Liposomal delivery of self-peptide and calcitriol as tolerogenic immunotherapy in rheumatoid arthritis: an exploration using sensory science.

Immunology research holds significant potential for enhanced inclusivity at the beginning of the science literacy journey, but persistent challenges stem from limited awareness that improvement is needed in this field. At the 2023 Monash Sensory Science Exhibition, we had the opportunity to present several tactile posters, using simple materials, for visually impaired participants to showcase our research on the pathogenesis of rheumatoid arthritis as a result of immune tolerance breakdown and liposome-based tolerogenic immunotherapy. The posters stimulated lively discussions about autoimmune arthritic diseases and our research. With consideration of the diversity of the participants, the efforts of scientists in promoting science literacy for the community can promote a more inclusive environment and engage and inspire a broader audience.

Mineralized tissues in hypophosphatemic rickets.

Hypophosphatemic rickets is caused by renal phosphate wasting that is most commonly due to X-linked dominant mutations in PHEX. PHEX mutations cause hypophosphatemia indirectly, through the increased expression of fibroblast growth factor 23 (FGF23) by osteocytes. FGF23 decreases renal phosphate reabsorption and thereby increases phosphate excretion. The lack of phosphate leads to a mineralization defect at the level of growth plates (rickets), bone tissue (osteomalacia), and teeth, where the defect facilitates the formation of abscesses. The bone tissue immediately adjacent to osteocytes often remains unmineralized ("periosteocytic lesions"), highlighting the osteocyte defect in this disorder. Common clinical features of XLH include deformities of the lower extremities, short stature, enthesopathies, dental abscesses, as well as skull abnormalities such as craniosynostosis and Chiari I malformation. For the past four decades, XLH has been treated by oral phosphate supplementation and calcitriol, which improves rickets and osteomalacia and the dental manifestations, but often does not resolve all aspects of the mineralization defects. A newer treatment approach using inactivating FGF23 antibodies leads to more stable control of serum inorganic phosphorus levels and seems to heal rickets more reliably. However, the long-term benefits of FGF23 antibody treatment remain to be elucidated.

Tumor-Targeted Nanoparticles Deliver a Vitamin D-Based Drug Payload for the Treatment of EGFR Tyrosine Kinase Inhibitor-Resistant Lung Cancer.

Mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor ( EGFR) gene drives the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR TKIs), including erlotinib and afatinib, are initially effective in treating EGFR mutant nonsmall cell lung cancer (NSCLC). However, drug resistance quickly develops due to several mechanisms, including induction of the epithelial-mesenchymal transition (EMT). No effective therapies are currently available for patients who develop EMT-associated EGFR TKI resistance. 1,25-Dihydroxyvitamin D3 (1,25D3) promotes epithelial differentiation and inhibits growth of NSCLC cells. 1,25D3 thus represents a promising agent for the treatment of EMT-associated EGFR TKI resistance. However, 1,25D3 induces the expression of 24-hydroxylase (24OHase), which decreases 1,25D3 activity. CTA091, a potent and selective 24OHase inhibitor, has been developed to attenuate this adverse effect. CTA091 also suppresses renal 24OHase activity and so may promote hypercalcemia. To exploit favorable effects of 1,25D3 plus CTA091 in tumor cells while avoiding problematic systemic effects of 24OHase inhibition, we developed EGFR-targeted, liposomal nanoparticles (EGFR-LP) to offer tumor-targeted co-delivery of 1,25D3 and CTA091. We then established an EMT-associated model of EGFR TKI resistance, and showed that such nanoparticles improved cellular uptake of 1,25D3 and CTA091, drove pro-epithelial signaling by upregulating E-cadherin ( CDH1), and significantly inhibited the growth of EGFR TKI resistant cells. Our results demonstrated that the delivery of vitamin D-based drug payloads via tumor-targeted EGFR-LP has promise as a new therapy for EFGR TKI resistant lung cancer. Future studies will focus on in vivo evaluation of biological activity, therapeutic benefits, and systemic toxicity prior to clinical translation.

Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats.

AIM: Vitamin D deficiency is considered to diminish bone regeneration. Yet, raising the serum levels takes months. A topic application of the active vitamin D metabolite, calcitriol, may be an effective approach. Thus, it becomes important to know the effect of vitamin D deficiency and local application on alveolar bone regeneration. MATERIAL AND METHODS: Sixty rats were divided into three groups; two vitamin depletion groups and a control group. Identical single defects (2 mm diameter) were created in the maxilla and mandible treated with calcitriol soaked collagen in one deficiency group while in the other two groups not. Histomorphometric analysis and micro CTs were performed after 1 and 3 weeks. Serum levels of 25(OH)D3 and PTH were determined. RESULTS: Bone formation rate significantly increased within the observation period in all groups. Bone regeneration was higher in the maxilla than in the mandible. However, bone regeneration was lower in the control group compared to vitamin depletion groups, with no significant effects by local administration of calcitriol (micro CT mandible p = 0.003, maxilla p < 0.001; histomorphometry maxilla p = 0.035, mandible p = 0.18). CONCLUSION: Vitamin D deficiency not necessarily impairs bone regeneration in the rat jaw and a single local calcitriol application does not enhance healing.

Topical Vitamin D Prevents Bone Loss and Inflammation in a Mouse Model.

There is a strong association between vitamin D levels and periodontal disease based on numerous epidemiological studies. We have previously shown that experimental deficiency of serum vitamin D in mice leads to gingival inflammation and alveolar bone loss. Treatment of cultured oral epithelial cells with the active form of vitamin D, 1,25(OH)2 vitamin D3 (1,25(OH)2D3), inhibits the extracellular growth and intracellular invasion of bacteria associated with periodontal disease. Maintenance of periodontal health may be due in part to the anti-inflammatory activities of vitamin D. Furthermore, this hormone can induce the expression of an antimicrobial peptide in cultured oral epithelial cells. We have shown that oral epithelial cells are capable of converting inactive vitamin D to the active form, suggesting that topical treatment of the oral epithelium with inactive vitamin D could prevent the development of periodontitis. We subjected mice to ligature-induced periodontitis (LIP), followed by daily treatment with inactive vitamin D or 1,25(OH)2D3. Treatment with both forms led to a reduction in ligature-induced bone loss and inflammation. Gingival tissues obtained from vitamin D-treated LIP showed production of specialized proresolving mediators (SPM) of inflammation. To examine the mechanism, we demonstrated that apical treatment of 3-dimensional cultures of primary gingival epithelial cells with vitamin D prevented lipopolysaccharide-induced secretion of proinflammatory cytokines and led to a similar production of SPM. Analysis of the oral microbiome of the mice treated with vitamin D showed significant changes in resident bacteria, which reflects a shift toward health-associated species. Together, our results show that topical treatment of oral tissues with inactive vitamin D can lead to the maintenance of periodontal health through the regulation of a healthy microbiome and the stimulation of resolution of inflammation. This strongly supports the development of a safe and effective vitamin D-based topical treatment or preventive agent for periodontal inflammation and disease.

Exclusive plaque psoriasis of the lips: efficacy of combination therapy of topical tacrolimus, calcipotriol, and betamethasone dipropionate.

A 16-year-old unmarried woman presented with recurrent cracking of the lips indicated by the appearance of grayish white flakes since October 2004, which, in due course, shed off leaving behind an apparently normal mucous membrane. Chewing roasted corn treated with salt and lemon (bhutta) initially caused the lesions. Ever since, it has been a cause of its exacerbation. She never had any relief with either systemic or topical treatment. In fact, an obsession had overtaken her, resulting in a psychological setback. She denied regular drug use for any other ailment. Her menstrual cycle was normal. There was a positive history of psoriasis in her mother. Examination of the lips was conspicuous. It was marked by the presence of a well-circumscribed, moist, raised plaque (Figure 1). Its surface was irregular, with elevation and depression. It was made up of thick, grayish white scales, which were arranged in layers; however, Grattage/Auspitz sign could not be elicited. Fissuring was prominent but the buccal mucosa, surface of the tongue, gingiva, and palate were normal. The clinical examination did not reveal any evidence of skin and/or nail psoriasis/psoriatic arthropathy or any other systemic abnormality. Blood examination including total and differential leukocyte count, complete hemogram, and liver and renal function tests were normal. Biopsy of the representative lesion was subjected to serial sections. They were stained with hematoxylin-eosin to work up microscopic pathology. It revealed the presence of mounds of parakeratosis with numerous neutrophilic Munro microabscesses (Figure 2). Submucosal vessels were dilated and congested. Periodic-acid-Schiff (PAS) stain revealed fungal hyphae and spores within the parakeratotic layer. Colonies of Gram-positive cocci were also demonstrated on the surface of the mucosa. She was administered combination therapy, comprising topical tacrolimus (0.1%) ointment and calcipotirol hydrate (50 microg/g) plus betmethasone dipropionate (0.5 mg/g) twice a day for 7 days. A single bolus dose of fluconazole 450 mg orally was also administered. The response to treatment was favorable and the lesions showed regression (Figure 3).

Evaluating the efficacy and safety of calcipotriene/betamethasone ointment occluded with a hydrogel patch: a 6-week bilaterally controlled, investigator-blinded trial.

Occlusive therapy with or without topical agents is effective in the treatment of psoriasis. This study assessed the efficacy and safety of an occlusive hydrogel dressing. Participants were treated with calcipotriene 0.005%-betamethasone dipropionate 0.064% ointment with and without a hydrogel patch. Thirty participants completed the 6-week, bilaterally controlled, investigator-blinded, single-center study. Substantial reductions in total modified psoriasis area and severity index (PASI) scores of occluded lesions versus nonoccluded lesions were seen as early as the first week of treatment and sustained through 4 weeks of the study. No adverse effects related to the study, including skin irritation, were observed or reported. Hydrogel dressings provide an effective and safe occlusive option to enhance topical therapy for psoriasis.

1α,25-dihydroxyvitamin D3 promotes early osteogenic differentiation of PDLSCs and a 12-year follow-up case of early-onset vitamin D deficiency periodontitis.

Periodontitis is a chronic periodontal disease that contributes to tooth loss. In recent years, many animal studies have reported that vitamin D (VitD) deficiency results in chronic periodontitis. However, no studies have reported cases of early-onset periodontitis with VitD deficiency. This study reports a 5-year-old male patient with early-onset periodontitis, VitD deficiency and VitD receptor (VDR) mutation. The patient was treated with VitD and calcium, and received systematic periodontal treatment. During the 12-year treatment, the periodontal conditions of this patient were stable. Our in vitro study found that VitD could promote the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP2), bone gamma-carboxyglutamate protein (BGLAP), and VDR in the early osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Meanwhile, VitD could downregulate mRNA expression levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-1ß (IL-1ß) and protein levels of IL-6 in the tumor necrosis factor-α (TNF-α) -induced inflammation of PDLSCs. Therefore, sufficient VitD supply can be a potential treatment for VitD deficiency induced early-onset periodontitis.

Intermittent therapy with 1,25 vitamin D and calcitonin prevents cyclosporin-induced alveolar bone loss in rats.

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 microg/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 microg/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

PD-L1- and calcitriol-dependent liposomal antigen-specific regulation of systemic inflammatory autoimmune disease.

Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.

Bioavailability of seocalcitol II: development and characterisation of self-microemulsifying drug delivery systems (SMEDDS) for oral administration containing medium and long chain triglycerides.

By constructing ternary phase diagrams it was possible to identify two self-microemulsifying drug delivery systems (SMEDDS) containing either medium chain triglycerides (MC-SMEDDS) or long chain triglycerides (LC-SMEDDS), with the same ratio between lipid, surfactant and co-surfactant. The SMEDDS ended up having a composition of 25% lipid, 48% surfactant and 27% co-surfactant, MC-SMEDDS: viscoleo, cremophor RH40, akoline MCM and LC-SMEDDS: sesame oil, cremophor RH40, peceol. Upon dilution with water both SMEDDS resulted in clear to bluish transparent microemulsions with a narrow droplet size of 30nm. The industrial usefulness of the developed SMEDDS was evaluated with regard to bioavailability and chemical stability using the vitamin D analogue, seocalcitol, as model compound. The absorption and bioavailability of seocalcitol in rats were approximately 45% and 18%, respectively, from both the MC-SMEDDS and LC-SMEDDS indicating similar in vivo behavior of the two formulations, despite the difference in nature of lipid component. There was no improvement in bioavailability by the use of SMEDDS, compared to the bioavailability achieved from simple MCT and LCT solutions (22-24%) (Grove, M., Pedersen, G.P., Nielsen, J.L., Mullertz, A., 2005. Bioavailability of seocalcitol. I. Relating solubility in biorelevant media with oral bioavailability in rats-effect of medium and long chain triglycerides. J. Pharm. Sci. 94, 1830-1838.). After 3 months' storage at accelerated conditions (40 degrees C/75% RH), a decrease in concentration of seocalcitol of 10-11% was found in MC-SMEDDS and LC-SMEDDS compared with a degradation of less than 3% for the simple lipid solutions of MCT and LCT. In this study the simple lipid solutions seem to be a better choice compared with the developed SMEDDS due to a slightly higher bioavailability and a better chemical stability of seocalcitol.

Localized sequence-specific release of a chemopreventive agent and an anticancer drug in a time-controllable manner to enhance therapeutic efficacy.

Combination chemotherapy with multiple drugs commonly requires several injections on various schedules, and the probability that the drug molecules reach the diseased tissues at the proper time and effective therapeutic concentrations is very low. This work elucidates an injectable co-delivery system that is based on cationic liposomes that are adsorbed on anionic hollow microspheres (Lipos-HMs) via electrostatic interaction, from which the localized sequence-specific release of a chemopreventive agent (1,25(OH)2D3) and an anticancer drug (doxorubicin; DOX) can be thermally driven in a time-controllable manner by an externally applied high-frequency magnetic field (HFMF). Lipos-HMs can greatly promote the accumulation of reactive oxygen species (ROS) in tumor cells by reducing their cytoplasmic expression of an antioxidant enzyme (superoxide dismutase) by 1,25(OH)2D3, increasing the susceptibility of cancer cells to the cytotoxic action of DOX. In nude mice that bear xenograft tumors, treatment with Lipos-HMs under exposure to HFMF effectively inhibits tumor growth and is the most effective therapeutic intervention among all the investigated. These empirical results demonstrate that the synergistic anticancer effects of sequential release of 1,25(OH)2D3 and DOX from the Lipos-HMs may have potential for maximizing DOX cytotoxicity, supporting more effective cancer treatment.

Nanocapsules with Polyelectrolyte Shell as a Platform for 1,25-dihydroxyvitamin D3 Neuroprotection: Study in Organotypic Hippocampal Slices.

Calcitriol (1,25-dihydroxyvitamin D3), an active metabolite of vitamin D3, besides the role in calcium and phosphorus metabolism, plays a role in maintaining the functions of the brain. Active forms of vitamin D3 stimulate neurotrophic factors' expression, regulate brain immune processes, and prevent neuronal damage. Therefore, a potential utility of vitamin D3 in a therapy of neurodegenerative disorders should be taken into account. On the other hand, systemic vitamin D3 treatment carries the risk of undesirable effects, e.g., hypercalcemia. Thus, 1,25-dihydroxyvitamin D3 targeting delivery by nanoparticles would be a tremendous advancement in treatment of brain disorders. Calcitriol was enclosed in emulsion-templated nanocapsules with different polymeric shells: PLL (Poly(L-lysine hydrobromide)), PLL/PGA (/Poly(L-glutamic acid)), and PLL/PGA-g-PEG (Poly(L-glutamic acid) grafted with polyethylene glycol). The average size of all synthesized nanocapsules ranged from -80 to -100 nm. Biocompatibilities of synthesized nanocarriers were examined in hippocampal organotypic cultures in basal conditions and after treatment with lipopolysaccharide (LPS) using various biochemical tests. We demonstrated that nanocapsules coated with PLL were toxic, while PLL/PGA- and PLL/PGA-g-PEG-covered ones were nontoxic and used for further experiments. Our study demonstrated that in LPS-treated hippocampal slices, both types of loaded nanoparticles have protective ability. Our findings underlined that the neuroprotective action of vitamin D3 in both free and nanoparticle forms seems to be related to the suppression of LPS-induced nitric oxide release.

Local administration of calcitriol positively influences bone remodeling and maturation during restoration of mandibular bone defects in rats.

The aim of this study was to investigate the influence of calcitriol on osteoinduction following local administration into mandibular bone defects. Calcitriol-loaded absorbable collagen membrane scaffolds were prepared using the polydopamine coating method and characterized by scanning electron microscopy. Composite scaffolds were implanted into rat mandibular bone defects in the following groups: no graft material (control), bare collagen membrane (CM group), collagen membrane bearing polydopamine coating (DOP/CM group), and collagen membrane bearing polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and 8weeks post-surgery, the osteogenic potential of calcitriol was examined by histological and immunohistochemical methods. Following in vivo implantation, calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic differentiation at weeks 1, and 2. Furthermore, statistical analysis revealed that the newly formed bone volume in the CAL/DOP/CM group was significantly higher than other groups at weeks 1, and 2. At weeks 4, and 8, the CAL/DOP/CM group showed more mineralized bone and uniform collagen structure. These data suggest that local administration of calcitriol is promising in promoting osteogenesis and mineralization for restoration of mandibular bone defects.

Histochemical examination of adipose derived stem cells combined with ß-TCP for bone defects restoration under systemic administration of 1α,25(OH)2D3.

The purpose of this study was to evaluate the effects of osteogenic differentiated adipose-derived stem cell (ADSC) loaded beta-tricalcium phosphate (ß-TCP) in the restoration of bone defects under intraperitoneal administration of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3). ADSCs were isolated from the fat tissue of 8 week old Wister rats and co-cultured with ß-TCP for 21 days under osteogenic induction. Then the ADSC-ß-TCP complexes were implanted into bone defects in the femora of rats. 1α,25(OH)2D3 (VD) or normal saline (NS) was administrated intraperitoneally every other day after the surgery. Femora were harvested at day 7, day 14 and day 28 post-surgery. There were 4 groups for all specimens: ß-TCP-NS group; ß-TCP-ADSC-NS group; ß-TCP-VD group and ß-TCP-ADSC-VD group. Alkaline phosphatase (ALP) was up-regulated obviously in ADSC groups compared with non-ADSC groups at day 7, day 14 and day 28, although high expression of runt-related transcription factor 2 (RUNX2) was only seen at day 7. Furthermore, the number of TRAP-positive osteoclasts and the expression of cathepsin K (CK) were significantly decreased in VD groups compared with non-VD groups at day 7 and day 14. As a most significant finding, the ß-TCP-ADSC-VD group showed the highest BV/TV ratio compared with the other three groups at day 28. Taken together, ADSC-loaded ß-TCP under the administration of 1α,25(OH)2D3 made a promising therapy for bone defects restoration.

Increased incidence of hip fracture in osteoporotic women treated with sodium fluoride.

There has been controversy as to whether fluoride therapy increases the risk of fracture in the appendicular skeleton. In the present study we compared the incidence of hip fracture in four groups of osteoporotic women: 22 treated with placebo, 17 with fluoride and calcium, 18 treated with fluoride and calcitriol, and 21 with calcitriol alone. Four hip fractures occurred in 3 patients on fluoride and calcitriol, and two hip fractures occurred in 2 patients on fluoride and calcium. No hip fractures occurred in patients receiving either calcitriol alone or placebo. The difference in fracture rates for fluoride versus nonfluoride treatment is significant (p = 0.006). Moreover, the six hip fractures occurring in patients receiving fluoride during 72.3 patient years of treatment is 10 times higher than would be expected in normal women of the same age. The probability of observing six fractures in 2 years is extremely small (0.0003). In four of the hip fracture cases, the history suggested a spontaneous fracture. These findings suggest that fluoride treatment can increase the risk of hip fracture in osteoporotic women.

Effects of one-year cyclical treatment with clodronate on postmenopausal bone loss.

We studied 60 women with postmenopausal bone loss randomly allocated to the following treatments: Group 1 (20 patients), no treatment; Group 2 (20 patients), clodronate 400 mg daily by mouth for 30 consecutive days, followed by 60 days of no treatment; Group 3 (20 patients) oral calcitriol 2 mcg by mouth for 5 days and oral clodronate 400 mg daily for additional 25 days, followed by 60 days of no treatment. The therapeutic cycles were repeated four times in the 12-month study period. In the 36 treated patients of Groups 2 and 3 who completed the study period we observed a progressive and significant increase in lumbar bone density both at 6 and 12 months of therapy, without significant differences between the two treatment protocols (+3.88 +/- 0.65%, P < 0.001 and +3.21 +/- 0.89%, P < 0.005 in Groups 2 and 3, respectively, at the end of the study). In contrast, there was a progressive and significant decline of bone mineral density in untreated patients (-2.34 +/- 0.49%, P < 0.001). After 12 months serum calcium values in treated subjects were higher than in untreated patients (P < 0.05). Serum phosphate was raised only in Group 2, mean values being higher after 12 months than before treatment (P < 0.05); parathyroid hormone (PTH) declined in all treated patients, the fall being significant in Group 2 (P < 0.02). No important side effects were observed with treatment and no patient withdrew because of these. We conclude that cyclical low dose clodronate therapy induced a gain in lumbar spine bone mass in patients with postmenopausal osteoporosis.

Effect of age on the rate of tooth movement in combination with local use of 1,25(OH)2D3 and mechanical force in the rat.

The purpose of this study was to compare the amount and rate of tooth movement in young and mature rats administered 1,25(OH)2D3 simultaneous with application of mechanical force. In 30 seven-week-old and 30 28-week-old male Wistar rats, the right maxillary first molar was moved buccally with a fixed appliance. The appliances delivered forces ranging from 5 to 20 g. Twenty microL of 1,25(OH)2D3 (10(-10) and 10(-8) mol/L) was injected locally into the submucosal palatal area of the root bifurcation of the right first molar. The left side was injected with phosphate-buffered saline (PBS). In young rats receiving 10(-10) mol/L 1,25(OH)2D3 every three days, tooth movement significantly increased to 126% of that in PBS-injected control rats on day 20. In 1,25(OH)2D3-injected mature rats, tooth movement was stimulated markedly and increased with 10(-10) mol/L to 245% and with 10(-8) mol/L to 154% of the amount of tooth movement seen in the PBS-injected controls by the end of the experiment. PBS-injected rats had a plateau stage where tooth movement did not occur at all, while there was no such lag-time in the 1,25(OH)2D3-injected group which showed continuous tooth movement. The local injection of 1,25(OH)2D3 did not change serum calcium, phosphate, and alkaline phosphatase activity, and there were no apparent clinical or microscopic side-effects.

1 alpha,25-dihydroxyvitamin D3 stimulation of osteopontin expression in rat clonal dental pulp cells.

Osteopontin (OPN) is a major phosphorylated non-collagenous protein isolated from bone. Rat clonal dental-pulp cell lines RPC-C2A and RDP4-1 produce and secrete OPN as a principal phosphoprotein. 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a potent calcitropic hormone which regulates calcified tissue metabolism including the synthesis of extracellular matrix proteins. The effects of 1,25(OH)2D3 on the expression of OPN mRNA and the synthesis of OPN protein by pulp cells in vitro were investigated. In RPC-C2A cells, 1,25(OH)2D3 markedly stimulated synthesis of both [32PO4]- and [35S]-methionine-labelled OPN. Phosphorylated OPN synthesis increased dose-dependently and showed a maximum level at 48 h after addition of 10(-11)-10(-7) M 1,25(OH)2D3. Similar stimulation was also observed in RDP4-1 cells. Northern hybridization analysis revealed that 1,25(OH)2D3 greatly increased the level of OPN mRNA in both pulp cell lines. Examination of the time course of the effects of 1,25(OH)2D3 on the level of OPN mRNA in RPC-C2A cells by dot-blot analysis showed that stimulation was detectable at 24 h and reached a maximum at 48 h after exposure to 10(-7)M 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 stimulates the production of dental-pulp OPN by a mechanism that involves de novo synthesis and transcriptional control.

1.25(OH)2 cholecalciferol pulse therapy and the effects of different dialysis membranes on serum PTH levels of haemodialysis patients.

Either oral, intravenous or subcutaneous 1.25(OH)2 cholecalciferol is used in the therapy of hyperparathyroidism, which is a serious complication in patients on haemodialysis. We studied a total of 30 patients (10 women and 20 men) and divided them into two groups depending on the different types of dialysis membranes used. In the polysulfone group, mean age was 43.7 +/- 0.97 years and the average dialysis period lasted 29.9 +/- 1.23 months. For the 15 cases in which we used cuprophane membrane the mean age was 40.2 +/- 1.31 years and the average dialysis period lasted 16.2 +/- 0.86 months. The calcium level of the dialysate in both groups was 1.5 mmol/l. According to the study protocol, the determined oral calcitriol dose was 0.07 mg/kg and it was administered intermittently. After one month on high dose calcitriol therapy, treatment was continued with a maintenance dose of 0.03 mg/kg for a further six months. As a phosphate binding agent, daily 3 g calcium carbonate was administered. Before starting this treatment protocol, patients went on a 1 mg/day calcitriol therapy, although the mean PTH level was 424.63 pg/ml and the mean serum alkaline phosphatase level was 290.2 U/l. During the pretreatment period, levels of PTH, alkaline phosphatase, ionized calcium, and total calcium remained significantly within normal limits as a result of the new therapy protocol applied. PTH and phosphorus clearance rates were compared in the patient groups in which different dialysis membranes had been used. PTH and phosphorus clearances were 15.2 +/- 3 ml/min and 239.1 +/- 19.2 ml/min, respectively, in the polysulfone membrane group, and 1.1 +/- 0.3 ml/min and 112.8 +/- 9.88 ml/min, respectively, in the cuprophane membrane group (p < 0.05).