Development of a DIVA (Differentiating Infected from Vaccinated Animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza

The present paper reports of the development and validation of a control strategy for avian influenza infections in poultry. The "DIVA" (Differentiating Infected from Vaccinated Animals) strategy is based on the use of an inactivated oil emulsion vaccine containing the same haemagglutinin (H) subtype as the challenge virus, but a different neuraminidase (N). The possibility of using the heterologous N subtype, to differentiate between vaccinated and naturally infected birds, was investigated through the development of an "ad hoc" serological test based on the detection of specific anti-N1 antibodies. This was achieved using a baculovirus expressing a recombinant N1 protein. The A/ck/Pakistan/H7N3 virus was used as a vaccine and birds were challenged with the HPAI A/ty/Italy/4580/V99/H7N1 strain. The homologous H group ensured a clinical protection of 93% regardless of the vaccination scheme used, and was able to prevent viraemia and muscle colonization in the clinically healthy challenged birds. However, it was not able to prevent viral shedding. The "ad hoc" serological assay was developed as an indirect immunofluorescence test, and was validated using 608 field sera, and showed an "almost perfect agreement" (Kappa value) with the HI test, with relative sensitivity and specificity values of 98.1 and 95.7, respectively. The results of the present investigation suggest that the "DIVA" control strategy may represent a tool for the control of avian influenza infections in poultry.     

Levothyroxine treatment in thyroid peroxidase antibody-positive women undergoing assisted reproduction technologies: a prospective study

BACKGROUND: Infertile women positive for thyroid antibodies suffer from a poor pregnancy/delivery outcome, although conflicting data have been published. Our objective was to investigate if levothyroxine (LT4) exerts any effect on pregnancy and/or delivery rates in thyroid peroxidase antibody (TPOAb)-positive (+) women undergoing assisted reproductive technologies. METHODS: Patients undergoing treatment were screened for TPOAb, thyroid-stimulating hormone (TSH) and free thyroxine (FT4). A total of 72 (15%) out of the 484 euthyroid women selected were TPOAb (+). These 72 patients were randomly divided into two groups: group A (n = 36) underwent LT4 treatment, group B (n = 36) placebo. Group C consisted of 412 women (85%) who were TPOAb negative (-). All patients received controlled ovarian stimulation. The endpoints of treatment were pregnancy rate, miscarriage rate and delivery rate. RESULTS: No differences in pregnancy rate were observed between the three groups. Miscarriage rate was higher in TPOAb (+) in comparison to TPOAb (-) [relative risk: 2.01 (95% CI = 1.13-3.56), P = 0.028]. CONCLUSIONS: The pregnancy rate is not affected either by presence of TPOAb or treatment with LT4. However, TPOAb (+) women show a poorer delivery rate compared to TPOAb (-). LT4 treatment in TPOAb (+) does not affect the delivery rate.     

Qualitative difference between the cytotoxic T lymphocyte responses to melanocyte antigens in melanoma and vitiligo

Vitiligo is a skin disorder characterized by depigmented macules secondary to melanocyte loss. An unusual facet is its relation to melanoma: cytotoxic T lymphocytes directed to melanocyte antigens are found in both conditions and imply a breakdown of tolerance, yet the resulting immune reaction is the opposite. The mechanisms at the basis of these opposite effects are not known. Here, we performed a direct comparison of whole melanocyte-specific T cell populations in the two diseases. We demonstrate that neither precursor frequencies of Melan-A/MART-1-specific T lymphocytes nor their status of activation differ significantly. However, by using a tetramer-based T cell receptor down-regulation assay, we documented a higher affinity of vitiligo T cells. We calculated that the peptide concentration required for 50% of maximal receptor down-regulation differed by 6.5-fold between the two diseases. Moreover, only vitiligo T cells were capable of efficient receptor down-regulation and IFN-gamma production in response to HLA-matched melanoma cells, suggesting that this difference in receptor affinity is physiologically relevant. The differences in receptor affinity and tumor reactivity were confirmed by analyzing Melan-A/MART-1-specific clones established from the two diseases. Our results suggest that the quality, and not the quantity, of the melanocyte-specific cytotoxic responses differs between the two pathologies.     

Intracellular antibodies for proteomics

The intracellular antibody technology has many applications for proteomics studies.

The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology.

We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins.

The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.     

A double cryptic chromosome imbalance is an important factor to explain phenotypic variability in Wolf-Hirschhorn syndrome

A total of five Wolf-Hirschhorn syndrome (WHS) patient with a 4p16.3 de novo microdeletion was referred because of genotype-phenotype inconsistencies, first explained as phenotypic variability of the WHS. The actual deletion size was found to be about 12 Mb in three patients, 5 Mb in another one and 20 Mb in the last one, leading us to hypothesize the presence of an extrachromosome segment on the deleted 4p. A der(4)(4qter --> p16.1::8p23 --> pter) chromosome, resulting from an unbalanced de novo translocation was, in fact, detected in four patients and a der(4)(4qter --> q32::4p15.3 --> qter) in the last. Unbalanced t(4;8) translocations were maternal in origin, the rec(4p;4q) was paternal. With the purpose of verifying frequency and specificity of this phenomenon, we investigated yet another group of 20 WHS patients with de novo large deletions (n = 13) or microdeletions (n = 7) and with apparently straightforward genotype-phenotype correlations. The rearrangement was paternal in origin, and occurred as a single anomaly in 19 out of 20 patients. In the remaining patient, the deleted chromosome 4 was maternally derived and consisted of a der(4)(4qter --> 4p16.3::8p23 --> 8pter). In conclusions, we observed that 20% (5/25) of de novo WHS-associated rearrangements were maternal in origin and 80% (20/25) were paternal. All the maternally derived rearrangements were de novo unbalanced t(4;8) translocations and showed specific clinical phenotypes. Paternally derived rearrangements were usually isolated deletions. It can be inferred that a double, cryptic chromosome imbalance is an important factor for phenotypic variability in WHS. It acts either by masking the actual deletion size or by doubling a quantitative change of the genome.     

A hyperpolarization-activated chloride current in xenopus laevis oocytes under voltage-clamp

Voltage-clamp experiments have been performed on ovulated Xenopus laevis oocytes. Two kinds of oocytes were studied: mature oocytes presenting the white spot due to germinal vesicle breakdown (GVBD) were found to be essentially unexcitable; oocytes without the white spot appeared to possess ionic channels which open transiently on hyperpolarization. The current is carried by chloride ions and, in Holtfreter's solution, has a reversal potential at approximately - 15 mV.     

CD1a and antitumour immune response

Primary immune response is based on the capacity of local professional antigen-presenting cells (whose prototype is represented by dendritic cells, DCs) to take up and present antigens to selected clones of T cells, but also to non-specific effector cells such as macrophages or natural killer cells. The four CD1 proteins, all of which share a limited homology to class I MHC proteins, are differently expressed in various cell types, of both mesenchymal and, as recently described, epithelial lineage. Regarding the role of CD1 molecules in the anti-tumour response, it has been reported that CD1+ dendritic cells are involved in the first steps of the primary immune response in a number of malignancies. Moreover, the presence of a high number of DCs in the tumoral or peritumoral area, as well as in the draining lymph nodes, has been shown to correlate with a better prognosis. A recent report on the presence of CD1a in metaplastic epithelial cells of Barrett esophagus introduced new questions about CD1a expression patterns. Moreover, the strong correlation between the lack of CD1a+ cells and the malignant evolution of the lesion may indicate a possible role of non-professional APCs in mediating and/or potentiating immune responses to tumours.     

Towards improved clinical characterization of Leber congenital amaurosis: neurological and systemic findings

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy that presents in infancy. LCA is both clinically and genetically heterogeneous. The aim of our study was to clarify the clinical aspects of LCA and to contribute to improved characterization of the disorder. We studied 40 children affected by LCA (mean age at first observation: 19 months, range: 8-50 months), who underwent a comprehensive evaluation that included: neurophthalmological evaluation, electroretinogram (ERG), and visual evoked potentials (VEPs), general and neurological examinations, developmental assessment using scales for visually impaired children, neuroradiological examinations, hepatic and renal function and metabolic investigations, brainstem auditory evoked potentials (BAEPs), EEG, and hand radiographs. Analyses of known LCA genes are ongoing. The subjects are still being followed up at 6-/12-month intervals. All the subjects fulfilled De Laey's criteria for LCA. The neurological examination was abnormal in 31 cases (hypotonia, ataxia with/without associated cerebellar signs). Cognitive development was normal in 24 cases, borderline in five, and subnormal in 11. Mild and nonspecific alterations on MRI were present in seven cases, and "molar tooth" sign in four; all the others had a normal neuroradiological picture. Among the subjects presenting with neurological signs, a subgroup (13 patients) emerged that was characterized by systemic (skin, kidney, liver) involvement. Our data confirm that LCA is a heterogeneous entity that can present as an isolated ocular manifestation, or in association with neurological and systemic abnormalities and support the need for a multidisciplinary approach to this entity and for genotype-phenotype studies.     

Echocardiography and Doppler sonography in the evaluation of cardiac structure and function

Physical activity increases the work load of the heart. The adjustments of the heart depend on the quality and quantity of the work performed. These adjustments concern the function and the morphology of the cardiovascular system. It is important to underline that these adjustments are not permanent and can disappear when physical activity is stopped. In young subjects the risks are very few while the benefits may be shown on a better and more armonic body structure. In the elderly the benefits can be achieved with a lesser cost for submaximal activities, but the risks are of course more frequent due the possible onset of cardiovascular disease. It is important to correctly recognize the limits whitin which the physical activity can be allowed because beneficial. Echocardiography has given an important contribution to evaluate the morpho-functional adaptions of the athlete's heart. Similarly, it has proven useful in the detection of pathological cardiovascular modifications, asymptomatic or pausymptomatic, that do not allow certification to practise sport at agonistic levels.     

Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP-specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA-DR2⁺, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16-38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86-99 was detected in the two other DR2⁺ patients. In each of the patients with other HLA-DR haplotypes (DR2^?), as well as in three DR2⁺ non-MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2⁺ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA-DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA-DRB1 gene in individuals with the HLA-DR2 serological specificity is also described. We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA-DR2 gene products alone do not account for the selection of the dominant MBP Tcell epitope.