Quantifying the accretion of hyperphosphorylated tau in the locus coeruleus and dorsal raphe nucleus: the pathological building blocks of early Alzheimer's disease.

AIMS: Hyperphosphorylated tau neuronal cytoplasmic inclusions (ht-NCI) are the best protein correlate of clinical decline in Alzheimer's disease (AD). Qualitative evidence identifies ht-NCI accumulating in the isodendritic core before the entorhinal cortex. Here, we used unbiased stereology to quantify ht-NCI burden in the locus coeruleus (LC) and dorsal raphe nucleus (DRN), aiming to characterize the impact of AD pathology in these nuclei with a focus on early stages. METHODS: We utilized unbiased stereology in a sample of 48 well-characterized subjects enriched for controls and early AD stages. ht-NCI counts were estimated in 60-µm-thick sections immunostained for p-tau throughout LC and DRN. Data were integrated with unbiased estimates of LC and DRN neuronal population for a subset of cases. RESULTS: In Braak stage 0, 7.9% and 2.6% of neurons in LC and DRN, respectively, harbour ht-NCIs. Although the number of ht-NCI+ neurons significantly increased by about 1.9× between Braak stages 0 to I in LC (P = 0.02), we failed to detect any significant difference between Braak stage I and II. Also, the number of ht-NCI+ neurons remained stable in DRN between all stages 0 and II. Finally, the differential susceptibility to tau inclusions among nuclear subdivisions was more notable in LC than in DRN. CONCLUSIONS: LC and DRN neurons exhibited ht-NCI during AD precortical stages. The ht-NCI increases along AD progression on both nuclei, but quantitative changes in LC precede DRN changes.

Lack of Control of Citrus Canker by Induced Systemic Resistance Compounds.

Induced systemic resistance compounds (ISRs), acibenzolar-S-methyl (Actigard), and harpin protein (Messenger) were assayed in the greenhouse against Xanthomonas axonopodis pv. citrumelo, the cause of citrus bacterial spot (CBS), and X. axonopodis pv. citri, the cause of Asiatic citrus canker. Actigard and Messenger applied as foliar sprays 3 to 7 days before inoculation reduced numbers of lesions when either bacterium at 103 or 104 CFU/ml was injection-infiltrated into Swingle citrumelo leaves. Based on this activity, the ISRs were evaluated in southern Brazil in orchards of sweet oranges with low to moderate canker disease incidence in spray programs with and without copper oxychloride (COC) and copper hydroxide (CuOH). Actigard and Messenger were applied full season or in the first two or three sprays of a six-spray program in an attempt to reduce early canker disease on foliage and thereby reduce subsequent fruit infection and premature drop. Sprays of COC and CuOH were moderately to highly effective in reducing canker disease incidence and preventing premature fruit drop. Actigard or Messenger in combination with COC and CuOH, respectively, did not significantly reduce citrus canker incidence on foliage or fruit drop compared with Cu alone. The lack of additional control with ISRs means they cannot be recommended at this time to augment Cu programs for management of citrus canker.

Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and 16S-23S intergenic spacer and rep-PCR fingerprinting.

The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.

First Report of Xylella fastidiosa on Catharanthus roseus in Brazil.

In 1998, plants of periwinkle (Catharanthus roseus L.) showing small leaves, short internodes, and dieback symptoms were observed in a garden at the Instituto Agronomico do Parana (IAPAR), Londrina, PR, Brazil. Stems of these plants were cut into short sections and the sap extracted from the tissue by squeezing with pliers. The sap was blotted onto a glass slide and examined for the presence of bacteria by light microscopy (×400). Microscopy observations revealed the presence of a large number of slender, rod-shaped bacterial cells. The bacteria present in the stems of periwinkle were isolated on buffered cysteine-yeast extract (BCYE) and periwinkle wilt (PW) agar media. Stems were disinfected in 70% alcohol and cut into short sections, and the sap extracted as described above. The sap was blotted directly onto the media and the plates were incubated at 28°C. Typical colonies of Xylella fastidiosa were observed 10 days after isolation on both media. Indirect immunofluorescence tests with antibody specific to X. fastidiosa and anti-IgG conjugated with tetrametylrhodamine isothiocyanate (TRITC) were carried out with xylem sap of periwinkle stem and the isolated bacteria. In both cases, immunofluorescence tests were positive for X. fastidiosa. These results confirm that periwinkle plants were infected with X. fastidiosa. This is the first report of the association of X. fastidiosa with periwinkle plants in Brazil. However, the symptoms observed for the X. fastidiosa-infected periwinkle plants differed from those described previously in the U.S. (1): those symptoms consisted of marginal chlorosis and occasional vein clearing of leaves and wilting of the plants. Reference: (1) R. E. McCoy et al. Plant Dis. Rep. 62:1022, 1978.

Differential expression of cell adhesion molecules in the functional compartments of lymph nodes and tonsils.

Aims-To analyse the topographical distribution of adhesion molecules involved in lymphocyte recirculation in human lymph nodes and tonsils. The study focused on the expression of LECAM-1 (CD62L), VLA-alpha4 (CD49d), VLA-beta1 (CD29), LFA-1 alphaL (CD11a), LFA-beta2 (CD18), VCAM-1 (CD106), ICAM-1 (CD54), and H-CAM (CD44).Methods-Reactive lymph nodes and palatine tonsils were studied using immunofluorescence methods with fluorescein isothiocyanate (FITC) labelled monoclonal antibodies directed against cell adhesion molecules. To investigate the expression patterns of these molecules in the T and B cell populations, double labelling experiments were performed using Texas Red labelled antibodies against CD2 or CD19, respectively. The images from each fluorochrome were then simultaneously analysed using a laser scanning confocal microscope.Results-LECAM-1, VLA-alpha4 and H-CAM were predominantly expressed by mantle zone B cells, VCAM-1 and ICAM-1 by germinal centre cells, most of which exhibited a reticular staining pattern suggestive of follicular dendritic cells, whereas LFA-1 alphaL and LFA-beta2 were mainly found in extrafollicular and germinal centre T cells. All high endothelial venules expressed VLA-beta1 and ICAM-1, whereas VCAM-1 was present in only a few, with variable intensity.Conclusions-The data show that all of these adhesion molecules are differentially distributed within the distinct functional microenvironments of both organs. The differences observed in the expression patterns among the B and T cells belonging to different compartments probably depend on the momentum of cell traffic, the stage of maturation/activation, as well as on their functional role in the immune response.

Genetic analysis of hrp-related DNA sequences of Xanthomonas campestris strains causing diseases of citrus.

The hrp gene cluster of strains of Xanthomonas campestris that cause diseases of citrus was examined by Southern hybridization of genomic DNA and by restriction endonuclease analysis of enzymatically amplified DNA fragments of the hrp gene cluster. The hrp genes were present in all strains of the pathovars of X. campestris tested in this study, including strains of the three aggressiveness groups of the citrus bacterial spot pathogen, X. campestris pv. citrumelo. X. campestris pv. citri strains in groups A, B, and C, which cause citrus canker A, B, and C, respectively, each produced characteristic restriction banding patterns of amplified hrp fragments. The restriction banding patterns of all strains within each group were identical. In contrast, restriction fragment length polymorphism was evident among strains of the moderately and weakly aggressive groups of X. campestris pv. citrumelo. X. campestris pv. citrumelo strains in the highly aggressive group had a homogeneous restriction banding pattern. The characteristic banding patterns obtained for each bacterial group indicate that X. campestris strains causing disease in citrus can be reliably differentiated and identified by restriction analysis of amplified DNA fragments of the hrp gene cluster. In addition, the phylogenetic analysis based on the restriction banding patterns of amplified fragments suggests a polyphyletic relationship of the hrp genes among the strains of X. campestris that cause disease in citrus.

Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria.

Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv. vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria. DNA sequences related to the hrp genes were successfully amplified from X. fragariae and from 28 pathovars of X. campestris. No DNA amplification occurred with genomic DNA from phytopathogenic strains of X. campestris pv. secalis, X. campestris pv. translucens, and X. albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella. The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis. DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads. However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains. The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads. We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads.

Randomized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers (Bothrops jararaca) in São Paulo, Brazil.

In São Paulo City, Brazil, 121 patients with moderately severe envenoming by Bothrops snakes (principally B. jararaca) were randomized for treatment with Brazilian polyspecific Bothrops antivenoms: Instituto Butantan (39 patients), Instituto Vital Brazil (41), Fundação Ezequiel Dias (FUNED) (41). The initial dose was four ampoules (40 ml) in 89 patients with less severe envenoming and eight ampoules (80 ml) in 32 patients with more severe envenoming. A second dose of four ampoules was required in 20 patients. Patients receiving the three antivenoms were comparable in all respects before treatment. There were no deaths. The majority showed rapid clinical improvement, resolution of local envenoming, cessation of bleeding and restoration of blood coagulability. No differences in the efficacy of the three antivenoms were revealed by clinical or laboratory observations, including measures of haematological, haemostatic and biochemical abnormalities. Twelve patients developed abscesses (Butantan 1, Vital Brazil 6, FUNED 5) and seven developed local necrosis (3,1,3). Of 88 patients followed up 20-30 days after the bite 33 (37.5%) still had symptoms or signs of local envenoming, especially swelling. Early (anaphylactic) reactions were unexpectedly frequent after all three antivenoms but were significantly more frequent with Butantan (87%) than with Vital Brazil (37%) or FUNED (56%) antivenoms (p < 0.001). A possible explanation was the higher total protein content and percentage immunoglobulin of Butantan antivenom. The doses of antivenom recommended in Brazil and used in this study may be unnecessarily high, resulting in an unacceptably high incidence of reactions. Results of the study should prompt a critical re-evaluation of antivenom production techniques and dosage recommendations in Brazil.

Comparison of the potency of three Brazilian Bothrops antivenoms using in vivo rodent and in vitro assays. BIASG (Butantan Institute Antivenom Study Group).

Three Brazilian polyspecific Bothrops antivenoms were compared using standard W.H.O. rodent in vivo and in vitro assays of their ability to neutralize the principal venom activities of pooled whole Bothrops jararaca venom. On a volume basis, the antivenoms were equally effective in neutralizing lethal activity in mice, and there were only minor differences in their ability to neutralize venom-induced haemorrhage, necrosis and procoagulant activity. Antivenom efficacy in neutralizing defibrinogenation varied. However, when equal amounts of antivenom IgG were compared, it was found that the FUNED antivenom best neutralized lethality, haemorrhage, necrosis and fibrinogen clotting activity. Vital Brazil and FUNED antivenoms were equally effective in neutralizing plasma coagulant activity but Vital Brazil antivenom was the more effective in neutralizing defibrinogenation.

[Lymnic molluscs from an Alto Araguaia area (Brazil)]. / Moluscos límnicos de uma área do Alto Araguaia (Brasil).

An analysis was carried out to determine the natural population of freshwater molluscs from 5 municipalities within the area of influence of the Couto Magalhães hydroelectric project. These municipalities cover a large area of the State of Goiás and Mato Grosso do Sul (Brazil). In this study 624 molluscs of the following species were examined: Drepanotrema anatinum, D. lucidum, D. depressissimum, Biomphalaria straminea, D. schrammi, Physa marmorata, Lymnaea columella, Pomacea and Eupera. One to their importance in public health, the discovery of Biomphalaria straminea and Lymnaea columella, intermediate hosts, respectively, of Shistosoma mansoni and Fasciola hepatica, deserves special mention.

Features of Turner's and DiGeorge's syndromes in a child with an X;22 translocation.

We describe the clinical and cytogenetic findings in an infant who presented with the features of both Turner's and DiGeorge's syndromes associated with a unique translocation between chromosomes X and 22.