Manipulating the pathway of ammonia assimilation in transgenic non-legumes and legumes

In this paper we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation has been increased by the use of non-leguminous and leguminous plants with genetically-altered capacities for ammonia assimilation. Using tobacco or Lotus as model plants, Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. In a few selected examples, the physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes. The use of such genetically-modified plants will allow us to better understand the molecular control of this metabolic pathway. It is also potentially of great importance in agriculture if such internal and stable modifications are beneficial in terms of nitrogen use efficiency, thus avoiding an excessive utilization of fertilizers or herbicides (GS inhibitors). Our current knowledge and prospects for future development are explored.     

Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus

The majority of human patients with pemphigus foliaceus (PF) have circulating IgG autoantibodies that target conformational epitopes on the desmosomal cadherin desmoglein-1 (dsg1). Limited studies using immunoblot techniques suggested that the principal autoantigen in dogs with PF might also be dsg1. It was the objective of this study to test this hypothesis. A comprehensive survey of canine PF sera was conducted using a novel screening strategy that detects conformational epitopes. This method consists of the ectopic expression of canine dsg1 at the surface of human 293T epithelial kidney cells and their live screening, i.e. prior to fixation. Out of seven control human PF sera that bound to canine epidermis, three (57%) contained IgG autoantibodies that recognized ectopically expressed canine dsg1 with a membrane and punctate pattern. Out of 83 canine PF sera only five (6%) contained IgG that recognized canine dsg1. Consistent with findings for human PF sera obtained in this study, autoantibody binding was conformation- and glycosylation-dependent as demonstrated by calcium chelation with EDTA and tunicamycin or wheat germ agglutinin treatment, respectively. In conclusion, these studies establish canine dsg1 as a minor autoantigen for canine PF. Antigenic epitopes appear to be conformation- and glycosylation-dependent.     

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria

Background – Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. Hypothesis/Objective – The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. Animals – Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. Methods – The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B cells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T‐helper 2 cytokines (interleukins IL‐4, IL‐5 and IL‐13), a T‐helper 1 cytokine (interferon‐γ), IL‐4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT‐PCR. Results – In subepidermal lesional skin of RU‐affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79‐positive (P ≤ 0.01), MAC387‐positive (P ≤ 0.01) and tryptase‐positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU‐affected horses contained more eosinophils (P ≤ 0.05) and tryptase‐positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL‐4 (P ≤ 0.01), IL‐13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL‐4 receptor α (P ≤ 0.05) was increased in lesional skin of RU‐affected horses compared with control horses. Expression of IL‐4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. Conclusions and clinical importance – Analysis of cytokine expression and inflammatory infiltrate suggests that T‐helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.     

Essential oil production increases value of Psiadia altissima fallows in Madagascar’s eastern forests

Fallow with Psiadia altissima is one of the most common post-‘slash and burn’ vegetation successions described in the evergreen forests of eastern Madagascar. Some fallows consist of almost pure stands of this species, of which the leaves produce an essential oil offering international commercial interest. The present research aims to evaluate the production potential of essential oil derived from different fallows rich in P. altissima. The study has revealed that fallows aged 4 and 6 years since the last crop abandonment produce the most essential oil (around 20 l ha⁻¹), but relative to fallow duration, the youngest fallows (1- or 2-year-old) are the most productive, respectively producing 12 and 6 l ha⁻¹ year⁻¹. Additionally, the trees from the youngest fallows have a substantial capacity for regeneration from coppice shoots, on condition that the cut is performed well above the root collar. Although farmers earn five times less from harvesting leaves than from cultivating rice from tavy, the possibility is there for them to complement their income and diversify their production. The overall results show that sustainable exploitation of fallows of P. altissima is a conceivable option. However, this can only be achieved through an integrated approach that takes into account the environmental and social constraints associated with the development of this new activity.     

Assessment of an optimized dog-culling program in the dynamics of canine Leishmania transmission

In Brazil, zoonotic visceral leishmaniasis (ZVL) control programs based on the mass elimination of seropositive dogs have failed to reduce the number of leishmaniasis cases. However, these programs have been done under sub-optimal conditions. We studied a cohort of dogs in an urban area in Brazil to determine, whether a dog-culling program optimized with: (i) replacement of a relatively low-sensitivity indirect immune-fluorescent test on blood eluate by a more sensitive enzyme-linked immunosorbent assay on serum blood samples; (ii) shortening of the time interval from serodiagnosis to removal of dogs; (iii) screening a high proportion of the dog population could reduce the incidence of canine Leishmania infection (CLI). The study ran from December 1997 to July 2000, with four follow-up assessments performed at approximately 8-month intervals. All dogs seropositive for anti-Leishmania antibodies were promptly eliminated. A large number of new dogs immigrated to the study area throughout the study period. They comprised 43.8-49.8% of the cohort at each follow-up assessment, and upto 15% of them already had Leishmania infection. Overall, 42 news cases of CLI were identified, for a crude incidence rate of 11.8 cases per 100 dog-years (95% CI 8.6-15.6). In the first, second, third and fourth follow-up assessments the incidence rates were 8.2 (95% CI 3.0-17.9), 12.2 (95% CI 6.3-21.2), 16.4 (95% CI 8.5-28.6) and 13.6 (95% CI 7.1-23.8), respectively. There was no statistically significant change in these rates throughout the study period. Our results suggest that dog-culling programs do not reduce the incidence of CLI, even with an optimized intervention. Possible reasons for this failure include: currently available serologic methods lack sufficient sensitivity and/or specificity to accurately identify all infected dogs warranting removal in order to prevent Leishmania transmission; destroyed dogs are immediately replaced by susceptible puppies, and quite often, by already infected dogs; and other reservoirs may be involved in maintaining canine infection. Further efforts on ZVL control should be directed to developing new strategies or to testing control methods already in place with properly designed trials.     

Advances in equine immunology: Havemeyer workshop reports from Santa Fe,New Mexico,and Hortobagy,Hungary

The horse has been human kind's most important partner throughout history. Similarly, in the field of immunology, many critical scientific advances have depended on the horse. Equine immunology today is an active and important field of study, with a focus on control of many common infectious diseases and immunopathologic conditions of broad comparative interest. In 2001 two major equine immunology workshops were held, in Santa Fe, USA, and in Hortobagy, Hungary, with major sponsorship from the Havemeyer Foundation. This report summarizes the scientific themes and foci of those meetings.     

In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells.We have analysed the ectopic expression of the human serum albumin gene in primary horse cells from different tissues. The vectors used are of pcDNA and pUC origin and include the cytomegalovirus (CMV) promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not.Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression in primary horse cells derived from skin, lung, duodenum and kidney.We report for the first time the significance of full consensus Kozak sequences for protein expression in horse cells in vitro.