Mechanisms of natural tolerance in the intestine: implications for inflammatory bowel disease

Tolerance, the regulated inability to respond to a specific immunologic stimulant, is a physiological event important to normal immune function. Just as loss of tolerance to self-proteins results in autoimmune diseases, we assert that loss of tolerance to commensal flora in the intestinal lumen leads to inflammatory bowel disease (IBD). Mechanisms through which the mucosal immune system establishes and remains hyporesponsive toward the presence of food proteins and commensal flora, which we define as natural tolerance, are discussed. In addition to the contributions by commensal flora, the innate host defense and the adaptive immune systems promote natural tolerance to sustain normal mucosal homeostasis. Understanding the molecular and cellular events that mediate natural tolerance will lead to more advanced insights into IBD pathogenesis and improved therapeutic options.     

Hyperhomocysteinemia exacerbates the development of intimal hyperplasia in Sprague-Dawley rats: Alleviation by rosiglitazone

The amino acid intermediate homocysteine (Hcy) is formed during the metabolism of methionine to cysteine. Hyperhomocysteinemia (HHcy) is recognized as an independent risk factor for coronary atherosclerosis. The circulating levels of total Hcy (tHcy) can increase due to intake of foods rich in methionine or deficiencies of vitamins such as folate, pyridoxine and cyanocobalamin, which are required for the metabolism of Hcy. In addition, mutations in the genes coding for Hcy metabolizing enzymes can contribute to an increase in tHcy levels. Clinical and epidemiological studies have shown that an elevated level of tHcy measured in serum or plasma is a strong predictor of cardiovascular disease risk, which appears to be greatest in patients who have HHcy following a methionine load. Intimal hyperplasia (IH) (intima/media [I/M] ratio) is the universal response of a vessel to injury and may result in vasoconstriction when left unattended. The effect of dietary HHcy on balloon catheter-injured carotid artery and its modulation (if any) by the peroxisome proliferator-activated receptor agonist gamma rosiglitazone was evaluated in 12-week-old female Sprague-Dawley rats fed either a control diet or a diet containing 1% L-methionine. Once the rats were established on the diet, the group that was fed 1% L-methionine was further subdivided and either given an aqueous preparation of 3 mg/kg/day rosiglitazone or the vehicle via oral gavage for one week. This was followed by surgically injuring the left carotid artery using a Maverick Over-The-Wire catheter (2.0 mm × 20 mm, 3.2F; Boston Scientific, USA). The rats were continued on their respective diets and drug regimen for 21 days postsurgery. On day 22 of the procedure, the rats were sacrificed for collection of blood, the carotid arteries and liver for biochemical and histological evaluation. Compared with controls there was a significant increase in both tHcy levels and I/M ratio in the rats fed 1% L-methionine (5.4±0.28 μM versus 32.8±3.01 μM, P<0.002; and 0.175±0.05 versus 1.05±0.23, P<0.005, respectively). The effect of rosiglitazone in rats fed the control diet was not prominent. On the other hand, administration of rosiglitazone to the rats on the 1% L-methionine diet significantly reduced the levels of serum tHcy (16.6±2.1 μM versus 32.8±3.01 μM, P<0.001); however, the tHcy levels remained significantly elevated compared with animals on the control diet (P<0.002). The group receiving the L-methionine diet plus rosiglitazone had an inhibition in the development of IH compared with those receiving the L-methionine diet alone (I/M of 0.278±0.041 versus 1.05±0.23, P<0.01). Moreover, the development of IH in the group receiving the L-methionine diet plus rosiglitazone treatment was not significantly different from that observed in the group on the control diet without rosiglitazone (0.278±0.041 versus 0.175±0.05, respectively). These findings may have important implications in deciphering the molecular mechanisms involved in the augmentation of IH in HHcy and modulation of this process by rosiglitazone.     

Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.     

High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.     

Infectious Diseases in Older Adults of Long‐Term Care Facilities: Update on Approach to Diagnosis and Management

The diagnosis, treatment, and prevention of infectious diseases in older adults in long‐term care facilities (LTCFs), particularly nursing facilities, remains a challenge for all health providers who care for this population. This review provides updated information on the currently most important challenges of infectious diseases in LTCFs. With the increasing prescribing of antibiotics in older adults, particularly in LTCFs, the topic of antibiotic stewardship is presented in this review. Following this discussion, salient points on clinical relevance, clinical presentation, diagnostic approach, therapy, and prevention are discussed for skin and soft tissue infections, infectious diarrhea (Clostridium difficile and norovirus infections), bacterial pneumonia, and urinary tract infection, as well as some of the newer approaches to preventive interventions in the LTCF setting.     

Dyslipidemia, but not hyperglycemia, induces inflammatory adhesion molecules in human retinal vascular endothelial cells

PURPOSE: The initial determinants of retinal microvascular damage in diabetic retinopathy are not well understood, but are likely to be induced by hyperglycemia and/or dyslipidemia. The purpose of this study was to examine the effect of fatty acids and hyperglycemia on human retinal vascular endothelial (hRVE) cells as a means of mimicking diabetic metabolic disorders. METHODS: The expression of adhesion molecules in hRVE and human umbilical vein endothelial cells (HUVECs) was assayed by Western blot analysis and confirmed by leukocyte adhesion assay. The mechanisms underlying the induction of adhesion molecules by fatty acids were further investigated by using cyclooxygenase (COX), lipoxygenase (LOX), and P450 monooxygenase (MOX) inhibitors. RESULTS: Treatment of hRVE cells with the n6 polyunsaturated fatty acids (PUFAs) 18:2n6 and 20:4n6 for up to 24 hours resulted in a significant induction of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 protein levels. In contrast, treatment with high glucose (22 mM) for 24 hours did not affect CAM expression. Induction of CAM by n6 PUFA correlated with enhanced leukocyte binding to hRVE cells. The effect of n6 PUFA on ICAM-1 and VCAM-1 was blocked by an inhibitor of LOX, but not by COX or MOX inhibitors. In contrast to hRVE cells, n6 PUFA did not induce ICAM-1 or VCAM-1 in HUVECs. CONCLUSIONS: The data obtained in this study demonstrate that acute exposure to linoleic or arachidonic acid, but not hyperglycemia, induces inflammatory adhesion molecule expression in the presence of LOX in microvascular hRVE cells, but not in HUVECs. These results are consistent with the emerging hypothesis recognizing early-stage diabetic retinopathy as a low-grade chronic inflammatory disease.     

Application of image-guided surface coil P-31 MR spectroscopy to human liver, heart, and kidney

Localized phosphorus-31 magnetic resonance (MR) spectroscopy in humans has previously been accomplished with surface coils by means of depth-resolved surface coil spectroscopy or rotating frame experiments, in which the extent of tissue sampled critically depends on surface coil placement. The authors' goal was to modify the surface coil image-selected in vivo spectroscopy (ISIS) experiment to accomplish three-dimensional volume selection through application of selective pulses in the presence of B0 gradients. Advantages of ISIS include the ability to use proton images to define the volume of interest (VOI) and reduced dependence on exact positioning of the surface coil. However, rapid replication of the surface coil ISIS experiment can cause spectral contamination from signals originating outside the VOI. A modified version of the ISIS experiment was developed to alleviate contamination under conditions of rapid replication. Applications of localized P-31 MR spectroscopy for observation of high-energy phosphorus metabolites are presented in human liver, heart, and transplanted and normal kidney.