Supplemental Material - Mental Health of Older Adults by Sexual Minority Status: Evidence From the 2021 National Health Interview Survey

Supplemental Material for Mental Health of Older Adults by Sexual Minority Status: Evidence From the 2021 National Health Interview Survey by Rainier Masa, Megumi Inoue, Lucas Prieto, Dicky Baruah, Sarah Nosrat, Samreen Mehak, and Don Operario in Journal of Applied Gerontology</p

<i>Schistosoma mansoni</i> infection suppresses the growth of <i>Plasmodium yoelii</i> parasites in the liver and reduces gametocyte infectivity to mosquitoes

<div><p>Malaria and schistosomiasis are major parasitic diseases causing morbidity and mortality in the tropics. Epidemiological surveys have revealed coinfection rates of up to 30% among children in Sub-Saharan Africa. To investigate the impact of coinfection of these two parasites on disease epidemiology and pathology, we carried out coinfection studies using <i>Plasmodium yoelii</i> and <i>Schistosoma mansoni</i> in mice. Malaria parasite growth in the liver following sporozoite inoculation is significantly inhibited in mice infected with <i>S</i>. <i>mansoni</i>, so that when low numbers of sporozoites are inoculated, there is a large reduction in the percentage of mice that go on to develop blood stage malaria. Furthermore, gametocyte infectivity is much reduced in mice with <i>S</i>. <i>mansoni</i> infections. These results have profound implications for understanding the interactions between <i>Plasmodium</i> and <i>Schistosoma</i> species, and have implications for the control of malaria in schistosome endemic areas.</p></div

Malaria parasite liver burden in BALB/c, C57BL/6, and CBA/J mice.

<p>(A) Copy number of <i>Plasmodium yoelii</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female BALB/c, B6, and CBA/J mice (N = 5) infected with 50 <i>Schistosoma mansoni</i>-cercaria 10 weeks previously were challenged with 1,500 SPZ of <i>P</i>. <i>yoelii</i> along with <i>S</i>. <i>mansoni-</i>non-infected controls. BALB/c: ***P<0.001, t = 6.283, df = 8; B6: *P<0.05, t = 2.511, df = 8; CBA: **P<0.01, t = 4.220, df = 7. (B) Copy number of <i>Plasmodium berghei</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female BALB/c, B6, and CBA/J mice (N = 5) infected with 50 <i>S</i>. <i>mansoni</i>-cercaria 10 weeks previously were challenged with 1,500 SPZ of <i>P</i>. <i>berghei</i> along with <i>S</i>. <i>mansoni-</i>non-infected controls. BALB/c: *P<0.05, t = 2.306, df = 8; B6: ***P<0.001, t = 5.336, df = 8; CBA: *P<0.05, t = 2.846, df = 7. All data were statistically examined using Student’s two-tailed t-test.</p

Information for PCR primers.

<p>Information for PCR primers.</p

Liver immunopathology in <i>Schistosoma mansoni</i>-cercariae infection and intraportal infusion of frozen <i>S</i>. <i>mansoni</i>-eggs.

<p>(A) Malaria parasite liver burden with/without intraportal infusion of frozen <i>S</i>. <i>mansoni</i>-eggs. Female B6 mice were intraportally inoculated 3,000/10,000 frozen <i>S</i>. <i>mansoni</i>-eggs and challenged with 1,500 sporozoites of <i>Plasmodium yoelii</i> along with controls inoculated with PBS (control: N = 3; 3,000 frozen eggs: N = 5, ***P<0.001, t = 1.943, df = 6; 10,000 frozen eggs: N = 3, *P<0.05, Student’s two-tailed t-test, t = 4.072, df = 4). (B) Macroscopic and microscopic images of liver pathology. The black lines indicate 250 micro meters. (C) The number of granulomas in the liver. Female BALB/c mice and B6 mice were inoculated with 50 <i>S</i>. <i>mansoni</i>-cercariae subcutaneously (naive: N = 5; 50 cercariae: N = 3) or inoculated with 3,000 frozen <i>S</i>. <i>mansoni</i>-eggs along with controls inoculated with PBS intraportaly (PBS: N = 5; 3,000 frozen eggs: N = 5). The numbers of granuloma were counted in 20 microscopic fields at 100 x magnification. *P<0.05, ***P<0.001. All data were statistically examined using Student’s two-tailed t-test. (D) The numbers of immune cells induced by intraportal infusion of 3,000 frozen <i>S</i>. <i>mansoni</i>-eggs. Female B6 mice (N = 4/group) were intraportaly inoculated 3,000 <i>S</i>. <i>mansoni</i>-eggs. (E) The numbers of immune cells induced by 50 <i>S</i>. <i>mansoni</i>-cercaria infection. Female BALB/c mice (naive: N = 9, 8 weeks: N = 9, 10 weeks N = 6) were infected with 50 <i>S</i>. <i>mansoni</i>-cercariae. *P<0.05, **P<0.01, ***P<0.001. All data were statistically examined using Student’s two-tailed t-test.</p

Gametocyte infectivity.

<p>(A) Parasitemia. Female BALB/c mice were each inoculated with 1 x 10⁶ <i>Plasmodium yoelii</i> parasitized erythrocytes intravenously with (n = 4 mice) or without (n = 5) pre-existing <i>Schistosoma mansoni</i> infection. Parasitaemia was determined by microscopic examination on days 3, 4, and 5 post-inoculation; day 3: Student’s two-tailed t-test; **P<0.01, t = -4.906, df = 7; Day5: *P<0.05, t = -2.922, df = 5 (<b>B</b>) Gametocyte density. Gametocyte density was determined on days 3 and 4 post-inoculation of 1 x 10⁶ <i>P</i>. <i>yoelii</i>-parasitized erythrocytes intravenously. Day 3: Student’s two-tailed t-test; **P<0.01, t = 3.813, df = 5; Day 4: **P<0.01, t = 3.608, df = 5. Error bars show the geometric mean with 95% confidence intervals. (C) Percentage of mosquitoes with one or more oocysts present on the midgut eight days post-feeding on infected mice. A minimum of eight mosquitoes were allowed to feed on each individual mouse in the group per day **P = 0.0003, (2-way ANOVA, F = 22.23, DFn = 1, DFd = 14). Error bars mar the standard error of the mean per mouse group. (D) Oocyst numbers per mosquito. The numbers of oocysts present on mosquito midguts were determined eight days post-mosquito feeding; day 3: Student’s two-tailed t-test, **P<0.01, t = 3.077, df = 25. Error bars show the geometric mean with 95% confidence intervals. Data is representative of three independent experiments.</p

Growth of malaria parasites in the liver and blood of mice following SPZ inoculation of <i>Plasmodium yoelii</i> with and without <i>Schistosoma mansoni</i> infection.

<p>(A) Copy number of <i>P</i>. <i>yoelii</i> 18s RNA gene per 1×10⁶mouse G3PDH gene measured at 42 h post SPZ inoculation. Female BALB/c mice (N = 6) infected with 50 <i>S</i>. <i>mansoni</i>-cercariae 10 weeks previously were challenged with 1,500 <i>P</i>. <i>yoelii</i> SPZ along with <i>S</i>. <i>mansoni</i>-non-infected controls. **P<0.01, Student’s two-tailed t-test, t = 4.362, df = 10. (B) Parasitaemia. Blood stage malaria parasites were monitored daily from day 2 to 8 post i.v. inoculation of 500 <i>P</i>. <i>yoelii</i> SPZ. (C) Percentage survival. Data from one representative experiment of three independent repeats are shown.</p

<i>Plasmodium yoelii</i> parasite density in the blood and liver.

<p>Copy number of <i>P</i>. <i>yoelii</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female BALB/c mice (N = 5) infected with 50 <i>Schistosoma mansoni</i>-cercaria 10 weeks previously were challenged with 1,500 SPZ of <i>P</i>. <i>yoelii</i> along with <i>S</i>. <i>mansoni</i>-non-infected controls. (A) <i>P</i>. <i>yoelii</i> parasitaemia in the blood. (B) <i>P</i>. <i>yoelii</i> parasite density and proliferation in the liver. ***P<0.001, Student’s two-tailed t-test, t = -6.316, df = 8.</p

Malaria outcomes with low dose SPZ challenge.

<p>Female BALB/c mice (N = 16) infected with 50 <i>Schistosoma mansoni</i>-cercariae 10 weeks previously were challenged with 50 sporozoites of <i>Plasmodium yoelii</i> along with <i>S</i>. <i>mansoni</i>-non-infected controls. (A) The percentage of mice that did not develop blood stage infection following inoculation of low does SPZ. Data were statistically examined using the log-rank test. ***P<0.001, x2 = 29.8, df = 1. (B) Parasitaemia. Mean parasitaemia in <i>S</i>. <i>mansoni</i> infected group was calculated only among blood stage malaria positive mice.</p

Malaria parasite liver burden in wild-type B6, IFN-gamma-deficient, and Interleukin-4-deficient mice.

<p>Copy number of <i>Plasmodium yoelii</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female IFN-γ^-/- mice (N = 8), IL-4^-/- mice (N = 8), and B6 WT mice (N = 4) were intraportally inoculated with 3,000 frozen <i>S</i>. <i>mansoni</i>-eggs and challenged with 1,500 SPZ of <i>P</i>. <i>yoelii</i> along with each control groups inoculated 100 μL PBS (N = 4). B6 WT mice: *P<0.05, t = 3.017, df = 5; IFN-γ^-/- mice: Not significant (NS), t = -1.303, df = 10; IL-4-/- mice: NS, t = -0.016, df = 10.</p