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Intracellular Antigen (intracellular + antigen)

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Medical Sciences75%

Selected Abstracts

Aggresome formation by anti-Ras intracellular scFv fragments

FEBS JOURNAL, Issue 2 2001
The fate of the antigen, antibody complex
Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett.,439, 197,202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem.267, 1196,205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen,antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen,antibody complex is naturally addressed to the ubiquitin,proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function. [source]

Extranodal NK/T-cell lymphoma, nasal type, presenting after 5 years of remission

INTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 3 2008
Tomonobu Ito MD
A 76-year-old woman with multiple edematous erythemas, erosions, and ulcers on the breast and abdomen was admitted to our hospital in June 2005. She had developed granulomatous bleeding lesions in the right nostril 6 years prior to her visit to our dermatology unit. She had been observed at the otorhinolaryngology department of our hospital, and a biopsy was taken from the nasal lesion. Computerized tomography and gallium scintigraphy (67Ga single-photon emission computed tomography) did not reveal any lesions corresponding to the diagnosis of malignant lymphoma. The histologic examination of the nasal specimen rendered a diagnosis of natural killer (NK)/T-cell lymphoma, nasal. Because imaging analysis indicated a small-sized tumor without metastases, oral prednisolone at 20 mg/day was administered for 1 month. The tumor decreased in size and disappeared after 19 months of low-dose steroid therapy. ,Five years after the initial treatment, the patient developed a fever of 38 °C with infiltrated erythemas and erosions on her breast. Erysipelas was initially suspected, but the antimicrobial agent did not show any effect and the multiple infiltrated erythemas and ulcers spread throughout her chest and abdomen (Fig. 1). The lymph nodes were not palpable. The right nasal cavity showed no granulomatous lesions or other signs of abnormality. The peripheral white blood cell count (3000/µL), red blood cell count (3.54 × 106/µL), and platelet count (112 × 103/µL) were reduced. Atypical lymphocytes were not observed. The serum lactic dehydrogenase (LDH; 1770 U/L; normal, 224,454 U/L), aspartate aminotransferase (AST; 140 U/L; normal, 10,30 U/L), and alanine aminotransferase (ALT; 57 U/L; normal, 3,29 U/L) levels were elevated. The soluble interleukin-2 (IL-2) receptor level was high (25,300 U/mL; normal, 167,497 U/mL). Epstein,Barr virus (EBV) serologic examination showed the immunoglobulin G (IgG) viral capsid antigen (VCA) at 1 : 320 and the EBV nuclear antigen (EBNA) at 1 : 40. IgM VCA and EBV early antigen-diffuse restricted antibody (EA) IgA and IgG were not detectable. Histologic findings from the left chest skin showed a distribution of atypical lymphocytes from the upper dermis to the subcutaneous tissue, and many foamy cells which had phagocytosed the hemocytes (Fig. 2a,b). Immunohistochemical analysis showed that the atypical lymphocytes were sCD3,, CD4,, CD8,, CD20,, CD56+, granzyme B+, and T-cell intracellular antigen (TIA-1) positive. Furthermore, EBV-encoded small RNAs (EBER), detected by in situ hybridization, exhibited a strong signal. The nasal lesions biopsied 6 years previously showed an identical staining pattern with the skin lesions immunohistochemically. Analysis of the T-cell receptor-, (TCR-,), TCR-,, and TCR-, gene did not reveal any clonal rearrangements, but the EBV gene was detected from the skin specimens by Southern blotting. Our patient's condition was diagnosed as a case of extranodal NK/T-cell lymphoma, nasal type, but the patient had concomitantly developed hemophagocytic syndrome (HPS). She was treated with a combination of steroid pulse therapy and chemotherapy (pirarubicin hydrochloride 30 mg/m2, cyclophosphamide 500 mg/m2, vincristine 1 mg/m2, prednisolone 30 mg/m2, etoposide 80 mg/m2). After the first session of chemotherapy, the lesions on the chest and abdomen diminished, but, 2 weeks later, the skin lesions recurred, and disseminated intravascular coagulation (DIC) induced by HPS supervened. The patient died as a result of multiple organ failure induced by HPS. Figure 1. Multiple infiltrated erythemas, erosions, and ulcers on the breast and abdomen Figure 2. Histologic findings of a skin biopsy specimen from the left chest (hematoxylin and eosin staining). (a) Dense infiltration of atypical lymphocytes from the upper dermis to the subcutaneous tissue (×40). (b) Many foamy cells had phagocytosed the hemocytes (×400) [source]

A clonal cutaneous CD30+ lymphoproliferative eruption in a patient with evidence of past exposure to hepatitis E

INTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 7 2000
Freddye M. Lemons-Estes CDR, MC USN
The patient was a 52-year-old white man who had worked in remote areas of the world during the past 2 years, including an extended period in rural areas of Central Africa and in Central and South America. He had no acute illnesses during the 2-year period except for rare, mild, upper respiratory tract infections. For approximately 1 year, however, he had developed recurrent, papular-vesicular, slightly painful lesions on the fingers and palms, that spontaneously healed over weeks to months ( Fig. 1). The patient had no other concurrent illnesses and no abnormal laboratory findings, except for positive enzyme-linked immunoabsorbent assay (ELISA) for immunoglobulin G (IgG) antibodies for hepatitis E virus (HEV) using a recombinant expressed HEV antigen (Genelabs Technologies, Inc., San Antonio). Prolonged treatment with minocycline did not appear to moderate the lesions. At approximately 2.5 years after the development of his first cutaneous lesion, however, the patient reported that he had had no new lesions for over 3 months. Figure 1. Vesicular ,lesion on the finger which regressed over a period of weeks A biopsy specimen showed an intraepidermal vesicle with prominent epidermal necrosis and reticular degeneration ( Fig. 2). Within the epidermis, there was a dense infiltrate of lymphoid cells. The majority of these cells were pleomorphic with prominent nucleoli and frequent mitotic figures ( Fig. 3). Sheets of atypical cells were found in the subjacent dermis. The infiltrate extended down into the reticular dermis. With extension into the dermis, the infiltrate became more polymorphous with more small lymphoid cells, large numbers of eosinophils, and some plasma cells located more deeply. Figure 2. Intraepidermal ,blister showing reticular degeneration and marked epidermotrophism of large atypical cells with extension into the dermis with a mixed infiltrate containing eosinophils and plasma cells (30×) Figure 3. Intraepidermal ,infiltrate of large atypical cells with extension into the dermis with a mixed infiltrate containing eosinophils and plasma cells (400×) Immunohistochemical stains for CD3 (DAKO), CD4 (Becton Dickinson), CD8 (Becton Dickinson), CD15 (LeuM1, Becton Dickinson), CD20 (L-26, DAKO), CD30 (Ber-H2, DAKO), CD45RO (UCHL1, DAKO), S-100 protein (DAKO), T-cell intracellular antigen (TIA) (Coulter), epithelial membrane antigen (EMA) (DAKO), KP-1 (CD68, DAKO), MAC-387 (DAKO), Epstein,Barr virus (EBV) latent membrane antigen-1 (LMP-1, DAKO), and EBV-encoded nuclear antigen 2 (EBNA2, DAKO) were performed on formalin-fixed tissue using the ABC method with DABA as the chromagen. CD3 showed diffuse membrane staining of the large atypical lymphoid cells, as well as the majority of the small lymphoid cells ( Fig. 4). CD4 showed positive membrane staining of the large atypical lymphoid cells and the majority of the small lymphoid cells. CD8 showed only scattered light membrane staining of small lymphoid cells. CD15 was negative, and CD20 showed foci of groups of small lymphoid cells mainly within the reticular dermis. CD30 showed positive membrane and paranuclear staining of the large atypical cells, most abundant within the epidermis and papillary dermis ( Fig. 5). CD45RO showed positive membrane staining of the large atypical cells and the majority of the small lymphoid cells. S-100 protein showed increased dendritic cells within the surrounding viable epidermis and the subjacent papillary dermis ( Fig. 6). TIA showed granular staining in the large atypical lymphoid cells and only rare staining in small lymphoid cells ( Fig. 7). EMA staining was essentially negative. KP-1 showed only scattered positive cells mainly in the lower papillary and the reticular dermis. MAC-387 showed membrane staining in the viable epidermis ( Fig. 8). LMP-1 and EBNA2 for EBV were negative within the lymphoid cells as well as within the overlying epidermis. Figure 4. Immunohistochemical ,staining for CD3 showing diffuse staining of lymphoid cells within the epidermis and dermis (150×) Figure 5. Immunohistochemical ,staining for CD30 showing membrane and paranuclear staining of large atypical lymphoid cells within the epidermis and papillary dermis (a, 150× b, 400×) Figure 6. Immunohistochemical ,staining for S-100 protein within the epidermis and in the papillary dermis (a, 150× b, 300×) Figure 7. Immunohistochemical ,granular staining of large atypical lymphoid cells for TIA (200×) Figure 8. Immunohistochemical ,staining for MAC-387 showing epidermal staining (100×) Gene rearrangement studies showed a ,-T-cell receptor gene rearrangement. The monoclonal band was detected with VJ1, VJ2, and D1J2 primer sets. The T-cell receptor , rearrangement assay has a sensitivity of 61% and a specificity of 94% for the detection of a monoclonal rearrangement in T-cell lymphomas for which amplifiable DNA can be recovered. Electron microscopy was performed on formalin-fixed material, positive-fixed with 2.5% phosphate-buffered glutaraldehyde and further with 1% osmium tetroxide by standard techniques. Intracellular, 50,60-nm, cytoplasmic, spherical, viral-like particles were identified ( Fig. 9). Figure 9. Electron ,microscopy showing 50,60-nm diameter, intracellular, viral-like particles (arrows) (70,000×) [source]

Effects of infliximab therapy on gene expression levels of tumor necrosis factor ,, tristetraprolin, T cell intracellular antigen 1, and Hu antigen R in patients with rheumatoid arthritis

ARTHRITIS & RHEUMATISM, Issue 7 2007
Makoto Sugihara
Objective Tristetraprolin (TTP), T cell intracellular antigen 1 (TIA-1), and Hu antigen R (HuR) are adenine/uridine-rich element binding proteins (ABPs) that affect the production of tumor necrosis factor , (TNF,) by binding to TNF messenger RNA (mRNA). TTP promotes deadenylation, TIA-1 inhibits translation, and HuR stabilizes TNF, mRNA. The aims of this study were to understand the posttranscriptional control of TNF, production in patients with rheumatoid arthritis (RA), and to identify parameters that may predict the efficacy of anti-TNF, therapy. Methods Peripheral blood mononuclear cells from 38 patients with RA were obtained before therapy and 2 weeks and 54 weeks after administration of the first dose of infliximab, and from 20 healthy control subjects. TNF,, TTP, TIA-1, and HuR gene expression levels were analyzed by real-time polymerase chain reaction. Results At baseline, TTP and HuR gene expression levels, as well as the TTP:TNF,, TTP:HuR, and TIA-1:TNF, gene expression ratios were lower in patients with RA than in control subjects, while expression of TNF,, TIA-1, and TIA-1:HuR was higher in patients with RA. The TTP:HuR expression ratio decreased significantly after administration of infliximab. Positive correlations were observed between TNF, and TTP, TNF, and TIA-1, TIA-1 and HuR, and TNF, and HuR gene expression in both healthy control subjects and patients with RA. At baseline, the TIA-1:HuR ratio tended to be higher in patients who achieved 50% improvement according to the American College of Rheumatology criteria (ACR50) at week 54 than in those who did not achieve at least an ACR20 response. Conclusion Differences in ABP gene expression may affect TNF, gene expression. A higher TIA-1:HuR expression ratio might correlate with the response to infliximab therapy. [source]

Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface-only versus intracellular antigens with the Fix & PermÔ reagent,,§

CYTOMETRY, Issue 1 2010
Elaine Sobral da Costa
Abstract Staining for intracellular markers with the Fix & PermÔ reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix & PermTM. Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix & Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for <24 h showed significant impact on the light scatter and fluorescence properties of PB leucocytes; similarly, the duration of the fixation period (once >15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques. © 2009 Clinical Cytometry Society [source]

Flow cytometric analysis of cell-surface and intracellular antigens in the diagnosis of acute leukemia

AMERICAN JOURNAL OF HEMATOLOGY, Issue 2 2001
Rogelio Paredes-Aguilera
Abstract To evaluate the usefulness of flow cytometric detection of intracellular antigens (Ags) in establishing proper lineage affiliation and its contribution to the diagnosis of acute leukemia, we studied 100 consecutive patients in whom acute leukemia was diagnosed between January 1997 and July 1998. Immunological classification was assessed using a three-line panel of monoclonal antibodies for phenotypic characterization of leukemic blast cells as proposed at the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. We found 74 cases of B-cell lineage acute lymphoblastic leukemia (ALL), seven cases of T-cell ALL, and 19 cases of acute myeloid leukemia (AML). In this study cytoplasmic (cy) CD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML. Applied in combination with immunophenotyping this knowledge led to improvement in diagnostic precision and refinement of immunological classification, ensuring the selection of the most appropriate therapy for the patients studied. In conclusion, intracellular Ags detection was of utmost importance in establishing correct lineage affiliation in cases lacking expression of B, T, or myeloid surface Ags or disclosing equivocal or ambiguous immunophenotypic features and in identifying biphenotypic acute leukemia. In combination with FAB morphology and immunophenotyping, we were able to reliably classify all patients with acute leukemia in this study. Am. J. Hematol. 68:69,74, 2001. © 2001 Wiley-Liss, Inc. [source]