Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity.

During 25 surveys of global Phytophthora diversity, conducted between 1998 and 2020, 43 new species were detected in natural ecosystems and, occasionally, in nurseries and outplantings in Europe, Southeast and East Asia and the Americas. Based on a multigene phylogeny of nine nuclear and four mitochondrial gene regions they were assigned to five of the six known subclades, 2a-c, e and f, of Phytophthora major Clade 2 and the new subclade 2g. The evolutionary history of the Clade appears to have involved the pre-Gondwanan divergence of three extant subclades, 2c, 2e and 2f, all having disjunct natural distributions on separate continents and comprising species with a soilborne and aquatic lifestyle and, in addition, a few partially aerial species in Clade 2c; and the post-Gondwanan evolution of subclades 2a and 2g in Southeast/East Asia and 2b in South America, respectively, from their common ancestor. Species in Clade 2g are soilborne whereas Clade 2b comprises both soil-inhabiting and aerial species. Clade 2a has evolved further towards an aerial lifestyle comprising only species which are predominantly or partially airborne. Based on high nuclear heterozygosity levels ca. 38 % of the taxa in Clades 2a and 2b could be some form of hybrid, and the hybridity may be favoured by an A1/A2 breeding system and an aerial life style. Circumstantial evidence suggests the now 93 described species and informally designated taxa in Clade 2 result from both allopatric non-adaptive and sympatric adaptive radiations. They represent most morphological and physiological characters, breeding systems, lifestyles and forms of host specialism found across the Phytophthora clades as a whole, demonstrating the strong biological cohesiveness of the genus. The finding of 43 previously unknown species from a single Phytophthora clade highlight a critical lack of information on the scale of the unknown pathogen threats to forests and natural ecosystems, underlining the risk of basing plant biosecurity protocols mainly on lists of named organisms. More surveys in natural ecosystems of yet unsurveyed regions in Africa, Asia, Central and South America are needed to unveil the full diversity of the clade and the factors driving diversity, speciation and adaptation in Phytophthora. Taxonomic novelties: New species: Phytophthora amamensis T. Jung, K. Kageyama, H. Masuya & S. Uematsu, Phytophthora angustata T. Jung, L. Garcia, B. Mendieta-Araica, & Y. Balci, Phytophthora balkanensis I. Milenkovic, Z. Tomic, T. Jung & M. Horta Jung, Phytophthora borneensis T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora calidophila T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora catenulata T. Jung, T.-T. Chang, N.M. Chi & M. Horta Jung, Phytophthora celeris T. Jung, L. Oliveira, M. Tarigan & I. Milenkovic, Phytophthora curvata T. Jung, A. Hieno, H. Masuya & M. Horta Jung, Phytophthora distorta T. Jung, A. Durán, E. Sanfuentes von Stowasser & M. Horta Jung, Phytophthora excentrica T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora falcata T. Jung, K. Kageyama, S. Uematsu & M. Horta Jung, Phytophthora fansipanensis T. Jung, N.M. Chi, T. Corcobado & C.M. Brasier, Phytophthora frigidophila T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora furcata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora inclinata N.M. Chi, T. Jung, M. Horta Jung & I. Milenkovic, Phytophthora indonesiensis T. Jung, M. Tarigan, L. Oliveira & I. Milenkovic, Phytophthora japonensis T. Jung, A. Hieno, H. Masuya & J.F. Webber, Phytophthora limosa T. Corcobado, T. Majek, M. Ferreira & T. Jung, Phytophthora macroglobulosa H.-C. Zeng, H.-H. Ho, F.-C. Zheng & T. Jung, Phytophthora montana T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora multipapillata T. Jung, M. Tarigan, I. Milenkovic & M. Horta Jung, Phytophthora multiplex T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora nimia T. Jung, H. Masuya, A. Hieno & C.M. Brasier, Phytophthora oblonga T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora obovoidea T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora obturata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora penetrans T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora platani T. Jung, A. Pérez-Sierra, S.O. Cacciola & M. Horta Jung, Phytophthora proliferata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora pseudocapensis T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pseudocitrophthora T. Jung, S.O. Cacciola, J. Bakonyi & M. Horta Jung, Phytophthora pseudofrigida T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora pseudoccultans T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pyriformis T. Jung, Y. Balci, K.D. Boders & M. Horta Jung, Phytophthora sumatera T. Jung, M. Tarigan, M. Junaid & A. Durán, Phytophthora transposita T. Jung, K. Kageyama, C.M. Brasier & H. Masuya, Phytophthora vacuola T. Jung, H. Masuya, K. Kageyama & J.F. Webber, Phytophthora valdiviana T. Jung, E. Sanfuentes von Stowasser, A. Durán & M. Horta Jung, Phytophthora variepedicellata T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora vietnamensis T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora ×australasiatica T. Jung, N.M. Chi, M. Tarigan & M. Horta Jung, Phytophthora ×lusitanica T. Jung, M. Horta Jung, C. Maia & I. Milenkovic, Phytophthora ×taiwanensis T. Jung, T.-T. Chang, H.-S. Fu & M. Horta Jung. Citation: Jung T, Milenkovic I, Balci Y, Janousek J, Kudlácek T, Nagy ZÁ, Baharuddin B, Bakonyi J, Broders KD, Cacciola SO, Chang T-T, Chi NM, Corcobado T, Cravador A, Dordevic B, Durán A, Ferreira M, Fu C-H, Garcia L, Hieno A, Ho H-H, Hong C, Junaid M, Kageyama K, Kuswinanti T, Maia C, Májek T, Masuya H, Magnano di San Lio G, Mendieta-Araica B, Nasri N, Oliveira LSS, Pane A, Pérez-Sierra A, Rosmana A, Sanfuentes von Stowasser E, Scanu B, Singh R, Stanivukovic Z, Tarigan M, Thu PQ, Tomic Z, Tomsovský M, Uematsu S, Webber JF, Zeng H-C, Zheng F-C, Brasier CM, Horta Jung M (2024). Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity. Studies in Mycology 107: 251-388. doi: 10.3114/sim.2024.107.04.

Extensive morphological and behavioural diversity among fourteen new and seven described species in Phytophthora Clade 10 and its evolutionary implications.

During extensive surveys of global Phytophthora diversity 14 new species detected in natural ecosystems in Chile, Indonesia, USA (Louisiana), Sweden, Ukraine and Vietnam were assigned to Phytophthora major Clade 10 based on a multigene phylogeny of nine nuclear and three mitochondrial gene regions. Clade 10 now comprises three subclades. Subclades 10a and 10b contain species with nonpapillate sporangia, a range of breeding systems and a mainly soil- and waterborne lifestyle. These include the previously described P. afrocarpa, P. gallica and P. intercalaris and eight of the new species: P. ludoviciana, P. procera, P. pseudogallica, P. scandinavica, P. subarctica, P. tenuimura, P. tonkinensis and P. ukrainensis. In contrast, all species in Subclade 10c have papillate sporangia and are self-fertile (or homothallic) with an aerial lifestyle including the known P. boehmeriae, P. gondwanensis, P. kernoviae and P. morindae and the new species P. celebensis, P. chilensis, P. javanensis, P. multiglobulosa, P. pseudochilensis and P. pseudokernoviae. All new Phytophthora species differed from each other and from related species by their unique combinations of morphological characters, breeding systems, cardinal temperatures and growth rates. The biogeography and evolutionary history of Clade 10 are discussed. We propose that the three subclades originated via the early divergence of pre-Gondwanan ancestors > 175 Mya into water- and soilborne and aerially dispersed lineages and subsequently underwent multiple allopatric and sympatric radiations during their global spread. Citation: Jung T, Milenkovic I, Corcobado T, et al. 2022. Extensive morphological and behavioural diversity among fourteen new and seven described species in Phytophthora Clade 10 and its evolutionary implications. Persoonia 49: 1-57. https://doi.org/10.3767/persoonia.2022.49.01.

Evaluating the efficiency of the Dental Teacher system as a digital preclinical teaching tool.

AIM: To investigate the use of a preparation evaluation system for enhancing the learning and performance of undergraduate dental students when cutting preparations. MATERIALS AND METHODS: Two groups of eighteen students each were randomly chosen from the fourth year of the dental programme. The task chosen for this study was to make a cavity in preparation for a mesio-occlusal ceramic onlay in a plastic tooth. The dimensions of the cavity were defined, and 2 burs of known size were used for preparation. For assessment, each tooth preparation was scanned with a digital scanner and analysed using the Dental Teacher software. In the control group, a second corrective preparation was made following the supervisor's instructions. In the test group, the second preparation was made based on Dental Teacher analysis. The final cavities were all scanned and assessed by Dental Teacher comparing the similarity of students' onlay cavity preparations to the ideal preparation. All data were recorded and analysed by the software, including cavity depth and width in the occlusal and proximal box, the extent of mesiobuccal cusp reduction and shoulder width around the mesiobuccal cusp. Finally, the data were statistically evaluated using a Wilcoxon matched pairs test and a Mann-Whitney U test. RESULTS: Three of the 6 cavity dimension parameters improved significantly in the test group whilst no improvement was found in the control group. A positive correlation was found between the improvement and the deviation measured for the first preparations, and it was stronger in the test group than in the control group. CONCLUSIONS: The use of Dental Teacher helped students to learn the preparation technique for onlay restorations more efficiently and seems to be a promising and useful method to facilitate their individual performance. Student feedback showed a great demand for digital aids in education.

Immune Class Regulation and Its Medical Significance Part II of a Report of a Workshop on Foundational Concepts of Immune Regulation.

We discussed different proposals for how the nature of the Th1/Th2 phenotype of an immune response is determined, and favoured one, the Threshold Hypothesis, as plausible and so useful as the basis for further discussions. The activation of a target CD4 T cell can be facilitated by helper CD4 T cells when the CD4 T cells interact via an antigen-presenting cell. The Threshold Hypothesis states that tentative and robust antigen-mediated CD4 T cell cooperation results in the target CD4 T cell, respectively giving rise, upon activation, to Th1 and Th2 cells. We primarily discussed four topics. We briefly discussed in the background section certain limitations of the Th1/Th2 paradigm in understanding immune class regulation, and the remarkable anti-inflammatory properties of human IgG4 antibody. Secondly, we assessed the role of class II MHC molecules in determining the number of mature CD4 T cells and so affecting the Th1/Th2 phenotype of immune responses. We also discussed the controversial role of CD8 T cells in affecting the Th1/Th2 phenotype of responses to MHC and other antigens, and the potential role of their relative scarcity in neonates in biasing responses towards an antibody, Th2 mode. Lastly, we examined the regulation of the Th1/Th2 phenotype of both primary and ongoing immune responses in the context of the intriguing proposal that antigen initially generates different classes/subclasses of immunity and then selects, by a feedback mechanism, the most effective class. We found this interesting idea difficult to reconcile with various observations.

Immunological Tolerance. Part I of a Report of a Workshop on Foundational Concepts of Immune Regulation.

This report, the first of two, arose from a one-week workshop directed at discussing concepts of immune regulation, and focuses on immunological tolerance. We first outline the major ideas we thought sufficiently plausible to provide a context for discussing more controversial issues around tolerance. We then report on our discussion of different experiments that appear paradoxical in terms of the different, contemporary models of CD4 T cell inactivation/activation, and how such observations might be resolved in terms of insights provided by these contemporary models. These discussions bear on the plausibility of the Pathogen-Associated Molecular Pattern (PAMP), Danger and Two Step, Two Signal Models for the activation of naïve CD4 T cells. Some of the observations considered appear paradoxical in terms of the PAMP and Danger Models, but not with the Two Step, Two Signal Model. For example, genetically immunodeficient mice have been given foreign, sterile ectopic grafts, and the immune system allowed to develop once these grafts were well-healed in, and so in the absence of PAMPs or danger. The grafts were rejected, unexpected on the PAMP or Danger Models. We also discussed considerations and observations bearing on the widely held idea that antigen must crosslink the membrane Ig receptors of a B cell to initiate the generation of signal 1, or the alternative possibility that monovalent binding by antigen can do so. We favored the latter possibility, and discussed a particular model, "the Elbow Model," for how this might be achieved.

First Report of Phytophthora × pelgrandis Causing Root Rot and Lower Stem Necrosis of Common Box, Lavender and Port-Orford-Cedar in Hungary.

In 2008 and 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown common box (Buxus sempervirens L.), lavender (Lavandula angustifolia Mill. 'Hidcote'), and Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Columnaris') in three ornamental nurseries in western Hungary. Number of affected plants ranged from approximately 100 (Port-Orford-cedar) to 250 (lavender). Isolations from necrotic root collars of each host plant species yielded four Phytophthora isolates developing uniform colonies on carrot agar with a maximum growth temperature of 35 to 36°C. The isolates were homothallic with smooth-walled oogonia (32.2 ± 2.3 to 35.9 ± 3.5 µm), aplerotic oospores (27.5 ± 1.8 to 32.1 ± 3.1 µm) and both amphigynous and paragynous antheridia, and produced chlamydospores (25.8 ± 3.9 to 29.1 ± 5.2 µm) and papillate sporangia (35.2 ± 2.5 to 43.5 ± 5.6 µm long and 27.6 ± 2.2 to 32.0 ± 3.8 µm wide), mostly obpyriform to nearly spherical or rarely distorted with two or three apices. In spring water, sporangia were both caducous with short pedicel and non-caducous. Multiplex ITS-PCR assay of DNA from all isolates, using primers specific for P. nicotianae (NICF1 and NICR2.1) and P. cactorum (CACTF1 and CACTR1) (1), amplified DNA fragments of the expected size for each Phytophthora species. In addition, isoenzyme analysis revealed a characteristic banding pattern of one heterodimer and two homodimer bands at both loci of the dimeric enzyme malate dehydrogenase. These bands comigrated with those of P. × pelgrandis (Gerlach et al.) (CBS 123385) and isolate PD 93/1339 (courtesy of W. A. Man in 't Veld), two natural hybrid strains of P. nicotianae and P. cactorum (2,3), proving that our four isolates can be referred to as this interspecific hybrid. Pathogenicity was tested on 1- or 3-year-old plants of the original host species and cultivars (for common box, cv. Faulkner was used). Cultures were grown for 4 to 6 weeks at 20°C on autoclaved millet grains moistened with V8 broth. Infested and uninfested grains were mixed with autoclaved soil in a ratio of 6% (w/v), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth chamber (20°C, 70% RH, 12-h photoperiod). Pots were flooded for 24 h on the 1st and 21st day after transplanting. All plants in infested potting mix showed symptoms of wilt associated with basal stem and root necrosis, similar to those observed on the plants from the field, within 2 and 3 months on lavender and both common box or Port-Orford-cedar, respectively. Additionally, a reduction of root weight ranging from 35 to 68% compared to the control was recorded. Growth reduction was significant at P ≤ 0.019 according to Mann Whitney test. Control plants remained healthy. The same Phytophthora hybrid was reisolated solely from inoculated plants. In Europe, hybrid isolates of P. nicotianae × P. cactorum have been reported on several ornamental plants, including lavender, in the Netherlands and Germany (2,3). However, to our knowledge, this is the first report of this hybrid in Hungary and as a pathogen of common box and Port-Orford-cedar in the world. References: (1) P. J. M. Bonants et al. Phytopathology 90:867, 2000. (2) W. A. Man in 't Veld et al. Phytopathology 88:922, 1998. (3) H. I. Nirenberg et al. Mycologia 101:220, 2009.

Alloreactivity: an old puzzle revisited.

Alloreactivity, defined as a strong primary T cell response against allelic variants of major histocompatibility complex (MHC) molecules in the species, has been a long-standing puzzle in immunology with some of its details remaining unclear up to now. Here I shall provide a historical overview of how our understanding of alloreactivity has evolved and propose an interpretation that considers alloreactivity to be a mixture of four mechanistically distinct prototypes of T cell response, namely, self-restricted peptide specific, allorestricted peptide specific, alloreactive peptide dependent and alloreactive peptide independent. The relative contribution of each prototype to a given alloresponse is dependent on the extent of disparity (i.e. the number and nature of amino acid substitutions in the docking surface for T cell receptor) between the MHC molecule that the T cell recognizes as self and the stimulating MHC molecule.

First Report of Phytophthora citrophthora Causing Root and Basal Stem Rot of Woody Ornamentals in Hungary.

From 2007 to 2009, necrotic bark lesions at the root collar and lower stem associated with root rot were observed on container-grown cork-bark fir (Abies lasiocarpa var. arizonica, Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Barabits Gold'), lavender (Lavandula angustifolia Mill. 'Hidcote'), flowering currant (Ribes sanguineum Pursh 'King Edward VII'), and lilac (Syringa vulgaris L. 'Belle de Nancy') in six ornamental nurseries in western Hungary. Symptoms also included reduced growth, wilting, and desiccation of branches. Mortality of affected plants ranged from a low level in flowering currant to a high frequency in cork-bark fir (1,000 plants; 50%) and lavender (2,500 plants; 50%). Isolations from necrotic tissues onto PARPB medium (1) yielded 13 isolates of a Phytophthora sp. None of the isolates produced sexual structures, sporangia, or chlamydospores but developed slightly stellate or patternless colonies with loose, aerial mycelium on nonselective carrot agar (CA) at 25°C. One isolate from each host species was further characterized and tested for pathogenicity. Growth on CA was fastest at 25°C (7.9 to 8.6 mm per day) and no growth occurred below 5°C or above 34°C. In nonsterile stream water, persistent, mono- and bipapillate, mostly ovoid, rarely distorted sporangia, measuring 40.5 to 49.4 × 29.8 to 37.3 µm were produced. In pairings on CA with A1 and A2 strains of P. cambivora and P. nicotianae, used as testers, none of our isolates produced gametangia. On the basis of these characteristics, the pathogen from ornamentals appeared to be P. citrophthora (Smith & Smith) Leon. (1). Species identity of all 13 isolates was determined in single-round PCR assays with the P. citrophthora-specific primer-pair Pc2B/Pc7 (2) and/or by sequencing the rDNA internal transcribed spacer (ITS) regions amplified with the universal ITS1/ITS4 primers. The primers Pc2B and Pc7 generated a single DNA fragment of the expected size (approximately 210 bp), and the rDNA ITS sequences (NCBI Accession Nos. GU723282, GU723284, GU723285, and GU723287) showed 99 to 100% homology with many GenBank sequences (e.g., HQ697232) of P. citrophthora as the closest match. Pathogenicity to the original host plant cultivars was tested on 2- or 3-year-old healthy plants potted in sterile soil and inoculated at the root collar (2 replicates per isolate). A 4-mm-diameter bark plug was removed and a mycelial disc of the same size from an actively growing CA culture was placed into the hole. Control plants received sterile CA plugs. Inoculation points were sealed with sterile moist cotton and Parafilm, covered with sterile soil, and then the plants were kept in a greenhouse at 24 ± 4°C. Symptoms identical to those observed on the naturally diseased hosts developed on inoculated plants within 3 months. Control plants remained healthy. P. citrophthora could be reisolated only from the infected plants. The pathogen is polyphagous, widely distributed (1), and has been associated with woody ornamentals in nurseries (3). However, to our knowledge, this is the first record of P. citrophthora in Hungary. The pathogen has to be considered as a threat to ornamental production within Hungary. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) A. Ippolito et al. Eur. J. Plant Pathol. 108:855, 2002. (3) B. W. Schwingle et al. Plant Dis. 91:97, 2007.

Evolutionary origin of the immune system: a dialogue with Rod Langman.

The need of a specific defence mechanism against intracellular pathogens is proposed to have arisen very early in evolution, perhaps already in protozoa, e.g. amoebae. The phagocytic machinery of amoebae lends itself as a possible starting point for the evolution of such a mechanism. The hypothetical evolutionary pathway described here has been constructed to demonstrate the feasibility of developing a defence system against pathogens in the amoeba, which bears resemblances to contemporary cell-mediated immunity, and can thus be considered as its ancestor.

First Report of Phytophthora citricola on False Cypress in Hungary.

In May 2005, an estimated 10 to 15% mortality of various cultivars of false cypress (also named Lawson cypress or Port-Orford-cedar [Chamaecyparis lawsoniana]) with severe wilt was observed in field stands of an ornamental nursery in western Hungary. Wilted plants had rot-associated reduction of their root system. Root discoloration and occasional chlorosis of lower leaves commenced on potted 3-year-old plants that were held in the open air for 10 to 12 months before planting. Four species of Phytophthora (P. lateralis, P. eriugena, P. hibernalis, and P. cinnamomi) have been reported on this host (2). Direct plating of discolored roots from the most susceptible cultivar (Silver Globus) onto a selective potato dextrose agar or carrot agar medium yielded pure cultures that developed white, stellate colonies with sparse aerial mycelia. The hyphal growth was optimal at 25°C, but the growth above 32°C and below 4°C was completely inhibited. Single, terminal sporangia on simple (occasionally sympodial) sporangiophores formed abundantly in nonsterile soil filtrate but not in agar. Sporangia, 31 to 67 µm (59.1 ± 9.3 µm) long and 25 to 39 µm (31.5 ± 4.0 µm) wide, were noncaducous and semipapillate, variable in shape, mostly obpyriform, rarely obovoid, ovoid-ellipsoid and spherical or bifurcated and distorted, and the exit pore was narrow (7.2 ± 0.8 µm). No external or internal proliferation and no hyphal swellings or chlamydospores were observed. The isolates were homothallic with smooth-walled oogonia (27.3 ± 3.4 µm in diameter) and paragynous antheridia. The oospores (24.7 ± 2.1 µm in diameter) were plerotic. The morpho-physiological features suggested that our isolates belonged to Waterhouse's Group III, and in particular, represented P. citricola. This was confirmed by cellulose acetate electrophoresis of malate dehydrogenase; the isozyme pattern of false cypress isolate was identical to that of the ITS-sequenced (NCBI Accession No. AY366193) P. citricola isolate from a Hungarian alder forest (1). Pathogenicity tests on four 3-year-old potted false cypress (cv. Silver Globus) plants in the greenhouse resulted in rapidly developing (within 2 weeks) sunken, necrotic lesions at the stem base around the site of wound inoculation with a 5-mm-diameter mycelial agar plug. After 12 weeks, each inoculated plant wilted and died. The causal agent was consistently reisolated from necrotic tissues. In Hungary, P. citricola was first isolated and identified from alder forest soil (1). Nonetheless that false cypress has been listed as the host of P. citricola in Norway and Poland (3,4), to our knowledge, this report is the first definitive description of this Phytophthora sp. on this host. References: (1) J. Bakonyi et al. Plant Pathol. 52:807, 2003. (2) D. C. Erwin and O. K. Ribeiro. Pages 282-287 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) V. Talgø V. and A. Stensvand. Grønn kunnskap e 7(101G):1, 2003. (4) K. Wiejacha et al. Page 45 in: Improvement and Unification of Plant Disease Diagnostics. Abstracts of International Workshop, Skierniewice, Poland, 2004.

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Binding affinity independent contribution of peptide length to the stability of peptide-HLA-DR complexes in live antigen presenting cells.

The effect of peptide length on the stability of peptide-HLR-DR1 (DR1) complexes was analyzed using two peptide series of increasing length, each containing a 7mer core with five DR1-binding anchors, extended stepwise with Ala residues at the N- and C-terminus, respectively. The Ala extensions, although did not affect binding affinity, significantly increased the half lives of peptide-DR1 complexes (from 1.5 h up to 10 h) in live antigen presenting cells (APC). Flanking residues from position -2 to 0 and 8 to 11 were involved in the affinity-independent increase of complex stability. The shortest (8mer and 9mer) peptides, with in vivo half lives of <2.5 h, were unable to form stable complexes with DR1 in presence of HLA-DM (DM) molecules, and were poor competitors of antigen presentation. Longer peptides were resistant to DM-mediated unloading, and were efficient competitors of antigen presentation. Thus, DM appears to limit short peptides in establishing biologically relevant DR occupancy, despite their high binding affinity. In APC, stable complexes can form only with high affinity peptides of >9 residues, and the longevity of complexes seems to depend on full of occupation of the binding site.

Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis.

Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A homology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease.

Peptide dependence of major histocompatibility complex class II specific alloreactive responses.

Splenic cells from transgenic mice, in which a single peptide is complexed to all major histocompatibility complex (MHC) class II molecules, are found to be incapable of triggering primary allogeneic mixed lymphocyte/leucocyte reactions (MLR) when co-cultured with lymphocytes from MHC class II congenic mouse strains. In addition, a single HLA-DR-blocking peptide can completely abrogate the capacity of splenocytes from chimeric HLA-DR/H2-E transgenic mice to stimulate primary MLR of T cells from wild-type mice. These results indicate that the primary alloreactive response is directed against a multitude of peptides presented by allogeneic MHC molecules.

Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires.

The role of HLA-DQ molecules in Ag presentation has, thus far, remained elusive. Here we report that two DQ allotypes, DQ7 (DQA1*0501/B1*0301) and DQ9 (DQA1*0201/B1*0303), are capable of binding peptide repertoires in complementarity with DR molecules. The results reflect fundamental differences in the binding modes of these two HLA class II isotypes, in that DQ7 and DQ9 but not DR molecules appear to have the capacity to bind peptide structures without type 1-like anchor residues. Consistent with this is our observation that none of the amino acid side chains of the class II-associated invariant chain peptides (CLIP) are required for association with DQ7 and DQ9, even though many of them are essential for CLIP-DR interaction. Together, these data reveal a functional complementarity of HLA-DR and -DQ molecules in Ag presentation.

The helper T-cell repertoire of mice expressing class II major histocompatibility complex beta chains in the absence of alpha chains.

Mutant mice generated by disrupting the H2-Aab major histocompatibility complex (Mhc) gene are demonstrated here to express Abetab chains in the absence of alpha chains. These mice possess a CD4(+) helper T cell (Th) repertoire that uses predominantly the Vbeta7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the alpha-free Abeta molecule. As an alloantigen, the Aalpha-free Abeta molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal alpha/beta heterodimers. Indeed, molecular modeling suggests a beta/beta homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vbeta usage, toward a series of foreign alpha/beta class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal alpha/beta heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature.

T cell repertoire: genomic or somatic bias toward recognition of major histocompatibility complex molecules?

The prevailing concept about a major influence of thymic positive selection on shaping the T cell repertoire during ontogeny is confronted with an old idea emphasizing a dominant role for genetic (evolutionary) factors in molding the recognition potential of mature T cells. Our recent results are not readily interpreted without introducing a new version of the old concept, according to which complementarity to the major histocompatibility complex peptide-binding site is a major evolutionary selective pressure on T cell antigen receptor variable genes, with alloreactivity being a reflection of this fact.

Influence of CD26 and integrins on the antigen sensitivity of human memory T cells.

The antigen sensitivity of class II MHC restricted human CD4 T-cell clones is demonstrated to increase gradually with time after restimulation. This is manifested in a requirement of less antigen in culture, as well as decreased numbers of peptide-MHC complexes per APC for T-cell activation, and in an increased resistance to inhibition by class II MHC blockade. The increase in antigen sensitivity is accompanied by increased cell-surface expression of CD26, LFA-1, and VLA-1, whereas the expression of TCR and a series of other cell-surface molecules remains unchanged. Using appropriate monoclonal antibodies, we have shown that CD26 and LFA-1 contribute directly to the increased antigen sensitivity of "late-stage" T-cell clones. The late-memory T-cell phenotype established in this study is shown to occur also among T cells activated in vivo. We suggest that increasing the antigen sensitivity via antigen-nonspecific molecules is a physiologic mechanism for maintaining T-cell memory in face of decreasing antigen concentration, and for ensuring preferential activation of memory T cells upon repeated encounter with antigen.

Induction of class II major histocompatibility complex blockade as well as T cell tolerance by peptides administered in soluble form.

Peptides binding to a particular class II major histocompatibility complex (MHC) molecule can inhibit the activation of T cells by other peptides binding to the same molecule, a phenomenon termed class II MHC blockade. All class II-binding peptides exert MHC blockade in vivo in depot form with adjuvant, and some also retain their blocking properties in soluble form. We demonstrate here that soluble peptides, when used at doses causing short-term MHC blockade, can also induce long-term antigen-specific T cell tolerance to themselves. The tolerogenicity of soluble peptides correlates with their antigenicity in adjuvant, but it is not necessarily related to their capacity to act as class II blockers in vivo. The tolerant state is manifested in a decreased production of both T helper cell 1 (Th1)-type and Th2-type lymphokines, and it cannot be reversed by interleukin-2. Once T cells are primed with a peptide in complete Freund's adjuvant, they are resistant to tolerization with the same peptide applied in soluble form. Tolerance induction is partially impaired in B cell-deficient mu MT-/- mice, suggesting a role for B cell antigen presentation in this process. The results suggest that the potential immunogenicity of class II MHC blockers could be circumvented by choosing a tolerogenic mode of application.