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Summary: Commercial cellular tests are used to diagnose Lyme borreliosis (LB), but studies on their clinical validation are lacking. This study evaluated the utility of an in‐house and a commercial enzyme‐linked immunosorbent spot (ELISpot) assay for the diagnosis of Lyme neuroborreliosis (LNB). Prospectively, peripheral blood mononuclear cells (PBMCs) were isolated from patients and controls and analysed using an in‐house Borrelia ELISpot assay and the commercial LymeSpot assay. B. burgdorferi B31 whole cell lysate and a mixture of outer surface proteins were used to stimulate the PBMCs and the numbers of interferon‐gamma‐secreting T cells were measured. Results were evaluated using receiver operating characteristic (ROC) curve analysis. Eighteen active and 12 treated LNB patients, 10 healthy individuals treated for an early (mostly cutaneous) manifestation of LB in the past and 47 untreated healthy individuals were included. Both assays showed a poor diagnostic performance with sensitivities, specificities, positive and negative predictive values ranging from 44.4–66.7%, 42.0–72.5%, 21.8–33.3% and 80.5–87.0%, respectively. The LymeSpot assay performed equally poorly when the calculation method of the manufacturer was used. Both the in‐house and the LymeSpot assay are unable to diagnose active LNB or to monitor antibiotic treatment success. [ABSTRACT FROM AUTHOR]

Recipients of allogeneic stem cell transplants (SCT) often show active Epstein–Barr virus (EBV) infection, which may progress to EBV-associated lymphoproliferative disorders. It is not known whether these EBV infections are true reactivations of the endogenous EBV strain or re-infections with an exogenous EBV strain. Fifty-three recipients of matched related or matched unrelated donor grafts were studied. EBV monitoring was based on a realtime TaqMan™ EBV DNA polymerase chain reaction (PCR) assay in plasma. In 17 patients, EBV DNA PCR monitoring was performed in peripheral blood mononuclear cells (PBMCs) as well. Mouth washings (MWs) were collected pre-transplant from all patients and family donors. Both pre-transplant EBV DNA from MWs and post-transplant EBV DNA from plasma or PBMCs were successfully obtained in 6 patients. A nested PCR targeting the EBV latent membrane protein-1 C-terminus gene was used to determine sequence variations enabling EBV strain typing. In 3 of 6 patients, the post-transplant EBV sequence pattern differed from the pre-transplant pattern, indicating a re-infection post-transplant with an exogenous strain instead of a reactivation of the original endogenous EBV strain. In the other 3 patients, the endogenous strain was identified. Active EBV infection resulting from re-infection was more severe compared with active EBV infection because of reactivation. In conclusion, active EBV infections after allogeneic SCT frequently result from re-infection with an exogenous EBV strain instead of a true reactivation of the endogenous strain and are potentially more severe. [ABSTRACT FROM AUTHOR]

Bryostatin-1 belongs to the family of macrocyclic lactones isolated from the marine bryozoan Bugula neritina and is a potent activator of protein kinase C (PKC). Bryostatin has been demonstrated to possess both in vivo and in vitro anti-leukaemic potential. In samples derived from chronic myeloid leukaemia (CML) patients, it has been demonstrated that bryostatin-1 induces a macrophage differentiation, suppresses colony growth in vitro and promotes cytokine secretion from accessory cells. We investigated the effect of bryostatin-1 treatment on colony-forming unit-granulocyte macrophage (CFU-GM) capacity in the presence of accessory cells, using mononuclear cells, as well as in the absence of accessory cells using purified CD34-positive cells. Cells were obtained from 14 CML patients as well as from nine controls. Moreover, CD34-positive cells derived from CML samples and controls were analysed for stem cell frequency and ability using the long-term culture initiating cell (LTCIC) assay at limiting dilution. Individual colonies derived from both the CFU-GM and LTCIC assays were analysed for the presence of the bcr-abl gene with fluorescence in situ hybridization (FISH) to evaluate inhibition of malignant colony growth. The results show that at the CFU-GM level bryostatin-1 treatment resulted in only a 1.4-fold higher reduction of CML colony growth as compared to the control samples, both in the presence and in the absence of accessory cells. However, at the LTCIC level a sixfold higher reduction of CML growth was observed as compared to the control samples. Analysis of the LTCICs at limiting dilution indicates that this purging effect is caused by a decrease in output per malignant LTCIC combined with an increase in the normal stem cell frequency. It is concluded that bryostatin-1 selectively inhibits CML growth at the LTCIC level and should be explored as a purging modality in CML. [ABSTRACT FROM AUTHOR]

The discovery of the Philadelphia chromosome and its consistent involvement in chronic myeloid leukemia (CML) was the first time that a relationship between a cytogenetic abnormality and malignancy was demonstrated. This review will try to provide an insight into the molecular mechanisms underlying this disease and outline the therapeutical options for patients with CML. [ABSTRACT FROM AUTHOR]

Although the mobilization and harvesting of Philadelphia chromosome-negative progenitors has been proven feasible by a number of groups, it is not clear which techniques should be used with regard to the monitoring of purging and evaluation of stem cell yield. Therefore, we isolated CD34-positive cells from leukapheresis samples of seven CML patients after induction therapy. These cells were analyzed using the colony-forming unit granulocyte–macrophage (CFU-GM) and long-term culture-initiating cell (LTCIC) assays. In addition, we analyzed frequency and clonogenicity of single stem cells using the LTCIC assay at limiting dilution. Individual colonies derived from these assays were subsequently analyzed for the presence of the bcr-abl gene with interphase fluorescence in situ hybridization (FISH) and for the presence of bcr-abl mRNA with RT-PCR. We compared these results with the cytogenetic analysis of the leukapheresis material. Molecular analysis of individual colonies correlated well with the results of cytogenetic analysis. However, in nine out of 18 samples, cytogenetic analysis exclusively demonstrated the presence of either Philadelphia chromosome-positive or -negative cells whereas with the molecular analysis of individual colonies tumor contamination or the presence of residual normal cells could be substantiated. We concluded that molecular analysis of individual colonies should be employed to further optimize induction protocols. With regard to stem cell mobilization we demonstrated that about 67 CFU-GM colonies and clusters were generated by one single LTCIC both for the CML samples and the samples obtained from patients with non-hematologic malignancy. This number is important since until now most centres use a number of four colonies and clusters generated by one LTCIC. Dividing the CFU-GM output in the LTCIC assay by four to determine LTCIC frequency could thus lead to an almost 20-fold overestimation of the LTCIC frequency. It is concluded... [ABSTRACT FROM AUTHOR]

In this study, we evaluated the effect of hyperthermia on hematopoietic progenitors from six chronic myeloid leukemia (CML) bone marrow (BM) samples at diagnosis and four peripheral blood stem cell (PBSC) samples from CML patients after stem cell mobilisation. CD34-positive cells, isolated from these samples, were incubated for 2 h at 37, 42 or 43 degrees C and were plated in the colony-forming unit granulocyte-macrophage (CFU-GM) and the long-term culture initiating cell (LTCIC) assay. To evaluate purging, individual colonies from these assays were analyzed for the presence of the bcr-abl gene with interphase fluorescence in situ hybridization (FISH) and/or RT-PCR. BM samples showed a significant higher sensitivity both at the CFU-GM and LTCIC level, after treatment at 42 degrees C, as compared to the control BM samples obtained from healthy volunteers. The four BM samples of CML patients with a low leukocyte number at diagnosis harbored a mixture of bcr-abl-negative and positive colonies and an increase in the percentage of bcr-abl-negative colonies was observed in all cases. CML patients with a high leukocyte count at diagnosis, however, showed only bcr-abl-positive progenitors even after hyperthermia. PBSCs showed a significant higher sensitivity at the LTCIC level but not at the CFU-GM level, after treatment at 42 degrees C, as compared to the control PBSC samples obtained from nonhematologic cancer patients. Molecular analysis of individual colonies demonstrated an increase of bcr-abl-negative progenitors after thermic treatment in two out of three samples. When comparing both stem cell sources, PBSCs showed a decreased thermic sensitivity as compared to the BM samples at the CFU-GM level, whereas at the LTCIC level an increased thermic sensitivity was observed, both for the controls and the CML samples. In conclusion, both for BM and PBSCs samples, CML progenitors are more sensitive to hyperthermia than control cells, especially at the LTCIC level. In agreement with these results, an increase of bcr-abl-negative progenitors in six out of seven samples could be demonstrated either at the CFU-GM level, LTCIC level or both. Hyperthermia should be explored further as a possible purging modality in CML. [ABSTRACT FROM AUTHOR]

In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte-macrophage (CFU-GM) colonies. One half was analyzed with I-FISH, for the presence of bcr-abl fusion gene. The other half was analyzed with RT-PCR for the presence of the bcr-abl mRNA. We wanted to address the following questions: (1) is the bcr-abl gene always expressed in CFU-GM colonies and (2) which technique has to be preferred to analyze individual CFU-GM colonies? In total, 133 colonies, derived from six CML patients, could be analyzed both with I-FISH and RT-PCR. We found a positive correlation in 89% of the cases: 118 colonies showed the same results with both techniques. However, 15 of the 106 I-FISH-positive colonies were negative in the RT-PCR. Serial analysis of the cDNA derived from 22 colonies showed in each round of amplification 21-29% RT-PCR-negative but I-FISH-positive colonies. However, all I-FISH-positive colonies showed at least one positive RT-PCR, either in the first, second or third round of amplification. These results indicate that the bcr-abl gene is probably always transcriptionally active in CFU-GM colonies. Reliable analysis with RT-PCR is possible but likely to generate false negative results. We conclude that: (1) I-FISH offers a reliable alternative to RT-PCR for analyzing individual hematopoietic colonies and (2) results obtained with RT-PCR should only be interpreted with caution. [ABSTRACT FROM AUTHOR]

Responds to questions put forth by Michele Baccarani on the data presented in the paper 'Hypersensitivity of bcr-abl-positive progenitors to hyperthermia in patients with chronic myeloid leukemia.' Explanation provided to various conclusions arrived at in the study.