Enhanced electrochemical sensing of leukemia cells using drug/lipid co-immobilized on the conducting polymer layer.

Electrochemical biosensors using five anticancer drug and lipid molecules attached on the conducting polymer layer to obtain the orientation of drug molecules toward cancer cells, were evaluated as sensing materials and their performances were compared. Conjugation of the drug molecules with a lipid, phosphatidylcholine (PC) has enhanced the sensitivity towards leukemia cells and differentiates cancer cells from normal cells. The composition of each layer of sensor probe was confirmed by electrochemical and surface characterization experiments. Both impedance spectroscopy and voltammetry show the enhanced interaction of leukemia cells using the drug/lipid modified sensor probe. As the number of leukemia cells increased, the charge transfer resistance (Rct) in impedance spectra increased and the amine oxidation peak current of drug molecules in voltammograms decreased at around 0.7-1.0V. Of test drug molecules, raltitrexed (Rtx) showed the best performance for the cancer cells detection. Cancer and normal cell lines from different origins were examined to evaluate the degree of expression of folate receptors (FR) on cells surface, where cervical HeLa cell line was found to be shown the highest expression of the receptor. Impedance and chronoamperometric experiments for leukemia cell line (Jurkat E6-1) showed linear dynamic ranges of 1.0×10(3)-2.5×10(5) cells/mL and 1.0×10(3)-8.0×10(3) cells/mL with detection limits of 68±5 cells/mL and 21±3 cells/mL, respectively.

Establishment of a bioluminescent imaging-based in vivo leukemia model by intra-bone marrow injection.

In vivo leukemia mouse models are usually generated by intraperitoneal (IP) or intravenous (IV) injection of leukemia cells. However, the pattern of leukemia development observed can be inconsistent. This study investigated injection directly into bone marrow [intra-bone marrow transplantation (IBMT)], the natural microenvironment of leukemia. A bioluminescent imaging-based leukemia animal model has been established by direct injection of a bioluminescent leukemia cells (CCRF-CEM/fLuc) into NOD/SCID mouse tibia bone marrow and compared with models established by IP and IV routes. The comparison revealed that a bioluminescent in vivo leukemia model established via IBMT could recapitulate leukemia more faithfully and facilitate improved quantification of leukemia engraftment kinetics with a wider range of bioluminescent intensity than IP or IV. IBMT of bioluminescent leukemic cells allowed quantification of dose-dependent responses to anti-leukemic drugs, thus validating this model as a potential preclinical anti-leukemic drug screening system. IBMT-leukemia cells isolated from peripheral blood of the model mice and then injected into new recipients successfully established a second generation IBMT in vivo model and demonstrated the reproducibility of the model. Bioluminescent imaging-based analysis of this IBMT-leukemia model could provide a means for the comprehensive evaluation of treatment responses with enhanced sensitivity in preclinical studies.

Leukemia diagnosis in murine bone marrow transplantation models.

The mouse is the most commonly used experimental animal, and a wide range of tumor types can arise in their hematopoietic system. Therefore, for research scientists and graduate students working in the field of experimental hematology, immunology, and cancer research, there is an urgent need for well-established protocols for the preparation of histology and cytology for leukemia diagnosis. Moreover, the criteria for the classification of hematopoietic neoplasms often vary between different laboratories. In this chapter, we describe diagnosis and analysis of leukemia in murine bone marrow transplantation models based primarily on the findings of the histology and cytology of hematopoietic and infiltrated tissues, peripheral blood smear, and immunophenotyping by FACS analysis.

Murine acute promyelocytic leukemia cells can be recognized and cleared in vivo by adaptive immune mechanisms.

BACKGROUND AND OBJECTIVES: In this study, we tested whether transgenic murine acute promyelocytic leukemia (APL) cells can be recognized and cleared by adaptive immune responses and/or vaccination strategies. DESIGN AND METHODS: Immunocompetent and SCID mice were examined for their ability to survive a challenge of APL cells. We also vaccinated immunocompetent mice with DNA vaccines encoding various portions of a bcr-1 PML-RARa fusion protein. RESULTS: In genetically compatible, immunocompetent animals, APL cells routinely engrafted and caused lethal leukemia; however, immunodeficient SCID mice required approximately 100-fold fewer APL cells to cause lethal disease. Massive doses of APL cells were efficiently eliminated in allogeneic recipients. Vaccination with a plasmid expressing a human PML-RARa cDNA conferred protection against leukemic cells in vivo; mice vaccinated with the human PML portion of the fusion gene demonstrated similar protection. Analysis of 10-mer peptides spanning the t(15;17) translocation-associated PML-RARa fusion breakpoint suggested that they were not involved in the generation of immune responses. INTERPRETATION AND CONCLUSIONS: These data show that tumor-specific immune clearance of APL cells does occur in mice. In this model system, the relevant immunogenic antigens may arise from the xenogenic PML portion of human PML-RARa, and not unique sequences derived from the breakpoint region. However, the study proves that APL cells are capable of being recognized and killed in vivo by adaptive immune responses, suggesting that therapeutic vaccines should be possible for this disease when relevant tumor-specific antigens are identified.

Triple basepair changes within and adjacent to the conserved YY1 motif upstream of the U3 enhancer repeats of SL3-3 murine leukemia virus cause a small but significant shortening of latency of T-lymphoma induction.

A highly conserved sequence upstream of the transcriptional enhancer in the U3 of murine leukemia viruses (MLVs) was reported to mediate negative regulation of their expression. In transient expression studies, negative regulation was reported to be conferred by coexpression of the transcription factor YY1, which binds to a motif in the upstream conserved region (UCR). To address the function of the UCR and its YY1-motif in an in vivo model of MLV-host interactions we introduced six consecutive triple basepair mutations into this region of the potent T-lymphomagenic SL3-3 MLV. We report that all mutants have retained their replication competence and that they all, like the SL3-3 wild type (wt), induce T-cell lymphomas when injected into newborn mice of the SWR strain. However, all mutants induced disease with slightly shorter latency periods than the wt SL3-3, suggesting that the YY1 motif as well as its immediate context in the UCR have a negative effect on the pathogenicity of the virus. This result may have implications for the design of retroviral vectors.

[Distribution of minimal residual leukemia in Brown Norway rats express LacZ gene].

OBJECTIVE: To investigate the distribution of minimal residual leukemia (MRL) in vivo. METHODS: The MRL model in BN rat was established by using LT12nl leukemia cell line carrying LacZ gene. The marker gene (LacZ) was detected by using reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR, X-gal staining, and cytomorphological and pathological examination. RESULTS: PCR assays showed positive result in humerus at day 4, femur at day 6, and spleen at day 9 after CTX treatment. The peak positive signal in humerus was stronger at day 9. X-gal staining showed negative in spleen at day 9. PCR showed negative results in peripheral blood and liver. CONCLUSION: PCR can be used as an effective tool to investigate the distribution pattern in experimental MRL.

Distinct in vivo engraftment and growth patterns of t(1;19)+/E2A-PBX1+ and t(9;22)+/BCR-ABL+ human leukemia cells in SCID mice.

The SCID mouse represents a valuable tool for assessing growth characteristics and drug sensitivity of human leukemic cells. We have examined differences in the engraftment patterns in SCID mice of primary human leukemic cells isolated from children (< 21 years old) with either t(1;19)+/E2A-PBX1+ or t(9;22)+/BCR-ABL+ acute lymphoblastic leukemia. Leukemic cells from 13/24 t(1;19)+/E2A-PBX1+ patients caused overt leukemia in SCID mice. Macroscopic lesions were evident in 6/13 cases, with multiple sites involved in some mice: hepatomegaly,(3) splenomegaly(4), thymic enlargement; liver tumors(1), kidney tumors(1), abdominal tumors(1). Microscopic lesions in SCID mouse organs were present in all 13 cases and involved the bone marrow, brain, heart, gut, liver, kidney, lung, ovary, pancreas, skeletal muscle, spleen, and thymus. Leukemic cells from 5/20 t(9;22)+/BCR-ABL+ patients caused overt leukemia in SCID mice. Notably, macroscopic lesions (splenomegaly; leukemic bones; hepatic tumors) were observed in only 1 case. In all 5 cases, microscopic lesions were found in the mouse bone marrow. Additional microscopic lesions were restricted to skeletal muscle, spleen, and mesentery (1 case) or thymus (1 case). These findings differ markedly from those of t(1;19)+/E2A-PBX1+ leukemic cells due to the lack of involvement of major organs such as liver, pancreas, kidney, skin, or brain. These data illustrate the biological heterogeneity of childhood ALL and suggest that the differential risks associated with t(1;19)+/E2A-PBX1+ and t(9;22)+/BCR-ABL ALL might arise from unique engraftment and proliferation capabilities of the respective leukemic cell populations.

[The nucleolar apparatus of the lymphoid cells in AKR mice at different stages of leukemogenesis]. / Nukleoliarnyi apparat limfoidnykh kletok myshei linii AKR na razlichnykh étapakh leikozogeneza.

Morphological features of nuclear apparatus in mice leukemic cells of the peripheral blood, lymph nodes, thymus, bone marrow and spleen were studied. These cells were taken from 12-months-old AKR mice. Enlarged percentage of cells with nucleolar lipid component in thymus characterizes preleukemic state for 6-months-old AKR line mice.

Course of murine leukemia retrovirus infection determined in vivo by magnetic resonance imaging.

Magnetic resonance imaging was applied to measure the volumes of spleens and lymph nodes of mice infected with three different leukemia retroviruses (LP-BM5 murine leukemia virus, Friend, and Rauscher) in vivo. Anesthesia by rapid intraperitoneal injection of Saffan was sufficient for magnetic resonance imaging and could be repeated at appropriate intervals. Eleven frontal magnetic resonance images through the abdomen with a center-to-center distance of 1.5 mm between adjacent slices were acquired simultaneously. To optimally demarcate spleens from the surrounding tissues, the magnetic resonance images were mildly T2-weighted for mice infected with LP-BM5 murine leukemia virus and mildly T1-weighted for those infected with Friend and Rauscher virus. Measurements requiring only 3 to 4 hours in groups of 24 to 28 mice were accomplished by using a standardized holder (i) accommodating two animals in the supine position and (ii) ensuring reproducible positioning in the magnetic resonance-instrument, and (iii) by reducing the number of phase-encoding steps of mildly T2-weighted magnetic resonance images from 256 to 128. Volumes of spleens and inguinal lymph nodes were calculated from the respective cross-sectional areas. The weights and magnetic resonance image-derived volumes of spleens and inguinal lymph nodes correlated well (r greater than 0.95). Despite large variations in the extent of splenomegaly and lymphadenopathy at any given time, the progression of the disease could easily be followed by repeating magnetic resonance imaging at intervals. Thus, statistically relevant results can be obtained in an infection model requiring the use of only a few animals.

Regression models for time to seroconversion following experimental bovine leukaemia virus infection.

This paper develops a parametric model for time to seroconversion after experimental bovine leukaemia virus (BLV) infection, and examines the effects of inoculation route, volume of inoculum, type of inoculation material, and antigen status of donor on seroconversion time. We used parametric and nonparametric statistical methodology to analyse interval data on 150 animals from 13 published reports. The log-logistic model fitted the observed times to seroconversion better than the log-normal or Weibull models, which were the considered alternatives.

Two murine monoclonal antibodies to peripheral blood monocyte differentiation antigens discriminate within M5 acute non-lymphoid leukemia (ANLL) cells.

Two murine monoclonal antibodies (MoAbs), LAM3 and LAM7 of the IgG1 isotype, which were produced by immunization with normal peripheral blood monocytes (PBM), were assayed in their specificity by indirect immunofluorescence against a panel of normal as well as leukemic cells. Both LAM3 and LAM7 were reactive with PBM while LAM3 also recognized platelets. Neither MoAb showed reactivity with erythrocytes, granulocytes, or resting and mitogen activated B and T lymphocytes. The reactivity with bone marrow cells correlated with the degree of monocyte contamination. Among the 62 cases of leukemia tested, which included three cases of B-CLL, 19 cases of ALL, and 40 cases of ANLL, both MoAbs reacted highly homogenously only with M5b ANLL cells. These findings indicate that the two MoAbs, which recognize two distinct epitopes, represent useful markers in the differential diagnosis of M5b ANLL.

Location of T-cell leukaemia cells in a model rat system by means of a fluorescent probe.

Fluorescence probes for the active centre of an enzyme associated with tumour cells have been used to locate leukaemia cells in a model rat system. These fluorescent techniques are inexpensive and rapid to carry out. The leukaemic cells can be located by fluorescence microscopy in frozen sections, wax embedded sections and resin embedded sections. The technique is illustrated with reference to sections of leukaemic rat kidney, epididymis and testis. These studies confirm earlier histological findings employing conventional staining techniques and have the advantage that individual leukaemia cells can be detected in leukaemic animals undergoing drug therapy. The evidence suggests that these techniques will be of value in further studies of the design of drugs directed to leukaemia cells.

[Mast cell-affinity lymphocytes as indicator cells of the forms of hemoblastoses]. / Mastotsitaffinnye limfotsity kak indikatornye kletki nekotorykh form gemoblastozov.

The formation of mastolymphocyte rosettes was used to determine that in chronic lymphocytic leukemia and bovine leukemia, unlike healthy people and animals as well as patients with solid tumours of different localization and Hodgkin's disease, a large amount of cells appear among peripheral blood lymphocytes having membrane affinity for mast cells. This may be used as an additional criterion in laboratory diagnosis of these diseases.

Detection of minimal residual disease in acute leukemia by flow cytometry.

Commercial flow cytometers can detect and enumerate rare cells at the level of 1 per 10(5) other cells within a reasonable measuring time, provided that the rare cells can be uniquely labeled with fluorescent marker. This detection level is sufficient for the enumeration of normal hematopoietic stem cells and committed progenitor cells. Detection at this level is useful for the quantitation of residual leukemic cells in remission bone marrow, for the analysis of the proliferative state of these cells as well as of normal stem cells, which are of importance in choosing the optimal chemotherapy regimen, and for monitoring the efficacy of maintenance chemotherapy. A further improvement in the speed of flow cytometers would be required, however, to make full use of the bone marrow samples.

[Demonstration of the expression of bovine leukemogenic virus (BLV) in sheep lymphocytes by an immunofluorescence technic using monoclonal antibodies]. / Mise en évidence de l'expression du virus leucémogène bovin (BLV) dans les lymphocytes de mouton par une technique d'immunofluorescence utilisant des anticorps monoclonaux.

An indirect immunofluorescence (IF) test was developed to detect bovine leukemia virus (BLV) antigen expression in infected sheep lymphocytes, using monoclonal antibodies anti BLV-major envelope glycoprotein gp51. Peripheral blood lymphocytes were cultivated for 48 h in presence of phytohemagglutinin (PHA) (50 micrograms/ml), and then fixed with acetone. The cells were assayed for the IF test. All experimentally infected sheep were positive with this test.