Autologous tooth for bone regeneration: dimensional examination of Tooth Transformer® granules.

OBJECTIVE: Since 1967, when the osteoinduction properties of autogenous demineralized dentin matrix were discovered, autologous tooth grafts have been advocated as a viable option to autologous or heterologous bone graft. Tooth graft materials may be extracted from the patient's whole tooth using a granulating device. The aim of this study was to examine the size of granules obtained by the Tooth Transformer (TT)® device, using a laser instrument with high precision. MATERIALS AND METHODS: The TT® device can obtain bone graft material in a short period from an extracted tooth. The resulting material can act as an osteoconductive scaffold, providing a mineral substrate during resorption, including platelet growth factors and morphogenetic proteins. Different studies have investigated the dimension and behavior of various graft material particles, since the size of the grafted particles may play a role in osteogenesis and bone regeneration. RESULTS: Different dimensions of granules are available: small (< 400 µm), medium (400 µm-1,000 µm) and large (1,000 µm-2,000 µm). From 4.03 µm to 100 µm the percentage of granules was 14.52 ± 1.93%. A larger part of the granules was up to 100 µm, while 85.47 ± 1.93% of the granules were from 100 µm to 1,000 µm. CONCLUSIONS: 85% of the granules produced were in accordance with the dimensions suggested in the literature.

Lack of Neutralization of 10-MDP Primers by Zirconia May Affect the Degree of Conversion of Dual-cure Resin Cement.

CLINICAL RELEVANCE: Use of zirconia primers with a low pH and a high acidic monomer concentration should be employed in combination with dual-cure resin cements that are less sensitive to an acidic environment. Primers with lower 10-MDP concentrations attain better outcomes.

Effects of Composites Containing Bioactive Glasses on Demineralized Dentin.

The aim of this study was to evaluate the degradation of completely demineralized dentin specimens in contact with a filler-free or 2 ion-releasing resins containing micrometer-sized particles of Bioglass 45S5 (BAG) or fluoride-containing phosphate-rich bioactive glass (BAG-F). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also used to evaluate the remineralization induced by the experimental ion-releasing resin-based materials. Dentin beams were totally demineralized in H3PO4 (10%) and placed in direct contact with a filler-free (RESIN) or 2 experimental ion-releasing resins (BAG or BAG-F) and immersed in artificial saliva (AS) up to 30 d. Further specimens were also processed and submitted to FTIR and SEM analysis to evaluate the remineralization induced by such ion-releasing resins before and after AS immersion. BAG and BAG-F alkalinized the incubation media. A significant decrease of the dry mass was observed between the specimens of all groups stored for 3 and 30 d in AS. However, the fluoride-containing phosphate-rich bioactive glass incorporated into a resin-based material (BAG-F) showed greater ability in reducing the solubilization of C-terminal cross-linked telopeptide (ICTP) and C-terminal telopeptide (CTX) after prolonged AS storage. Moreover, after 30 d of AS storage, BAG-F showed the greatest remineralizing effect on the stiffness of the completely demineralized dentin matrices. In conclusion, fluoride-containing phosphate-rich bioactive glass incorporated as micrometer-sized filler in dental composites may offer greater beneficial effects than Bioglass 45S5 in reducing the enzyme-mediated degradation and remineralization of demineralized dentin.

Novel hydroxyapatite nanorods improve anti-caries efficacy of enamel infiltrants.

OBJECTIVES: Enamel resin infiltrants are biomaterials able to treat enamel caries at early stages. Nevertheless, they cannot prevent further demineralization of mineral-depleted enamel. Therefore, the aim of this work was to synthesize and incorporate specific hydroxyapatite nanoparticles (HAps) into the resin infiltrant to overcome this issue. METHODS: HAps were prepared using a hydrothermal method (0h, 2h and 5h). The crystallinity, crystallite size and morphology of the nanoparticles were characterized through XRD, FT-IR and TEM. HAps were then incorporated (10wt%) into a light-curing co-monomer resin blend (control) to create different resin-based enamel infiltrants (HAp-0h, HAp-2h and HAp-5h), whose degree of conversion (DC) was assessed by FT-IR. Enamel caries lesions were first artificially created in extracted human molars and infiltrated using the tested resin infiltrants. Specimens were submitted to pH-cycling to simulate recurrent caries. Knoop microhardness of resin-infiltrated underlying and surrounding enamel was analyzed before and after pH challenge. RESULTS: Whilst HAp-0h resulted amorphous, HAp-2h and HAp-5h presented nanorod morphology and higher crystallinity. Resin infiltration doped with HAp-2h and HAp-5h caused higher enamel resistance against demineralization compared to control HAp-free and HAp-0h infiltration. The inclusion of more crystalline HAp nanorods (HAp-2h and HAp-5h) increased significantly (p<0.05) the DC. SIGNIFICANCE: Incorporation of more crystalline HAp nanorods into enamel resin infiltrants may be a feasible method to improve the overall performance in the prevention of recurrent demineralization (e.g. caries lesion) in resin-infiltrated enamel.

Polyaspartic acid enhances dentine remineralization bonded with a zinc-doped Portland-based resin cement.

AIM: To ascertain whether biomimetic phosphoprotein analogues (polyaspartic acid (PAS) and sodium trimetaphosphate (TMP)) improve bonding efficacy and dentine remineralization ability of a novel zinc-doped Portland-based resinous sealing cement. METHODOLOGY: Bonding procedures were performed on phosphoric acid-etched dentine, and several groups were established regarding biomimetic analogue application: (1) no application, (2) PAS-treated dentine and (3) dentine treated with a mixture of PAS and TMP. Raman spectroscopy and microtensile bond strength (MTBS) with fracture analysis by scanning electron microscopy were carried out. MTBS values were compared by anova, Student-Newman-Keuls and Student's t-tests (P < 0.05 and P < 0.01, respectively). RESULTS: Twenty-four hour MTBS values were not affected by the different bonding procedures. After 6 months, MTBS decreased in those groups in which the phosphoproteins analogues were not applied (P < 0.05). When PAS was applied, MTBS was maintained after 6 months (P > 0.05). The novel material bonded without primer application induced bioactive crystal (calcium carbonate and Ettringite) precipitation onto the etched dentine and augmented the degree of crystallinity at the hybrid layer. Mineral-to-matrix ratio was increased at the hybrid layer of the PAS-treated specimens; this primer was also able to catalyse dentine remineralization, without an increase in crystallinity. CONCLUSIONS: PAS application onto demineralized dentine produced an inhibition or delay of mineral phase crystallization, enhancing the remineralization potential of the Portland microfillers at the resin-dentine bonded interface.

Impact of smoking on guided tissue regeneration using a biocomposite poly (lactic-co-glycolic) acid/sub-micron size hydroxyapatite with a rubber dam as an alternative barrier.

The purpose of our study was to critically evaluate the results obtained from a guided tissue regeneration technique after 12 months using a bocomposite poly (lactic-co-glycolic) acid/sub-micron size hydroxyapatite (PLGA/HA) with a rubber dam as a barrier in smoking and non-smoking patients. We selected 36 patients (18 current smokers and 18 non-smokers) diagnosed with chronic advanced periodontitis with a periodontal site (probing depth [PD] >5) amenable to regenerative surgery. Twelve months after surgery, the periodontal parameters were found to have statistically improved, when non-smokers were compared with smokers, in: PD reduction (6.3 ± 2.1 mm vs. 3.6 ± 1.9 mm); CAL gain (4.4 ± 1.1 vs. 2.8 ± 2.2 mm); recession (1.8 ± 1.4 mm vs. 0.8 ± 0.9 mm); and hard tissue fill (4.7 ± 0.8 mm vs. 2.8 ± 2.1 mm). Furthermore, since we found PD baseline differences between groups, smoking seemed not to influence the outcomes achieved (CAL gain and ΔREC) 12 months post surgery with respect to PD baseline. The use of PLGA/HA with a rubber dam significantly improved the periodontal parameters in both smoking and non-smoking subjects. This improvement was nevertheless lower in smokers than the non-smokers, confirming the negative impact of smoking on periodontal regeneration.

Zoledronate and ion-releasing resins impair dentin collagen degradation.

This study analyzed the amounts of solubilized telopeptides cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and C-terminal crosslinked telopeptide of type I collagen (CTX) derived from matrix-metalloproteinases (MMPs) and cysteine cathepsins (CTPs) subsequent to application of a filler-free (Res.A) or an ion-releasing resin (Res.B) to ethylenediaminetetraacetic acid (EDTA)-demineralized dentin with or without zoledronate-containing primer (Zol-primer) pre-treatment. The chemical modification induced following treatments and artificial saliva (AS) storage was also analyzed through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Totally EDTA-demineralized specimens were infiltrated with Res.A or Res.B with or without Zol-primer pre-treatment, light-cured, and immersed in AS for up to 4 wk. ICTP release was reduced following infiltration with Res.B and further reduced when Res.B was used with Zol-primer; remarkable phosphate mineral uptake was attained after AS storage. CTX release was increased in Res.A- and Res.B-treated dentin. However, when Zol-primer was used with Res.A, the CTX release fell significantly compared to the other tested resin-infiltration methods. In conclusion, zoledronate offers an additional inhibitory effect to the ion-releasing resins in MMP-mediated collagen degradation. However, Zol-primer induces a modest reduction in CTX release only when used with resin-based systems containing no ion-releasing fillers.

Magnesium phosphate cements for endodontic applications with improved long-term sealing ability.

AIM: To characterize three radiopaque Magnesium Phosphate Cements (MPCs) developed for endodontic purposes. METHODOLOGY: Three experimental MPCs containing Bi2 O3 were formulated. The experimental cements, which consisted of mixtures of magnesium oxide with different phosphate salts, were characterized for setting time, injectability, porosity, compressive strength and phase composition. The long-term sealing ability of the experimental MPCs applied in single-rooted teeth as root canal filling material or as sealer in combination with gutta-percha was also assessed using a highly sensitive fluid filtration system. A mineral trioxide aggregate (MTA) cement was used as control. Statistical analysis was performed with two- or three-way analysis of variance (anova) and Tukey's test was used for comparisons. RESULTS: The addition of 10 wt% Bi2 O3 within the composition of the MPCs provided an adequate radiopacity for endodontic applications according to ISO 6876 standard. The reaction products resulting from the MPCs were either struvite (MgNH4 PO4 ·6H2 O) or an amorphous sodium magnesium phosphate. The porosity of the three MPCs ranged between 4% and 11%. The initial setting time of the experimental cements was between 6 and 9 min, attaining high early compressive strength values (17-34 MPa within 2 h). All MPC formulations achieved greater sealing ability than MTA (P < 0.05) after 3 months, which was maintained after 6 months for two of the experimental cements (P < 0.05). CONCLUSIONS: These MPCs had adequate handling and mechanical properties and low degradation rates. Furthermore, a stable sealing ability was demonstrated up to 6 months when using the cement both as root filling material and as sealer in conjunction with gutta-percha.

Can the hydrophilicity of functional monomers affect chemical interaction?

The number of carbon atoms and/or ester/polyether groups in spacer chains may influence the interaction of functional monomers with calcium and dentin. The present study assessed the chemical interaction and bond strength of 5 standard-synthesized phosphoric-acid ester functional monomers with different spacer chain characteristics, by atomic absorption spectroscopy (AAS), ATR-FTIR, thin-film x-ray diffraction (TF-XRD), scanning electron microscopy (SEM), and microtensile bond strength (µTBS). The tested functional monomers were 2-MEP (two-carbon spacer chain), 10-MDP (10-carbon), 12-MDDP (12-carbon), MTEP (more hydrophilic polyether spacer chain), and CAP-P (intermediate hydrophilicity ester spacer). The intensity of monomer-calcium salt formation measured by AAS differed in the order of 12-MDDP=10-MDP>CAP-P>MTEP>2-MEP. FTIR and SEM analyses of monomer-treated dentin surfaces showed resistance to rinsing for all monomer-dentin bonds, except with 2-MEP. TF-XRD confirmed the weaker interaction of 2-MEP. Highest µTBS was observed for 12-MDDP and 10-MDP. A shorter spacer chain (2-MEP) of phosphate functional monomers induced formation of unstable monomer-calcium salts, and lower chemical interaction and dentin bond strength. The presence of ester or ether groups within longer spacer carbon chains (CAP-P and MTEP) may affect the hydrophilicity, µTBS, and also the formation of monomer-calcium salts.

Experimental etch-and-rinse adhesives doped with bioactive calcium silicate-based micro-fillers to generate therapeutic resin-dentin interfaces.

OBJECTIVES: This study aimed at evaluating the therapeutic bioactive effects on the bond strength of three experimental bonding agents containing modified Portland cement-based micro-fillers applied to acid-etched dentin and submitted to aging in simulated body fluid solution (SBS). Confocal laser (CLSM) and scanning electron microscopy (SEM) were also performed. METHODS: A type-I ordinary Portland cement was tailored using different compounds such as sodium-calcium-aluminum-magnesium silicate hydroxide (HOPC), aluminum-magnesium-carbonate hydroxide hydrates (HCPMM) and titanium oxide (HPCTO) to create three bioactive micro-fillers. A resin blend mainly constituted by Bis-GMA, PMDM and HEMA was used as control (RES-Ctr) or mixed with each micro-filler to create three experimental bonding agents: (i) Res-HOPC, (ii) Res-HCPMM and (iii) Res-HPCTO. The bonding agents were applied onto 37% H3PO4-etched dentin and light-cured for 30s. After build-ups, they were prepared for micro-tensile bond strength (µTBS) and tested after 24h or 6 months of SBS storage. SEM analysis was performed after de-bonding, while CLSM was used to evaluate the ultra-morphology/nanoleakage and the mineral deposition at the resin-dentin interface. RESULTS: High µTBS values were achieved in all groups after 24h. Only Res-HOPC and Res-HCPMM showed stable µTBS after SBS storage (6 months). All the resin-dentin interfaces created using the bonding agents containing the bioactive micro-fillers tested in this study showed an evident reduction of nanoleakage and mineral deposition after SBS storage. CONCLUSION: Resin bonding systems containing specifically tailored Portland cement micro-fillers may promote a therapeutic mineral deposition within the hybrid layer and increase the durability of the resin-dentin bond.

Prolonged curing time reduces the effects of simulated pulpal pressure on the bond strength of one-step self-etch adhesives.

The aim of this study was to evaluate the effects of extended light-curing procedures on the microtensile bond strength (µTBS) of one-step self-etch adhesives (1-SEAs) submitted to simulated pulpal pressure. Coronal deep-dentin specimens were bonded using Clearfil S3 Bond (S3), Adper Easy Bond (EB), or G-Bond Plus (GB) following the manufacturers' recommendations and light-cured for 10 seconds or 40 seconds. The dentin-bonded specimens were stored in distilled water for 24 hours without pulpal pressure (control) or submitted to 20 cm H2O simulated pulpal pressure for 24 hours. The specimens were cut into matchsticks and subjected to µTBS testing. The data were statistically analyzed using the three-way analysis of variance and Tukey's tests (p<0.05). Debonded sticks were investigated through scanning electron microscopy. EB obtained higher bond strengths than GB and S3. However, prolonged light activation (40 seconds) provided higher µTBS for all adhesives when submitted to pulpal pressure. Conversely, pulpal pressure caused a drop in µTBS in EB and S3 when light-cured for 10 seconds. A mixed failure mode was mainly attained for the control groups, whereas the specimens submitted to pulpal pressure failed in the adhesive mode. The µTBS of GB was not affected by pulpal pressure when light-cured for 10 seconds. Adhesive was the most prevalent failure mode, except when light-cured for 40 seconds, which showed predominantly cohesive failure. Extended curing times improved the resistance of 1-SEAs to simulated pulpal pressure.

Microbiochemical analysis of carious dentine using Raman and fluorescence spectroscopy.

The aim of this study was to evaluate and correlate objectively the microspectroscopically derived biochemical components of sound, infected and affected carious dentine with their microhardness and autofluorescence (AF) characteristics. Over 3 million high-resolution Raman spectra from 8 extracted human carious teeth were recorded using Raman spectrometer with parallel spectrum acquisition. Green AF signals across each carious lesion from all samples were acquired with a similar spatial resolution using confocal fluorescence microscopy. The Knoop microhardness (KHN) from a total of 233 co-localized areas was recorded from the same samples and allocated subjectively into the three zones. Cluster analysis of the Raman data, performed using in-house software, produced five independent spectral components representing mineral content, protein content, porphyrin fluorescence (PF), putative infected dentine signal (IDS) and affected dentine signal (ADS). The distributions of the 5 Raman components and the AF signal were matched across all samples and their average values were calculated for each corresponding KHN area. The infected dentine was defined significantly by the KHN, AF and by the relative contribution of the mineral, PF and IDS clusters. Protein cluster was not statistically related to the KHN or AF. A delineation between affected and sound dentine was observed using the KHN, AF, PF and ADS parameters. This study concludes that micro-Raman spectroscopy can provide a non-invasive and objective evaluation of different carious dentine zones. Being able to detect and assess clinically the caries-affected dentine during minimally invasive operative caries management is important to control the risk of unnecessary tissue removal.

Effects of MMP inhibitors incorporated within dental adhesives.

Matrix metalloproteinase (MMP) inhibition has been shown to reduce adhesive bond degradation when applied as a pre-conditioner, adding to clinical steps in the placement of adhesives, but their incorporation within dental adhesives has not been fully explored. This study examined the effect of including 2 MMP inhibitors (BB94 and GM6001) within the primers of 3 commercially available adhesives. Fluorometric assay and zymography showed that adhesives with MMP inhibitors had high affinity toward both synthetic fluorogenic FRET peptides (95%) and dentin powder substrates, respectively. The immediate microtensile bond strength was enhanced for 2 types of adhesives following the addition of both inhibitors. However, no changes were detected between the control and the inhibitor groups following 3-month storage. The modified two-step etch-and-rinse and single-step systems showed less Rhodamine B penetration to the "hybrid layer" and to the "adhesive", respectively. The incorporation of BB94 and GM6001 within the primers resulted in the inhibition of dentin MMPs with improved initial bond strength and enhanced sealing ability.

The dentine remineralization activity of a desensitizing bioactive glass-containing toothpaste: an in vitro study.

BACKGROUND: Dentine hypersensitivity occurs easily on exposed dentine. This study aimed to evaluate the effectiveness of a novel bioactive glass-containing toothpaste on dentine permeability and remineralization. METHODS: Thirty dentine discs were divided into three groups of 10 specimens each. The groups corresponded to the following brushing treatments: no brush, distilled water, and bioactive glass-containing toothpaste (Novamin). The toothpaste was applied twice a day for 7 days. Dentine permeability was measured after ethylene-diamine-tetraacetic acid (EDTA) etching, initial application, 3-day application, 7-day application and citric acid challenge, respectively. The dentine morphology was investigated using scanning electron microscopy (SEM). Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy was performed to monitor the mineral variation on demineralized dentine. Qualitative information of elemental variation before and after treatments on completely demineralized dentine was detected by energy dispersive X-ray (EDX) analysis. RESULTS: The bioactive glass-containing toothpaste significantly reduced dentine permeability after the 7-day treatment and showed, under SEM, excellent resistance to acid challenge compared to the other groups. ATR/FTIR and EDX revealed increased mineral content after treatment with Novamin. CONCLUSIONS: As the innovative bioactive glass-containing toothpaste occlude dentinal tubules and resist acid challenge, it may be useful for the treatment of dentine hypersensitivity and dentine remineralization.

Ultramorphology and dentine permeability changes induced by prophylactic procedures on exposed dentinal tubules in middle dentine.

OBJECTIVES: The purpose of this study was to evaluate the changes in dentinal permeability (i.e. hydraulic conductance) after prophylactic treatment performed using prophy-powders with air-polishing system or prophy-pastes applied on exposed middle dentine. The changes in dentine morphology were evaluate by SEM. STUDY DESIGN: Several prophylactic pastes or air-polishing powders were tested in this study. Dentine discs from human third molars were used to study the quantitative reduction of the dentine permeability under simulated pulpal pressure (20 cm H2O). Further specimens were gold-coated and observed with the use of a SEM. RESULTS: The results of this study showed different reductions of dentin permeability effects based on the type of product employed (i.e. prophylactic paste or air-polishing powders). The use of Sylc bioactive glass and sodium bicarbonate were the most effective in reducing dentine permeability of the specimens etched with the phosphoric. The air-polishing procedures performed with Sylc bioactive glass created a dentine surface devoid of exposed dentinal tubules due to the presence of a compact multilayered smear layer. Colgate Sensitive Pro-Relief and Nupro NU-Solution reduced the dentine permeability up to 69.8% and 66.9% respectively. CONCLUSION: Although all the tested products are able to statistically reduce dentine permeability, Sylc bioactive glass is an innovative and effective product suitable for the reducing of the dentine permeability during prophylactic procedures of air-polishing.

Micro-computed tomography of tooth tissue volume changes following endodontic procedures and post space preparation.

AIM: To compare the volume of hard tooth tissue lost after caries removal, access cavity preparation, root canal preparation, fibre post space and cast post preparation in carious premolar teeth. The null hypothesis tested was that there is no difference between the volumes of hard tooth tissue lost expressed as a percentage of the preoperative hard tooth tissue volume, after each operative procedure. METHODOLOGY: Twelve extracted human premolars with mesial or distal carious cavities penetrating into the pulp chamber were selected. Teeth were scanned using a microCT scanner. After each operative procedure the loss of hard tooth tissue volume was measured. The data were statistically analysed using one-way analysis of variance and Fisher's PLSD test with statistical significance set at alpha = 0.01. RESULTS: The percentage of preoperative hard tooth tissue volume lost after caries removal was 8.3 +/- 5.83, after access cavity preparation the loss of volume reached 12.7 +/- 6.7% (increase of 4.4%). After root canal preparation, fibre post space and cast post preparation the hard tissue volume lost reached, 13.7 +/- 6.7 (increase of 1%), 15.1 +/- 6.3 (increase of 1.4%) and 19.2 +/- 7.4 (increase of 4.1%) respectively. Each procedure performed after caries removal significantly increased (P < 0.01) the amount of hard tissue volume lost with the exception of the root canal preparation. CONCLUSIONS: Access cavity and post space preparation are the procedures during root canal treatment which result in the largest loss of hard tooth tissue structure. Cast post space preparation causes a larger loss of tooth structure than fibre post space preparation. This should be taken into account when planning root canal treatment and restoration of root filled teeth that are to be restored with cuspal coverage restorations.

In situ enamel morphology evaluation after acidic soft drink consumption: protection factor of contemporary toothpaste.

OBJECTIVE: The enamel erosion induced by acidic soft drinks is an increasingly important problem. The aim of this study was to investigate the effect of soft drinks on enamel erosion and the protection offered by representative modern toothpastes using a new 'in situ scanning electron microscopy (SEM)-replica technique'. METHODS: Six patients were selected to receive in vivo enamel replicas, fabricated with a polyvinyl-siloxane/polyether impression material. Scanning electron microscopy was used to evaluate the morphology of enamel surface before and after exposure to lemon juice and SPRITE. The protective effectiveness of toothpaste was further evaluated with the same method. Furthermore, to validate the effectiveness of the in situ SEM-replica technique, we compared it to a direct in vitro SEM investigation on extracted teeth. RESULTS: Scanning electron microscopy investigation of the in situ replicas showed severe enamel morphology alterations after acidic soft drink exposure. On the other hand, it was also showed the protective effectiveness of toothpaste in preventing enamel erosion induced by acidic soft drinks. The direct in vitro SEM investigation provided similar enamel erosion results and proved the effectiveness of the in situ SEM-replica technique. CONCLUSION: Acidic soft drinks induce enamel erosion but regular use of toothpaste might reduce the risk for enamel erosion. The in situ SEM-replica technique provides an accurate method for tracing enamel morphology alterations and erosion induced by acidic soft drinks.

Severe thrombocytopenia in an acquired immunodeficiency syndrome patient associated with pentamidine-dependent antibodies specific for glycoprotein IIb/IIIa.

Severe thrombocytopenia developed in a patient with acquired immunodeficiency syndrome during treatment with intravenous pentamidine for Pneumocystis carinii pneumonia. The patient's bone marrow contained adequate numbers of megakaryocytes, suggesting peripheral platelet destruction. Platelet counts ranged between less than 3 and 20 x 10(9)/L for 2 weeks despite cessation of pentamidine, platelet transfusions, high-dose intravenous IgG, and 2 mg/kg/d prednisone. Thereafter, the platelet count increased to prepentamidine levels (95 x 10(9)/L0, permitting rapid withdrawal of steroids. Testing by immunofluorescence disclosed a high-titer, pentamidine-dependent IgG antibody in the patient's acute-phase serum that almost entirely disappeared by the time the patient's platelet count returned to baseline levels. This antibody reacted only with platelet glycoprotein (GP) IIb/IIIa as shown by antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies specific for various GPs, and was absorbable by normal, but not by GPIIb/IIIa-deficient platelets (from a patient with Glanzmann's thrombasthenia). The pentamidine-dependent antibody could not be demonstrated by immunoprecipitation using the patient's serum and 125I-labeled normal platelets, although a separate pentamidine-independent antibody was detected by this method. This latter antibody reacted with two GPs having molecular weights consistent with GPIIb/IIIa, and was present in postrecovery as well as acute-phase sera. However, only the pentamidine-dependent antibody was temporally associated with the severe thrombocytopenia. Therefore, we believe that these studies demonstrate, for the first time, that intravenous pentamidine therapy can provoke formation of drug-dependent antibodies that induce immunologic thrombocytopenia.

Detection of clonal platelet antibodies in immunologically-mediated thrombocytopenias: association with circulating clonal/oligoclonal B cells.

Aware that T and B cells in autoimmune thrombocytopenia are abnormal, including the existence of clonal B cell populations, we sought to characterize this clonal phenomenon in various immunological thrombocytopenias using platelet antibody light chain analysis, flow cytometry, Southern blot analysis, and PCR. Using a monoclonal antibody-antigen capture ELISA, we analysed sera from 21 of 26 patients with autoimmune, alloimmune, or drug-induced immunological thrombocytopenia for the light chain phenotypes of their platelet antibodies. Alloantibodies and drug-dependent antibodies from four and 14 patients, respectively, were found that expressed a predominant type of light chain, suggesting that these platelet-reactive antibodies were monoclonal or oligoclonal in nature. 14 of the 26 patients were available for light chain B cell phenotyping studies. Of these 14 patients, thrombocytopenia was due to autoimmunity in two, drug-induced immunity in four, and alloimmunity in eight. We detected clonal populations of B cells in all 14 patients by flow cytometry. Although six of these latter patients possessed platelet antibodies with clonal characteristics, light chain phenotypes of antibodies in five patients were opposite to those of their B cells. Eight of these patients were further examined for immunoglobulin gene rearrangement using Southern and/or polymerase chain reaction analysis. In all eight patients we detected clonal or oligoclonal B cell populations. Only two of these patients had malignancies (chronic lymphocytic leukaemia) that would be expected to have detectable clonal B cells, and thus the mechanism for clonal expansion in the other six patients did not appear to be related to an obvious neoplastic process. Prior to these studies, detection of clonal B cells in thrombocytopenic patients without known malignancies was limited to individuals with autoimmune thrombocytopenia, prompting the speculation that this particular autoimmune disorder arises from B cell dysregulation, rather than from expansion of specific autoantibody producing B cell clones. In contrast, the current studies provide evidence that clonal B cells are common to patients with any form of immunologically-mediated thrombocytopenia. Moreover, the majority of the platelet antibodies (86%) present in these disorders exhibited monoclonal characteristics in that there was an apparent restriction in light chain usage.

Treatment of refractoriness to platelet transfusion by protein A column therapy.

Ten thrombocytopenic patients (platelets < 10-24 x 10(9)/L) who were refractory to platelet transfusion were investigated for their responsiveness to staphylococcal protein A column therapy. Nine patients had previously been treated with steroids, intravenous immune globulin, and/or other forms of immunosuppressive therapy without improvement in their transfusion response. All patients were receiving multiple platelet transfusions without achieving 1-hour corrected count increments (CCIs) > or = 7500. Eight patients had antibodies that reacted with platelets and were directed against HLA class I antigens, ABO antigens, and/or platelet-specific alloantigens. Plasma (500-2000 mL) from each patient was passed over a protein A silica gel column and then returned to the patient. Patients received from 1 to 14 treatments. A positive response to protein A therapy was defined as at least a doubling of the pretreatment platelet count and/or two successive 10- to 120-minute posttransfusion CCIs > or = 7500. Following plasma treatments, 6 of 10 patients responded with daily platelet counts that averaged 48 +/- 11 x 10(9) per L as compared with counts of 16 +/- 7 x 10(9) per L (p < 0.0005) before treatment. Posttransfusion CCI values determined in four of these patients averaged 2480 +/- 810 and 10,010 +/- 3540 (p < 0.005) before and after treatment, respectively. In contrast, among the four unresponsive patients, platelet counts averaged 10 +/- 9 and 13 +/- 10 x 10(9) per L (p = NS), respectively, while posttransfusion CCIs were 700 +/- 1410 and 1520 +/- 2460 (p = NS), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)