Influence of nanostructured calcium aluminate and calcium silicate on the liver: histological and unbiased stereological analysis.

AIM: To examine the potential systemic toxicity of nanostructured materials based on calcium silicate and calcium aluminate, for potential application in Dentistry. METHODOLOGY: Twenty-four Albino Wistar rats aged 2 months were used as an in vivo animal model for subcutaneous implantation of the investigated materials, placed in polyethylene tubes. Thirty days after implantation, the livers of the rats were analysed and following histological and stereological parameters were evaluated for volume density of hepatocytes and blood sinusoids, number and numerical density of hepatocytes, surface of hepatocytes and their nucleuses, nucleocytoplasmic ratio and mitotic index of hepatocytes. Stereological measurements were achieved using Cavalieri's principle, with grid P2 and unbiased analysis. Additionally, immunohistochemistry studies were performed to further analyse changes in liver tissue. Several haematological and biochemical parameters of blood of experimental animals were also analysed, as well as local tissue reactions around the implants. Statistical analysis was performed using parametric (anova and t-test) and nonparametric tests (Kruskal-Wallis and Mann-Whitney U-test) depending on data distribution. RESULTS: Implanted dental cements led to an increase in stereological and histological parameters in liver tissue compared to control rats. Although the investigated parameters mostly showed significant differences between control and experimental animals, the liver tissue of the experimental animals did not have visible signs of pathological changes. This was supported by the analysis of blood parameters which were not significantly different between control and experimental animals. Also, the subcutaneous tissues had minimal inflammatory reactions. Immunohistochemistry studies revealed that nanostructured materials induced proliferation of hepatocytes, but that the immunological response to the materials was not strong enough to induce proliferation of immunoreactive cells in liver in the observed time period. CONCLUSIONS: This study was performed as a contribution to the attestation of the biocompatibility of dental cements based on calcium silicate and calcium aluminate. Although these materials induced several changes in the liver structure, they were not clinically relevant and represent a normal and reversible response of the liver to the presence of biocompatible materials in the body. Blood and immunohistochemistry analyses and local tissue reactions further confirmed that these materials possess good biocompatible potential.

Spinal epidural abscess following treatment with rituximab.

OBJECTIVE: To describe spinal epidural abscess after treatment with rituximab. METHODS: Report of a new case of cervical epidural spinal abscess after treatment with rituximab and review of the literature. RESULTS: Biologic agents are associated with an increased risk of serious infections, there are only few reports of spinal epidural abscesses following immunomodulatory treatment with biologic agents, including only one other case of an epidural abscess after the use of rituximab. The risk may be greater with the use of other immunosuppressive medications, or with combined immunotherapy. CONCLUSION: Spinal epidural abscess is a rare complication with potential severe morbidity, and continued vigilance is needed as timely intervention may prevent severe morbidity and potentially fatal outcome.

Intravenous immunoglobulin in the treatment of neurologic disorders.

Intravenous immunoglobulins (IVIGs) are often used in the treatment of autoimmune disorders and immunodeficiencies, and it has been estimated that neurologic indications can account for up to 43% of IVIG used in clinical practice. In neurologic clinical practice, IVIG is used for acute therapy of newly diagnosed autoimmune disorders or exacerbations of pre-existing conditions, or as long-term maintenance treatment for chronic disorders. IVIG exerts its effects on humoral and cell-based immunity through multiple pathways, without a single dominant mechanism. Clinical use of IVIG has been supported by guidelines from American Academy of Neurology and European Federation of Neurologic Societies. IVIG is generally recommended for the treatment of Guillain-Barre syndrome and chronic inflammatory demyelinating polyneuropathy in adults, multifocal motor neuropathy and myasthenia gravis, and should be considered as a treatment option for dermatomyositis in adults and Lambert-Eaton myasthenic syndrome. Additional potential indications include stiff person syndrome, multiple sclerosis during pregnancy or while breastfeeding, refractory autoimmune epilepsy, and paraneoplastic disorders. Clinical use of IVIG is mostly safe but few adverse effects may still occur with potentially severe complications, including aseptic meningitis and thromboembolism. In addition to intravenous route (IVIG), subcutaneous immunoglobulins have been used as an alternative treatment option, especially in patients with limited intravenous access. Treatment with IVIG is effective in various autoimmune diseases, but its broader use is constrained by limited supply. This review evaluates the use of immunoglobulins in treatment of neurologic diseases.

Control of human and plant fungal pathogens using pentaene macrolide 32, 33-didehydroroflamycoin.

AIMS: The aim of this study was to address the toxicity of recently described polyene macrolide 32, 33-didehydroroflamycoin (DDHR) on a wide range of fungal pathogens and its potential to control plant fungal diseases. METHODS AND RESULTS: The antifungal activity of DDHR in vitro was examined against common human and plant pathogenic fungi using a broth microdilution assay and a disk diffusion assay. Minimum inhibitory concentrations ranged from 12·5 to 35 µg ml(-1) . A radial growth inhibition assay showed that DDHR inhibited mycelia growth, inducing mycelial necrosis and affecting sporulation. During the in vivo assay on apple fruits administration of DDHR 1 h before fungal inoculation inhibited spreading of the infection. Importantly, DDHR exhibited no phytotoxic effects on the model plant, Capsicum annum, verified by the plant growth rate and chlorophyll content. CONCLUSIONS: DDHR inhibits growth of various plant pathogens in vitro with the strongest activity against Alternaria alternata, Colletotrichum acutatum and Penicillium expansum, and protects apple fruits from decay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the inhibitory effect of DDHR on important pathogenic fungal isolates. DDHR could be a good scaffold for developing new antifungal agents for fruit and vegetable protection.

Biocompatibility of new nanostructural materials based on active silicate systems and hydroxyapatite: in vitro and in vivo study.

AIM: To evaluate in vitro cytotoxicity and in vivo inflammatory response to new nanostructural materials based on active calcium silicate systems (CS) and hydroxyapatite (HA-CS). METHODOLOGY: Cytotoxicity of eluates of new nanostructural noncommercial materials CS and HA-CS, and MTA (White MTA, Angelus(®) Soluções Odontológicas, Londrina, Brazil) as a control, were tested using the MTT assay on MRC-5 cells. Eluates of set materials were tested in 100% and 50% concentrations, 24 h, 7 days and 21 days post-elution. The pH values were determined for undiluted eluates of set materials. Polyethylene tubes containing the test materials (CS, HA-CS, MTA) were implanted in subcutaneous tissue of Wistar rats. Histopathological examinations were conducted at 7, 15, 30 and 60 days after the implantation. Data were statistically analyzed using three-way and one-way anova Tukey's post hoc test as well as Kruskall-Wallis test with Dunn's post hoc test at α = 0.05. RESULTS: All materials significantly reduced cell viability; especially when undiluted eluates were used (P < 0.001). After 24 h elution, cell viability was 10 ± 1.8%, 49.5 ± 4.2% and 61 ± 7.4%, for MTA, and HA-CS, respectively. However, CS and HA-CS were significantly less toxic than the control material MTA (P < 0.05). Cytotoxicity could be at least partially attributed to pH kinetics over time. Dilution of eluates of all tested materials resulted in better cell survival. Histopathological examination indicated similar inflammatory reaction, vascular congestion and connective tissue integrity associated with CS, HA-CS and MTA at each observation period (P > 0.05). The only significant difference was found for capsule thickness, that is thicker capsule was associated with HA-CS compared to MTA at 60 days (P = 0.0039). HA-CS induced moderately thick capsules (median score 3, score range 2-3), whereas MTA resulted in thin capsule formation (median score 2, score range 1-3). CONCLUSIONS: Evaluation of cytotoxicity and inflammatory response indicated better biocompatibility of CS and HA-CS, in comparison with MTA (White MTA, Angelus(®) Soluções Odontológicas, Londrina, Brazil).

First Report of Anthracnose on Alfalfa Caused by Colletotrichum linicola in Serbia.

Alfalfa (Medicago sativa L.) is economically the most important forage crop in Serbia. In July 2009, alfalfa plants showed symptoms characteristic of anthracnose disease ("shepherd's crook") including wilting and death of the upper portion of the stems. Anthracnose of alfalfa has been reported to be caused by Colletotrichum trifolii or C. destructivum (2). Alfalfa plants with anthracnose symptoms were collected in Srpska Crnja, South Banat District, Serbia. Infected tissue samples were surface disinfected with 5% sodium hypochlorite for 2 min and washed three times for 5 min in sterile distilled water. Surface sterilized tissue was transferred to sterile filter paper and placed on potato dextrose agar (PDA), and incubated at 24°C in the dark for 10 days (1). Developing colonies were light to dark olive green. In cultures on PDA medium, acervuli were formed. Conidia from acervuli were released in mucous masses that were orange to cream-pink in color. Conidia were hyaline, aseptate, straight with one end pointed and the other slightly rounded, measuring 12.5 to 25.0 × 2.5 to 7.5 µm (mean 19.83 × 4.42 µm). After 5 days, numerous setae were formed. The setae were slightly darker at the bottom and lighter at the top, septate with 3 septa. Setae dimensions were 100 to 185.5 × 2.5 to 5 µm (average 160.9 × 3.12 µm). The isolated fungus was designated Coll-44. Stems of 30 7-week-old plants were spray-inoculated in the laboratory with an aqueous suspension of conidia (106 spores per ml; 10 ml per plant) harvested from 7-day-old cultures grown on PDA. The plants and two non-inoculated check plants were placed in a greenhouse and a covered with plastic bags at 25°C in darkness. After 48 h, plastic bags were removed from the all plants. All plants were watered once a day. Symptoms were observed 10 days after inoculation. No symptoms were observed on non-inoculated plants. In the greenhouse, all 30 inoculated plants became diseased with anthracnose symptoms after 10 days. Coll-44 was consistently re-isolated from diseased stem tissue. Koch's postulates were fulfilled by re-isolation from inoculated alfalfa plants. Pure culture of the Coll-44 isolate was deposited in the public collection of CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (specimen no. CBS 3263). Partial sequences of the internal transcribed spacer regions-ITS (GenBank Accession No. JX908364) and betatubulin-TUB2 gene (KJ556347) were amplified and sequenced from extracted fungal DNA with primer pairs ITS1-ITS4 (4) and T1-Bt2b (3), respectively. ITS sequence of the Coll-44 isolate showed 100% nucleotide identity to the GenBank accessions JQ005765 and AB046609 of C. linicola. TUB2 sequence of isolate Coll-44 showed 99.6% nucleotide identity with the GenBank accession JQ005849 of C. linicola isolate CBS 172.51. To our knowledge, this is the first report of C. linicola causing alfalfa anthracnose in Serbia. References: (1) A. P. Baxter et al. S. Afr. J. Bot. 2:259, 1983. (2) K. D. Hyde et al. Fungal Divers. 39:1, 2009. (3) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Colletotrichum clavatum Causing Quince Anthracnose in Serbia.

Quince (Cydonia oblonga Mill.) tree is traditionally grown in Serbia. The fruits are used for compote, marmalade, and brandy production. In December 2012, quince fruits cv. Leskovacka with symptoms of postharvest anthracnose were collected in a storage facility in the area of Sabac, western Serbia. The symptoms were observed on fruits approximately 2 months after harvest. The incidence of the disease was about 3%, but the symptoms were severe. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. Small pieces (3 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphology characteristics of three isolates were observed after growth on PDA for 7 days at 25°C under a 12-h photoperiod. Fungal colonies developed white to gray dense aerial mycelium with orange conidial masses in the center of the colony. Conidia were hyaline, aseptate, clavate with rounded distal apices, 15.2 (12.8 to 16.8) × 4.5 (4.0 to 5.2) µm (mean L/W ratio = 3.3, n = 100). Morphological characteristics are consistent with the description of Colletotrichum clavatum (2). Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1/IT4 primers and ß-tubuline 2 gene using T1/T2 primers. The nucleotide sequences were deposited in GenBank (ITS Accession Nos. KF908866, KF908867, and KF908868; ß-tubuline 2 gene KF908869, KF908870, and KF908871). BLAST analyses of ITS and ß-tubuline 2 gene sequences showed that isolates from quince were 100% identical to other C. clavatum in GenBank (ITS JN121126, JN121130, JN121132, and JN121180; ß-tubuline 2 gene JN121213 to 17, JN121219, JN121228, JN121261 to 62, and JN121266 to 69), thus confirming the morphological identification. To fulfill Koch's postulates, asymptomatic fruits of quince cv. Leskovacka (five fruits per isolate) were surface sterilized with 70% ethanol, wounded with a sterile needle, and inoculated with 50 µl of a spore suspension (1 × 106 conidia/ml). Five control fruits were inoculated with 50 µl of sterile distilled water. The experiment was repeated twice. After 10 days of incubation in plastic containers, under high humidity (>90% RH) at 25°C, typical anthracnose symptoms developed on inoculated fruits, while control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as original isolates. C. clavatum previously indicated as group B (3), or genetic group A4 within the C. acutatum sensu lato complex (4), is responsible for olive anthracnose in some Mediterranean countries (1,2), and has been reported as causal agent of anthracnose on a wide range of other hosts including woody and herbaceous plants, ornamentals, and fruit trees worldwide (4). To our knowledge, this is the first report of C. clavatum in Serbia, and the first report of quince anthracnose caused by this pathogen in Europe. Anthracnose caused by C. clavatum can endanger the production and storage of quince in the future, and may require investigation of new disease management practices to control this fungus. References: (1) S. O. Cacciola et al. J. Plant Pathol. 94:29, 2012. (2) R. Faedda et al. Phytopathol. Mediterr. 50:283, 2011. (3) R. Lardner et al. Mycol. Res. 103:275, 1999. (4) S. Sreenivasaprasad and P. Talhinhas. Mol. Plant Pathol. 6:361, 2005.

IGFBP-2 nuclear translocation is mediated by a functional NLS sequence and is essential for its pro-tumorigenic actions in cancer cells.

IGFBP-2 is highly expressed in both the serum and tumor tissues of most cancers, and is considered one of the most significant genes in the signature of major cancers. IGFBP-2 mainly modulates IGF actions in the pericellular space; however, there is considerable evidence to suggest that IGFBP-2 may also act independently of the IGFs. These IGF-independent actions of IGFBP-2 are exerted either via interactions at the cell surface or intracellularly, via interaction with cytoplasmic or nuclear-binding partners. The precise mechanism underlying the intracellular/intranuclear localization of IGFBP-2 remains unclear. In this study, we investigated IGFBP-2 nuclear localization in several common cancer cells with the aim of dissecting the mechanism of its nuclear trafficking. IGFBP-2 is detected in the nuclei of common cancer cells, including breast, prostate and several neuroblastoma cell lines, using cell fractionation and confocal microscopy. Via nuclear import assays, we show that nuclear entry of IGFBP-2 is mediated by the classical nuclear import mechanisms, primarily through importin-α, as demonstrated by the use of blocking, competition and co-immunoprecipitation assays. Bioinformatics analysis of the IGFBP-2 protein sequence with PSORT II identified a classical nuclear localization signal (cNLS) sequence at 179PKKLRPP185, within the IGFBP-2 linker domain, mutagenesis of which abolishes IGFBP-2 nuclear import. Accordingly, the NLSmutIGFBP-2 fails to activate the VEGF promoter, which would otherwise occur in the presence of wild-type IGFBP-2. As a consequence, no activation of angiogenic processes were observed in NLSmutIGFBP-2 expressing SHEP cells when implanted onto our in vivo quail chorio-allantoic membrane model. Taken together, these data show for the first time that IGFBP-2 possesses a functional NLS sequence and that IGFBP-2 actively translocates into the nucleus by a classical nuclear import mechanism, involving formation of IGFBP-2 complexes with importin-α. Nuclear IGFBP-2 is required for the activation of VEGF expression and consequent angiogenesis.

Optimization of the monolayer growth in adsorption-desorption processes.

Kinetics of the deposition process of dimers in the presence of desorption is studied by Monte Carlo method on a one-dimensional lattice. The aim of this work is to investigate how do various temporal dependencies of the desorption rate hasten or slow down the deposition process. The growth of the coverage θ(t) above the jamming limit to its steady-state value θ(∞) is analyzed when the desorption probability P(des) decreases both stepwise and linearly (continuously) over a certain time domain. We report a numerical evidence that the time needed for a system to reach the given coverage θ can be significantly reduced if P(des) decreases in time. Finally, a self-consistent optimization procedure, when the probability P(des) depends on the current coverage density θ(t), is formulated and tested. The present model reproduces qualitatively the densification kinetics and the memory effects of vibrated granular materials. Our results suggest that the process of vibratory compaction of granular materials can be optimized by using a time dependent intensity of external excitations.

First Report of Brenneria nigrifluens as the Causal Agent of Shallow-Bark Canker on Walnut Trees (Juglans regia) in Serbia.

In late summer 2011, shallow, irregular cankers were observed on trunks and branches of non-chemically-treated walnut trees (Juglans regia L.) on a 30-year-old orchard in the region of Fruska Gora (Vojvodina, Serbia). Disease incidence was ~80% and yield loss was ~50%. For pathogen isolation, small pieces (~5 mm diameter) of wood tissue collected at the edge of the cankers were macerated in sterile distilled water and streaked onto nutrient agar with 5% sucrose. Plates were then incubated at 28°C for 2 days. The prevalent bacterial colonies and those similar in appearance to Brenneria nigrifluens (Wilson et al.) Hauben et al. were purified on nutrient agar (NA). Eight gram-negative, oxidasenegative, catalase-positive strains, showing oxidative and fermentative metabolism, were selected for further characterization. To identify the bacteria on a molecular basis, we analyzed the 16S rDNA and gyr B gene sequences. The 16S rDNA partial sequences of analyzed strains were amplified using the primers P0 (5'-GAGAGTTTGATCCTGGCTCAG-3') and P6 (5'-CTACGGCTACCTTGTTACGA-3') (3). Additionally, the gyr B gene sequences were generated with primers GyrB-F (5'-MGGCGGYAAGTTCGATGACAAYTC-3') and GyrB-R (5'-TRATBKCAGTCARACCTTCRCGSGC-3') (2). All amplicons were purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions and sequenced by Macrogen Inc. (Seoul, South Korea) using the same primers used for amplification. The sequences were edited using FinchTV v.1.4.0, assembled using the Clustal W program integrated into MEGA5 software (4), and deposited in NCBI GenBank under accessions JX484738 to 40 for the 16S rDNA gene and KC571240 to 47 for the gyr B gene. The 1,359-bp 16S rDNA sequences obtained for the eight strains were compared to the reference 16S rDNA sequences retrieved from GenBank. BLAST analysis revealed 100% homology of Serbian strains with sequences of B. nigrifluens (Z96095 and FJ611884). The gyr B gene sequences of our strains were 100% homologous to the sequences of B. nigrifluens deposited in GenBank (JF311612 to 15). Pathogenicity of all strains was confirmed on young fruits by infiltration of bacterial suspensions (108 CFU ml-1 from a 48 h NA culture) with syringe into the mesocarp of walnut fruits and by stem infiltration with syringes without needles into branch wounds (1). Inoculated fruits were incubated in plastic boxes for 8 days at 20°C, 80 to 100% RH, with a 12-h photoperiod. Inoculated plants were maintained for 3 months at 22 to 28°C with continuous light and at 70 to 80% RH in plastic tunnels. Inoculated fruits developed bark canker symptoms at the inoculation sites, which became necrotic and released a reddish brown exudate. Necrotic lesions were observed on inoculated branches. B. nigrifluens was reisolated from the margins of necrotic fruit and stem tissue. Physiological and biochemical tests showed that strains grew at 36°C and did not produce arginine dihydrolase, H2S, indole, nitrate, nor a fluorescent pigment on King's B medium. They did not induce a hypersensitive reaction on tobacco leaves and did not hydrolyse gelatin and starch. They produced acid without gas from glucose, inositol, sorbitol, arabinose, and sucrose, but not from maltose and lactose (1). Results of pathogenicity and biochemical tests were also the same for reisolated strains. This is the first report of B. nigrifluens as the causal agent of shallow-bark canker on walnut trees in Serbia. References: (1) E. G. Biosca and M. M. López. J. Plant Pathol. 94:105, 2012. (2) P. Ferrente and M. Scotrichini. Plant Pathol. 59:954, 2010. (3) A. Grifoni et al. FEMS Microbiol. Lett. 127:85, 1995. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

New nanostructural biomaterials based on active silicate systems and hydroxyapatite: characterization and genotoxicity in human peripheral blood lymphocytes.

AIM: To characterize and investigate the genotoxic effect of a new endodontic cement based on dicalcium- and tricalcium-silicate (CS) with hydroxyapatite (HA) on human lymphocytes. METHODOLOGY: Hydrothermal treatment was applied for synthesis of CS and HA. The final mixture HA-CS, with potential to be used in endodontic practice, is composed of CS (34%) and HA (66%). Human lymphocytes were incubated with HA, HA-CS and CS for 1 h, at 37 °C and 5% CO2. Cell viability was determined using the trypan blue exclusion assay. To evaluate the level of DNA damage comet assay (single cell gel electrophoresis) was performed. For the statistical analysis anova and Duncan's Post Hoc Test were used. RESULTS: The SEM analysis indicated that CS consisted mostly of agglomerates of several micrometers in size, built up from smaller particles, with dimensions between 117 and 477 nm. This is promising because dimensions of agglomerates are not comparable with channels inside the cell membranes, whereas their nano-elements provide evident activity, important for faster setting of these mixtures compared to MTA. Values of DNA damage obtained in the comet assay indicated low genotoxic risk of the new endodontic materials. CONCLUSIONS: The significantly improved setting characteristics and low genotoxic risk of the new material support further research.

First Report of the Cereal Cyst Nematode Heterodera filipjevi on Wheat in Serbia.

The most globally recognized and economically important nematode on wheat is the cereal cyst nematode (CCN) complex (1). One of the most important species of this group is Heterodera filipjevi (Madzidov, 1981) Mulvey and Golden, 1983. During regular soil quarantine control in September 2010, Heterodera sp. cysts were found in soil samples originating from a wheat field in Gunaros, Vojvodina Province, in northern Serbia. The wheat was a winter crop grown in a dryland production system and had an average cyst density of 2.50/100 cm3 of soil. Morphologically, the cysts were golden brown and lemon shaped with a posterior protuberance. The vulval cone was bifenestrate with horseshoe-shaped semifenestra, bullae, and underbridge. Cyst measurements (n = 30) ranged as follows: cyst length (without neck): 511.50 to 899.00 µm, cyst width: 201.50 to 682.00 µm, fenestral length: 44.80 to 65.60 µm, fenestral width: 24.00 to 40.00 µm, vulval bridge length: 12.80 to 20.80 µm, vulval bridge width: 6.40 to 14.40 µm, vulval slit: 6.00 to 12.80 µm, and underbridge length: 60.00 to 112.00 µm. The second-stage juveniles had an offset head, stylet with characteristic anchor-shaped basal knobs, four incisures, and a conical tail with a rounded tip. The J2 morphometrics (n = 30) were: length: 447.30 to 611.10 µm, width: 22.40 to 25.60 µm, stylet: 20.80 to 24.00 µm, tail length: 56.00 to 68.80 µm, tail width: 14.40 to 19.20 µm, and hyaline length: 35.20 to 44.80 µm. The ITS region was used for molecular analysis. Each DNA sample was extracted from a single cyst. Sequencing was done with primers TW81 and AB28 (2). In comparison with other H. filipjevi populations, the obtained sequence (GenBank Accession No. JX235959) revealed 99 to 100% similarity. Morphological and molecular data confirmed the existence of H. filipjevi. This is, to our knowledge, the first report of H. filipjevi from Serbia. Since wheat has important socioeconomic value for Serbia, after extensive surveys, additional phytosanitary measures may be necessary to prevent the spread of this parasite. References: (1) J. M. Nicol et al. Current Nematode Threats to World Agriculture. Genomics and Molecular Genetics of Plant-Nematode Interactions, Springer, New York, 2011. (2) A. M. Skantar et al. J. Nematol. 39:133, 2007.

Identification and Characterization of Eutypa lata on Grapevine in Serbia.

In the period from late May 2004 to late May 2010, grapevine (Vitis vinifera L.) between 11 and 22 years old was observed for the incidence of symptoms of shortened shoots and zigzag internodes, with tiny, chlorotic leaves, torn and bended edges, with necrosis along the edges of leaves and dead internervous tissue. These symptoms are conspicuous especially when the vine is in the F phases of development. Later, in the course of vegetation, the dying of infected shoots and branches or covering of symptoms by a new foliage mass is perceived. Foliar symptoms are initially confined to one arm of infected vines; however, as the disease progresses, symptoms may spread throughout the entire vine. If a cross-section is made of an infected trunk, the canker appears as a wedge-shaped area of discolored wood spreading to the center of the trunk. In the period from 2004 to 2010, disease incidence was high, approaching 20%, and decline-affected cordons, vine branches, or whole plants was very high, resulting in losses of more than 35%. The loss created by a decline in grapevine quality is not included in this estimate. In this study, the causal agent was diagnosed as Eutypa lata (anamorph Libertella blepharis), on the basis of morphology of conidia of a Libertella anamorph on a 4- to 6-week-old culture on PDA (1) and by molecular identification. Molecular identification was performed by PCR and RFLP analysis and supplemented by sequence analysis. Total DNA was isolated from cultured mycelia of fungi using CTAB extraction protocol. PCR reaction was performed by universal ITS1/ITS4; the primer pair and RFLP patterns were determined after restriction with AluI (3). For specific identification of E. lata, the primer pair Lata 1/Lata 2.2 (2) were used and the 385-bp fragment was detected from analyzed isolates. Five selected isolates were purified and a fragment encompassing ITS1, ITS2, and 5.8S rDNA gene was sequenced. Sequences were deposited in the NCBI database under Accession Nos. JQ041699, JQ041700, JQ041701, JQ041702, and JQ041703. Sequence comparisons revealed high nucleotide identity among isolates (99.6 to 100%). When aligned with other E. lata isolates retrieved from the NCBI database, Serbian isolates show the highest nucleotide identity with the isolates from North America (AY462541, AY462540, AY662393, AY662392) and Australia (EU835166, EU835163, EU835162, EU835161, EU835160, EU835159, EU835156). A pathogenicity test was performed in February 2006 in a greenhouse at room temperature (approximately 22°C) and included inoculation rooted cuttings of grapevine (cultivars Cabernet Sauvignon, Prokupac, and Drenak) plants by mycelium. Agar plugs containing mycelium were inserted into 5 mm diameter holes drilled in the main stem of the rootlings and sealed by wrapping with Parafilm. Uninoculated control vines treated with a sterile agar plug were included in the experiment. Foliar symptoms and discoloring of wood beneath and above the inoculation site, inoculated plants, was observed. Reisolation and reinoculation were performed 27 months later, and 54 months later the pathogenicity test was confirmed (4). To our knowledge, this is the first report of death of infected cordons of grapevine by E. lata in Serbia. References: (1) D. A. Glawe et al. Mycotaxon 2:123, 1982. (2) P. Lecomte et al. Appl. Environ. Microbiol. 66:4475, 2000. (3) P. E. Rolshausen et al. Plant Dis. 88:925, 2004. (4) M. Sosnowski et al. Aust. N.Z. Grapegrower Winemaker 493:14, 2005.

Chronic inflammatory demyelinating polyneuropathy and ventilatory failure: report of seven new cases and review of the literature.

BACKGROUND: Ventilatory involvement is rarely reported in chronic inflammatory demyelinating polyneuropathy (CIDP), but small prospective studies showed frequent involvement of phrenic nerves, which is usually overshadowed by severe limb weakness. OBJECTIVES: To report the clinical features of CIDP associated with ventilatory failure. RESULTS: There were seven patients (43% women), with a mean age of 58.6 (range 38-82). The clinical courses were relapsing in five and progressive in two. Four patients had an initial event simulating Guillain-Barre syndrome (GBS). Ventilatory failure was recurrent in three patients. Five patients had full or nearly complete recoveries; one still requires nocturnal ventilation; and one died (14%) of myocardial infarction while still requiring mechanical ventilation. CONCLUSIONS: Clinical ventilatory dysfunction in CIDP is usually not an indicator of poor prognosis, and many patients recover without significant permanent disability. The mortality rate is similar to intubated patients with GBS. Patients with cardiopulmonary comorbidities and acute GBS-like onset of CIDP may be at higher risk of ventilatory failure which typically responds to 'standard' treatments of CIDP. Larger prospective studies are needed to define the prevalence, clinical spectrum and significance of ventilatory involvement in CIDP and to establish guidelines for evaluation and treatment.

Evaluation of biological debris on endodontic instruments after cleaning and sterilization procedures.

AIM: To examine the presence of biological debris and the level of contamination on reusable endodontic instruments those were subjected to different cleaning methods prior to sterilization. METHODOLOGY: One hundred and eighty endodontic instruments from eighteen dental practices were analysed. These practices used different decontamination protocols for reusable instruments. The presence of organic debris was detected by the use of Van Gieson's stain. Forty-eight new stainless steel hand instruments were used as controls. The samples were examined by light microscopy. RESULTS: Residual biological debris was observed in 96% of the samples. The mean value of maximum biological contamination was 34% in the group in which the instruments were brushed manually and immersed in alcohol, 25% in the group in which commercially available disinfectants were used and 5% in the group in which the instruments were cleaned ultrasonically. There was a statistically significant difference in the mean values with respect to the cleaning protocol applied (P < 0.001). CONCLUSIONS: The methods used to clean endodontic instruments appear to be generally ineffective for the removal of biological debris. The best method was the one that included mechanical, chemical and ultrasonic cleaning of instruments.

Treatment of tardive dyskinesia with levetiracetam in a transplant patient.

OBJECTIVE: To describe successful treatment of tardive dyskinesia with levetiracetam. BACKGROUND: Tardive dyskinesia is a late-onset movement disorder caused by exposure to dopamine receptor blocking agents, most commonly neuroleptics. Metoclopramide is frequently used to treat gastrointestinal dysmotility. It has antidopaminergic properties, and is estimated to be responsible for two-thirds of drug-related movement disorders. DESIGN/METHODS: Case report. RESULTS: A 68-year-old woman presented with a history of intestinal transplantation (12 years ago; short gut syndrome related to bowel resection for rectal carcinoma) and renal transplantation (1 year ago; diabetes). She developed involuntary movements with stereotypic oro-buccal-lingual dyskinesias and right-sided choreiform movements. Her Abnormal Involuntary Movement Scale score (AIMS) score was 27. She has been treated with metoclopramide for gastrointestinal dysmotility for more than 10 years and was diagnosed with tardive dyskinesia. Treatment with levetiracetam 250 mg orally b.i.d. led to a significant improvement of abnormal movements within a week. Her AIMS score decreased to 8. DISCUSSION: Tardive dyskinesia may be quite disabling and options include withdrawal of offending medication, or use of tetrabenazine or reserpine. Several reports also suggested improvement of tardive movement disorders with levetiracetam. In our patient, levetiracetam relieved symptoms of tardive dyskinesia and allowed continuous use of metoclopramide. Larger studies are needed to confirm its efficacy.

Vasculitic neuropathy--electrodiagnostic findings and association with malignancies.

BACKGROUND: Vasculitic neuropathies occur in the context of systemic disorders or in isolation. Histopathologic evaluation remains the gold standard for diagnosis, but certain electrodiagnostic findings may heighten suspicion of vasculitic neuropathy and improve the yield of nerve and muscle biopsy. AIM OF THE STUDY: Description of electrodiagnostic patterns associated with vasculitic neuropathies, and a report of a possible association with malignancies. METHODS: Retrospective review of medical records of patients with histopathologically proven vasculitic and non-vasculitic axonal neuropathies evaluated at the University of Pittsburgh Medical Center from November 1995 to November 2003. RESULTS: The most distinctive electrodiagnostic patterns associated with vasculitic neuropathy were mononeuritis multiplex (27.5% vs 4% in controls; P = 0.003) and axonal sensorimotor polyneuropathy with side-to-side amplitude asymmetry (50% vs 32%, P > 0.05). Additionally, six patients (15% vs 2%; P = 0.034) developed various malignancies within 2 years of onset of vasculitic neuropathy. CONCLUSIONS: While generalized polyneuropathy was the most common presentation of nerve vasculitis, our study affirms side-to-side amplitude asymmetry and mononeuritis multiplex as the most distinctive electrodiagnostic features. The frequent occurrence of malignancies suggests a possible association with the vasculitic neuropathy and warrants additional investigation.

Granulomatous amebic encephalitis in a multivisceral transplant recipient.

A 40-year-old man with multivisceral allograft developed acutely right-sided numbness 9 months after transplantation. Cranial magnetic resonance imaging (MRI) showed a small left parietal lesion, and cerebrospinal fluid analysis was unremarkable. Stereotactic brain biopsy was non-diagnostic. The patient continued to deteriorate, developed cerebral edema and died at 13 days after the onset of symptoms. Unexpectedly, autopsy demonstrated acanthamebic encephalitis. This case highlights diagnostic difficulties encountered with amebic encephalitis and expands the spectrum of opportunistic central nervous system (CNS) infections in solid and visceral organ transplant recipients.

High titers of CA-125 may be associated with recurrent ischemic strokes in patients with cancer.

In addition to etiologies common in the general population, strokes in cancer patients may be caused by hypercoagulable states, hyperviscosity, cardiogenic embolism, and neoplastic vessel infiltration. Intravascular mucins were reported in patients with recurrent thromboembolism. The authors report four patients with metastatic cancer, brain infarcts, and other thromboembolic disease with markedly elevated levels of the tumor marker CA-125 and explore possible associations between this mucinous protein and strokes.

Subacute rhombencephalitis optica responsive to intravenous immunoglobulins.

A 53-year-old man presented with optic neuritis, followed within 10 weeks by a subacute progression of weakness, ataxia, and multiple cranial nerve palsies. Cranial magnetic resonance imaging demonstrated multiple T2-hyperintense lesions extending from the ponto-medullary junction to the thalamus and internal capsule. Cerebrospinal fluid analysis showed lymphocytic pleocytosis with elevated protein content. A brain biopsy revealed inflammatory changes. After a month long period of inexorable worsening, the patient was treated with intravenous immunoglobulins (2 g/kg). He responded to treatment, and recovered over the subsequent 3 months. The patient's response to treatment suggests that intravenous immunoglobulins should be considered in patients with inflammatory brain disorders.