Immunosorbent Electron Microscopy Research Articles

In Vivo Evolution of a Klebsiella pneumoniae Capsule Defect With wcaJ Mutation Promotes Complement-Mediated Opsonophagocytosis During Recurrent Infection.

Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.

Mitochondrion-NLRP3 inflammasome activation in macrophages: A novel mechanism of the anti-inflammatory effect of Notopterygium in rheumatoid arthritis treatment

ObjectiveThe mechanism by which Notopterygium (NE) regulates the nucleotide‐binding, oligomerization domain (NOD)‐like receptor family and pyrin domain-containing 3 (NLRP3) inflammasome to treat rheumatoid arthritis (RA) was investigated to reveal the scientific implications of NE in RA treatment. MethodsAdjuvant arthritis (AA) rats were replicated. After NE intervention, the anti-inflammatory efficacy of NE in vivo was determined. The mechanism of NE in RA treatment was predicted by network pharmacology, and the key target for further experiments was found through the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG). The effect of NE on the NLRP3 inflammasome in AA rats was verified. Furthermore, with the induction of inflammation in RAW264.7 cells by lipopolysaccharide (LPS), several techniques, such as Griess assay, enzyme linked immunosorbent assays, electron microscopy, and fluorescence probe technology, were used to investigate the anti-inflammatory and related mechanisms of NE in RA treatment. ResultsNE could inhibit inflammation in AA rats. KEGG results showed that NLRP3 participated in the top three pathways of NE in RA treatment. Through Western blotting and immunofluorescence assays, this study demonstrated that NE can regulate NLRP3, pro-Caspase-1, Caspase-1, and CD11b in the ankle joint of AA rats. NE may significantly reduce the LPS-induced inflammatory response of RAW264.7 cells by alleviating mitochondrial damage, reducing the number of mitochondrial deoxyribonucleic Acid and mitochondrial reactive oxygen species, inhibiting NLRP3 inflammasome activation. ConclusionThe anti-inflammatory and antirheumatic effect of NE may involve regulating NLRP3 inflammasome activation through mitochondria. NLRP3 is probably the key target molecule of NE in the treatment of RA.

Fungi Fibrinolytic Compound 1 Plays a Core Role in Modulating Fibrinolysis, Altering Plasma Clot Structure, and Promoting Susceptibility to Lysis.

Fibrin clot structure and function are major determinants of venous and arterial thromboembolic diseases, as well as the key determinants of the efficiency of clot lysis. Studies have revealed that fungi fibrinolytic compound 1 (FGFC1) is a novel marine pyranisoindolone natural product with fibrinolytic activity. Here, we explore the impacts of FGFC1 on clot structure, lysis, and plasminogen activation in vitro using turbidimetric, enzyme-linked immunosorbent assay, confocal and electron microscopy, urokinase, or plasmin chromogenic substrate. Clots formed in the presence of FGFC1 expressed reduced fibrin polymerization rate and maximum turbidity; however, they did not influence the lag phase of fibrin polymerization. In the absence of scu-PA (single-chain urokinase plasminogen activator), microscopy revealed that FGFC1 increased the number of protofibrils within fibrin fiber and the pore diameter between protofibrils, inducing clots to form a region of thinner and looser networks separated by large pores. The effects of FGFC1 on scu-PA-mediated plasma clot structure were similar to those in the absence of scu-PA. In addition, FGFC1 promoted the lysis of clots and increased the D-dimer concentration in lysate. FGFC1 increased the generation rate of p-nitroaniline in plasma. These results show that FGFC1 has fibrinolytic activity in plasma, leading to interference with the release of fibrinopeptide B to affect lateral aggregation of protofibrils and increase clot susceptibility to fibrinolysis by altering its structure.

Detection of Mandarivirus and greening bacterium using electron microscopy, PCR and RT-PCR in Kinnow mandarin nurseries in Punjab

Kinnow mandarin is one of the most important fruit crops in northern India particularly in Punjab. Indian citrus ringspot virus (ICRSV), Citrus yellow vein clearing virus (CYVCV) and citrus greening bacterium (CGB) are very important graft transmissible pathogens causing reduced productivity in Kinnow mandarin. A survey was undertaken in 11 kinnow mandarin nurseries, 2 to 4 star rated by National Horticultural Board in Punjab. Samples collected from these nurseries were indexed by electron microscopy, immunosorbent electron microscopy (ISEM), PCR and RT-PCR. In RT-PCR test, out of 33 samples of kinnow mandarin mother plants, 3 samples were found positive for CYVCV and 6 samples for ICRSV. In two nurseries, both the viruses were detected in the same plant indicating mixed infection in the mother plants. In ISEM test, 4 samples out of 12 samples were positive for ICRSV and 2 samples were positive for CYVCV. CGB was detected in two plants out of nine plant leaf samples collected from four nurseries. In view of these studies it is evident that molecular diagnostics as indexing tool need to be applied for the production of clean planting material of kinnow mandarin.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

​Techniques for Detection and Diagnosis of Plant Viruses: A Review

Plant viruses are famed for serious economic losses to most of the agricultural and horticultural crops all around the world. The losses caused by plant viruses are estimated to be several billion bucks per year and are therefore a major threat to global crop production and food security. Traditional diagnostic methods based on biological and physical properties of virus such as appearance of symptoms, bioassay on indicator plants, vector borne and virus particle morphology by electron microscopy are cumbersome, time-consuming and expensive. Recognition of disease based on the symptoms indicated by virus affected plants, can invariably be the first process in several cases, however it’s confusing additionally as a result of the symptoms are changeable and depends on several factors like abiotic conditions. There are several sensitive and effective diagnostic methods developed during the last four decades based on virus protein e.g., immunosorbent electron microscopy (ISEM), enzyme-linked immunosorbent assay (ELISA) and nucleic acid e.g., nucleic acid hybridization, isothermal and thermostable PCRs, for plant virus detection, each having its own advantage and disadvantage. This review provides an update on the progress made on the techniques that are useful for early, sensitive, accurate and reliable detection and diagnosis of plant viruses.

Detection of Begomovirus in chilli and tomato plants using functionalized gold nanoparticles

Begomoviruses are a major class of Geminiviruses that affects most dicotyledonous plants and causes heavy economic losses to farmers. Early detection of begomovirus is essential to control the spread of the disease and prevent loss. Many available detection methods like ELISA, immunosorbent electron microscopy, PCR or qPCR require expertise in handling sophisticated instruments, complex data interpretation and costlier chemicals, enzymes or antibodies. Hence there is a need for a simpler detection method, here we report the development of a visual detection method based on functionalized gold nanoparticles (AuNP assay). The assay was able to detect up to 500 ag/µl of begomoviral DNA (pTZCCPp3, a clone carrying partial coat protein gene) suspended in MilliQ water. Screening of chilli plants for begomoviral infection by PCR (Deng primers) and AuNP assay showed that AuNP assay (77.7%) was better than PCR (49.4%). The AuNP assay with clccpi1 probe was able to detect begomoviral infection in chilli, tomato, common bean, green gram and black gram plants which proved the utility and versatility of the AuNP assay. The specificity of the assay was demonstrated by testing with total DNA from different plants that are not affected by begomoviruses.

Discovery and Characterization of a Novel Bipartite Botrexvirus From the Phytopathogenic Fungus Botryosphaeria dothidea.

In this study, we describe a novel positive, single-stranded (+ss) RNA mycovirus, named Botryosphaeria dothidea botrexvirus 1 (BdBV1), from a phytopathogenic fungus Botryosphaeria dothidea showing abnormal morphology and attenuated virulence. BdBV1 is phylogenetically related to Botrytis virus X (BotVX) and is the second potential member of the proposed genus Botrexvirus in the family Alphaflexiviridae. However, it differs from the monopartite BotVX in that BdBV1 possesses a bipartite genome comprised of two ssRNA segments (RNA1 and RNA2 with lengths of 5,035 and 1,063 nt, respectively). BdBV1 RNA1 and RNA2 encode putative RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes, which share significant identity with corresponding genes in both fungal and plant viruses. Moreover, open reading frames (ORFs) 2–4 of BdBV1 RNA1 shared no detectable identity with any known viral proteins. Immunosorbent electron microscopy (ISEM) analysis using an antibody against the virus CP generated in vitro revealed that BdBV1 is encapsidated in filamentous particles. A comparison of the biological effects of BdBV1 infection on symptoms and growth in isogenic lines of virus-free and virus-infected B. dothidea revealed that BdBV1 is probably involved in reduced growth and virulence of the host fungus. This study describes and characterizes a novel bipartite botrexvirus, which is closely related to uni- and multi-partite fungal and plant viruses and contributes useful information to a better understanding of virus evolution.

Cellular mechanisms of impaired mineralocorticoid and glucocorticoid production by endocrine disruptor DDT

Developmental exposure to persistent endocrine disruptors is of a great concern. Affection of adrenal hormones production by low-dose exposure to dichlorodiphenyltrichloroethane (DDT) has been revealed recently. Objective of the research – investigation of ultrastructural mechanisms of impaired mineralocorticoid and glucocorticoid production after prenatal and postnatal exposure to endocrine disruptor DDT. Male Wistar rats exposed to low doses of DDT during prenatal and postnatal development were studied. Aldosterone and corticosterone levels in serum were measured by enzyme-linked immunosorbent assay, histological examination and electron microscopy of the adrenals were performed. Pubertal rats, developmentally exposed to low doses of DDT, demonstrated lowered aldosterone and elevated corticosterone serum levels. After puberty the rats showed tendency to normalization of hormones’ production. Morphological examination of the adrenals revealed less developed zona glomerulosa in pubertal period and its relative hyperplasia after puberty. Microcirculatory disorders and focal cell death were observed in outer zona fasciculata. Electron microscopy of glomerulosa cells found signs of suppressed secretory activity in pubertal period and no significant reduction of mitochondria size in adult rats. Total number of mitochondria in 1 µm2 of cytoplasm and percent of mitochondria with swollen matrix were diminished compared to the control. Cells of inner zona fasciculata demonstrated increased functional activity. Age-dependent changes in fine structure of fasciculata cells were similar to the control, but were more pronounced. In this way, cellular mechanism of impaired mineralocorticoid production in rats prenatally and postnatally exposed to low doses of endocrine disruptor DDT, are insufficient mitochondrion function and impaired reorganization of mitochondrial apparatus, which occurs during pubertal period. Mechanisms of elevated glucocorticoid secretion are attributed to enhanced function of mitochondria in fasciculata cells.

Cardiopulmonary bypass increases endothelial dysfunction after pulmonary ischaemia-reperfusion in an animal model.

Endothelial dysfunction during ischaemia-reperfusion (IR) is a major cause of primary graft dysfunction during lung transplantation. The routine use of cardiopulmonary bypass (CPB) during lung transplantation remains controversial. However, the contribution of CPB to pulmonary endothelial dysfunction remains unclear. The objective was to investigate the impact of CPB on endothelial dysfunction in a lung IR rat model. Rats were allocated to 4 groups: (i) Sham, (ii) IR, (iii) CPB and (iv) IR-CPB. The primary outcome was the study of pulmonary vascular reactivity by wire myograph. We also assessed glycocalyx degradation by enzyme-linked immunosorbent assay and electron microscopy and both systemic and pulmonary inflammation by enzyme-linked immunosorbent assay and immunohistochemistry. Rats were exposed to 45 min of CPB and IR. We used a CPB model allowing femoro-femoral support with left pulmonary hilum ischaemia for IR. Pulmonary endothelium-dependent relaxation to acetylcholine was markedly reduced in the IR-CPB group (10.7 ± 9.1%) compared to the IR group (50.5 ± 5.2%, P < 0.001), the CPB group (54.1 ± 4.7%, P < 0.001) and the sham group (80.8 ± 6.7%, P < 0.001), suggesting that the association of pulmonary IR and CPB increases endothelial dysfunction. In IR-CPB, IR and CPB groups, vasorelaxation was completely abolished when inhibiting nitric oxide synthase, suggesting that this relaxation process was mainly mediated by nitric oxide. We observed higher syndecan-1 plasma levels in the IR-CPB group in comparison with the other groups, reflecting an increased degradation of glycocalyx. We also observed higher systemic inflammation in the IR-CPB group as shown by the increased plasma levels of IL-1β, IL-10. CPB significantly increased the IR-mediated effects on pulmonary endothelial dysfunction. Therefore, the use of CPB during lung transplantation could be deleterious, by increasing endothelial dysfunction.

Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins.

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.

Orexin-A exacerbates Alzheimer’s disease by inducing mitochondrial impairment

Alzheimer’s disease (AD) is a progressive neurodegenerative disease which is characterized by the accumulation of amyloid-β peptide (Aβ). Orexin-A is a neuropeptide which has been reported to participate in the pathogenesis of AD. Thus, we aimed to investigate the possible mechanism by which Orexin-A acts in AD. APP/PS1 transgenic mice, an animal model of AD, were intracerebroventricularly injected with Orexin-A. Aβ-treated SH-SY5Y cells were used as a cell model of AD and treated with Orexin-A. The Morris water maze test, fluorescence microscopy, enzyme-linked immunosorbent assay (ELISA), electron microscopy, real-time PCR, and other biochemical assays were conducted. The Morris water maze test showed that Orexin-A aggravated cognitive deficit in APP/PS1 mice. Using thioflavine-S staining and ELISA, we found that Orexin-A promoted Aβ accumulation in APP/PS1 mice. By evaluating mitochondrial morphology, cytochrome c oxidase activity, ATP level, mitochondrial DNA copy number, and reactive oxygen species, we found that Orexin-A aggravated mitochondrial impairment in APP/PS1 mice and Aβ-treated SH-SY5Y cells. Our results indicate that Orexin-A exacerbates AD by inducing mitochondrial impairment. This is a new mechanism that explains how Orexin-A participates in the pathogenesis of AD.

Development of RT-PCR degenerate primers for the detection of two mandariviruses infecting citrus cultivars in India

Indian citrus ringspot virus (ICRSV) and Citrus yellow vein clearing virus (CYVCV) are the mandariviruses infecting various citrus cultivars in India and around the world. In the fields, it was observed that citrus plants infected by both the viruses and frequently expressed only ringspot symptoms. The ICRSV-specific polyclonal-antibody used in immuno-sorbent electron microscopy (ISEM) and enzyme linked immuno-sorbent assay (ELISA) could detect only ICRSV in mixed infections. Therefore, the conserved sequences of the RNA dependent RNA polymerase (RdRP) gene of the alphaflexiviruses were exploited for developing a RT-PCR based assay for detection of both the mandariviruses simultaneously, if present. A degenerate primer pair was designed to amplify a ∼435bp fragment by multiple alignments of the RdRP gene sequences of the members of genera Mandarivirus, Potexvirus and Allexivirus. The developed RT-PCR assay was validated for detecting both, CYVCV and ICRSV in mixed infections as well as in single virus-infected citrus plants. The presence of ICRSV or CYVCV or both of them together in such plants were confirmed by using primer pair specific to each of these viruses. Further, the identity of the amplicons was confirmed by sequencing and the virus species were determined with BLASTN analysis. The degenerate primers also amplified the corresponding target sequences of an allexivirus and a potexvirus from the respective infected garlic/ onion and tobacco plants. The use of the degenerate primers for the detection of these virus species of the genus Mandarivirus will be useful in citrus certification programmes.

Antitumor Effects of a Recombinant Baculovirus Displaying Anti-HER2 Scfv Expressing Apoptin in HER2 Positive SK-BR-3 Breast Cancer Cells

Aim: Since HER2 targeted therapies have shown clinical benefit in breast cancer, in the present study recombinant baculovirus (BV) displaying anti-HER2 single-chain variable domain fragment (scFv) expressing Apoptin was generated. Methods: The binding specificity and surface display of anti-HER2 scFv were evaluated using enzyme-linked immunosorbent assay (ELISA) and electron microscopy, respectively. The targeting properties and cytotoxic effect on breast cancer cells determined by fluorescence microscopy and MTT assays. Results: The results demonstrated that recombinant BV could specifically bind to HER2-overexpressing SK-BR-3 cells but not to the HER2 negative MCF-7 cells and reduced the viability of SK-BR-3 cells by expressing Apoptin. Conclusion: These results suggest that the antitumor effect of Apoptin in combination with HER2 targeting of this recombinant BV makes it a promising vector in targeted cancer therapy.

First Report of physostegia chlorotic mottle virus on Tomato (Solanum lycopersicum) in Germany

In September 2015, a tomato sample collected in the German state of Hesse was sent to the Julius Kuhn-Institut for analysis. While the fruits showed marbling and discoloration, the leaf samples from this plant did not show any obvious symptoms. Transmission electron microscopy (TEM) revealed the presence of bullet-shaped virus particles indicating the presence of a rhabdovirus. However, immunosorbent electron microscopy using antiserum JKI-1073 for Eggplant mottled dwarf virus (EMDV) could not confirm EMDV infection. The virus was mechanically transmitted to Nicotiana benthamiana, N. clevelandii, and Chenopodium quinoa inducing yellowing and leaf deformation, while mechanical transmission to N. occidentalis (P1 and 37b) failed. Extraction of double stranded-RNA (dsRNA) followed by random-PCR (Froussard 1992), cloning of PCR products, and sequencing failed to reveal any virus sequences. Total RNA was extracted from infected N. benthamiana, followed by ribo-depletion, library preparation and submission for next-generation sequencing (NGS) using an Illumina MiSeq platform as described by Knierim et al. (2017). De novo assembly of the trimmed reads was done with Geneious v 10.1.3 (Biomatters LTD, NZ). Using MEGA BLAST, 13 contigs showed between 95.6 and 98.5% similarity with physostegia chlorotic mottle virus (PhCMoV) isolate PV-1182 (accession no. KX636164). The complete PhCMoV genome (13,321 nt length) was assembled by mapping reads to this reference genome and used to design PhCMoV-specific RT-PCR primers for detection (HZ-343 5′-CGGTGAGTGGGGCAACTAAT-3′/HZ-344 5′-AGCGATGGGGTCTAGTGTCT-3′). RT-PCR confirmed the presence of PhCMoV in the test plants resulting in amplicons of approximately 875 bp. In August 2016, similar symptoms on tomato fruits were observed by a different grower in Hesse. The presence of PhCMoV was confirmed by TEM and RT-PCR. Additionally, the PCR products were sequenced and showed 97% identity to KX636164. Surprisingly, reanalysis of a tomato sample from 2003 that was infected by a hitherto unknown rhabdovirus using NGS also confirmed infection with PhCMoV. This sample also originated from Hesse although the original grower is unknown. The complete genome of the 2003 PhCMoV sample was assembled following the same methods described above. Pairwise comparison between the genomes of 2015 and 2003 isolates resulted in 99.7% nucleotide identity and 96.9% when compared with KX636164. These findings indicate the presence of PhCMoV in tomato in Germany for a long time albeit isolated occurrences in different production areas. PhCMoV was recently identified from Physostegia virginiana plants showing leaf deformation and severe chlorotic and mottle symptoms in Austria (Menzel et al. 2016). However, it is not known if there is a link between PhCMoV isolates infecting P. virginiana and tomato as the routes of transmission and dissemination are currently unknown. The sequences from this report were deposited in GenBank (accession nos. KY706238 and KY859866 [full-length sequences], KY882263 and KY882264 [partial sequences]). To our knowledge, this is the first host record of PhCMoV in tomato and a new country record for Germany.

Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein.

Citrus yellow mosaic virus (CYMV) is a member of genus Badnavirus of the family Caulimoviridae. It is the causal agent of citrus yellow mosaic disease in citrus and causes reduction in yield. As the virus is vegetative propagated by grafting, development of high-throughput diagnosis methods based on serological techniques is a prerequisite for production of healthy virus-free planting material. The current study describes the development of polyclonal antibodies raised in rabbits against purified recombinant virion-associated protein (rVAP) encoded by ORF-II of CYMV. The specificity of developed antiserum was evaluated in immunosorbent electron microscopy (ISEM), antigen-coated plate-enzyme linked immunosorbent assay (ACP-ELISA) and immunocapture PCR (IC-PCR). The antiserum specifically reacted up to a dilution of 1:2000 in ACP-ELISA for detection of CYMV-infected plants. The antiserum was validated by screening CYMV-infected plants maintained in the glass house through ACP-ELISA. To the best for our knowledge, this is the first report on production of polyclonal antiserum using recombinant virion-associated protein as fusion protein, which could be used for screening CYMV-infected plants by ELISA and IC-PCR. These immunodiagnostic methods can be effectively employed in routine indexing of citrus and in quarantine process.

Occurrence and Transmission of Grapevine Virus A in South African grapevines

Antiserum prepared to a local isolate of grapevine virus A (GVA) was used in immunosorbent electron microscopy (ISEM) with decoration and in an unlabelled antibody enzyme-linked immunosorbent assay (ELISA) for the screening of grapevine sources for the presence of virus. GV A occurred extensively in grapevine plantings showing visual symp· toms of leafroll or indexing positive for leafroll. GV A was found not to be associated with either stem pitting or fleck symptoms. It occurred mostly in association with undecorated closterovirus (CV)-like particles. Limited ISEM with decoration of the CV-like particles using antiserum to a Swiss isolate of CV 2 200 nm, (CV type I) confirmed the presence of a second CV in local grapevine sources. In addition, it indicated the presence of a third CV -like particle longer than GVA. GVA together with undecorated CV-like particles were also detected in initially GVA-free LN-33 grapevines growing under field conditions and naturally infected with Ieafroll. GV A and CV type I were also present in the vine mealybug Planococcus ficus, following exposure to a grapevine source carrying these viruses. H'lwever, due to lack of CV type I antiserum, only GV A was confirmed in grapevine following controlled transmissions with P. ficus although undecorated CV -like particles were present. The reliability of the ELISA detection procedure appears to be influenced by seasonal fluctuations in GV A concentrations.

The Elimination of Fanleaf Virus from Grapevines Using in vitro Somatic Embryogenesis Combined with Heat Therapy

Somatic embryos were successfully regenerated from callus tissue of anthers and ovaries excised from inflorescences of grapevines infected with grapevine fanleaf virus (GFLV). Production of pro-embryogenic masses (PEMS) was controlled by specific growth regulators and culture conditions, including heat incubation at 35°C. Somatic embryos (containing roots and cotyledons) and plantlets were subjected to immunosorbent electron microscopy (ISEM) and serological tests (ELISA). Results show that somatic embryogenesis in combination with heat therapy of the cultures is an effective procedure to eliminate GFLV from anther and ovary source material.

The Effectiveness of in vitro Somatic Embryogenesis in Eliminating Fanleaf Virus and Leafroll Associated Viruses from Grapevines

Somatic embryos were successfully regenerated from callus tissue of anthers and ovaries extracted from inflorescences of grapevines infected with grapevine fanleaf virus (GFLV) and grapevine leafroll associated viruses (GLR) respectively. Production of pro-embryogenic masses (PEMS) was controlled by specific growth regulators and culture conditions. Somatic embryos (containing roots and cotyledons) and plantlets were subjected to immunosorbent electron microscopy (ISEM) as well as serological tests (ELISA). Results indicated that somatic embryogenesis derived from ovary tissue of infected grapevines is an effective technique to eliminate grapevine Ieafroll associated viruses from grapevines but the procedure was not successful in the elimination of GFL V from anther source material.

Platelet-Derived S100A8/A9 and Cardiovascular Disease in Systemic Lupus Erythematosus.

Levels of S100A8/A9, a proinflammatory and prothrombotic protein complex, are increased in several diseases, and high levels predispose to cardiovascular disease (CVD). Recently, platelet S100A8/A9 synthesis was described in mice and humans in relation to CVD. The aim of this study was to investigate the role of platelet S100A8/A9 in systemic lupus erythematosus (SLE), a disease with markedly increased cardiovascular morbidity, as well as the exact platelet distribution of the S100A8/A9 proteins. The occurrence and distribution of platelet S100A8/A9 protein were detected by enzyme-linked immunosorbent assay, electron microscopy, Western blotting, and flow cytometry in healthy controls (n = 79) and in 2 individual cohorts of SLE patients (n = 148 and n = 318, respectively) and related to cardiovascular morbidity. We observed that human platelets expressed S100A8/A9 proteins, and that these were localized in close proximity to intracellular membranes and granules as well as on the cell surface upon activation with physiologic and pathophysiologic stimuli. Interestingly, S100A8/A9 was enriched at sites of membrane interactions, indicating a role of S100A8/A9 in cell-cell communication. S100A8/A9 levels were highly regulated by interferon-α, both in vivo and in vitro. Patients with SLE had increased platelet S100A8/A9 content compared with healthy individuals. Increased levels of platelet S100A8/A9 were associated with CVD, particularly myocardial infarction (odds ratio 4.8, 95% confidence interval 1.5-14.9, P = 0.032 [adjusted for age, sex, and smoking]). Platelets contain S100A8/A9 in membrane-enclosed vesicles, enabling rapid cell surface deposition upon activation. Furthermore, platelet S100A8/A9 protein levels were increased in SLE patients, particularly in those with CVD, and may be a future therapeutic target.