Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring

The authors developed a standardized approach for immune monitoring of antigen-specific CD8+ T cells within peripheral blood lymphocytes (PBLs) that combines direct ex vivo analysis of Melan-A/MART-1 and influenza-specific CD8+ T cells with HLA-A2/peptide multimers and interferon-gamma ELISPOT assays. Here the authors assessed the quality of results obtained with 180 PBLs from healthy donors and melanoma patients. Reproducibility of the multimer assay was good (average of 15% variation). In the absence of in vivo antigen-specific T-cell responses, physiologic fluctuations of multimer-positive T cells was low, with variation coefficients of 20% for Melan-A and 28% for influenza-specific T cells. In contrast, patients with vaccination-induced T-cell responses had significantly increased T-cell frequencies clearly exceeding physiologic fluctuations. Comparable results were obtained with ELISPOT assays. In conclusion, this approach is well suited to assess T-cell responses as biologic endpoints in clinical vaccine studies.     

Lack of BRAF mutations in uveal melanoma

RAF proteins are serine/threonine kinases that mediate cellular responses to growth signals by activating the mitogen-activated protein kinase pathway. Mutations in the BRAF gene causing a V599E amino acid substitution that enhance the kinase activity have been described in >60% of cutaneous melanomas and premalignant melanocytic lesions. We have investigated the frequency of BRAF mutations at the expression level in melanomas of the uveal tract. None of the 30 metastases and 10 primary uveal melanomas tested expressed the V599E mutation. In contrast, this mutation was expressed by 65% of cutaneous melanoma samples, confirming previous results. In addition, a double mutation resulting in V599K substitution was detected in two suspect ocular metastases of cutaneous melanoma. Analysis of exon 11, the second common site of BRAF mutations, revealed only wild-type sequences in uveal melanomas. Analysis of tumor lysates showed the presence of phosphorylated mitogen-activated protein kinase, kinase, and mitogen-activated protein kinase in 50% of uveal and 100% of cutaneous melanoma metastases. Taken together, these results suggest that although the common BRAF mutations found in cutaneous melanoma do not play a role in tumorigenesis of uveal tract melanocytes, activation of the RAF/mitogen-activated protein kinase pathway may nevertheless play an important role in uveal melanoma.     

21-Hydroxylase-deficient nonclassic adrenal hyperplasia is a progressive disorder: a multicenter study

OBJECTIVE: Our aim was to determine whether the clinical features of 21-hydroxylase-deficient nonclassic adrenal hyperplasia are correlated with either age at symptom onset or age at presentation, or both, and with the degree of adrenocortical abnormality. STUDY DESIGN: In a multicenter cohort design 220 women with nonclassic adrenal hyperplasia, with a basal or adrenocorticotropic hormone-stimulated 17-hydroxyprogesterone level >30.3 nmol/L, were studied, either prospectively (n = 39) or retrospectively (n = 181). Patients were stratified by age of presentation into 5 groups: (1) <10 years (n = 25), (2) 10 to 19 years (n = 64), (3) 20 to 29 years (n = 83), (4) 30 to 39 years (n = 30), and (5) 40 to 49 years (n = 16). Two patients >50 years old were excluded from the analysis because of age. RESULTS: Ninety-two percent of patients <10 years old had premature pubarche at presentation, whereas clitoromegaly and acne were each present in only 20% of these younger subjects. With only patients > or =10 years old considered, presenting clinical features included hirsutism (59%), oligomenorrhea (54%), acne (33%), infertility (13%), clitoromegaly (10%), alopecia (8%), primary amenorrhea (4%), and premature pubarche (4%). Among the patients >/=10 years old, the prevalence but not the degree of hirsutism increased significantly with age. Basal levels of 17-hydroxyprogesterone in adolescents were significantly higher than the levels found either in children (<10 years old) or women 40 to 49 years old (P <.01 and P <.03, respectively), although no difference was noted in the stimulated 17-hydroxyprogesterone levels between age groups. The adrenocorticotropic hormone-stimulated levels but not the basal levels of 17-hydroxyprogesterone were significantly higher in patients with clitoromegaly than in women without clitoromegaly. Alternatively, there were no differences in either basal or stimulated 17-hydroxyprogesterone levels between patients with and those without hirsutism, acne, or alopecia. CONCLUSION: In children <10 years old the most common presenting complaint was premature pubarche, whereas hirsutism and oligomenorrhea were more common in older patients. The prevalence of hirsutism increased with age, suggesting the progressive nature of nonclassic adrenal hyperplasia. Furthermore, the adrenocorticotropic hormone-stimulated levels of 17-hydroxyprogesterone were higher in patients with clitoromegaly, which suggests that the degree of adrenocortical dysfunction in nonclassic adrenal hyperplasia determines, at least in part, the clinical presentation.     

Production and characterization of a monoclonal antibody reactive with a specific neoantigenic determinant (comprising B beta 54-118) in degradation products of fibrin and of fibrinogen

Balb/c mice were immunized with a mixture of fibrin degradation products (XDPs) prepared by complete lysis of a human blood clot by tissue-type plasminogen activator and purified by immunoaffinity chromatography. Spleen cells of the mice were fused with P3 X 63 Ag 8653 myeloma cells. A clone (FDP 14) was selected that produces monoclonal antibodies (MoAbs) of the IgG1 kappa type that react with a neoantigenic determinant exposed in these XDPs, but not in intact fibrinogen or in fibrin monomers. Furthermore, the MoAb is reactive with some pure, individual degradation products of fibrinogen (fragments X, Y, E, and the N-terminal disulphide knot) and with the fibrinogen B beta-chain but not with A alpha- and gamma-chains or with fragments D, FCB-2 and FCB-3. Comparison of the known primary structures of these fibrinogen fragments indicates that the stretch B beta 54-118 comprises at least an important part of the epitope recognized by FDP-14. Apparently, this stretch contributes importantly to a neoantigenic determinant that is not functional in intact fibrinogen and fibrin monomer and that can be made functional by reduction of fibrinogen, or by digestion with plasmin or CNBr.     

Sandwich ELISA for t-PA antigen employing a monoclonal antibody

A sandwich enzyme-linked immunosorbent assay (ELISA) for determination of tissue-type plasminogen activator (t-PA) was developed. 96-well flat-bottom polystyrene plates were coated with polyclonal (goat) anti t-PA IgG (10 micrograms/ml). After addition of samples monoclonal (murine) anti t-PA IgG (1.5 micrograms/ml) was added. Finally, peroxidase labelled anti-mouse IgG (goat) was used to quantify the bound second antibody. The assay can be used for determination of t-PA antigen in purified systems, in cell culture supernatants, and in human plasma, provided that EDTA (0.005 M) is present in the sample. In 78 healthy volunteers, t-PA antigen levels at rest were 0.4 - 15.2 ng/ml (4.3 +/- 2.7, means +/- S.D.); A significant positive correlation between t-PA antigen and age could be demonstrated.