Expression of IFNalpha-inducible genes and modulation of HLA-DR and thyroid stimulating hormone receptors in Graves' disease.

Interferon alpha (IFNalpha) plays an important role in the pathogenesis of different autoimmune diseases. IFNalpha is widely used for the treatment of chronic viral infections, particularly chronic hepatitis C virus infection; however, several case reports have emerged describing autoimmune conditions, such as Graves' disease (GD), that have developed in the patients receiving IFNalpha. The mechanism by which IFNalpha is involved in GD remains poorly understood. We investigated the expression of IFNalpha and IFNalpha-inducible genes (IFIGs) in GD and found that IFIGs were overexpressed in 60% of 54 clinical diagnostic GD patients. These elevated IFIGs correlated with serological levels of autoantibody to thyroid stimulating hormone receptor (TSHR). Recombinant human IFNalpha stimulated primary cultured thyrocytes resulted in not only high level expression of IFIGs, but also, more importantly, expression of MHC-II antigens (HLA-DR3 and HLA-DR5) and TSHR in GD subjects. Furthermore, thyroid gland tissues from GD patients over express HLA-DR, TSHR and IFNalpha receptors at both messenger RNA and protein levels. Taken together, these data indicated that in GD patients, IFNalpha can function on thyroid tissue to induce a number of genes, particularly MHC class II molecules which may enhance autoantigen presentation of TSHR on thyrocytes.

Allergic bronchopulmonary aspergillosis: a US perspective.

PURPOSE OF REVIEW: The present article is an update of allergic bronchopulmonary aspergillosis. Although a rare condition, allergic bronchopulmonary aspergillosis does affect a number of patients with asthma and cystic fibrosis. Prompt recognition and treatment of the disease is critical to improving patient outcomes. RECENT FINDINGS: There is currently much active research being performed in the area of allergic bronchopulmonary aspergillosis. Fascinating insights are being made into the pathophysiology and genetics of the disease. Additionally, research is ongoing on the use of recombinant Aspergillus allergens as an aid to the diagnosis of allergic bronchopulmonary aspergillosis. SUMMARY: These new insights into the genetics and pathophysiology of allergic bronchopulmonary aspergillosis and the development of these new diagnostic techniques could ultimately lead to improved patient treatment. These areas form an important basis for further research.

Studies of HLA associations in male and female patients with Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

HLA associations are found to differ with the gender of the patient in some autoimmune diseases. Here we have investigated whether there are gender-related HLA associations in Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), both of which occur more frequently in male patients than in females. In GBS, no particular HLA associations were noted, except for a slight negative association in both males and females for carriage of HLA-DR5. In CIDP, the gene frequency and the frequency of individuals positive for HLA-DR2 were greater in female patients than female controls, although this was statistically significant only for the gene frequency. Furthermore more female CIDP patients were homozygous for DR2, than male CIDP patients, or male or female controls and patients with GBS. This suggests that sex-related factors may interact with the risk associated with carriage of HLA-DR2 for development of CIDP.

Identification of two Melan-A CD4+ T cell epitopes presented by frequently expressed MHC class II alleles.

Because of its expression pattern restricted to cells of the melanocytic lineage and to melanoma cells, Melan-A is an important target of immunotherapeutic approaches for the treatment of melanoma. Identification of Melan-A derived sequences recognized by specific T cells is therefore of great interest for the development of these therapeutic strategies. Using circulating CD4(+) T cells from healthy donors, we identified two Melan-A-derived CD4(+) T cell epitopes mapping to the 1-20 and 91-110 regions of the protein and restricted by HLA-DR11 and HLA-DR52 molecules, respectively. CD4(+) T cells specific for the identified epitopes were able to recognize the native antigen when endogenously expressed by antigen presenting cells and tumor cells. In addition, CD4(+) T cells specific for Melan-A 91-110 recognized the epitope after exogenous processing and presentation of Melan-A recombinant protein. Identification of these epitopes will be instrumental for the evaluation of the immune response to Melan-A in cancer patients.

T-cell receptor bias in patients with allergic bronchopulmonary aspergillosis.

CD4(+) Th2 helper cell mediated immune responses have been shown to play a crucial role in the pathogenesis of ABPA. HLA and TCR are the candidate genes, which can influence the specificity of these responses. We have previously established a strong association of HLA DR2/5 in ABPA susceptibility. The study was designed to determine whether allergen specific T cell express a limited usage of T cell receptor (TCR) Vbeta gene repertoire in ABPA and to find an association of susceptible HLA-DR determinants with the identified TCR gene segments. TCR Vbeta typing was performed on antigen specific T cell lines from 14 ABPA and 12 nonABPA patients. The majority of ABPA patients (86%) expressed allergen specific T cells with Vbeta13 genes indicating its role in susceptibility, whereas in nonABPA controls, Vbeta1 genes T cell repertoires were predominantly expressed. The unrestricted pattern of Vbeta gene amplification seen before antigen stimulation suggests an oligoclonal expansion of a specific T cell population in response to the allergen Asp f 1 in ABPA and nonABPA patients. The increased usage of Vbeta13 in ABPA and Vbeta1 in nonABPA indicates their importance in susceptibility and resistance, respectively.

Hashimoto's disease during interferon-alpha therapy in a patient with pre-treatment negative anti-thyroid autoantibodies and with the specific genetic susceptibility to the thyroid disease.

OBJECTIVES: The authors described a case of Hashimoto's disease during interferon-alpha (IFN-alpha) treatment for chronic viral C hepatitis in a patient with the specific genetic susceptibility associated with the thyroid disease. RESULTS: A 60-year-old woman with chronic active viral C hepatitis (HCV genotype = 3a) started IFN-alpha therapy in November '96. Before treatment thyroid function tests were normal and anti-thyroid (anti-thyroglobulin and anti-thyroid peroxidase) Abs were negative. During IFN therapy, serum aminotransferases fell within the normal range and viremia (serum HCV-RNA) became negative after one year. After 20 months, the patient presented clinical features of primary hypothyroidism. Anti-thyroid Abs were found positive. Hormonal, ultrasonographic, radioiodine scanning and fine needle aspiration findings were consistent with the diagnosis of Hashimoto's thyroiditis. The tissutal typing of the patient showed the presence of Human Leukocyte Antigen (HLA) DRB1*11 gene (corresponding to DR5 antigen). IFN-alpha therapy was suspended and a treatment with l-T4 started. Chronic viral infection relapsed after the suspension of the IFN-alpha therapy. CONCLUSIONS: This case report showed that the clinical appearance of Hashimoto's disease after IFN-alpha therapy for chronic C hepatitis in our patient was associated with a specific genetic predisposition (DR5) for this pathology. Further studies are necessary to evaluate whether the study of HLA antigens may be a very useful tool to detect the patients with a predisposition to develop autoimmune thyroiditis, in order to make a early diagnosis of thyroid disorders during the IFN-alpha treatment.

HLA class II phenotype controls diversification of the autoantibody response in primary Sjögren's syndrome (pSS).

The coexistence of anti-La (SS-B) and anti-Ro (SS-A) autoantibodies in pSS is probably explained by intermolecular spreading of autoimmunity toward different components of the La/Ro ribonucleoprotein (RNP). In order to evaluate the role of the HLA class II phenotype in controlling diversification of this autoantibody response, 80 patients with pSS were typed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) at the HLA class II loci DRB1, DQA1 and DQB1. Serum samples were examined for anti-La and anti-Ro by counterimmunoelectrophoresis and by ELISA using purified recombinant La and 60-kD Ro proteins. Patient sera were classified according to the extent of diversification of the anti-La, anti-Ro response including the presence or absence of precipitating anti-La antibodies. Immunogenic characteristics of these stratified groups were then studied. All patients with pSS, with or without autoantibodies to Ro and La, were found to have at least one of the HLA-DRB1 types DR2, DR3 or DR5. The HLA DR3-DQA1*0501-DQB1*02 (DR3-DQ2) haplotype was primarily associated with a diversified La/Ro RNP response containing precipitating autoantibodies to La (P<0.001); whereas the haplotype HLA DR2-DQA1*0102-DQB1*0602 (DR2-DQ1) was associated with a less diversified La/Ro RNP response containing non-precipitating (restricted epitope) anti-La autoantibodies (P<0.001). Anti-La-positive patients lacking both HLA-DR2 and HLA-DR3 all expressed the HLA-DQA1*0501 allele, which was present at increasing frequency with greater diversification of the anti-La/Ro autoantibody response. The association of distinct HLA haplotypes with different degrees of autoantibody diversification in patients with pSS suggests a model of HLA-restricted presentation of La/Ro peptide determinants to autoreactive helper T cells. We propose that non-precipitating anti-La responses are driven by limited intermolecular help from DR2-DQ1-restricted T helper cells recognizing Ro determinants. On the other hand, we speculate that the more diversified, precipitating anti-La responses obtain more efficient cognate T help from DR3-DQ2-restricted T helper cells recognizing La determinants, where HLA-DQA1*0501 may be a critical determinant for antigen presentation.

Isolation of scFv fragments to polymorphic and monomorphic HLA-DR epitopes from a synthetic phage library.

The possibility of producing human recombinant antibodies by the phage display libraries technology has opened up new perspectives in the fields of immunological research and therapeutic applications. Despite the interest for a potential use of this technology in the HLA field, no information is so far available about selection and screening of libraries with HLA antigens. In this study we report our first experience with expression and characterization of human single-chain antibody fragments (scFvs) against HLA-DR epitopes selected from a synthetic phage library.

A novel approach to human anti-HLA mABs production: use of phage display libraries.

The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DR beta chain. Western blot analysis indicated that the 7 scFvs react with the DR1 alpha/beta-dimer but not with free alpha- or beta- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.

Human leukocyte antigens in occupational allergy: a possible protective effect of HLA-B16 in laboratory animal allergy.

Human leukocyte antigens (HLA-A, -B, -C, and -DR loci) and possible associations with occupational allergy to laboratory animals and atopy indicators were studied in laboratory animal workers with airway symptoms (n = 92) and in those who were symptom free (n = 27), as well as in a population reference group of blood donors in good health (n = 123). The laboratory animal workers, but not the population reference group, were allergologically examined with skin prick testing to common environmental and animal allergens together with measurement of total serum IgE levels. Seven HLA antigens, i.e., HLA-A9, -B5, -B12, -B16, -DR4, -DR5, and -Drw6, suggested possible associations with symptoms and/or atopy indicators. When correcting the p-values for the number of studied antigens, only the HLA-B16 differences remained statistically significant. HLA-B16 was elevated in symptom-free subjects compared to the population reference group and in subjects with serum IgE < 10 kU/L. Subjects with serum IgE > 100 kU/L and sensitized against environmental and/or laboratory animals, including LAA asthmatics, lacked HLA-B16. It is suggested that HLA-B16 or an immunosuppressive gene linked to HLA-B16 reduce the risk of producing IgE antibodies against animal protein allergens. However, our a priori hypothesis of a possible risk associated with HLA B15-DR4 could not be confirmed.

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line.

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.

Certain HLA-DR5 and -DR6 major histocompatibility complex class II alleles are associated with a CD8 lymphocytic host response to human immunodeficiency virus type 1 characterized by low lymphocyte viral strain heterogeneity and slow disease progression.

Either of two structurally related major histocompatibility complex class II alleles, DRB1*1102, which encodes a DR5 specificity, or DRB1*1301, which encodes a DR6 specificity, was found in 67% of individuals responding to human immunodeficiency virus type 1 (HIV-1) infection with a syndrome characterized by persistent circulating and diffusely infiltrative CD8 lymphocytosis (DILS), slow progression to opportunistic infections, and delayed CD4 T-cell depletion. These alleles were present in only 28% of ethnically matched HIV-positive controls (P = 0.001). The frequency of DRB1*1301 was increased in both Blacks and Caucasians with this syndrome, while that of DRBI*1102 was increased only in Blacks, where 80% had either of these alleles. To investigate whether the host response associated with these alleles influences the evolutionary divergence of the HIV-1 genome, sequencing of the envelope V3 loop was performed. This revealed a significantly diminished lymphocyte viral heterogeneity compared with random HIV+ controls matched for CD4 T-cell levels. These results suggest that the immunogenetics of the host influence the nature of the immune response to HIV-1, which may lead to constrained evolution of HIV-1 gene products. Of possible relevance, the alpha-helical third diversity region common to both the DRB1*1102 and DRB1*1301 allelic products was noted to have homology with the C-terminal region of the HIV-1 envelope V3 loop at six of nine consecutive residues. This suggests the possibility that these alleles may bias the anti-HIV T-cell receptor repertoire through a mimicry mechanism.

[Otoneurologic examination of patients with AIDS]. / Exploraciones otoneurologicas en pacientes con SIDA.

The influence of the HIV virus on the central nervous system tracts has been definitively shown. Over the past year we have affected otoneurologic tests based on elicited auditive potentials and tests of oculomotility on a total of 30 patients affected by stages II and III of the disease. The results show that around 27 percent of cases present alterations in the provoked auditory potentials and 20 percent in the oculomotility tests. The results are compared with those of previous studies.

Self-reactive and antigen-specific T cell clones derived from a HLA-DR4+/DR5+ donor: T cell receptors and MHC-restriction patterns.

The relationship of heat shock proteins and rheumatoid arthritis as well as the relevance of autoreactivity in this disease is unclear. T cells of six individuals (four expressing the DRB1*0401 allele, one harboring DRB1*0404 and one the DRB1*0407 allele) were cloned in the presence of 65kD mycobacterial heat shock protein (HSP60) in order to determine T cell receptors (TcR) used and the MHC class II restriction patterns of potentially relevant T cell clones (TcC). All TcC obtained were not specific for HSP60, but six TcC of one donor (HLA-DR4/HLA-DR5) were responsive towards autologous antigen-presenting cells. One TcC displayed authentic autoreactivity whereas five TcC reacted specifically to serum proteins. The amino acids (aa) of the MHC molecule, crucial for immune recognition were mapped to aa #71 or #86 of either maternal or paternal origin. The strictly autoreactive TcC did not recognize transfected L cells implicating specificity for self-peptides not presented by L cells or the involvement of adhesion molecules. Correlations between autoreactivity and TcR V(D)J sequences or N nucleotides of various "autoreactive" TcC were not evident.

The association of HLA-DR antigens with acute steroid-resistant rejection and poor kidney graft survival.

The association of an early steroid-resistant rejection (SRR) with HLA-DR antigens was analyzed in 410 kidney transplantations. A severe SRR leading to a poor (45%) 1-year graft survival (GS) occurred in 22 transplantations (5%). An acute reversible rejection (ARR) with a GS of 94% was found in 80 transplantations (20%). For the 308 (75%) transplantations with no early rejection episodes the GS was 91%. HLA-DR5 and -DR8 present in the donor as incompatible antigens were strongly associated with SRR. Further, a mismatched DR1 from the kidney donor predicted a rejection, either reversible or irreversible. These findings may have practical implications for an early diagnosis of SRR and for considering of rescue therapy whenever transplantation with disparities in these loci has been performed.

Allorecognition of HLA DR2 and DR5 molecules by V-beta-8-positive T-cell clones.

V-beta-8-positive T cells were isolated from human peripheral blood lymphocytes using a monoclonal antibody specific for the V beta 8 family. Alloreactive T-cell lines were generated by stimulation with mononuclear cells from individuals homozygous for HLA DR1-DR9. Cloned V beta 8-positive T cells were then assayed for alloreactivity based on a proliferative assay using irradiated B-cell lines. V beta 8-positive T-cell clones alloreactive to DR2 and DR5 molecules were chosen for further study based on the association of these MHC antigens with autoimmune disease. DNA sequence analysis confirmed the use of the V beta 8 gene family as well as providing information on the use of the V, D, J and N segments in these alloreactive T-cell clones.