OBJECTIVEThe aim of this study was to compare the efficacy of root canal cleanliness with and without Nd:YAP laser and to assess the effect of the laser on the mineral content of the dentin.BACKGROUND DATAA high degree of cleanliness of the canal when using the Nd:YAG laser has been shown while the laser is in contact with the canal wall. A new Nd:YAP laser has been studied recently, which is considered to be superior to the Nd:YAG with regard to antibactericidal ability due to its 1.34 micro m wavelength, which is in the infra-red range. This wavelength is absorbed better in water than that of Nd:YAG.MATERIALS AND METHODSFourteen extracted single rooted premplars were divided into two groups. In group 1, canals were cleaned, instrumented, and shaped with K files. In group 2, initial preparation was done using K files and completed with a Nd:YAP laser. Teeth were then split longitudinally and submitted to scanning electron microscopy.RESULTSThe cleanliness of the laser-treated teeth was significantly greater than teeth treated with K files alone (p < 0.05). No difference in Ca and P content was detected when the use of K files was compared to the use of laser.CONCLUSIONIt appears that the Nd:YAP laser improves the cleanliness of the root canal. However, since the Nd:YAP laser serves as an addition to K files, its clinical value for replacing conventional root canal instrumentation remains to be determined.
The use of human umbilical cord (UC) blood as a source of transplantable hematopoietic stem cells and progenitor cells may present some advantages over the use of BM. For example, it has been suggested that the degree of HLA matching may be less stringent, and the risk of GvHD may be lower. We have been studying the ex vivo expansion of UC blood T lymphocytes with a view to their use in the adoptive immunotherapy of cancer, autoimmunity, and infectious disease. We have developed a new method involving the use of a conditioned medium (XLCM) that consistently results in levels of UC blood T cell expansion not hitherto possible. Primary cultures of unfractionated low-density MNC (LDMNC) derived from UC blood treated with 5% XLCM routinely show expansions greater than 10,000-fold within 4 weeks. By contrast, similar FBS-free cultures treated with IL-2 expand less than 10-fold and not after 1 week, and cultures treated with IL-2 and concanavalin A (ConA) expand to a maximum of only 300-500-fold over 2 weeks and fail to continue to proliferate thereafter. The MAb, OKT3, which, when combined with IL-2 and FBS, is known to stimulate proliferation of adult peripheral blood lymphocytes, permitted only a 17-fold expansion of UC blood lymphocytes under the same conditions. Thus, XLCM, which can also stimulate adult peripheral blood lymphocyte expansion to levels exceeding 100,000-fold in 3-4 weeks, is uniquely able to stimulate proliferation of UC blood lymphocytes to high levels. From initiation of the UC blood or adult peripheral blood LDMNC/XLCM cultures up to approximately 2 weeks, the cultures are dominated by CD4+ T lymphocytes. By 4 weeks, >80% of the cultured cells bear the CD8+ phenotype, whereas UC blood T lymphocytes cultured in the presence of IL-2 are predominantly CD8+. Thus, XLCM not only allows high levels of expansion of UC blood T lymphocytes not heretofore possible but also permits the selective expansion of different T lymphocyte subsets from a single source.
Although PBSC transplantation has an advantage over BM transplantation in that fewer burdens are placed on the patient at the time of stem cell collection, the number of collected cells decreases when leukapheresis is done repeatedly. We examined the relation between the number of times leukapheresis is performed and the number of mononuclear cells (MNC), CD34+ cells, and colony-forming unit-granulocyte-macrophages (CFU-GM) collected. The percentage of CD34+ cells was measured by flow cytometry and the number of CFU-GM was measured by a progenitor assay. The number of cells collected was significantly decreased by the third collection. Therefore, to secure enough cells for transplantation, leukapheresis ideally should be performed no more than twice if PBSC collection is to be efficient.
Many controversies still exist about the timing of leukapheresis procedures for PBSC transplantation. Thirty-nine patients were followed daily by monitoring the absolute PB WBC count and CD34+ cell enumeration prior to apheresis. These determinations were compared with the apheresis cell content (nucleated cells and CD34+ cells yield). There was a highly significant correlation between PB CD34+ cells and apheresis CD34+ cell yield (r = 0.921, p < 0.001). A small but significant correlation was found between the PB WBC count and the apheresis nucleated cell content (r = 0.383, p < 0.001), but no correlation was found between PB WBC count and apheresis CD34+ cell yield (r = -0.065, p = 0.460). A target value of 20 X 10 6 CD34+ cells/L was determined to be the most reliable predictor to collect at least 1.0 X 10 6 CD34+ cells/kg in a single apheresis. Of the 39 patients, 20 could be followed after transplantation, and a good correlation was found for total number of CD34+ cells reinfused and platelet and neutrophil engraftment. No correlation was found for nucleated cells infused and engraftment. CD34+ cell determination is a better predictor than WBC count for timing leukapheresis and is thus recommended for monitoring the quality of the product.
Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.
In a previous study, we speculated that the early phase of hematopoietic recovery after PBSC transplantation (PBSCT) is rapid because of the increased production of endogenous cytokines by co-transfused monocytes and lymphocytes (Kawano Y, et al. Blood 81:856, 1993). To clarify this point, the:serum level of G-CSF was measured using an ELISA, and various other cytokines, including GM-CSF, macrophage-CSF (M-CSF), SCF, IL-6, IFN-gamma, and soluble IL-2 receptor (IL-2R), were tested for comparison in children receiving conventional or high-dose chemotherapy and autologous transplantation with unmanipulated or purified PBSC. Serum G-CSF levels in patients receiving conventional chemotherapy (n = 21) or PBSCT without exogenous G-CSF treatment (n = 19) increased to 1245 +/- 2337 pg/ml and 2741 +/- 2331 pg/ml, respectively. Likewise, the peak level of G-CSF in patients who did not receive G-CSF was Statistically equivalent to the trough level in those who did. There was no significant difference in the speed of hematopoietic recovery with or without G-CSF treatment in both the conventional chemotherapy and PBSCT cohorts. In addition, no meaningful change was observed in the kinetics of other tested factors in either conventional therapy: or PBSCT settings, regardless of whether the:patient did or did not receive G-CSF. Endogenously produced serum peak G-CSF levels after PBSCT with purified CD34(+) cells were identical to those after the same procedure with unmanipulated cells. These results confirm that children receiving intense chemotherapy followed by autologous PBSCT produce a high level of G-CSF during the cytopenic period that is not due to the infusion of a large amount of facilitating cells capable of producing G-CSF.
Journal of HematotherapyVol. 8, No. 3 Changing Approaches to Transplant Conditioning for Hematologic MalignancyEndre KelemenEndre KelemenSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/106161299320235AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Changing Approaches to Transplant Conditioning for Hematologic Malignancy." , 8(3), pp. 231–232FiguresReferencesRelatedDetails Volume 8Issue 3Jun 1999 To cite this article:Endre Kelemen.Changing Approaches to Transplant Conditioning for Hematologic Malignancy.Journal of Hematotherapy.Jun 1999.231-232.http://doi.org/10.1089/106161299320235Published in Volume: 8 Issue 3: July 9, 2004PDF download
Journal of HematotherapyVol. 8, No. 1 Editorial New Frontiers in HematotherapyDenis K. EnglishDenis K. EnglishSearch for more papers by this authorPublished Online:25 Aug 2004https://doi.org/10.1089/106161299320514AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Editorial New Frontiers in Hematotherapy." , 8(1), pp. 1–2FiguresReferencesRelatedDetails Volume 8Issue 1Feb 1999 To cite this article:Denis K. English.Editorial New Frontiers in Hematotherapy.Journal of Hematotherapy.Feb 1999.1-2.http://doi.org/10.1089/106161299320514Published in Volume: 8 Issue 1: August 25, 2004PDF download
Journal of HematotherapyVol. 8, No. 2 Expanding Mobilized Progenitors for HematotherapyDenis EnglishDenis EnglishSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/106161299320361AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Expanding Mobilized Progenitors for Hematotherapy." , 8(2), pp. 91–92FiguresReferencesRelatedDetails Volume 8Issue 2Apr 1999 To cite this article:Denis English.Expanding Mobilized Progenitors for Hematotherapy.Journal of Hematotherapy.Apr 1999.91-92.http://doi.org/10.1089/106161299320361Published in Volume: 8 Issue 2: July 9, 2004PDF download
Six patients who were to undergo autologous PBSC transplantation with positively selected CD34+ cells were included in this study to compare the efficiency of two devices for clinical grade stem cell selection, the Isolex 300i (Baxter, Munich, Germany) and CEPRATE SC (CellPro, Bothell, WA). PBSC were mobilized by chemotherapy and G-CSF and were collected by leukapheresis on a CS3000 cell separator on 2 consecutive days. The two apheresis products were pooled for CD34 selection. The pooled apheresis products from each patient were divided into two equal portions to be separated on each of the two devices. Cell selection was performed according to the manufacturers' instructions. Enumeration of CD34+ cells was performed by flow cytometry using the HPCA-2 MAb. Purity and yield were significantly better with Isolex than with CEPRATE. Median purity was 93.0% (range 80%-98%) for Isolex and 61.5% (range 27%-72%) for CEPRATE (p = 0.03); median yields for Isolex and for CEPRATE were 48.0% (range 18%-73%) and 23.0% (range 17%-29%), respectively (p = 0.03). The number of CD34 + cells/kg body weight was also significantly higher with Isolex (median 3.8 X 106, range 1.7-5.2) compared with CEPRATE (median 2.35 X 106, range 0.7-4.3) (p = 0.03). Thus, the Isolex 300i device gave products of higher purity and recovered a higher proportion of the CD34+ cells in the harvest before separation. The yield was still poor with both devices, however, and further optimization of the technique for clinical grade stem cell selection is warranted.
The present study was performed to investigate the character of hematopoietic progenitor cells in fetal cord blood (CB). Thirty blood samples from fetuses at a median of 24 weeks of gestation (range 19-29) and 30 neonatal CB samples were analyzed for their immunophenotype by three-color flow cytometry and examined for the presence of female cells by fluorescence in situ hybridization (FISH). We tested the effects of different cytokine combinations (rhIL-1beta, rhIL-3, rhIL-6, rh erythropoietin [rhEPO], rhGM-CSF plus rhSCF, and rhSCF plus rhflt3-ligand) on the differentiation of 100 CD34+-enriched neonatal CB cells for up to 21 days. Ex vivo expansion of 32 unselected fetal blood samples cells was performed in the presence of rhSCF and rhflt3-ligand. The percentage of CD34+ cells in fetal blood was significantly higher compared with neonatal CB (1.24%+/-0.82% versus 0.33%+/-0.18%, p = 0.0001) and inversely correlated with the age of gestation. The contamination of fetal and neonatal CB with maternal cells was low (1.72%+/-0.89%, range 1.0%-4.0%). By using rhflt3-ligand we were able to expand committed progenitor cells while maintaining cells with stem cell function. The use of expanded fetal immature progenitors might have implications for in utero transplantation and autologous gene therapy.
Activation of peripheral blood mononuclear cells (PBMC) with IL-2 generates lymphokine-activated killer (LAK) cells that show a broad target cell range. In adoptive immunotherapy using in vitro-generated LAK cells, the intensity and specificity of their cytotoxic activity affect the prognosis of cancer patients. The present study was designed to examine the tumor-specific spectrum of T lymphocytes generated from the PBMC of patients with recurrent glioblastoma by in vitro propagation with IL-2 plus either soluble or solid-phase anti-CD3 monoclonal antibody (MAb) in short-term or long-term cultures. Both short-term and long-term culturing with solid-phase anti-CD3 MAb plus IL-2 yielded broad-reactivity CD8+ alphabetaT and gammadeltaT lymphocytes, both of which were non-MHC restricted, as shown by the fact that they were able to lyse autologous glioblastoma cells, MHC class I+II- allogeneic glioblastoma cells, and MHC class I-II-NK-sensitive K562 target cells. More importantly, these cells from patients failed to lyse fresh autologous PBMC. These results demonstrate that cells generated using this approach are non-MHC-restricted LAK cells and exhibit marked tumor specificity. In contrast, incubation with soluble anti-CD3 MAb generated T lymphocytes that after long-term culture, were either CD4+ or CD8+. These caused significant lysis of both allogeneic and autologous glioblastoma target cells, the extent of lysis being greater than that using cells produced by culturing with the solid-phase MAb. However, both the CD4+ and CD8+ cells also caused greater lysis of autologous normal PBMC, indicating that cells generated using this approach may cause significant adverse reactions in cancer patients if used for immunotherapy.
A major hindrance to the use of PBSC in allogeneic transplantation is the high rate of contamination with T lymphocytes, resulting in a considerable risk of GvHD. Natural killer (NK) cells are active against tumor cells but do not contribute to the development of GvHD. After adsorption of CD34+ cells of mobilized allogeneic leukapheresis products on a Ceprate column, we studied the separation of CD34 unadsorbed cells by counterflow centrifugal elutriation (CCE). Up to 1.0 x 10(10) cells were clearly separated into lymphocytes (fractions 110 and 140 ml/min), monocytes, and polymorphonuclear cells (fraction rotor off). Characterized by flow cytometry, T cells were distributed nearly equal to fractions 110 and 140. NK cells were concentrated 3.4-fold in fraction 140 as compared with the unseparated cells. The ratio of NK cells/T cells was improved by 33%. These results indicate that CCE is an effective method to enrich NK cells and to reduce T cells in stem cell separation products. Therefore, it is an option for adoptive therapy of cancer patients after transplantations (e.g., CML in relapse).
Several studies have clearly documented a more rapid hematopoietic recovery with growth factor-mobilized PBSC than with bone marrow. Time to engraftment for neutrophils and platelets average 8-12 days in contrast to 2-4 weeks after bone marrow. This rapid hematopoietic recovery with PBSC has decreased the duration of hospitalization, transfusion requirements, and costs. Although growth factors alone may mobilize enough PBSC for high-dose chemotherapy, administration of growth factor after submyeloablative chemotherapy increases the yield of CD34(+) cells. Based on the car rent data, CD34(+) cell content of PBSC appears to be the single most powerful predictor of hematopoietic recovery. Infusion of greater than or equal to 5 x 10(6) CD34(+) cells/kg is associated with a rapid engraftment of neutrophils and platelets, although successful engraftment has also been reported with infusion of 2.5-5 x 10(6) CD34(+) cells/kg. Age, prior radiotherapy,marrow involvement, and prior chemotherapy regimens are important factors influencing the yield of stem cells. Therefore, using these parameters, we may identify the patients who will fail to mobilize sufficient numbers of PBSC before collection and use new strategies for stem cell mobilization. Because of the ease of collection and rapid engraftment after myeloablative therapy, PBSC have replaced bone marrow for autologous transplantation and may supplant bone marrow for allogeneic transplantation in the near future.
Chimerism analysis by DNA-based methods is a valuable diagnostic tool for monitoring engraftment and leukemic relapse after allogeneic BMT or PBPC transplantation (PBPCT). We investigated the chimerism after T-cell-depleted BMT (n = 32) in comparison with T-cell-depleted PBPCT (n = 39). BM grafts were T-cell depleted using the Campath-IgM antibody plus complement. For T-cell depletion of the PBPC grafts, a selection of CD34+ cells with or without a subsequent CD2/3 depletion was performed. In all patients, the T-cell dose of the transplant was < 10(6)/kg body weight. Between day 13 and day 120 after transplantation, chimerism analysis was done by RFLP or amplified fragment length polymorphism (PCR-AFLP), with a detection limit of 1%-5% recipient cells. In the BMT group, 8 of 32 (25%) patients showed a mixed chimerism, but only one graft rejection and no leukemic relapse occurred after a median follow-up of 41 (3-84) months. All patients with PBPCT revealed a complete chimerism of their granulocytes, and 38 of 39 patients showed complete chimerism of their lymphocytes. Follow-up time in these patients is 7 (2-21) months, with no graft rejection and two leukemic relapses. G-CSF-mobilized PBPC are superior to BM cells for full engraftment even after T-cell-depleted transplantation. The more relevant factor for developing complete chimerism seems to be the quantity and possibly the quality of the stem cells rather than the residual T-cell load of the graft. However, a mixed chimerism of the lymphocytes early after transplantation does not predict a higher rate of graft rejection or leukemic relapse.
Proliferative responses and cytokine secretion were compared when AML blasts were cultured in the three serum-free media, X-Vivo 10, X-Vivo 15, and defined serum-free medium (IMDM with mercaptoethanol, low-density lipoprotein, albumin, and transferrin) and in media containing 10% inactivated fetal bovine serum (FBS). The following AML blast functions were investigated: (a) constitutive cytokine secretion, (b) autonomous and cytokine-dependent proliferation, and (c) accessory cell function during T cell activation. Constitutive cytokine secretion and accessory cell function differed markedly when using different culture media. For the constitutive AML blast secretion of IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, no qualitative differences were seen, but quantitative differences were observed with decreased cytokine levels for cells cultured in X-Vivo 10 and X-Vivo 15. The accessory cell function of AML blasts was also decreased in the X-Vivo media, whereas differences were less pronounced when comparing AML blast proliferation. Our results clearly demonstrate that a well-characterized culture system is essential for in vitro studies of AML blast functions.
Twenty-five G-CSF-mobilized leukapheresis products (mLP) were screened for cellular composition, including CD34+DR-, CD34+DR+ and leukocyte profile, to compare with 5 native (unstimulated) LP (nLP) and 16 BM inoculi. G-CSF stimulation led to an increase in CD34+ cells and CD15+ cells but did not influence the lymphocyte content of mLP. Two groups of 14 and 16 patients were allografted with phenotypically defined mLP (1-4 mLP were used for each patient) and BM, respectively. mLP used for allografting had significantly more CD34+ cells, including CD34+DR- cells, monocytes, T cells, and B cells as compared with BM inoculi. Patients were followed for median observation time of 289 days and 409 days for the mLP (PBPC) and BM groups, respectively. The two groups were well matched in regard to age, sex, and stage of disease, with a slight prevalence of major blood group incompatibility (7 of 14 versus 3 of 16) and a lower donor/recipient weight ratio (0.8+/-0.2 vs 1.5+/-0.6, p = 0.002) in the PBPC group. Granulocyte and platelet recovery was faster in the PBPC group than in the BM group. The time of reaching 20,000/microl platelets but not 500/microl granulocytes correlated with the number of CD34+ cells in each inoculum. The survival curves of the PBPC and BM groups were similar, as was the incidence of acute GvHD (aGvHD). This was also valid for aplastic anemia cases (7 and 5 patients in the PBPC and BM group, respectively), who benefited from a high number of CD34+ grafted cells but did not experience aGvHD. Thus, mLP do not appear to elicit aGvHD with higher frequency than BM and may be preferable for hematotherapy.
Following two workshops on standardization of enumeration of CD34+ cells in blood and leukapheresis products, the Nordic Stem Cell Laboratory Group (NSCL-G) evaluated the Milan/Mulhouse/Nordic standard in clinical practice during the third workshop (WS-III). This report documents an acceptable interlaboratory variation in the most clinically active laboratories, with a coefficient of variation (CV) below 0.19 in 7 of 8 analyses performed. The introduction of a pan-CD45 antibody in the analysis did not improve the CV. Comparison of two different CD34 class II antibodies on a total of 99 samples and procedures with and without washing on a total of 96 samples revealed a significant correlation (r2 >0.99) for all analyses. Finally, subset analysis of uncommitted and lineage-specific progenitors revealed major gating difficulties, indicating that further improvements are necessary. In an analysis of more than 600 patients undergoing mobilization and harvest of blood progenitors, with about 500 patients autografted, we found a significant correlation between blood levels of CD34+ cells and recovery of CD34+ cells from each harvest as well as between CD34+ cell number reinfused and time to neutrophil and platelet recovery. This report documents for the first time that the very simple Milan/Mulhouse method (termed The Nordic Standard) can be used by a group of laboratories to obtain important clinical information. Consequently, we consider this method as the conventional method in quality assessment of autografts, which should provide a benchmark for development of second-generation improvements.
Megakaryocytes (MK) were expanded from purified human CD34+ cells obtained from three sources, bone marrow (BM), mobilized peripheral blood progenitor cells (PB), and umbilical cord (UC) blood. CD34+-selected cells were cultured for 12 days with 10 ng/ml thrombopoietin (TPO), 10 ng/ml IL-3, 10 ng/ml TPO + 10 ng/ml IL-3, or 200 ng/ml promegapoietin (PMP), a chimeric dual agonist of the c-Mpl and human IL-3 receptors. MK production was compared in serum-free versus human serum-supplemented liquid media. PMP and the combination of TPO and IL-3 (TPO + IL-3) increased MK production similarly. Culturing CD34+ cells with PMP in serum-free medium resulted in a twofold increase in MK yield compared with serum-supplemented medium. CD34+ cells from UC proliferated more than those from either BM or PB in liquid culture, resulting in much greater MK production under all conditions. Phenotypic analysis of the uncultured CD34+ cells showed that BM had a higher frequency of CD34+/CD41+ cells than PB or UC. TPO + IL-3 or PMP produced larger and greater numbers of BFU-MK and CFU-MK per seeded CD34+/CD41+ cell from UC than from either BM or PB. Thus, although uncultured CD34+-selected BM cells contained a higher frequency of committed mature MK progenitors, UC CD34+ cells had a greater proliferative capacity and, therefore, were more productive. PMP induced megakaryocytopoietic activity comparable to that achieved with TPO + IL-3 and may be useful for ex vivo expansion of MK for clinical trials.
