An enterococcal phage-derived enzyme suppresses graft-versus-host disease.

Changes in the gut microbiome have pivotal roles in the pathogenesis of acute graft-versus-host disease (aGVHD) after allogenic haematopoietic cell transplantation (allo-HCT)1-6. However, effective methods for safely resolving gut dysbiosis have not yet been established. An expansion of the pathogen Enterococcus faecalis in the intestine, associated with dysbiosis, has been shown to be a risk factor for aGVHD7-10. Here we analyse the intestinal microbiome of patients with allo-HCT, and find that E. faecalis escapes elimination and proliferates in the intestine by forming biofilms, rather than by acquiring drug-resistance genes. We isolated cytolysin-positive highly pathogenic E. faecalis from faecal samples and identified an anti-E. faecalis enzyme derived from E. faecalis-specific bacteriophages by analysing bacterial whole-genome sequencing data. The antibacterial enzyme had lytic activity against the biofilm of E. faecalis in vitro and in vivo. Furthermore, in aGVHD-induced gnotobiotic mice that were colonized with E. faecalis or with patient faecal samples characterized by the domination of Enterococcus, levels of intestinal cytolysin-positive E. faecalis were decreased and survival was significantly increased in the group that was treated with the E. faecalis-specific enzyme, compared with controls. Thus, administration of a phage-derived antibacterial enzyme that is specific to biofilm-forming pathogenic E. faecalis-which is difficult to eliminate with existing antibiotics-might provide an approach to protect against aGVHD.

Identification of clinical factors related to antibody-mediated immune response to the subfornical organ.

OBJECTIVE: We recently reported cases of adipsic hypernatremia caused by autoantibodies against the subfornical organ in patients with hypothalamic-pituitary lesions. This study aimed to clarify the clinical features of newly identified patients with adipsic hypernatremia whose sera displayed immunoreactivity to the mouse subfornical organ. DESIGN: Observational cohort study of patients diagnosed with adipsic hypernatremia in Japan, United States, and Europe. METHODS: The study included 22 patients with adipsic hypernatremia but without overt structural changes in the hypothalamic-pituitary region and congenital disease. Antibody response to the mouse subfornical organ was determined using immunohistochemistry. The clinical characteristics were compared between the patients with positive and negative antibody responses. RESULTS: Antibody response to the mouse subfornical organ was detected in the sera of 16 patients (72.7%, female/male ratio, 1:1, 12 pediatric and 4 adult patients). The prolactin levels at the time of diagnosis were significantly higher in patients with positive subfornical organ (SFO) immunoreactivity than in those with negative SFO immunoreactivity (58.9 ± 33.5 vs. 22.9 ± 13.9 ng/ml, p < .05). Hypothalamic disorders were found in 37.5% of the patients with positive SFO immunoreactivity. Moreover, six patients were diagnosed with rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation/neural tumor syndrome after the diagnosis of adipsic hypernatremia. Plasma renin activity levels were significantly higher in patients with serum immunoreactivity to the Nax channel. CONCLUSIONS: The patients with serum immunoreactivity to the SFO had higher prolactin levels and hypothalamic disorders compared to those without the immunoreactivity. The clinical characteristics of patients with serum immunoreactivity to the subfornical organ included higher prolactin levels and hypothalamic disorders, which were frequently associated with central hypothyroidism and the presence of retroperitoneal tumors.

Bexarotene-induced central hypothyroidism assessed by TRH stimulation test in cutaneous T-cell lymphoma patients.

Bexarotene-induced central hypothyroidism (CH), for which levothyroxine (LT4) replacement is recommended, has been shown to be caused by pituitary but not hypothalamic disorder experimentally, though the underlying mechanism in humans remains unclear. Here, the pathophysiology of bexarotene-induced CH was examined using a TRH stimulation test in cutaneous T-cell lymphoma (CTCL) patients. In this retrospective longitudinal observational study, serum TSH and free T4 (F-T4) levels were measured in 10 euthyroid patients with CTCL during 24 weeks of bexarotene treatment. TRH stimulation testing was performed following CH diagnosis, with LT4 replacement dosage adjusted to maintain F-T4 at the pre-treatment level. After one week of bexarotene administration, all 10 patients developed CH, based on combined findings of low or low-normal F-T4 with low or normal TSH levels. TSH peak response after a stimulation test at one week was reached at 30 minutes. However, that was <4 µIU/mL in all patients, indicating a blunted though not exaggerated and delayed TSH response. In eight who continued bexarotene for 24 weeks, median LT4 replacement dosage was 125 (range, 75-150) µg/day. TSH level at 30 as well as 15, 60, 90, and 120 minutes after TRH stimulation was significantly correlated with LT4 replacement dosage (ρ = -0.913, p = 0.002), whereas TSH and F-T4 basal levels at one week were not. These results suggest that pituitary hypothyroidism is responsible for bexarotene-induced CH, while TSH levels after TRH stimulation precisely reflect residual pituitary-thyroid function in patients receiving bexarotene.

Etelcalcetide decreases the PTH-calcium setpoint without changing maximum and minimum PTH secretion in mice with primary hyperparathyroidism.

INTRODUCTION: Etelcalcetide binds to the extracellular domain of the calcium-sensing receptor (CaSR), while cinacalcet binds to the 7-transmembrane domain of the CaSR; however, it is unknown, whether etelcalcetide has similar effects to cinacalcet on parathyroid hormone (PTH) secretion. MATERIALS AND METHODS: The PTH-calcium setpoint and maximum and minimum PTH secretion were determined using an 'in vivo setpoint analyses.' The PTH-calcium setpoint was obtained in a mouse model of primary hyperparathyroidism (PC) and wild-type (WT) mice, with PC mice divided into two groups. The setpoint was obtained after 7 days of etelcalcetide (3.0 mg/kg BW/day) or vehicle administration via anosmotic pump. After 7 days of crossover administration, the setpoint was obtained again. Parathyroid glands were obtained after crossover administration, and CaSR expression was analyzed by immunohistochemistry. RESULTS: Etelcalcetide administration significantly decreased the setpoint from 9.03 ± 0.56 mg/dL to 6.80 ± 0.28 mg/dL, which was restored to 8.81 ± 0.38 mg/dL after vehicle administration. In the second group of mice, vehicle administration did not alter the setpoint (8.84 ± 0.69 mg/dL to 8.98 ± 0.63 mg/dL), but subsequent etelcalcetide administration significantly decreased it to 7.10 ± 0.72 mg/dL. There was no significant change in maximum and minimum PTH secretion. Expression levels of parathyroid CaSR were lower in PC mice than in WT mice; however, no significant differences were observed between the two mouse groups. CONCLUSION: Etelcalcetide decreased the PTH-calcium setpoint without changing maximum and minimum PTH secretion in PC mice, suggesting that like cinacalcet, etelcalcetide has calcimimetic potency.

Effect of etelcalcetide on parathyroid hormone secretion by primary hyperparathyroidism patient-derived primary parathyroid cells.

INTRODUCTION: Etelcalcetide (Parsabiv®, AMG 416/ONO-5163) is a novel allosteric modulator for the calcium-sensing receptor approved for hemodialysis patients with secondary hyperparathyroidism of uremia. Etelcalcetide reduced parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism of uremia in clinical studies. However, its direct effect on parathyroid hormone secretion in human parathyroid cells remains unknown. This study aimed to determine if etelcalcetide suppresses parathyroid hormone secretion by human parathyroid cells in vitro. MATERIALS AND METHODS: We prepared primary cell cultures from human parathyroid tissue and determined calcium-sensing receptor expression levels by immunohistochemistry. Pathyroid tumors were removed from fourteen patients with primary hyperparathyrodism. Parathyroid tissue was dispersed with collagenase, resuspended in culture medium, incubated for 2 h with etelcalcetide and Ca2+, and the medium was then collected. Final etelcalcetide concentrations in the medium were 0.005-50 µmol/L. Levels of human parathyroid hormone in the medium were determined by enzyme-linked immunosorbent assay. RESULTS: In eight of the fourteen parathyroid cell cultures, extracellular Ca2+ reduced parathyroid hormone levels. In four of the eight parathyroid cell cultures which responded extracellular Ca2+, etelcalcetide reduced hormone secretion with the 50% effective concentrations of 0.57, 20.8, 0.42, and 0.57 µmol/L. Expression levels of the calcium-sensing receptor were significantly lower in primary hyperparathyroidism patient-derived parathyroid tissues compared with controls. CONCLUSION: This is the first report that etelcalcetide directly reduced parathyroid hormone secretion from the primary cultured human parathyroid cells from patients with primary hyperparathyroidism. To verify this conclusion, further studies are needed using secondary hyperparathyroidism patient-derived parathyroid cells.

Effects of burosumab on osteocalcin and bone mineral density in patient with 15-year history of nonremission tumor-induced osteomalacia initially treated with conventional therapy: Case report.

Excess fibroblast growth factor 23 (FGF23) causes hypophosphatemic osteomalacia, which is associated with impaired bone matrix mineralization. Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome caused by over-secretion of FGF23 from a tumor. Burosumab, a fully human monoclonal antibody with activities against FGF23, was initially approved in Japan before the rest of the world for treatment of FGF23-associated hypophosphatemic osteomalacia by TIO. We report here a patient with a 15-year history of non-remission TIO initially treated with conventional therapy who was then switched to burosumab treatment. Persistent hypophosphatemia and a relative low level of osteocalcin (bone Gla protein, BGP) compared with bone alkaline phosphatase (BAP) level, indicating poor matrix mineralization, developed during long-term conventional therapy. Repeated surgical and stereotactic body radiation treatments did not result in complete resection of the causable tumor, and bone mineral density (BMD) gradually decreased. Ultimately, burosumab treatment was administered and the serum Pi concentration immediately normalized, while both BGP and BMD also showed a good response. This is first known case report of the detailed efficacy of burosumab for nonremission TIO as an alternative to conventional therapy.

Effects of denosumab as compared with parathyroidectomy regarding calcium, renal, and bone involvement in osteoporotic patients with primary hyperparathyroidism.

PURPOSE: To evaluate the effects of denosumab (Dmb) on calcium, renal, and bone involvement in osteoporotic patients with primary hyperparathyroidism (PHPT) and compare with those who underwent a parathyroidectomy (PTX) procedure. METHODS: This retrospective, longitudinal study included patients treated with Dmb (60 mg) once every 6 months (n = 19) and those who successfully underwent a PTX procedure (n = 19) corrected calcium (cCa), eGFR, bone mineral density (BMD) in the lumbar spine (LS), total hip (TH), and femoral neck (FN) and LS-trabecular bone score (TBS) changes at 1 year after beginning Dmb or undergoing PTX were measured. RESULTS: Dmb group had older age, and showed milder disease activity and lower eGFR as compared with PTX group. In PTX group, cCa and eGFR were significantly decreased following surgery, while those were stable in Dmb group. There were significant increases in LS, TH, and FN-BMD in both Dmb (LS: 6.0 ± 0.8%, TH: 3.7 ± 1.0%, FN: 4.3 ± 1.5%) and PTX (LS: 11.2 ± 1.5%, TH: 7.5 ± 1.5%, FN: 7.9 ± 2.1%) groups. In Dmb group, LS-TBS was significantly improved by 3.0 ± 1.0%, while TBS change in PTX group approached significance (2.8 ± 1.5%). Percent change in TH-BMD was significantly correlated with baseline tartrate-resistant acid phosphatase-5b (TRACP-5b) in both groups. CONCLUSIONS: Dmb treatment not only increased BMD, dependent on bone turnover status, the same as PTX, but also improved LS-TBS. In addition, it did not decrease the level of eGFR, whereas PTX did. These results suggest that Dmb treatment help in the clinical management of osteoporotic patients with PHPT who do not undergo surgery as alternative to PTX.

Denosumab Improves Glomerular Filtration Rate in Osteoporotic Patients With Normal Kidney Function by Lowering Serum Phosphorus.

Higher serum phosphorus (Pi) increases the risk for chronic kidney disease (CKD). It was reported that a single administration of denosumab or zoledronate significantly suppressed serum Pi levels as well as those of bone resorption markers in serum. Also, previous evidences suggest a link between bone anti-resorptive therapy and vasoprotective/renoprotective effects through mechanisms that remain unexplored. The aim of this study is to assess the renoprotective effect of denosumab and involvement of denosumab-induced reduction in serum Pi in osteoporotic patients. Osteoporotic patients (n = 73) without overt proteinuria in dipstick test results were treated with denosumab (60 mg) every 6 months during the study period (24 months). Estimated glomerular filtration rate based on serum cystatin C (eGFRcys) was used as a filtration marker and tartrate-resistant acid phosphatase-5b (TRACP-5b) as a bone resorption marker. For analysis of non-CKD patients (n = 56), those with eGFRcys <60 mL/min/1.73 m2 were excluded. A single injection of denosumab suppressed serum Pi as well as TRACP-5b during the first 6 months, whereas age-related decline in eGFRcys was significantly reversed, with an increase of 2.75 ± 1.2 mL/min/1.73 m2 after 24 months noted. Multivariate analysis showed that serum Pi reduction following the initial denosumab injection was positively associated with serum TRACP-5b suppression during that same period (ß = 0.241, p = 0.049). In addition, a positive association of serum Pi suppression, but not of corrected calcium or TRACP-5b, with eGFRcys increase after 24 months (ß = 0.321, p = 0.014) was found after adjustments for gender, age, BMI, antihypertensive drug use, albumin, and eGFRcys. The same was observed in osteoporotic cases restricted to non-CKD patients. In conclusion, serum Pi reduction resulting from phosphorus load decrement from bone induced by denosumab is a determinant for eGFRcys increase. Early introduction of bone antiresorptive therapy can retain glomerular filtration in osteoporosis cases, including non-CKD patients. © 2019 American Society for Bone and Mineral Research.

Attenuated Dentin Matrix Protein 1 Enhances Fibroblast Growth Factor 23 in Calvaria in a Primary Hyperparathyroidism Model.

Fibroblast growth factor 23 (FGF23) secretion is facilitated by the PTH, particularly in hyperparathyroidism. The PTH also attenuates dentin matrix protein 1 (DMP1), which is produced by osteocytes to contribute to bone mineralization and suppress FGF23 expression. Nevertheless, it remains unknown whether attenuated DMP1 affects FGF23 expression in hyperparathyroidism. We examined their expression in bone tissue using a mouse model of primary hyperparathyroidism (PHPT). PHPT mice increased serum FGF23 levels, along with a high level of serum PTH. Fgf23 expression increased, and Dmp1 decreased significantly in the calvaria of PHPT mice compared with wild-type mice and primary osteoblasts treated with PTH. In UMR106 mature osteoblasts, PTH increased Fgf23 expression and decreased Dmp1 expression, and stimulation of protein kinase A (PKA) signaling by forskolin also increased Fgf23 expression and decreased Dmp1 expression in a dose-dependent manner, whereas inhibition of PKA signaling with 10-5 M H89 reversed the changes in Fgf23 and Dmp1 expression when cells were stimulated with PTH. Silencing Dmp1 along with PTH treatment led to an additive increase in Fgf23 expression, accompanied by additive phosphorylation of the cAMP-response element-binding protein. These results indicate that persistent and high levels of PTH lead to the continuous activation of PKA signaling in osteoblasts/osteocytes, resulting in an increase in FGF23 and a decrease in DMP1 in bone. Moreover, suppression of DMP1 enhanced FGF23 expression in PHPT, besides having a direct effect on PTH. These mechanisms may describe one of the pathogeneses behind the increase in FGF23 transcription in bone tissue in patients with PHPT.