Anti-Interleukin-23 Autoantibodies in Adult-Onset Immunodeficiency.

BACKGROUND: Autoantibodies against interleukin-12 (anti-interleukin-12) are often identified in patients with thymoma, but opportunistic infections develop in only some of these patients. Interleukin-12 (with subunits p40 and p35) shares a common subunit with interleukin-23 (subunits p40 and p19). In a patient with disseminated Burkholderia gladioli infection, the identification of both anti-interleukin-23 and anti-interleukin-12 prompted further investigation. METHODS: Among the patients (most of whom had thymoma) who were known to have anti-interleukin-12, we screened for autoantibodies against interleukin-23 (anti-interleukin-23). To validate the potential role of anti-interleukin-23 with respect to opportunistic infection, we tested a second cohort of patients with thymoma as well as patients without either thymoma or known anti-interleukin-12 who had unusual infections. RESULTS: Among 30 patients with anti-interleukin-12 who had severe mycobacterial, bacterial, or fungal infections, 15 (50%) also had autoantibodies that neutralized interleukin-23. The potency of such neutralization was correlated with the severity of these infections. The neutralizing activity of anti-interleukin-12 alone was not associated with infection. In the validation cohort of 91 patients with thymoma, the presence of anti-interleukin-23 was associated with infection status in 74 patients (81%). Overall, neutralizing anti-interleukin-23 was detected in 30 of 116 patients (26%) with thymoma and in 30 of 36 patients (83%) with disseminated, cerebral, or pulmonary infections. Anti-interleukin-23 was present in 6 of 32 patients (19%) with severe intracellular infections and in 2 of 16 patients (12%) with unusual intracranial infections, including Cladophialophora bantiana and Mycobacterium avium complex. CONCLUSIONS: Among patients with a variety of mycobacterial, bacterial, or fungal infections, the presence of neutralizing anti-interleukin-23 was associated with severe, persistent opportunistic infections. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

IFNγ regulates NAD+ metabolism to promote the respiratory burst in human monocytes.

Interferon γ (IFNγ) is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher-than-normal OCR, whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh syndrome patient monocytes) or NADPH oxidase (diphenyleneiodonium treatment or chronic granulomatous disease [CGD] patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.

Single Cell Mass Spectrometry Quantification of Anticancer Drugs: Proof of Concept in Cancer Patients.

In clinical cancer medicine, the current inability to quantify intracellular chemotherapy drug concentrations in individual human cells limits the personalization and overall effectiveness of drug administration. New bioanalytical methods capable of real-time measurement of drug levels in live single cancer cells would allow for more adaptive and personalized administration of chemotherapy drugs, potentially leading to better clinical outcomes with fewer side effects. In this study, we report the development of a new quantitative single cell mass spectrometry (qSCMS) method capable of providing absolute drug amounts and concentrations in single cancer cells. Using this qSCMS system, quantitative analysis of the intracellular drug gemcitabine present in individual bladder cancer cells is reported, including in bladder cancer cells isolated from patients undergoing standard-of-care gemcitabine chemotherapy. The development of single cell pharmacology bioanalytical methods can potentially lead to more effective and safely administered drug medications in patients, especially in the treatment of cancer.

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

Mass Spectrometry Measurement of Single Suspended Cells Using a Combined Cell Manipulation System and a Single-Probe Device.

Existing single cell mass spectrometry (SCMS) sampling platforms are largely designed to work only with immobilized cells and not the suspended cells isolated from patient samples. Here, we present a novel method that integrates a commercially available cell manipulation system commonly used for in vitro fertilization with the Single-probe SCMS sampling technology. The combined Single-probe SCMS/cell manipulating platform is capable of rapidly analyzing intracellular species in real time from a suspension leukemia cell line. A broad range of molecular species was detected, and species of interest were verified using tandem MS (MS/MS). Experimental results were analyzed utilizing statistical analyses such as principle component analysis (PCA) and  t-tests. The developed SCMS/cell manipulation system is a versatile tool to provide rapid single cell analysis of broad types of patient cell samples.