The evolution of organic material on Asteroid 162173 Ryugu and its delivery to Earth.

The recent return of samples from asteroid 162173 Ryugu provides a first insight into early Solar System prebiotic evolution from known planetary bodies. Ryugu's samples are CI chondrite-like, rich in water and organic material, and primarily composed of phyllosilicate. This phyllosilicate surrounds micron to submicron macromolecular organic particles known as insoluble organic matter. Using advanced microscopy techniques on Hayabusa-2 samples, we find that aqueous alteration on Ryugu produced organic particles richer in aromatics compared to less altered carbonaceous chondrites. This challenges the view that aromatic-rich organic matter formed pre-accretion. Additionally, widespread diffuse organic material occurs in phyllosilicate more aliphatic-, carboxylic-rich, and aromatic-poor than the discrete organic particles, likely preserving the soluble organic material. Some organic particles evolved to encapsulate phyllosilicate, indicating that aqueous alteration on Ryugu led to the containment of soluble organic matter within these particles. Earth therefore has been, and continues to be, delivered micron-sized polymeric organic objects containing biologically relevant molecules.

Familial hypocalciuric hypercalcemia in an index male: grey zones of the differential diagnosis from primary hyperparathyroidism in a 13-year clinical follow up.

Familial hypocalciuric hypercalcemia (FHH) type 1, caused by a heterozygous inactivating mutation of the gene encoding the calcium-sensing receptor (CaSR), is characterized by mild to moderate hypercalcemia, hypocalciuria and inappropriately normal or elevated parathyroid hormone (PTH). FHH must be differentiated from primary hyperparathyroidism (PHPT) because parathyroidectomy is ineffective in the former. Herein, we report a 39-year-old male patient with a 13-year history of asymptomatic PTH-dependent hypercalcemia (mean calcium of 2.88 mmol/l; reference range 2.15-2.55 mmol/l) and calcium-to-creatinine clearance ratio (Ca/Cr) ranging from 0.007 to 0.0198, which is consistent with either FHH or PHPT. Although a family history of hypercalcemia was negative, and PET-CT with fluorocholine was suggestive of a parathyroid adenoma, genetic analysis of the CaSR gene identified a heterozygous inactivating mutation NM_000388.4:c.1670G>A p. (Gly557Glu) in exon 6 and a polymorphism NM_000388.4:c.1192G>A p. (Asp398Asn) in exon 4. The G557E mutation has been previously reported in a Japanese family in which all family members with the mutation had Ca/Cr below 0.01 consistent with FHH. The biochemical profile of FHH and PHPT may overlap. Our FHH patient with a G557E CaSR mutation illustrates that the differential diagnosis can be difficult in an index case with no family history, (false) positive parathyroid imaging and higher calciuria than expected for FHH. Calcium intake, vitamin D status and bone resorption might have contributed to the Ca/Cr variations over a 13-year clinical follow up. This case thus emphasizes the irreplaceable role of genetic testing of the CaSR gene when clinical evaluation is inconclusive.

[Plasma cell separation algorithm from bone marrow samples]. / Algoritmus separace plazmatických bunek ze vzorku kostní drene.

BACKGROUNDS: The aim of this paper is to present an algorithm for plasma cell separation from bone marrow samples of multiple myeloma patients. The main prerequisite for applying modern research methods in this disease is gaining pure cell populations. MATERIAL AND METHODS: Bone marrow samples were collected from outpatients or inpatients of the Internal Haematology and Oncology Clinic of the Faculty Hospital Brno, after they had signed an Informed Consent Form. The bone marrow was first depleted of red cells (by density gradient centrifugation or erythrolysis), plasma cells were labelled by monoclonal antibody against syndecan-1 (CD138) and separated either magnetically or by cell sorter. The purity of separated population was evaluated by flow cytometry or, alternatively, morfologically. RESULTS: We processed 28 bone marrow samples, in parallel, by magnetic or fluorescence-based separation, and we evaluated the purity of the separated fractions. Based on a statistical evaluation of resulting purities in the entire sample set as well as the individual groups divided according to the initial plasma cell content, a separation algorithm was proposed with a cut-off value of 5% of plasma cells in mononuclear fraction of bone marrow: samples with less than 5% of plasma cells are henceforth separated on cell sorter, samples with more than 5% are separated magnetically.The effectiveness of this algorithm was evaluated after the first year of its application on a dataset of 210 bone marrow samples: median purity of the separated plasma cells increased from 62.4% (0.4-99.6%) to 94.0% (23.9-100%). CONCLUSION: The introduction of a fluorescence-based separation markedly increased the effectiveness of plasma cell separation from bone marrow samples, mainly in samples with low plasma cell content where magnetic separation used thus far is not sufficient. This finding also opened a door for plasma cell separation of bone marrow samples from patients with monoclonal gammopathy of undetermined significance, where plasma cell count is typically below or just over one percent.

Dasatinib in imatinib-resistant or -intolerant CML patients: data from the clinical practice of 6 hematological centers in the Czech Republic.

Dasatinib is effective second line treatment for patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib. We report here the first experiences with dasatinib therapy in 71 CML patients resistant or intolerant to imatinib from the real clinical practice of 6 hematological centers in the Czech Republic. Dose 100 mg daily and 70 mg twice daily was administered to patients with chronic phase (CP) and advanced phases (AP) CML. In chronic phase (n=46), complete hematological reponse (CHR) was achieved in 97%, major cytogenetic reponse (MCgR) in 77% and complete cytogenetic response (CCgR) in 67%. Major molecular reponse (MMR) was achieved in 19/31 patients in median of 10 months. In advanced phase (n=25), CHR was attained in 77%, MCgR in 39%, CCgR in 33% and MMR in 2/18 patients. Eleven different baseline mutations were followed up in 15 patients. Dasatinib eliminated mutations in most of the patients, but 3 patients acquired a new one. Novel mutations were detected under dasatinib therapy in 2 patients. Dasatinib was well tolerated, cytopenias were common and was managed by dose modification. The estimated progression free survival (PFS) at 12 months was 97+/-3% in CP and 62+/-21% in AP. The median time to treatment failure was 605 days in AP while it was not reached in CP patients. Our clinical experiences, described here, confirmed that dasatinib is associated with high response rates especially in imatinib resistant or intolerant CML patients in chronic phase.

[The preparation of anticancer vaccine for patients with multiple myeloma on the base of monoclonal immunoglobulin loaded dendritic cells]. / Príprava protinádorové vakcíny pro pacienty s mnohocetným myelomem na bázi dendritických bunek nalozených monoklonálním imunoglobulinem.

BACKGROUND: On June 2006, phase II clinical trial focused on anticancer vaccination of multiple myeloma patients, was started. On September 2007, the immune and clinical response evaluation of first four patients was finished.The anticancer vaccine contained dendritic cells loaded with monoclonal immunoglobulin produced by myeloma cells. METHODS AND PATIENTS: Within the frame of phase II clinical trial were vaccinated four myeloma patients with stable disease. It was administered six vaccines for each patient, monthly. The dendritic cells were cultured from the patient's peripheral blood mononuclear cells and loaded with autologous monoclonal immunoglobulin under the good manufacturing practice conditions. After the safety and quality control, the satisfactory vaccine was administered to the patient. The functional characteristic of dendritic cells was evaluated using flow cytometry, the immune response was evaluated using ELISpot. The clinical response was monitored using monoclonal immunoglobulin concentration in patient's sera. RESULTS AND CONCLUSION: The immune response detected using ELISpot was observed in 3/4 patients. The monoclonal immunoglobulin concentration was changeable for all twelve months, but never exceeded the range of 25% for minimal clinical response achievement. During the vaccination, no significant toxicities or negative side-effects were observed. The clinical trial is going on with vaccination other patients with multiple myeloma.

Prognosis of pediatric patients transplanted for Ph+ chronic myeloid leukemia in the period from 1989 to 2006 in the Czech Republic.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by clonal proliferation of primitive hematopoietic stem cell. The median age at diagnosis is 55 to 60 years with less than 10% of patients younger 20 years. Incidence of CML in children in the Czech Republic is 0.106 cases/100 thousands per year. Here we report outcome of 38 pediatric patients (median age 12.5 years; range 1.8 - 17.3) with Ph-positive CML diagnosed between years 1989 to 2006. Primarily chronic phase of the disease was diagnosed in 32 (84%) patients. 32 (84.2%) patients underwent hematopoietic stem cell transplantation (HSCT) with the median age at transplantation of 14.9 years (range 6.9 - 20.5 years). Out of transplanted patients 16 (50%) obtained graft from unrelated donor, 13 (41%) from matched sibling donor, 2 from haploidentical family donor and autologous transplantation has been performed in one case. 6 patients were not transplanted, 4 of them died (median 1.2 years from diagnosis), 2 are alive 0.6 and 17.8 years from the diagnosis. Overall survival (OS) in 25 patients after HSCT at our department during the whole period is 66.7% with 15/16 being in stable continuous molecular-genetic remission (94%). During the period of time results of transplantations have been significantly improved (p=0.0071). OS after HSCT until year 1997 is 25% while from year 1998 until now is 87.5%. All centers OS of patients after HSCT is 71%. Results of HSCT in children with CML obtained from the year 1998 at our center are fully comparable with results achieved in large and experienced centers. HSCT remains the only proven and effective method for the treatment of CML. Clinical studies assessing the role of tyrosine kinase inhibitors in children instead of early HSCT should be planned carefully in order to avoid sub-optimal outcomes.

Supercritical fluid extraction (SFE) of 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI) from ground-coffee with high-performance liquid chromatographic-electrospray mass spectrometric quantification (HPLC/ESI-MS).

Two polar analytes, 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI), were extracted with supercritical carbon dioxide (CO2) modified with aqueous methanol. The method was applied to a roasted coffee powder with good recovery rates. Method efficiency was compared with that of solid-phase extraction using SCX Disc cartridges and validated for spiked solid matrix. The analytes were determined using isocratic liquid chromatography-mass spectrometry (LC/MS) on an Atlantis HILIC Silica column (150 x 2.1 mm, 3 microm) with 80% methanol and 20% 0.01 mol l-1 ammonium formate as the mobile phase. The limit of quantification was around 1.5 pg for 4-MeI and 2.0 pg for THI. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of >0.999. The coefficient of variation for the intra-day and inter-day precisions was <4% (n = 6). Accuracy was in the range 98-101%; recovery rates were > or = 98 and > or = 99% for THI and 4-MeI, respectively. Several samples of Arabica coffee from various locations and commercially available 'off-the-shelf' coffee products (Arabica/Robusta mixtures) were analysed to test the method.

Disease status in patients with chronic myeloid leukemia is better characterized by BCR-ABL/BCR transcript ratio than by BCR-ABL transcript level, which may suggest a role of normal BCR gene in the disease pathogenesis.

Monitoring of BCR-ABL transcript level is widely used in chronic myeloid leukemia (CML) to follow up response to therapy. In this study we compare abilities of two quantitative RT-PCR assays to characterize the disease status in CML patients: RT-PCR quantifying the BCR-ABL transcript concentration and RT-PCR determining the BCR-ABL/BCR transcript ratio (R). We demonstrate that in non-responders only R, but not BCR-ABL, unambiguously characterizes the state of disease. Moreover, R values >1 found in all poor responders indicate lower BCR expression compared to BCR- ABL in these patients. Our results demonstrate the importance of BCR-ABL/BCR transcript ratio for the disease status and the disease prognosis characterization and suggest a possible role of the normal BCR gene expression in CML pathogenesis.

[Ten years of molecular monitoring of chronic myeloid leukemia by quantitative RT-PCR]. / Deset let molekulárního monitorování chronické myeloidní leukémie metodou kvantitativní RT-PCR.

Chronic myeloid leukemia (CML) is characterized by the presence of BCR-ABL fusion gene resulting from the reciprocal chromosome translocation t(9;22)(q34;q 11), karyotypically detected as Ph chromosome. BCR-ABL gene was proved to play the principal role in CML pathogenesis. It is a hallmark of CML used in diagnostics and monitoring of the response to the therapy. The most sensitive method of detecting BCR-ABL aberration is RT-PCR which is able to find a single in leukemic cell between 10(6) normal leukocytes. Monitoring of BCR-ABL transcript level by quantitative RT-PCR is of the high prognostic value. High or increasing BCR-ABL transcript number signalizes bad response to treatment and a bad prognosis. On the contrary RT-PCR negativity, low level, or decreasing BCR-ABL transcript number denotes good response to treatment and good prognosis. Q-RT-PCR can detect changes in disease status several weeks or even months earlier than other methods. In 1994 the Q-RT-PCR was introduced at the Institute of Hematology and Blood Transfusion in Prague and was used for early detection of relapse after transplantation. At present it is used in all patients with CML for monitoring of response to the treatment. We have confirmed that this precise, sensitive and non-invasive method is of the principal importance for monitoring of disease status in CML patients.

[Imatinib mesylate (Glivec) in treatment of chronic phase chronic myeloid leukemia]. / Imatinib mesylát (Glivec) v lécbe chronické fáze chronické myeloidní leukémie.

The new knowledge in molecular biology and pathophysiology of chronic myeloid leukemia enabled the development of imatinib mesylate (Glivec, formerly STI571). Imatinib potently inhibits several protein tyrosine kinases, including BCR-ABL, c-Kit, and PDGF receptor. Imatinib blocks the phosphorylation of downstream target proteins and interrupts the malignant transformation leading to the development of CML. Phase I and II studies demonstrated that imatinib is highly effective and well tolerated in all phase of CML. We got our experience with imatinib on more than two-year monitoring 34 patients within the Expanded Access Study CST1571 0113. Imatinib 400 mg/d was administered orally to 10 women and 24 men in median age of 53 years (22-70) who were hematologically (n = 9) or cytogenetically (n = 13) resistant, cytogenetically refractory (n = 3) or intolerant (n = 9) to interferon alpha. The median follow-up time was 97.5 weeks (23-115), the median time from CML diagnosis to the start of the study was 32.3 months (6-140.5). Complete hematologic response was achieved in 33 of 34 (97%) pts, total major cytogenetic response (complete plus major) in 21 of 33 (63%) pts. Cytogenetic relapse was observed in 2 of 33 pts (6%), cytogenetic progression in 4 (12%) pts. Non-hematologic toxicity was mild (grade 1 or 2) and no patient was excluded from the study due to it. Hematological toxicity grade 3 limited dose of imatinib in 26% of patients and probably caused lower rate of cytogenetic responses in heavy pretreated patients. Both quantitative RT-PCR methods (competitive RT-PCR and real-time RT-PCR Light-Cycler) were found useful to monitor patients with CML on imatinib therapy. Our results confirmed high efficacy and safety of imatinib in late-chronic phase CML patients failing prior interferon therapy. The lower incidence of hematological toxicity and higher rate of cytogenetic responses in patients treated with imatinib in early-chronic phase CML justify according to our opinion the recommendation to administer imatinib early after the diagnosis of CML in patients who are not indicated for allogeneic transplantation.

Location of the BCR/ABL fusion genes on both chromosomes 9q34 in Ph negative chronic myeloid leukemia.

We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.

Transdermal absorption of phytoestrogens.

The transdermal absorption of the isoflavones, daidzein and genistein, applied on the skin in olive oil was studied in vivo. The concentrations of the isoflavones and their metabolites were monitored in plasma and urine by GC-MS methods. It was found that the concentration of genistein in plasma was 3-fold higher than the plasma concentration of daidzein. In contrast, daidzein excretion was 2-3-fold higher than that of genistein in urine. The excretion rate of the studied phytoestrogens in urine and their concentration in plasma was significantly decreased after repeated transdermal application. The urinary recovery of administered daidzein and genistein after the first application was 15.9% and 7.7%, respectively and this dropped to 1.6% and 0.7% after the second application. The results obtained might suggest that daidzein and genistein are captured in the skin following repeated transdermal application.

Prediction and reversion of post-transplant relapse in patients with chronic myeloid leukemia using mixed chimerism and residual disease detection and adoptive immunotherapy.

In the prospective study, we examined hematopoietic mixed chimerism (using polymerase chain reaction (PCR) of variable number of tandem repeat-VNTR sequences) and minimal residual disease (MRD) status (using qualitative and in the case of positivity quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the BCR/ABL fusion mRNA) in serial peripheral blood samples taken from 25 patients after bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Increasing mixed chimerism in correlation with increasing signal of MRD was detected in 10 patients. In two patients mixed chimera status and BCR/ABL rearrangement led to hematologic relapse, in five patients molecular relapse was followed by reappearance of Ph chromosome and three patients developed molecular relapse only. Adoptive immunotherapy-donor lymphocyte infusion (DLI), interferon (INF) and discontinuation of post-transplant immunosupression-separately or in different combinations was used in nine patients with molecular, cytogenetic or hematologic relapse of CML. The results demonstrate that significant response at the molecular level can be achieved for a majority of CML patients and that using of all forms of adoptive immunotherapy controlled by MC and MRD is more efficient in patients treated in early molecular relapse-with minimal disease burdens.

[Interferon alpha--the drug of choice for patients with chronic myeloid leukemia]. / Interferon alfa--lék volby pro nemocné s chronickou myeloidní leukémií.

BACKGROUND: The conventional, or standard, treatment of chronic myeloid leukaemia (CML) with hydroxyurea and busulfan has no marked influence on its course or duration. Interferon (IFN) alpha administration has, on the other hand, been shown to induce not only a haematological but also cytogenetic response, i.e. partial or complete bone marrow repopulation by Ph-negative cells. This has triggered studies comparing IFN and conventional chemotherapy. The present work had the purpose to gain experience with IFN alpha treatment and compare the results with hydroxyurea and busulfan treatment, in the chronic phase of CML. METHODS AND RESULTS: Therapeutic results obtained in 30 patients given IFN alpha and 30 others given conventional chemotherapy were evaluated retrospectively. In spite of the short time of IFN administration (4-27 months), significantly more complete haematological responses (83%) were observed in this than the conventional chemotherapy group (57%). Cytogenetic responses were achieved in 36%, complete cytogenetic remission in 20% of IFN-treated patients. Conventional chemotherapy produced no cytogenetic effect. CONCLUSION: The results obtained confirm the value and efficacy of IFN-alpha treatment in CML patients, especially if it is started early and the dose is effective. Regular cytogenetic monitoring is necessary. Longer follow-up of the patients will be necessary for evaluation of the IFN effect on the length of their survival.

Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation.

Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.