Oral Mesenchymal Stem/Progenitor Cells: The Immunomodulatory Masters.

Oral mesenchymal stem/progenitor cells (MSCs) are renowned in the field of tissue engineering/regeneration for their multilineage differentiation potential and easy acquisition. These cells encompass the periodontal ligament stem/progenitor cells (PDLSCs), the dental pulp stem/progenitor cells (DPSCs), the stem/progenitor cells from human exfoliated deciduous teeth (SHED), the gingival mesenchymal stem/progenitor cells (GMSCs), the stem/progenitor cells from the apical papilla (SCAP), the dental follicle stem/progenitor cells (DFSCs), the bone marrow mesenchymal stem/progenitor cells (BM-MSCs) from the alveolar bone proper, and the human periapical cyst-mesenchymal stem cells (hPCy-MSCs). Apart from their remarkable regenerative potential, oral MSCs possess the capacity to interact with an inflammatory microenvironment. Although inflammation might affect the properties of oral MSCs, they could inversely exert a multitude of immunological actions to the local inflammatory microenvironment. The present review discusses the current understanding about the immunomodulatory role of oral MSCs both in periodontitis and systemic diseases, their "double-edged sword" uniqueness in inflammatory regulation, their affection of the immune system, and the underlying mechanisms, involving oral MSC-derived extracellular vesicles.

Effect of Inflammation on Gingival Mesenchymal Stem/Progenitor Cells' Proliferation and Migration through Microperforated Membranes: An In Vitro Study.

BACKGROUND: In the field of periodontal guided tissue regeneration, microperforated membranes have recently proved to be very promising periodontal regenerative tissue engineering tools. Regenerative periodontal approaches, employing gingival mesenchymal stem/progenitor cells in combination with these novel membranes, would occur mostly in inflamed microenvironmental conditions intraorally. This in turn entails the investigation into how inflammation would affect the proliferation as well as the migration dynamics of gingival mesenchymal stem/progenitor cells. Materials and Methods. Clones of human gingival mesenchymal stem/progenitor cells (GMSCs) from inflamed gingival tissues were characterized for stem/progenitor cells' characteristics and compared to clones of healthy human GMSCs (n = 3), to be subsequently seeded on perforated collagen-coated poly-tetra-floro-ethylene (PTFE) membranes with a pore size 0.4 and 3 microns and polycarbonic acid membranes of 8 microns pore size in Transwell systems. The population doubling time and the MTT test of both populations were determined. Fetal bovine serum (FBS) was used as a chemoattractant in the culturing systems, and both groups were compared to their negative controls without FBS. Following 24 hours of incubation period, migrating cells were determined on the undersurface of microperforated membranes and the membrane-seeded cells were examined by scanning electron microscopy. RESULTS: GMSCs demonstrated all predefined stem/progenitor cell characteristics. GMSCs from inflamed gingival tissues showed significantly shorter population doubling times. GMSCs of inflamed and healthy tissues did not show significant differences in their migration abilities towards the chemoattractant, with no cellular migration observed in the absence of FBS. GMSCs from healthy gingival tissue migrated significantly better through larger micropores (8 microns). Scanning electron microscopic images proved the migratory activity of the cells through the membrane pores. CONCLUSIONS: Inflammation appears to boost the proliferative abilities of GMSCs. In terms of migration through membrane pores, GMSCs from healthy as well as inflamed gingival tissues do not demonstrate a difference in their migration abilities through smaller pore sizes, whereas GMSCs from healthy gingival tissues appear to migrate significantly better through larger micropores.

Stem/progenitor cell-mediated pulpal tissue regeneration: a systematic review and meta-analysis.

BACKGROUND: Stem/progenitor cell-mediated pulpal regeneration could represent a promising therapeutic alternative in the field of clinical endodontics. AIM: The present study aimed to systematically assess and meta-analyse dental pulpal tissue regeneration, pulpal vitality and apical healing after the transplantation of stem/progenitor cells versus no transplantation. DATA SOURCES: MEDLINE, Cochrane CENTRAL and EMBASE were searched up to January 2019 for animal experiments and human trials evaluating the pulpal transplantation of stem/progenitor cells. Cross-referencing and hand search were additionally performed. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS AND INTERVENTIONS: Based on randomized controlled clinical trials (RCTs) or controlled clinical trials (CCTs), conducted in animals or humans, the effect of the transplantation of stem/progenitor cells compared to no transplantation on pulpal tissue regeneration, pulpal vitality and apical healing was examined. STUDY APPRAISAL AND SYNTHESIS METHODS: The primary outcome was histologically determined pulpal tissue regeneration, whilst pulpal vitality and apical healing were secondary outcomes. The SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) guidelines and the revised Cochrane risk of bias tool (RoB 2.0) were used for risk-of-bias assessment. Pooled standardized differences in means (SDM) and 95% confidence intervals (95% CI) were calculated using random-effects meta-analyses. RESULTS: From 2834 identified articles, eight animal experiments (82 animals with 336 experimental pulpal defects) and one human trial (40 humans with 40 pulpal defects) were included. Risk of bias of most animal studies was high, whilst the human trial revealed 'some concerns'. Stem/progenitor cell-transplanted pulps demonstrated significantly increased pulpal tissue regeneration compared with controls (SDM [95%CI]: 6.29 [3.78-8.80]). LIMITATIONS: Data on pulpal vitality and apical healing were sparse and inconsistent. Heterogeneity across studies was substantial, publication bias was present, and mainly indirect, surrogate outcome measures were applied. The overall strength of evidence was very low. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: The transplanation of stem/progenitor cells shows promise for pulp regeneration, whilst clinical routine application is still not in reach. Further investigations, employing a comprehensive set of outcomes including those demonstrating functional pulp regeneration relevant for patient-centred care, are required.

Effect of total sonicated Aggregatibacter actinomycetemcomitans fragments on gingival stem/progenitor cells.

BACKGROUND: Aggregatibacter-actinomycetemcomitans (A.actinomycetemcomitans) are strongly associated with localized-aggressive-periodontitis (LAgP). The study's aim was to test for the first time the effect of total sonicated A.actinomycetemcomitans-bacterial-fragments on gingival mesenchymal stem/progenitor cells' (G-MSCs) proliferation and regenerative gene expression in-vitro. MATERIAL AND METHODS: G-MSCs were isolated, characterized, expanded and stimulated by total sonicated A.actinomycetemcomitans-bacterial-fragments (0 (negative-control), 15, 60, 120 and 240µg/ml; serovar-b; n=6/group). Cellular proliferation and NF-κß (NFKB1), Alkaline Phosphatase (ALPL), Collagen-I (COL1A1), Collagen-III (COL3A1), Osteonectin (SPARC) and Osteopontin (SPP1) m-RNA expression were assessed via reverse-transcription-polymerase-chain-reaction (RT-PCR) at 24, 48 and 72 hours and CFUs-ability evaluated at twelve days. RESULTS: G-MSCs demonstrated stem/progenitor cells' characteristics. A.actinomycetemcomitans-bacterial-fragments (up to 72 hours) resulted in marked G-MSCs' proliferation over-time (p<0.001) and elevated NFKB1 (p=0.017), COL1A1 (p=0.025), SPARC (p=0.025), decreased ALPL (p=0.017), with no significant differences for COL3A1 and SPP1 expression or stimulation times (p>0.05; Friedman-test). Longer-term stimulation for twelve days reduced G-MSCs' CFUs. CONCLUSIONS: Sonicated A.actinomycetemcomitans-bacterial-fragments' exert beneficial short-term effects on G-MSCs' proliferative and non-mineralized tissue forming aptitude. Results shed new light on the importance of periodontal treatment for LAgP patients, using power driven sonic/ultrasonic devices, which, in addition to reducing the subgingival microbial load, produces cell-stimulatory A.actinomycetemcomitans-bacterial-fragments, with positive attributes on tissue reparative/regenerative responses of tissue resident stem/progenitor cells in their niche.

Adult mesenchymal stem cells explored in the dental field.

During the last decade it was realized that stem cell-based therapies hold an enormous therapeutic potential, improving the life of patients with conditions ranging from neurodegenerative and traumatic diseases to regenerative medicine requiring replacement of complex structures such as bones and teeth. Based on their ability to regenerate and/or repair damaged tissue and eventually restore organ function, multiple types of stem/progenitor cells have been discovered. In the field of periodontal regeneration and tooth engineering, several types of adult multipotent mesenchymal stem cells from various sources are currently being investigated. These include the bone marrow stromal stem cells (BMSSCs), adipose-derived stromal cells (ADSCs), dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from human exfoliated deciduous teeth (SHEDs), stem cells from the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone proper-derived stem cells, and gingival stem cells. The potential of these different MSCs as precursors for regenerative purposes in the dental field is discussed in this chapter.

PGA-associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair.

The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non-articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell-cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid- (PGA) associated porcine heterotopic mono- and cocultures were assessed for cell vitality, types II, I, and total collagen-, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only ß1-integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species-dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan-rich hyaline-like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time-dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair.

In vitro and in vivo neo-cartilage formation by heterotopic chondrocytes seeded on PGA scaffolds.

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3 weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12 weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6 weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12 weeks in vivo construct size reductions were higher compared with 6 weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.

Transpalatal distraction osteogenesis prior to alveolar bone grafting in cleft lip and palate patients.

Alveolar bone grafting is a standard method for treating alveolar cleft. To ensure the best outcome, improving the arch form as well as soft tissue quality in the area around the cleft is recommended. In this study, 11 patients who presented with alveolar cleft and collapsed maxillary arch were treated in the following sequence: transpalatal distraction osteogenesis followed by soft tissue surgery in some cases and by cancellous bone graft. In all cases, transpalatal distraction osteogenesis successfully corrected the transverse maxillary deficiency. One case showed a complete loss of the bone graft. Other minor complications were reported but they did not affect the final outcome.

Cytokine gene polymorphisms in egyptian cases with brain tumors.

BACKGROUND: Cytokines are proposed to play important roles in brain tumor biology as well as neurodegeneration or impaired neuronal function. OBJECTIVES: This work aimed to check the association of polymorphisms of cytokine genes in Egyptian cases with brain tumors. METHODS: This work included 45 cases affected by brain tumors diagnosed as 24 benign and 21 malignant. Their median age was 45 years, and they were 20 males and 25 females. These cases were taken randomly from the Neurosurgery Department of Mansoura University Hospital, Egypt. Case genotypes were compared to 98 healthy unrelated controls from the same locality. DNA was amplified using PCR utilizing sequence specific primers (SSP) for detection of polymorphisms related to TNF-a-308 (G/A), IL-10-1082 (G/A), IL-6-174 (G/C) and IL-1Ra (VNTR) genes. RESULTS: Cases affected with benign brain tumors showed a significant higher frequency of IL-10-1082 A/A [odds ratio (OR=8.0), p<0.001] and IL-6-174 C/C (OR=6.3, p=0.002) homozygous genotypes as compared to controls. Malignant cases, on the other hand, showed significantly higher frequency of IL-6-174 C/C (OR =4.8, p=0.002) homozygous genotype and TNF-a-308 A/A (OR=4.9, p<0.001) homozygous genotype when compared to controls. In the meantime, all cases showed no significant difference regarding the distribution of IL-1Ra VNTR genotype polymorphism compared to controls. CONCLUSIONS: Cytokine gene polymorphisms showed a pattern of association with brain tumors which may have potential impact on family counseling and disease management.

Effects of a selection of histone deacetylase inhibitors on mast cell activation and airway and colonic smooth muscle contraction.

Studies of histone deacetylase (HDAC) inhibitors, novel anticancer drugs, in models of autoimmune diseases, asthma, and inflammatory bowel disease suggest that HDAC inhibitors may also have useful anti-inflammatory effects. Accordingly, in vitro studies relevant to asthma and inflammatory bowel disease were conducted using a selection of HDAC inhibitors: suberoylanilide hydroxamic acid (SAHA, Vorinostat), and a related branched hydroxamic acid, diamide (1), MGCD0103 and two short chain fatty acid derivatives: sodium butyrate (of use in inflammatory bowel disease) and sodium valproate. The ability of those HDAC inhibitors to modulate antigen- or agonist-induced contraction of isolated guinea pig tracheal rings and colon, agonist-induced contraction of rat colon, and histamine release from rat peritoneal mast cells was examined. Pre-incubation (up to 6 h) with 10-40 microM of SAHA, diamide (1), or MGCD0103 caused significant inhibition of the antigen-induced contraction of sensitised guinea pig tracheal rings as well as inhibition of the contraction induced by histamine, 5-hydroxytryptamine and carbachol (G-protein coupled receptor agonists), while sodium butyrate (1 mM) and sodium valproate (100 microM) were weak inhibitors. Contraction of tracheal rings by sodium fluoride (NaF, a non-selective G-protein activator), KCl and a peroxyl radical generator was blocked by MGCD0103. Additionally, MGCD0103 significantly inhibited antigen-induced histamine release from IgE antibody-sensitised rat peritoneal mast cells, and NaF-induced histamine release, as well as inhibiting NaF-induced colon contraction. Those various effects appear to involve modulation of cell signaling, probably involving G-protein coupled pathways, and further support the development of HDAC inhibitors as anti-inflammatory agents.

Temporomandibular joint reconstruction with costochondral graft using modified approach.

12 patients presenting with long standing temporomandibular joint (TMJ) ankylosis were treated with a costochondral graft inserted through a modified approach. The age of the patients ranged from 5 to 17 years. A preauricular incision was made for resection of the ankylosed condyle. After release of the ankylosis the contralateral rib was harvested with costal cartilage. An intra-oral incision was made along the external oblique ridge to the mucobuccal fold and was used for resection of the coronoid process and insertion and fixation of the graft. The graft was fixed with a minimum of three titanium screws. The patients were instructed to start physiotherapy 1 week postoperatively and were followed up clinically and radiographically using 3D CT. Postoperative results were encouraging, the graft took well in all patients without postoperative infection or graft rejection. The graft was properly positioned in all cases. There were no visible scars as the preauricular scar is relatively hidden, no possibility of damaging the facial nerve or the marginal mandibular branch and shorter operating time.

The impact of haemodialysis-associated variables on lipid profile in Egyptian haemodialysis population.

INTRODUCTION: Haemodialysis (HD) patients appear to have particular susceptibility for cardiovascular (CV) diseases with lipid abnormalities among its significant contributors. However, there is controversy concerning the combined effect on lipid constituents during HD of the three commonly used variables; the type of heparin, dialysis membrane and the constituent of dialysate buffer bases. Consequently, this controlled prospective study was thought of. PATIENTS: Randomly 63 patients were assigned from Urology and Nephrology haemodialysis (HD) unit, Mansoura, Egypt for the planned study. Their mean age was 45.79 +/- 13.11 years. Fourteen patients with end-stage renal disease (ESRD) served as control group for the remaining 49 HD patients. They were subdivided according to the HD duration (< and > 1 year), anticoagulant used (unfractionated [UFH] and low-molecular weight heparin [LMWH, Enoxaparin), membrane type (Hemophane [HP] and polysulfone [PS] membrane) and dialysate buffer bases (bicarbonate versus acetate based). METHODS: Determining the fasting lipid value of total cholesterol (TC) and triglycerides (TG) as well as lipoproteins including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and lipoprotein (a) [Lp (a)] was completed. RESULTS: Bicarbonate dialysate was associated with significantly lower TG (134.7 +/- 11 mg/dl vs. 153 +/- 14 mg/dl, p = 0.004), higher HDL-C (33.1 +/- 3 vs. 28.3 +/- 2, p = 0.0002) and subsequently better atherosclerosis risk ratio [TC/HDL-C (ARR)] (6.02 +/- 0.09 vs. 5.3 +/- 0.9, p = 0.001) despite its insignificant effect on TC and LDL-C. However, logarithm (log) Lp (a) level was significantly higher (1.92 +/- 0.05 vs. 1.82 +/- 0.04 p = 0.001) in comparison with acetate dialysate. Membrane type was not influential in those dialyzed for < 1 year before intervention while after a year of HD, PS (n = 11) compared to HP filters (n = 11) significantly lowered TC (151.7 +/- 16 vs. 172.6 +/- 12, p = 0.003), TG (127.8 +/- 15 vs. 155.7 +/- 15, p = 0.004), LDL-C (122.1 +/- 5 vs. 130.6 +/- 7, p = 0.006) levels as well as ARR (5.9 +/- 0.5 vs. 5.4 +/- 0.3, p = 0.02). Likewise was the reduction in log Lp (a) (1.9 +/- 0.03 vs. 1.8 +/- 0.04, p = 0.002) with insignificant effect on HDL-C. After 6 months, Enoxaparin caused significant improvement of TC (0.0004), TG (p = 0.018), LDL-C (p = 0.006), HDL-C (0.041) and Lp (a) (0.047) compared to UFH. Patients who continued on Enoxaparin for 3 more months displayed an even better attenuation in most of lipid parameters whilst continuation of UFH was insignificant. Switching few patients (n = 4) from UFH to LMWH for 3 months resulted in significant lowering of TC (153 +/- 7 vs. 177.7 +/- 3, p = 0.01), TG (127.5 +/- 5 vs. 137.3 +/- 4, p = 0.03) and LDL-C (124.7 +/- 5 vs. 127.5 +/- 5, p = 0.005). However, switching equal number of patients from LMWH to UFH caused no significant change. CONCLUSION: Dyslipidaemia in Egyptian haemodialysis patients was improved when bicarbonate-based haemodialysis, the use of polysulfone membrane, and more so when the low-molecular weight heparin Enoxaparin were used.

Effect of cyclosporin and tacrolimus (FK506) on the antigen-induced mediator release, membrane potential and 86Rb+/K+ and Ca2+ fluxes in the RBL-2H3 cell line.

The immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) inhibit the activation by antigen of T-lymphocytes as well as mast cells. The mechanism of their action on mast cells has yet to be elucidated. We, therefore, assessed their effect on antigen-induced histamine and beta-hexosaminidase release, membrane potential changes (bis-oxonol fluorescent probe), 86RB+ (marker for K+)-efflux, the intracellular free calcium concentration ([Ca2+]i in single cells) and 45Ca2+ uptake (CsA only) in RBL-2H3 cells, a mucosal-type mast cell line, passively sensitized with monoclonal mouse IgE antibody. Antigen addition induced depolarization within 1-2 min, followed by slower repolarization, reaching a steady state (approximately 90% repolarization) after 7-9 min. CsA and FK506 each dose-dependently inhibited antigen-induced histamine and beta-hexosaminidase secretion and the membrane repolarization phase, with similar IC50s for both actions, approximately 20 nM for CsA and approximately 2 nM for FK506. Antigen-induced 86Rb+-efflux was also significantly inhibited. Antigen-evoked increase in [Ca2+]i (area under the curve, AUC) was reduced by 35% and 52% in the presence of CsA or FK506 (1 microM each), respectively. However, 45Ca2+-uptake was not inhibited by CsA. These results suggest that both CsA and FK506 may inhibit mediator release from mast cells via blocking two interrelated processes, which are involved in the secretory process: 1. Membrane repolarization phase, which is essential for optimal mediator secretion and is mediated by a Ca2+-sensitive K+-efflux, yet to be further characterized, and (2) Increase in [Ca2+]i, probably via reduction of Ca(+2)-release from intracellular stores, [Ca2+]s.

Natural products as angiogenesis modulators.

Cancer remains one of the major causes of death worldwide. Anti-angiogenic therapy is one of the new approaches to anticancer therapy. Out of 22 angiogenesis inhibitors currently under clinical trials there are 11 natural products or were modeled on a natural product parent. This review shows the potential of natural products for the discovery of new anti-angiogenic leads.

Biochemical and histological studies on H2-receptor antagonist ranitidine-induced hepatotoxicity in rats.

This study was designed to investigate the hepatotoxicity of ranitidine treatment in dose levels of 10, 30, and 50 mg/kg b.wt. for 3 weeks period in male rats. The results showed some adverse changes in rats treated with either 10 or 30 mg/kg. Treatment with dose of 50 mg/kg produced marked increase in the activity of both acid phosphatase in liver and aspartate aminotransferase in serum and liver, with a tendency for increase in serum alanine aminotransferase activity. Also, a significant decrease in the serum activity of both amylase and alkaline phosphatase was noted. Microscopic examination of livers of the same animals revealed absence of some hepatic cells, pyknotic nuclei, dilatation of blood sinusoids, binucleated cells, and infiltration of lymphocytes. These biochemical and histological changes indicate that ranitidine when given chronically in high dose could produce hepatotoxicity in rats.

An office-based immunodiagnostic assay for detecting urinary nuclear matrix protein 52 in patients with bladder cancer.

OBJECTIVE: To report the rapid (5 min) and simple detection of a nuclear matrix protein (NMP) in the urine of patients with bladder cancer, using a newly developed office-based dot-enzyme-linked immunosorbent assay (ELISA). PATIENTS AND METHODS: Western blot and specific immunoglobulin-G antibody were used to identify the urinary NMP marker. Urine samples from 149 patients with bladder cancer and 72 controls were evaluated using the developed dot-ELISA. The initial responses of 43 patients treated by irradiation were followed using the assay. RESULTS: The NMP marker was identified in the urine of patients with bladder cancer at 52 kDa (NMP-52) by Western blot. The dot-ELISA detected the urinary NMP-52 marker in 92% of patients with squamous cell carcinoma, 98% with transitional cell carcinoma, and all six of those with adenocarcinoma of the bladder, with a specificity of 94%. The positive and negative predictive values (97% and 94%, respectively) and efficiency (96%) of the dot-ELISA were high. In addition, the NMP-52 tumour marker was not detected in the urine of patients who showed a response after radiotherapy. CONCLUSION: Detecting the urinary NMP-52 marker using dot-ELISA would be helpful in the rapid diagnosis and follow-up of patients with bladder cancer.

Inhibition of the antigen-induced activation of RBL-2H3 cells by charybdotoxin and cetiedil.

Quinidine and Ba(2+), non-selective K(+)-channel blockers, have previously been shown to inhibit antigen-induced mediator (beta-hexosaminidase) release from RBL-2H3 cells, a mucosal-type mast cell line. We therefore used selective blockers of Ca(2+)-activated and other K(+) channels to determine if there was a role for these channels in antigen-induced mediator release. Charybdotoxin and cetiedil dose-dependently inhibited beta-hexosaminidase release with IC(50) values of 133 nM and 84 microM, respectively. Charybdotoxin also inhibited the repolarization phase of the antigen-induced biphasic change in the membrane potential (IC(50) 84 nM), antigen-stimulated 86Rb(+)-efflux and increase in free intracellular calcium, [Ca(2+)](i). Iberiotoxin, margatoxin, apamin and tetraethylammonium had no effect on beta-hexosaminidase release. These results suggest that K(+) conductances play a significant role in mediator release from RBL-2H3, that these conductances are of the intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca)) type, and that they are somewhat similar to those which have been described in red blood cells, though they are much less sensitive to clotrimazole.

Inhibition of the antigen-induced activation of RBL-2H3 cells by cetiedil and some of its analogues.

Our previous studies on rat basophilic leukaemia (RBL-2H3) cells suggested that IK(Ca) channels similar to those in red blood cells (RBC) may be involved in the antigen-induced beta-hexosaminidase release. Since cetiedil blocks these channels in both cell types, we studied the inhibition by a selection of the synthetic analogues of cetiedil (UCL compounds) of antigen-induced beta-hexosaminidase release and 86Rb(+)-efflux from RBL-2H3 cells. We tested the (+)- and (-)-enantiomers of cetiedil (UCL 1348 and UCL 1349), the more lipophilic triphenylacetic acid derivatives (UCL 1495 and UCL 1617) and (9-benzyl-fluoren)-9-yl derivatives (UCL 1608 and UCL 1710). They all inhibited antigen-induced beta-hexosaminidase release and 86Rb(+)-efflux. Their relative potency in inhibiting antigen-induced beta-hexosaminidase release was UCL 1608>1710>1617>1348>1349>1495, with IC(50) values of 9.6+/-0.6, 14.4+/-2.2, 23.4+/-1.4, 29.8+/-1.1, 77.5+/-11.8 and 104.6+/-14.7 (microM), respectively. These IC(50)s suggest some dissimilarity between IK(Ca) in RBL-2H3 cells and RBC. Lipophilicity and potency were well correlated in RBC, but not in RBL-2H3 cells.

Spectrofluorimetric determination of manzamine A in spiked human urine and plasma.

The native fluorescence of manzamine A (a biologically active beta-carboline marine-derived alkaloid) has been studied under different conditions. The highest fluorescence intensity was obtained in methanol. Two wavelength settings were found to be suitable for excitation, 280 nm and 340 nm; while lambdamax emission was constant in both cases at 387 nm. The fluorescence intensity at 340/387 nm setting was 1.6 greater than that obtained at 280/387 nm settings. The calibration curves were rectilinear over the range 0.1-2.0 and 0.5-2.5 microg/ml for the two settings, respectively. The detection limits were 0.05 microg/ml (9.1 x 10(-9) M) and 0.1 microg/ml (1.82 x 10(-8) M) at 340/387 nm and 280/387 nm, respectively. The proposed method was applied to the determination of manzamine A in spiked human urine and plasma samples adopting the 340/387 nm wavelength setting, the % recoveries (n = 6) were 99.61 +/- 0.90 and 100.25 +/- 1.63, respectively.