Inhibition of HERV-K (HML-2) in amyotrophic lateral sclerosis patients on antiretroviral therapy.

Reactivation of Human Endogenous Retrovirus K (HERV-K), subtype HML-2, has been associated with pathophysiology of amyotrophic lateral sclerosis (ALS). We aimed to assess the efficacy of antiretroviral therapy in inhibiting HML-2 in patients with ALS and a possible association between the change in HML-2 levels and clinical outcomes. We studied the effect of 24-weeks antiretroviral combination therapy with abacavir, lamivudine, and dolutegravir on HML-2 levels in 29 ALS patients. HML-2 levels decreased progressively over 24 weeks (P = 0.001) and rebounded within a week of stopping medications (P = 0.02). The majority of participants (82%), defined as "responders", experienced a decrease in HML-2 at week 24 of treatment compared to the pre-treatment levels. Differences in the evolution of some of the clinical outcomes could be seen between responders and non-responders: FVC decreased 23.69% (SE = 11.34) in non-responders and 12.71% (SE = 8.28) in responders. NPI score decreased 91.95% (SE = 6.32) in non-responders and 53.05% (SE = 10.06) in responders (P = 0.01). Thus, participants with a virological response to treatment showed a trend for slower progression of the illness. These findings further support the possible involvement of HML-2 in the clinical course of the disease.

Comparative Assessment of Cytokine Pattern in Early and Late Onset of Neonatal Sepsis.

Neonatal sepsis is a significant health issue associated with high mortality. Immune responses associated with neonatal sepsis, such as proinflammatory cytokine production, are believed to play a central role in the pathogenesis of this disease. In the present study, serum levels of the proinflammatory cytokines TNF-α, IL1-ß, and IL-6 and the anti-inflammatory cytokines IL-4 and IL-10 were evaluated for 25 subjects with neonatal sepsis. We observed that subjects with late onset of sepsis (LOS), as well as those with early onset of sepsis (EOS), had a substantial increase in serum TNF-α. In contrast to EOS, subjects with LOS demonstrated a significant increase in serum levels IL-6 and IL-10. Additionally, we observed a significant difference in cytokine profiles between acute and postacute cases of neonatal sepsis. For instance, the level of proinflammatory cytokines, such as TNF-α and IL-6, was elevated in the acute phase, whereas the production of anti-inflammatory cytokines, such as IL-10, became substantially upregulated during the postacute phase. Additionally, no correlation was observed between cytokine levels and CRP levels or lymphocyte counts. Thus, in contrast to CRP levels and lymphocyte counts, examination of the cytokine profile can provide valuable information when determining the most effective therapy for treating neonatal sepsis. This information may be useful to physicians when determining if anti-inflammatory or immune stimulatory therapy is warranted.

Physical performance in newly diagnosed hypothyroidism: a pilot study.

OBJECTIVE: Hypothyroidism is complicated by neuromuscular symptoms (myalgias, slowness of movements, and tiredness) and signs (easy fatigability and cramps), which may have a negative impact on general well-being and quality of life. In a pilot, prospective, controlled study, we investigated the features of muscle dysfunction in hypothyroidism by disease questionnaire, biochemical measures, and physical performance tests. MATERIALS AND METHODS: Fifty-seven consecutive patients with newly diagnosed hypothyroidism were enrolled, 27 subclinical (S-Hypo) and 30 overt (O-Hypo). A series of 30 euthyroid subjects, with similar demographic characteristics, served as controls. Patients were administered a short disease questionnaire and underwent laboratory exams and standardized physical tests, both at baseline and after restoration of biochemical euthyroidism. RESULTS: Compared to euthyroid controls, the O-Hypo group showed significantly higher prevalence of neuromuscular symptoms and significantly higher serum creatine phosphokinase (CPK) levels (p value < 0.0001). S-Hypo had slightly higher CPK levels and prevalence of neuromuscular symptoms than controls. Both S-Hypo and O-Hypo patients performed worse than controls in the six-minute walking test. Differences between patients and controls in handgrip strength test and timed chair standing test failed to reach statistical significance (although a trend was noticeable), possibly due to the small sample size. In O-Hypo, an inverse correlation was found between CPK levels and the handgrip strength test (p value < 0.001). Restoration of euthyroidism was associated with normalization of questionnaire responses, six-minute walking test, as well as serum CPK levels. CONCLUSION: In addition to neuromuscular symptoms, hypothyroidism is associated with abnormalities of physical performance. The six-minute walking test is the most valuable test to assess this aspect. In the pilot study, levothyroxine therapy could reverse muscle functional abnormalities.

Enhancing Allergen-Presentation Platforms for Sublingual Immunotherapy.

Sublingual immunotherapy (SLIT) relies on high doses of allergens to treat patients with type I allergies. Although SLIT is commonly performed without any adjuvant or delivery system, allergen(s) could be further formulated with allergen-presentation platforms to better target oral dendritic cells eliciting regulatory immune responses. Improving the availability of allergens to the immune system should enhance SLIT efficacy, while allowing to decrease allergen dosing. Herein, we present an overview of adjuvants and vector systems that have been, or could be, considered as candidate allergen-presentation platforms for the sublingual route. Such platforms encompass adjuvants capable of stimulating allergen-specific TH1 and/or regulatory CD4+ T-cell responses, including 1,25-dihydroxy vitamin D3, glucocorticoids, Toll-like receptor ligands as well as selected bacterial probiotic strains. A limiting factor for SLIT efficacy is the number of dendritic cells capturing the allergens in the upper layers of oral tissues. Thus, adsorption or encapsulation of the allergen(s) within mucoadhesive particulate vector (or delivery) systems also has the potential to significantly enhance SLIT efficacy due to a facilitated allergen uptake by tolerogenic oral dendritic cells.

An AT-barrier mechanically controls DNA reannealing under tension.

Regulation of genomic activity occurs through the manipulation of DNA by competent mechanoenzymes. Force-clamp optical tweezers that allow the structural dynamics of the DNA molecule to be measured were used here to investigate the kinetics of mechanically-driven strand reannealing. When the force on the torsionally unconstrained λ-phage DNA is decreased stepwise from above to below the overstretching transition, reannealing occurs via discrete shortening steps separated by exponentially distributed time intervals. Kinetic analysis reveals a transition barrier 0.58 nm along the reaction coordinate and an average reannealing-step size of ∼750 bp, consistent with the average bp interval separating segments of more than 10 consecutive AT bases. In an AT-rich DNA construct, in which the distance between segments of more than 10 consecutive AT is reduced to ∼210 bps, the reannealing step reduces accordingly without changes in the position of the transition barrier. Thus, the transition barrier for reannealing is determined by the presence of segments of more than 10 consecutive AT bps independent of changes in sequence composition, while the length of the reannealing strand changes according to the distance between poly-AT segments at least 10 bps long.

Genome-wide association analysis identifies genetic variations in subjects with myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis, also known as chronic fatigue syndrome or ME/CFS, is a multifactorial and debilitating disease that has an impact on over 4 million people in the United States alone. The pathogenesis of ME/CFS remains largely unknown; however, a genetic predisposition has been suggested. In the present study, we used a DNA single-nucleotide polymorphism (SNP) chip representing over 906,600 known SNPs to analyze DNA from ME/CFS subjects and healthy controls. To the best of our knowledge, this study represents the most comprehensive genome-wide association study (GWAS) of an ME/CFS cohort conducted to date. Here 442 SNPs were identified as candidates for association with ME/CFS (adjusted P-value<0.05). Whereas the majority of these SNPs are represented in non-coding regions of the genome, 12 SNPs were identified in the coding region of their respective gene. Among these, two candidate SNPs resulted in missense substitutions, one in a pattern recognition receptor and the other in an uncharacterized coiled-coil domain-containing protein. We also identified five SNPs that cluster in the non-coding regions of T-cell receptor loci. Further examination of these polymorphisms may help identify contributing factors to the pathophysiology of ME/CFS, as well as categorize potential targets for medical intervention strategies.

Evaluation of preformed endodontic postretention force using post-BRUSH technique.

AIM: In this work it has been evaluated how the use of an endocanalar brush (post-brush) is able to clean the post-space macroscopically and eliminate permanently any debris, leading to the determination of an enhancement of the dentine-cement interface adhesion. The retentive power of cylindrical-grooved in two different groups of dental elements (A GROUP and B GROUP) will be compared, cementing A group according to standard protocol and B group with treatment of Post-Brush. METHODS: Forty monoradicular elements were selected to carry out this experimental work. The roots were treated endodontically. Samples were divided randomly into two groups. For each group was used a different type of cementation. These tests were performed through the use of an electronic dynamometer. The samples were subjected to tensile strength to obtain a axial traction shear-stress and a subsequent separation of sample postreconstruction from root canal, at a loading maximum value recorded by the dynamometer right after the detachment. RESULTS: B GROUP, which included cemented cylindrical-rubbed posts before treatment of Post-Brush, has shown a tensile shear-stress at break performances higher than that A GROUP, in which cylindrical-rubbed posts have been cemented according to the standard protocol. CONCLUSION: Post-brush allows to obtain a better mechanical cleaning, going to remove gutta-percha residues, smear-layer and endodontic cement, which remain adhere on the canal walls otherwise they will not be removed completely by the use of the etching and the washing techniques as it tends to adhere to the canal walls.

Pituitary apoplexy during pregnancy: a rare, but dangerous headache.

Pituitary apoplexy is a rare endocrine emergency that occurs in a small number of patients with a pituitary tumor. It is a clinical syndrome characterized by the sudden onset of headache, nausea, vomiting, visual impairment, and decreased consciousness, caused by hemorrhage and/or infarction of the pituitary gland. Pituitary apoplexy has very rarely been described during pregnancy, when it is potentially life-threatening to both the mother and the fetus, if unrecognized. Only a few cases have been published to date. The review of the existing literature underlines that pituitary apoplexy, although rare, should be borne in mind when a pregnant woman presents with severe headache and visual defects of sudden onset. After initial management, which includes intravenous glucocorticoid therapy, fluid and electrolyte replacement, the final selection of medical or surgical treatment should result from a multidisciplinary approach involving expert specialists, keeping into account both severity of clinical presentation and gestational week.

The transition mechanism of DNA overstretching: a microscopic view using molecular dynamics.

The overstretching transition in torsionally unconstrained DNA is studied by means of atomistic molecular dynamics simulations. The free-energy profile as a function of the length of the molecule is determined through the umbrella sampling technique providing both a thermodynamic and a structural characterization of the transition pathway. The zero-force free-energy profile is monotonic but, in accordance with recent experimental evidence, becomes two-state at high forces. A number of experimental results are satisfactorily predicted: (i) the entropic and enthalpic contributions to the free-energy difference between the basic (B) state and the extended (S) state; (ii) the longitudinal extension of the transition state and (iii) the enthalpic contribution to the transition barrier. A structural explanation of the experimental finding that overstretching is a cooperative reaction characterized by elementary units of approximately 22 base pairs is found in the average distance between adenine/thymine-rich regions along the molecule. The overstretched DNA adopts a highly dynamical and structurally disordered double-stranded conformation which is characterized by residual base pairing, formation of non-native intra-strand hydrogen bonds and effective hydrophobic screening of apolar regions.

Upregulation of IFN-γ and IL-12 is associated with a milder form of hantavirus hemorrhagic fever with renal syndrome.

Hantavirus hemorrhagic fever with renal syndrome (HFRS) is a zoonotic disease characterized by acute onset, fever, malaise, and back pain. As the disease progresses, hemorrhagic disturbances and kidney dysfunctions predominate. The examination of tissue collected postmortem supports the premise that virus replication is not responsible for this pathology; therefore, it is widely believed that virus-induced immune responses lead to the clinical manifestations associated with HFRS. The overproduction of inflammatory cytokines is commonly reported in subjects with HFRS and has given rise to the hypothesis that a so-called "cytokine storm" may play a pivotal role in the pathogenesis of this disease. Currently, supportive care remains the only effective treatment for HFRS. Our data show that serum levels of interferon (IFN)-γ, interleukin (IL)-10, CCL2, and IL-12 are upregulated in HFRS cases when compared to healthy controls and the level of upregulation is dependent on the phase and severity of the disease. Furthermore, we observed an association between the mild form of the disease and elevated serum levels of IFN-γ and IL-12. Collectively, these observations suggest that the administration of exogenous IFN-γ and IL-12 may provide antiviral benefits for the treatment of HFRS and, thus, warrants further investigations.

Role of plasmacytoid dendritic cell subsets in allergic asthma.

Plasmacytoid dendritic cells (pDCs) are major type-I interferon-producing cells that play important roles in antiviral immunity and tolerance induction. These cells share a common DC progenitor with conventional DCs, and Fms-like tyrosine kinase-3 ligand is essential for their development. Several subsets of pDCs have been identified to date including CCR9(+) , CD9(+) , and CD2(+) pDCs. Recently, three subsets of pDCs were described, namely CD8α(-) ß(-) , CD8α(+) ß(-) , and CD8α(+) ß(+) subsets. Interestingly, CD8α(+) ß(-) and CD8α(+) ß(+) but not CD8α(-) ß(-) pDCs were shown to have tolerogenic effects in experimentally induced allergic asthma. These tolerogenic effects were shown to be mediated by the generation of FOXP3(+) regulatory T cells through retinoic acid and the induction of retinaldehyde dehydrogenase enzymes. These newly described subsets of pDCs show high potentials for novel therapeutic approaches for the treatment of allergic diseases. In this review, we will address the new progress in our understanding of pDC biology with respect to allergic disease, in particular allergic asthma.

CD8α⁺ß⁻ and CD8α⁺ß⁺ plasmacytoid dendritic cells induce Foxp3⁺ regulatory T cells and prevent the induction of airway hyper-reactivity.

Dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self-antigens. Here, we demonstrate for the first time that mouse plasmacytoid DCs (pDCs) can be segregated into three distinct populations, exhibiting phenotypic and functional differences, according to their surface expression of CD8α or CD8ß as CD8α⁻ß⁻, CD8α⁺ß⁻, or CD8α⁺ß⁺. In a mouse model of lung inflammation, adoptive transfer of CD8α⁺ß⁻ or CD8α⁺ß⁺ pDCs prevents the development of airway hyper-reactivity. The tolerogenic features of these subsets are associated with increased production of retinoic acid, which leads to the enhanced induction of Foxp3⁺ regulatory T cells compared with CD8α⁻ß⁻ pDCs. Our data thus identify subsets of pDCs with potent tolerogenic functions that may contribute to the maintenance of tolerance in mucosal sites such as the lungs.

Mechanics of myosin function in white muscle fibres of the dogfish, Scyliorhinus canicula.

The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (C(f)) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa(−1). This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa(−1). The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm(−1)) is smaller than that of frog (2.78 ± 0.30 pN nm(−1)). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog.

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle.

A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ.With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4-30◦C) and substrate concentration. VF was 8.88 ± 0.26 µms⁻¹ at 30.6◦C and decreased to 1.60 ± 0.09 µms⁻¹ at 4.5◦C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40-50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5◦C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads.

Does psychomotor retardation define a clinically relevant phenotype of unipolar depression?

BACKGROUND: The recognition and assessment of psychomotor retardation may have implications for better definition of the clinical phenotypes of depression. The aim of this study was to assess the clinical correlates of psychomotor retardation endorsed at any time during the patients' lifetime (LPR). METHODS: The study sample included 291 patients with non-psychotic major depressive disorder (MDD) participating in the clinical trial, "Depression: The Search for Treatment-Relevant Phenotypes." Psychomotor retardation was measured using a factor derived from the Mood Spectrum Self-Report (MOODS-SR) assessment. Using a pre-defined cut-off score on the lifetime psychomotor retardation (LPR) factor of the MOODS-SR, participants were classified into high and low scorers. Logistic regression analysis was used to evaluate the relationship between LPR and subthreshold bipolarity. RESULTS: Compared to low scorers, participants with high scores on the LPR factor had greater severity of depression and more bipolarity indicators. CONCLUSIONS: The MOODS-SR appears to be helpful to identify clinical phenotypes of unipolar depression and to highlight the usefulness of a lifetime approach to the assessment of psychopathology in the characterisation of patients with unipolar depression.

Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

PD-L1 and PD-L2 modulate airway inflammation and iNKT-cell-dependent airway hyperreactivity in opposing directions.

Interactions of the inhibitory receptor programmed death-1 (PD-1) with its ligands, programmed death ligand (PD-L)1 and PD-L2, regulate T-cell activation and tolerance. In this study, we investigated the role of PD-L1 and PD-L2 in regulating invariant natural killer T (iNKT)-cell-mediated airway hyperreactivity (AHR) in a murine model of asthma. We found that the severity of AHR and airway inflammation is significantly greater in PD-L2(-/-) mice compared with wild-type mice after either ovalbumin (OVA) sensitization and challenge or administration of alpha-galactosylceramide (alpha-GalCer). iNKT cells from PD-L2(-/-) mice produced significantly more interleukin (IL)-4 than iNKT cells from control mice. Moreover, blockade of PD-L2 interactions of wild-type iNKT cells in vitro with monoclonal antibodies (mAbs) resulted in significantly enhanced levels of IL-4 production. In contrast, PD-L1(-/-) mice showed significantly reduced AHR and enhanced production of interferon-gamma (IFN-gamma) by iNKT cells. iNKT-deficient Jalpha18(-/-) mice reconstituted with iNKT cells from PD-L2(-/-) mice developed high levels of AHR, whereas mice reconstituted with iNKT cells from PD-L1(-/-) mice developed lower levels of AHR compared with control. As PD-L2 is not expressed on iNKT cells but rather is expressed on lung dendritic cells (DCs), in which its expression is upregulated by allergen challenge or IL-4, these findings suggest an important role of PD-L2 on lung DCs in modulating asthma pathogenesis. These studies also indicate that PD-L1 and PD-L2 have important but opposing roles in the regulation of AHR and iNKT-cell-mediated activation.

Diagnostic imaging in the study of human hepatobiliary fascioliasis.

PURPOSE: Fascioliasis is a rare zoonotic disease caused by the trematode Fasciola hepatica. We present the typical patterns of hepatobiliary fascioliasis observed in ten patients studied with multimodality imaging. MATERIALS AND METHODS: Between 2002 and 2005, ten women with fascioliasis were admitted to the Brigham and Women's Hospital, Harvard Medical School (BWH), with abdominal pain and mild fever. All imaging modalities, including ultrasound (US), computed tomography (CT), magnetic resonance (MR) imaging (n = 2) and endoscopic retrograde cholangiopancreatography (ERCP) (n = 1) were reviewed by two expert radiologists working in consensus. RESULTS: In all patients (10/10, 100%), US showed parenchymal heterogeneity characterised by multiple subcapsular and peribiliary hypoechoic nodular lesions that were ill-defined and coalesced into tubular or tortuous structures. In six patients (6/10, 60%), the lesions appeared hypoechoic, whereas in four patients (4/10, 40%), there was an alternation of hyperechoic and hypoechoic nodules. On CT, all patients (10/10, 100%) showed hypodense patchy lesions in subcapsular, peribiliary or periportal locations, which coalesced to form tubular structures and were more evident during the portal phase. Lesion diameter ranged from 2 cm to 7 cm. Capsular enhancement was seen in four cases on CT (4/10, 40%) and in one also at MR imaging. MR imaging, performed in two patients, confirmed the presence of the lesions, which appeared hyperintense on T2-weighted images and were characterised by mild peripheral enhancement after gadolinium administration. Four patients had gallbladder wall thickening (4/10, 40%), with parasites in the gallbladder lumen. CONCLUSIONS: Although rare, hepatobiliary fascioliasis should be considered in the differential diagnosis in the appropriate clinical scenario, especially in patients coming from endemic areas. The typical imaging pattern of fascioliasis is the presence of subcapsular, peribiliary or periportal nodules that are usually ill-defined and coalesce, giving rise to a tubular or tortuous appearance.