Gonadal hormones impart male-biased behavioral vulnerabilities to immune activation via microglial mitochondrial function.

There is a strong male bias in the prevalence of many neurodevelopmental disorders such as autism spectrum disorder. However, the mechanisms underlying this sex bias remain elusive. Infection during the perinatal period is associated with an increased risk of neurodevelopmental disorder development. Here, we used a mouse model of early-life immune activation that reliably induces deficits in social behaviors only in males. We demonstrate that male-biased alterations in social behavior are dependent upon microglial immune signaling and are coupled to alterations in mitochondrial morphology, gene expression, and function specifically within microglia, the innate immune cells of the brain. Additionally, we show that this behavioral and microglial mitochondrial vulnerability to early-life immune activation is programmed by the male-typical perinatal gonadal hormone surge. These findings demonstrate that social behavior in males over the lifespan are regulated by microglia-specific mechanisms that are shaped by events that occur in early development.

Hofbauer cells and fetal brain microglia share transcriptional profiles and responses to maternal diet-induced obesity.

Maternal immune activation is associated with adverse offspring neurodevelopmental outcomes, many mediated by in utero microglial programming. As microglia remain inaccessible throughout development, identification of noninvasive biomarkers reflecting fetal brain microglial programming could permit screening and intervention. We used lineage tracing to demonstrate the shared ontogeny between fetal brain macrophages (microglia) and fetal placental macrophages (Hofbauer cells) in a mouse model of maternal diet-induced obesity, and single-cell RNA-seq to demonstrate shared transcriptional programs. Comparison with human datasets demonstrated conservation of placental resident macrophage signatures between mice and humans. Single-cell RNA-seq identified common alterations in fetal microglial and Hofbauer cell gene expression induced by maternal obesity, as well as sex differences in these alterations. We propose that Hofbauer cells, which are easily accessible at birth, provide novel insights into fetal brain microglial programs, and may facilitate the early identification of offspring vulnerable to neurodevelopmental disorders in the setting of maternal exposures.

The Non-Specific Drp1 Inhibitor Mdivi-1 Has Modest Biochemical Antioxidant Activity.

Mitochondrial division inhibitor-1 (mdivi-1), a non-specific inhibitor of Drp1-dependent mitochondrial fission, is neuroprotective in numerous preclinical disease models. These include rodent models of Alzheimer's disease and ischemic or traumatic brain injury. Among its Drp1-independent actions, the compound was found to suppress mitochondrial Complex I-dependent respiration but with less resultant mitochondrial reactive oxygen species (ROS) emission compared with the classical Complex I inhibitor rotenone. We employed two different methods of quantifying Trolox-equivalent antioxidant capacity (TEAC) to test the prediction that mdivi-1 can directly scavenge free radicals. Mdivi-1 exhibited moderate antioxidant activity in the 2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS) assay. Half-maximal ABTS radical depletion was observed at ~25 µM mdivi-1, equivalent to that achieved by ~12.5 µM Trolox. Mdivi-1 also showed antioxidant activity in the α, α-diphenyl-ß-picrylhydrazyl (DPPH) assay. However, mdivi-1 exhibited a reduced capacity to deplete the DPPH radical, which has a more sterically hindered radical site compared with ABTS, with 25 µM mdivi-1 displaying only 0.8 µM Trolox equivalency. Both assays indicate that mdivi-1 possesses biochemical antioxidant activity but with modest potency relative to the vitamin E analog Trolox. Future studies are needed to evaluate whether the ability of mdivi-1 to directly scavenge free radicals contributes to its mechanisms of neuroprotection.

DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling.

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

Novel genetic variants in MAPT and alterations in tau phosphorylation in amyotrophic lateral sclerosis post-mortem motor cortex and cerebrospinal fluid.

Although the molecular mechanisms underlying amyotrophic lateral sclerosis (ALS) are not yet fully understood, several studies report alterations in tau phosphorylation in both sporadic and familial ALS. Recently, we have demonstrated that phosphorylated tau at S396 (pTau-S396) is mislocalized to synapses in ALS motor cortex (mCTX) and contributes to mitochondrial dysfunction. Here, we demonstrate that while there was no overall increase in total tau, pTau-S396, and pTau-S404 in ALS post-mortem mCTX, total tau and pTau-S396 were increased in C9ORF72-ALS. Additionally, there was a significant decrease in pTau-T181 in ALS mCTX compared controls. Furthermore, we leveraged the ALS Knowledge Portal and Project MinE data sets and identified ALS-specific genetic variants across MAPT, the gene encoding tau. Lastly, assessment of cerebrospinal fluid (CSF) samples revealed a significant increase in total tau levels in bulbar-onset ALS together with a decrease in CSF pTau-T181:tau ratio in all ALS samples, as reported previously. While increases in CSF tau levels correlated with a faster disease progression as measured by the revised ALS functional rating scale (ALSFRS-R), decreases in CSF pTau-T181:tau ratio correlated with a slower disease progression, suggesting that CSF total tau and pTau-T181 ratio may serve as biomarkers of disease in ALS. Our findings highlight the potential role of pTau-T181 in ALS, as decreases in CSF pTau-T181:tau ratio may reflect the significant decrease in pTau-T181 in post-mortem mCTX. Taken together, these results indicate that tau phosphorylation is altered in ALS post-mortem mCTX as well as in CSF and, importantly, the newly described pathogenic or likely pathogenic variants identified in MAPT in this study are adjacent to T181 and S396 phosphorylation sites further highlighting the potential role of these tau functional domains in ALS.

Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis.

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer's disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

COVID-19 mRNA vaccines drive differential antibody Fc-functional profiles in pregnant, lactating, and nonpregnant women.

Substantial immunological changes occur throughout pregnancy to render the mother immunologically tolerant to the fetus and allow fetal growth. However, additional local and systemic immunological adaptations also occur, allowing the maternal immune system to continue to protect the dyad against pathogens both during pregnancy and after birth through lactation. This fine balance of tolerance and immunity, along with physiological and hormonal changes, contributes to increased susceptibility to particular infections in pregnancy, including more severe coronavirus disease 2019 (COVID-19). Whether these changes also make pregnant women less responsive to vaccination or induce altered immune responses to vaccination remains incompletely understood. To define potential changes in vaccine response during pregnancy and lactation, we undertook deep sequencing of the humoral vaccine response in a group of pregnant and lactating women and nonpregnant age-matched controls. Vaccine-specific titers were comparable between pregnant women, lactating women, and nonpregnant controls. However, Fc receptor (FcR) binding and antibody effector functions were induced with delayed kinetics in both pregnant and lactating women compared with nonpregnant women after the first vaccine dose, which normalized after the second dose. Vaccine boosting resulted in high FcR-binding titers in breastmilk. These data suggest that pregnancy promotes resistance to generating proinflammatory antibodies and indicates that there is a critical need to follow prime-boost timelines in this vulnerable population to ensure full immunity is attained.

Maternal SARS-CoV-2 infection elicits sexually dimorphic placental immune responses.

There is a persistent bias toward higher prevalence and increased severity of coronavirus disease 2019 (COVID-19) in males. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of COVID-19 disease in adults and play a key role in the placental antiviral response. Moreover, the interferon response has been shown to alter Fc receptor expression and therefore may affect placental antibody transfer. Here, we examined the intersection of maternal-fetal antibody transfer, viral-induced placental interferon responses, and fetal sex in pregnant women infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Placental Fc receptor abundance, interferon-stimulated gene (ISG) expression, and SARS-CoV-2 antibody transfer were interrogated in 68 human pregnancies. Sexually dimorphic expression of placental Fc receptors, ISGs and proteins, and interleukin-10 was observed after maternal SARS-CoV-2 infection, with up-regulation of these features in placental tissue of pregnant individuals with male fetuses. Reduced maternal SARS-CoV-2­specific antibody titers and impaired placental antibody transfer were also observed in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.

Placental Expression of ACE2 and TMPRSS2 in Maternal Severe Acute Respiratory Syndrome Coronavirus 2 Infection: Are Placental Defenses Mediated by Fetal Sex?

BACKGROUND: Expression of angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2), host molecules required for viral entry, may underlie sex differences in vulnerability to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We investigated whether placental ACE2 and TMPRSS2 expression vary by fetal sex in the presence of maternal SARS-CoV-2 infection. METHODS: Placental ACE2 and TMPRSS2 expression was quantified by quantitative reverse transcription polymerase chain reaction (RT-PCR) and by Western blot in 68 pregnant women (38 SARS-CoV-2 positive, 30 SARS-CoV-2 negative) delivering at Mass General Brigham from April to June 2020. The impact of fetal sex and maternal SARS-CoV-2 exposure on ACE2 and TMPRSS2 was analyzed by 2-way analysis of variance (ANOVA). RESULTS: Maternal SARS-CoV-2 infection impacted placental TMPRSS2 expression in a sexually dimorphic fashion (2-way ANOVA interaction, P = .002). We observed no impact of fetal sex or maternal SARS-CoV-2 status on ACE2. TMPRSS2 expression was significantly correlated with ACE2 expression in males (Spearman ρ = 0.54, P = .02) but not females (ρ = 0.23, P = .34) exposed to maternal SARS-CoV-2. CONCLUSIONS: Sex differences in placental TMPRSS2 but not ACE2 were observed in the setting of maternal SARS-CoV-2 infection, which may have implications for offspring vulnerability to placental infection.

COVID-19 mRNA vaccines drive differential Fc-functional profiles in pregnant, lactating, and non-pregnant women.

Significant immunological changes occur throughout pregnancy to tolerize the mother and allow growth of the fetal graft. However, additional local and systemic immunological adaptations also occur, allowing the maternal immune system to continue to protect the dyad against foreign invaders both during pregnancy and after birth through lactation. This fine balance of tolerance and immunity, along with physiological and hormonal changes, contribute to increased susceptibility to particular infections in pregnancy, including more severe COVID-19 disease. Whether these changes also make pregnant women less responsive to vaccination or induce altered immune responses to vaccination remains incompletely understood. To holistically define potential changes in vaccine response during pregnancy and lactation, we deeply profiled the humoral vaccine response in a group of pregnant and lactating women and non-pregnant age-matched controls. Vaccine-specific titers were comparable, albeit slightly lower, between pregnant and lactating women, compared to non-pregnant controls. Among pregnant women, we found higher antibody titers and functions in those vaccinated with the Moderna vaccine. FcR-binding and antibody effector functions were induced with delayed kinetics in both pregnant and lactating women compared to non-pregnant women. Antibody boosting resulted in high FcR-binding titers in breastmilk. These data point to an immune resistance to generate highly inflammatory antibodies during pregnancy and lactation, and a critical need to follow prime/boost timelines in this vulnerable population to ensure full immunity is attained.

Sexually dimorphic placental responses to maternal SARS-CoV-2 infection.

There is a persistent male bias in the prevalence and severity of COVID-19 disease. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of disease in adults, and play a key role in the placental anti-viral response. Moreover, the interferon response has been shown to alter Fc-receptor expression, and therefore may impact placental antibody transfer. Here we examined the intersection of viral-induced placental interferon responses, maternal-fetal antibody transfer, and fetal sex. Placental interferon stimulated genes (ISGs), Fc-receptor expression, and SARS-CoV-2 antibody transfer were interrogated in 68 pregnancies. Sexually dimorphic placental expression of ISGs, interleukin-10, and Fc receptors was observed following maternal SARS-CoV-2 infection, with upregulation in males. Reduced maternal SARS-CoV-2-specific antibody titers and impaired placental antibody transfer were noted in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.

Coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study.

BACKGROUND: Pregnant and lactating women were excluded from initial coronavirus disease 2019 vaccine trials; thus, data to guide vaccine decision making are lacking. OBJECTIVE: This study aimed to evaluate the immunogenicity and reactogenicity of coronavirus disease 2019 messenger RNA vaccination in pregnant and lactating women compared with: (1) nonpregnant controls and (2) natural coronavirus disease 2019 infection in pregnancy. STUDY DESIGN: A total of 131 reproductive-age vaccine recipients (84 pregnant, 31 lactating, and 16 nonpregnant women) were enrolled in a prospective cohort study at 2 academic medical centers. Titers of severe acute respiratory syndrome coronavirus 2 spike and receptor-binding domain immunoglobulin G, immunoglobulin A, and immunoglobulin M were quantified in participant sera (n=131) and breastmilk (n=31) at baseline, at the second vaccine dose, at 2 to 6 weeks after the second vaccine, and at delivery by Luminex. Umbilical cord sera (n=10) titers were assessed at delivery. Titers were compared with those of pregnant women 4 to 12 weeks from the natural infection (n=37) by enzyme-linked immunosorbent assay. A pseudovirus neutralization assay was used to quantify neutralizing antibody titers for the subset of women who delivered during the study period. Postvaccination symptoms were assessed via questionnaire. Kruskal-Wallis tests and a mixed-effects model, with correction for multiple comparisons, were used to assess differences among groups. RESULTS: Vaccine-induced antibody titers were equivalent in pregnant and lactating compared with nonpregnant women (pregnant, median, 5.59; interquartile range, 4.68-5.89; lactating, median, 5.74; interquartile range, 5.06-6.22; nonpregnant, median, 5.62; interquartile range, 4.77-5.98, P=.24). All titers were significantly higher than those induced by severe acute respiratory syndrome coronavirus 2 infection during pregnancy (P<.0001). Vaccine-generated antibodies were present in all umbilical cord blood and breastmilk samples. Neutralizing antibody titers were lower in umbilical cord than maternal sera, although this finding did not achieve statistical significance (maternal sera, median, 104.7; interquartile range, 61.2-188.2; cord sera, median, 52.3; interquartile range, 11.7-69.6; P=.05). The second vaccine dose (boost dose) increased severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G, but not immunoglobulin A, in maternal blood and breastmilk. No differences were noted in reactogenicity across the groups. CONCLUSION: Coronavirus disease 2019 messenger RNA vaccines generated robust humoral immunity in pregnant and lactating women, with immunogenicity and reactogenicity similar to that observed in nonpregnant women. Vaccine-induced immune responses were statistically significantly greater than the response to natural infection. Immune transfer to neonates occurred via placenta and breastmilk.

COVID-19 vaccine response in pregnant and lactating women: a cohort study.

BACKGROUND: Pregnant and lactating women were excluded from initial COVID-19 vaccine trials; thus, data to guide vaccine decision-making are lacking. We sought to evaluate the immunogenicity and reactogenicity of COVID-19 mRNA vaccination in pregnant and lactating women. METHODS: 131 reproductive-age vaccine recipients (84 pregnant, 31 lactating, and 16 non-pregnant) were enrolled in a prospective cohort study at two academic medical centers. Titers of SARS-CoV-2 Spike and RBD IgG, IgA and IgM were quantified in participant sera (N=131), umbilical cord sera (N=10), and breastmilk (N=31) at baseline, 2nd vaccine dose, 2-6 weeks post 2nd vaccine, and delivery by Luminex, and confirmed by ELISA. Titers were compared to pregnant women 4-12 weeks from native infection (N=37). Post-vaccination symptoms were assessed. Kruskal-Wallis tests and a mixed effects model, with correction for multiple comparisons, were used to assess differences between groups. RESULTS: Vaccine-induced immune responses were equivalent in pregnant and lactating vs non-pregnant women. All titers were higher than those induced by SARS-CoV-2 infection during pregnancy. Vaccine-generated antibodies were present in all umbilical cord blood and breastmilk samples. SARS-CoV-2 specific IgG, but not IgA, increased in maternal blood and breastmilk with vaccine boost. No differences were noted in reactogenicity across the groups. CONCLUSIONS: COVID-19 mRNA vaccines generated robust humoral immunity in pregnant and lactating women, with immunogenicity and reactogenicity similar to that observed in non-pregnant women. Vaccine-induced immune responses were significantly greater than the response to natural infection. Immune transfer to neonates occurred via placental and breastmilk.

Compromised SARS-CoV-2-specific placental antibody transfer.

SARS-CoV-2 infection causes more severe disease in pregnant women compared to age-matched non-pregnant women. Whether maternal infection causes changes in the transfer of immunity to infants remains unclear. Maternal infections have previously been associated with compromised placental antibody transfer, but the mechanism underlying this compromised transfer is not established. Here, we used systems serology to characterize the Fc profile of influenza-, pertussis-, and SARS-CoV-2-specific antibodies transferred across the placenta. Influenza- and pertussis-specific antibodies were actively transferred. However, SARS-CoV-2-specific antibody transfer was significantly reduced compared to influenza- and pertussis-specific antibodies, and cord titers and functional activity were lower than in maternal plasma. This effect was only observed in third-trimester infection. SARS-CoV-2-specific transfer was linked to altered SARS-CoV-2-antibody glycosylation profiles and was partially rescued by infection-induced increases in IgG and increased FCGR3A placental expression. These results point to unexpected compensatory mechanisms to boost immunity in neonates, providing insights for maternal vaccine design.

Assessment of Maternal and Neonatal SARS-CoV-2 Viral Load, Transplacental Antibody Transfer, and Placental Pathology in Pregnancies During the COVID-19 Pandemic.

Importance: Biological data are lacking with respect to risk of vertical transmission and mechanisms of fetoplacental protection in maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objective: To quantify SARS-CoV-2 viral load in maternal and neonatal biofluids, transplacental passage of anti-SARS-CoV-2 antibody, and incidence of fetoplacental infection. Design, Setting, and Participants: This cohort study was conducted among pregnant women presenting for care at 3 tertiary care centers in Boston, Massachusetts. Women with reverse transcription-polymerase chain reaction (RT-PCR) results positive for SARS-CoV-2 were recruited from April 2 to June 13, 2020, and follow-up occurred through July 10, 2020. Contemporaneous participants without SARS-CoV-2 infection were enrolled as a convenience sample from pregnant women with RT-PCR results negative for SARS-CoV-2. Exposures: SARS-CoV-2 infection in pregnancy, defined by nasopharyngeal swab RT-PCR. Main Outcomes and Measures: The main outcomes were SARS-CoV-2 viral load in maternal plasma or respiratory fluids and umbilical cord plasma, quantification of anti-SARS-CoV-2 antibodies in maternal and cord plasma, and presence of SARS-CoV-2 RNA in the placenta. Results: Among 127 pregnant women enrolled, 64 with RT-PCR results positive for SARS-CoV-2 (mean [SD] age, 31.6 [5.6] years) and 63 with RT-PCR results negative for SARS-CoV-2 (mean [SD] age, 33.9 [5.4] years) provided samples for analysis. Of women with SARS-CoV-2 infection, 23 (36%) were asymptomatic, 22 (34%) had mild disease, 7 (11%) had moderate disease, 10 (16%) had severe disease, and 2 (3%) had critical disease. In viral load analyses among 107 women, there was no detectable viremia in maternal or cord blood and no evidence of vertical transmission. Among 77 neonates tested in whom SARS-CoV-2 antibodies were quantified in cord blood, 1 had detectable immunoglobuilin M to nucleocapsid. Among 88 placentas tested, SARS-CoV-2 RNA was not detected in any. In antibody analyses among 37 women with SARS-CoV-2 infection, anti-receptor binding domain immunoglobin G was detected in 24 women (65%) and anti-nucleocapsid was detected in 26 women (70%). Mother-to-neonate transfer of anti-SARS-CoV-2 antibodies was significantly lower than transfer of anti-influenza hemagglutinin A antibodies (mean [SD] cord-to-maternal ratio: anti-receptor binding domain immunoglobin G, 0.72 [0.57]; anti-nucleocapsid, 0.74 [0.44]; anti-influenza, 1.44 [0.80]; P < .001). Nonoverlapping placental expression of SARS-CoV-2 receptors angiotensin-converting enzyme 2 and transmembrane serine protease 2 was noted. Conclusions and Relevance: In this cohort study, there was no evidence of placental infection or definitive vertical transmission of SARS-CoV-2. Transplacental transfer of anti-SARS-CoV-2 antibodies was inefficient. Lack of viremia and reduced coexpression and colocalization of placental angiotensin-converting enzyme 2 and transmembrane serine protease 2 may serve as protective mechanisms against vertical transmission.

Establishment of a pediatric COVID-19 biorepository: unique considerations and opportunities for studying the impact of the COVID-19 pandemic on children.

BACKGROUND: COVID-19, the disease caused by the highly infectious and transmissible coronavirus SARS-CoV-2, has quickly become a morbid global pandemic. Although the impact of SARS-CoV-2 infection in children is less clinically apparent, collecting high-quality biospecimens from infants, children, and adolescents in a standardized manner during the COVID-19 pandemic is essential to establish a biologic understanding of the disease in the pediatric population. This biorepository enables pediatric centers world-wide to collect samples uniformly to drive forward our understanding of COVID-19 by addressing specific pediatric and neonatal COVID-19-related questions. METHODS: A COVID-19 biospecimen collection study was implemented with strategic enrollment guidelines to include patients seen in urgent care clinics and hospital settings, neonates born to SARS-CoV-2 infected mothers, and asymptomatic children. The methodology described here, details the importance of establishing collaborations between the clinical and research teams to harmonize protocols for patient recruitment and sample collection, processing and storage. It also details modifications required for biobanking during a surge of the COVID-19 pandemic. RESULTS: Considerations and challenges facing enrollment of neonatal and pediatric cohorts are described. A roadmap is laid out for successful collection, processing, storage and database management of multiple pediatric samples such as blood, nasopharyngeal and oropharyngeal swabs, sputum, saliva, tracheal aspirates, stool, and urine. Using this methodology, we enrolled 327 participants, who provided a total of 972 biospecimens. CONCLUSIONS: Pediatric biospecimens will be key in answering questions relating to viral transmission by children, differences between pediatric and adult viral susceptibility and immune responses, the impact of maternal SARS-CoV-2 infection on fetal development, and factors driving the Multisystem Inflammatory Syndrome in Children. The specimens in this biorepository will allow necessary comparative studies between children and adults, help determine the accuracy of current pediatric viral testing techniques, in addition to, understanding neonatal exposure to SARS-CoV-2 infection and disease abnormalities. The successful establishment of a pediatric biorepository is critical to provide insight into disease pathogenesis, and subsequently, develop future treatment and vaccination strategies.

Neonatal immune challenge induces female-specific changes in social behavior and somatostatin cell number.

Decreases in social behavior are a hallmark aspect of acute "sickness behavior" in response to infection. However, immune insults that occur during the perinatal period may have long-lasting consequences for adult social behavior by impacting the developmental organization of underlying neural circuits. Microglia, the resident immune cells of the central nervous system, are sensitive to immune stimulation and play a critical role in the developmental sculpting of neural circuits, making them likely mediators of this process. Here, we investigated the impact of a postnatal day (PND) 4 lipopolysaccharide (LPS) challenge on social behavior in adult mice. Somewhat surprisingly, neonatal LPS treatment decreased sociability in adult female, but not male mice. LPS-treated females also displayed reduced social interaction and social memory in a social discrimination task as compared to saline-treated females. Somatostatin (SST) interneurons within the anterior cingulate cortex (ACC) have recently been suggested to modulate a variety of social behaviors. Interestingly, the female-specific changes in social behavior observed here were accompanied by an increase in SST interneuron number in the ACC. Finally, these changes in social behavior and SST cell number do not appear to depend on microglial inflammatory signaling, because microglia-specific genetic knock-down of myeloid differentiation response protein 88 (MyD88; the removal of which prevents LPS from increasing proinflammatory cytokines such as TNFα and IL-1ß) did not prevent these LPS-induced changes. This study provides novel evidence for enduring effects of neonatal immune activation on social behavior and SST interneurons in females, largely independent of microglial inflammatory signaling.

Isolation of Microglia from Mouse or Human Tissue.

Microglia are the innate immune cells of the central nervous system. Although numerous methods have been developed to isolate microglia from the brain, the method of dissociation and isolation can have a profound effect on the function of these highly dynamic cells. Here, we present an optimized protocol to isolate CD11b+ cells (microglia) from mouse or human brain tissue using magnetic bead columns. Isolated microglia can be used to model diseases with neuroinflammatory components for potential therapeutic discoveries. For complete details on the use and execution of this protocol, please refer to Hanamsagar et al. (2017), Rivera et al. (2019), and Edlow et al. (2019).