In vitro, monocyte 1␣-hydroxylase converts 25-hydroxyvitamin D [25(OH)D] to 1,25-dihydroxyvitamin D to regulate local innate immune responses, but whether 25(OH)D repletion affects vitamin D-responsive monocyte pathways in vivo is unknown. Here, we identified seven patients who had 25(OH)D insufficiency and were undergoing long-term hemodialysis and assessed changes after cholecalciferol and paricalcitol therapies in both vitamin D-responsive proteins in circulating monocytes and serum levels of inflammatory cytokines. Cholecalciferol therapy increased serum 25(OH)D levels four-fold, monocyte vitamin D receptor expression three-fold, and 24-hydroxylase expression; therapy decreased monocyte 1␣-hydroxylase levels. The CD16 ϩ "inflammatory" monocyte subset responded to 25(OH)D repletion the most, demonstrating the greatest increase in vitamin D receptor expression after cholecalciferol. Cholecalciferol therapy reduced circulating levels of inflammatory cytokines, including IL-8, IL-6, and TNF. These data suggest that nutritional vitamin D therapy has a biologic effect on circulating monocytes and associated inflammatory markers in patients with ESRD.

The failure to reject the semiallogenic fetus by maternal T lymphocytes suggests that potent mechanisms regulate these cells. PDCD1 is a CD28 family receptor expressed by T cells, and its ligand CD274 is strongly expressed by trophoblast cells of the human placenta. In this study, we examined whether human maternal T cells express PDCD1. Immunofluorescence examination of uterine tissues revealed PDCD1 expression on CD3+ cells was low in nonpregnant endometrium but increased in first-trimester decidua and remained elevated in term decidua (P < 0.05). In addition, higher relative proportions of term decidual CD8bright, CD4+, and regulatory T cells expressed PDCD1 in comparison to autologous peripheral blood (P < 0.05). Term decidual T cells also expressed full-length and soluble PDCD1 mRNA isoforms more abundantly than their peripheral blood counterparts (P ≤ 0.05). We also examined the effects of PDCD1:CD274 interactions in decidual T cells. Jar choriocarcinoma cells were transfected with CD274 and cocultured with activated decidual CD4+ or CD8bright T cells for 72 h followed by analysis of cytokine concentration and decidual T cell apoptosis. Compared with empty vector-transfected cells, CD274-transfected Jar cells caused a significant suppression of interferon gamma and tumor necrosis factor alpha production by CD4+ (P < 0.05) but not CD8bright T cells, while having no effect on secretion of IL10 or T cell apoptosis. These results suggest that the PDCD1:CD274 pathway functions in modification of maternal decidual lymphocyte cytokine secretion during pregnancy.

Tularemia is a zoonotic infectious disease caused by the facultative intracellular bacterium Francisella tularensis. The initial clinical presentation and dominant features of this condition reflect the mode of transmission and the route of infection. The ulceroglandular form, most frequently acquired from bites of arthropods, is characterized by an initial papular skin lesion that can progress to an eschar or ulcer with regional lymphadenopathy. Inhalation of aerosolized bacteria or secondary hematogenous dissemination of infection to the lungs results in pneumonic tularemia characterized by fever, a broad range in the severity of respiratory symptoms, and a high mortality rate. In the so-called "typhoidal" form of tularemia, patients present with similar systemic symptoms without a clear focus of primary infection. They may show sepsis and rhabdomyolysis with multiorgan system failure, including renal impairment. Following pulmonary and gastrointestinal infection, systemic dissemination of the pathogen leads to the colonization of the liver, where organisms infect both macrophages and hepatocytes (4, 6). At autopsy, the livers of infected human beings often show areas of focal coagulation necrosis throughout the parenchyma of the organ (18).The infection of mice with the live vaccine strain (LVS) of F. tularensis subsp. holarctica is a commonly used animal model and is associated with the formation of multifocal hepatic microgranluomas containing CD11b ϩ macrophages and Gr-1 ϩ

We developed a single-plasmid-based regulatable protein expression system for Borrelia burgdorferi. Expression of a target gene is driven by P ost , a hybrid B. burgdorferi ospA-tetO promoter, from a recombinant B. burgdorferi plasmid constitutively expressing TetR. The system was tested using the green fluorescent protein (GFP) as a reporter. Under noninducing conditions, recombinant B. burgdorferi cells were nonfluorescent, no GFP protein was detected, and residual, small amounts of transcript were detectable only by reverse transcription-PCR but not by Northern blot hybridization. Upon induction with anhydrotetracycline, increasing levels of GFP transcript, protein, and fluorescence were observed. This tight and titratable promoter system will be invaluable for the study of essential borrelial proteins. Since target protein, operator, and repressor are carried by a single plasmid, the system's application is independent of a particular strain background.

SummaryHistamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I 2 (PGI 2 ), PGE 2 and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [ 3 H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPSinduced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10-and 15-fold increases in PGI 2 , PGE 2 and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI 2 , PGE 2 and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.