Transcriptomic and cellular decoding of functional brain connectivity changes reveal regional brain vulnerability to pro- and anti-inflammatory therapies.

BACKGROUND: Systemic inflammation induces acute changes in mood, motivation and cognition that closely resemble those observed in depressed individuals. However, the mechanistic pathways linking peripheral inflammation to depression-like psychopathology via intermediate effects on brain function remain incompletely understood. METHODS: We combined data from 30 patients initiating interferon-α treatment for Hepatitis-C and 20 anti-tumour necrosis factor (TNF) therapy for inflammatory arthritis and used resting-state functional magnetic resonance imaging to investigate acute effects of each treatment on regional global brain connectivity (GBC). We leveraged transcriptomic data from the Allen Human Brain Atlas to uncover potential biological and cellular pathways underpinning regional vulnerability to GBC changes induced by each treatment. RESULTS: Interferon-α and anti-TNF therapies both produced differential small-to-medium sized decreases in regional GBC. However, these were observed within distinct brain regions and the regional patterns of GBC changes induced by each treatment did not correlate suggesting independent underlying processes. Further, the spatial distribution of these differential GBC decreases could be captured by multivariate patterns of constitutive regional expression of genes respectively related to: i) neuroinflammation and glial cells; and ii) glutamatergic neurotransmission and neurons. The extent to which each participant expressed patterns of GBC changes aligning with these patterns of transcriptomic vulnerability also correlated with both acute treatment-induced changes in interleukin-6 (IL-6) and, for Interferon-α, longer-term treatment-associated changes in depressive symptoms. CONCLUSIONS: Together, we present two transcriptomic models separately linking regional vulnerability to the acute effects of interferon-α and anti-TNF treatments on brain function to glial neuroinflammation and glutamatergic neurotransmission. These findings generate hypotheses about two potential brain mechanisms through which bidirectional changes in peripheral inflammation may contribute to the development/resolution of psychopathology.

Toward the unity of pathological and exertional fatigue: A predictive processing model.

Fatigue is a common experience in both health and disease. Yet, pathological (i.e., prolonged or chronic) and transient (i.e., exertional) fatigue symptoms are traditionally considered distinct, compounding a separation between interested research fields within the study of fatigue. Within the clinical neurosciences, nascent frameworks position pathological fatigue as a product of inference derived through hierarchical predictive processing. The metacognitive theory of dyshomeostasis (Stephan et al., 2016) states that pathological fatigue emerges from the metacognitive mechanism in which the detection of persistent mismatches between prior interoceptive predictions and ascending sensory evidence (i.e., prediction error) signals low evidence for internal generative models, which undermine an agent's feeling of mastery over the body and is thus experienced phenomenologically as fatigue. Although acute, transient subjective symptoms of exertional fatigue have also been associated with increasing interoceptive prediction error, the dynamic computations that underlie its development have not been clearly defined. Here, drawing on the metacognitive theory of dyshomeostasis, we extend this account to offer an explicit description of the development of fatigue during extended periods of (physical) exertion. Accordingly, it is proposed that a loss of certainty or confidence in control predictions in response to persistent detection of prediction error features as a common foundation for the conscious experience of both pathological and nonpathological fatigue.

Interactions between perceptions of fatigue, effort, and affect decrease knee extensor endurance performance following upper body motor activity, independent of changes in neuromuscular function.

Prior exercise has previously been shown to impair subsequent endurance performance in non-activated muscles. Declines in the neuromuscular function and altered perceptual/affective responses offer possible mechanisms through which endurance performance may be limited in these remote muscle groups. We thus conducted two experiments to better understand these performance-limiting mechanisms. In the first experiment, we examined the effect of prior handgrip exercise on the behavioral, perceptual, and affective responses to a sustained, sub-maximal contraction of the knee extensors. In the second experiment, transcranial magnetic stimulation was used to assess the neuromuscular function of the knee extensors before and after the handgrip exercise. The results of the first experiment demonstrated prior handgrip exercise increased the perceptions of effort and reduced affective valence during the subsequent knee extensor endurance exercise. Both effort and affect were associated with endurance performance. Subjective ratings of fatigue were also increased by the preceding handgrip exercise but were not directly related to knee extensor endurance performance. However, perceptions of fatigue were correlated with heightened effort perception and reduced affect during the knee extensor contraction. In the second experiment, prior handgrip exercise did not significantly alter the neuromuscular function of the knee extensors. The findings of the present study indicate that motor performance in the lower limbs following demanding exercise in the upper body appears to be regulated by complex, cognitive-emotional interactions, which may emerge independent of altered neuromuscular function. Subjective fatigue states are implicated in the control of perceptual and affective processes responsible for the regulation of endurance performance.

PET imaging shows no changes in TSPO brain density after IFN-α immune challenge in healthy human volunteers.

Depression is associated with peripheral inflammation, but its link with brain microglial activity remains unclear. In seven healthy males, we used repeated translocator protein-Positron Emission Tomography (TSPO-PET) dynamic scans with [11C]PBR28 to image brain microglial activation before and 24 h after the immune challenge interferon (IFN)-α. We also investigated the association between changes in peripheral inflammation, changes in microglial activity, and changes in mood. IFN-α administration decreased [11C]PBR28 PET tissue volume of distribution (Vt) across the brain (-20 ± 4%; t6 = 4.1, p = 0.01), but after correction for radioligand free-plasma fraction there were no longer any changes (+23 ± 31%; t = 0.1, p = 0.91). IFN-α increased serum IL-6 (1826 ± 513%, t6 = -7.5, p < 0.001), IL-7 (39 ± 12%, t6 = -3.6, p = 0.01), IL-10 (328 ± 48%, t6 = -12.8, p < 0.001), and IFN-γ (272 ± 64%, t6 = -7.0, p < 0.001) at 4-6 h, and increased serum TNF-α (49 ± 7.6%, t6 = -7.5, p < 0.001), IL-8 (39 ± 12%, t6 = -3.5, p = 0.013), and C-reactive protein (1320 ± 459%, t6 = -7.2, p < 0.001) at 24 h. IFN-α induced temporary mood changes and sickness symptoms after 4-6 h, measured as an increase in POMS-2 total mood score, confusion and fatigue, and a decrease in vigor and friendliness (all p ≤ 0.04). No association was found between changes in peripheral inflammation and changes in PET or mood measures. Our work suggests that brain TSPO-PET signal is highly dependent of inflammation-induced changes in ligand binding to plasma proteins. This limits its usefulness as a sensitive marker of neuroinflammation and consequently, data interpretation. Thus, our results can be interpreted as showing either that [11C]PBR28 is not sensitive enough under these conditions, or that there is simply no microglial activation in this model.

Blueberries as an additive to increase the survival of Lactobacillus johnsonii N6.2 to lyophilisation.

Effective cultivation methods, total cost, and biomass preservation are key factors that have a significant impact on the commercialisation and effectiveness of probiotics, such as Lactobacillus. Sugar polymers, milk and whey proteins have been suggested as good additives for industrial preparations. Alternative compounds, such as phytophenols, are a more attractive option, given their potential benefits to human health. The overall goal of this study was to determine if the addition of blueberry phytophenols improves the survival of Lactobacillus johnsonii N6.2 during the freeze-drying process. The addition of blueberry aqueous extract (BAE) stimulated the growth of L. johnsonii under aerobic conditions and improved the stationary phase survival of the bacteria. Furthermore, the addition of BAE to the culture media improved the endurance of L. johnsonii N6.2 to freeze-drying stress, as well as to storage at 4 °C for up to 21 weeks. Moreover, blueberry extract performed more effectively as a lyophilising additive compared to skim milk and microencapsulation with whey protein/sodium alginate. In sum, this study demonstrates that BAE is an effective additive to increase the growth and survival of L. johnsonii N6.2 when added to the culture medium and/or used as a lyophilising preservative. Moreover, BAE or other polyphenols sources might likely enhance growth and increase survival of more probiotic lactic acid bacterial strains.

Interferon-alpha-Induced Changes in NODDI Predispose to the Development of Fatigue.

Interferon-alpha (IFN-α) is an important mediator of antiviral immune responses. It is also used clinically in the treatment of hepatitis-C infection. Though effective, IFN-α-based therapies can often impair mood, motivation and cognition, which when severe can appear indistinguishable from major depression. In susceptible patients, fatigue and motivational impairment emerge early and have been linked to changes in basal ganglia (striatal) metabolism, neurochemistry and microstructural integrity. Here we use neurite orientation dispersion and density imaging (NODDI) modeling of multi-shell diffusion MRI to investigate whether changes in orientation-dispersion index (ODI) or neurite density index (NDI) can predict the later emergence of IFN-α-induced fatigue. Eighteen patients initiating IFN-α-based treatment for hepatitis-C underwent diffusion MRI and blood sampling at baseline and 4 h after their first IFN-α injection. They were then followed up with regular psychological assessments for 12 weeks of treatment. IFN-α injection stimulated an acute inflammatory cytokine response and evoked acute fatigue that peaked between 4 and 12 weeks of treatment. Within the brain, IFN-α induced an acute increase in NDI in patients that experienced a simultaneous increase in IFN-α-induced fatigue but not in patients that did not. Acute changes in striatal microstructure additionally predicted the continued development of fatigue but not mood symptoms 4 and 8 weeks later into treatment. Our findings highlight the value of NODDI as a potential in vivo biomarker of the central effects of peripheral inflammation. We highlight the exquisite sensitivity of the striatum to IFN-α and further implicate striatal perturbation in IFN-α-induced fatigue.

Motivation and value influences in the relative balance of goal-directed and habitual behaviours in obsessive-compulsive disorder.

Our decisions are based on parallel and competing systems of goal-directed and habitual learning, systems which can be impaired in pathological behaviours. Here we focus on the influence of motivation and compare reward and loss outcomes in subjects with obsessive-compulsive disorder (OCD) on model-based goal-directed and model-free habitual behaviours using the two-step task. We further investigate the relationship with acquisition learning using a one-step probabilistic learning task. Forty-eight OCD subjects and 96 healthy volunteers were tested on a reward and 30 OCD subjects and 53 healthy volunteers on the loss version of the two-step task. Thirty-six OCD subjects and 72 healthy volunteers were also tested on a one-step reversal task. OCD subjects compared with healthy volunteers were less goal oriented (model-based) and more habitual (model-free) to reward outcomes with a shift towards greater model-based and lower habitual choices to loss outcomes. OCD subjects also had enhanced acquisition learning to loss outcomes on the one-step task, which correlated with goal-directed learning in the two-step task. OCD subjects had greater stay behaviours or perseveration in the one-step task irrespective of outcome. Compulsion severity was correlated with habitual learning in the reward condition. Obsession severity was correlated with greater switching after loss outcomes. In healthy volunteers, we further show that greater reward magnitudes are associated with a shift towards greater goal-directed learning further emphasizing the role of outcome salience. Our results highlight an important influence of motivation on learning processes in OCD and suggest that distinct clinical strategies based on valence may be warranted.

Sensations of skin infestation linked to abnormal frontolimbic brain reactivity and differences in self-representation.

Some patients experience skin sensations of infestation and contamination that are elusive to proximate dermatological explanation. We undertook a functional magnetic resonance imaging study of the brain to demonstrate, for the first time, that central processing of infestation-relevant stimuli is altered in patients with such abnormal skin sensations. We show differences in neural activity within amygdala, insula, middle temporal lobe and frontal cortices. Patients also demonstrated altered measures of self-representation, with poorer sensitivity to internal bodily (interoceptive) signals and greater susceptibility to take on an illusion of body ownership: the rubber hand illusion. Together, these findings highlight a potential model for the maintenance of abnormal skin sensations, encompassing heightened threat processing within amygdala, increased salience of skin representations within insula and compromised prefrontal capacity for self-regulation and appraisal.

Impulsivity in disorders of food and drug misuse.

BACKGROUND: Evidence suggests some overlap between the pathological use of food and drugs, yet how impulsivity compares across these different clinical disorders remains unclear. Substance use disorders are commonly characterized by elevated impulsivity, and impulsivity subtypes may show commonalities and differences in various conditions. We hypothesized that obese subjects with binge-eating disorder (BED) and abstinent alcohol-dependent cohorts would have relatively more impulsive profiles compared to obese subjects without BED. We also predicted decision impulsivity impairment in obesity with and without BED. METHOD: Thirty obese subjects with BED, 30 without BED and 30 abstinent alcohol-dependent subjects and age- and gender-matched controls were tested on delay discounting (preference for a smaller immediate reward over a larger delayed reward), reflection impulsivity (rapid decision making prior to evidence accumulation) and motor response inhibition (action cancellation of a prepotent response). RESULTS: All three groups had greater delay discounting relative to healthy volunteers. Both obese subjects without BED and alcohol-dependent subjects had impaired motor response inhibition. Only obese subjects without BED had impaired integration of available information to optimize outcomes over later trials with a cost condition. CONCLUSIONS: Delay discounting appears to be a common core impairment across disorders of food and drug intake. Unexpectedly, obese subjects without BED showed greater impulsivity than obese subjects with BED. We highlight the dissociability and heterogeneity of impulsivity subtypes and add to the understanding of neurocognitive profiles across disorders involving food and drugs. Our results have therapeutic implications suggesting that disorder-specific patterns of impulsivity could be targeted.

Disorders of compulsivity: a common bias towards learning habits.

Why do we repeat choices that we know are bad for us? Decision making is characterized by the parallel engagement of two distinct systems, goal-directed and habitual, thought to arise from two computational learning mechanisms, model-based and model-free. The habitual system is a candidate source of pathological fixedness. Using a decision task that measures the contribution to learning of either mechanism, we show a bias towards model-free (habit) acquisition in disorders involving both natural (binge eating) and artificial (methamphetamine) rewards, and obsessive-compulsive disorder. This favoring of model-free learning may underlie the repetitive behaviors that ultimately dominate in these disorders. Further, we show that the habit formation bias is associated with lower gray matter volumes in caudate and medial orbitofrontal cortex. Our findings suggest that the dysfunction in a common neurocomputational mechanism may underlie diverse disorders involving compulsion.

Gray matter textural heterogeneity as a potential in-vivo biomarker of fine structural abnormalities in Asperger syndrome.

Brain imaging studies contribute to the neurobiological understanding of Autism Spectrum Conditions (ASC). Herein, we tested the prediction that distributed neurodevelopmental abnormalities in brain development impact on the homogeneity of brain tissue measured using texture analysis (TA; a morphological method for surface pattern characterization). TA was applied to structural magnetic resonance brain scans of 54 adult participants (24 with Asperger syndrome (AS) and 30 controls). Measures of mean gray-level intensity, entropy and uniformity were extracted from gray matter images at fine, medium and coarse textures. Comparisons between AS and controls identified higher entropy and lower uniformity across textures in the AS group. Data reduction of texture parameters revealed three orthogonal principal components. These were used as regressors-of-interest in a voxel-based morphometry analysis that explored the relationship between surface texture variations and regional gray matter volume. Across the AS but not control group, measures of entropy and uniformity were related to the volume of the caudate nuclei, whereas mean gray-level was related to the size of the cerebellar vermis. Similar to neuropathological studies, our study provides evidence for distributed abnormalities in the structural integrity of gray matter in adults with ASC, in particular within corticostriatal and corticocerebellar networks. Additionally, this in-vivo technique may be more sensitive to fine microstructural organization than other more traditional magnetic resonance approaches and serves as a future testable biomarker in AS and other neurodevelopmental disorders.

Autism attenuates sex differences in brain structure: a combined voxel-based morphometry and diffusion tensor imaging study.

BACKGROUND AND PURPOSE: It has been proposed that autism spectrums condition may represent a form of extreme male brain (EMB), a notion supported by psychometric, behavioral, and endocrine evidence. Yet, limited data are presently available evaluating this hypothesis in terms of neuroanatomy. Here, we investigated sex-related anatomic features in adults with AS, a "pure" form of autism not involving major developmental delay. MATERIALS AND METHODS: Males and females with AS and healthy controls (n = 28 and 30, respectively) were recruited. Structural MR imaging was performed to measure overall gray and white matter volume and to assess regional effects by means of VBM. DTI was used to investigate the integrity of the main white matter tracts. RESULTS: Significant interactions were found between sex and diagnosis in total white matter volume, regional gray matter volume in the right parietal operculum, and fractional anisotropy (FA) in the body of the CC, cingulum, and CR. Post hoc comparisons indicated that the typical sexual dimorphism found in controls, whereby males have larger FA and total white matter volume, was absent or attenuated in participants with AS. CONCLUSIONS: Our results point to a fundamental role of the factors that underlie sex-specific brain differentiation in the etiology of autism.

Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences.

The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

Fusarium oxysporum f. sp. palmarum, a Novel Forma Specialis Causing a Lethal Disease of Syagrus romanzoffiana and Washingtonia robusta in Florida.

A new disease of Syagrus romanzoffiana (queen palm) and Washingtonia robusta (Mexican fan palm) has spread across the southern half of Florida during the past 5 years. The initial foliar symptom is a one-sided chlorosis or necrosis of older leaf blades, with a distinct reddish-brown stripe along the petiole and rachis and an associated discoloration of internal tissue. Within 2 to 3 months after onset of symptoms, the entire canopy becomes desiccated and necrotic but the leaves do not droop or hang down around the trunk. Based on pathogenicity and morphological and molecular characterization, the etiological agent has been identified as a new forma specialis of Fusarium oxysporum, designated f. sp. palmarum. Sequence analysis of a portion of the translation elongation factor 1-α gene (EF-1α) separated 27 representative isolates into two EF-1α groups, which differed by two transition mutations. Members of both EF-1α groups are pathogenic on both species of palm. A phylogenetic analysis inferred from partial EF-1α sequences from a genetically diverse set of F. oxysporum isolates, including three other formae speciales pathogenic on palm (i.e., f. sp. albedinis, f. sp. canariensis, and f. sp. elaeidis), suggested that f. sp. palmarum and f. sp. albedinis may be more closely related to one another than either is to the two other palm pathogens.

Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

First Report of a 16SrIV Group Phytoplasma Associated with Declining Coyol Palms in Honduras.

Lethal yellowing (LY) of coconut palm (Cocos nucifera L.) caused by a subgroup 16SrIV-A phytoplasma has been present along the northern coast and adjacent Bay Islands of Honduras since 1996. In the southern municipalities of San Esteban and Guanaco, approximately 150 km from the Atlantic coast, substantial numbers of coyol palm (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) and several coconut palms growing nearby were either dead or in an advanced stage of decline during 2005. Declining palms of both species exhibited loss of fruit, withered inflorescences, and dried, discolored (grayish brown) leaves. Most or all leaves on declining palms had collapsed and hung downward around the stem. Samples (3 to 5 g) of tissue were excised from stems of 20 coyol and 2 coconut palms. DNA was extracted from each sample using a previously described protocol (2) and assayed for phytoplasma DNA using polymerase chain reaction (PCR) employing phytoplasma universal rRNA primer pair P1 (1) and P7 (4). A product of expected size (approximately 1.8 kb) was amplified from 12 of 20 diseased coyol palms, 2 of 2 diseased coconut palms and from DNA of a Florida-grown Chinese fan palm (Livistona chinensis (Jacq.) R. Br. ex Mart.) with LY symptoms included as a known positive control. Amplified P1/P7 products used in a nested PCR assay with 16SrIV-specific primer pair LY16Sf (2) and LY16Sr (5'-GCTTACGCA-GTTAGGCTGTC-3') yielded a product of approximately 1.39 kb. Neither primary nor nested PCRs generated a product from DNA of healthy coconut palm. Poor quality of tissues within stems of declining palms may have contributed to lack of amplification of any product from eight coyol samples. A reevaluation of DNA samples using PCR employing nonribosomal primer pair LYF1/LYR1, which specifically detects subgroup 16SrIV-A phytoplasmas (3), failed to amplify an expected 1-kb product from any palm sample other than the LY positive control. Digestion of nested amplification products (1.39 kb) with AluI endonuclease and electrophoresis of digests through 8% polyacrylamide gels revealed the same three-fragment restriction pattern for all phytoplasmas detected in coyol and coconut palms. The pattern differed from the five-fragment profile of LY phytoplasma rDNA included for comparative purposes. These results indicate that coyol palm is a new phytoplasma host and that decline symptoms on coyol and adjacent coconuts were not a consequence of natural dispersion of subgroup 16SrIV-A phytoplasmas from the northern coast. The 16S rDNA sequences amplified from coyol palm decline (CPD) (GenBank Accession No. DQ321818) and coconut decline (CLDO) (GenBank Accession No. DQ321819) phytoplasmas were coidentical and most similar (99.87%) to that of Yucatan coconut lethal decline (LDY), a known subgroup 16SrIV-B strain. This relationship was further supported by phylogenetic analysis of rDNA sequences. On the basis of these findings, we have tentatively concluded that strains CPD and CLDO represent new members of the coconut lethal yellows subgroup 16SrIV-B. References: (1) S. Deng and C. Hiruki, J. Microbiol. Methods 14:53, 1991 (2) N. A. Harrison et al. Plant Dis. 86:676, 2002 (3) N. A. Harrison et al. Plant Pathol. 43:998, 1994. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

First Report of "Candidatus Phytoplasma Asteris"-Related Strains Infecting Chinaberry Trees with Leaf Yellowing Symptoms in Vietnam.

Although no loss of crown shape or unusual growth were evident on two mature Chinaberry trees (Melia azedarach L.) located near the citadel in central Hué city, Vietnam, leaves on both trees displayed distinctive interveinal yellowing during September 2003. This symptom was reminiscent in appearance to foliar discoloration previously observed on mature Chinaberry trees in El Torno, Santa Cruz, Bolivia that was subsequently attributed to phytoplasma infection of these trees (2). Eight samples of yellowed leaves were collected from affected trees and preserved by pressing and drying for later analysis. Total nucleic acids were extracted from 0.5 g of each leaf sample and assayed for phytoplasma DNA using a polymerase chain reaction (PCR) assay with phytoplasma universal rRNA primer pair P1 and P7 (4). No visible product was generated from any Chinaberry sample while a product of expected size (1.8 kb) was obtained from DNA of a periwinkle plant (Catharanthus roseus (L.) G. Don) infected with "Candidatus Phytoplasma asteris"-related strain eastern aster yellows (EAY) and included as a known positive control in the assay. After P1/P7-primed products were reamplified by PCR with nested phytoplasma universal 16S rRNA primer pair R16mF2/R16mR1 (1), a 1.4-kb product of predicted size was obtained from the eight samples and EAY positive control, whereas no product was obtained from DNA of seed-grown healthy periwinkle included as a negative control. Digests of nested PCR products (1.4 kb) with HaeIII or MseI endonuclease, and electrophoresis of digests through 8% polyacrylamide gels, revealed no apparent differences in restriction fragment patterns among products from Chinaberry samples. However, HaeIII and MseI patterns differed from those obtained by digestion of nested PCR products from EAY, a known 16SrI-A subgroup phytoplasma (3), with these enzymes. Chinaberry phytoplasmas were definitively identified as group 16SrI strains after reevaluation of samples by a PCR incorporating ribosomal protein (rp) gene primer pair rpF1/rpR1 and reamplification of resulting products with nested 16SrI group-specific primer pair rp(I)F1A/rp(I)R1A (3). A 1.2-kb product of expected size was obtained from all Chinaberry samples and EAY positive control only. Restriction fragment length polymorphism patterns produced by DraI or SspI endonuclease digests of nested PCR products (1.2 kb) revealed no differences among Chinaberry samples, although patterns associated with each enzyme differed from those observed for the EAY positive control. Sequence comparison and phylogenetic analysis of Chinaberry yellows phytoplasma (CbY-V) 16Sr DNA (GenBank Accession No. AY863003) determined this strain to be most closely related (99.65%) to Epilobium phyllody phytoplasma, a 16SrI-B subgroup strain (3). However, based on analysis of rp gene sequences (GenBank Accession No. DQ321823), strain CbY-V was judged most similar (99.59%) to cabbage proliferation, a well characterized 16SrI-B subgroup, rpI-B subgroup phytoplasma (3). To our knowledge, this is the first record of phytoplasma infection of Chinaberry, a common urban shade tree in Vietnam. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004 (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Late recurrences of Sydenham's chorea are not associated with anti-basal ganglia antibodies.

Anti-basal ganglia antibodies (ABGA) have been associated with 100% of acute cases and 69% of persistent cases of Sydenham's chorea. We describe two cases of late recurrences of Sydenham's chorea with absence of ABGA. Both patients had several childhood episodes of Sydenham's chorea. MRI imaging of the basal ganglia and exhaustive investigations for other causes of chorea were normal or negative. The absence of ABGA may be evidence against an autoimmune pathology in late and some persistent recurrences. We suggest the likely pathophysiology to be dopamine hypersensitivity of chronically damaged basal ganglia neurones possibly following induction of an autoimmune antibody response in childhood.

Atypical herpes type 2 encephalitis associated with normal MRI imaging.

We describe a case of chronic atypical herpes simplex type 2 encephalitis in an immunocompromised 68 year old man presenting with headache and cognitive changes without focal neurological or MRI findings. To our knowledge this is the first described case of herpes simplex encephalitis associated with normal MRI brain imaging and non-focal neurological examination. This further expands the range of clinical presentations that may be associated with herpes simplex encephalitis and emphasises the value of PCR for herpes simplex virus in the investigation of encephalitis regardless of imaging findings.

Detection and Characterization of a Lethal Yellowing (16SrIV) Group Phytoplasma in Canary Island Date Palms Affected by Lethal Decline in Texas.

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in Canary Island date (Phoenix canariensis) palms displaying symptoms similar to lethal yellowing (LY) disease in Corpus Christi, TX. An rDNA product (1.8 kb) was amplified consistently from 10 of 11 palms by PCR employing phytoplasma universal rRNA primer pair P1/P7. Also, AluI endonu-clease digests and sequencing of P1/P7 products revealed that nontarget Bacillus megaterium-related rDNA sequences of similar size were co-amplified along with phytoplasma rDNA from 10 palms. A 1,402-bp product was obtained from all 11 symptomatic palms when initial P1/P7 products were reamplified by PCR employing nested LY phytoplasma group-specific 16S rRNA primer pair LY16Sf/LY16Sr. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products revealed that palm-infecting phytoplasmas were uniform and most similar to strains composing the coconut lethal yellowing phytoplasma (16SrIV) group. Sequence analysis of 16S rDNA determined the Texas Phoenix palm decline (TPD) phytoplasma to be phylogenetically closest to the Carludovica palmata leaf yellowing (CPY) phytoplasma. rDNA profiles of strains TPD and CPY obtained with AluI were co-identical and distinct from other known 16SrIV group phytoplasmas. On this basis, both strains were classified as members of a new subgroup, 16SrIV-D.