Defensin-driven viral evolution.

Enteric alpha-defensins are potent effectors of innate immunity that are abundantly expressed in the small intestine. Certain enteric bacteria and viruses are resistant to defensins and even appropriate them to enhance infection despite neutralization of closely related microbes. We therefore hypothesized that defensins impose selective pressure during fecal-oral transmission. Upon passaging a defensin-sensitive serotype of adenovirus in the presence of a human defensin, mutations in the major capsid protein hexon accumulated. In contrast, prior studies identified the vertex proteins as important determinants of defensin antiviral activity. Infection and biochemical assays suggest that a balance between increased cell binding and a downstream block in intracellular trafficking mediated by defensin interactions with all of the major capsid proteins dictates the outcome of infection. These results extensively revise our understanding of the interplay between defensins and non-enveloped viruses. Furthermore, they provide a feasible rationale for defensins shaping viral evolution, resulting in differences in infection phenotypes of closely related viruses.

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection.

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.

The role of NOD2 in murine and human melioidosis.

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low-dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared with wild-type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis, and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole-blood stimulation with the NOD2 ligand, muramyl dipeptide, or B. pseudomallei. To our knowledge, these findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.

Impaired TLR5 functionality is associated with survival in melioidosis.

Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1ß in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.

Directed evolution of mutator adenoviruses resistant to antibody neutralization.

We incorporated a previously identified mutation that reduces the fidelity of the DNA polymerase into a human adenovirus vector. Using this mutator vector, we demonstrate rapid selection of resistance to a neutralizing anti-hexon monoclonal antibody due to a G434D mutation in hexon that precludes antibody binding. Since mutator adenoviruses can accumulate compound mutations that are unattainable using traditional random mutagenesis techniques, this approach will be valuable to the study of antivirals and host factor interactions.

Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei.

Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of the Gram-negative soil saprophyte Burkholderia pseudomallei. We developed a reproducible experimental murine model of pneumonic melioidosis induced by inhalation of aerosolized B. pseudomallei 1026b. In a series of experiments performed to bracket the lethal dose, we found that C57BL/6 mice were modestly more resistant than BALB/c mice (median lethal dose 334 CFU/lung vs 204 CFU/lung). We further characterized infection and pulmonary inflammation in C57BL/6 mice infected with a sublethal dose. We observed pulmonary replication and dissemination of bacteria to distant organs in the first days after infection, followed by bacterial containment by day 4 and no evidence of recrudescent infection for up to 2 months. We measured a robust host inflammatory response notable for a neutrophilic bronchoalveolar lavage fluid profile, elevated cytokines and chemokines in the lung and serum and scattered foci of neutrophilic infiltrates in the alveoli and in a perivascular distribution on histological analysis. We previously noted a similar pattern of inflammation in mice infected with aerosolized B. thailandensis. This report builds on the limited literature describing experimental murine pneumonic melioidosis induced by aerosol and characterizes pulmonary infection and resultant inflammation in C57BL/6 mice infected with aerosolized B. pseudomallei. This model has utility for the study of bacterial and host factors that contribute to the virulence of melioidosis.

[Experimental and pathological changes of the neuromuscular junction]. / Remaniements expérimentaux et pathologiques de la jonction neuromusculaire.

The objective of our study was to investigate the cellular communication between the axon and its postsynaptic targets in the synapse. We used the neuromuscular junction (NMJ) model, which is a highly specialized structure between the nerve, the muscle, and the Schwann cell terminal where the motor neuron orders the muscle to contract. We used experimental models of motor nerve reimplantation in a denervated muscle to determine whether 1) the formation of new NMJ could participate in reinnervation of the muscle necessary to contraction or 2) the blockage of neurotransmitter release using botulinum toxin could be compensated by the formation of new NMJ. We also studied human genetic diseases that affect neuromuscular transmission--congenital myasthenic syndromes--to identify the mutations in the genes coding for synaptic molecules and to analyze the compensatory processes involved in NMJ dysfunction so that muscle contraction can occur in these conditions.

Flagellin-independent effects of a Toll-like receptor 5 polymorphism in the inflammatory response to Burkholderia pseudomallei.

BACKGROUND: Toll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation. CONCLUSIONS/SIGNIFICANCE: The effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required.

Separate neural pathways for the visual analysis of object shape in perception and prehension.

BACKGROUND: Earlier work with neurological patients has shown that the visual perception of object size and orientation depends on visual pathways in the cerebral cortex that are separate from those mediating the use of these same object properties in the control of goal-directed grasping. We present evidence suggesting that the same dissociation between perception and action is evident in the visual processing of object shape. In other words, discrimination between objects on the basis of their shape appears to be mediated by visual mechanisms that are functionally and neurally distinct from those controlling the pre-shaping of the hand during grasping movements directed at those same objects. RESULTS: We studied two patients with lesions in different parts of the cerebral visual pathways. One patient (RV), who had sustained bilateral lesions of the occipitoparietal cortex, was unable to use visual information to place her fingers correctly on the circumference of irregularly shaped objects when asked to pick them up, even though she had no difficulty in visually discriminating one such object from another. Conversely, a second patient (DF), who had bilateral damage in the ventrolateral occipital region, had no difficulty in placing her fingers on appropriate opposition points during grasping, even though she was unable to discriminate visually amongst such objects. CONCLUSIONS: This double dissociation lends strong support to the idea that the visual mechanisms mediating the perception of objects are functionally and neurally distinct from those mediating the control of skilled actions directed at those objects. It also supports the recent proposal of Goodale and Milner that visual perception depends on a ventral stream of projections from the primary visual cortex to the inferotemporal cortex, whereas the visual control of skilled actions depends on a dorsal stream from the primary visual cortex to the posterior parietal cortex.

Off-host development and survival of Rhipicephalus (Boophilus) microplus in the Brazilian semiarid.

Rhipicephalus (Boophilus) microplus is the most economically important tick of the world, including Brazil. Whereas the epidemiology of R. microplus is well known in most of the Brazilian land, virtually nothing is known from the Brazilian semiarid region, the Caatinga biome. Considering the relevance of R. microplus for the cattle industry within the Caatinga, this study aimed to evaluate for the first time the off-host development of this tick species under natural conditions of the Caatinga. During 2011-2015, engorged females of R. microplus were exposed to field conditions of native Caatinga, where female oviposition, egg incubation and hatching, and larval survival were quantified. In parallel, counterpart ticks (control group) were evaluated under optimal conditions in the laboratory. During the study, nearly 100% of the engorged females of the control group successfully oviposited fertile egg masses, from which most of the eggs yielded larvae (mean % hatching usually>90%). Under field conditions, while almost 100% of the engorged females oviposited, in most of the times no larvae hatched from these eggs. Furthermore, when larvae hatched, mean % hatching was most of the times <50%. Soil temperatures did not oscillate drastically through the study, with mean temperature between 30 and 35°C in most of the time. In contrast, rainfall was irregular throughout the study. Correlation analyses indicated that either the number of egg masses that yielded larvae, or the mean % hatching of the egg masses were positively associated with higher rainfall months. Our results showed that in most of the time, field conditions were unfavorable for R. microplus, indicating that cattle would have a low exposure to R. microplus infestations when raised in Caatinga fields like those of the present study.

Bilateral blindness in temporal arteritis with skip areas.

A 73-year-old woman received systemic corticosteroids for suspected temporal arteritis after blindness developed in the right eye during the previous day. Because a right temporal artery biopsy specimen was reported as normal, a diagnosis of arteriosclerotic ischemic optic neuropathy was made and the corticosteroid therapy was discontinued. Two weeks later the patient rapidly lost vision in her left eye. A left temporal artery biopsy specimen showed granulomatous arteritis adjacent to normal artery (skip area). Deeper sections of the original right temporal artery biopsy specimen also demonstrated a small focus of granulomatous arteritis adjacent to normal artery. In patients with suspected temporal arteritis, numerous step sections of a long segment of temporal artery should be examined; if the results are normal, the contralateral temporal artery should be studied. A nonspecific inflammatory reaction within or adjacent to the artery should alert the pathologist to the possible presence of a nearby focus of granulomatous arteritis.

Eccrine sweat gland carcinoma of the eyelid with orbital involvement.

A 70-year-old man had a sweat gland carcinoma in his right upper eyelid that was slowly growing, but it clinically remained confined to the lid tissues for a period of 25 years. The tumor ultimately extended deeper into the orbit and invaded the sclera, the optic nerve sheath, and the ethmoidal sinuses. Histopathologic examination disclosed malignant features with a variety of cellular patterns. The results of electron microscopic studies confirmed the origin of the tumor from the eccrine sweat glands.

Rotation of Listing's plane by horizontal, vertical and oblique prism-induced vergence.

We examined the changes in Listing's plane resulting from prismatically induced vergence. The three-dimensional angular positions of the two eyes were compared in normal subjects wearing search coils and gazing at targets 1.9 m away with and without prisms. For horizontal base-out prisms each degree of convergence in one eye yielded 0.72 deg of temporal rotation of Listing's plane in that eye. The results from vertical prisms were not what was expected from the horizontal results. A base-up prism on the right eye induced a downward and temporal rotation of Listing's plane. A base-down prism on the right eye induced an upward and nasal rotation of Listing's plane. The effects of oblique prisms were those expected from combining the effects of horizontal and vertical prisms. Thus in addition to producing a horizontal or vertical misalignment of the gaze line, prisms induce an unexpected position-dependent torsional disparity.

Task-dependent changes in the shape and thickness of Listing's plane.

We examined the 2D surface formed by 3D eye positions of normal subjects to determine whether the shape and thickness changed in tasks that differed in saccadic directions: random, horizontal, vertical, radial, clockwise and counter-clockwise. Eye positions during the random task did not lie precisely on Listing's plane but on a surface with a small twist. This twist was present before, during, and after saccades. The degree of twist changed with the task; becoming less twisted for horizontal tasks and more twisted in the vertical tasks. The surface thickness changed with the task becoming thicker for multidirectional tasks. This greater thickness may occur because surfaces obtained in multidirectional tasks are the composite of surfaces with slightly different shapes.

Using a synoptophore to test Listing's law during vergence in normal subjects and strabismic patients.

The synoptophore was used to measure torsional interocular disparity. This, in turn, was used to compute how much the angle between the Listing's plane (LP) of the two eyes changes as a function of the vergence angle. The ratio of these two angles was defined as G. We measured G in normals and in patients suffering from intermittent horizontal strabismus. Consistent with previous search-coil experiments and with our previous visual test measures, the results using the synoptophore suggest that, for normals, G is less than 1. In the patient group the mean G was similar in magnitude but more variable. The variations in G did not appear to be related to the patient's measurement of ocular deviation. This result suggests that the vergence-related rotation of LP in these patients may be related to other factors besides the effort required to fuse the lines of sight.

Terson's syndrome: a reversible cause of blindness following subarachnoid hemorrhage.

Terson's syndrome refers to the occurrence of vitreous hemorrhage with subarachnoid hemorrhage (SAH), usually due to a ruptured cerebral aneurysm. Although it is a well-described entity in the ophthalmological literature, it has been only rarely commented upon in the neurosurgical discussion of SAH. Fundus findings are reported in a prospective study of 22 consecutive patients with a computerized tomography- or lumbar puncture-proven diagnosis of SAH. Six of these patients had intraocular hemorrhage on initial examination. In four patients vitreous hemorrhage was evident on presentation (six of eight eyes). In the subsequent 12 days, vitreous hemorrhage developed in the additional two patients (three of four eyes) due to breakthrough bleeding from the original subhyaloid hemorrhages. The initial amount of intraocular hemorrhage did not correlate with the severity of SAH. Two of the six patients with intraocular hemorrhage died, whereas five of the 16 remaining SAH patients without intraocular hemorrhage died. Of the four survivors with intraocular hemorrhage, three showed gradual but significant improvement in their visual acuity by 6 months. The fourth underwent vitrectomy at 8 months after presentation and had a good visual result. With modern and aggressive medical and microsurgical management, Terson's syndrome should be recognized as an important reversible cause of blindness in patients surviving SAH.

Polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) and their contaminants have definite ocular and dermatologic side effects, often directly related to the serum level of these compounds. These are usually the first symptoms and signs to appear. Other side effects are far more variable, and the carcinogenic role of PCBs and their contaminants remains controversial.

Clinical application of multiplanar computerized tomography of the orbit.

Multiplanar image reconstruction involves the reconstruction of sagittal, coronal or oblique views form a series of transverse computerized tomography images without exposing the patient to more radiation. The technique, especially when used in conjunction with high-resolution scanning, is invaluable in the diagnosis of lesions within the orbit. This paper deals with the clinical application of this new technique.

Sarcoidosis of the eyelid skin.

A 64-year-old white woman presented with nodular lesions on the arms and face, including both medial canthi and the left upper eyelid. Histopathological examination of the biopsied eyelid lesion and other facial lesions disclosed the presence of noncaseating granulomas strongly suggestive of sarcoidosis. No other evidence of ocular sarcoidosis was present. The patient had clinical, radiologic and laboratory findings consistent with a diagnosis of systemic sarcoidosis. Therapy with chloroquine phosphate led to regression of the cutaneous lesions.