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

Lupus pernio. Presentación de una serie de 8 pacientes / Lupus pernio. A report of a series of 8 patients

Objetivos. Aunque el lupus pernio (LP) es la lesión cutánea más característica de la sarcoidosis crónica, en nuestro país se han comunicado muy pocos casos. El objetivo del estudio fue revisar la frecuencia y características clínicas de los pacientes con LP en una serie amplia de pacientes con sarcoidosis. Métodos. Revisión retrospectiva de la frecuencia y características de los pacientes diagnosticados de LP de la serie de sarcoidosis de nuestro centro durante un periodo de 35 años. Resultados. De 507 pacientes con sarcoidosis, 8 (1,6%) presentaron LP. La edad media fue de 42 años. En 6 casos el LP fue la forma de presentación de la sarcoidosis. Cinco pacientes mostraron afectación de la piel nasal y un caso presentó afectación severa de la mucosa nasal. Todos los pacientes fueron tratados con antimaláricos, 4 con corticoides, 2 con láser o con combinaciones con otros fármacos. Resultados. Ningún paciente con afectación cutánea nasal presentó remisión del LP. Conclusiones. El LP es poco frecuente en las formas clínicas de la sarcoidosis de nuestro país. La afectación cutánea nasal no responde al tratamiento. La reciente introducción del infliximab puede representar un avance en el tratamiento del LP(AU)

Objectives. Although lupus pernio (LP) is the most characteristic cutaneous lesion of chronic sarcoidosis, only a few cases have been reported in our country. The aim of this study was to review the frequency and clinical characteristics of patients with LP in a large series of patients with sarcoidosis. Methods. A retrospective review of the frequency and characteristics of patients diagnosed as having LP from the series of sarcoidosis of our institution for a period of 35 years was performed. Results. Eight (1.6%) out of 507 patients with sarcoidosis were diagnosed of LP. Mean age was 42 years. In 6 patients, LP was the presentation form of sarcoidosis. Five patients had involvement of the nasal skin and one patient severe involvement of the nasal mucosa. All the patients were treated with antimalarial drugs, 4 with oral corticosteroids, 2 with laser therapy, or with combinations with other drugs. None of the patient having nasal skin involvement showed remission of LP. Conclusions. LP is a rare clinical form of sarcoidosis in our country. No treatment is effective for nasal skin involvement. The recent introduction of infliximab may represent an advance in the treatment of LP(AU)

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.

Valor actual del test de Kveim en el diagnóstico de la sarcoidosis / Current value of the kveim test in the diagnosis of sarcoidosis

Introducción. La sarcoidosis es una enfermedad multisistémica para cuyo diagnóstico es preciso demostrar granulomas en uno o más órganos. El test de Kveim-Siltzbach puede ser útil para casos en los que no se puede demostrar inflamación granulomatosa en órganos afectados. En los últimos años su uso se ha restringido debido a la dificultad para obtenerlo y algunos autores lo han desaconsejado por el riesgo de transmisión de enfermedades infecciosas como la encefalopatía espongiforme bovina. Pretendemos analizar el valor actual del test Kveim-Siltzbach en los pacientes con especiales dificultades diagnósticas.Métodos. Se ha practicado el test de Kveim-Siltzbach a 20 pacientes remitidos al Servicio de Medicina Interna del Hospital de Bellvitge de Barcelona con sospecha de sarcoidosis en los que el diagnóstico no pudo ser confirmado mediante otros métodos. Las reacciones positivas se clasificaron en tres categorías según la cantidad de granulomas y la presencia de necrosis. Los pacientes fueron controlados durante un mínimo de 2 años.Resultados. El test de Kveim-Siltzbach fue positivo en 10 casos (ligeramente en 4 casos, moderadamente en cuatro e intensamente positivo en dos). En nueve de ellos el diagnóstico de sarcoidosis se confirmó mediante otros procedimientos. Únicamente en un paciente con test positivo el diagnóstico de sarcoidosis no pudo confirmarse por otros métodos.Conclusiones. El test de Kveim-Siltzbach sigue siendo útil como procedimiento diagnóstico en pacientes con especiales dificultades en la demostración histológica de granulomas (AU)

Moratoria para el test de Kveim / Moratorium on Kveim test

No disponible

Fiebre de origen desconocido como forma de presentación del seudotumor inflamatorio del hígado / Inflammatory seudotumor of the liver presenting as fever of unknown origin

No disponible

[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.