Microbial Lineages in Sarcoidosis. A Metagenomic Analysis Tailored for Low-Microbial Content Samples.

RATIONALE: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives.

On the nature of sarcoidosis.

More than 140 years since its recognition as a clinical entity, sarcoidosis remains enigmatic. Its classification as a disease vs. a syndrome is uncertain. Its etiology remains undefined. The "immune paradox" (delayed type hypersensitivity anergy in a setting of exuberant systemic granulomatous response) resists explanation. Its relationship to the Kveim test is poorly understood. Its prognostic determinants and treatment indications are among the unsolved or disputed problems. Immunological investigations generated the thesis that the characterizing systemic granuloma arise as a fallback reaction to inefficient cellular immune processing, due most often to impaired myeloid dendritic cell function of unknown cause. The concept that sarcoidosis represents a (genetically conditioned) default to a more primitive immunological response provides a unifying explanation for its development in persons with a variety of antigenic exposures and in individuals with cellular immune deficiencies. It furnishes a coherent explanation for the apparent paradox that individuals exhibiting the most intense cellular response experience the most favorable outcomes and for the adverse effect of corticosteroid-suppression in recent onset sarcoidosis.

A novel immunogen to modulate cytokine production and promote immune system reconstitution in HIV-AIDS.

This 'proof of concept' study was implemented in anticipation of identifying and testing a novel antigen of human origin as a potential immunogen in a paradigm that emphasizes immunomodulation and immune system reconstitution as requisites to the development of an effective human immunodeficiency virus (HIV)-acquired immune deficiency syndrome vaccine. Fifteen HIV-infected, highly active antiretroviral therapy (HAART) naive, otherwise healthy male seropositive patients were stratified by [CD4+] into 3 groups of 5 patients: group 1 >500/mm; group 2 > 250/mm but <500/mm; and group 3 < 250/mm. Five healthy male subjects were used as controls. Replicate peripheral blood mononuclear cell (PBMC) [H]thymidine uptake phytohemaglutinin-stimulated proliferation studies, and serum cytokine assays were carried out in the presence or absence of Kveim antigen at dilutions ranging from 0.001 to 100 µg/mL. Serum cytokines [interleukin-2 (IL-2), IL-4, IL-6, interferon gamma, and tumor necrosis factor alpha] were assayed using standardized methodology. Nonparametric statistical analyses and linear regression analysis were used to test for statistical significance and strength of associations. PBMCs harvested from HIV-infected patients and incubated, ex vivo, demonstrated reproducible, antigen concentration-dependent changes in cytokine production over a range of antigen concentrations (0.001-100 µg/mL) in contrast to antigen-naive PBMCs and controls. Significant correlations were demonstrated between antigen concentration and the amount of cytokines secreted. The magnitude of the cytokine response and the patterns of cytokine secretion were HIV group-specific and could be used to identify and distinguish between the 3 groups of HIV-infected subjects. A shift toward the production of type 1-like (Th1) cytokines characteristically seen in systemic sarcoidosis and associated with effective HAART was seen when patterns of cytokine secretion were compared between antigen exposed and antigen-naive PBMCs. PBMCs harvested from seropositive HIV-infected patients and exposed to the Kveim antigen have the following properties: (1) They demonstrate proliferation and exhibit an antigen concentration-dependent secretion of cytokines. The magnitude of the cytokine response can be used to identify and distinguish between groups of seropositive patients stratified by [CD4+]. (2) These PBMCs secrete cytokines in patterns suggestive of a shift to a type 1-like (Th1) response characteristic of HAART and sarcoidosis as opposed to the type 2-like (Th2) cytokine profile characteristic of HIV-acquired immune deficiency syndrome.

Sarcoidose: o que interessa na prática médica? / Sarcoidosis: what matters in medical practice?

Os autores, baseados na vivência clínica em Pneumologia e em revisão bibliográfica, repassam os principais tópicos às dificuldades no diagnóstico de sarcoidose. Abordam critérios para o diagnóstico, resultantes da somação de vários fatores, dando ênfase ao quadro clínico compatível, confirmação histopatológica, teste de Kveim-Siltzbach positivo e resposta clínico-radiográfica eficaz à corticoterapia sistêmica. Ilustram o tema em pauta com apresentação de três casos, os quais se revelaram por demais exuberantes pelos seus elementos apresentados com os sintomas respiratórios dominando toda a cena

The authors, based on the clinical practice in Pneumology and in review of literature, revise the main topics of the difficulties in the diagnosis of sarcoidosis. They approach criteria for the diagnosis, resultants of the addition of several factors, giving emphasis to the compatible clinical picture, histopathologic confirmation, positive Kveim-Siltzbach's test and clinical radiographic answer effective to the systemic corticotherapy. They illustrate the topic under discussion with presentation of three cases, which were revealed for too much exuberant for your elements presented with the breathing symptoms dominating the whole scene

Potential etiologic agents in sarcoidosis.

The etiology of sarcoidosis remains uncertain. The hallmark of sarcoidosis is the epithelioid granuloma, which serves as a necessary starting point for considering disease etiology. Any etiologic agent of sarcoidosis must also explain the typical clinical behaviors and characteristic immunopathologic features of the disease. One clinical observation that serves as a bridge to the etiology of sarcoidosis is the Kveim reaction. In this reaction, local epithelioid granulomas develop several weeks after the intradermal injection of homogenates of sarcoidosis tissue. Our group capitalized on the known properties of the Kveim reagent to search for candidate pathogenic tissue antigens in sarcoidosis without other a priori hypotheses regarding possible microbial or autoimmune etiologies. Using a limited proteomics approach based on the physicochemical properties of Kveim reagent, we detected a limited number of poorly soluble antigenic proteins in sarcoidosis tissues by protein immunoblotting, using sarcoidosis sera. Matrix-associated laser desorption/ionization-time of flight mass spectrometry identified one of these antigens to be the Mycobacterium tuberculosis catalase-peroxidase protein (mKatG). We found IgG responses to recombinant mKatG in more than 50% of patients with sarcoidosis but rarely in purified protein derivative (PPD)-negative control subjects. These findings support the conclusion that mKatG is a tissue antigen and target of the adaptive immune response in sarcoidosis, providing further evidence of a mycobacterial etiology in a subset of sarcoidosis. More generally, the approach used in these studies might be employed to discover and validate other candidate pathogenic antigens in sarcoidosis or other granulomatous disorders.

Seasonality of the onset of symptoms, tuberculin test anergy and Kveim positive reaction in a large cohort of patients with sarcoidosis.

BACKGROUND AND OBJECTIVES: Sarcoidosis is a systemic granulomatous disease of unknown aetiology and pathogenesis. This study evaluated the seasonal variation in the onset of symptoms, Tuberculin anergy and Kveim positive reaction in a cohort of 492 patients with sarcoidosis and in a subgroup of 248 patients with known Kveim test responses. METHODS: The medical records of 492 patients with sarcoidosis were retrospectively reviewed. Roger's test for cyclic variation was used to assess the statistical significance of the observed seasonal variation. RESULTS: For all sarcoidosis patients (n = 492) the onset of symptoms was most frequent in spring (61.8%) and least frequent in summer (31.7%) (P < 0.001). For patients with Tuberculin anergy (n = 364) the onset of symptoms was most frequent in spring and least frequent in autumn (P < 0.001); there was no seasonal variation among Tuberculin positive patients (n = 128). Of those patients with a Kveim test result (n = 248), the onset of symptoms was most frequent in spring and least frequent in summer (P < 0.001); there was no seasonal variation for patients with a negative Kveim results (n = 50 patients). CONCLUSIONS: The onset of the symptoms was most frequent in spring and least frequent in the second half of the year (summer or autumn) in patients with sarcoidosis, Tuberculin anergy and a positive Kveim reaction. The significance of this finding in relation to aetiology and clinical utility needs to be further assessed.

Pathology of sarcoidosis.

The role of pathology in the diagnosis of sarcoidosis is identification of granulomas in tissue specimens and performance of studies to exclude known causes of granulomatous inflammation. The granulomas of sarcoidosis are nonspecific lesions that, by themselves and in the absence of an identifiable etiologic agent, are not diagnostic of sarcoidosis or any other specific disease. Among the diseases to be excluded are mycobacterial, fungal, and parasitic infections, chronic beryllium disease and other pneumoconiosis, hypersensitivity pneumonitis, and Wegener's granulomatosis. Even after extensive workup a substantial number of granulomas will remain unclassified. Not every disease that features nonnecrotizing granulomas of undetermined etiology is sarcoidosis. The granulomas of sarcoidosis may exhibit focal necrosis of minimal amount. In cases with granulomas that exhibit a greater degree of necrosis an infectious or other nonsarcoid etiology should be strongly suspected. Strict clinical, radiological, and pathological criteria must be used for diagnosis. In cases that exhibit necrotizing granulomas with more than minimal, focal necrosis, extrathoracic involvement only, and/or incompatible clinical and radiological findings, the diagnosis of sarcoidosis should be approached with great caution. The diagnosis is most secure when compatible clinical and radiological findings are supported by the demonstration of microorganism-negative, nonnecrotizing granulomas in a biopsy specimen accompanied by biopsy evidence or strong clinical evidence of multisystem involvement, and negative cultures for bacteria, mycobacteria, and fungi. A positive Kveim-Siltzbach test provides strong support for the diagnosis of sarcoidosis.

Sarcoidosis / Sarcoidosis

La sarcoidosis es una afección sistémica, de naturaleza granulomatosa y de etiología desconocida, en cuya patogenia intervienen, probablemente mecanismos inmunológicos. La afección puede comprometer prácticamente todos los órganos y sistemas siendo los mas frecuentemente afectados el aparato respiratorio, la piel y el ojo. Las formas agudas regresan generalmente en forma espontánea; las crónicas se tratan con corticoides y/o inmunosupresores.

Analysis of differentially regulated mRNAs in monocytic cells induced by in vitro stimulation with Kveim-Siltzbach test reagent.

Sarcoidosis is a granulomatous disorder of unknown origin. The sarcoid spleen-derived Kveim-Siltzbach test reagent (KSTR) elicits a sarcoid-specific, granulomatous response and was used to diagnose sarcoidosis. The active component and the pathomechanism by which KSTR induces granuloma are still unknown. This study investigated the KSTR-associated gene expression pattern in cells of patients with sarcoidosis or chronic beryllium disease (CBD). The monocytic-like cell line U937, alveolar macrophages of sarcoidosis patients, and peripheral blood monocytes of patients with CBD were stimulated with KSTR and other granuloma-associated stimuli. The KSTR-associated gene expression pattern was analyzed by means of differential display reverse transcription-polymerase chain reaction in combination with a multiple comparison of expressed sequence tags induced in response to KSTR and other granuloma-associated stimuli. Depending on the origin of cells tested, 3.7% to 14.6% of the analyzed sequence tags showed differential regulation induced by granuloma-associated stimuli. Alterations restricted to KSTR stimulation could be observed in 1.2% for the cell line U937, in 2.8% for blood monocytes of patients with CBD, and 1.3% for alveolar macrophages of sarcoidosis patients. These data are comparable to those achieved for the other granuloma-associated stimuli tested in this study. Therefore, it can be assumed that KSTR induces pathomechanisms for granuloma formation in sarcoidosis as beryllium in CBD.

Older sarcoidosis patients: experience of a medical center in Turkey.

OBJECTIVES: Although sarcoidosis is classically defined to be a disease of young adults, it might also be seen at older ages. There are very few clinical studies which focus on the features of patients diagnosed at older ages. In this study, we tried to determine the frequency of patients diagnosed at or above 50 years of age and to compare the clinical and demographic features of these subjects with other sarcoidosis patients. METHODS: We evaluated the general clinical features of sarcoidosis patients more than 50 years of age who were diagnosed at our center within a 36-year period. We also compared the clinical features of older sarcoidosis patients with the features of other patients. RESULTS: Of 579 sarcoidosis patients being followed up at our center, 102 (17.7%) were older than 50 years of age at the time of initial diagnosis. The female to male ratio in this group was higher than the ratio in other sarcoidosis patients (3.43 versus 1.85, P = 0.015). When the features of older patients were compared with other sarcoidosis patients, extrapulmonary involvement was observed to be more common in this group (P < 0.001). By contrast, arthritis or arthralgia (P < 0.001), clinical presentation in the form of Löfgren syndrome (P < 0.001), erythema nodosum (P < 0.001), and uveitis (P = 0.006) were less frequent. CONCLUSIONS: Although generally presenting as a disease of the young, in many subjects sarcoidosis is diagnosed at older ages, and this study indicates that the clinical features of sarcoidosis in older subjects differ from those found among younger patients.

[Ocular manifestations in sarcoidosis]. / Objawy oczne w przebiegu sarkoidozy.

PURPOSE: The ophthalmic examination of patients with diagnosed systemic sarcoidosis. MATERIAL AND METHODS: 33 patients (17 women, 16 men), aged 22-60 years had ophthalmic examination (visual acuity, anterior and posterior segment evaluation, applanation tonometry). In 8 patients repeated examination was performed (duration of observation: 2-31 months). RESULTS: In 27 patients no characteristic features of ocular sarcoidosis were found. In 6 persons (18.2%) variety of ocular lesions was recognized (nodular infiltrations of the eyelids, chronic uveal inflammation, signs of anterior and posterior uveitis in the past, optic disc oedema). In 3 cases ocular lesions preceded the signs of systemic sarcoidosis. This emphasizes the importance of the routine ophthalmic examination of patients with suspected or proven sarcoidosis.

Analysis of the Kveim-Siltzbach test reagent for bacterial DNA.

The sarcoid spleen-derived reagent for the Kveim-Siltzbach test (KST) elicits a sarcoid-specific, granulomatous, cutaneous response used to establish the diagnosis of sarcoidosis. In the context of the ongoing discussion of a bacterial cause of sarcoidosis we asked the question whether bacterial DNA could be found in the KST reagent. For this purpose two different KST reagents, an identical preparation from a normal spleen, and a native sarcoid spleen were analyzed by polymerase chain reaction (PCR) employing universal primers detecting conserved DNA sequences coding for bacterial ribosomal 16S RNA. Neither KST reagents, the control preparation, nor the spleen yielded a positive signal, indicating that the preparations are free of bacterial contamination. Because the KST reagent elicits granuloma, these results do not support the hypothesis of a bacterial cause of sarcoid granuloma.

New monoclonal antibodies against the epithelioid cells in sarcoid granulomas.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. Extensive investigations of granulomas have suggested several possible causes, but these are still controversial. We previously developed an anti-Kveim monoclonal antibody, IHY-1, which reacts with sarcoid granulomas as well as with epithelioid cells of various granulomatous diseases including tuberculosis. In the present study, we developed 2 new anti-Kveim monoclonal antibodies, IHY-2 and IHY-3, which react with epithelioid cells in sarcoidosis but not in tuberculosis. These antibodies reacted with a small population of alveolar macrophages in sarcoidosis and hypersensitivity pneumonitis by flow cytometry, as well as with most epithelioid cells in sarcoid granulomas by immunoperoxidase technique, suggesting that these cells expressed the antigen present in the Kveim reagent. Although the antigens recognized by these antibodies have not yet been identified, these monoclonal antibodies might become useful tools to elucidate the etiology of sarcoidosis.

Kveim antigen: what does it tell us about causation of sarcoidosis?

This article explores the role of the Kveim-Siltzbach (KS) test in finding the cause of sarcoidosis. Experimental granulomas are formed by a T-cell mediated immunologic response to particulate agents which resist degradation and persist in tissues for prolonged periods. There is no animal model for human sarcoidosis. However, the KS test is an in vivo model of sarcoidosis. KS homogenates incite a tissue response in patients with sarcoidosis histologically identical to disease-caused granulomas. The suspensions are particulate and maintain activity when exposed to a variety of chemical and physical stresses. Studies of the monocyte and T-cell host response confirm that KS reagent provokes a sarcoidosis-like antigen driven granuloma. KS suspensions contain an antigen(s) that incite a granuloma identical with that occurring in sarcoidosis. Identification of the active principle in KS suspensions should aid in the search for the cause of sarcoidosis.

Mantoux test site granuloma: an appraisal of the diagnostic value in sarcoidosis.

Out of 146 cases, 92 (64%) cases with active sarcoidosis showed granulomas in biopsy at 4-6 weeks after Mantoux test (MT). No granuloma was seen in anyone of the 162 sites injected with other allergens in 27 of MT site positive cases, serving as self-control. False positive biopsies were not seen. Another control group of 40 patients with other respiratory disorders like tuberculosis failed to produce any granulomatous response at the site of MT. Mantoux test site biopsy may prove a valuable diagnostic test in sarcoidosis in India.

[Evaluation of different pathologic methods for the diagnosis of sarcoidosis].

The biopsy of different tissues were reviewed in 34 patients with intrathoracic sarcoidosis for determining their value on the diagnosis of sarcoidosis. Biopsy of different tissues was performed by routine method. The transbronchial lung biopsy (TBLB) was done under bronchoscope. The diagnosis was confirmed in 90.9% (10/11) of the biopsies of periphery lymph nodes, 75.0% (3/4) of the scalene nodes' biopsies, 68.4% (13/19) of the transbronchial lung biopsies and 70.6% (12/17) of the bronchial mucosal biopsies (BMB) through fibroptic bronchoscopy. Only 4 in 7 patients were diagnosed through skin biopsies. Kveim test was positive in 5 of 6 patients. Both TBLB and BMB were undertaken in 16 patients. The coincidence ratio between them was as high as 81.3%. The total diagnostic percentage through bronchoscopy was 87.5%. Significant differences were found between the results of TBLB or SMB before and after 1990 (P < 0.05 and 0.01 separately proved by chi 2 test). After 1990, 85.7% of TBLBs and 92.3% of BMBs were diagnostic. According to the study, TBLB and BMB were the methods that were repeatable, highly diagnostic, less invasive, and mutually compensable. They were superior over the other methods in the diagnosis of sarcoidosis. The diagnostic yield could be elevated by the accumulation of experiences and the improvement of techniques. They deserved recommending in the clinical practice.

[The Kveim-Siltzbach test in sarcoidosis]. / La prueba de Kveim-Siltzbach en la sarcoidosis.

BACKGROUND: The Kveim-Siltzbach test has been widely used in the diagnosis of sarcoidosis. This reactive is currently available in the authors' hospitals. Previous experience with this test in the same centers was reviewed to know its diagnostic usefulness. The present is the first series described in Spain. METHODS: From 1977 to 1988 the Kveim-Siltzbach test was performed in 79 patients diagnosed or suspected of having sarcoidosis. The study was carried out as cooperation of the validation process of sarcoid spleen suspensions (lots K12, K12 1/2, K32, K41, K42, K42 1/2 and K50 of the Colindale antigen) prepared in the Standards Laboratory for Serological Reagents in the Royal Brompton Hospital in London. RESULTS: The results of test positivity were determined following the criteria established by Siltzbach. The global sensitivity of the Kveim-Siltzbach test was 78% (CI 95% = 67.8-86.9). Positivity ranged from 84% in subacute sarcoidosis to 61% in chronic sarcoidosis (p = 0.08). Likewise, it was higher in patients with radiologic stage I (84%) and II (81%) with respect to stage III (62%) or the cases with exclusive extrathoracic sarcoidosis (stage 0) (57%) (p = 0.11). No association was found between the Kveim-Siltzbach test positivity and the elevation in serum values of the angiotensin-converting enzyme (p = 0.575). CONCLUSIONS: In the present study the Kveim-Siltzbach test showed high sensitivity in the diagnosis of sarcoidosis. This fact, together with the high specificity observed in other studies, makes the test a very useful tool for the non-invasive diagnosis of sarcoidosis.

Selection of oligoclonal V beta-specific T cells in the intradermal response to Kveim-Siltzbach reagent in individuals with sarcoidosis.

Sarcoidosis is a multiorgan granulomatous disorder of unknown etiology characterized by noncaseating granulomas in involved tissues. A positive Kveim-Siltzbach reaction is a granulomatous response to an intradermal injection of a suspension of sarcoid tissue extract in individuals with sarcoidosis. The protracted time course and granulomatous features of this reaction have a striking resemblance to the Mitsuda reaction in tuberculous leprosy, which suggests that the Kveim-Siltzbach reaction is a response to an unknown Ag(s). To evaluate whether this reaction is Ag-driven, an analysis of the TCR V beta repertoire in 15 Kveim-Siltzbach reaction sites was performed using a PCR technique and primers specific for 20 V beta gene families. Results of this analysis demonstrated a pattern of V beta expression dominated by expression of V beta 2, V beta 3, V beta 6, or V beta 8 to levels > 20% of total V beta gene expression in nine of 15 individuals. Analysis of paired biopsy and blood specimens revealed a preferential expression of specific V beta genes, such as V beta 3, V beta 5, and V beta 8, at sites of Kveim-Siltzbach reactions to levels four to seven times that of the corresponding peripheral blood. Sequence analysis demonstrated that preferential expression of specific V beta genes at Kveim-Siltzbach reaction sites is oligoclonal. Furthermore, the dominant V beta 8 sequence present at one of the reaction sites contained a sequence motif in the variable-diversity-joining junctional region previously identified in sarcoid lung and blood T cell populations. These results suggest that the Kveim-Siltzbach reaction is characterized by a limited TCR beta-chain repertoire consistent with an Ag-driven T cell immune response.