Characterization of SARS-CoV-2 antibodies in human milk from 21 women with confirmed COVID-19 infection.

BACKGROUND: One potential mechanism for protection from SARS-CoV-2 in children is through passive immunity via breast milk from a mother infected with the novel coronavirus. The primary objectives of this study were to establish the presence of SARS-CoV-2-specific IgA and IgG and to characterize the antigenic regions of SARS-CoV-2 proteins that were reactive with antibodies in breast milk. METHODS: Between March 2020 and September 2020, 21 women with confirmed SARS-CoV-2 infection were enrolled in Mommy's Milk. Participants donated serial breast milk samples around their time of illness. Breast milk samples were used to probe a multi-coronavirus protein microarray containing full-length and variable-length overlapping fragments of SARS-CoV-2 proteins. Samples were also tested against S and N proteins by electrochemiluminescence assay. RESULTS: The breast milk samples contained IgA reactive with a variety of SARS-CoV-2 antigens. The most IgA-reactive SARS-CoV-2 proteins were N (42.9% of women responded to ≥1 N fragment) and S proteins (23.9% responded to ≥1 fragment of S1 or S2). IgG responses were similar. A striking observation was the dissimilarity between mothers in antibody recognition, giving distinct antibody reactivity and kinetic profiles. CONCLUSIONS: Individual COVID-19 cases had diverse and unique milk IgA profiles following the onset of symptoms. IMPACT: In this observational longitudinal case series of 21 women with confirmed SARS-CoV-2 infection, IgA binding to SARS-CoV-2 proteins detected by orthologous proteome microarray and electrochemiluminescence assays was observed in >75% of women, but there was heterogeneity in which antigens and how many were reactive between women. Immunological profiles of protein regions recognized by each woman were distinct. Diverse repertoires of mucosal breast milk antibody to SARS-CoV-2 reflect heterogeneous passive transfer of maternal antibody to exposed breastfeeding infants.

Methamphetamine signals transcription of IL1ß and TNFα in a reactive oxygen species-dependent manner and interacts with HIV-1 Tat to decrease antioxidant defense mechanisms.

Methamphetamine (Meth) abuse is a common HIV co-morbidity that is linked to aggravated Central Nervous System (CNS) inflammation, which accentuates HIV- associated neurological disorders, triggered both directly or indirectly by the drug. We used the well-established human innate immune macrophage cell line system (THP1) to demonstrate that Reactive Oxygen Species (ROS) immediately induced by Meth play a role in the increased transcription of inflammatory genes, in interaction with HIV-1 Tat peptide. Meth and Tat, alone and together, affect early events of transcriptional activity, as indicated by changes in RNA polymerase (RNAPol) recruitment patterns throughout the genome, via ROS-dependent and -independent mechanisms. IL1ß (IL1ß) and TNF α (TNFα), two genes with defining roles in the inflammatory response, were both activated in a ROS-dependent manner. We found that this effect occurred via the activation of the activator protein 1 (AP-1) comprising cFOS and cJUN transcription factors and regulated by the SRC kinase. HIV-1 Tat, which was also able to induce the production of ROS, did not further impact the effects of ROS in the context of Meth, but promoted gene activity independently from ROS, via additional transcription factors. For instance, HIV-1 Tat increased NFkB activation and activated gene clusters regulated by Tata box binding peptide, ING4 and IRF2. Importantly, HIV-1 Tat decreased the expression of anti-oxidant genes, where its suppression of the detoxifying machinery may contribute to the aggravation of oxidative stress induced by ROS in the context of Meth. Our results provide evidence of effects of Meth via ROS and interactions with HIV Tat that promote the transcription of inflammatory genes such as IL1ß and TNFα.

Elucidating Human Milk Oligosaccharide biosynthetic genes through network-based multi-omics integration.

Human Milk Oligosaccharides (HMOs) are abundant carbohydrates fundamental to infant health and development. Although these oligosaccharides were discovered more than half a century ago, their biosynthesis in the mammary gland remains largely uncharacterized. Here, we use a systems biology framework that integrates glycan and RNA expression data to construct an HMO biosynthetic network and predict glycosyltransferases involved. To accomplish this, we construct models describing the most likely pathways for the synthesis of the oligosaccharides accounting for >95% of the HMO content in human milk. Through our models, we propose candidate genes for elongation, branching, fucosylation, and sialylation of HMOs. Our model aggregation approach recovers 2 of 2 previously known gene-enzyme relations and 2 of 3 empirically confirmed gene-enzyme relations. The top genes we propose for the remaining 5 linkage reactions are consistent with previously published literature. These results provide the molecular basis of HMO biosynthesis necessary to guide progress in HMO research and application with the goal of understanding and improving infant health and development.

Longitudinal Changes in Human Milk Oligosaccharides (HMOs) Over the Course of 24 Months of Lactation.

BACKGROUND: Human milk oligosaccharides (HMOs) are complex glycans that are highly abundant in human milk. While over 150 HMOs have been identified, it is unknown how individual HMOs change in concentration over 24 months of lactation. OBJECTIVES: To understand how HMO concentrations change over 24 months of lactation. METHODS: Breast milk samples were collected from participants in a longitudinal cohort study of Hispanic mother-infant pairs at 1, 6, 12, 18, and 24 months postpartum. Concentrations of 19 of the most abundant HMOs were measured using HPLC. Because the parent study is ongoing and not all participants have finished all time points yet, the sample sizes ranged per time point (n = 207 at 1 month; n = 109 at 6 months; n = 83 at 12 months; n = 59 at 18 months; and n = 28 at 24 months). Approximately 88% of participants were classified as HMO secretors-a genetic factor that affects concentrations of HMOs such as 2'fucosyllactose (2'FL) and lacto-N-fucopentaose I-while the remaining 12% were classified as nonsecretors. Mixed models were used to examine changes in HMO concentrations and relative abundances over the course of lactation. RESULTS: The majority of HMOs significantly decreased in concentration over the course of lactation. The exceptions were 2'FL, sialyl-lacto-N-tetraose b, and disialyl-lacto-N-tetraose, which did not change with time, and 3-fucosyllactose (3FL) and 3'-sialyllactose (3'SL), which significantly increased. The concentration of 3FL increased 10-fold, from 195 (IQR 138-415) µg/mL at 1 month to 1930 (1100-2630) µg/mL at 24 months, while 3'SL increased 2-fold, from 277 (198-377) µg/mL to 568 (448-708) µg/mL over the same time period. CONCLUSIONS: These results indicate that HMOs do not decrease in concentration uniformly across lactation. In particular, 3FL and 3'SL increased over the course of lactation in this cohort. Future studies are required to fully understand the functions of these HMOs.

Brain Reward Function after Chronic and Binge Methamphetamine Regimens in Mice Expressing the HIV-1 TAT Protein.

BACKGROUND: Methamphetamine abuse and human immunodeficiency virus (HIV) are common comorbidities. HIV-associated proteins, such as the regulatory protein TAT, may contribute to brain reward dysfunction, inducing an altered sensitivity to methamphetamine reward and/or withdrawal in this population. OBJECTIVE: These studies examined the combined effects of TAT protein expression and, chronic and binge methamphetamine regimens on brain reward function. METHODS: Transgenic mice with inducible brain expression of the TAT protein were exposed to either saline, a chronic, or a binge methamphetamine regimen. TAT expression was induced via doxycycline treatment during the last week of methamphetamine exposure. Brain reward function was assessed daily throughout the regimens, using the intracranial self-stimulation procedure, and after a subsequent acute methamphetamine challenge. RESULTS: Both methamphetamine regimens induced withdrawal-related decreases in reward function. TAT expression substantially, but not significantly increased the withdrawal associated with exposure to the binge regimen compared to the chronic regimen, but did not alter the response to acute methamphetamine challenge. TAT expression also led to persistent changes in adenosine 2B receptor expression in the caudate putamen, regardless of methamphetamine exposure. These results suggest that TAT expression may differentially affect brain reward function, dependent on the pattern of methamphetamine exposure. CONCLUSION: The subtle effects observed in these studies highlight that longer-term TAT expression, or its induction at earlier stages of methamphetamine exposure, may be more consequential at inducing behavioral and neurochemical effects.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

Sirtuin 1-Chromatin-Binding Dynamics Points to a Common Mechanism Regulating Inflammatory Targets in SIV Infection and in the Aging Brain.

Microglia and macrophages are the main non-neuronal subsets of myeloid origin in the brain, and are critical regulators in neurodegenerative disorders, where inflammation is a key factor. Since HIV infection results in neurological perturbations that are similar to those in aging, we examined microglial and infiltrating myeloid subsets in the search for changes that might resemble the ones in aging. For that, we used the SIV infection in rhesus macaques to model neuroAIDS. We found that Sirt-1, a molecule that impacts survival and health in many models, was decreased in cell preparations containing a majority of microglia and myeloid cells from the brain of infected macaques. The role of Sirt-1 in neuroAIDS is unknown. We hypothesized that Sirt-1 silencing functions are affected by SIV. Mapping of Sirt-1 binding patterns to chromatin revealed that the number of Sirt-1-bound genes was 29.6% increased in myeloid cells from infected animals with mild or no detectable neuropathology, but 51% was decreased in severe neuropathology, compared to controls. Importantly, Sirt-1-bound genes in controls largely participate in neuroinflammation. Promoters of type I IFN pathway genes IRF7, IRF1, IFIT1, and AIF1, showed Sirt-1 binding in controls, which was consistently lost after infection, together with higher transcription. Loss of Sirt-1 binding was also found in brains from old uninfected animals, suggesting a common regulation. The role of Sirt-1 in regulating these inflammatory markers was confirmed in two different in vitro models, where Sirt-1 blockage modulated IRF7, IRF1 and AIF1 levels both in human macrophage cell lines and in human blood-derived monocytes from various normal donors, stimulated with a TLR9 agonist. Our data suggests that Sirt-1-inflammatory gene silencing is disturbed by SIV infection, resembling aging in brains. These findings may impact our knowledge on the contribution of myeloid subsets to the neurological consequences of HIV infection, aggravated and overlapping with the aging process.

Modeling the Function of TATA Box Binding Protein in Transcriptional Changes Induced by HIV-1 Tat in Innate Immune Cells and the Effect of Methamphetamine Exposure.

Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Methamphetamine abuse affects gene expression in brain-derived microglia of SIV-infected macaques to enhance inflammation and promote virus targets.

BACKGROUND: Methamphetamine (Meth) abuse is a major health problem linked to the aggravation of HIV- associated complications, especially within the Central Nervous System (CNS). Within the CNS, Meth has the ability to modify the activity/function of innate immune cells and increase brain viral loads. Here, we examined changes in the gene expression profile of neuron-free microglial cell preparations isolated from the brain of macaques infected with the Simian Immunodeficiency Virus (SIV), a model of neuroAIDS, and exposed to Meth. We aimed to identify molecular patterns triggered by Meth that could explain the detection of higher brain viral loads and the development of a pro-inflammatory CNS environment in the brain of infected drug abusers. RESULTS: We found that Meth alone has a strong effect on the transcription of genes associated with immune pathways, particularly inflammation and chemotaxis. Systems analysis led to a strong correlation between Meth exposure and enhancement of molecules associated with chemokines and chemokine receptors, especially CXCR4 and CCR5, which function as co-receptors for viral entry. The increase in CCR5 expression was confirmed in the brain in correlation with increased brain viral load. CONCLUSIONS: Meth enhances the availability of CCR5-expressing cells for SIV in the brain, in correlation with increased viral load. This suggests that Meth is an important factor in the susceptibility to the infection and to the aggravated CNS inflammatory pathology associated with SIV in macaques and HIV in humans.

Opsin expression in human epidermal skin.

Human skin is constantly exposed to solar light containing visible and ultraviolet radiation (UVR), a powerful skin carcinogen. UVR elicits cellular responses in epidermal cells via several mechanisms: direct absorption of short-wavelength UVR photons by DNA, oxidative damage caused by long-wavelength UVR, and, as we recently demonstrated, via a retinal-dependent G protein-coupled signaling pathway. Because the human epidermis is exposed to a wide range of light wavelengths, we investigated whether opsins, light-activated receptors that mediate photoreception in the eye, are expressed in epidermal skin to potentially serve as photosensors. Here we show that four opsins­OPN1-SW, OPN2, OPN3 and OPN5­are expressed in the two major human epidermal cell types, melanocytes and keratinocytes, and the mRNA expression profile of these opsins does not change in response to physiological UVR doses. We detected two OPN3 splice variants present in similar amounts in both cell types and three OPN5 splice isoforms, two of which encode truncated proteins. Notably, OPN2 and OPN3 mRNA were significantly more abundant than other opsins and encoded full-length proteins. Our results demonstrate that opsins are expressed in epidermal skin cells and suggest that they might initiate light-induced signaling pathways, possibly contributing to UVR phototransduction.

UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes.

While short exposure to solar ultraviolet radiation (UVR) can elicit increased skin pigmentation, a protective response mediated by epidermal melanocytes, chronic exposure can lead to skin cancer and photoaging. However, the molecular mechanisms that allow human skin to detect and respond to UVR remain incompletely understood. UVR stimulates a retinal-dependent signaling cascade in human melanocytes that requires GTP hydrolysis and phospholipase C ß (PLCß) activity. This pathway involves the activation of transient receptor potential A1 (TRPA1) ion channels, an increase in intracellular Ca(2+), and an increase in cellular melanin content. Here, we investigated the identity of the G protein and downstream elements of the signaling cascade and found that UVR phototransduction is Gαq/11 dependent. Activation of Gαq/11/PLCß signaling leads to hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). We found that PIP2 regulated TRPA1-mediated photocurrents, and IP3 stimulated intracellular Ca(2+) release. The UVR-elicited Ca(2+) response appears to involve both IP3-mediated release from intracellular stores and Ca(2+) influx through TRPA1 channels, showing the fast rising phase of the former and the slow decay of the latter. We propose that melanocytes use a UVR phototransduction mechanism that involves the activation of a Gαq/11-dependent phosphoinositide cascade, and resembles light phototransduction cascades of the eye.

UVA phototransduction drives early melanin synthesis in human melanocytes.

Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen [1] comprising ~95% ultraviolet A (UVA) and ~5% ultraviolet B (UVB) at the Earth's surface, promotes melanin synthesis in epidermal melanocytes [2, 3], which protects skin from DNA damage [4, 5]. UVB causes DNA lesions [6] that lead to transcriptional activation of melanin-producing enzymes, resulting in delayed skin pigmentation within days [7]. In contrast, UVA causes primarily oxidative damage [8] and leads to immediate pigment darkening (IPD) within minutes, via an unknown mechanism [9, 10]. No receptor protein directly mediating phototransduction in skin has been identified. Here we demonstrate that exposure of primary human epidermal melanocytes (HEMs) to UVA causes calcium mobilization and early melanin synthesis. Calcium responses were abolished by treatment with G protein or phospholipase C (PLC) inhibitors or by depletion of intracellular calcium stores. We show that the visual photopigment rhodopsin [11] is expressed in HEMs and contributes to UVR phototransduction. Upon UVR exposure, significant melanin production was measured within one hour; cellular melanin continued to increase in a retinal- and calcium-dependent manner up to 5-fold after 24 hr. Our findings identify a novel UVA-sensitive signaling pathway in melanocytes that leads to calcium mobilization and melanin synthesis and may underlie the mechanism of IPD in human skin.