A monoclonal antibody that recognizes a unique 13-residue epitope in the cytoplasmic tail of HLA-E.

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E.

Relevância clínica e frequência de padrões citoplasmático e pontilhado fino denso observados em FAN-HEp-2 / Clinical relevance and frequency of cytoplasmic and nuclear dense fine speckled patterns observed in ANA-HEp-2

OBJETIVOS: O presente estudo procurou determinar a frequência e relacionar o título sorológico dos padrões de imunofluorescência para citoplasma celular e nuclear pontilhado fino denso com possível correlação clínica. MÉTODOS: No período entre 2007 a 2009 foram avaliados os resultados de 2.788 testes sorológicos para pesquisa de autoanticorpos pela técnica de imunofluorescência indireta (IFI), utilizando como substrato células HEp-2, realizados no LAC-HUSM/UFSM. RESULTADOS: Entre as amostras analisadas, 1.998 resultaram não reagentes para a pesquisa de autoanticorpos. Entre as amostras reagentes (n = 790) foram encontradas 57 (7,2 por cento) amostras apresentando padrão de reatividade descrita como pontilhado fino denso (SFD) (3,8 por cento) ou fluorescência citoplasmática (Cit) (3,4 por cento). Nas amostras com padrão SFD (n = 29), nove apresentaram título < 1/160, onde apenas um paciente apresentava doença autoimune (DAI). Entre os pacientes com título > 1/160 apenas um não apresentava DAI. Entre as amostras com padrão Cit (n = 27), 20 apresentavam título < 1/160, onde apenas oito não tinham DAI associada. Todos os outros 7 pacientes com título > 1/160 tinham relato de DAI. CONCLUSÃO: Os resultados encontrados ratificam o valor de 1/160 como melhor ponto de corte para definição de presença de DAI, para qualquer um dos padrões de fluorescência avaliados. Contudo, deve-se prestar atenção a títulos inferiores, principalmente para IFI Cit, uma vez que apenas 40 por cento não apresentavam relato de DAI presente.

OBJECTIVES: This study aimed to determine the frequency and antibody titers of nuclear dense fine and cytoplasmic patterns with possible clinical correlation. METHODS: From 2007 to 2009, the results of 2,788 autoantibody serological tests were assessed by indirect immunofluorescence (IIF) at LAC-HUSM/UFSM, using as substrate HEp-2. RESULTS: Among the analyzed samples, 1,998 of them were negative for autoantibodies. Among the positive samples (n = 790), we found 57 (7.2 percent) showing reactivity pattern described as dense fine speckled (DFS) (3.8 percent), or cytoplasmic (Cit) fluorescence (3.4 percent). In samples with standard DFS (n = 29), nine had titers of 1/160, and only one patient had autoimmune disease (AID). Among patients with titers > 1/160, only one patient did not have AID. Among samples with standard Cit (n = 27), 20 had titers of 1/160, and only eight were not associated with AID. The other seven patients with titers > 1/160 reported AID. CONCLUSION: The results confirm the value of 1/160 as the best cut-off point for defining AID presence, for any of the fluorescence assessed patterns. However, attention should be given to lower titers, especially for Cit IIF, since only 40 percent did not report the presence of AID.

Interleukin-1 beta and interleukin-8 in healthy and inflamed dental pulps

After aggression to the dental pulp, some cells produce cytokines in order to start and control the inflammatory process. Among these cytokines, interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) emerge as important ones. OBJECTIVE: The purpose of this study was to analyze the location, distribution and concentration of these cytokines in healthy and inflamed dental pulps. MATERIAL AND METHODS: Twenty pulps, obtained from healthy third molars (n=10) and from pulpectomies (n=10) were used for the study, with half of each group used for immunohistochemistry and half for protein extraction and ELISA assays. Fibroblasts obtained from healthy dental pulps, stimulated or not by Escherichia coli lipopolysaccharide (LPS), in order to simulate aggression on the cell cultures, were also used and analyzed by ELISA for IL-1β and IL-8 as complementary information. Data obtained from immunohistochemistry were qualitatively analyzed. Data obtained from ELISA assays (tissue and cells) were statistically treated by the t-test (p<0.05). RESULTS: Immunohistochemically, it was observed that inflamed pulps were strongly stained for both cytokines in inflammatory cells, while healthy pulps were not immunolabeled. ELISA from tissues quantitatively confirmed the higher presence of both cytokines. Additionally, cultured pulp fibroblasts stimulated by LPS also produce more cytokines than the control cells. CONCLUSIONS: It may be concluded that inflamed pulps present higher amounts of IL-1β and IL-8 than healthy pulps and that pulp fibroblasts stimulated by bacterial LPS produce higher levels of IL-1β and IL-8 than the control group.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques...

Autoanticuerpos anticitoplasma de neutrófilos / Neutrophil anticytoplasma autoantibodies

Los ANCA son anticuerpos detectados frecuentemente en enfermedades autoinmunes, especialmente vasculitis. Su actividad se dirige contra diferentes estructuras citoplasmáticas de los neutrófilos. El presente artículo es una revisión de sus especificidades antigénicas, sus métodos de detección, sus correlaciones clínicas y su posible rol etiopatogénico en estas enfermedades

Síndrome pulmón-riñón / Lung-kidney syndrome

El síndrome pulmón riñón se caracteriza por hemorragia alveolar severa y glomerulonefritis. Obedece a variadas etiologías, siendo la vasculitis la causa más frecuente. Recientemente se ha establecido que aquellas vasculitis que comprometen a los capilares, alveolares y glomerulares, cursan con síndrome pulmón riñón. Histológicamente se expresan con una inflamación necrotizante de los capilares alveolares y con glomerulonefritis necrotizante y crescéntica en el riñón. El descubrimiento de los ANCA (Antineutrophil Cytoplasmic Antibodies) a permitido mejorar nuestra comprensión del síndrome pulmón riñón. En este artículo se revisa a la luz de los conocimientos actuales, el diagnóstico etiológico de este síndrome. Considerando que las alteraciones histológicas a nivel pulmón son inespecíficas, el diagnóstico etiológico debe realizarse en base a las manifestaciones clinicopatológicas del compromiso extrapulmonar y/o de las alteraciones serológicas características

Estudio clínico y anatomopatológico en cinco casos de vasculitis portadores de anticuerpos anticitoplasmáticos de neutrófilos con patrón citoplasmático / Clinical and anatomopathological study in 5 cases of vasculitis carrying neutrophil anticytoplasmatic antibodies with cytoplasmatic pattern

We report five patients with vasculitis and antineutrophil cytoplasmic antibodies with cytoplasmic pattern. All had severe upper and lower respiratory tract necrotizing lesions. Three had kidney failure due to rapidly progressive glomerulonephritis. The pathological study showed a crescentic glomerulonephritis, a chronic granulomatous inflammation in the lungs and in the nasal mucosa, an acute nonspecific inflammation or a chronic granulomatous inflammation and focal blood vessel fibrinoid necrosis. All patients with simultaneous involvement of lungs and kidneys had high titers of antineutrophil cytoplasmic antibodies. The nomenclature and classification of these diseases is discussed