Characterization of ANA immunofluorescence patterns sharing nuclear speckles with variable intensity

• Grant number: 09/04063-1
• Support Opportunities: Scholarships in Brazil - Master
• Start date: September 01, 2009
• End date: August 31, 2011
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Luiz Eduardo Coelho Andrade
• Grantee: Rossana Rassi Alvarenga
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

The history of autoimmune diseases, specially those ones with rheumatology interest, is closely linked to autoantibodies. Some of these autoantigens express their activity in the dependence of the cell cycle and show a pleomorphic pattern in AAN-HEp-2. In these cases, what is observed is the variability in the morphological pattern shown by several cells in the same microscopic field. In our practice, we have observed some samples which present a pattern in which the main feature is the variability in fluorescence intensity among the cells. One of these pattern is the target of special interest due to its relatively high frequency and it has been called among us as Fine Speakled Nuclear Pattern with Variable Intensity (PF-IV). This study wants to set the immunological features of the autoantibodies involved in this indirect immunofluorescence pattern (PF-IV) and the partial molecular characterization of their respective target autoantigens. Methods: For this purpose we selected sera samples of this pattern, both from the Rheumatology Division of Unifesp and from the Fleury Group. We used different commercial kits of HEp-2 cells, in house cell cultures, with different fixation protocols and some other cell lines as substrate for the indirect immunofluorescence technique to assure this pattern reproducibility. Following, we characterized the isotype distribution of PF-IV reactivity and the autoantigens behaviour associated with PF-IV after the cell substrate pretreatment with proteases, hipermolar buffers and nucleases. Finally, we characterized the expression of the target autoantigens associated with the PF-IV pattern in the different cell cycle phases, using cell synchronization and flow citometry. Summary of the results: The selected sera samples showing the pattern PF-IV with title e 1:320 were submitted to IIF using comercial kit (Bion) and had three experient readers to assure this pattern reproductibility. Until now, 200 sera previasly classified as cell cycle dependent were tested, and 128 of them, reclassified as PF-IV. After reclassification, these sera were reagrupated in five different subgroups called PF-IV Clássico, CENP-F Like, PF-IV with Synchronized nucleolus, QH-IV, and PF-IV with Pleomorfism, due to their morfological variability. Among these subgroups, we selected two of them for later tests, PF-IV Clássico (19) and CENP-F Like (67). From all sera tested in other comercial HEp-2 kits, few ones showed some difference in the reading. We characterized the isotype distribution of PF-IV reactivity of the sera reclassified in PF-IV, and all of them were positive for IgG. Cell synchronization tests with sera from the two selected subgroups showed that using Timidine, most of the cells in PF-IV Clássico and CENP-F like were positive (G2 phases). Flow citometry tests showed that both subgroups have the curve of cell cycle. Using anti-cyclins we demonstrated that proteins in sera PF-IV Clássico and CENP-F like are mainly expressed in G2 phase of the cell cycle. Conclusions: The present study demostrated that those patterns, PF-IV Clássico and CENP-F Like, are really related with a specific protein, which expression is dependent of the phase of the cell cycle. It was demonstrated that it is not artefact. Isotype caracterization showed that the majority are IgG positives. With cell synchronization tests we observed that the protein expressed both in sera PF-IV Clássico and sera CENP-F like appears in G2 phase. Flow cytometry only confirmed these results, using specific cell cycle anti-cyclins. We yet do not have clinical correlation, but these findings may have great clinical relevance and may bring good benefits to cell biology and molecular studies.