if there is a difference in the C-terminus of CYP2E1 in these species, this region of marmoset CYP2E1 was sequenced following amplification of marmoset liver cDNA with primers selected according to conserved regions identified in human and cynomolgus monkey CYP2E1. It was found that the deduced amino acid sequence of marmoset CYP2E1 in this region is very similar to human CYP2E1, but due to two base differences in the marmoset nucleic acid sequence, the C-terminus of marmoset CYP2E1 is extended by 2 amino acids, i.e. Val-Ile-Pro-Arg-Ser-Ser-Val. This difference is sufficient to prevent the binding of an antibody raised against the C-terminus of human CYP2E1. The expression of CYP2E1 in the marmoset was confirmed by raising an antibody against the deduced C-terminus of marmoset CYP2E1 (Pro-Arg-Ser-Ser-Val). In immunoblotting, this antibody bound to a single protein of 54 kDa in marmoset liver microsomal fraction. The intensity of the band was increased in isoniazid-treated marmosets, consistent with induction of CYP2E1. The antibody did not recognise human or cynomolgus monkey CYP2E1. This was expected since the immunising peptide sequence does not occur in these enzymes. The results demonstrate the presence of CYP2E1 in marmoset liver and illustrate the importance of the C-terminus for the production of specific antibodies against P450 enzymes.

An inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome P-450 enzymes CYP1A1 and CYP1A2.

A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does not bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-Gln-Pro-Arg-Leu: residues 356-363 and 350-357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did not bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O-deethylase) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450, whilst binding of the monoclonal antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450. These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies.

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach.

BACKGROUND: Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event. RESULTS: A combined bioinformatics approach of motif prediction and evolutionary and structural analyses identified tyrosines163 and 1856 of the skeletal muscle heavy chain as the leading candidate for the sites of insulin-mediated tyrosine phosphorylation. CONCLUSION: Our work is suggestive that tyrosine phosphorylation of myosin heavy chain, whether in skeletal muscle or in platelets, is a significant event that may initiate cytoskeletal reorganization of muscle cells and platelets. Our studies provide a good starting point for further functional analysis of MHC phosphor-signalling events within different cells.

Thromboxane release by lymphokine-differentiated U937 human monocytic cells: response to platelet-activating factor (PAF) and chemotactic peptide (FMLP) but not to low affinity IGE-receptor (Fc epsilon RII/CD23) occupation.

The primary objective of this study was to explore if the CD23 antigen is a functional low affinity IgE receptor on macrophages for the release of thromboxane B2 (TXB2). The responsiveness of U937 monocytic cells and their macrophage-like inducible forms to platelet-activating factor (Paf), the chemotactic peptide fMLP, and low affinity IgE-receptor occupation was examined. Differentiation of U937 cells by phorbol myristate acetate (PMA) and a cancer cell line (HBT 5637) conditioned medium (5637-CM), but not INFg or IL4, resulted in a macrophage-like cell line which released TXB2. A high basal release of TXB2 with no significant response to Paf or fMLP challenge was seen following culture of cells with PMA. In 5637-CM-differentiated cells, Paf and fMLP induced a rapid release of TXB2, about 10 fold above basal activity. There was a slow Ca-independent response to short-term treatment with PMA and a rapid Ca-dependent response to the ionophore A23187. Both stimulants acted synergistically on TXB2 synthesis in 5637-CM differentiated cells. Although low affinity receptors for IgE (Fc epsilon RII/CD23) were induced by 5637-CM, no TXB2 was released in response to soluble or latex-bound IgE-antigen complexes or to anti-Fc epsilon RII/CD23-antibodies. IL4 and to a lesser extent INFg both induced Fc epsilon RII/CD23 receptor expression, but inhibited release of TXB2 in response to Paf, fMLP, or PMA. We conclude that the functional receptors for IgE on mature macrophages are most probably not Fc epsilon RII/CD23.

Head injury from falls in children younger than 6†years of age.

UNLABELLED: The risk of serious head injury (HI) from a fall in a young child is ill defined. The relationship between the object fallen from and prevalence of intracranial injury (ICI) or skull fracture is described. METHOD: Cross-sectional study of HIs from falls in children (<6 years) admitted to UK hospitals, analysed according to the object fallen from and associated Glasgow Coma Score (GCS) or alert, voice, pain, unresponsive (AVPU) and CT scan results. RESULTS: Of 1775 cases ascertained (median age 18 months, 54.7% boys), 87% (1552) had a GCS=15/AVPU=alert. 19.3% (342) had a CT scan: 32% (110/342) were abnormal; equivalent to 5.9% of the overall population, 16.9% (58) had isolated skull fractures and 13.7% (47) had ICI (49% (23/47) had an associated skull fracture). The prevalence of ICI increased with neurological compromise; however, 12% of children with a GCS=15/AVPU=alert had ICI. When compared to falls from standing, falls from a person's arms (233 children (mean age 1 year)) had a significant relative OR for a skull fracture/ICI of 6.94 (95% CI 3.54 to 13.6), falls from a building (eg, window or attic) (mean age 3 years) OR 6.84 (95% CI 2.65 to 17.6) and from an infant or child product (mean age 21 months) OR 2.75 (95% CI 1.36 to 5.65). CONCLUSIONS: Most HIs from a fall in these children admitted to hospital were minor. Infants, dropped from a carer's arms, those who fell from infant products, a window, wall or from an attic had the greatest chance of ICI or skull fracture. These data inform prevention and the assessment of the likelihood of serious injury when the object fallen from is known.