Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis.

Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.

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Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy.

The intracellular localization of viroids has been investigated by viroid-specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine--rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC-conjugated antibody. In plants infected with either a lethal or an intermediate PSTVd strain, the highest intensity of fluorescence that arose from hybridization with the probe specific for the viroid (+)strand was found in the nucleoli, confirming results of previous fractionation studies. A similar distribution was found for (-)strand replication intermediates of PSTVd using specific (+)strand transcripts as hybridization probes. In order to determine if viroids are located at the surface or in the interior of the nucleoli, the distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM). It was shown by three-dimensional reconstruction that viroids are neither restricted to the surface of the nucleoli nor to a peripheral zone, but are instead homogeneously distributed throughout the nucleolus. The functional implications of the intranucleolar location of viroids and their replication intermediates are discussed with respect to proposed mechanisms of viroid replication and pathogenesis.

HLA-DQ8 transgenic mice lacking endogenous class II molecules respond to house dust allergens: identification of antigenic epitopes.

We have introduced HLA-DQ8 (HLADQB*0302 and HLA-DQA*0301) genes into A beta 0 knockout mice. Transgenic animals were immunized with a whole body extract of Dermatophagoides pteronyssinus (Der p), one of the causative agents of house dust mite allergy. Transgenic mice expressing HLA-DQ8 genes elicited HLA-DQ8-restricted responses driven by CD4+ T cells. Synthetic-overlapping peptides representing a major allergen of house dust mite (Der p 2) were synthesized and used as immunogens. HLA-DQ8+ mice responded to three peptides: 8 (residues 61-80), 11 (residues 91-110), and 13 (residues 111-129). The mice produced IL-2, IL-4, and IL-6 response to Der p challenge, suggesting a mixed Th1/Th2 response. These mice represent a new model for studies of the immune basis of allergy.

Immune response of HLA-DQ transgenic mice to house dust mite allergen p2: identification of HLA-DQ restricted minimal epitopes and critical residues.

HLA-DQ8 (HLA-DQA1*0301; HLA-DQB1*0302) and HLA-DQ6 (HLA-DQA1*0103; HLA-DQB1*0602) genes were introduced into mouse class II (H-2A(o)(beta)) knockout mice. Transgenic HLA-DQ8 and HLA-DQ6 mice were individually immunized and challenged using synthetic peptides representing HDM (Dermatophagoides pteronyssinus) allergen p2. HLA-DQ8 mice responded to p2 peptides 1-20, 41-60, 51-70, 61-80, 91-110, and 101-120. HLA-DQ6 mice responded to peptides 1-20, 11-30, 21-40, 41-60, and 51-70. Using single amino acid truncated 30-mer peptides, residues necessary for HLA-DQ8 recognition were identified spanning regions 3-12, 50-70, and 91-120. A synthetic peptide comprising residues 3-12 was synthesized and a series of single alanine substitutions was introduced into the minimal peptide. Introduction of alanine residues at positions 3, 11, and 12 resulted in a significant loss of immune recognition. It was concluded that residues 4, 5, 7, 11, and 12 are critical for immune recognition by HLA-DQ8 mice.

HLA-DQ6 and HLA-DQ8 transgenic mice respond to ragweed allergens and recognize a distinct set of epitopes on short and giant ragweed group 5 antigens.

We have investigated the genetic and molecular basis of immune responsiveness to short ragweed (SRW) (Ambrosia artemisiifolia) extract, and group 5 allergens from short and giant (Ambrosia trifida) ragweed using transgenic mice expressing DQ6 (HLA-DQA1*0103, HLA-DQB1*0601) and DQ8 (HLA-DQA1*0301, HLA-DQB1*0302) genes in class II knockout (A beta0) mice. Panels of overlapping peptides spanning the Amb a 5 and Amb t 5 Ags were synthesized. Mice were immunized with whole SRW extract or individual peptides s.c. and lymph node cells (LNC) were challenged in vitro. Strong T cell responses to SRW extract were measured in both HLA-DQ transgenic mice, while control, HLA-DQ6-/DQ8-/H-2A beta0, mice were unresponsive. IL-5 and IL-10 were the primary cytokines produced by in vitro challenged LNC of SRW-primed transgenic mice. HLA-DQ6-restricted T cell responses were detected to all three peptides of Amb t 5 and two determinants (residues 1-20 and 11-30) on Amb a 5. In contrast, LNC of HLA-DQ8 mice did not recognize peptide 11-30 of Amb t 5 Ag, but recognized several Amb a 5 determinants. The immune response in transgenic mice was dependent upon CD4+ T cells and was HLA-DQ restricted. Primed with purified Amb t 5, both transgenics recognized peptide 21-40, and an additional DQ6-restricted epitope was found within residue 1-20. SRW-immunized HLA-DQ6 mice respond to peptide 11-30 of Amb a 5, while HLA-DQ8 mice strongly recognize peptide 1-20. These results demonstrate the specificity of HLA class II polymorphism in allergen sensitivity and pave the way for developing antagonistic peptides for desensitization.

Identification of T cell determinants on human type II collagen recognized by HLA-DQ8 and HLA-DQ6 transgenic mice.

HLA-DQA1*0301 and HLA-DQB1*0302 genes encoding the HLA-DQ8 molecule and HLA-DQA1*0103 and HLA-DQB1*0601 genes encoding the HLA-DQ6 molecule were introduced into H-2Abetao knockout mice. Three lines of transgenic mice were established: HLA-DQ8, HLA-DQ6, and HLA-DQ8beta6alpha. HLA-DQ8 mice are susceptible to collagen-induced arthritis, while HLA-DQ6 mice are resistant. HLA-DQ8beta6alpha mice develop polychrondritis in addition to arthritis. Transgenic mice were primed and challenged with individual synthetic peptides representing human type II collagen. A total of 101 synthetic peptides were tested in each transgenic line of mice. HLA-DQ8 mice responded to 15 synthetic peptides representing all cyanogen bromide fragments. In contrast, HLA-DQ6 mice responded to a subset of the peptides recognized by HLA-DQ8 T cells. HLA-DQ8beta6alpha mice, although exhibiting diminished responses to the majority of HLA-DQ8-restricted determinants, elicited enhanced responses to two peptides. In addition, HLA-DQ8beta6alpha mice respond to two unique peptide determinants contained within cyanogen bromide fragments CB10 and CB11 showing the significance of mixed isotype dimers in the immune response. The determinants recognized by the HLA-DQ transgenic mice are distinct from those previously identified using conventional laboratory mice. These results suggest that human class II transgenic mice offer a means of identifying human class II-restricted epitopes associated with potential human autoantigens.

Characterization of the antigenic structure of human type II collagen.

A series of 101 peptides each 20 amino acids in length (10-residue overlap) spanning the helical portion of the mature alpha-chain of human type II collagen (CII) was synthesized. DBA/1 (H-2q) mice were immunized with individual peptides, and draining lymph node cells were challenged in vitro. Strong responses were measured to three peptides: peptide I (residues 74-93), peptide 14 (residues 254-273), and peptide 81 (residues 924-943). B10.Q (H-2q) mice were responsive to peptides I and 81 but not to peptide 14. B10.RIII (H-2r) mice, which are resistant to arthritis induction following immunization with human CII, were unresponsive to peptides I, 14, and 81. Using single amino acid truncated peptides, we determined minimal immunostimulatory lengths for peptides I and 81. Residues critical to antigenicity were identified by introducing alanine and glycine substitutions into minimal length immunostimulatory peptides. The determinants within peptides I and 81 are 100% homologous to mouse CII and are autoantigens. Peptide 81 has homology to viral proteins. Peptide 14 is 90% homologous to mouse CII and has homology to heat shock proteins.