Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

<p>Abstract</p> <p>Purpose</p> <p>In a departure from conventional strategies to improve treatment outcome for myeloid malignancies, we report the isolation of leukemia-specific peptides using a phage display library screened with freshly obtained human myeloid leukemia cells.</p> <p>Results</p> <p>A phage display library was screened by 5 rounds of biopanning with freshly isolated human AML cells. Individual colonies were randomly picked and after purification, biologic activity (growth and differentiation) on fresh AML cells was profiled. Ten peptides were synthesized for further biological studies. Multiple peptides were found to selectively bind to acute myeloid leukemia (AML) cells. The peptides bound to leukemia cells, were internalized and could induce proliferation and/or differentiation in the target patient cells. Two of the peptides, HP-A2 and HP-G7, appeared to have a novel mechanism of inducing differentiation since they did not cause G1 arrest in cycling cells even as the expression of the differentiation marker CD11b increased.</p> <p>Conclusion</p> <p>Peptide induced differentiation of leukemia cells offers a novel treatment strategy for myeloid malignancies, whereas their ability to induce proliferation could be harnessed to make cells more sensitive to chemotherapy. Conceptually, these leukemia specific peptides can also be used to refine diagnosis, document minimal residual disease, and selectively deliver toxins to malignant cells.</p

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)-0

by streptavidin-FITC. For this specimen, HP-A2 was detected in AML but not normal cells while HP-C8 was detected in both populations.<p><b>Copyright information:</b></p><p>Taken from "Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)"</p><p>http://www.jhoonline.org/content/1/1/8</p><p>Journal of Hematology and Oncology 2008;1():8-8.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2494998.</p><p></p

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)-3

Days, cell morphology was evaluated. The percentage of each cell population was determined by counting 200 cells.<p><b>Copyright information:</b></p><p>Taken from "Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)"</p><p>http://www.jhoonline.org/content/1/1/8</p><p>Journal of Hematology and Oncology 2008;1():8-8.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2494998.</p><p></p

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)-2

Number of colonies was assessed.<p><b>Copyright information:</b></p><p>Taken from "Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)"</p><p>http://www.jhoonline.org/content/1/1/8</p><p>Journal of Hematology and Oncology 2008;1():8-8.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2494998.</p><p></p

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)-4

Lture. The numbers of colonies and cell morphology were evaluated for each specimen. The probability of the AML specimen to respond to the peptide (increase of colony number, decrease of colony number and cell differentiation) was assessed. The numbers on the bars indicate the number of patients evaluated.<p><b>Copyright information:</b></p><p>Taken from "Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)"</p><p>http://www.jhoonline.org/content/1/1/8</p><p>Journal of Hematology and Oncology 2008;1():8-8.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2494998.</p><p></p

Association of Posterior Reversible Encephalopathy Syndrome and Transient Apical Ballooning Syndrome (Takotsubo): First Case Report of a Man and Review of the Literature

Introduction: An association of posterior reversible encephalopathy syndrome (PRES) and takotsubo is rare. We present the first case of a male patient. Case Report: A 69-year-old man presented to the hospital in a persistent comatose state following a generalized tonic-clonic seizure with high blood pressure. The electrocardiogram revealed transient left bundle branch block. Troponin and BNP were elevated. Cardiac ultrasound showed large apical akinesia with altered left ventricular ejection fraction, and the left ventriculogram showed characteristic regional wall motion abnormalities involving the mid and apical segments. Brain MRI showed bilateral, cortical, and subcortical vasogenic edema predominant in the posterior right hemisphere. The lumbar puncture and cerebral angiography were normal. Paraclinical abnormalities were reversible within 2 weeks with a clinical recovery in 3 months, confirming the takotsubo and the PRES diagnoses. Discussion: Several theories hypothesize the underlying pathophysiology of takotsubo or PRES. Circulating catecholamines are up to 3 times higher in patients with takotsubo causing impaired microcirculation and apical hypokinesia. An association of both takotsubo and asthma crisis and PRES and asthma crisis underlines the role of catecholamines in the occurrence of these disorders. Conclusion: Early recognition of this rare association, in which heart and neurological damage may require rapid intensive care support, is needed

Vitamin D induces interleukin-1β expression: paracrine macrophage epithelial signaling controls M. tuberculosis infection.

Although vitamin D deficiency is a common feature among patients presenting with active tuberculosis, the full scope of vitamin D action during Mycobacterium tuberculosis (Mtb) infection is poorly understood. As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response. Transcriptome profiling also revealed that the profile of target genes regulated by 1,25D is substantially altered by infection, and that 1,25D generally boosts infection-stimulated cytokine/chemokine responses. We further focused on the role of 1,25D- and infection-induced interleukin 1β (IL-1β) expression in response to infection. 1,25D enhanced IL-1β expression via a direct transcriptional mechanism. Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome. The impact of IL-1β production was investigated in a novel model wherein infected macrophages were co-cultured with primary human small airway epithelial cells. Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells. These effects were independent of 1,25D-stimulated autophagy in macrophages but dependent upon epithelial IL1R1 signaling and IL-1β-driven epithelial production of the antimicrobial peptide DEFB4/HBD2. These data provide evidence that the anti-microbial actions of vitamin D extend beyond the macrophage by modulating paracrine signaling, reinforcing its role in innate immune regulation in humans

Control of <i>Mtb</i> infection by co-cultured epithelial cells is dependent on epithelial IL1R1 and DEFB4 expression.

<p>(<b>A</b>) CFU of macrophages (Mφ) infected with H37Rv at an MOI of 5 for 4 hours and treated with vehicle control or 100 nM 1,25D (+D), with and without the additional presence of SAECs in transwell co-culture (CC). Data are from three experimental replicates (mean and SD) and representative of three independent experiments using different donors of primary cells. Statistical significance was determined by one-way ANOVA. (*P<0.05). (<b>B</b>) Validation of siRNA-mediated knockdown of <i>IL1R1</i> expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siIL1R1. Data are from three experimental replicates (mean and SD), **P<0.01 as determined by student's T-test relative to respective siCTL controls. (<b>C</b>) CFU quantification of <i>Mtb</i> in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to <i>IL1R1</i>. 1,25D (D) was added as indicated. Data are from three experimental replicates (mean and SD) and representative of two independent experiments. Statistical significance was determined by one-way ANOVA. (**P<0.01). (<b>D</b>) Validation of siRNA-mediated knockdown of <i>DEFB4</i> expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siDEFB4. Data are from three experimental replicates (mean and SD) and representative to two independent replicates. **P<0.01 as determined by student's T-test relative to siCTL control. (<b>E</b>) CFU quantification of <i>Mtb</i> in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to <i>IL1R1</i> or <i>DEFB4</i>. Data are from three experimental replicates (mean and SD) and representative of two independent experiments using separate donors of SAECs. Statistical significance was determined by one-way ANOVA. (*P<0.05).</p

1,25D alters the host macrophage transcriptomic response to <i>Mtb</i> infection.

<p>(<b>A</b>) Intensity heat map of genes regulated 5-fold or more during infection in the absence or presence of 1,25D. THP-1 cells were either not infected (NI) or infected with H37Rv (I) and treated with vehicle (DMSO) or 100 nM 1,25D (+D) for 24 hours. Each vertical line represents one gene that was either up-regulated (red), down-regulated (blue) or not affected (white) under each of the conditions relative to uninfected cells not treated with 1,25D, as indicated in the scale. Group 1 represents those genes that were only detected in the NI+D condition, group 2 were those genes that were commonly expressed in both infected conditions, and group 3 represents those genes that were expressed in only one of the infected conditions as a result of 1,25D treatment. (<b>B</b>) Functional clustering heat map of genes selected for a >5-fold change in either the I or I+D condition relative to the NI control as well as having a >1.5-fold difference between the two. Increasing brightness for red and blue denote up- and down-regulation respectively. (<b>C</b>) Highest-rated functions associated with relative expression of I+D/I in genes from <b>B</b>, with a Fisher's exact test p-value threshold set at 0.01 (red line) using Ingenuity Pathways Analysis (IPA) software. (<b>D</b>) Network clustering analysis of genes from <b>B</b> using IPA software. Solid and hashed lines denote known direct and indirect actions between two proteins as determined by IPA. Red and blue denote relative up- or down-regulation of their expression in the I+D condition as compared to the I condition, respectively. Data is derived from analysis of Affymetrix Human Gene 1.0ST microarray chips. mRNA samples for analysis were prepared in triplicate, and data presented is representative of two independent replicates.</p