A novel conditional platelet depletion mouse model reveals the importance of platelets in protection against Staphylococcus aureus bacteremia.

BACKGROUND: Platelets are critical cells for maintaining vascular hemostasis, but their activities in other processes are becoming apparent. Specifically, the ability of platelets to recognize and respond to infectious agents is an important area of investigation. To understand the physiologic roles of platelets in vivo, most researchers have used antibody-mediated platelet depletion, which has certain limitations. OBJECTIVE: To develop an optimal system with which to study the contribution of platelets to protection against S. aureus blood infection. METHODS: Here, we describe a novel experimental model of conditional platelet depletion based on the Cre-recombinase cell ablation system. With this technology, the simian diphtheria toxin receptor was expressed in platelet factor 4-positive cells (megakaryocytes and platelets). RESULTS: Systemic administration of diphtheria toxin every 48 h resulted in reduced platelet numbers that became undetectable after 6 days. Although platelets were depleted, no other blood cells were affected. With this newly developed model, the functional contributions of platelets to protection against Staphylococcus aureus bacteremia was examined. Platelet-depleted mice succumbed to infection more rapidly than wild-type mice, and had a significantly higher bacterial burden in kidneys, elevated levels of serum markers of kidney damage, and increased levels of cytokines indicative of septic shock. CONCLUSIONS: Here, we illustrate a new mouse model for conditional platelet depletion, and implicate platelets as important participants in the immune response to bacterial blood infections.

Seeding life on the moons of the outer planets via lithopanspermia.

Material from the surface of a planet can be ejected into space by a large impact and could carry primitive life-forms with it. We performed n-body simulations of such ejecta to determine where in the Solar System rock from Earth and Mars may end up. We found that, in addition to frequent transfer of material among the terrestrial planets, transfer of material from Earth and Mars to the moons of Jupiter and Saturn is also possible, but rare. We expect that such transfers were most likely to occur during the Late Heavy Bombardment or during the ensuing 1-2 billion years. At this time, the icy moons were warmer and likely had little or no ice shell to prevent meteorites from reaching their liquid interiors. We also note significant rates of re-impact in the first million years after ejection. This could re-seed life on a planet after partial or complete sterilization by a large impact, which would aid the survival of early life during the Late Heavy Bombardment.

Analysis of FcgammaRIIA cytoplasmic tail requirements in signaling for serotonin secretion: evidence for an ITAM-dependent, PI3K-dependent pathway.

The human Fc receptor, FcgammaRIIA, is known to mediate phagocytosis and endocytosis, yet the greatest numbers of these receptors are expressed on the surface of non-phagocytic platelets, where they are involved in serotonin secretion. FcgammaRIIA harbours three tyrosine (Y) residues within its cytoplasmic domain. Y1 is upstream of both Y2 and Y3, which are contained within an immunoreceptor tyrosine-based activation motif (ITAM), required for many signaling events. We have demonstrated that the two ITAM tyrosines are required for phagocytic signaling and that mutation of a single ITAM tyrosine decreases but does not abolish phagocytic signaling. Furthermore, we have identified that the YMTL motif is required for endocytosis. These observations suggest that FcgammaRIIA utilizes different sequences for various signaling events. Therefore, we investigated the sequence requirements for another important FcgammaRIIA-mediated signaling event, serotonin secretion, using Rat Basophilic Leukemia (RBL-2H3) cells transfected with wildtype (WT) FcgammaRIIA or mutant FcgammaRIIA. Stimulation of cells expressing WT FcgammaRIIA induced release of serotonin at a level 7-fold greater than that in nonstimulated WT FcgammaRIIA-transfected cells or nontransfected RBL cells. Mutation of either ITAM tyrosine (Y2 or Y3) to phenylalanine was sufficient to abolish serotonin secretion. Further, while inhibition of Syk with piceatannol blocked phagocytosis as expected, it did not inhibit serotonin secretion. Additionally, inhibition of phosphoinositol-3-kinase (PI3K) with wortmannin only had a partial effect on serotonin signaling, despite the fact that the concentrations used completely abolished phagocytic signaling. These data suggest that the requirements for serotonin secretion differ from those for phagocytosis mediated by FcgammaRIIA.

Real-time seizure prediction from local field potentials using an adaptive Wiener algorithm.

Approximately 30% of individuals with epilepsy have refractory seizures that cannot be controlled by current pharmacological treatment measures. For such patients, responsive neurostimulation prior to a seizure may lead to greater efficacy when compared with current treatments. In this paper, we present a real-time adaptive Wiener prediction algorithm implemented on a digital signal processor to be used with local field potential (LFP) recordings. The hardware implementation of the algorithm enables it to be a miniaturized portable system that could be used in a hand-held device. The adaptive nature of the algorithm allows the seizure data to be compared with baseline data occurring in the recent past rather than a preset value. This enhances the sensitivity of the algorithm by accounting for the time-varying dynamics of baseline, inter-ictal and ictal activity. The Wiener algorithm was compared to two statistical-based naïve prediction algorithms. ROC curves, area over ROC curves, predictive power, and time under false positives are computed to characterize the algorithm. Testing of the algorithm via offline Matlab analysis on kainate-treated rats results in prediction of seizures about 27 s before clinical onset, with 94% sensitivity and a false positive rate of 0.009 min(-1). When implemented on a real-time TI C6713 signal processor, the algorithm predicts seizures about 6.7s before their clinical onset, with 92% sensitivity and a false positive rate of 0.08 min(-1). These results compare favorably with those obtained in similar studies in terms of sensitivity and false positive rate.

A rare ganglioneuroblastoma secreting dopamine and the value of its measurement in diagnosis and prognosis.

A case is described of a patient with a ganglioneuroblastoma, initially located in the right adrenal, which produced an excess of dopamine (7646 and 7959 nmol/24 h), approximately two and a half times the upper limit of the normal daily urine output. The urinary excretion of noradrenaline, adrenaline and methylated derivatives was always within the normal reference ranges. The patient was generally well, with normal blood pressure and only mild flushes. Two years after surgical resection, recurrence was indicated by an increase in urinary dopamine (8507 nmol/24 h); it was located in the tumour bed and left side of the neck by CT and (123)I MIBG scans. The patient was treated with a high dose of (131)I MIBG, with subsequent reduction in dopamine production. This was repeated on four other occasions, the latest being in January 2005. The output of dopamine was thus used as a marker of tumour diagnosis and progression and it is recommended that the assay of dopamine be included in the screening of catecholamine-secreting tumours to avoid possible misdiagnosis.

Aneurysmal subarachnoid haemorrhage: guidance in making the correct diagnosis.

BACKGROUND: The natural history of untreated aneurysmal subarachnoid haemorrhage carries a dismal prognosis. Case fatalities range between 32% and 67%. Treatment with either surgical clipping or endovascular coiling is highly successful at preventing re-bleeding and yet the diagnosis is still missed. METHODS: Based on the national guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage and a review of other available literature this study has compiled guidance in making the diagnosis. CONCLUSION: In patients presenting with a suspected non-traumatic subarachnoid haemorrhage, computed tomography within 12 hours will reliably show 98% of subarachnoid haemorrhage. In patients who present after 12 hours with a negative computed tomogram, formal cerebrospinal fluid spectophotometry will detect subarachnoid haemorrhage for the next two weeks with a reliability of 96%. Between the early diagnosis with the aid of computed tomography and the later diagnosis with the added benefit of spectophotometry in the period where computed tomograms become less reliable, it should be possible to diagnose most cases of subarachnoid haemorrhage correctly.

Temporal lobe epilepsy: analysis of failures and the role of reoperation.

PURPOSE: To analyze failures and reoperations in temporal lobe epilepsy (TLE), and compare these patients with those seizure-free, and to determine any significant differences between the groups. METHODS: A total of 262 patients with TLE, treated surgically between 1984 and 2002, were followed at 3, 6 and 12 months and yearly thereafter. Sixty-five percent became seizure-free (class I), 19% had rare seizures (class II), and 16% continued to have seizures (classes III and IV). Patients in classes III and IV underwent re-evaluation, and were compared with seizure-free patients. RESULTS: Analysis of failures (n = 41): 12% had febrile seizures (FS), 29% head trauma, 7% encephalitis, 52% abnormal imaging, 34% bitemporal spiking, and 20% posterior temporal localization. Post-surgical MRI (available in 30 of 41 patients) showed residual posterior mesial temporal structures (PMTS) in 86.6%, PMTS and posterior temporal lesions (PTLs) in 6.6%, and PTLs in another 6.6%. Twenty-one had reoperation, 14 had resection of the PMTS, five of the PMTS and basal posterior temporal cortex, and two of the PMTS, and PTLs. There was no surgical mortality or morbidity; 57% became seizure-free and 24% had rare seizures. Seizure-free patients (n = 170): 45% had FS, 12% head trauma and 70% abnormal imaging studies. CONCLUSION: When compared with seizure-free patients, patients who failed TLE surgery were less likely to have a history of FS and abnormal imaging, and more likely to have a history of head trauma, encephalitis and posterior temporal localization, suggesting larger epileptogenic zones. Following reoperation, 57% became seizure-free. Predictors of a good outcome after reoperation were anterior temporal localization and abnormal imaging studies.

Angiography postclipping and coiling of cerebral aneurysms.

Postclipping cerebral angiography is generally not practised in the UK. The International Subarachnoid Trial (ISAT) data show that coiling compared favourably with clipping in the early posttreatment phase. We present a 4-year, single unit experience comparing cerebral angiography at 6 months postclipping and postcoiling, defining the proportion of aneurysms in either group, which were incompletely excluded from the cerebral circulation after treatment. There were 4 'dog-ear' remnants (4.6%) in the clipping group of 86 aneurysms, one of which required further surgery. Thirty-one out of 82 (37.8%) coiled aneurysms that underwent check angiography were inadequately excluded from the cerebral circulation at 6 months. Of these, to date, four patients have undergone re-coiling. Although the immediate complications of coiling may be less than those of clipping (ISAT), it seems that the degree and permanence of exclusion of an aneurysm from the cerebral circulation may be more secure with surgery. In summary, the rates of incomplete aneurysmal exclusion from the cerebral circulation, the requirement for reintervention and the requirement for continuing surveillance were all higher in the coiled population than in the clipped population.

A parallel approach: the impact of schuss radiography of the degenerate knee on clinical management.

Schuss radiographs are PA weight bearing views of the knee taken in 30 degrees of flexion. Several studies have shown them to be more sensitive detectors of osteoarthritic changes in the knee than standard extension AP views. We compared the plans of management proposed by eight consultant orthopaedic surgeons to two case presentations of each of a series of 50 patients with tibiofemoral osteoarthritis. The clinical and radiological information provided on each patient was identical in the two presentations except that on one occasion the surgeons were shown the extension AP radiograph while on the other it was replaced by the schuss view. The panel altered their management plan in over 40% of cases. This represented a reduction of almost 50% in arthroscopies in the schuss group with a move towards definitive surgery. The total number of procedures proposed was also reduced. The radiograph was useful determining the management of patients with predominantly lateral, as well as medial or generalised symptoms. We conclude that the schuss radiograph is a valuable tool in the assessment of knee osteoarthritis the use of which can alter clinical management. By reducing non-therapeutic arthroscopies it may significantly reduce total number of operations to be performed in this patient group.

Temporal lobe epilepsy: analysis of patients with dual pathology.

OBJECTIVES: To determine the frequency and types of dual pathology in patients with temporal lobe epilepsy (TLE) and to analyze the clinical manifestations and surgical outcome. MATERIAL AND METHODS: A total of 240 patients with TLE underwent temporal resections following a comprehensive pre-surgical evaluation. Thirty-seven (15.4%) of these had hippocampal sclerosis (HS) or temporal lobe gliosis in association with another lesion (dual pathology). RESULTS: Eighteen of 37 patients with dual pathology had heterotopia of the temporal lobe, nine had cortical dysplasia, four had cavernous angiomas or arteriovenous malformations, one had a dysembryoplastic neuroepithelial tumor, one had a contusion and four patients had cerebral infarctions in childhood. 68.5% had abnormal head magnetic resonance imagings, 91.3% had abnormal positron emission tomography scans, and 96% had abnormal ictal SPECT. The intracarotid amobarbital procedure (IAP) showed impaired memory of the epileptogenic side in 72% of the patients. Twenty patients had left and 17 had right-sided en bloc temporal resections, including the lesion and mesial temporal structures. Twenty-six (70.2%) became seizure-free, eight (21.6%) had rare seizures, two (5.4%) had worthwhile seizure reduction and one (2.7%) had no improvement (range of follow-up 1-16 years, mean = 7.4 years). CONCLUSIONS: 15.4% had dual pathology. The dual pathology was almost exclusively seen in patients whose lesions were congenital, or occurred early in life, suggesting that the hippocampus is more vulnerable and more readily develops HS in early childhood. Resections, including the lateral and mesial temporal structures led to a favorable outcome with no mortality and little morbidity.

An unusual cause of diabetic ketoacidosis and fulminant septicaemia.

BACKGROUND: Diabetic ketoacidosis (DKA) is a common medical emergency. Resistant and recurrent DKA can be due to underlying infection, and a detailed travel history may be important in determining the cause in such cases. We report here a case of unusual DKA and fulminant septicaemia in a Caucasian male with Type 1 diabetes 2 years after returning from living in Thailand. CASE REPORT: A 39-year-old Caucasian male was diagnosed with Type 1 diabetes whilst working in Thailand where he also subsequently developed a cavitating lung lesion diagnosed locally as pulmonary tuberculosis. Two years after returning to the UK he was admitted with DKA and septicaemia. Despite correction of his DKA his condition deteriorated and he developed a fluid collection anterior to the left hip on computed tomography scanning. Blood and fluid aspirate cultures confirmed a diagnosis of melioidosis, a rare fulminant septicaemia in the UK, but endemic in South-east Asia and tropical Australia. Full recovery followed changing antibiotics to intravenous ceftazidime with no relapse 3 years after acute episode. CONCLUSIONS: Physicians as well as microbiologists should consider melioidosis in anyone presenting with septicaemia and/or resistant DKA, especially if the history includes travel to endemic areas or if the cultures suggest Pseudomonas-like organism. With increasing international travel, it is crucial to remember that good travel history could be life-saving in some cases of septicaemia.

Temporal lobe epilepsy surgery: outcome, complications, and late mortality rate in 215 patients.

PURPOSE: We studied the surgical outcome, complications, and the late mortality rate in a large group of patients with medically refractory temporal lobe epilepsy (TLE). METHODS: Two-hundred fifteen patients with TLE were treated surgically between 1984 and 1999 after a comprehensive presurgical evaluation. Patients were followed up at 6 weeks, 3-6 months, and yearly thereafter. In addition, questionnaires were sent on the anniversary of their surgery. Surgical outcome (Engel's classification), complication rate, and factors contributing to late mortality were analyzed. Standardized mortality ratios (SMRs) were calculated. RESULTS: There was no surgical mortality. Two (0.9%) had mild hemiparesis, one (0.4%) had a hemianopia, seven (3.2%) had transient cranial nerve palsies, and eight (3.7%) had transient postoperative language difficulties. One hundred forty-eight (69%) became seizure free, 43 (20%) had rare seizures, 14 (6.5%) had worthwhile seizure reduction, and 10 (4.6%) had no improvement (follow-up, 1-15 years). Three (2%) of 148 seizure-free patients died during follow-up, compared with eight (11.9%) of 67 not seizure-free patients. The mean duration of epilepsy before surgery for the surviving patients was 17.8 years, and for those patients who died, 25.9 years (p < 0.05). Six (5.7%) of 104 patients with right-sided resections died during follow-up, compared with five (4.5%) of 111 with left-sided resections. CONCLUSIONS: Eighty-nine percent of patients became seizure free or had rare seizures, with low morbidity, and no surgical mortality. The late mortality occurred predominantly in patients with persistent seizures (SMR, 7.4). Those patients who died had a longer duration of epilepsy before surgery. In contrast, among those patients who became seizure free, the mortality rate was much lower, and similar to the general population of Indiana (SMR, 1.7).

The cytoplasmic domain of FcgammaRIIA (CD32) participates in phagolysosome formation.

Signaling motifs located within the cytoplasmic domain of certain receptors contribute to lysosome fusion. Most studies have described lysosome fusion with respect to endocytic receptors. Phagolysosome fusion has not been extensively studied. To test the hypothesis that the tail of FcgammaRIIA participates in phagolysosomal fusion, a "reverse" genetic complementation system was used. It was previously shown that complement receptor type 3 (CR3) can rescue the phagocytic activity of a mutant FcgammaRIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fcgamma receptor-dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of FcgammaRIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type FcgammaRIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of FcgammaRIIA containing a full-length cytoplasmic tail with the 2 ITAM tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2) CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that observed with the wild-type receptor. In contrast, in the presence of CR3 and the tail-minus form of FcgammaRIIA, internalized particles did not colocalize with dextran. Electron microscopy revealed that the lysosomal enzyme acid phosphatase colocalized with immunoglobulin G-coated targets internalized by wild-type FcgammaRIIA but not by tail-minus FcgammaRIIA and CR3. Thus, the tail of FcgammaRIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence.

Focal functional deficits in temporal lobe epilepsy on PET scans and the intracarotid amobarbital procedure: comparison of patients with unitemporal epilepsy with those requiring intracranial recordings.

PURPOSE: To analyze the relationship between the intracarotid amobarbital procedure (IAP) and positron emission tomography (PET) and study the lateralizing value of these tests in patients with unitemporal epilepsy and those requiring intracranial recordings. METHODS: We compared 51 patients with unitemporal epilepsy (group1) with 26 patients in whom surface recordings failed to reveal a distinct unitemporal focus, necessitating invasive recordings (group 2). RESULTS: The brain magnetic resonance imaging (MRI) scans for group 1 showed mesial temporal sclerosis in 70.5% of the patients. PET showed unilateral temporal hypometabolism in 88%. In addition, 74.5% of the patients in group 1 had impaired memory on the epileptogenic side on the IAP, and 89.4% of those patients also had ipsilateral temporal hypometabolism on PET scans. All the group 1 patients underwent temporal resections. The pathologic examination showed hippocampal sclerosis in 72% of the patients. Eighty percent of group 1 patients became seizure free, and 16% had rare seizures (follow-up, 2-7 years). MRIs for group 2 showed mesial temporal sclerosis in 31% of the patients; PET scans showed temporal hypometabolism in 39%. The IAP was lateralized in 47.8%. Sixty-nine percent had temporal lobe resections. The pathologic examination showed hippocampal sclerosis in 44% of the patients. Forty-four percent of group 2 patients became seizure free, and 27.7% had rare seizures (follow-up, 2-8 years). CONCLUSIONS: Ninety-six percent of the patients with unitemporal foci had focal functional deficits on the epileptogenic side on 18-fluorodeoxyglucose-(FDG) PET scans, the IAP, or both. The results of the FDG-PET were predictive of impaired memory on the IAP. Memory impairment contralateral to the temporal hypometabolism found on the PET scans was never seen. These patients had an excellent outcome. In contrast, <50% of the patients requiring intracranial recordings had focal functional deficits, suggesting that more a diffuse pathology may account for their less favorable outcome.

Mercury inhibition of neutrophil activity: evidence of aberrant cellular signalling and incoherent cellular metabolism.

Exposure to environmental heavy metals has been reported to affect the immune system. Here, we tested the hypothesis that Hg(+2), acting through membrane proteins, disrupts metabolic dynamics and downstream cell functions in human neutrophils. We found that HgCl(2) inhibited: (1) polarization and (2) immunoglobulin (Ig)G-mediated phagocytosis of sheep erythrocytes in a dose-dependent manner from 2.5 to 10 microM. Because these activities have been linked with pro-inflammatory signalling, we also studied the effects of HgCl(2) on intracellular signalling by measuring protein tyrosine phosphorylation. HgCl(2) at doses = 1 microM increased tyrosine phosphorylation. We also studied the effect of HgCl(2) on neutrophil metabolism by measuring NAD(P)H autofluorescence as an indicator of intracellular NAD(P)H concentration. After HgCl(2) treatment, we found that normal sinusoidal NAD(P)H oscillations became incoherent. We recently reported that the NAD(P)H oscillation frequency is affected by cell migration and activation, which can in turn be regulated by integrin-mediated signalling. Therefore, we examined the effects of HgCl(2) on cell surface distribution of membrane proteins. After exposure to environmentally relevant concentrations of HgCl(2) we found that CR3, but not other membrane proteins (e.g. uPAR, Fc gamma RIIA and the formyl peptide receptor), became clustered on cell surfaces. We suggest that HgCl2 disrupts integrin signalling/functional pathways in neutrophils.

Imaging sustained dissipative patterns in the metabolism of individual living cells.

Theoretical studies have predicted spatiotemporal organization of cell metabolism. Using a rapidly gated CCD camera, we demonstrate for the first time sustained traveling waves of NAD(P)H autofluorescence and protons in individual morphologically polarized living cells. Chemical concentration fronts moved in the direction of cell orientation, thus correlating dissipative structures with cell shape.

Mercuric chloride damages cellular DNA by a non-apoptotic mechanism.

Mercury is a xenobiotic metal that is well known to adversely affect the immune system, however, little is known as to the molecular mechanism. Recently, it has been suggested that mercury may induce immune dysfunction by triggering apoptosis in immune cells. Here, we studied the effects of Hg(2+) (HgCl(2)) on U-937 cells, a human cell line with monocytic characteristics. We found that these cells continued to proliferate when exposed to low doses of mercury between 1 and 5 microM. Using the single cell gel electrophoresis (SCGE) or 'comet' assay, we found that mercury damaged DNA at these levels. Between 1 and 50 microM Hg(2+), comet formation was concentration-dependent with the greatest number of comets formed at 5 microM mercury. However, the appearance of mercury-induced comets was qualitatively different from those of control cells treated with anti-fas antibody, suggesting that although mercury might damage DNA, apoptosis was not involved. This was confirmed by the finding that cells treated with 5 microM mercury were negative for annexin-V binding, an independent assay for apoptosis. These data support the notion that DNA damage in surviving cells is a more sensitive indicator of environmental insult than is apoptosis, and suggests that low-concentrations of ionic mercury may be mutagenic.

Preliminary experience in the treatment of choroidal melanoma with gamma knife radiosurgery.

OBJECT: The authors report their early results from an ongoing experience treating patients with choroidal melanoma by using gamma knife radiosurgery (GKS). METHODS: Between September 1998 and March 2000, 11 patients were treated for choroidal melanoma. Treatment was facilitated with specialized frame placement. Eye immobilization was accomplished with supra- and infraorbital nerve block and tethering sutures to the periorbital tissue. Magnetic resonance imaging was performed to localize the tumor for treatment planning. Plugging patterns were used to steer fall-off radiation away from the fovea, optic nerve, or lens. Tumor volume, tumor location relative to critical structures, and dose to critical structures were determined using GammaPlan. Tumor response was determined using ultrasonography. Toxicity was determined by clinical assessment, visual acuity testing, and ophthalmoscopy. All 11 patients successfully completed the treatment. In every case, 40 Gy was prescribed to the 50% isodose, which completely encompassed all visible tumor. Tumor height ranged from 2.9 to 7 mm. The tumor diameter ranged from 6 to 13 mm. The range of follow up was 2 to 19 months. No tumor has progressed. One patient had improvement in vision because of improvement in retinal detachment. Two patients experienced visual decline. One patient's visual decline was due to a vitreous hemorrhage, and the other's was due to hard exudates encroaching on the macula. One patient has developed a dry eye that is managed effectively with topical eye lubricants. CONCLUSIONS: This preliminary experience demonstrates that GKS is a feasible treatment option for small- to medium-sized choroidal melanomas. Longer follow up and additional patients will be required to improve the assessment and the ultimate tumor control and toxicity in this ongoing series.