Phenotype, function, and differentiation potential of human monocyte subsets.

Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1ß in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

Long-term effects of alemtuzumab on regulatory and memory T-cell subsets in kidney transplantation.

BACKGROUND: Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes. METHODS: We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied. RESULTS: We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells. CONCLUSIONS: Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

Hereditary pancreatitis amlodipine trial: a pilot study of a calcium-channel blocker in hereditary pancreatitis.

OBJECTIVES: Hereditary pancreatitis (HP) is a form of recurrent acute pancreatitis (AP) mediated by mutations in cationic trypsinogen (PRSS1). Mutations cluster in the calcium-associated regulator regions of PRSS1. In rats, calcium-channel blockers (CCB) prevent hyperstimulation-associated AP. Because of the potential importance of hyperstimulation in triggering episodes of AP in HP, we designed a pilot study to evaluate the safety and potential benefit of CCB use in HP. METHODS: Subjects 6 years or older had a PRSS1 mutation, recurrent AP, and pain. Total study duration was 16 weeks. Amlodipine was given during weeks 0 to 11. Dose (2.5, 5, or 10 mg) was based on weight (range, 0.08-0.17 mg x kg(-1) x d(-1)). Subjects filled a daily diary including pain (0-10 scale) and blood pressure reading. Clinical assessments occurred at weeks -4, 0, 1, 2, 6, 10, 11, and 12. Subjects filled a Medical Outcomes Study Short-Form Survey version 2 (SF-10 for children <14 years old) at weeks -4, 0, 6, and 10. Data were compared for weeks -4 to 0 and 6 to 10. RESULTS: Nine subjects signed informed consent (4 males; 12-52 years old). Four were excluded during the screening phase. Drug was discontinued in one due to development of unilateral lower-extremity numbness. Four subjects (12-31 years old) completed the study. Mean blood pressure, laboratory tests, physical findings, and daily pain scores did not clinically significantly differ before and during drug therapy, but all reported reduced symptoms. Three reduced analgesic use. Three had improved scores on the Medical Outcomes Study Short-Form Survey version 2. CONCLUSIONS: Amlodipine is generally safe in subjects with HP and does not increase pain or episodes of AP. Further research into the mechanism of CCB on pancreatitis would be important to provide a pathophysiologic basis to support further trials in HP.