ifn γ neutralization antibody  (Proteintech)

Journal: iScience

Article Title: Elimination of intra-hepatocytic malaria parasites is driven by non-canonical autophagy but not nitric oxide production

doi: 10.1016/j.isci.2025.112052

Figure Lengend Snippet: Inducible nitric oxide synthase (iNOS) is dispensable for elimination of liver stages by protective innate and adaptive immune responses (A) Schematic diagram of the experimental design. Mice were either mock-infected with the debris from uninfected mosquito salivary glands (Mock) or with 100,000 Py LARC GAP sporozoites on day zero (0). Three days later, mice were re-infected with 50,000 Py GFPluc sporozoites. Liver stage burden of the secondary infection was monitored by bioluminescent imaging (total flux) 42–44 h later. (B) Quantification of liver stage burden via bioluminescent imaging. Liver stage burden of the secondary infection is reduced by 2.5-logs in WT C57BL6/J (B6 WT) mice previously infected with Py LARC GAP. IFN-γ neutralization (αIFN-γ) at the time of the secondary infection completely reverses LARC GAP-induced innate immune elimination of the secondary LS infection. NOS2 −/− mice however exhibit 2.5-log reduction in the LS burden of the secondary infection, comparable to WT mice. (C) Schematic of Py LARC GAP immunization in WT C57BL6/J mice with an isotype control antibody (αIgG2a) and WT mice with IFN-γ neutralization (αIFNγ) and NOS2 −/− mice. All mice were immunized thrice with 50,000 Py LARC GAP sporozoites and then challenged with 10,000 Py GFP luc sporozoites 30 days after the last Py LARC GAP immunization. Liver stage burden was measured by bioluminescence imaging at 44 hpi. (D) Quantification of liver stage burden via bioluminescent imaging in LARC GAP immunized mice, 42–44 h after challenge. IFN-γ blocking impairs adaptive immune elimination of LS infection. NOS2 −/− mice however exhibit LS burden comparable to WT control mice. Bar graphs are expressed as mean ± SD. Asterisks indicate significant differences in total flux (bioluminescence) using a two-tailed Mann-Whitney U test (∗∗ p < 0.01,∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Each dot represents a single mouse. Data from panels B and D are compiled from at least two or more independent experiments with two or more mice per group for each experiment.

Article Snippet: 4-5 weeks after the third immunization, cohorts of WT mice were treated either with an isotype control antibody or an IFN-γ neutralizing antibody (clone XMG1.2, cat#: I-1119, Leinco Technologies) and then subsequently infected with 10,000 Py GFP-luc sporozoites intravenously one day later.

Techniques: Infection, Imaging, Neutralization, Control, Blocking Assay, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Elimination of intra-hepatocytic malaria parasites is driven by non-canonical autophagy but not nitric oxide production

doi: 10.1016/j.isci.2025.112052

Figure Lengend Snippet: IFN-γ-mediated LS elimination in vitro does not depend on inducible nitric oxide synthase (A) Live confocal microscopy images of HepG2-CD81 cells infected with Py UIS4-GFP parasites. Cells were not treated (NT) or treated with IFN-γ for 4 h starting at 24 hpi. Parasites were visualized by the GFP fluorescence of the UIS4-GFP fusion protein. Nuclei were labeled by using the DNA probe Hoechst 33342. Bar 500μm. (B) Quantification of LS infection rate. HepG2-CD81 cells were infected with Py UIS4-GFP parasites and not treated (NT) or treated at 12 hpi, 24 hpi, or 36 hpi with IFN-γ for 4 h. LS infection rates were quantified at 48 h by visual counting under the microscopy. Three independent experiments were performed. Three replicas were considered for each experiment. Statistical analysis was done by performing ANOVA with Bonferroni posttest (∗∗∗∗: p < 0.0001). Bar graphs expressed the mean ± SD. (C) HepG2-CD81 cells were treated and infected as Figure 2 C. The size of the parasites was calculated by determining the area of the LS in a confocal section as shown in Figure 2 A. Three independent experiments were performed, and 100 LS were considered for each experiment. Statistical analysis was done by performing ANOVA with a Bonferroni posttest (∗ and ∗∗: p < 0.01). Bar graphs are expressed as mean ± SD. (D) HepG2-CD81 cells infected with the Py UIS4-GFP parasites and not treated (NT) or treated with IFN-γ at 24 hpi and with/without the NOS2 inhibitor L-NAME. Liver stage numbers were determined by microscopy at 48hpi as in Figure 2 B. Three intendent experiments were performed. Every experiment consists of three replicas. Statistical analysis was done by performing ANOVA with Bonferroni posttest (∗∗∗: p < 0.001). Bar graphs are expressed as mean ± SD.

Article Snippet: 4-5 weeks after the third immunization, cohorts of WT mice were treated either with an isotype control antibody or an IFN-γ neutralizing antibody (clone XMG1.2, cat#: I-1119, Leinco Technologies) and then subsequently infected with 10,000 Py GFP-luc sporozoites intravenously one day later.

Techniques: In Vitro, Confocal Microscopy, Infection, Fluorescence, Labeling, Microscopy

Journal: iScience

Article Title: Elimination of intra-hepatocytic malaria parasites is driven by non-canonical autophagy but not nitric oxide production

doi: 10.1016/j.isci.2025.112052

Figure Lengend Snippet: Non-canonical autophagy induced by exogenous IFN-γ drives elimination of liver stages (A) Quantification of LS infection rates in gene-specific siRNA-treated HepG2-CD81 cells infected with Py UIS4-GFP parasite. Cells were transfected with a specific pool of siRNAs 24 h before infection and not treated (NT) or treated with INF-γ at 24 hpi for 4 h. LS infection rate was quantified by live microscopy at 48 hpi by visually counting the LS parasites. Three independent experiments were performed. Three replicas for each experiment were conducted. Statistical analysis was done by performing ANOVA with Bonferroni posttest (∗∗: p < 0.01). Bar graphs are expressed as mean ± SD. NST: Not specific target. (B) Quantification of LS size in siRNA treated HepG2-CD81 cells infected with Py UIS4-GFP parasites. Cells were treated as above. Quantification of LS size was determined as in Figure 2 C. Statistical analysis was done by performing ANOVA with Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed as mean ± SD. NST: Not specific target. (C) ATG16L1 localization was determined by confocal microscopy. HepG2-CD81 cells infected with Py UIS4-GFP parasite were not treated (NT) or treated with INF-γ at 24 hpi. Cells were fixed at 30 hpi and stained with a specific antibody for ATG16L1. Parasites were visualized by the GFP signal of the parasite UIS4-GFP fusion protein. Nuclei were labeled by using the DNA probe Hoechst 33258. Bar 100μm. (D) Quantification of PVM labeled with ATG16L1. Confocal images as in Figure 2 C were scored for the percentage of PVM labeled with ATG16L1. Three independent experiments were conducted. Each experiment consisted of >50 parasites. Statistical analysis was performed by using a nonparametric t-test. Bar graphs are expressed a mean ± SD. (E) Quantification of ATG16L1 localization at the PVM. Confocal images as shown in Figure 2 C were used to quantify the percentage of PVM perimeter labeled with ATG16L1. Three independent experiments were conducted. Each experiment consisted of >50 parasites. Statistical analysis was performed by using a nonparametric t-test. (F) Quantification of LS infection rates in ATG16L1-siRNA treated HepG2-CD81 cells infected with Py UIS4-GFP parasites. Method as described in Figure 2 A. Three independent experiments were performed. Each experiment consisted of three replicas. Statistical analysis was done by performing ANOVA with a Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed as mean ± SD. NST: Not specific target. (G) Quantification of LS size for ATG16L1-siRNA treated HepG2-CD81 cells infected with Py UIS4-GFP parasite. Methods and statistics as described in Figure 2 B.

Article Snippet: 4-5 weeks after the third immunization, cohorts of WT mice were treated either with an isotype control antibody or an IFN-γ neutralizing antibody (clone XMG1.2, cat#: I-1119, Leinco Technologies) and then subsequently infected with 10,000 Py GFP-luc sporozoites intravenously one day later.

Techniques: Infection, Transfection, Microscopy, Confocal Microscopy, Staining, Labeling

Journal: iScience

Article Title: Elimination of intra-hepatocytic malaria parasites is driven by non-canonical autophagy but not nitric oxide production

doi: 10.1016/j.isci.2025.112052

Figure Lengend Snippet: NOX2/gp91 phox is important for IFN-γ-mediated LS elimination (A) Quantification of LS infection rates in NOX2/gp91 phox -siRNA treated HepG2-CD81 cells infected with Py UIS4-GFP parasites. Method as described in Figure 2 A. Three independent experiments were performed. Each experiment consisted of three replicas. Statistical analysis was done by performing ANOVA with a Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed as mean ± SD. NST: Not specific target. (B) Quantification of LS size in NOX2/gp91 phox -siRNA treated HepG2-CD81 cells infected with Py UIS4-GFP parasites. Method as described in Figure 2 B. Statistical analysis was done by performing ANOVA with a Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed as mean ± SD. NST: Not Specific Target. (C) NOX2/gp91 phox localization determined by confocal microscopy. HepG2-CD81 cells infected with Py WT parasite were not treated (NT) or treated with INF-γ at 24 hpi. Cells were fixed at 25 hpi and stained with a specific antibody for NOX2/gp91 phox and UIS4. Nuclei were labeled by using the DNA probe Hoechst 33258. Bar 100μm. (D) Quantification of PVM labeled with NOX2/gp91 phox . Confocal images as shown in Figure 4 C were used to quantify the number of PVM labeled in control not treated (NT) and INF-γ treated infected cells. Three independent experiments were conducted. Each experiment consisted of >50 parasites. Statistical analysis was performed by using a nonparametric t-test (∗∗: p < 0.01). Bar graphs are expressed as mean ± SD. (E) Control of a secondary infection in WT C57BL/6J, IFN-γ receptor-deficient mice ( IFNγR −/− ) and NOX2/gp91 phox−/− mice. Plasmodium liver stage burden of the secondary infection is reduced by 3-logs in WT C57BL6/J mice previously infected with LARC GAP. Loss of the IFNγR completely reverses Py LARC GAP induced innate immune control of the secondary infection. NOX2/gp91 phox−/− mice exhibit 1.5-log reduction in the liver stage burden of the secondary infection. Asterisks indicate significant differences in total flux (bioluminescence) using a two-tailed Mann-Whitney U test (∗∗ p < 0.01,∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Each dot represents a single mouse. Data are compiled from at least two independent experiments with two or more mice per group per experiment.

Article Snippet: 4-5 weeks after the third immunization, cohorts of WT mice were treated either with an isotype control antibody or an IFN-γ neutralizing antibody (clone XMG1.2, cat#: I-1119, Leinco Technologies) and then subsequently infected with 10,000 Py GFP-luc sporozoites intravenously one day later.

Techniques: Infection, Confocal Microscopy, Staining, Labeling, Control, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Elimination of intra-hepatocytic malaria parasites is driven by non-canonical autophagy but not nitric oxide production

doi: 10.1016/j.isci.2025.112052

Figure Lengend Snippet: GABARAP family protein is required for IFN-γ-mediated LS elimination (A) Quantification of LS infection rates in gene-specific siRNA-treated HepG2-CD81 cells infected with Py UIS4-GFP parasites. Cells were transfected with siRNAs 24 h before infection and treated with INF-γ at 24 hpi for 4 h. Liver stage infection rate was quantified by live microscopy at 48 hpi by visually counting parasites. Three independent experiments were performed. Each experiment consists of three replicas. Statistical analysis was performed by ANOVA with a Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed as mean ± SD. NST: Not Specific Target. (B) Quantification of LS size in siRNA treated HepG2-CD81 cells infected with Py UIS4-GFP parasites. Cells were treated as described above. Quantification of LS size was determined as described in Figure 2 C. Statistical analysis was performed by ANOVA with a Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed a mean ± SD. NST: Not Specific Target. (C) HepG2-CD81 cells infected with Py UIS4-GFP parasites were not treated (NT) or treated with IFN-γ at 24 hpi. After fixation at 30 hpi, cells were stained with a pan- anti -GABARAP antibody and the DNA probe Hoechst 33258 to visualize the nuclei. Parasite PVM was visualized by the GFP fluorescence of the parasite protein UIS4-GFP. Bar 100μm. (D) Quantification of PVM labeled with GABARAPs antibody. Cells were treated and infected as in Figure 5 C. >50 PVM were scored for positive GABARAP labeling in three independent experiments. Statistical analysis was performed by using nonparametric t-test (∗∗: p < 0.01). Bar graphs are expressed as mean ± SD. (E) Quantification of GABARAP localization at the PVM. Confocal images as shown in Figure 5 C were used to quantify the localization of GABARAP at the PVM for control not treated and INF-γ treated cells. The percentages of the labeling for PVM perimeters were determine. Three independent experiments were conducted. Each experiment consisted of >50 parasites. Statistical analysis was performed by using nonparametric t-test (∗∗: p < 0.01). Bar graphs are expressed as mean ± SD.

Article Snippet: 4-5 weeks after the third immunization, cohorts of WT mice were treated either with an isotype control antibody or an IFN-γ neutralizing antibody (clone XMG1.2, cat#: I-1119, Leinco Technologies) and then subsequently infected with 10,000 Py GFP-luc sporozoites intravenously one day later.

Techniques: Infection, Transfection, Microscopy, Staining, Fluorescence, Labeling, Control

Journal: iScience

Article Title: Elimination of intra-hepatocytic malaria parasites is driven by non-canonical autophagy but not nitric oxide production

doi: 10.1016/j.isci.2025.112052

Figure Lengend Snippet: INF-γ induces lysosomal interaction with the LS parasites (A) Lysosomal localization determined by confocal microscopy. Hepg2-CD81 cells were infected with pyUIS4-GFP and not treated (NT) or treated with INF-γ at 24hpi. Cells were fixed at 30 hpi and stained with a specific antibody for the lysosomal marker LAMP1. Parasite PVMs were visualized by expression of the parasite UIS4-GFP and DNA stained with the probe Hoechst 33258. Bar 100 μm (B) Quantification of PVMs/lysosome interaction. Cells were treated and stained as in Figure 7 A. The PVMs were scored for contacting nor not lysosomes. Three independent experiments were conducted, and >100 PVM for experiments were scored. Statistical analysis was performed by using nonparametric t-test (∗∗: p < 0.01). Bar graphs are expressed as mean ± SD. (C) Quantification of percentage of PVM perimeter in contact with lysosomes. Cells were treated and stained as in Figure 6 A. Three independent experiments were performed and >50 PVMs were consider for each experiment. Statistical analysis was performed by using nonparametric t-test (∗∗: p < 0.01). Bar graphs are expressed a mean ± SD. (D) Quantification of LS infection rates in RAB7A siRNA-treated HepG2-CD81 cells infected with Py UIS4-GFP parasite. Cells were transfected with siRNAs 24h before infection and treated with INF-γ at 24 hpi for 4 h. Liver stage infection was quantified by live microscopy at 48hpi by visually counting parasite. Three independent experiments were performed. Each experiment consists of three replicas. Statistical analysis was performed by ANOVA with Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed as mean ± SD. NST: Not Specific Target. (E) Quantification of LS size In RAB7A siRNA-treated HepG2-CD81 cells infected with Py UIS4-GFP parasite. Cells were transfected with siRNAs 24 h before infection and treated with IFN-γ at 24hpi for 4 h. Quantification of LS size was determined as in Figure 2 B. Statistical analysis was performed by ANOVA with Bonferroni posttest (∗: p < 0.05). Bar graphs are expressed a mean ± SD. NST: Not Specific Target.

Article Snippet: 4-5 weeks after the third immunization, cohorts of WT mice were treated either with an isotype control antibody or an IFN-γ neutralizing antibody (clone XMG1.2, cat#: I-1119, Leinco Technologies) and then subsequently infected with 10,000 Py GFP-luc sporozoites intravenously one day later.

Techniques: Confocal Microscopy, Infection, Staining, Marker, Expressing, Transfection, Microscopy