Kaplan-Meier (time-to-event) plots of the preoviposition period and the incubation periods, and boxplots representing egg hatching rate, and the number of larvae from engorged <i>Ixodes pacificus</i> females.

<p>2A) A Kaplan-Meier plot where each colored line corresponds to the fraction of adult female ticks that had not started laying eggs on a particular day after the ticks were injected with antibiotics or water (control). Vertical dashes on the colored lines represent censorship, where a tick that had not started to lay eggs died and was dropped from the study. The tetracycline group (red) took significantly longer to start laying eggs compared to the rest (<i>P</i><0.05) 2B) A Kaplan-Meier plot where the colored lines represent the proportion of egg samples laid by individual ticks whose eggs had not finished hatching into larvae a certain number of days after oviposition began. 2C) A boxplot showing the hatching rate, or the fraction of tick eggs that successfully hatched into larvae, in the four treatment groups. Each individually colored box represents the hatching rate distribution of eggs laid by each female tick in the group. 2D) A boxplot specifying the distribution of the total number of larvae each female yielded. No significant difference in (C) hatching rate or (D) the number of larvae was present between treatment groups (<i>P</i><0.05). Circles outside the boxes in (C) and (D) represent outliers. The colors in all the plots in the figure correspond to specific treatment groups: blue - ampicillin, yellow - ciprofloxacin, red - tetracycline, green - injection with water (control).</p

Prevalence of infection with <i>Rickettsia</i> species phylotype G021 in <i>Ixodes pacificus</i> egg cohorts of 10 from each of the 14 ticks in the ciprofloxacin-injected group.

<p>Prevalence of infection with <i>Rickettsia</i> species phylotype G021 in <i>Ixodes pacificus</i> egg cohorts of 10 from each of the 14 ticks in the ciprofloxacin-injected group.</p

<i>In vivo</i> tumor targeting evaluation.

<p>(A) T₂WI and T₂-mapping images of mice bearing Bel-7402 subcutaneous tumors before and after tail vein administration of RGD-PEG-<i>g</i>-PEI-SPION or PEG-<i>g</i>-PEI-SPION. (B) The normalized MR signal intensity of the tumors before and after tail vein administration of the various complexes. (C) T₂ values of the tumors before and after tail vein administration of the various complexes. (means±SD; n = 5).</p

T2-mapping image of the Bel-7402 cells.

<p>(A) T₂-mapping image of the Bel-7402 cells incubated with PEG-<i>g</i>-PEI-SPION or RGD-PEG-<i>g</i>-PEI-SPION at different Fe concentrations. (B) T₂ values of the cells incubated with various complexes at different Fe concentration. (means±SD; n = 3).</p

The apoptotic response to different treatments in Bel-7402 cells.

<p>(A) Images show Bel-7402 cells incubated with: RGD-PEG-<i>g</i>-PEI-SPION/siRNA, PEG-<i>g</i>-PEI-SPION/siRNA, RGD-PEG-<i>g</i>-PEI-SPION/siNC or PEG-<i>g</i>-PEI-SPION/siNC (×200; scale bar:100 µm) (B) Percentage of apoptotic cells induced by various complexes at N/P ratio of 10 as quantified by TUNEL analysis (means±SD; n = 3; *<i>p</i><0.05, compared with RGD-PEG-<i>g</i>-PEI-SPION/siRNA; Control: the cells without transfection).</p

Tumor growth inhibition analysis.

<p>The tumor growth inhibition analysis in nude mice bearing Bel-7402 tumors after injection of various complexes formed at a N/P ratio of 10. (means±SD; n = 10; *<i>p</i><0.05, compared with RGD-PEG-<i>g</i>-PEI-SPION/siRNA; Control: the mice injected with PBS).</p

<i>In vitro</i> cell transfection efficiency analysis and cell uptake analysis.

<p>(A) The percentage of FITC-positive cells incubated with RGD-PEG-<i>g</i>-PEI-SPION/siRNA, PEG-<i>g</i>-PEI-SPION/siRNA or RGD-PEG-<i>g</i>-PEI-SPION/siRNA in the presence of free RGD at various N/P ratios. (means±SD, n = 3; *<i>p</i><0.05, compared with RGD-PEG-<i>g</i>-PEI-SPION/siRNA at the same N/P ratio). (B) The laser confocal microscope images of Bel-7402 cells transfected with RGD-PEG-<i>g</i>-PEI-SPION/siRNA, PEG-<i>g</i>-PEI-SPION/siRNA or RGD-PEG-<i>g</i>-PEI-SPION/siRNA in the presence of free RGD at a N/P ratio of 10.(×630; scale bar:10 µm).</p

Simultaneous Diagnosis and Gene Therapy of Immuno-Rejection in Rat Allogeneic Heart Transplantation Model Using a T‑Cell-Targeted Theranostic Nanosystem

As the final life-saving treatment option for patients with terminal organ failure, organ transplantation is far from an ideal solution. The concomitant allograft rejection, which is hardly detectable especially in the early acute rejection (AR) period characterized by an intense cellular and humoral attack on donor tissue, greatly affects the graft survival and results in rapid graft loss. Based on a magnetic resonance imaging (MRI)-visible and T-cell-targeted multifunctional polymeric nanocarrier developed in our lab, effective co-delivery of pDNA and superparamagnetic iron oxide nanoparticles into primary T cells expressing CD3 molecular biomarker was confirmed <i>in vitro</i>. In the heart transplanted rat model, this multifunctional nanocarrier showed not only a high efficiency in detecting post-transplantation acute rejection but also a great ability to mediate gene transfection in T cells. Upon intravenous injection of this MRI-visible polyplex of nanocarrier and pDNA, T-cell gathering was detected at the endocardium of the transplanted heart as linear strongly hypointense areas on the MRI <i>T</i>₂*-weighted images on the third day after cardiac transplantation. Systematic histological and molecular biology studies demonstrated that the immune response in heart transplanted rats was significantly suppressed upon gene therapy using the polyplex bearing the DGKα gene. More excitingly, the therapeutic efficacy was readily monitored by noninvasive MRI during the treatment process. Our results revealed the great potential of the multifunctional nanocarrier as a highly effective imaging tool for real-time and noninvasive monitoring and a powerful nanomedicine platform for gene therapy of AR with high efficiency

L-Wnt3a stimulates the survival, proliferation, and engraftment of bone marrow cells.

<p>(A) Aged BMT were treated with L-PBS or (B) L-Wnt3a with an effective concentration = 150 ng/ml, then transplanted into a skeletal defect and after 12 h, analyzed for DNA fragmentation associated with cell apoptosis using TUNEL. (C) Quantification of caspase activity in aged BM treated with L-PBS (grey bar; N = 4) or L-Wnt3a (blue bar; N = 4). (D) Quantification of Ki67 immunostaining for aged BM treated with (E) L-PBS or (F) L-Wnt3a, then transplanted into a skeletal defect and after 12 h analyzed for cell proliferation. (G) FACS analyses of L-PBS treated BMT, harvested from the defect site on post-transplant day 5. (H) FACS analyses of L-Wnt3a treated BMT, harvested from the defect site on post-transplant day 5. (I) GFP immunostaining identifies the L-PBS treated BMT on post-transplant day 7 (J) GFP immunostaining identifies the L-Wnt3a treated BMT on post-transplant day 7. (K) GFP immunostaining shown in higher magnification demonstrating L-PBS treated BMT on post-transplant day 7 (L) GFP immunostaining shown in higher magnification demonstrating L-Wnt3a treated BMT on post-transplant day 7 (M) Histomorphometric quantification of GFP immunopositive cells in the defect site on post-transplant day 7 (N = 5 for each condition). Abbreviations: GFP: green fluorescent protein; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; Scale bars: A,B: 50 µm E,F: 100 µm; I,J: 200 µm; K,L: 50 µm.</p

Wnt3a associates with the liposomal surface.

<p>(A) The rate of Wnt activity partitioning into the liposomal pellet is shown; initially, all Wnt activity (blue lines) is found in the supernatant but within 30 min, the majority of Wnt activity is associated with the liposomal pellet and by 6 h, 90% of Wnt activity is found in the liposomal pellet. (B) Immunoblot analysis using Wnt3a antibody shows that similar to the Wnt activity, initially the majority of the Wnt is found in the supernatant (supnt); within 30 min majority of the majority of Wnt3a segregates into the liposomal pellet and by 6 h 90% of the protein is associated with the liposomal pellet. (C) Following ultra-centrifugation, the majority of the protein is found in the liposomal pellet. (D) Wnt activity (blue bar) is also found in the pellet. Although some Wnt3a is found in the aqueous supernatant, it is inactive (figure C). (E) Sucrose density gradient centrifugation and phospholipid quantification assay (orange line), demonstrate that PBS liposomes migrate to higher density fractions. (F) Sucrose density gradient centrifugation, phosphatidyl choline lipid quantification and Wnt reporter assay, demonstrate that Wnt activity (blue line), the lipids (orange line), co-fractionate on a sucrose density gradient. (G) Immunoblotting analyses using Wnt3a antibody show that Wnt co-migrates with fractions showing maximum Wnt activity and lipid concentration. (H) The stability of L-Wnt3a is measured. At 4°C, L-Wnt3a retains >80% of its activity after extended storage. (I) An anti-Wnt3a immunoblot reveals no evidence of degradation products of L-Wnt3a after extended storage. Data are mean ±SEM from, or are representative of, at least three independent replicates.</p