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.

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.

Diagnosis of sarcoidosis.

During the last decade, many biochemical and immunologic advances have been made in the treatment and understanding of sarcoidosis. These studies have helped us to understand the basic mechanisms involved in granuloma formation, and many clinicians have used the information to diagnose and assess the activity of sarcoidosis. Further studies are needed to clearly establish the role of these advances in the everyday management of patients with sarcoidosis.

Quantitation of cutaneous Langerhans cells of sarcoidosis patients.

Langerhans cells play a role in cell-mediated immune reactions which are often depressed in sarcoidosis. We examined the epidermis of 17 anergic patients with sarcoidosis (Kveim-reactive and/or biopsy-proved) for the number of Langerhans cells in noninvolved skin and in any cutaneous sarcoidal lesions. Skin biopsies of 10 healthy volunteers served as controls. In comparison to controls, the epidermis overlying noninvolved (p less than 0.05), sarcoidal (p less than 0.0005), and Kveim-reactive (p less than 0.005) skin contained significantly fewer detectable Ia and T6 antigen-bearing Langerhans cells. The reductions within noninvolved skin were most pronounced in patients with multisystem disease. Lower epidermal Langerhans cell densities, in comparison to controls, were detected in both prednisone-treated and untreated patients. Epidermis overlying sarcoidal skin of untreated patients contained significantly fewer Ia and T6 antigen-bearing Langerhans cells (p less than 0.05, p less than 0.0025, respectively) than epidermis from noninvolved skin. Whether reduced numbers of cutaneous Langerhans cells are due to either a local and/or systemic effect of sarcoidosis, or reflect the anergic state of these patients is unknown.

Immunology of sarcoidosis.

The cardinal immunologic changes in sarcoidosis consist of depression of delayed-type hypersensitivity, hyperreactive circulating antibody responses and the Kveim-Siltzbach skin test phenomenon. Depression of delayed-type hypersensitivity is demonstrated by skin tests using tuberculin, mumps, pertussis, trichophytin, oidiomycin, dinitrochlorobenzene and Californian keyhole limpet hemocyanin. The cultured lymphocytes from patients with depression of delayed-type hypersensitivity react poorly to phytohemagglutinin, and there is a close correlation between anergy of lymphocytes in culture and by cutaneous anergy. In vivo cutaneous anergy mirrors in vitro cellular hyporeactivity. Other technics used to expose immunologic defects in peripheral lymphocytes of patients with sarcoidosis include tests of T and B cell function, rosetie formation and migration inhibition. Whereas there is cutaneous anergy and impaired cellular immunity in patients with sarcoidosis, the reverse holds for circulating factors. There are increased circulating immunoglobulin levels, increased circulating antibody levels to Epstein-Barr, herpes simplex, rubella, measles and parainfluenza viruses, increase antibody response to mismatched blood and occasional false-positive Wassermann reactions, but there is no increase in circulating autoan tibodies. There is no evidence that patients with sarcoidosis belong predominantly to any particular histocompatibility locus. Worldwide figures for the Kveim-Siltzbach skin test are presented. They provide evidence of its specificity in various international series. The causes of nonspecific reactions are discussed.

Positive Kveim test in patients with coexisting sarcoidosis and human immunodeficiency virus infection.

We present two cases of sarcoidosis complicated by HIV infection. Each case had a different level of sarcoidosis activity and coexisted with either an AIDS-related infection or a HIV-positive state. Manifestations of sarcoidosis were not apparent in the patient with the AIDS-defining opportunistic infection, but were active in the patient with asymptomatic HIV infection. Both patients had granulomatous reactions to Kveim antigen, and one had such a reaction following an AIDS-defining infection. These findings suggest that non-T-cell mechanisms may be involved in granuloma formation in sarcoidosis.

Anti-Kveim monoclonal antibody. New monoclonal antibody reacting to epithelioid cells in sarcoid granulomas.

A monoclonal antibody to the sarcoid granulomagenic agent contained in Kveim suspension was prepared by immunizing mice with Kveim suspension. One monoclonal antibody (IHY-1) that reacted with the epithelioid cells in sarcoid granulomas on immunoperoxidase technique was selected. The immunoperoxidase technique was used to compare this monoclonal antibody's binding to sarcoidosis- or tuberculosis-affected lymph nodes. IHY-1 is a monoclonal antibody of IgM class. This antibody did not react to erythrocytes, lymphocytes, monocytes, alveolar macrophages, or the macrophage-derived cell lines such as U-973 and KG-1. It reacted to granuloma epithelioid cells of sarcoidosis-affected lymph nodes. The monoclonal antibody also reacted positively to epithelioid cells in tuberculous granulomas although the reaction was not as strong. Since IHY-1 was found to bind to both types of granulomas, this suggests that the epithelioid cells in sarcoidosis have antigenicity common to the epithelioid cells in tuberculosis.

Course of chronic hilar sarcoidosis in relation to markers of granulomatous activity.

Studies of the course of sarcoidosis have emphasized that patients with hilar or mediastinal adenopathy usually recover within several years or develop dissemination to the lungs. Chronic hilar and mediastinal adenopathy persisting with little or no change for many decades is an important subgroup that has not received adequate attention. Twelve such patients have been studied. Seven remained asymptomatic, despite persistent adenopathy, for a mean period of 16 years; two with disfiguring facial sarcoids received corticosteroids for 18 and 27 years, respectively, and three patients after ten years of stable adenopathy developed pulmonary infiltrates. Tests performed on patients with hilar adenopathy to evaluate cellular activity after a mean interval of over 16 years included Kveim reaction (positive in nine of ten), serum angiotensin converting enzyme (elevated in eight of 12), and gallium-67 scanning (hilar uptake in all eight tested). Results were similar for patients who remained well and for those who had symptomatic or progressive disease, indicating that these parameters of granulomatous activity do not reflect the duration of the disease, its outcome, or the need for treatment.

Validation and standardization of Kveim test suspensions prepared from two human sarcoid spleens.

Single lots of a Chase-Siltzbach type I Kveim test material from each of two sarcoid spleens and designated lot 5 of spleen K12 and lot 1 of spleen K13 have been validated alongside a single lot (lot 10) of a 'standard' suspension provided by Dr L. E. Siltzbach and prepared identically from the spleen of patient J (SPLEEN J) in New York. Additionally, a half-dilution of lot 5, K12, was included in this comparison. The reactivity of each suspension was assessed among patients with active and inactive sarcoidosis. The selectivity of each suspension for sarcoidosis was assessed similarly by comparison with results in patients with active and quiescent pulmonary parenchymal tuberculosis and in healthy subjects. All patients were closely matched and two Kveim tests were made in each subject according to a prearranged statistical design. The reactivity of the K12, K12 1/2 dilution, and K13 suspensions among patients with active and inactive sarcoidosis was closely similar to that with the 'standard' S10 suspension and in accordance with the expected proportions of reactions in patients at different stages of sarcoidosis. The K12, K13, and 'standard' S10 suspensions yielded a negligible proportion of positive reactions among patients with active and quiescent pulmonary tuberculosis and among healthy subjects: thus, as judged by these tests each suspension showed a high degree of selectivity for sarcoidosis. The results of this validation study are discussed in relation to the results of other studies in which lots 5 and 14 of K12 and early and late batches of a suspension prepared from another sarcoid spleen at the Commonwealth Serum Laboratories designated CSL and provided by Dr T.H. Hurley in Melbourne were employed. Using lot 5 of K12 positive reactions were found in an appreciable proportion of patients with Crohn's disease, ulcerative colitis, and tuberculous lymphadenitis. A closely similar rate of positive reactions was encountered among patients with Crohn's disease following tests with batch 0025 of CSL suspension and with another lot (lot 14) derived from spleen K12. A close concordance of results was obtained with lot 5(K12) and with batch 0042 CSL among patients with ulcerative colitis, but at a lower rate of reactivity. We conclude that positive reactions also occur in some diseases other than sarcoidosis and consider that the difficulties in determining the criteria for an acceptable test suspension become increasingly apparent as additional Kveim tests are made with one particular lot and with seqential lots of material from a 'validated' tissue source.

Sarcoidosis: histopathological definition and clinical diagnosis.

Sarcoidosis is best defined in histopathological terms as 'a disease characterised by the presence in all of several affected organs and tissues of non-caseating epithelioid-cell granulomas, proceeding either to resolution or to conversion into hyaline connective tissue'. Although the defining characteristics are thus histopathological, diagnosis during life depends largely upon clinical, radiological, and immunological findings. The amount of support required from histology varies greatly from case to case. Though histology from one site cannot in itself establish the diagnosis of sarcoidosis, a generalised disease, detailed histological study of biopsy tissue makes an important and often essential contribution. In many instances, complete lack of necrosis, an intact reticulin pattern, and failure to demonstrate infective agents permit an unequivocal statement of compatibility with this diagnosis; however, a compatible clinical picture and absence of evidence of known causes of local granulomatous reactions or of other generalised granulomatous diseases are required for definitive diagnosis. In some, the histological pattern deviates in some particular from the accepted 'typical' pattern; there may be a little necrosis, the follicular pattern of the granuloma may be less than perfect, and exclusion of known infective agents can never be absolute. In such instances, subsequent surveillance, including possible response to treatment, may show a clinical course justifying a diagnosis of sarcoidosis, and necropsy may establish it; but it must be recognised that in a few cases, particularly those in which the clinical evidence of disease is confined to one organ, diagnosis is likely to remain in doubt for long periods. Reports on the histology of the Kveim test should be made without knowledge of clinical findings and in terms of the presence and quality of granulomatous response. A granulomatous reaction to a validated test suspension makes a contribution to diagnosis similar to the finding of granulomas in an additional organ or tissue.

Early cellular responses to intradermal injection of Kveim suspension in normal subjects and those with sarcoidosis.

In a detailed controlled study of the cellular response to Kveim suspension in vivo we used immunohistological and histochemical methods to examine cryostat sections of immature Kveim biopsy specimens in subjects with sarcoidosis and normal controls. Changes seen at 48 hours, at which time papular reactions have sometimes been reported, are described. Eight cases of sarcoidosis previously confirmed by a positive Kveim test were studied, in five of whom the test remained positive; plus two subjects with sarcoidosis studied prospectively; and four healthy controls. There were two main features of the 48 hour response: collagen disruption with associated histiocytes, which showed increased acid phosphatase activity; and perivascular infiltrates of lymphocytes and small groups of dendritic cells. The T4:T8 ratios in the infiltrates were similar to those found in the peripheral blood of the subjects, and few lymphocytes showed evidence of activation. T lymphocytes were also seen free in the dermis and migrating to the epidermis. Small juxtacapillary clumps of dendritic cells, identified by NA1/34 (= OKT6; Langerhans' cells) and RFD1 (interdigitating cell) monoclonal antibodies, were found. The Langerhans' cells in the epidermis were, however, normal in number and distribution. These features, which were found in all groups, are not consistent with pre-existing hypersensitivity to Kveim suspension in sarcoidosis. Subsequent differences between sarcoid and normal subjects in the development of granulomas in the Kveim response may therefore relate to the different handling of the foreign material by the cells affected, rather than to differences in the early non-specific recruitment of the cells to the test site.

The Edinburgh spleen: source of a validated Kveim-Siltzbach test material.

Although it is true that some Kveim-Siltzbach test suspension may for reasons unknown behave in a totally nonspecific way and so be useless in the confirmation of active sarcoidosis, the experience with the Edinburgh spleen has shown that it is also true that a preparation can be made which acts specifically in the sarcoid context and fulfils all the Siltzbach criteria. The active principle probably resides in the membrane components of sarcoid tissue cells.

Experience with a Dutch kveim suspension in men and guinea pigs.

A Kveim antigen was prepared according to the Chase-Siltzbach method from the spleen of a patient with sarcoidosis. This antigen was tested in a number of patients with sarcoidosis or possible sarcoidosis and also in a number of non-sarcoid patients. Several lots prepared from this spleen were compared, yielding differences in reactogenicity. Maybe this can be explained by heterogeneity of the spleen. An animal model (the BCG-presensitized guinea pig) is presented that may be applicable for a preclinical evaluation of lots of Kveim antigen.

The Kveim test: analysis of results of tests using colindale (K 12) materials.

Results of an analysis are presented of the salient clinical, pathological, and Kveim-test data of importance in the diagnostic assessment of 839 patients in whom a Kveim test was made as part of their routine clinical investigation. From these analyses the following significant observations can be made: 1) The age distribution and negative tuberculin results in patients with sarcoidosis were in keeping with the diagnosis of sarcoidosis, whereas for those in whom a diagnosis other than sarcoidosis was made, they were not. 2) The findings of 64% positive tuberculin results in cases of confirmed sarcoidosis of less than 2 yr known duration and only 39% in those of greater than 2 yr duration are closely similar to those found in international Kveim-test studies reported by Siltzbach in 1966 and Hurley and Bartholomeusz in 1971. 3) The use of the material was helpful in confirming a diagnosis of sarcoidosis in patients with extrathoracic lesions, for positive results were obtained in 33% of those with uveitis 40% of those with erythema nodosum only (findings which are in keeping with those of other studies in the United Kingdom, and 60% among 27 patients presenting with lesions in various sites, including 2 with cranial nerve lesions and one with cardiac arrhythmia. 4) We found only 2 positive reactions (less than 1%) among 221 patients in whom a diagnosis other than sarcoidosis was reached. Clearly these findings show that the Kveim test material. Lots 19 and 22, of spleen K 12 exhibited not only a high degree of reactivity and selectivity for sarcoidosis, but also that they were of considerable practical value as an aid to diagnosis. The finding of only 2(0.9%) positive Kveim tests among 221 patients with diseases other than sarcoidosis is of special interest. Of the different diseases that were ultimately diagnosed only 6 patients tested had lymphoma, only 11 had either pulmonary or lymphatic tuberculosis, and only one had Crohn's disease. Although these numbers are small, it is relevant to compare the Kveim-test data of Lots 19 and 22 of K 12 with those of Lot 5 and of Lot 14 of the same spleen. In validation studies Lot 5 yielded the expected proportion of positive reactions at different stages of sarcoidosis and a negligible proportion of positive reactions with active or quiescent pulmonary tuberculosis or among healthy subjects. However, in subsequent studies in special groups of patients, those with Crohn's disease, ulcerative colitis, or lymphatic tuberculosis, a proportion of the Kveim tests performed with these lots were positive. Within the context of the present field study, the results of these analyses confirm that the last lots of K 12 material exhibited a high degree of selectivity for sarcoidosis and that they emphasize again the practical value of the Kveim tests with suspensions that have undergone careful validation.

Immunological studies in sarcoidosis: a comparison of in vivo and in vitro Kveim tests.

No migration inhibition was observed in the 46 patients or the 12 controls when the Danish Kveim antigen was used in the LMAT. No migration inhibition was found in 23 of the same patients when the LMT was employed using the same antigens (Table 3).

Patterns of sarcoidosis in three population groups in New York City.

Caucasians in this study group presented in the chronic stage of sarcoidosis more frequently than the Blacks and Puerto-Rican-born. 2) Twenty-eight percent of the Caucasians and 26% of the Blacks who presented in the chronic stage of sarcoidosis, died of pulmonary insufficiency. 3) Puerto-Rican-born women are especially prone to present with erythema nodosum and Stage I radiographic sarcoidosis. 4) Sarcoidosis with extrathoracic organ involvement was more common in Black and Puerto-Rican-born patients than in Caucasians. 5) The size of the Kveim papules was larger in the Black and Puerto-Rican-born patients than among the Caucasians. 6) Two-thirds of all groups were insensitive to tuberculin. 7) The extent and duration of disease at presentation is a major determinant prognosis. 8) Ethnic factors influence the clinical patterns in sarcoidosis, but socioeconomic factors may preclude generalizations based solely upon clinic populations in New York City.

Flexible-bronchoscope biopsy of lung and bronchial wall in intrathoracic sarcoidosis.

1) Twenty of 25 patients (80%) with sarcoidosis had positive lung biopsies obtained by flexible-bronchoscope biopsy. 2) Three of the 5 negative biopsies were in patients with chronic sarcoidosis whose chest radiograph was unchanged for 1 yr or more. 3) Two insignificant pneumothoraces occurred as a complication of the bronchoscopic biopsy procedures. 4) Noncaseating epithelioid-cell granulomas found on fiberoptic bronchoscopic biopsy are no more specific for the diagnosis of sarcoidosis than similar findings in any other organ biopsy.

Further observations on the transmissibility of Crohn's disease.

In controlled experiments normal and immunologically deficient CBA mice were inoculated with whole fresh, fresh autoclaved, fresh supernatant, or filtered supernatant (0.2 mu) of whole Crohn's or non-Crohn's homogenate into footpads, intraperitoneally or intravenously. Epithelioid and giant-cell granulomas were present in a substantial proportion the footpads and in bowel or mesenteric lymph nodes of a proportion of given each fresh Crohn's homogenate by any of these routes 15-17 months after inoculation, but were not present in mice given non-Crohn's or autoclaved Crohn's homogenate. Successful passages were achieved following the inoculation of Crohn's mouse tissue homogenates, including passage from mice receiving filtered supernatant (0.2mu) or whole Crohn's homogenate, into footpads or intravenously. The epithelioid- and giant-cell granulomas evolved slowly over a period of many months following the inoculation of Crohn's tissue or passage homogenates and persisted thereafter. The transmissible agent is inactivated when homogenate from Crohn's tissue is autoclaved, can be passaged successfully into footpads or intravenously, and has been shown to pass an 0.2 mu membrane filter. It is therefore presumably viable and must approximate to the size of a virus or be capable of being deformed to pass a filter of such a pore size (0.2mu).

Comparative in vitro reactivities of leukocytes from sarcoids and normals to different Kveim preparations.

Comparative studies of in vivo and in vitro reactivities to Kveim preparations have been carried out in 45 sarcoid and 30 normal subjects. Lymphocytes of 14 of 45 sarcoids and 4 of 30 normals proliferated significantly in response to at least 1 of the 4 Kveim suspensions used for in vitro studies. The prevalence of positive responses were significantly greater in sarcoids than normals in cultures containing Kveim-CSL and Kveim-Edinburgh. The Kveim-reactive cells were less stimulated by PHA, but no other significant alterations were found, including the presence or absence of in vivo Kveim reactivity in the cell donors. Reactivity to more than 1 Kveim preparation occurred in 9 of 14 cases. Kveim-induced leukocyte migration inhibition occurred more commonly in sarcoids than normals, but differences were not as striking. There was not a precise correlation between proliferative and migration-inhibition responses to Kveim.

Macrophage reactivity in skin window of sarcoidosis patients.

We investigated 582 cases of sarcoidosis by the skin-window (Rebuck) method. The number of macrophages appearing in skin windows was significantly increased in 432 patients with sarcoidosis in the active phase. Further, increased numbers of macrophages in the windows were found in patients with a positive Kveim-Siltzbach test, high immunoglobulin levels, increased B lymphocytes, and decreased T lymphocytes. Passive transfer of delayed-type hypersensitivity was weaker and shorter in sarcoidosis patients with increased presence of macrophages than in healthy controls. A close correlation between high macrophage reactivity and the other sepcific immunological factors in sarcoidosis is suggested.