Effects of Extracorporeal Blood Flow Rates on Patient Tolerance for LIXELLE® Treatment during Outpatient Hemodialysis.

INTRODUCTION: Accumulation of ß2-microglobulin (B2M) in dialysis patients contributes to several comorbidities of end-stage kidney disease (ESKD). The LIXELLE® device adsorbs B2M from blood using sorbent bead technology. Studies in Japan showed that LIXELLE treatment during hemodialysis (HD) at blood flow rates up to 250 mL/min removes B2M above HD alone and is well tolerated. We investigated tolerance for LIXELLE treatment during HD at higher HD blood flow rates standard in the USA. METHODS: A prospective, open-label, non-randomized, single-arm, early-feasibility study (EFS) assessed tolerance and safety of LIXELLE treatment during HD at blood flow rates up to 450 mL/min. ESKD patients (40-75 years old) on thrice weekly outpatient HD were eligible. After a 1-week HD run-in, patients received LIXELLE plus HD at a blood flow rate of 250 mL/min (1 week), followed by LIXELLE plus HD at a blood flow rate up to 450 mL/min (1 week). These blood flow rates were tested with three LIXELLE column sizes in sequence (treatment = 6 weeks). B2M removal was assessed for each combination. RESULTS: Ten patients with a historic intradialytic hypotension (IDH) rate of 0.42 events/HD session/patient were enrolled. Nine patients completed all combinations without IDH events (treatment IDH rate: 0.56 events/HD session/patient). No treatment-emergent serious adverse events or significant changes in red blood cell, platelet, or complement indices except haptoglobin were reported. B2M reduction ratios and removal of select proteins (<40 kDa) increased with escalating column size and blood flow rate. CONCLUSION: LIXELLE plus HD across all column sizes was safe and well tolerated at blood flow rates up to 450 mL/min. Extent of B2M removal corresponded to column size-blood flow rate combinations. This EFS provides a risk profile to guide further studies of LIXELLE in ESKD patients at US-standard blood flow rates.

CCR3 plays a role in murine age-related cognitive changes and T-cell infiltration into the brain.

Targeting immune-mediated, age-related, biology has the potential to be a transformative therapeutic strategy. However, the redundant nature of the multiple cytokines that change with aging requires identification of a master downstream regulator to successfully exert therapeutic efficacy. Here, we discovered CCR3 as a prime candidate, and inhibition of CCR3 has pro-cognitive benefits in mice, but these benefits are not driven by an obvious direct action on central nervous system (CNS)-resident cells. Instead, CCR3-expressing T cells in the periphery that are modulated in aging inhibit infiltration of these T cells across the blood-brain barrier and reduce neuroinflammation. The axis of CCR3-expressing T cells influencing crosstalk from periphery to brain provides a therapeutically tractable link. These findings indicate the broad therapeutic potential of CCR3 inhibition in a spectrum of neuroinflammatory diseases of aging.

Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice.

Mice null for the Vitamin D receptor (VdrKO) have a disrupted first hair follicle cycle and aborted subsequent hair follicle cycling. We examined the expression of different markers and mediators of hair follicle cycling in the hair follicle of the VdrKO mouse during days 13-22 when the hair follicle normally initiates and completes the first catagen. We compared the expression of those genes in mice with a nonsense mutation in hairless (Rhino), which have a similar alopecia phenotype, and to Cyp27b1 null mice which are deficient in the production of 1,25(OH)2D3, the Vdr ligand, but display normal hair follicle cycling. Our results demonstrate the down regulation of hair follicle markers and the alteration of expression of hedgehog (Hh), Wnt, Fgf, and Tgfbeta pathways in VdrKO and Rhino mice, but not in Cyp27b1KO mice. Treatment of VdrKO mice with an agonist to the Hh pathway partially restored hair follicle cycling, suggesting a role of this pathway in the regulation of hair follicle cycling by VDR. These results suggest that Vdr regulates directly or indirectly the expression of genes required for hair follicle cycling, including Hh signaling, independent of 1,25(OH)2D3.

Quantification of the vitamin D receptor-coregulator interaction.

The vitamin D receptor (VDR) regulates a diverse set of genes that control processes including bone mineral homeostasis, immune function, and hair follicle cycling. Upon binding to its natural ligand, 1alpha,25(OH)(2)D(3), the VDR undergoes a conformational change that allows the release of corepressor proteins and the binding of coactivator proteins necessary for gene transcription. We report the first comprehensive evaluation of the interaction of the VDR with a library of coregulator binding motifs in the presence of two ligands, the natural ligand 1alpha,25(OH)(2)D(3) and a synthetic, nonsecosteroidal agonist LG190178. We show that the VDR has relatively high affinity for the second and third LxxLL motifs of SRC1, SRC2, and SRC3 and second LxxLL motif of DRIP205. This pattern is distinct in comparison to other nuclear receptors. The pattern of VDR-coregulator binding affinities was very similar for the two agonists investigated, suggesting that the biologic functions of LG190178 and 1alpha,25(OH)(2)D(3) are similar. Hairless binds the VDR in the presence of ligand through a LxxLL motif (Hr-1), repressing transcription in the presence and absence of ligand. The VDR binding patterns identified in this study may be used to predict functional differences among different tissues expressing different sets of coregulators, thus facilitating the goal of developing tissue- and gene-specific vitamin D response modulators.

The Transient Role for Calcium and Vitamin D during the Developmental Hair Follicle Cycle.

The role for 1,25-dihydroxyvitamin D3 and/or calcium in hair follicle cycling is not clear despite their impact on keratinocyte differentiation. We found that calbindin-D9k null (knockout) pups generated from calbindin-D9k knockout females fed a vitamin D-deficient, low-calcium (0.47%) diet develop transient alopecia. The pups appear phenotypically normal until 13 days of age, after which the hair progressively sheds in a caudocephalic direction, resulting in truncal alopecia totalis by 20-23 days, with spontaneous recovery by 28 days. Histological studies showed markedly dystrophic hair follicles, loss of hair shafts with increased apoptosis, and hyperplastic epidermis during this time. Ha1 expression is lost during catagen in all mice but recovers more slowly in the knockout pups on the vitamin D-deficient, low-calcium diet. Keratin 1 expression is reduced throughout days 19-28. The expressions of involucrin, loricrin, and cathepsin L is initially increased by day 19 but subsequently falls below those of controls by day 23, as does that of desmoglein 3. Feeding the mothers a high-vitamin D/high-calcium (2%)/lactose (20%) diet lessens the phenotype, and knockout pups fostered to mothers fed a normal diet do not develop alopecia. Our results show that in calbindin-D9k knockout pups, a maternal vitamin D-deficient/low-calcium diet leads to transient noncicatricial alopecia.

Protective role of vitamin D signaling in skin cancer formation.

Vitamin D sufficiency is associated with protection against malignancy in a number of tissues clinically, and a strong body of evidence from animal and cell culture studies supports this protective role. Cancers in the skin differ, however, in that higher serum levels of 25OHD are associated with increased basal cell carcinomas (BCC), the most common form of epidermal malignancy. This result may be interpreted as indicating the role of UVR (spectrum 280-320) in producing vitamin D in the skin as well as causing those DNA mutations and proliferative changes that lead to epidermal malignancies. Recent animal studies have shown that mice lacking the vitamin D receptor (VDR) are predisposed to developing skin tumors either from chemical carcinogens such as 7,12-dimethylbenzanthracene (DMBA) or chronic UVR exposure. Such studies suggest that vitamin D production and subsequent signaling through the VDR in the skin may have evolved in part as a protective mechanism against UVR induced epidermal cancer formation. In this manuscript we provide evidence indicating that vitamin D signaling protects the skin from cancer formation by controlling keratinocyte proliferation and differentiation, facilitating DNA repair, and suppressing activation of the hedgehog (Hh) pathway following UVR exposure. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

1α,25(OH)2-dihydroxyvitamin D3/VDR protects the skin from UVB-induced tumor formation by interacting with the ß-catenin pathway.

Ultra violet (UV) irradiation, in particular UVB, is the single most important carcinogen for skin tumor formation. UVB induces genetic mutations and immune suppression, which lead to abnormal cell proliferation and eventually tumor formation. Previously studies from our group and others demonstrated that both global and epidermal specific VDR knock out mice are predisposed to either chemical (DMBA)- or long-term UVB-induced skin tumor formation, paralleled by an increase in ß-catenin signaling. Using primary cultured human keratinocytes, we further demonstrated that 1,25(OH)2-dihydroxyvitamin D3 (1,25(OH)2D3) suppresses cyclin D1 and Gli1 which are regulated by ß-catenin/TCF signaling and have a critical role in epidermal carcinogenesis. Blockage of VDR by siRNA resulted in hyperproliferation of keratinocytes, and increased expression of cyclin D1 and Gli1. In addition, we also showed that 1,25(OH)2D3/VDR directly regulates transcriptional activity of ß-catenin/TCF signaling using the -catenin reporter TopGlow. Using K14 driven tamoxifen-induced cre recombinase to delete both VDR and ß-catenin in keratinocytes of mice following the first hair follicle cycle, we found that ablation of epidermal specific ß-catenin cannot rescue VDR null mice from UVB-induced skin tumor formation. Further study using VDR or ß-catenin single null mice is necessary to compare with the data from double null mice. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Overexpression of hedgehog signaling is associated with epidermal tumor formation in vitamin D receptor-null mice.

The vitamin D receptor (VDR) ligand, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), reduces proliferation and enhances differentiation, and thus has been investigated for a role in preventing or treating cancer. Mice deficient for the VDR display a hyperproliferative response in the hair follicle and epidermis and decreased epidermal differentiation. Unlike their wild-type littermates, when treated with 7,12 dimethylbenzanthracene (DMBA) or UVB, they develop skin tumors, including some characteristic of overexpression of the hedgehog (Hh) pathway. Both the epidermis and utricles of the VDR-null animals overexpress elements of the Hh pathway (sonic hedgehog (Shh) 2.02-fold, patched1 1.58-fold, smoothened 3.54-fold, glioma-associated oncogene homolog (Gli)1 1.17-fold, and Gli2 1.66-fold). This overexpression occurs at an age (11 weeks) at which epidermal hyperproliferation is most visible and is spatially controlled in the epidermis. DMBA- or UVB-induced tumors in the VDR-null mice also overexpress elements of this pathway. Moreover, 1,25(OH)(2)D(3) downregulates the expression of some members of the Hh pathway in an epidermal explants culture system, suggesting a direct regulation by 1,25(OH)(2)D(3). Our results suggest that increased expression of Shh in the keratinocytes of the VDR-null animal activates the Hh pathway, predisposing the skin to the development of both malignant and benign epidermal neoplasms.

Vitamin D2 formation from post-harvest UV-B treatment of mushrooms (Agaricus bisporus) and retention during storage.

The objectives of this research were to study the effects of high intensity (0.5, 0.75, and 1.0 mW/cm (2)), dose (0.5, 1.0, and 1.5 J/cm (2)), and postharvest time (1 and 4 days) on the vitamin D 2 formation in Portabella mushrooms ( Agaricus bisporus) as a result of UV-B exposure, as well as the vitamin D 2 degradation in treated mushrooms during storage. Within each intensity application, dose had the largest effect where more exposure converted more vitamin D 2 from ergosterol. Similar dose across each intensity application resulted in similar vitamin D 2 concentration. Practical commercial production requires as short a treatment time as possible, and intensity was a major factor from this standpoint where the time it took to achieve a similar vitamin D 2 concentration for similar dose exposure was significantly reduced as intensity increased. By using an intensity of 1.0 mW/cm (2) at a dose of 0.5 J/cm (2), the concentration of vitamin D 2 produced was 3.83 microg/g dry solids of mushrooms in 8 min, whereas using an intensity of 0.5 mW/cm (2) at a dose of 0.5 J/cm (2), the concentration of vitamin D 2 produced was 3.75microg/g dry solids of mushrooms in 18 min. Also, postharvest time did not have a significant effect on vitamin D 2 formation in mushrooms that were treated 1 and 4 days after harvest. Vitamin D 2 degraded in treated mushrooms during storage by apparent first-order kinetics, where the degradation rate constant was 0.025 h (-1). The information provided in this study will help mushroom producers develop commercial-scale UV treatment processes to add value to their crop while improving consumer health.