Pregranutomatous phase of sarcoidosis: Immunohistochemical diagnosis
Department of Ophthalmology, Medical University of Gdansk, Danzig, Pomeranian Voivodeship, PolandActa Histochemica (Impact Factor: 1.71). 02/2006; 107(6):473-7. DOI: 10.1016/j.acthis.2005.09.003
Histopathological confirmation of clinical suspicion of sarcoidosis is based on the finding of non-caseating granulomas in biopsy material, usually in prescalene lymph nodes or in transbronchial lung biopsies. Lymph node reactive sinus histiocytosis (RSH) seen in relation to various inflammatory and non-inflammatory diseases can mimic the pregranulomatous phase of sarcoidosis (PSH). Differentiation of sinus histiocytosis based on histopathological features alone is limited. The purpose of this study is immunohistochemical determination of lymph node cellular response in granulomatous sarcoidosis, the PSH and RSH using a immunohistochemistry employing a panel of antibodies. Patient groups under study each contained 25 patients and included: those with clinical picture of sarcoidosis and non-caseating granulomatous lymphadenitis; those with confirmed sarcoidosis and with sinus histiocytosis without granuloma formation in lymph nodes; and finally, those without sarcoidosis and with “reactive” sinus histiocytois in lymph nodes. Lymph node biopsy tissue was fixed in buffered formaldehyde, routinely processed to paraffin wax blocks, cut into 4-μm-thick sections, stained with hematoxylin and eosin and immunohistochemically labelled using a triple-layer APAAP protocol with purified polyclonal antibodies directed against SP 70 and SP90 from Mycobacterium tuberculosis and monoclonal antibodies against CD22, CD4, CD8, CD56, and CD68. Intensity of immunolabelling was assessed semiquantitatively by two independent observers.
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ABSTRACT: Sarcoidosis (SA) is a granulomatous disorder of an unknown etiology. Mycobacterium tuberculosis heat shock proteins (Mtb-hsp), considered as causative agents, play an important role in apoptosis. A role for apoptosis has been proposed in pathogenesis of SA and tuberculosis (TB) granuloma formation but results remain controversial. Differences in Mtb-hsp-induced apoptosis between SA, TB, and healthy subjects found in this study might put some light on the etiology of SA. Early apoptotic peripheral blood mononuclear cells (PBMC) were determined in 22 SA patients, 20 TB patients, and 20 healthy volunteers by flow cytometry (Annexin-V-FITC). Our results revealed that spontaneous apoptosis of monocytes and CD8+ T-cells was comparable between tested groups. Apoptosis of unstimulated CD4+ T-cells was significantly lower in TB versus controls and insignificantly lower versus SA. Mtb-hsp- and PHA (Phytohemagglutinin)-induced monocytes apoptosis was significantly lower in TB versus controls and SA. Mtb-hsp-induced CD4+ T-cell apoptosis was significantly lower in TB versus controls and SA. There were no differences of PHA-induced CD4+ T-cell and CD8+ T-cell apoptosis between tested groups. Apoptosis of Mtb-hsp-induced CD8+ T-cells was significantly lower in TB and SA versus controls. Analysis of PBMC apoptosis before and after stimulation in each tested group revealed that, in contrast to TB, sarcoid monocytes were resistant to Mtb-hsp- and PHA-induced apoptosis and CD4+ T-cells were resistant to PHA- but not Mtb-hsp-induced apoptosis. CD8+ T-cell apoptosis, before and after Mtb-hsp or PHA stimulation, was significantly increased in all tested groups. It seems likely that dysregulated apoptosis of CD4+ T-cells and resistant apoptosis monocytes may be involved in pathogenesis of SA.
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ABSTRACT: Pathologic similarities between sarcoidosis (SA) and tuberculosis (TB) suggest M. tuberculosis antigen(s) as causative agents. It seems likely that in the genetically different predisposed hosts, the same antigen(s) may cause the development of sarcoid or tuberculous Th1 response. AIM AND MATERIALS/METHODS: To test a difference in haplotypes associated with both diseases, we compared the distribution of DRB1, DQA1 and DQB1 alleles in 45 SA patients, 62 TB patients and in 143 healthy volunteers, using a PCR-SSP method. Our results revealed that DRB1*03/*11, DQB1*02, DQA*0501 in Stage I of SA with Löfgren's syndrome (Ls) and DRB1*15, DQA1*0102/*0103 in Stage II of SA were more common, whereas DQA1*0102 (Ls) and DRB1*16/*04/*08, DQB1*03/*04/*05/*06, DQA1*0301 (Ls, Stage II) were less common than in the controls. Nevertheless, after Bonferroni correction, only DRB1*04, DQB1*02/*03/*05/*06, DQA1*0102/*0301/*0501 differed significantly. In TB group, DRB1*16/*14, DQB1*05, DQA1*0303 were more frequent and DRB1*11, DQB1*02, DQA1*0201/*0505 less frequently present as compared to the controls, but frequency of DRB1*16, DQB1*02/*05 and DQA1*0303/*0505 only was significantly different after correction. After correction in both Stages of SA, DRB1*11 was more common and DRB1*16/*04/*14, DQB1*03/*05, DQA1*0301/0302/*0303 were less frequent than in the TB group. DQB1*02, DQA1*0201/*0501 (Ls) and DRB1*15/*13 (Stage II) were more frequently present in SA than in TB, but after correction, only DRB1*15, DQB1*02, DQA1*0501 were significantly different. We identified associations of HLA class II alleles in SA and TB with expression pattern specific and different for each group. In most cases, in SA patients frequency of HLA class II alleles occurrence is opposite to the frequency in TB patients.
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ABSTRACT: The affinity of antibody to antigen, in addition to providing the possibility of measuring the antigen in tissue extracts through methods such as RIA (Radioimmunoassay) and EIA (Enzymimmunoassay) and possibility of isolating and analyzing dispersed cell colonies using flowcytometry, makes it possible to determine the site of antigen in tissues (Immunohistochemistry) or in cells (Immunocytochemistry). Production of APAAP complexes and comparing them with similar foreign products to determine the site of antigen in tissues or in cells. Secreted antibodies of the two hybridomas (A(1)G(9)G(3) and A(1)G(8)F(7) produced in our laboratory) were concentrated, purified and characterized. Then the monoclonal antibodies were mixed with alkaline phosphatase enzyme (ALP) to use in immunocytochemistry (ICC) and immunohistochemistry (IHC) staining. Both of the cell colonies had the potentiality of producing anti- alkaline phosphatase monoclonal antibody with high affinity. The complex from mAb and enzyme - for the third phase of APAAP technique - was very effective and its sensitivity was comparable to that of the similar foreign kit. Considering the high affinity of the mAb of the two hybridomas and the stability of the complex resulted from mixing mAb and the enzyme ALP for a long time, it is possible to use the obtained APAAP complex in the immunocyto (or histo) chemistry - as the third phase.
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