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Biomedical subjects

Michael Levin

Publications and source records attributed to Michael Levin.

At least 55 records · Page 3Linked to original sources
PubMed2005

Efficient Ex vivo stimulation of Mycobacterium tuberculosis-specific T cells by genetically detoxified Bordetella pertussis adenylate cyclase antigen toxoids.

Mycobacterium tuberculosis is a significant threat to global health. Mycobacterium bovis BCG vaccine provides only partial protection, and the skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), lacks specificity and sensitivity. The use of genetically detoxified Bordetella pertussis adenylate cyclase toxin (CyaA) as a delivery system for two immunodominant proteins of M. tuberculosis that are of greater specificity than PPD, early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10), was therefore investigated. CyaA toxoids incorporating these antigens were able to restimulate T cells from more than 91% tuberculosis patients and healthy sensitized donors. Delivery of antigen by CyaA decreased by 10-fold the amount of ESAT-6 and CFP-10 required to restimulate T cells, and in low responders, the overall frequency of gamma interferon-producing cells detected by enzyme-linked immunospot assay was increased (P < 0.01 for both antigens). Delivery of ESAT-6 and CFP-10 by CyaA enabled the detection of both CD4(+) and CD8(+) T cells: these responses could be blocked by inhibition of major histocompatibility complex class II or class I, respectively. Covalent linkage of antigen to the CyaA vector was required for enhancement to occur, as a mixture of mock CyaA toxoid plus recombinant ESAT-6 did not lead to enhancement. In a simplified whole-blood model to detect tuberculosis infection, the frequency of positive responses to CFP-10 was increased by CyaA delivery, a potentially important attribute that could facilitate the identification of latent infection.

Adenylyl Cyclases↗
PubMed2005

Serotonin transporter function is an early step in left-right patterning in chick and frog embryos.

The neurotransmitter serotonin has been shown to regulate a number of embryonic patterning events in addition to its crucial role in the nervous system. Here, we examine the role of two serotonin transporters, the plasma membrane serotonin transporter (SERT) and the vesicular monoamine transporter (VMAT), in embryonic left-right asymmetry. Pharmacological or genetic inhibitors of either SERT or VMAT specifically randomized the laterality of the heart and viscera in Xenopus embryos. This effect takes place during cleavage stages, and is upstream of the left-sided gene XNR-1. Targeted microinjection of an SERT-dominant negative construct confirmed the necessity for SERT function in embryonic laterality and revealed that the descendants of the right ventral blastomere are the most dependent upon SERT signaling in left-right patterning. Moreover, the importance of SERT and VMAT in laterality is conserved in chick embryos, being upstream of the early left-sided gene Shh. Endogenous transcripts of SERT and VMAT are expressed from the initiation of the primitive streak in chick and are asymmetrically expressed in Hensen's node. Taken together our data characterize two new right-sided markers in chick gastrulation, identify a novel, early component of the left-right pathway in two vertebrate species, and reveal a new biological role for serotonin transport.

Animals↗
PubMed2005

[Occupational aspects of low back pain].

Low back pain is a very common ailment among workers of all ages. Approximately 60-90% of the adult population suffers from low back pain at least once during their life time. The yearly incident rate of low back pain is about 1-2%. This is one of the major causes of occupational illnesses and absence from work. A high rate of low back pain is found among workers involved with heavy physical labour and also among truck drivers. Risk factors for low back pain are intense heavy labour, lifting heavy objects, protracted static positions, repetitive movements and awkward body postures accompanied by vibration. Prolonged standing and sitting are not risk factors for low back pain. Work dissatisfaction, low wages and low levels of education are possible predictors for low back pain. The only reliable predictor of future low back pain is the evidence of previous back pain as disclosed during the preemployment health examination.

Automobile Driving↗
PubMed2004

Putative vaccine antigens from Neisseria meningitidis recognized by serum antibodies of young children convalescing after meningococcal disease.

Serum samples from 31 children < or = 4 years old who were convalescing after meningococcal disease were used in a quantitative hybridization assay to establish antibody reactivity to 94 candidate meningococcal vaccine antigens. Genes encoding 22 of 23 strongly recognized proteins were found in > or = 94% of the patients' meningococcal strains, and most were also widely prevalent in Neisseria lactamica and other commensal Neisseria species. Similar antibody reactivity was found in serum samples from healthy control children, suggesting that these antibodies arose from asymptomatic colonization. The 23rd protein, NadA, elicited strong reactivity solely in convalescent patients previously infected with a nadA+ strain. nadA was not present in any of 29 diverse N. lactamica strains, suggesting that reactivity in these children arose from meningococcal infection. In contrast, serum samples from healthy adults contained anti-NadA immunoglobulin G at high levels. The correlation of NadA antibody level with natural acquisition of protective immunity suggests that NadA may be a valuable component of a childhood antimeningococcal vaccine.

Antibodies, Bacterial↗
PubMed2004

Size and charge characteristics of the protein leak in dengue shock syndrome.

BACKGROUND: he mechanism underlying the transient vascular leak syndrome of dengue hemorrhagic fever (DHF) is unknown. We aimed to determine whether molecular size and charge selectivity, which help restrict plasma proteins within the intravascular space, are altered in patients with DHF and whether a disturbance of the anionic glycosaminoglycan (GAG) layer on the luminal endothelial surface contributes to disease pathogenesis. METHODS: We measured serial plasma levels and fractional clearances of proteins with different size and charge characteristics in 48 children with dengue shock syndrome (DSS) and urinary excretion profiles of heparan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate in affected children and healthy control subjects. RESULTS: Compared with convalescent values, acute plasma concentrations of all proteins were reduced, with increased fractional clearances. Smaller proteins were more affected than larger molecules. Albumin, which is normally protected from leakage by its strong negative charge, demonstrated a clearance pattern similar to that of transferrin, a neutral molecule of similar size. Urinary heparan sulfate excretion was significantly increased in children with DSS. CONCLUSIONS: The endothelial size-dependent sieving mechanism for plasma proteins is at least partially retained, whereas selective restriction based on negative charge is impaired. The increased heparan sulfate excretion suggests a role for GAGs in the pathogenesis of the vascular leak.

Adolescent↗
PubMed2004

The embryonic origins of left-right asymmetry.

The bilaterally symmetric body plan of vertebrates features several consistent asymmetries in the placement, structure, and function of organs such as the heart, intestine, and brain. Deviations from the normal pattern result in situs inversus, isomerisms, or heterotaxia (independent randomization), which have significant clinical implications. The invariance of the left-right (LR) asymmetry of normal morphology, neuronal function, and phenotype of several syndromes raises fascinating and fundamental questions in cell, developmental, evolutionary, and neurobiology. While a pathway of asymmetrically expressed signaling factors has been well-characterized in several model systems, very early steps in the establishment of LR asymmetry remain poorly understood. In particular, the origin of consistently oriented asymmetry is unknown. Recently, a candidate for the origins of asymmetry has been suggested: bulk transport of extracellular morphogens by rotating primary cilia during gastrulation. This model is appealing because it 'bootstraps' morphological asymmetry of the embryo from the intrinsic structural (molecular) chirality of motile cilia. However, conceptual and practical problems remain with this hypothesis. Indeed, the genetic data are also consistent with a different mechanism: cytoplasmic transport roles of motor proteins. This review outlines the progress and remaining questions in the field of left-right asymmetry, and focuses on an alternative model for 'Step 1' of asymmetry. More specifically, based on wide-ranging data on ion fluxes and motor protein function in several species, it is suggested that laterality is driven by pH/voltage gradients across the midline, which are established by chiral movement of motor proteins with respect to the cytoskeleton.

Animals↗
PubMed2004

A novel immunohistochemical method for evaluation of antibody specificity and detection of labile targets in biological tissue.

Immunohistochemistry is a key tool for analyzing target molecule localization within tissues. However, accurate results require an antibody that can distinguish between similar compounds. We present a simple immunohistochemical method that can also be used to rapidly evaluate antibodies' specificities. We demonstrate this technique with serotonin, an extremely labile compound. Serotonin (5-hydroxytryptamine, 5HT) is an important neurotransmitter regulating normal cognition and several mental disorders, as well as tumor growth, cardiopathology, and embryogenesis. Immunohistochemical detection of serotonin is commonly used as a neuronal cell marker and to provide crucial information on serotonin's role as an embryonic morphogen. It is necessary to be able to distinguish serotonin from closely related molecules with significantly different biological activity. Using our method, we identify antibodies that are specific for serotonin and show that some commercial 5HT antibodies often used to identify serotonergic cells in published papers are not 5HT-specific. These data demonstrate the necessity of specifically testing antibodies (especially in areas of high clinical relevance such as 5HT). We also illustrate detection of serotonergic cells in embryonic tissue using our technique. This method offers a number of general advantages for testing specificity of antibodies to any biological molecule, and helps avoid false positives and negatives during immunohistochemistry.

Animals↗
PubMed2004

Role of interleukin 6 in myocardial dysfunction of meningococcal septic shock.

BACKGROUND: Myocardial failure has a central role in the complex pathophysiology of septic shock and contributes to organ failure and death. During the sepsis-induced inflammatory process, specific factors are released that depress myocardial contractile function. We aimed to identify these mediators of myocardial depression in meningococcal septic shock. METHODS: We combined gene-expression profiling with protein and cellular methods to identify a serum factor causing cardiac dysfunction in meningococcal septic shock. We identified genes that were significantly upregulated in blood after exposure to meningococci. We then selected for further analysis those genes whose protein products had properties of a myocardial depressant factor--specifically a 12-25 kDa heat-stable protein that is released into serum shortly after onset of meningococcal infection. FINDINGS: We identified 174 significantly upregulated genes in meningococcus-infected blood: six encoded proteins that were of the predicted size and had characteristics of a myocardial depressant factor. Of these, interleukin 6 caused significant myocardial depression in vitro. Removal of interleukin 6 from serum samples of patients with meningococcaemia and from supernatants of inflammatory cells stimulated by meningococci in vitro abolished the negative inotropic activity. Furthermore, concentrations in serum of interleukin 6 strongly predicted degree of myocardial dysfunction and severity of disease in children with meningococcal septic shock. INTERPRETATION: Interleukin 6 is a mediator of myocardial depression in meningococcal disease. This cytokine and its downstream mediators could be a target for future treatment strategies.

Adult↗
PubMed2004

Use of recombinant tissue plasminogen activator in children with meningococcal purpura fulminans: a retrospective study.

OBJECTIVE: Meningococcal disease causes septic shock with associated disseminated intravascular coagulation and hemorrhagic skin necrosis. In severe cases, widespread vascular thrombosis leads to gangrene of limbs and digits and contributes to morbidity and mortality. Uncontrolled case reports have suggested that thrombolytic therapy may prevent some complications, and the use of tissue plasminogen activator (t-PA) has been widespread. Our aim was to summarize the clinical outcome and adverse effects where systemic t-PA has been used to treat children with fulminant meningococcemia. DESIGN: International, multiple-center, retrospective, observational case note study between January 1992 and June 2000. SETTING: Twenty-four different hospitals in seven European countries and Australia. PATIENTS: A total of 62 consecutive infants and children with severe meningococcal sepsis in whom t-PA was used for the treatment of predicted amputations and/or refractory shock (40 to treat severe ischemia, 12 to treat shock, and ten to treat both). INTERVENTIONS: t-PA was administered with a median dose of 0.3 mg.kg(-1).hr(-1) (range, 0.008-1.13) and a median duration of 9 hrs (range, 1.2-83). MAIN RESULTS: Twenty-nine of 62 patients died (47%; 95% confidence interval, 28-65). Seventeen of 33 survivors had amputations (11 below knee/elbow or greater loss; six less severe). In 12 of 50 patients to whom t-PA was given for imminent amputation, no amputations were observed. Five developed intracerebral hemorrhages (five of 62, 8%; 95% confidence interval, 0.5-16). Of these five, three died, one developed a persistent hemiparesis, and one recovered completely. CONCLUSIONS: The high incidence of intracerebral hemorrhage in our study raises concerns about the safety of t-PA in children with fulminant meningococcemia. However, due to the absence of a control group in such a severe subset of patients, whether t-PA is beneficial or harmful cannot be answered from the unrestricted use of the drug that is described in this report. Our experience highlights the need to avoid strategies that use experimental drugs in an uncontrolled fashion and to participate in multiple-center trials, which are inevitably required to study rare diseases.

Amputation, Surgical↗
PubMed2004

Hypokalemia in children with severe falciparum malaria.

OBJECTIVES: Acidosis is now recognized as an important component of the severe malaria syndrome and a predictor of fatal outcome. Alterations in plasma potassium concentrations are commonly associated with acidosis. To date, there is little information about the changes in potassium in severe malaria. DESIGN: Prospective study examining the changes in plasma potassium in the first 24 hrs following admission in children with severe malaria. Urinary fractional excretion of potassium and the transtubular gradient of potassium were examined at admission. SETTING: High-dependency unit on the coast of Kenya. PATIENTS: Kenyan children admitted to hospital with clinical features of severe malaria (impaired consciousness or deep breathing) complicated by acidosis (base deficit >8). INTERVENTIONS: Children received standard therapy for severe malaria; in addition, they received boluses of either 0.9% saline or 4.5% human albumin solution to correct hypovolemia, and intravenous potassium replacement was prescribed to children who developed hypokalemia (plasma potassium <3 mmol/L). MEASUREMENTS AND MAIN RESULTS: Thirty-eight Kenyan children were recruited with severe malaria and acidosis. At admission, serum potassium was normal (3-5.5 mmol/L) in 31 (81.6%) and low (<3 mmol/L) in four (11%) children, and three (6.3%) children had hyperkalemia (>5.5 mmol/L). Plasma potassium decreased rapidly within 4-8 hrs of admission: 15 (40%) patients were hypokalemic (<3 mmol/L); of these, five (13%) had plasma potassium of <2.5 mmol/L. Fractional excretion of potassium and the transtubular gradient of potassium were above normal range, indicating renal potassium loss. CONCLUSIONS: Hypokalemia is a common complication of severe malaria; however, it is often not apparent on admission. On correction of acidosis, plasma potassium decreases precipitously, and thus careful, serial monitoring of serum potassium is suggested in patients with severe malaria complicated by acidosis.

Acidosis↗
PubMed2004

Novel human in vitro system for evaluating antimycobacterial vaccines.

Major research efforts are directed towards the development of a better antimycobacterial vaccine. But progress in the field of tuberculosis vaccine development has been hampered by the lack of human in vitro models to assess vaccine immunogenicity and efficacy. New candidate vaccines will have to be evaluated against the existing Mycobacterium bovis BCG "gold standard." It is therefore important to understand the type of immune responses elicited by BCG vaccination to enable comparisons with potential new candidates. We used a novel human in vitro whole-blood model, which measures immune responses to mycobacteria by use of reporter gene-tagged BCG (BCG lux), to study immune responses to BCG vaccination in 50 neonates in a setting in Cape Town, Republic of South Africa, where tuberculosis is endemic. BCG vaccination significantly reduced growth of BCG lux in whole blood (prevaccination median growth ratio [GR], 9.6; range, 1.3 to 24; postvaccination median GR, 3.9; range, 0.6 to 12.2 [P < 0.0001]). Growth of BCG lux was better restricted in vaccinated infants than in unvaccinated age-matched controls (n = 4). BCG vaccination induced significantly higher gamma interferon production in response to BCG lux (P < 0.0001) and to purified protein derivative (P = 0.0001). No significant changes in either growth of BCG lux or cytokine production occurred in an adult control group (n = 6) over the study period. The whole-blood luminescence model detects changes in cellular immune responses to mycobacteria induced by BCG vaccination. It is therefore a useful new tool in studying the immunogenicity of newly developed vaccine candidates prior to large field trials assessing efficacy.

BCG Vaccine↗
PubMed2004

Gamma interferon-based immunodiagnosis of tuberculosis: comparison between whole-blood and enzyme-linked immunospot methods.

The gamma interferon secretion levels of 52 adults whose T cells had been stimulated with the Mycobacterium tuberculosis antigens CFP-10 and ESAT-6 were measured by enzyme-linked immunospot methods and a whole-blood-based assay. The test results correlated well (r = 0.689, P < 0.0001). Its simple format and use of only small volumes of blood make the whole-blood assay suitable for pediatric practice and field trials.

Adult↗
PubMed2004

Safety, pharmacokinetics, and pharmacodynamics of drotrecogin alfa (activated) in children with severe sepsis.

OBJECTIVE: In a phase 3 trial, recombinant human activated protein C (drotrecogin alfa [activated]) significantly reduced mortality in adult patients with severe sepsis. We have now performed a preliminary analysis of the safety, pharmacokinetics, and pharmacodynamics of drotrecogin alfa (activated) in pediatric patients with severe sepsis. DESIGN AND SETTING: Open-label, nonrandomized, sequential, 2-part study conducted in 11 medical centers in the United States and United Kingdom. PATIENTS: Eighty-three pediatric patients with severe sepsis aged term newborn (>or=38 weeks' gestation) to <18 years old. INTERVENTION: In part 1, drotrecogin alfa (activated) was administered as escalating doses of 6, 12, 24, and 36 micro g/kg per hour for 6 hours for each patient (n = 21). In part 2, drotrecogin alfa (activated) was infused at a rate of 24 micro g/kg per hour for 96 hours in 62 patients. MAIN OUTCOME MEASURES: Plasma clearance, plasma concentration, D-dimer, protein C, and antithrombin levels were measured, and adverse events were monitored. RESULTS: The trial enrolled 83 pediatric patients with severe sepsis, aged term newborn (>or=38 weeks' gestation) to <18 years. In part 1, a dose of 24 micro g/kg per hour produced steady-state plasma concentrations of activated protein C similar to those attained in equivalently dosed adult severe sepsis patients. For all pediatric patients dosed at 24 micro g/kg per hour, the median weight-normalized clearance was 0.45 L/hour/kg and the median steady-state concentration was 51.3 ng/mL. The mean plasma half-life was 30 minutes. Weight-normalized clearance in pediatric and adult patients did not differ significantly with age or weight. D-dimer levels decreased 26% from baseline to end of infusion. Baseline levels of protein C and antithrombin increased 79% and 24%, respectively, over the 96-hour treatment period in part 2. The incidence of serious bleeding during infusion and during the entire study period was 2.4% and 4.8%, respectively. CONCLUSIONS: Pediatric patients with severe sepsis manifest sepsis-induced coagulopathy including protein C deficiency comparable to that seen in adults with severe sepsis. The pharmacokinetics, pharmacodynamic effects, and safety profile of drotrecogin alfa (activated) in pediatric patients are similar to those previously published for adult patients. A large, phase 3, randomized, placebo-controlled study is ongoing to confirm these results and formally assess the safety and efficacy of drotrecogin alfa (activated) in children.

Adolescent↗
PubMed2003

Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis.

To gain insight into the molecular mechanisms underlying the control of morphogenetic signals by H+ flux during embryogenesis, we tested Fusicoccin-A (FC), a compound produced by the fungus Fusicoccum amygdali Del. In plant cells, FC complexes with 14-3-3 proteins to activate H+ pumping across the plasma membrane. It has long been thought that FC acts on higher plants only; here, we show that exposing frog embryos to FC during early development specifically results in randomization of the asymmetry of the left-right (LR) axis (heterotaxia). Biochemical and molecular-genetic evidence is presented that 14-3-3-family proteins are an obligate component of Xenopus FC receptors and that perturbation of 14-3-3 protein function results in heterotaxia. The subcellular localization of 14-3-3 mRNAs and proteins reveals novel cytoplasmic destinations, and a left-right asymmetry at the first cell division. Using gain-of-function and loss-of-function experiments, we show that 14-3-3E protein is likely to be an endogenous and extremely early aspect of LR patterning. These data highlight a striking conservation of signaling pathways across kingdoms, suggest common mechanisms of polarity establishment between C. elegans and vertebrate embryos, and uncover a novel entry point into the pathway of left-right asymmetry determination.

14-3-3 Proteins↗
PubMed2003

Assay of locus-specific genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility.

As the central component of the human endotoxin sensor, Toll-like receptor 4 (TLR4) functions in the early detection and response to Gram-negative infection. We therefore examined a large collection of patients with meningococcal sepsis, comparing the frequency of rare TLR4 coding changes to those in an ethnically matched control population. TLR2 sequences were also acquired and compared. Total nucleotide variation at TLR4 and TLR2 loci was assayed by using a novel computational method. A total of 3.01 megabases of coding sequence was captured at these loci from white subjects with or without meningococcal disease. Authentic mutations were found and high-quality, bidirectional coverage was measured across the coding region by using mutationseeker, a program specifically designed to assay locus-specific genetic load. Using a method that obviates the confounding effect of linkage disequilibrium, we observed that rare heterozygous missense mutations of TLR4 contribute to the development of systemic meningococcal disease among white populations of the southern United Kingdom (P = 0.02; odds ratio 8.2). When results from all white populations were pooled, an overwhelmingly significant excess of such mutations was observed among individuals with disease (P = 2 x 10(-6); odds ratio 27.0). The common white TLR4 variant (TLR4B), synonymous TLR4 substitutions, and variant TLR2 alleles were not significantly over-represented among patients with systemic meningococcal infections. No single variant of TLR4 was significantly over-represented in the meningococcal population. Collectively, however, rare TLR4 coding variants were markedly over-represented. Sensing via TLR4 probably contributes to the early containment of meningococcal infection, and sensing defects create increased risk of disease.

Alleles↗
PubMed2003

Bioelectromagnetics in morphogenesis.

Understanding the factors that allow biological systems to reliably self-assemble consistent, highly complex, four dimensional patterns on many scales is crucial for the biomedicine of cancer, regeneration, and birth defects. The role of chemical signaling factors in controlling embryonic morphogenesis has been a central focus in modern developmental biology. While the role of tensile forces is also beginning to be appreciated, another major aspect of physics remains largely neglected by molecular embryology: electromagnetic fields and radiations. The continued progress of molecular approaches to understanding biological form and function in the post genome era now requires the merging of genetics with functional understanding of biophysics and physiology in vivo. The literature contains much data hinting at an important role for bioelectromagnetic phenomena as a mediator of morphogenetic information in many contexts relevant to embryonic development. This review attempts to highlight briefly some of the most promising (and often underappreciated) findings that are of high relevance for understanding the biophysical factors mediating morphogenetic signals in biological systems. These data originate from contexts including embryonic development, neoplasm, and regeneration.

Animals↗
PubMed2003

Motor protein control of ion flux is an early step in embryonic left-right asymmetry.

The invariant left-right asymmetry of animal body plans raises fascinating questions in cell, developmental, evolutionary, and neuro-biology. While intermediate mechanisms (e.g., asymmetric gene expression) have been well-characterized, very early steps remain elusive. Recent studies suggested a candidate for the origins of asymmetry: rotary movement of extracellular morphogens by cilia during gastrulation. This model is intellectually satisfying, because it bootstraps asymmetry from the intrinsic biochemical chirality of cilia. However, conceptual and practical problems remain with this hypothesis, and the genetic data is consistent with a different mechanism. Based on wide-ranging data on ion fluxes and motor protein action in a number of species, a model is proposed whereby laterality is generated much earlier, by asymmetric transport of ions, which results in pH/voltage gradients across the midline. These asymmetries are in turn generated by a new candidate for "step 1": asymmetric localization of electrogenic proteins by cytoplasmic motors.

Animals↗