The Ketogenic Diet and Its Effect on Bone Mineral Density: A Retrospective Observational Cohort Study.

BACKGROUND: During long-term follow-up of children treated with the ketogenic diet therapy (KDT) have an increased incidence of bone fractures. However, the exact contribution of KDT to a decreased bone mineral density (BMD) remains unclear. OBJECTIVE: This study aimed to evaluate (changes in) BMD in children treated with KDT and to evaluate whether intravenous bisphosphonate therapy may be effective. DESIGN: In this retrospective, observational cohort study, all children treated with KDT from 2010 until 2018 at the Radboudumc Amalia Children's hospital were included. Patients who were on KDT for more than 6 months and who had at least two dual-energy X-ray (DXA)-scans were eligible for inclusion for longitudinal analysis. Z-scores of DXA-scans were compared over the course of time. RESULTS: In 34 out of 68 patients, one or more lumbar DXA-scans were performed, with a mean lumbar Z-score of -1.32 ± 1.74. Of these 68 patients, 8.8% got a fracture during KDT, and also 8.8% got kidney stones. In 20 patients, more than one DXA-scan was performed. A statistically not significant decrease in BMD (0.22 Z-score/year) was found. However, there was an increase in BMD in the five patients treated with intravenous bisphosphonate therapy. This was statistically significant in comparison to the nonbisphosphonate treated group (p = 0.034). CONCLUSION: Children on KDT have low normal BMD which may decrease further during KDT. For this reason monitoring of BMD is crucial, as is monitoring of kidney stones and hypercalciuria. Intravenous bisphosphonate therapy may have a positive effect, when other therapies have failed.

Acquired skewing of Lyonization remains stable for a prolonged period in healthy blood donors.

The pattern of X-chromosome inactivation (XCIP), or Lyonization, can be used to distinguish monoclonal from polyclonal cell populations in females. However, a skewed XCIP exists in hematopoietic cells in approximately 40% of healthy elderly females, interfering with interpretation of clonality assays. In hematopoiesis, an active stem cell pool is assumed to be present within a larger population of inactive stem cells, with a continuous exchange of cells between the two compartments. The assumption that the active stem cell pool size decreases with age may explain the phenomenon of acquired skewing occurring by chance and predicts the XCIP of this population to fluctuate. This fluctuation should be reflected in the XCIP of peripheral granulocytes. We examined the XCIP for fluctuations in time in peripheral granulocytes, monocytes and T cells of young, middle-aged and elderly healthy females. We used an optimized HUMARA PCR assay that eliminates unbalanced DNA amplification. We found no fluctuations in XCIP in any age group in up to 18 months follow-up. We conclude that acquired skewing arises gradually in life without fluctuations in XCIP and that analysis at multiple time points cannot distinguish monoclonal hematopoiesis from normal, skewed hematopoiesis.

Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR.

We used a recently developed system for real-time quantitative polymerase chain reaction (PCR) to determine residual disease in patients with chronic myeloid leukaemia. The expression of the Bcr-Abl hybrid oncogene was determined and normalized by using the PBGD housekeeping gene product as endogenous reference. The sensitivity and reproducibility of the assay was tested on cell line K562. A dilution of Bcr-Abl-positive cell line K562 remained positive at up to 250 fg of RNA. 10 copies of Bcr-Abl DNA in water could still be detected. The dynamic range of the method spanned six orders of magnitude. Analysis of 10 identical assays on K562 RNA resulted in a variation of 15%. To test the feasibility of normalization of Bcr-Abl dosage by the PBGD product, we compared the efficiencies of the RT-PCRs in 150 patient analyses. We concluded that PBGD was a suitable and stringent quality control standard. Three patients who were treated with donor leucocyte infusions for chronic myeloid leukaemia who had relapsed after bone marrow transplantation were followed over time. The normalized Bcr-Abl dosage was compared to the results of cytogenetics. Cytogenetic analysis was negative below a normalized Bcr-Abl dose of about 3 x 10(-2). This semi-automated method is fast, sensitive and accurate and enables a high throughput of samples.

An antisense Bcr-Abl phosphodiester-tailed methylphosphonate oligonucleotide reduces the growth of chronic myeloid leukaemia patient cells by a non-antisense mechanism.

The specificity of antisense oligonucleotides targeted to the mRNA breakpoint region of the Bcr-Abl oncogene, found in leukaemic cells from patients with chronic myeloid leukaemia, remains controversial due to non-specific effects. To prevent protein binding of oligonucleotides we designed and tested a methylphosphonate oligonucleotide with an attached 3' soluble phosphodiester tail. Growth of chronic myeloid leukaemia (CML) cell lines BV173, KCL-22 and cells of CML patients tested was inhibited by the b2a2 type antisense Bcr-Abl oligonucleotide and not with controls. Also the growth of control CD34+ cells of two healthy donors, control cell lines and cells from AML patients was only moderately affected or not affected. Bcr-Abl protein studies in combination with growth-determination experiments indicated that the antisense methylphosphonate Bcr-Abl oligonucleotide tested is a potent inhibitor of the growth of CML cells but works in a non-antisense manner.

Bax mutations in cell lines derived from hematological malignancies.

Many genes are involved in cell cycle control, DNA repair and induction of cell death. Alterations in these genes have been responsible for the development of cancer as well as for resistance to cancer therapy. Recently, an emerging family of bcl2-like genes has been identified that plays a role in the regulation of cell death. Its members are highly conserved in several domains which have been shown to be important for homodimerization or heterodimerization. The ratio between BAX/BCL2 heterodimers and BAX/BAX homodimers appears to be pivotal in deciding the life of death of a cell. We recently detected mutations in evolutionary highly conserved domains of the bax gene in cell lines derived from hematologic malignancies. Similar artificially generated mutations in other bcl2-like family members bcl2, bclxl, or ced9 have been shown to alter their function. This suggests a role for bax mutations in the multi-step pathogenesis of hematological malignancies.

Residual normal, highly proliferative progenitors can be isolated from the CD34+/33- fraction of AML with a more differentiated phenotype (CD33+).

Since in AML differentiation is abnormal but not absent, a hierarchy of stem cells, progenitor cells and more differentiated cells is postulated. The leukemic stem cell might also be characterized by the expression of CD34 and the absence of differentiation markers. Bone marrow samples of 33 AML patients, including 10 patients both at presentation and after relapse, were double labeled for CD34 and CD33. In 14/33 AML less than 1% of the labeled cells were found in the CD34+/33- fraction. After relapse a certain shift towards a more primitive phenotype was observed, but in 4/5 relapsed AML the CD34+/33- fraction remained below 1%. Single cells from the different subfractions were cultured and showed heterogeneous cluster and colony growth in both the CD34-/33+ and CD34+/33+ fraction. More colonies were observed in the CD34+/33- fraction. In AML with a more 'mature' phenotype (low number of CD34+/CD33- cells), highly proliferative myeloid, erythroid and mixed colonies could be cloned exclusively from this small CD34+/33- fraction. In five patients with numerical chromosomal abnormalities all these highly proliferative colonies appeared disomic using in situ hybridization (ISH) with centromeric probes. Based on these data we conclude that the CD34+/33- cell fraction in AML with a more mature immunophenotype (small fraction of cells CD34+/33-) comprise residual normal progenitors, while no primitive leukemic progenitors could be identified.

Donor leukocyte infusions for chronic myeloid leukemia relapsed after allogeneic bone marrow transplantation.

PURPOSE: Treatment options for patients with chronic myeloid leukemia (CML) who relapse after allogeneic bone marrow transplantation (BMT) are limited. Treatment with lymphocytes from the original marrow donor and the influence on the malignant clone was studied in these patients. PATIENTS AND METHODS: Seven patients with CML that had relapsed after BMT with T-cell-depleted grafts were treated. Six patients received leukocyte infusions from the original marrow donor. One patient received a second BMT with unseparated marrow from the same sibling donor. Chimerism was studied using erythrocyte and cytogenetic markers. Residual leukemic cells were monitored by cytogenetic analysis of the Philadelphia (Ph) chromosome and by polymerase chain reaction (PCR) of the breakpoint cluster region/Abelson (BCR-ABL) fusion gene. RESULTS: In five patients with hematologic relapse, the Ph chromosome disappeared 1 to 3 months after the leukocyte infusions. Cytogenetic analysis and in situ hybridization (ISH) showed only donor cells during further follow-up. Four to five patients became negative for the BCR-ABL translocation by PCR. Graft-versus-host disease (GVHD) always preceded response and was severe in two patients. One patient with cytogenetic relapse showed no response after leukocyte infusions. GVHD after second BMT was of moderate severity. One year after second BMT, PCR for the BCR-ABL translocation was negative. CONCLUSION: Infusion of donor leukocytes is an effective treatment with a low mortality in patients with CML relapsed after BMT with a T-cell-depleted graft. Longer follow-up and more patients will be needed to know whether cure will be permanent.

Karyotyping urine sediment cells confirms trisomy 12 mosaicism detected at amniocentesis.

A newborn is described in whom trisomy 12 mosaicism was detected prenatally at third trimester amniocentesis during the fourth pregnancy of a 34-year-old woman. After birth, trisomy 12 cells were found in placental tissue and in cultured urine sediment cells. A sample of cord blood and a skin biopsy revealed only normal (46,XX) cells. Both parents had a normal karyotype. After a difficult start with unexplained hypoglycaemias and convulsion equivalents, the girl is doing well at the age of 9 months: there are no signs of central motor disturbance. The importance of the use of cultured urine sediment cells in confirming chromosomal mosaicism is stressed.