Experimental verification of principal losses in a regulatory particulate matter emissions sampling system for aircraft turbine engines.

A sampling system for measuring emissions of nonvolatile particulate matter (nvPM) from aircraft gas turbine engines has been developed to replace the use of smoke number and is used for international regulatory purposes. This sampling system can be up to 35 m in length. The sampling system length in addition to the volatile particle remover (VPR) and other sampling system components lead to substantial particle losses, which are a function of the particle size distribution, ranging from 50 to 90% for particle number concentrations and 10-50% for particle mass concentrations. The particle size distribution is dependent on engine technology, operating point, and fuel composition. Any nvPM emissions measurement bias caused by the sampling system will lead to unrepresentative emissions measurements which limit the method as a universal metric. Hence, a method to estimate size dependent sampling system losses using the system parameters and the measured mass and number concentrations was also developed (SAE 2017; SAE 2019). An assessment of the particle losses in two principal components used in ARP6481 (SAE 2019) was conducted during the VAriable Response In Aircraft nvPM Testing (VARIAnT) 2 campaign. Measurements were made on the 25-meter sample line portion of the system using multiple, well characterized particle sizing instruments to obtain the penetration efficiencies. An agreement of ± 15% was obtained between the measured and the ARP6481 method penetrations for the 25-meter sample line portion of the system. Measurements of VPR penetration efficiency were also made to verify its performance for aviation nvPM number. The research also demonstrated the difficulty of making system loss measurements and substantiates the E-31 decision to predict rather than measure system losses.

Endothelial interleukin-21 receptor up-regulation in peripheral artery disease.

In most patients with symptomatic peripheral artery disease (PAD), severe stenosis in or occlusion of the major blood vessels that supply the legs make the amount of distal blood flow dependent on the capacity to induce angiogenesis and collateral vessel formation. Currently, there are no medications that improve perfusion to the ischemic limb, and thus directly treat the primary problem of PAD. A recent report from our group in a pre-clinical mouse PAD model showed that interleukin-21 receptor (IL-21R) is up-regulated in the endothelial cells from ischemic hindlimb muscle. We further showed that loss of IL-21R resulted in impaired perfusion recovery in this model. In our study, we sought to determine whether IL-21R is present in the endothelium from ischemic muscle of patients with PAD. Using human gastrocnemius muscle biopsies, we found increased levels of IL-21R in the skeletal muscle endothelial cells of patients with PAD compared to control individuals. Interestingly, PAD patients had approximately 1.7-fold higher levels of circulating IL-21. These data provide direct evidence that the IL-21R pathway is indeed up-regulated in patients with PAD. This pathway may serve as a therapeutic target for modulation.

ADAM12: a genetic modifier of preclinical peripheral arterial disease.

In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 (ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2.

Loss of interleukin-21 receptor activation in hypoxic endothelial cells impairs perfusion recovery after hindlimb ischemia.

OBJECTIVE: Surgical hindlimb ischemia (HLI) in mice has become a valuable preclinical model to study peripheral arterial disease. We previously identified that the different phenotypic outcomes after HLI across inbred mouse strains is related to a region on the short arm of mouse chromosome 7. The gene coding the interleukin-21 receptor (IL-21R) lies at the peak of association in this region. APPROACH AND RESULTS: With quantitative real-time polymerase chain reaction, we found that a mouse strain with a greater ability to upregulate IL-21R after HLI had better perfusion recovery than a strain with no upregulation after HLI. Immunofluorescent staining of ischemic hindlimb tissue showed IL-21R expression on endothelial cells (ECs) from C57BL/6 mice. An EC-enriched fraction isolated from ischemic hindlimb muscle showed higher Il-21R levels than an EC-enriched fraction from nonischemic limbs. In vitro, human umbilical vein ECs showed elevated IL-21R expression after hypoxia and serum starvation. Under these conditions, IL-21 treatment increased cell viability, decreased cell apoptosis, and augmented tube formation. In vivo, either knockout Il21r or blocking IL-21 signaling by treating with IL-21R-Fc (fusion protein that blocks IL-21 binding to its receptor) in C57BL/6 mice resulted in less perfusion recovery after HLI. Both in vitro and in vivo modulation of the IL-21/IL-21R axis under hypoxic conditions resulted in increased signal transducer and activator of transcription 3 phosphorylation and a subsequent increase in the B-cell lymphoma leukemia-2/BCL-2-associated X protein ratio. CONCLUSION: Our data indicate that IL-21R upregulation and ligand activation in hypoxic ECs may help perfusion recovery by limiting/preventing apoptosis and favoring cell survival and angiogenesis through the signal transducer and activator of transcription 3 pathway.

MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway.

BACKGROUND: MicroRNAs are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease. Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hindlimb ischemia between inbred mouse strains to identify key microRNAs involved in perfusion recovery from hindlimb ischemia. METHODS AND RESULTS: From comparative microRNA profiling between inbred mouse strains that display profound differences in their extent of perfusion recovery after hindlimb ischemia, we found that the mouse strain with higher levels of microRNA-93 (miR-93) in hindlimb muscle before ischemia and the greater ability to upregulate miR-93 in response to ischemia had better perfusion recovery. In vitro, overexpression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells and enhanced proliferation in both cell types. In addition, miR-93 overexpression enhanced endothelial cell tube formation. In vivo, miR-93 overexpression enhanced capillary density and perfusion recovery from hindlimb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in vitro and in vivo modulation of miR-93 resulted in alterations in the expression of >1 cell cycle pathway gene in 2 different cell types. CONCLUSIONS: Our data indicate that miR-93 enhances perfusion recovery from hindlimb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue.

Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery.

Targeting therapeutic gene expression to the skeletal muscle following intravenous (IV) administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. As adeno-associated virus serotype 9 (AAV-9) transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell-surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to the skeletal muscle was also examined by comparing it with the cytomegalovirus (CMV) promoter after systemic administration following surgically induced HLI. Despite reduced blood flow to the ischemic limbs, CK6 promoter-driven luciferase activities in the ischemic gastrocnemius (GA) muscles were ∼34-, ∼28- and ∼150-fold higher than in the fully perfused contralateral GA, heart and liver, respectively, 10 days after IV administration. Furthermore, luciferase activity from the CK6 promoter in the ischemic GA muscles was ∼twofold higher than with CMV, while in the liver CK6-driven activity was ∼42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with the muscle-specific promoters. Studies with Evans blue dye and fluorescently labeled lectins revealed that vascular permeability and desialylation of the cell-surface glycans were increased in the ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ∼sixfold higher in the ischemic muscle compared with the non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect.

Atmospheric measurements of the physical evolution of aircraft exhaust plumes.

Drawing from a series of field measurement activities including the Alternative Aviation Fuels Experiments (AAFEX1 and AAFEX2), we present experimental measurements of particle number, size, and composition-resolved mass that describe the physical and chemical evolution of aircraft exhaust plumes on the time scale of 5 s to 2-3 min. As the plume ages, the particle number emission index initially increases by a factor of 10-50, due to gas-to-particle formation of a nucleation/growth mode, and then begins to fall with increased aging. Increasing the fuel sulfur content causes the initial increase to occur more rapidly. The contribution of the nucleation/growth mode to the overall particle number density is most pronounced at idle power and decreases with increasing engine power. Increasing fuel sulfur content, but not fuel aromatic content causes the nucleation/growth mode to dominate the particle number emissions at higher powers than for a fuel with "normal" sulfur and aromatic content. Particle size measurements indicate that the observed particle number emissions trends are due to continuing gas-to-particle conversion and coagulation growth of the nucleation/growth mode particles, processes which simultaneously increase particle mass and reduce particle number density. Measurements of nucleation/growth mode mass are consistent with the interpretation of particle number and size data and suggest that engine exit plane measurements may underestimate the total particle mass by as much as a factor of between 5 and 10.

Angiogenesis in skeletal muscle precede improvements in peak oxygen uptake in peripheral artery disease patients.

OBJECTIVE: Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, causing claudication and exercise intolerance. The mechanism(s) by which exercise training improves functional capacity is not understood. This study tested the hypothesis that in PAD patients who undergo supervised exercise training, increases in capillary density (CD) in calf muscle take place before improvements in peak oxygen uptake (VO(2)). METHODS AND RESULTS: Thirty-five PAD patients were randomly assigned to 12 weeks of directly supervised or home-based exercise training. Peak VO(2) testing and gastrocnemius muscle biopsies were performed at baseline and after training. CD (endothelial cells/mm(2)) was measured using immunofluorescence staining. After 3 weeks of directly supervised training, patients had an increase in CD (216±66 versus 284±77, P<0.01) but no increase in peak VO(2). However, after 12 weeks, peak VO(2) increased (15.3±2.8 versus 16.8±3.8, P<0.01), whereas in muscle, CD remained increased over baseline, but there were no changes in markers of oxidative capacity. Within subjects, CD was related to peak VO(2) before and after directly supervised training. CONCLUSION: Changes in CD in ischemic muscle with training may modulate the response to training, and those changes precede the increase in VO(2).

Adeno-associated virus serotype 9-mediated overexpression of extracellular superoxide dismutase improves recovery from surgical hind-limb ischemia in BALB/c mice.

OBJECTIVE: Neovascularization is a physiologic repair process that partly depends on nitric oxide. Extracellular superoxide dismutase (EcSOD) is the major scavenger of superoxide. It is an important regulator of nitric oxide bioavailability and thus protects against vascular dysfunction. We hypothesized that overexpression of EcSOD in skeletal muscle would improve recovery from hind-limb ischemia. METHODS: Adeno-associated virus serotype 9 (AAV9) vectors expressing EcSOD or luciferase (control) from the cytomegalovirus promoter were cross-packaged into AAV9 capsids and injected intramuscularly into the hind-limb muscles (1 × 10(11) viral genomes/limb) of 12-week-old mice. Ischemia was induced after intramuscular injections. Laser Doppler was used to measure limb perfusion on days 0, 7, and 14 after injection. Values were expressed as a ratio relative to the nonischemic limb. EcSOD expression was measured by Western blotting. Capillary density was documented by immunohistochemical staining for platelet endothelial cell adhesion molecule. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated biotin-deoxy uridine triphosphate nick-end labeling and necrosis was visually evaluated daily. RESULTS: EcSOD expression was twofold upregulated in EcSOD treated vs control ischemic muscles at day 14. Capillary density (capillaries/fiber) was 1.9-fold higher in treated (1.65 ± 0.02) vs control muscle (0.78 ± 0.17, P < .05). Recovery of perfusion ratio at day 14 after ischemia was 1.5-fold greater in EcSOD vs control mice (P < .05). The percentage of apoptotic nuclei was 1.3% ± 0.4% in EcSOD-treated mice compared with 4.2% ± 0.2% in controls (P < .001). Limb necrosis was also significantly lower in EcSOD vs control mice. CONCLUSION: AAV9-mediated overexpression of EcSOD in skeletal muscle significantly improves recovery from hind-limb ischemia in mice, consistent with improved capillary density and perfusion ratios in treated mice.

p-MAPK1/3 and DUSP6 regulate epididymal cell proliferation and survival in a region-specific manner in mice.

A fully developed, functional epididymis is important for male fertility. In particular, it is apparent that without the most proximal region, the initial segment (IS), infertility results. Therefore, it is important to understand the development and regulation of this crucial epididymal region. We have previously shown that many functions of the IS are regulated by luminal fluid factors/lumicrine factors from the testis. This study provides evidence that lumicrine factors activated the ERK pathway only in epithelial cells of the IS from Postnatal Day (P) 14 to P19 and sustained this activation into adulthood. The activated ERK pathway promoted cell proliferation and differentiation in the developing IS, although in the adult, its role was switched to maintain cell survival. To understand further the regulation of cell proliferation in the IS, we examined the role of DUSP6, an MAPK1/3 (ERK1/2) preferred phosphatase that is also regulated by lumicrine factors in the IS. Utilizing Dusp6(-/-) mice, our studies, surprisingly, revealed that Dusp6 was a major regulator of cell proliferation in the caput and corpus regions, whereas components of the ERK pathway, together with PTEN and SRC, were the major regulators of cell proliferation in the IS. We hypothesize that region-specific regulation of cell proliferation is caused by differences in the balance of activities between pro- and antiproliferation signaling pathway components for each epididymal region. An understanding of the mechanisms of cell proliferation may provide clues as to why the epididymis rarely succumbs to cancer.

Adaptation of a proton transfer reaction mass spectrometer instrument to employ NO+ as reagent ion for the detection of 1,3-butadiene in the ambient atmosphere.

A proton transfer reaction mass spectrometer (PTR-MS) instrument was adapted to employ NO+ as a chemical reagent ion without any hardware changes by switching the reagent ion source gas from water vapor to dry air. Ionization of dry air within the hollow cathode ion source generates a very intense source of NO+ with only a minor impurity of NO2+. The intensities of the primary NO+ reagent ion and the unwanted impurity NO2+ are controllable and dependent on the operational conditions of the hollow cathode ion source. Ion source tuning parameters are described, which maintain an intense source of NO+ while keeping the impurity NO2+ signal to less than 2% of the total reagent ion intensity. This method is applied to the detection of 1,3-butadiene. NO+ reacts efficiently with 1,3-butadiene via a charge exchange reaction to produce only the molecular ion, which is detected at m/z 54. Detection sensitivities of the order of 45 pptv for a 1-s measurement of 1,3-butadiene are demonstrated. We present the first real-time on-line sub parts per billion measurement of 1,3-butadiene in the ambient atmosphere. The only likely interference is from 1,2-butadiene. Concurrent measurements of benzene are provided and suggest that the vehicular emissions are the predominant source of 1,3-butadiene in a suburban Boston area monitoring location.

Intermediate chain subunit as a probe for cytoplasmic dynein function: biochemical analyses and live cell imaging in PC12 cells.

Cytoplasmic dynein 1 is a multi-subunit motor protein responsible for microtubule minus end-directed transport in axons. The cytoplasmic dynein intermediate chain subunit has a scaffold-like role in the dynein complex; it directly binds to four of the other five subunits, the heavy chain and the three light chains. The intermediate chain also binds the p150 subunit of dynactin, a protein that is essential for many dynein functions. We reexamined the generation of rat cytoplasmic dynein intermediate chain isoforms by the alternative splicing of the two genes that encode this subunit and identified an additional splicing site in intermediate chain gene 1. We reinvestigated the expression of the intermediate chain 1 isoforms in cultured cells and tissues. The Loa mouse, which is homozygote lethal, contains a missense mutation in the region of the cytoplasmic dynein heavy chain gene that binds the intermediate chain. Protein binding studies showed that all six intermediate chains were able to bind to the mutated heavy chain. GFP-tagged intermediate chains were constructed and PC12 cell lines with stable expression of the fusion proteins were established. Live cell imaging and comparative immunocytochemical analyses show that dynein is enriched in the actin rich region of growth cones.

Technologies for the study of epididymal-specific genes.

Sperm maturation occurs during transit through the epididymis. Interactions between the epididymal epithelium and the sperm are crucial for the maturation process. Analyses of existing male-infertile mouse lines have begun to enumerate some of the genes involved. Recent advances in transgenic technologies to generate temporally and spatially restricted targeted gene disruptions show promise for progress in understanding sperm maturation. Gene silencing agents, such as RNAi, to manipulate gene expression may prove useful for the analysis of epididymal genes involved in the maturation process.

Male contraception-a topic with many facets.

In addition to the scientific issues associated with male contraception, there are a variety of other concerns that must be addressed before new male contraceptives reach the market. Cultural attitudes toward contraception will play a role both in the acceptability of any contraceptive and in compliance and usage. Delivery methods must also be considered; different methods are favored depending on the social context. Prevention of sexually transmitted diseases by a combined contraceptive/microbicidal treatment is a laudable goal, and may enhance public acceptance of a male contraceptive. This review is the result of a workshop that was convened to address these topics.

Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation.

The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.

Spontaneous spinal cord herniation.

Spontaneous spinal cord herniation is a rare and under recognized condition. The commonest presentation is a Brown-sequard syndrome. It most commonly occurs in the thoracic region through an anterolateral dural defect, and the pathophysiology is ill understood. We present a case of spontaneous spinal cord herniation along with a review of literature.

Growth factor regulation of cytoplasmic dynein intermediate chain subunit expression preceding neurite extension.

Cytoplasmic dynein is a motor protein responsible for intracellular movements toward the minus ends of microtubules. The intermediate chains are one of the subunits important for binding dynein to cargo. The intermediate chains are encoded by two genes and are translated into at least five different polypeptide isoforms in rat brain. In rat optic nerve, dynein with only one of the intermediate chain polypeptides is found associated with membrane bounded organelles in fast anterograde transport. Dynein containing the other intermediate chain polypeptides associates with a different set of proteins, in the slow transport component. To determine if the intermediate chain expression levels are regulated during neurite differentiation, we analyzed the protein levels by two-dimensional SDS-PAGE and intermediate chain mRNA by RT-PCR in cultured rat pheochromocytoma (PC12) cells. In the absence of nerve growth factor, the major intermediate chain isoform is the IC74-2C polypeptide. IC74-2C is ubiquitous and is utilized for constitutive dynein function and association with membrane bounded organelles. Within 24 hr of the addition of nerve growth factor to the cultures, there is an increased expression of the developmentally regulated isoforms that are associated with the actin cytoskeleton. This change in intermediate chain isoform expression preceded neurite growth. Nerve growth factor induced differentiation also results in increased light intermediate chain phosphorylation. The growth factor induced changes in the expression of dynein intermediate chains suggests that specific intermediate chain isoforms are utilized during axon growth.

Sural nerve fibre pathology in diabetic patients with mild neuropathy: relationship to pain, quantitative sensory testing and peripheral nerve electrophysiology.

Nerve fibre pathology is poorly described in diabetic patients with mild neuropathy and has not been adequately related to clinical evaluation, quantitative sensory examination and neurophysiology. Sural nerve myelinated and unmyelinated fibre pathology was morphometrically quantified and related to the presence of pain and conventional measures of neuropathic severity in 15 diabetic patients with mild neuropathy and 14 control subjects. Diabetic patients demonstrated a significant (P < 0.01) reduction in myelinated fibre density, but no change in fibre/axonal area, or g-ratio, compared to control subjects. Unmyelinated fibre degeneration was evidenced by an increase in the percentage of unassociated Schwann cell profiles (P < 0.0001) and a reduction in axon density (P < 0.0008) in diabetic patients. This was associated with a significant reduction in unmyelinated axon diameter (P < 0.001) with a shift of the size frequency distribution to the left (P < 0.02). Neurophysiology, quantitative sensory testing and nerve fibre pathology failed to differentiate diabetic patients with painful and painless neuropathy and failed to correlate with any measure of unmyelinated fibre pathology.

Transgenic technologies for the study of epididymal function.

Sperm mature and acquire the capacity for fertilization during their transit through the epididymis, however little is known of the molecular events that comprise sperm maturation. Recent advances in transgenic mouse technology hold promise for illumination of this process. Most of the existing infertile, transgenic mouse lines seem to have defects in epithelial structure or sperm transport rather than direct defects in the maturation of sperm. Temporally and spatially restricted targeted disruptions of epididymal specific genes should provide great insight into the epididymal contribution to sperm maturation.

Probability of bilateral disease in people presenting with a unilateral vestibular schwannoma.

BACKGROUND: Some 4%-5% of those who develop vestibular schwannomas have neurofibromatosis type 2 (NF2). Although about 10% of these patients present initially with a unilateral vestibular schwannoma, the risk for a patient with a truly sporadic vestibular schwannoma developing contralateral disease is unknown. METHODS: A United Kingdom survey of 296 patients with NF2 was reviewed for laterality of vestibular schwannoma at presentation and the presence of other NF2 related features. The time to presentation of bilateral disease was calculated for patients presenting with a unilateral tumour. Mutation analysis of the NF2 gene was carried out on all available cases presenting initially with unilateral disease. RESULTS: Of 240 patients with NF2 with vestibular schwannomas, 45 (18%; 32 sporadic, 13 familial) had either a unilateral tumour or delay in detection between the first and contralateral tumours. Among those tested for NF2 mutations, eight of 27 and nine of 13 were identified among sporadic and familial cases respectively. Sporadic cases showed a high female to male ratio and 19 of 32 have not as yet developed a contralateral tumour (mean 4.1 years after diagnosis of the first). Thirteen of 32 sporadic patients developed a contralateral tumour (mean 6.5 years after the first tumour diagnosis, range 0-22 years) compared with 11 of 13 familial patients (mean delay 5 years, range 0-16 years). Seven of the 45 patients had neither a family history of NF2 nor evidence of related tumours at initial presentation (six before the age of 35 years). CONCLUSION: The risk of patients with sporadic unilateral vestibular schwannomata developing a contralateral tumour in the absence of family history or other features of NF2 is low, but those presenting with other neurogenic tumours in addition to vestibular schwannoma are at high risk of harbouring an NF2 mutation in at least a proportion of their somatic cells.