OVERALL HYPOTHESIS
The authors’ overall hypothesis states that a fatal myeloproliferative disorder, MPD is evoked in all hematopoietic cells following expression of Ptpn11D61Y. The research study provides a mouse model illustrating MDP evoked by Ptpn11D61Y. Results indicate that the disorder is caused by lineage-specific and cell-autonomous effects that mutant Ptpn11 has on several hematopoiesis stages.
A rare and lethal MPD common in the early childhood stage is known as Juvenile myelomonocytic leukemia, JMML. Some of the characteristics associated with this disorder include discriminatory hyper-reactivity of progenitors hematopoiesis to granulocyte-macrophage colony stimulating factor, macrocytic anemia accompanies with fatal hemoglobinemia as well as hepatosplenomegaly. About seventy five to eighty five percent of all cases of JMML result from either function gain mutations in KRAS, NRAS or PTPN11 or function loss mutations in a gene called NF1 whose function is encoding of an activating protein for Ras GTPase. All the mentioned genes are essential components of a signaling cascade referred to as RAS/ERK. This cascade in combination with pharmacologic and biochemical analyses function in deregulation of the signaling cascade during pathogenesis of JMML. The most probable cause of JMML is somatic mutations in PTPN11, whose role is encoding the tryrosine phosphatase, which is non-receptive in nature. The abstract of the research paper, fails to cover all the research findings obtained from different models. However, there is an adequate development of the overall conceptual framework, methods, design and analyses. The title of the paper clearly describes the study, which entails to examine the causes of fatal MPD via cell-autonomous effect that it has on multiple hematopoiesis stages.
There were seven figures employed in the research study. The hypothesis for the first figure states that mice that express Ptpn11D61Y develop a fatal myeloproliferative disorder, MPD. The method used was introducing a mutation of D61Y into the Ptpn11 locus of the mouse as well as a transcriptional STOP cassette that was loxP-bracketed. There was also injection of properly targeted ES Clones into blastocytes to obtain a germ-line transmission of inducible Ptpn11D61Y. The results obtained indicated that there were no viable Ptpn11D61Yexpressing progeny when there was crossing between LSL- Ptpn11D61Y mice and EIIS-Cre transgenic mice which at early stages of embryogenesis, express mice. There was complete deletion of the STOP cassette in common myeloid progenitors, LSK Cells, granulocyte-macrophage progenitors and megakaryocytic-erythroid progenitors from mice that were induced with Mx1-Cre; LSL- Ptpn11D61Y. Though at the initial stages Ptpn11D61Y mice seemed healthy, they all died prematurely. The median survival was forty five weeks following injection. The overall is that MPD developed in Ptpn11D61Y mice, which clearly supports the hypothesis states earlier.
The hypothesis for the second figure is the MPD is evoked by the histopathology of Ptpn11D61Y. According to the results obtained in from this figure, the bone marrow of diseased mice illustrated increased amount of predominantly granulocytic cells that were immature. Extramedullary hematopoiesis was exhibited in mutant genes accompanied by disruption of the normal architecture of the spleen as a result of mature myeloid cells accumulation into the red pulp. According to flow cytometry an increase by nine to ten folds was revealed in reference to the ratio of Mac+ and Gr+ cells. The Ter 119+ erythroid progenitors in mutant spleens, increased by six to seven fold. Another result that was observed was the presence of periportal cuffing of liver sinusoids with infiltrating granulocytes in all diseased animals that were examined. The final result was that no skin tumors, lung adenomas and any other obvious neoplasm were detected in Ptpn11D61Ymice, contrary to knocking mice which had induced expression of oncogenic Kras G12D. In conclusion, Ptpn11D61Y mice revealed a marked rise in number of mature myeloid elements contained in the bone marrow, peripheral blood and the spleen. This clearly supports the hypothesis that MPD is evoked by the histopathology of Ptpn11D61Y.
The third figure illustrates the abnormal distribution of myeloid precursors and phenotypic HSCs in the spleen and bone marrow of diseased Ptpn11D61Y mice. The hypothesis states that expression of Ptpn11D61Y changes progenitor compartments and the phenotypic HSC. The method entailed examination of splenic and bone marrow hematopoietic progenitor cells in Ptpn11D61Ymice by utilizing a combination of cell surface markers that are used to differentiate between short term HSCs and long-term HSCs from common lymphoid progenitors, multipotent progenitors and GMPs. There was a decreased total bone marrow cellularity as well as diminished LSK and LK compartments in mice with myeloproliferative disorder. The number of CD34–Flk2– cells within the LSK compartment was lower in mutant mice that with control littermates. Additionally, there were a lower proportion of CD150+cd48– LSK cells in mutant bone marrow. Normal bone marrow had LT-HSCs that were highly pure. An indication of the mentioned data is the loss of LT-HSCs in bone marrow mutant of Ptpn11D61Y. Therefore the figure clearly indicates an abnormal distribution of both myeloid precursors and phenotypic HSCs in the spleen and bone marrow of diseased Ptpn11D61Ymice.
The title of the fourth figure is adoptive transfer of spleen and bone marrow cells from mice with Ptpn11D61Y. The main question that the experiment sought to answer was if adoptive transfer of spleen or bone marrow cells from mice with Ptpn11D61Y could confer myeloproliferative disorder to the recipients. Spleen cells and bone marrow cells from Ptpn11D61Ymice were moved in combination with CD45 WT bone marrow cells into CD45 that were lethally irradiated. Flow cytometry was used to quantify the percentage of peripheral blood cells that expressed CD45. After a period of 24 weeks, representative spleen cells from a recipient following transplantation showed the presence of B220+, Gr+and CD3+/CD4+/CD8+ that were derived from the donor.
Some recipients contacted T-ALL/lymphoma-like syndrome which is characterized by splenomegaly and increased number of white blood cells. Other mice succumbed to death most likely as a result of a similar syndrome. One limitation is that it was not possible to recover tissue and cells in order to carry out a detailed analysis. There is no single time that recipients show symptoms of MPD or progressive monocytosis/granulocytosis. Moreover, there was no persistent multilineage donor cells-derived reconstitution. Both spleen and bone marrow cells expressed RNA components of Ptpn11D61Y . From the results obtained, it is clear that expression of Ptpn11D61Y results to compromising of the number of bone marrow stem cells as well as their function.
The hypothesis for the fifth figure is that CICs are evoked by committed progenitors from Ptpn11D61Y. GMPs, CMPs, LSK Cells and MEPs from Ptpn11D61Y mice were purified in order to determine if phenotypic HSCs and/or committed progenitors lead to CICs. These mice as well as control littermates were purified by sorting that was fluorescence-activated and placed in media of methylcellulose without growth factors. Virtually all GMPs and to a lesser degree, CMPs resulted to CICs. The use of purified progenitors obtained from preleukemic animals in which expression of Ptpn11D61Y had been induced shortly produced similar results. The overall finding was that the differentiation ability of GMPs and CMPs was not changed by Ptpn11D61Y expression.
The hypothesis for figure six is that progenitors of Ptpn11D61Y show reduced CFU-E activity and enhanced BFU-E activity. Though showing marked increase in Ter 119+ progenitors of the spleen, Ptpn11D61Y mice developed anemia. Despite the fact that anemia could partly be caused by hypersplenism, a question that arises is if Ptpn11D61Y mice had perturbed erythroid differentiation. The researchers plated bone marrow and MEPs from both diseased and controlled mice in methylcellulose media that contained CFU-E, EPO and BFU-E colonies and observations made after two or eight days. Results indicated that there was consistent production of decreased numbers of CFU-E colonies by MEPs while the BFU-colonies generated were larger and greater in number.
Figure seven is titled, signaling aberrations in Ptpn11D61Y cells. There is a possibility of mutant expression having unique, cell-type dependent outcomes on cytokine or growth factor signaling. A robust protocol for analysis using flow cytometry indicated that Erk and Akt activation were evoked by SCF and this was significantly enhanced in LK and LSK cells derived from Ptpn11D61Y mice. One limitation of this experiment was lack of available antibodies, which prevented assessment of expression of GM-CSFR alpha. The overall indication of the data obtained was that mutant GMPs, LSK cells and CMPs Showed unique cell-intrinsic changes in the responsiveness of cytokine and growth factor.
Generally, the figures were correctly labeled and the experiments done, precisely supported their specific hypotheses. According to the general findings, distinct and multiple effects on various hematopoietic cell components are associated with leukemogenic Ptpn11, and this is thought to cause MPD. It is evident that Ptpn11D61Y mice have significantly high numbers of LSK cells. A Higher number of such cells are witnessed in the spleen rather than in the bone marrow. There is depletion of LSK Cells in the bone marrow of diseased mice. Despite the fact that the size of LSK Compartment is big, there is decreased activity of stem cell in Ptpn11D61Ymice. Transplantation of Ptpn11D61Y bone marrow cells do not result to persistent multilineage reconstitution.
There are many probable explanations to justify failure of transplanting Ptpn11D61Y-evoked myeloproliferative disorder. The first explanation is that there could be rare cases of MPD-initiating stem cell, implying the necessity to have another heat besides the expression of Ptpn11D61Y. Another probable reason is the possibility of MPD Being evoked solely via Ptpn11D61Y expression in a significantly small subpopulation of HSC.
Despite the fact that increased size of LSK compartment plays a fundamental role in formation of MPD evoked by Ptpn11D61Y a mere increase in the number of LSK is not adequate enough to lead to MPD following an intact homeostatic control. Further research is required in order to verify if it is possible for competitor cells that are co-transplanted with diseased cells to compete for the niche of stem cells and inhibit development of MPD.
Reference
Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2676094/?tool=pmcentrez
On May 2, 2011
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