Cell and platelet composition assays by flow cytometry: basis for new platelet-rich fibrin methodologies.

Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are selective blood fractions obtained by cen¬trifugation. They act locally on inflammation and immunity as adjuvant homeostatic modulators during tissue regeneration. In recent years, many methods for achieving these blood concentrates have emerged, whose parameters of time and force of centrifugation presented themselves as critical, conflicting, and poorly understood points. Thus, the present study aimed to evaluate the effect of different centrifugal experimental parameters on the concentration of cells and platelets in samples of anticoagulated blood. Blood samples were centrifuged by forces of 200, 400 and 800 x g for 5, 10 and 15 minutes of centrifugation times to obtain three fractions: a) platelet-poor plasma (PPP), b) leukocyte-rich plasma (L- PRP) and c) red blood cell sed¬iment (RBC). The leukocyte and platelet content of each centrifuged fraction was measured by automated flow cytometry associated with the peroxidase reaction for differential leukocyte count. The application of 200 x g generated a more significant dispersive content of leukocytes and platelets in the supernatant fraction of PPP when compared to the other two strength ranges. However, it presented the highest concentration of platelets in the sediment (P <0.05 ANOVA), representing a loss of total mass during processing. The 400 and 800 x g forces showed leukocytes and platelets condensed in the L-PRP fraction and lower levels in the sedi¬ment, demonstrating the greater effectiveness of buoyancy in the resuspension of these sedimented elements. Our experimental data showed that the concentration and organization of leukocytes and platelets in the centrifuged blood matrices are very sensitive to variations in g force and centrifugation time, thus generating products with different biological composition and characteristics, and with specific potential therapeutic effects. The present study did not focus on comparing authoring methods, but on presenting the impact of methodological variations on the biological nature of centrifuged blood matrices. Further in vivo studies are needed to assess the specific clinical effect of each methodological change.

Phase II/III study of second-line chemotherapy comparing irinotecan-alone with S-1 plus irinotecan in advanced gastric cancer refractory to first-line treatment with S-1 (JACCRO GC-05).

BACKGROUND: In Japan, S-1 plus cisplatin has been used as first-line therapy for advanced gastric cancer (AGC). Patients with no response to first-line treatment with S-1 often receive a taxane-alone or irinotecan-alone as second-line treatment. However, second-line treatment with S-1 plus irinotecan is widely used in patients with AGC resistant to first-line S-1-based chemotherapy. The goal of this trial was to determine whether the consecutive use of S-1 plus irinotecan improves survival when compared with irinotecan-alone as second-line treatment for AGC. PATIENTS AND METHODS: Patients who had disease progression during first-line S-1-based chemotherapy were randomly assigned to receive S-1 plus irinotecan or irinotecan-alone. The S-1 plus irinotecan group received oral S-1 (40-60 mg/m(2)) on days 1-14 and intravenous irinotecan (150 mg/m(2)) on day 1 of a 21-day cycle. The irinotecan-alone group received the same dose of irinotecan intravenously on day 1 of a 14-day cycle. The primary end point was overall survival (OS). RESULTS: From February 2008 to May 2011, a total of 304 patients were enrolled. The median OS was 8.8 months in the S-1 plus irinotecan group and 9.5 months in the irinotecan-alone group. This difference was not significant (hazard ratio for death, 0.99; 95% confidence interval 0.78-1.25; P = 0.92). Grade 3 or higher toxicities were more common in the S-1 plus irinotecan group than in the irinotecan-alone group. CONCLUSION: The consecutive use of S-1 plus irinotecan is not recommended as second-line treatment in patients who are refractory to S-1-based first-line chemotherapy. ClinicalTrials.gov ID: NCT00639327.

16th IHIW: international histocompatibility working group in hematopoietic cell transplantation.

The International Histocompatibility Working Group is a collaborative international effort to understand the HLA and non-HLA genetics of the transplantation barrier. The Working Group is comprised of experts in the fields of histocompatibility and immunogenetics, hematopoietic cell transplantation and outcomes research. Data for 25 855 unrelated donor transplants were submitted in support of research studies for the 16th International Histocompatibility Workshop. Active investigation is in progress in seven key areas: the impact of HLA matching, role of race and ethnicity, identification of permissible HLA mismatches, haplotype-associated determinants, minor histocompatibility antigens, immune response genes and KIR genetics. New hypotheses for the 16th workshop were developed for immunogenetic studies in cord blood and haploidentical-related donor transplantation.

High-resolution inkjet printing of all-polymer transistor circuits.

Direct printing of functional electronic materials may provide a new route to low-cost fabrication of integrated circuits. However, to be useful it must allow continuous manufacturing of all circuit components by successive solution deposition and printing steps in the same environment. We demonstrate direct inkjet printing of complete transistor circuits, including via-hole interconnections based on solution-processed polymer conductors, insulators, and self-organizing semiconductors. We show that the use of substrate surface energy patterning to direct the flow of water-based conducting polymer inkjet droplets enables high-resolution definition of practical channel lengths of 5 micrometers. High mobilities of 0.02 square centimeters per volt second and on-off current switching ratios of 10(5) were achieved.

Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness.

DFN3, an X chromosome-linked nonsyndromic mixed deafness, is caused by mutations in the BRN-4 gene, which encodes a POU transcription factor. Brn-4-deficient mice were created and found to exhibit profound deafness. No gross morphological changes were observed in the conductive ossicles or cochlea, although there was a dramatic reduction in endocochlear potential. Electron microscopy revealed severe ultrastructural alterations in cochlear spiral ligament fibrocytes. The findings suggest that these fibrocytes, which are mesenchymal in origin and for which a role in potassium ion homeostasis has been postulated, may play a critical role in auditory function.

Combined Endovascular and Microsurgical Hybrid Management of Cerebral Aneurysms: The Preliminary Fujita Experience.

INTRODUCTION: A subgroup of complex aneurysms demands multimodal treatment by microscopic and endovascular means. Partial obliteration or remnant postprocedure demands to go further to the other modality. MATERIALS AND METHODS: All patients between July 2016 and January 2017 who had to undergo multimodality measures for complete obliteration of the aneurysms were included in the study. The patients who had either undergone clipping or coiling for their aneurysms, but with incomplete obliteration of the aneurysm, were also included in the study. RESULTS: Between July 2016 and January 2017, a total of three patients had to undergo coiling after clipping of the aneurysm for complete obliteration. Two patients had to go for clipping following coiling. All five patients had complete obliteration of the aneurysm sac. CONCLUSION: Both microscopic and endovascular means are complementary measures instead of competing procedures. It is important to realize the technical difficulties when surgical therapy follows initial endovascular treatment. Similarly, coiling a previously clipped aneurysm can be difficult, if the clip obscures normal working projections.

Staged Deep Brain Stimulation of Ventral Intermediate Nucleus of the Thalamus for Suppression of Essential Tremors.

Deep brain stimulation (DBS) of the ventral intermediate nucleus (VIM) of the thalamus is a powerful surgical option in the treatment of essential tremors (ETs). However, its therapeutic efficacy depends on the tremor distribution. DBS is highly efficient in the relief of distal appendicular tremor but not other types of tremor. We report a case of staged DBS of ventral intermediate nucleus (VIM) of thalamus for the suppression of ETs.

The role of frozen section biopsy for parotid gland tumour with benign fine-needle aspiration cytology.

OBJECTIVES: This study focused on parotid gland tumours diagnosed as benign by fine-needle aspiration cytology and investigated the necessity of frozen section biopsy. METHODS: There were 104 cases of parotid gland tumour where fine-needle aspiration cytology was benign and frozen section biopsy was subsequently performed, between April 2006 and June 2016. In this retrospective study, the results of frozen section biopsy were analysed and compared with the final histological diagnosis. RESULTS: Among the 104 cases diagnosed as benign by fine-needle aspiration cytology, 102 cases and 2 cases were diagnosed as benign and malignant, respectively, by frozen section biopsy. The final histological diagnoses showed that 98 cases were benign and 6 cases were malignant. The sensitivity and specificity values of frozen section biopsy in detecting malignant tumours were 33 per cent and 100 per cent, respectively. CONCLUSION: The necessity of frozen section biopsy in cases with benign fine-needle aspiration cytology may be low in parotid gland surgery.

Clinical characteristics of chronic graft-versus-host disease following umbilical cord blood transplantation for adults.

Chronic GVHD is a significant complication following allogeneic hematopoietic stem cell transplantation; however, the clinical characteristics of chronic GVHD following cord blood transplantation (CBT) in adults have not been well described. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers of the Nagoya Blood and Marrow Transplantation Group. Of 77 patients, 29 survived without graft failure or progression of underlying diseases for at least 100 days after transplantation. The median age of the 29 patients was 42 years (range, 18-67 years). Seven patients developed chronic GVHD (extensive, n=4; limited, n=3) disease. The cumulative incidence of chronic GVHD 1 year after day 100 was 24% (95% confidence interval (CI), 11-41%), and the organs involved were the skin (n=6), oral cavity (n=4), liver (n=1) and gastrointestinal tract (n=1). In three patients, chronic GVHD was resolved with supportive care. The remaining four were successfully treated with additional immunosuppressive therapy. Event-free survival rates of the 29 patients with and without chronic GVHD 3 years after day 100 were 83 (95% CI, 27-97%) and 36% (95% CI, 17-56%), respectively (P=0.047). These results suggest that chronic GVHD following CBT is mild and has a graft-versus-malignancy effect.

Successful third kidney transplantation with intensive immunosuppression in a highly sensitized recipient.

HLA sensitization associated with previous kidney transplantation is a major drawback to retransplantation. Recently we successfully performed a third graft using intensive immunosuppression for a highly sensitized recipient. The patient was a 31-year-old man who had previously undergone a living donor graft from his father at our institute in 1999. His kidney graft function had deteriorated due to chronic allograft nephropathy, returning to hemodialysis therapy in 2005. He received a second graft from a deceased donor in another country on August 14, 2006. It rejected on postoperative day 3 possibly due to acute accelerated rejection. He was offered a third kidney from his brother. Panel-reactive antibody (PRA) tested before the third procedure revealed positive class I (88%) and class II (96%) PRAs. Mycophenolate mofetil (MMF) was started 3 weeks before the third transplantation, and preoperative plasmapheresis performed thrice. He underwent the living donor graft on March 9, 2007. Immunosuppression consisted of tacrolimus, MMF, methylprednisolone, and basiliximab. Immediately afterward there was a sudden decrease in allograft blood flow and urine output, implying hyperacute rejection. Following treatment with plasmapheresis and a single dose of rituximab (200 mg), the kidney allograft function recovered, although the PRA at 3 weeks was still positive. Six months posttransplantation, he is well with a creatinine of 0.9 mg/dL. Our protocol may reduce the risk for graft loss in a highly sensitized transplant recipient.

Endodermal cyst of the cranio-cervical junction.

We reviewed 36 patients with endodermal cysts occurring at the craniocervical junction. They were aged between 3 and 66 years. Headache, motor weakness, and neck pain were commonly observed symptoms. Radiographically, T1-weighted magnetic resonance imaging of the tumours demonstrated a hypointense, isointense, or hyperintense signal according to the cystic content. In most cases, the cyst walls did not enhance after gadolinium administration. Histologically, the cysts were found to be lined by a single layer of epithelium. Histochemical and immunohistochemical studies showed that almost all were reactive to periodic acid schiff stain, epithelial membrane antigen, and carcino-embryonic antigen, but negative to glial fibrillary acidic protein. Mainly, the suboccipital approach with or without a laminectomy, or the trans-oral approach were selected for surgical excision of these tumours. In 17 of the 36 patients, total or gross total resections were performed, and subtotal resections were achieved in sixteen. Three patients developed recurrences.

Four subtypes of petroclival meningiomas: differences in symptoms and operative findings using the anterior transpetrosal approach.

BACKGROUND: Petroclival meningiomas are vaguely defined as tumours arising from the antero-medial zone to the internal auditory meatus. This report subclassifies petroclival meningiomas based on their origin determined by using radiological and intra-operative findings. METHOD: Ninety-one patients with petroclival meningioma underwent surgery via the anterior transpetrosal approach. The Meckel's cave was routinely opened. Tumour origin was classified into four subtypes according to the main attachment and trigeminal nerve deviation into, upper clivus (UC), cavernous sinus (CS), tentorium (TE), and petrous apex (PA). Their characteristic clinical symptoms and anatomical features were investigated. FINDINGS: The characteristic symptom was ataxia in the UC type (37.5%), abducens nerve palsy in the CS type (64.3%) and trigeminal neuropathy, mainly neuralgia in the PA type (80.0%) with a higher statistical difference from other subtypes. The rate of tumour invasion into Meckel's cave reached 70.3% in average, with the lowest rate in the PA type (25.0%). The rate of middle fossa extension was the highest in the TE type (59.5%). The middle fossa approach was considered to be ideal for UC and TE types because of easier access to the Meckel's cave. Radical dissection without complications was difficult in the CS type. Both the anterior transpetrosal approach and the lateral suboccipital approach could be indicated in the PA type due to the rare invasion of Meckel's cave and middle fossa, and frequent extension into the internal auditory meatus. CONCLUSIONS: This classification is useful to predict the relation between the tumour and the cranial nerves based on symptoms and images. The anterior transpetrosal approach could be used for all four subtypes and with an absolute indication in the UC and TE types showing middle fossa extension.

Variation of dose distribution of stereotactic radiotherapy for small-volume lung tumors under different respiratory conditions.

PURPOSE: To clarify the effects of respiratory condition on dose calculation for stereotactic radiotherapy of small lung tumors. METHODS AND MATERIALS: Computed tomography (CT) data were obtained for nine tumors (diameter, 2.1-3.6cm; mean, 2.7cm) during the stable state, deep expiration, and deep inspiration breath-hold states. Rotational Irradiation with 3 non-coplanar arcs (Rotational Irradiation) and static irradiation with 18 non-coplanar ports (Static Irradiation) using 6-MV photons were evaluated using Fast Fourier Transform (FFT) convolution and Multigrid (MG) superposition algorithms. Dose-volume histograms (DVHs), mean path-length (PL) and mean effective path-length (EPL) were calculated. RESULTS: Although the PL was larger for the inspiration state than for the stable state and the expiration state, the EPL was 0.4-0.5cm smaller in the inspiration state than in the expiration state (p=0.01 for Rotational Irradiation; p=0.03 for Static Irradiation). The isocenter dose obtained by the FFT convolution algorithm was 7-12% higher than that obtained with the MG superposition algorithm. A leftward shift of the DVH obtained by MG superposition was noted for the inspiration state compared with the expiration state. CONCLUSIONS: The choice of the proper algorithm is important to accounting for changes in respiration state. Differences in isocenter dose were not large among the respiratory states analyzed. EPL was a little shorter for inspiration than for expiration, although there were larger and reverse trends in path length. A leftward shift of the DVH obtained for the inspiration state when MG superposition was used.

Olfactory schwannoma.

We report a case of olfactory schwannoma with calcification. Intraoperative findings indicated that the tumour originated from the olfactory groove. Intraoperative findings of previous studies have not indicated a clear relationship between subfrontal schwannoma and the olfactory nerve, which seems strange, given the association between tumours and cranial nerves at other sites. We suggest this observation has not been reported because the growing olfactory schwannoma changes the local morphology, affecting the appearance of the olfactory nerve.

How I do it: underwater endoscopic ear surgery for plugging in superior canal dehiscence syndrome.

BACKGROUND: Underwater endoscopic ear surgery does not require suction and so protects the inner ear from unexpected aeration that may damage its function in the treatment of labyrinthine fistula. A method of underwater endoscopic ear surgery is proposed for the treatment of superior canal dehiscence. METHODS: Underwater endoscopic ear surgery was performed for plugging of the superior semicircular canal through the transmastoid approach. Saline solution was infused into the mastoid cavity through an Endo-Scrub Lens Cleaning Sheath. The tip of the inserted endoscope was filled completely with saline water. RESULTS: Using this underwater endoscopic view, the canal was clearly dissected to expose the semicircular canal membranous labyrinth and dehiscence area. No particular complication occurred during the surgical procedure. CONCLUSION: The underwater endoscopic ear surgery technique for plugging in superior canal dehiscence secures an excellent visual field and protects the inner ear from unexpected aeration.

Effect of phorbol myristate acetate on release of arachidonic acid and its metabolites in the osteoblastic MOB 3-4 cell line and its subclone, MOB 3-4-F2.

We examined the effect of phorbol 12-myristate 13-acetate (PMA) on release of arachidonic acid (AA) and its metabolites in osteoblastic cells in an attempt to study mechanism of the regulation of phospholipase A2 (PLA2) activity. In the MOB 3-4-F2 cell line, a subclone of the clonal osteoblastic MOB 3-4 cell line, PMA (0.1-100 nM) changed its appearance and increased AA release in a dose- and time-dependent manner, whereas 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) did not show a significant affect on the release. The addition of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, greater than or equal to 1.5 mM), a Ca2+ chelator, almost completely inhibited the PMA-induced AA release without affecting the intrinsic AA release. Preincubation with staurosporine (5-20 nM), an inhibitor of protein kinase C (PKC), partially (approximately 60%) blocked the AA release. However, 30-min preincubation with H-7 (50-200 microM), an inhibitor of PKC, failed to block the AA release. PMA, thus, appeared to stimulate AA release partially by a staurosporine-sensitive mechanism, probably an activation of PKC, in an external Ca(2+)-dependent manner. On the other hand, MOB 3-4 cells responded to PMA with an increased AA release but not with a drastic change of its shape. Both staurosporine and BAPTA exerted similar inhibitory effects. Prolonged exposure (48 h) to PMA (0.1-10 nM) enhanced DNA synthesis of MOB 3-4-F2 cells, but not MOB 3-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol ester-like action of staurosporine on the cAMP response to prostaglandin E2 in two macrophage-like cell lines at distinct differentiation stages.

The purpose of this study is to investigate the involvement of protein kinase C (PKC) in prostaglandin E2 (PGE2)-stimulated cAMP production of two macrophage-like cell lines (G3 and XC). XC cells are thought to be placed at a more differentiated stage than G3 cells [Orikasa et al. (1991) Cell Immunol. 132, 350-365]. In RPMI 1640 containing 10% (v/v) heat-inactivated foetal calf serum (FCS), in which the cAMP response of both cells to PGE2 increased with duration of culture, XC cells showed greater response than G3 cells at 2 days of culture. In alpha-minimum essential medium (alpha-MEM) containing 20% (v/v) heat-inactivated horse serum (HS), the cAMP response of both cells was apparently greater than that in RPMI 1640 containing 10% (v/v) FCS. These cells increased cAMP production also in response to PGE1 and PGF2 alpha, and the order of potency in increase was PGE1 > PGE2 >> PGF2 alpha. Interestingly, a short-term (20 min) treatment with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC or staurosporine, a relatively specific inhibitor of PKC, augmented the PGE2-stimulated cAMP production in these cells cultured in alpha-MEM containing 20% (v/v) HS. However, a long-term (24 h) treatment with these compounds did not alter the cAMP response. In G3 cells, PMA appeared more potent than staurosporine in terms of augmentation, whereas in XC cells, the former appeared less potent than the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

Starvation of a clonal osteoblast-like cell line, MOB 3-4-F2, down-regulates prostaglandin E2 receptors but increases cAMP response to prostaglandin E2.

Prostaglandin E2 (PGE2) stimulated cAMP production in the MOB 3-4-F2 cell line, a subclone of the osteoblast-like MOB 3-4 cell line. After being cultured in alpha-minimum essential medium supplemented with 10% heat-inactivated foetal calf serum (HIFCS), cells responded to PGE2 (greater than or equal to 50 ng/ml) with a small, but significant, increase in cAMP production. This response did not vary with duration of culture. In 2% HIFCS-containing medium, despite their lower basal cAMP level, cells responded to PGE2 (greater than or equal to 5 ng/ml) with strikingly increased cAMP production. In addition, prolonged culture in this serum-deficient medium enhanced this response. On the other hand, culture of cells in 2% HIFCS-containing medium decreased the apparent number of PGE2 receptors, which was also enhanced by prolonged culture, without effect on their apparent affinity. Their number in 10% HIFCS-containing medium, more than that in 2% HIFCS-containing medium, was almost constant, independent of the culture period. Starvation of MOB 3-4-F2 cells in serum-deficient medium, therefore, appeared to down-regulate PGE2 receptors but increase the cAMP response to PGE2. Moreover, prolonged starvation of cells appeared to facilitate these phenomena. Our findings suggest that cAMP response to PGE2 does not always reflect the number of available PGE2 receptors in the cells.

The calcium-buffering phase of bone mineral: some clues to its form and formation.

Devitalized, neonatal, rat calvariae incubated in a buffered solution release Ca2+ and Pi during the first day into the medium until levels reach 1.2 mM and 2.6 mM, respectively. Thereafter levels gradually decrease to stabilize by the fifth day at 0.5 mM and 2 mM. Comparison of these solubility changes with those of known calcium phosphate salts suggests that calvarial solubility is controlled by a mixture of impure, soluble, apatite precursors, which at cell death transform to an impure apatite. Increasing concentrations of Mg2+ increased the rate of Ca2+ release and, at 3 mM, stabilized Ca2+ by the fifth day at 0.9 mM. Decreasing the pH from 7.4 to 7.2 produced similar solubility changes as did the addition of citrate at 0.3 mM, phosphocitrate at 0.3 mM, ATP at 0.1 mM, and the bisphosphonate, HEBP, at 0.1 mM. Osteocalcin did not increase calvarial solubility but was able to slow the rate of calcium phosphate phase transition if present at the time of in vitro crystal formation. Phosphocitrate and HEBP were also more effective when present during in vitro crystal formation. HEBP was most effective when present during biological crystal formation, as shown by the increased solubility of devitalized calvariae removed after in vivo administration of HEBP. In vivo manipulations of osteoclast activity produced changes in plasma calcium which correlated with the solubility of the corresponding calvariae after removal and devitalization. Low milk intake increased calvarial solubility. Increasing doses of 1,25(OH)2D3 increased plasma calcium and calvarial solubility, both of which were reversed by injection of acetazolamide. It was concluded from this survey of devitalized bone solubility that calcium exchange between bone and body fluids can buffer calcium homeostasis in the young rat. The exchange is passive. The active components appear to be osteoblastic formation of soluble apatite precursors and their stabilizers and, in reverse, osteoclastic transformation of apatite to precursors by H2CO3 secretion.

Characteristics of NaF-induced differentiation of HL-60 cells.

Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells. When human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10nM, 0-4 days) augmented this antiproliferative effect. NaF increased cellular reduction of nitroblue tetrazolium (NBT), and this effect was strongly augmented by 1,25(OH)2D3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)2D3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)2D3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E2 (PGE2) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)2D3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha, and 1,25(OH)2D3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NBT reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)2D3; adding exogenous PGE2 (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-48 h) of NaF and 1,25(OH)2D3 interaction strongly suggest that 1,25(OH)2D3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE2 itself. Such a mechanism for NaF and 1,25(OH)2D3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)2D3 and PGE1 on the differentiation of HL-60 cells.