Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Ectopically expressed leaf and bulb lectins from garlic (Allium sativum L.) protect transgenic tobacco plants against cotton leafworm (Spodoptera littoralis).

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.

Expression of garlic leaf lectin under the control of the phloem-specific promoter Asus1 from Arabidopsis thaliana protects tobacco plants against the tobacco aphid (Myzus nicotianae).

BACKGROUND: To check for correlation between the insecticidal properties and the specificity of lectins, a comparative study was made of the insecticidal activities of two garlic lectins with different biological activities. RESULTS: The insecticidal activity of the garlic (Allium sativum L.) leaf lectin ASAL and bulb lectin ASAII towards the tobacco aphid Myzus nicotianae Blackman was studied using bioassays with transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38). Bioassays were started with newborn nymphs of the tobacco aphid. Although during the first 7-8 days when nymphs developed to adults there were no apparent effects, part of the nymphal population was found to develop into winged (alate) forms. Later it became clear that transgenic plants expressing ASAL and ASAII had a significant effect on the reproduction capacity of the resulting adults, with a reduction of up to 40%. Different life table parameters such as prereproductive time, intrinsic rate of natural increase, generation time and doubling time were significantly affected (P < 0.05) in aphids grown on transgenic plant material expressing ASAL and ASAII. CONCLUSION: Bioassays with tobacco plants expressing ASAL and ASAII demonstrated a significant impact on the population growth of M. nicotianae. Therefore, both lectins can be considered as valuable candidate aphid control agents.

Jekyll encodes a novel protein involved in the sexual reproduction of barley.

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal-filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.