Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone,the murine model of X‐linked hypophosphatemia

X‐linked hypophosphatemia (XLH/HYP)—with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses—is caused by mutations in the zinc‐metallopeptidase PHEX gene (phosphate‐regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization‐inhibiting, acidic serine‐ and aspartate‐rich motif (ASARM)‐containing peptides, which are proteolytically derived from the mineral‐binding matrix proteins of the SIBLING family (small, integrin‐binding ligand N‐linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif—osteopontin (OPN) and bone sialoprotein (BSP)—as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full‐length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an ～35 kDa OPN fragment that was not present in wild‐type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild‐type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full‐length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization‐inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. © 2013 American Society for Bone and Mineral Research.     

Plasma Cytokine Analysis in Patients with Advanced Extremity Melanoma Undergoing Isolated Limb Infusion

Background

Preprocedure clinical and pathologic factors have failed to consistently differentiate complete response (CR) from progressive disease (PD) in patients after isolated limb infusion (ILI) with melphalan for unresectable in-transit extremity melanoma.

Methods

Multiplex immunobead assay technology (Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel, Millipore Corp., Billerica, MA; and Magpix analytical test instrument, Luminex Corp., Austin, TX) was performed on pre-ILI plasma to determine concentrations of selected cytokines (MIP-1α, IL-1Rα, IP-10, IL-1β, IL-1α, MCP-1, IL-6, IL-17, EGF, IL-12p40, VEGF, GM-CSF, and MIP-1β) on a subset of patients (n = 180) who experienced CR (n = 23) or PD (n = 24) after ILI. Plasma from normal donors (n = 12) was also evaluated.

Results

Of 180 ILIs performed, 28 % (95 % confidence interval 22–35, n = 50) experienced a CR, 14 % (n = 25) experienced a partial response, 11 % (n = 21) had stable disease, 34 % (n = 61) had PD, and 13 % (n = 23) were not evaluable for response. Tumor characteristics and pharmacokinetics appeared similar between CR (n = 23) and PD (n = 24) patients who underwent cytokine analysis. Although there were no differences in cytokine levels between CR and PD patients, there were differences between the melanoma patients and controls. MIP-1α, IL-1Rα, IL-1β, IL-1α, IL-17, EGF, IL-12p40, VEGF, GM-CSF, and MIP-1β were significantly higher in normal controls compared to melanoma patients, while IP-10 was lower (p < 0.001) in controls compared to melanoma patients.

Conclusions

Patients with unresectable in-transit melanoma appear to have markedly decreased levels of immune activating cytokines compared to normal healthy controls. This further supports a potential role for immune-targeted therapies and immune monitoring in patients with regionally advanced melanoma.     

Efficacy of Crocus sativus L. on reduction of cadmium‐induced toxicity on spermatogenesis in adult rats

Cadmium is a toxic heavy metal element, which probably cause infertility by impairment in spermatogenesis. The present work aimed (i) to study the toxic effect of cadmium on spermatogenesis in rat, as well as (ii) the protective effect of Crocus sativus L. on cadmium‐intoxicated rats. Cadmium chloride was administered intraperitoneally during 16 days at intervals of 48 h between subsequent treatments. Crocus sativus L. was pre‐treated in both of control and cadmium‐injected rats. Animals were sacrificed on day 17 after the first treatment. The left cauda epididymis was removed and immediately immersed into Hank's balanced salt solution for the evaluation of sperm count and viability, and left testis was fixed in 10% formalin for histological evaluation. Following contamination with cadmium, a decrease was observed in the number and viability of cauda epididymis sperm, which were increased by Crocus sativus L. pre‐treatment (P < 0.05). In addition, cadmium decreased both cell proliferation and Johnsen Scores in the seminiferous tubules, which were reversed by Crocus sativus pre‐treatment (P < 0.05). Furthermore, cadmium‐induced decrease in the amount of free serum testosterone as well as an increase in lipid peroxidation activity in the testicular tissue was reversed by Crocus sativus L. (P < 0.05). These findings may support the concept that Crocus sativus L. can improve the cadmium toxicity on spermatogenesis.     

Gastroesophageal pH step-up inaccurately locates proximal border of lower esophageal sphincter

Limiting the widespread use of 24-hr pH monitoring is the necessity of manometrically placing the pH probe 5 cm above the proximal lower esophageal sphincter (LES) border. Therefore, we prospectively compared LES localization by gastroesophageal pH step-up with manometry in 71 patients and 14 asymptomatic volunteers. The gastroesophageal pH step-up significantly correlated with the proximal LES border in patients (r=0.53, P<0.0001) and volunteers (r=0.91, P<0.0001). Based on previously published criteria, the pH step-up value was considered acceptably accurate if it was within ±3 cm (6 cm total span) of the manometrically determined proximal LES border. In 58% of patients and 29% of volunteers the pH step-up occurred outside this accuracy range. Esophagitis (P=0.015) and abnormal reflux parameters (P=0.002) were variables contributing to this error. Subsequent analysis found that the pH step-up overestimated the proximal LES border and occurred at the midportion of the sphincter. The pH step-up still inaccurately located the mid LES in 34% of patients. Therefore, manometry should remain the standard for accurate LES localization prior to placing the pH probe.     

Sensitivity and reproducibility of chemical tests for fecal occult blood with an emphasis on false-positive reactions

In 20 healthy volunteers ingesting 5 to 50 ml of⁵¹Cr-red cells, reaction intensities obtained with four chemical methods for fecal occult blood were compared with the “true” blood loss simultaneously determined by radioassay of each stool. Dilute tincture of guaiac reagent was found to have the same sensitivity and high frequency of false-positive reactions as the saturated guaiac reagent, but was more reproducible. Hematest^TM was slightly less sensitive but was poorly reproducible and yielded frequent false-negative as well as false-positive reactions. False-positive reactions by both methods were not eliminated by a meat-free diet; they were increased with guaiac reagents if stools were stored for 3 or more days. A new guaiac method (Hemoccult^TM) was found to be one-fourth as sensitive as the older tests, but was virtually free from false-positive reactions, even on an unrestricted diet and after storage of the stool specimens. It is recommended that the use of Hematest be abandoned and that Hemoccult be used preferentially if future studies confirm that its sensitivity is sufficient to detect most gastrointestinal lesions which are yielding occult blood.     

Lateral spread response monitoring during microvascular decompression for hemifacial spasm

Aim

The aim of the present study was to determine (1) whether successful intraoperative electromyography monitoring for lateral spread response (LSR) is possible with partial neuromuscular blockade (NMB) in subjects undergoing microvascular decompression (MVD) for hemifacial spasm and (2) the adequate level of NMB to achieve that goal.

Material and methods

A total of 61 patients in whom LSR was monitored during MVD were enrolled in the study. Patients were randomly allocated to two groups: group TOF in which the NMB target was maintenance of two train-of-four (TOF) counts and group T1 in which the NMB target was maintenance of a T₁/Tc ratio of 50?% (T₁: first twitch height of TOF and Tc: control twitch height). The adductor pollicis brevis muscle was used to monitor TOF responses. The frequency of successful LSR monitoring, defined as successful baseline establishment and maintenance of LSR until surgical decompression, was compared between the two groups.

Results

Of the 61 patients 2 were excluded from the study so that 30 patients in group TOF and 29 patients in group T1 were analyzed. The success rate of LSR monitoring was clinically acceptable and significantly higher in group T1 than in group TOF, i.e. n?=?15 (50.0?%) in group TOF versus n?=?24 (82.8?%) in group T1 (P?=?0.008), corresponding to a 32.8?% higher success rate in group T1 than group TOF (95?% CI: 13.9–51.7?%). Mean vecuronium infusion dose was smaller and mean TOF count was higher in group T1 than group TOF with a TOF count =?2 (1) in group TOF versus 3 (1) in group T1 (P?=?0.003). Mean sevoflurane and remifentanil infusion doses were not different between groups. There was no incidence of spontaneous movement during microscopy in either group.

Conclusion

Maintenance of partial NMB with a target T₁/Tc ratio of 50?% resulted in a clinically acceptable success rate of LSR monitoring and surgical condition during MVD. Maintenance of partial NMB with a target T₁/Tc ratio of 50?% rather than TOF count of two during LSR monitoring for MVD can therefore be recommended.     

Moderate acute exercise (70% VO2 peak) induces TGF‐β, α‐amylase and IgA in saliva during recovery

Strenuous exercise promotes changes in salivary IgA and can be associated with a high incidence of upper respiratory tract Infections. However, moderate exercise enhances immune function. The effect of exercise on salivary IgA has been well studied, but its effect on other immunological parameters is poorly studied. Thus, this study determined the effect of moderate acute exercise on immunological salivary parameters, such as the levels of cytokines (TGF‐β and IL‐5), IgA, α‐amylase and total protein, over 24 h. Ten male adult subjects exercised for 60 min at an intensity of 70% VO₂ peak. Saliva samples were collected before (‘basal’) and 0, 12 and 24 h after an exercise session. The total salivary protein was lower after 12 and 24 h than immediately after exercise, whereas α‐amylase increased at 12 and 24 h after exercise compared with basal levels. The IgA concentration was increased at 24 h after exercise relative to immediately after exercise, and there was no difference in the IL‐5 while TGF‐β concentration increased in recovery. In conclusion, 70% VO₂ peak exercise does not induce changes immediately after exercise, but after 24 h, it produces an increase in salivary TGF‐β without changing IL‐5.