12/5/2023 0 Comments India ink staining whole blood![]() ![]() A repeat chest CT in July 2012 demonstrated expansion of this infiltrate, with multiple new similar regions identified bilaterally bronchoalveolar lavage revealed periodic acid-Schiff (PAS)–positive material on cytology, consistent with a diagnosis of PAP. The patient was maintained on fluconazole and has had no evidence of recurrence of infection however, a chest CT scan performed in April 2012 demonstrated a new small region with ground-glass opacification. Amphotericin B was given for 14 d but was changed to fluconazole because of renal toxicity. Lumbar puncture showed cryptococcal Ag positive at 1:16, with 241 WBCs/mm 3, lymphocyte predominant, glucose of 36 mg/dl, and protein of 75 mg/dl. Approximately 1 wk later, he complained of weakness and tremors. His serum cryptococcal Ag was 1:512, and fluconazole was initiated. A large perihilar mass led to bronchoscopic biopsy that showed C. Seizures, homonymous hemianopsia, normal pressure hydrocephalus, chronic brain syndromeĪ previously healthy HIV-negative 47-y-old Mexican man living in California since age 12 presented in 2008 with persistent cough prompting chest x-ray. Recovered remains on maintenance fluconazole Required whole-lung lavage for PAP with subsequent improvement Plasma inhibition of GM-CSF–induced MIP-1αįever, headache, neck pain, diplopia, confusionīlurred vision, nausea, vomiting, fever, chillsĪsymptomatic ground-glass opacities on chest CTĪmB then fluconazole antituberculous therapy Data were collected using a FACSCalibur (BD Biosciences), analyzed using FlowJo software (TreeStar), and graphed with Prism5 (GraphPad). Monocytes were identified by CD14 (BD Pharmingen) surface staining before being fixed and permeabilized for intracellular staining with p-STAT5 (Y694) Ab (BD Pharmingen), as described previously ( 22). To demonstrate that patient plasma blocked GM-CSF signaling, PBMCs (5 × 10 5 cells) from healthy controls were cultured in complete RPMI 1640 media containing control or patient plasma (10%) and left unstimulated or stimulated with GM-CSF (10 ng/ml) or IL-3 (10 ng/ml) (both from R&D Systems) for 30 min at 37☌. To demonstrate that patient cells had intact GM-CSF signaling when washed free of autologous plasma, PBMCs (5 × 10 5 cells) from patients were cultured in complete RPMI media containing control or patient plasma (10%) and were left unstimulated or stimulated with GM-CSF (10 ng/ml) or IL-3 (10 ng/ml) (both from R&D Systems) for 30 min at 37☌. Plasma isolation, cell culture, and stimulation ![]() Stored cerebrospinal fluid from patients identified as having anti–GM-CSF autoantibodies was examined in the same manner using neat fluid. Fluorescence intensity for each bead type was plotted as a function of Ab titer (GraphPad Prism, version 5.0c). Beads were washed again and incubated with Streptavidin-PE (Bio-Rad) before being run in a multiplex assay on the Bio-Plex (Bio-Rad) instrument. ![]() Beads were combined and incubated for 1 h with subject or control plasma at 1:100 dilution, washed, and incubated with biotinylated mouse anti-human total IgG, as well as IgG subclasses, and IgA, IgM, and IgE (Sigma). Briefly, six sets of differentially fluorescing magnetic beads (Bio-Rad) were conjugated to 2.5 μg recombinant human GM-CSF, IFN-α, IFN-γ, IL-12p70, IL-17A, or IL-22 (R&D Systems). Plasma from patients and healthy controls was screened for anti-cytokine autoantibodies using a particle-based technology that was described previously ( 32). These cases need not have associated PAP. Anti–GM-CSF autoantibodies are associated with some cases of cryptococcal meningitis in otherwise immunocompetent patients. ![]() This effect was limited to their IgG fraction. Plasma from all patients prevented GM-CSF–induced STAT5 phosphorylation and MIP-1α production in normal PBMCs. Two of the seven later developed evidence of PAP. We identified seven HIV-negative patients with cryptococcal meningitis who tested positive for high-titer anti–GM-CSF autoantibodies. Her diagnosis prompted screening of patients with cryptococcal meningitis for anticytokine autoantibodies. We recognized anti–GM-CSF autoantibodies in an otherwise healthy female with cryptococcal meningitis who later developed pulmonary alveolar proteinosis (PAP). We assessed the ability of anti–GM-CSF autoantibody–containing plasmas to inhibit GM-CSF signaling. We performed clinical histories, immunological evaluation, and anticytokine autoantibody screening in four current patients with cryptococcal meningitis and identified and tested 103 archived plasma/cerebrospinal fluid samples from patients with cryptococcal meningitis. GM-CSF regulates the function of phagocytes and pulmonary alveolar macrophages, critical elements in cryptococcal control. Cryptococcal meningitis has been described in immunocompromised patients, as well as in those for whom no immune defect has been identified. ![]()
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