Open field and light/dark box test.

<p>Inner (A) and outer (B) locomotion in the open field test, time spent in the white compartment (C) and number of transitions between compartments (D) in the light/dark box test displayed by SLA16 and SHR rats from both sexes. Bars and vertical lines represent the means and S.E.M. of animals grouped by sex and strain (n = 18–28/sex/strain). (*), (**) and (***) represent significant (p<0.05, p<0.01 and p<0.001, respectively) overall effect of strain (two-way ANOVA).</p

Effects of the estrous cycle phases on behaviors in the open field.

<p>Inner (A) and outer (B) locomotion displayed by SLA16 and SHR female rats. Squares and vertical lines represent the means and S.E.M. of animals grouped by estrous cycle phase and strain (n = 4–12/phase/strain). (***) represent significant (p<0.001) overall effect of strain (two-way ANOVA).</p

T-maze.

<p>Inhibitory avoidance and one way escape times (s) displayed by SLA16 and SHR male rats in the T-maze. Squares and vertical lines represent the means and S.E.M. of animals grouped by strain and trial (n = 12/strain). (*) represent significant (p<0.05) overall effect of strain (repeated-measure ANOVA).</p

Construction of the SLA16 congenic strain.

<p>After obtaining an F1 (or N1), the animals generated were continuously backcrossed to SHR rats for 10 generations and genotyped for three markers (two flanking and one in the middle of <i>Anxrr16</i>) for selecting heterozygous animals for the <i>Anxrr16</i> region, after which <i>Anxrr16</i> heterozygous rats were intercrossed. The congenic strain is homozygous for LEW alleles in the <i>Anxrr16</i> region, and about 99.9% SHR in the rest of the genome.</p

Schematic representation of allele frequencies in SLA16 animals from generations N10F4 to N10F7.

<p>The black areas represent portions of the genome where over 97% of the alleles came from the SHR strain, while the white areas are completely constituted by LEW alleles, and the hatched areas (transition) indicate regions where both alleles are found in variable proportions. (*) indicates two molecular markers for which positions in Mpb are not available on the Rat Genome Database, with their locations being estimated based on their position in Centimorgan.</p

Behavioral profile of the SLA16 congenic strain during its construction.

<p>Inner locomotion displayed by animals during the development of SLA16 (generations N3 to N10) in the open field test. In each of these generations, males and females that were either homozygous for SHR alleles or heterozygous in the <i>Anxrr16 locus</i> were tested. Bars and vertical lines represent the means and S.E.M. of animals grouped by genotype. (*) represents significant (p<0.05) overall effect of genotype (two-way ANOVA for each generation separately).</p

Set-shifting procedure.

<p>Number of trials to criterion (A) and number and types of errors (B) of SLA16 and SHR male rats during the visual cue discrimination task, in the set-shifting procedure. Bars and vertical lines represent the means and S.E.M. of rats grouped by strain (n = 9–12/strain). No significant (p>0.05) effect of strain was detected for the number of trials to criterion and number of perseverative (PE), regressive (RE), never-reinforced (NR) or total errors (Student’s t-test).</p

Table_2_High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil.XLSX

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.</p

Table_1_High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil.DOCX

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.</p