11 research outputs found
Factors associated with incident syphilis.
<p><u>Legend:</u> n (%): number (percentage) of participants; HR: hazard ratio; IQR: interquartile range; 95% CI: 95% confidence intervals; HIV: Human immunodeficiency virus, ART: antiretroviral therapy; CMV: cytomegalovirus; EBV: Epstein-Barr virus; HSV-1 and -2: Herpes simplex virus type 1 and type 2; HHV-6/-7/-8: human herpesvirus type 6/type 7/type 8. In <b>bold:</b> significant <i>p</i>-values (<i>p</i><0.1).</p><p>Factors associated with incident syphilis.</p
Genital MCP-1 levels, CMV shedding and syphilis acquisition.
<p>CMV shedding (panel A) and syphilis acquisition (panel B) were both associated with LOWER seminal MCP-1 levels (Mann Whitney test).</p
Demographics and co-infections at baseline.
<p><u>Legend</u>: n (%): number (percentage) of participants; MSM: men who have sex with men; HIV: Human immunodeficiency virus; ART: antiretroviral therapy; IQR: interquartile range; HHV: Human Herpesviruses; CMV: cytomegalovirus; EBV: Epstein-Barr virus; HSV-1 and -2: Herpes simplex virus type 1 and type 2; HHV-6/-7/-8: human herpesvirus type 6/type 7/type 8.</p><p>Demographics and co-infections at baseline.</p
Leukemia regenerative capacity of the CD45<sup>+</sup>CD34<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup> population.
<p>(A) Representative photographs of hematopoietic organs following intrahepatic transplantation of 1 500 FACS purified CD34<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup>Lin<sup>−</sup> cells from a NOTCH1<sup>Mutated</sup> T-ALL (Patient 05) sample demonstrates serial transplantation potential of this refined LIC population, as shown by the presence of an enlarged thymus, spleen and pale marrows over several transplantation generations. (B, C) FACS analysis of the tertiary (3°) transplant recipients of 30000 CD34<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup>Lin<sup>−</sup> cells sorted from a NOTCH1<sup>Mutated</sup> T-ALL (Patient 11) revealed the persistence of an expanded human CD45<sup>+</sup>CD34<sup>+</sup>CD2<sup>+</sup> (including CD7<sup>+</sup> and CD7<sup>−</sup>) population in the transplanted mouse hematopoietic organs (bone marrow, spleen, thymus and liver).</p
Patient characteristics.
<p>Diagnostic blast immunophenotypic analysis (in bulk patient samples) and DNA sequencing (in the CD34<sup>+</sup> cell population) were performed for 12 pediatric T-ALL patient samples. Patient age range was 2–23 and mean age was 11.3 years. Diagnostic samples included patients 1–11 while sample 12 was donated by a patient with relapsed T-ALL. Diagnostic blast immunophenotypes were not available (N/A) for 2 of 12 patients. Treatment was performed according to protocol COG 05-01 for patients 1 to 7; protocol COG 9404 for patients 8 to 10; the Larson protocol for patient 11, and patient 12 received COG-ALL043 treatment. Targeted exon sequencing performed on T-ALL CD34<sup>+</sup> LIC revealed NOTCH1 HD or PEST domain mutations in 6 of 12 T-ALL (patients 01, 03, 05, 08, 11, 12) samples. PTEN mutations and/or deletions were detected in 4 of 12 samples (patients 01, 05, 06, 11). Patient 5 also had a PIK3R1 mutation. <sup>#</sup> Indicates <i>NOTCH1<sup>Mutated</sup></i> patient samples, and * indicates NOTCH1 activation in the absence of NOTCH1 mutation (<i>NOTCH1<sup>High</sup></i>) by qRT-PCR.</p
<i>NOTCH1<sup>Mutated</sup></i> LIC serially transplant T-ALL.
<p>(A) Schema of T-ALL LIC mouse model. Equivalent numbers of human CD34<sup>+</sup> and CD34<sup>−</sup> cells derived from both NOTCH1 Mutated (NOTCH1<sup>Mutated</sup> and NOTCH1 wild-type (NOTCH1<sup>WT</sup>) samples, as defined by DNA sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039725#pone-0039725-t001" target="_blank">Table 1</a>), were selected using immunomagnetic beads from T-ALL blood or bone marrow, transduced with lentiviral luciferase and transplanted (50000 cells/mouse) intrahepatically into RAG2<sup>−/−</sup>γ<sub>c</sub><sup>−/−</sup> mice within 48 hours of birth. Engraftment was monitored over 10 weeks via non-invasive bioluminescent imaging system (IVIS 200, Caliper LifeSciences). After 10 weeks, mice were sacrificed and secondary transplants were performed with 50000 immunomagnetically-purified human CD34<sup>+</sup> or CD45<sup>+</sup> cells from primary CD34<sup>+</sup> and CD34<sup>−</sup> T-ALL engrafted mouse marrow, respectively. (B) T-ALL CD34<sup>+</sup> cells from 12 of 12 T-ALL samples engrafted leukemia to varying extents in the marrow, spleen and thymus in all primary but only 10 of 11 serial transplant recipients. Representative bioluminescent images (captured on an IVIS 200 system) demonstrating engraftment of CD34<sup>+</sup> (50000) NOTCH1<sup>Mutated</sup> T-ALL (1B, upper) compared with an equivalent number of CD34<sup>−</sup> cells (1B, lower). (C) Quantitative bioluminescent imaging (photons/second/cm<sup>2</sup>/sr) of CD34<sup>+</sup> cell and CD34<sup>−</sup> cell engraftment over 10 weeks following transplantation of NOTCH1<sup>Mutated</sup> T-ALL samples (red, n = 4 patients) compared with NOTCH1<sup>WT</sup> samples (blue, n = 4 patients). Mice (n = 76) transplanted with lentiviral luciferase-transduced NOTCH1<sup>WT</sup> patient samples (Patients 04, 07, 09, 10) and mice (n = 79) transplanted with NOTCH1<sup>Mutated</sup> patient samples (Patients 03, 05, 08, 11) were included in the bioluminescent imaging study (error bars, mean ± SEM. **, P = 0.0005, Student’s t-test). (D) Photographs depicting marrow, thymic and splenic size following NOTCH1<sup>Mutated</sup> T-ALL CD34<sup>+</sup> cell (upper panel) and CD34<sup>−</sup> cell (middle panel) transplants compared with no transplant control mice (lower panel). (E) Representative FACS analysis demonstrating human CD45<sup>+</sup> and CD34<sup>+</sup> leukemic engraftment in the marrow, spleen and thymus in primary transplant recipients of NOTCH1<sup>Mutated</sup> T-ALL Pt 05 CD34<sup>+</sup> cells (CD34<sup>+</sup>1° transplant, upper panel) and CD34<sup>−</sup> cells (CD34<sup>−</sup> 1° transplant, middle panel) compared with no transplant control mice (lower panel). (F) Representative FACS analysis demonstrating human CD34<sup>+</sup>CD45<sup>+</sup> serial leukemic engraftment in the marrow (upper), spleen (middle) and thymus (lower) following transplantation of secondary recipients (2° transplant) with 50000 CD34<sup>+</sup> cells isolated from NOTCH1<sup>Mutated</sup> T-ALL (Pt 05) primary recipients.</p
hN1 mAb treatment inhibits <i>NOTCH1<sup>Mutated</sup></i> LIC burden.
<p>(A) Comparative FACS analysis of human CD34<sup>+</sup>CD45<sup>+</sup> cells and CD34<sup>+</sup>CD2<sup>+</sup> leukemic burden in the bone marrows from NOTCH1<sup>Mutated</sup> LIC (Patient 11) engrafted mice following treatment with control mAb (left panel) or hN1 mAb (right panel). (B) FACS analysis of human CD34<sup>+</sup> and NOTCH1<sup>+</sup> cell survival in the mouse spleens following control mAb (n = 9) or hN1 mAb treatment (n = 9) of NOTCH1<sup>Mutated</sup> LIC (Patients 05, 08, 11) engrafted mice (upper panel). Representative FACS plots show the reduction in both CD34<sup>+</sup> and NOTCH1<sup>+</sup> cell populations. (C) Representative FACS analysis demonstrating engraftment of CD34<sup>+</sup>CD45<sup>+</sup> cells in the bone marrows of secondary (2°) transplant recipients following transplantation of control mAb (left panel) or hN1 mAb (right panel) treated bone marrow (Patient 11). (D) Graph of percent human T-ALL total CD45<sup>+</sup> (blue) and CD34<sup>+</sup>CD45<sup>+</sup> (red) cells in the bone marrows of 2° transplant recipients of control mAb (n = 6) and hN1 mAb (n = 6) treated NOTCH1-driven LIC (Patients 02, 11) (error bars ± SEM; P = 0.16, and P = 0.086, respectively, by Student’s t-test). All results reflect data collected from two independent experiments.</p
hN1 mAb treatment inhibits NOTCH1-driven LIC self-renewal.
<p>(A) Graph depicting average number of caspase 3 (red) and NOTCH1 (blue) positive cells (mean ± SEM), measured by marrow immunohistochemistry (cell number/randomized 40× fields) following hN1 versus control mAb treatment of T-ALL LIC (Patient 08) engrafted mice (**, P = 0.005, *, P = 0.046, respectively by two tailed Student’s t test with unequal variance). (B) Immunoperoxidase analysis of human NOTCH1, human CD45 and human Caspase 3 expression in no transplant control (left panel), compared with control mAb (middle panel) and hN1 mAb (right panel) treated NOTCH1<sup>Mutated</sup> LIC (Patient 08) engrafted mice (40× magnification). (C) Following hN1 mAb treatment, immunoperoxidase staining of marrow sections was used to compare NOTCH1 intracellular domain (ICN1) expression (Patient 08) in control mAb (left) and hN1mAb (right) treated mice (40× magnification). (D) Quantitative RT-PCR assessment of relative reduction in HPRT normalized NOTCH1 transcript levels in human CD34<sup>+</sup> cells derived from NOTCH1<sup>Mutated</sup> (Patients 05, 08, 11) T-ALL LIC engrafted bone marrows following hN1 mAb or control mAb treatment (**, P<0.01; *, P<0.05, Student’s t-test). All results reflect data collected from two independent experiments.</p
hN1 mAb treatment inhibits <i>NOTCH1<sup>Mutated</sup></i> T-ALL LIC burden.
<p>(A) Schema of T-ALL LIC mouse model treatment with a selective anti-NOTCH1-NRR (hN1) mAb. Within 10 to 12 weeks of NOTCH1<sup>Mutated</sup> T-ALL CD34<sup>+</sup> (50000) cell transplantation into post-natal day 2 RAG2<sup>−/−</sup>γ<sub>c</sub><sup>−/−</sup> mice, intraperitoneal treatment was instituted for 3 weeks with a NOTCH1 (hN1, 10 mg/kg every 4 days) mAb or control mAb for 6 doses. Mice were sacrificed within one day of completion of dosing followed by further studies. (B) The average percentage of CD34<sup>+</sup> engraftment was compared by FACS analysis of hematopoietic tissues (bone marrow, spleen and thymus) from mice that received transplants of T-ALL LIC from NOTCH1<sup>Mutated</sup> patient samples (03, 05, 08, 11; n = 29 mice) and NOTCH1<sup>WT</sup> patient samples (04, 06, 09, 10; n = 22 mice) (error bars ± SEM. ***, P<0.001, unequal variance Student’s t-test). (C) The average percentage of human CD34<sup>+</sup> T-ALL LIC burden in the bone marrow (red, Patient 05, n = 6; blue, Patient 08, n = 7; green, Patient 11, n = 6) in control mAb treated mice (n = 19) and hN1 mAb treated mice (red, Patient 05, n = 7; blue, Patient 08, n = 7; green, Patient 11, n = 7) (**, P = 0.003 by Wilcoxon test) was compared by FACS analysis. Response in the bone marrow of mice transplanted with LIC from patients 05, 08 and 11 varied (Patient 05, P = 0.8308; Patient 08, P = 0.007; Patient 11, P = 0.017, by Wilcoxon test). In addition to NOTCH1 activating mutations, patient 05 harbored both PTEN and PI3KR1 mutations and patient 11 had a PTEN frameshift mutation, while patient 08 was wild-type at these loci (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039725#pone-0039725-t001" target="_blank">Table 1</a>). (D) Average percentage of T-ALL CD34<sup>+</sup> LIC burden in the mouse spleens following control mAb treatment (n = 19) compared to hN1 mAb treatment (n = 21) (***, P<0.001, Wilcoxon test). (E) Average percentage of T-ALL CD34<sup>+</sup> LIC burden in the mouse thymus after control mAb treatment (n = 19) when compared to hN1 mAb treatment (n = 21) (no significant differences between the two groups). All results reflect data collected from two independent experiments.</p
An expanded CD45<sup>+</sup>CD34<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup> population in <i>NOTCH1<sup>Mutated</sup></i> T-ALL LIC is sensitive to hN1 mAb treatment.
<p>(A) Total human CD45<sup>+</sup> cells including CD34<sup>+</sup>CD45<sup>+</sup> and CD34<sup>-</sup>CD45<sup>+</sup> in secondary (2°) transplant recipients were summarized by graphing the results of CD45 FACS analysis. Human cord blood CD34<sup>+</sup> progenitors were used as a normal progenitor control, where the engraftment of human CD45<sup>+</sup> cells in bone marrow was 7.13% ±1.3 (n = 6). (B) FACS analysis of pediatric T-ALL engrafted bone marrows revealed an expanded human CD45<sup>+</sup>CD34<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup> population in secondary (2°) transplant recipients that was more prominent in NOTCH1<sup>Mutated</sup> T-ALL (Patient 05, n = 9; Patient 08, n = 8; Patient 11, n = 8; Patient 12, n = 3) and NOTCH1<sup>High</sup> T-ALL (Patient 02, n = 10) transplanted mice than NOTCH1<sup>WT</sup> T-ALL transplanted mice (Patient 09, n = 4; Patient 10, n = 3). Human cord blood CD34<sup>+</sup> progenitors were used as a normal progenitor control (n = 6). The CD34<sup>+</sup>CD45<sup>+</sup>, CD34<sup>+</sup>CD45<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup>, and CD34<sup>+</sup>CD45<sup>+</sup>CD2<sup>+</sup>CD7<sup>−</sup> populations were significantly higher in the bone marrows of both NOTCH1<sup>Mutated</sup> and NOTCH1<sup>High</sup> T-ALL LIC transplanted mice (**, P<0.01; ***, P<0.001, Student’s t test) when compared with NOTCH1<sup>WT</sup> T-ALL LIC transplanted mice. (C) FACS analysis of pediatric T-ALL LIC (Patient 05, n = 5 in control group, n = 6 in hN1 group; Patient 08, n = 5 in control group, n = 6 in hN1 group; Patient 11, n = 4 in control group, n = 5 in hN1 group) engrafted bone marrows showing a reduction in the human CD45<sup>+</sup>CD34<sup>+</sup>CD2<sup>+</sup>CD7<sup>+</sup> cell population following hN1 mAb treatment compared to control IgG1 mAb (***, P<0.001, Student’s t test).</p