Monitoring of systemic and local immune signatures in response to HCMV reactivation during lactation

The human herpesvirus cytomegalovirus (HCMV) persists in the host in latent form for a lifetime and may periodically reactivate. During lactation, reactivation of HCMV occurs locally in the mammary gland without detectable systemic DNAemia. Preterm infants are at risk of infection through ingesting breast milk and may develop life-threatening disease with sepsis-like symptoms. In this thesis, the modulation of the immune signature in response to HCMV reactivation in the mammary gland, investigated non-invasively by analyzing breast milk, was compared to corresponding simultaneously drawn blood samples of HCMV-seropositive, as well as HCMV-seronegative mothers. This “BlooMil” study was performed on longitudinal samples taken from birth through 60 days postpartum over four time ranges. The viral load in breast milk whey of HCMV-seropositive mothers showed a unimodal course and revealed high differences in peak viral loads between individuals (104 – 106 copies/ml). Breast milk whey of only one of 18 HCMV-seropositive mothers did not contain reactivated virus during the observation period. The humoral immune response against HCMV assessed as IgG in breast milk was very low. However, six of 18 mothers showed an increase of HCMV-specific IgGs after peak viral load. Five of 18 mothers had no measurable HCMV-IgG concentrations in breast milk using an electro-chemiluminescence immunoassay. Nevertheless, recomLine blots for six prominent HCMV antigens (IE1; CM2, fusion protein of pUL44 and UL57; p150; p65 and gB glycoprotein 1 and 2) detected anti-p150-IgG reactivity with intensities at least around the cut-off level in all breast milk samples. Anti-gB1 IgG was detected in all plasma samples, but only in 5 of 18 milk samples. Plasma HCMV-IgG titers increased over the analyzed time range. Immunomonitoring of mothers revealed a significant increase of CD3+ T cells in breast milk of HCMV-seropositive but not seronegative mothers over time. The CD4+/CD8+ T cell ratio was significantly lower in breast milk of HCMV-seropositive than seronegative mothers. Additionally, most T cells were activated (HLA-DR+) memory T cells, which increased over time. Naïve T cells were present only in very limited numbers in breast milk. HCMV-specific T cells were measured by quantifying CD8+ T cells binding to pp65 and IE1 peptides using MHC class I tetramers. Most breast milk samples displayed slightly higher frequencies of HCMV-specific T cells than the corresponding blood samples. The analysis of 92 inflammatory cytokines revealed elevated levels of CC- and CXC- chemokines in HCMV-seropositive compared to -seronegative mothers’ breast milk. The humoral IgG immune response in breast milk of most HCMV-seropositive mothers resulted in no evidence of interrelation to the observed decreasing viral load in the milk during the unimodal course of reactivation. However, a compartmentalization in the mammary gland regarding immune cells, such as CD3+ T cells and activated (HLA-DR+) CD8+ T cells, was observed. These immune cells showed increasing frequencies in breast milk over time compared to corresponding blood samples or seronegative mothers’ breast milk and might therefore contribute to the decrease of HCMV loads in breast milk after the peak

Frequencies of activated T cell populations increase in breast milk of HCMV-seropositive mothers during local HCMV reactivation

BackgroundHuman cytomegalovirus (HCMV) can reactivate in the mammary gland during lactation and is shed into breast milk of nearly every HCMV-IgG-seropositive mother of a preterm infant. Dynamics of breast milk leukocytes during lactation, as well as blood leukocytes and the comparison between both in the context of HCMV reactivation is not well understood.MethodsHere, we present the BlooMil study that aimed at comparing changes of immune cells in blood and breast milk from HCMV-seropositive- vs -seronegative mothers, collected at four time ranges up to two months post-partum. Viral load was monitored by qPCR and nested PCR. Multiparameter flow cytometry was used to identify leukocyte subsets.ResultsCD3+ T cell frequencies were found to increase rapidly in HCMV-seropositive mothers’ milk, while they remained unchanged in matched blood samples, and in both blood and breast milk of HCMV-seronegatives. The activation marker HLA-DR was more strongly expressed on CD4+ and CD8+ T cells in all breast milk samples than matched blood samples, but HCMV-seropositive mothers displayed a significant increase of HLA-DR+ CD4+ and HLA-DR+ CD8+ T cells during lactation. The CD4+/CD8+ T cell ratio was lower in breast milk of HCMV-seropositive mothers than in the blood. HCMV-specific CD8+ T cell frequencies (recognizing pp65 or IE1) were elevated in breast milk relative to blood, which might be due to clonal expansion of these cells during local HCMV reactivation. Breast milk contained very low frequencies of naïve T cells with no significant differences depending on serostatus.ConclusionTaken together, we conclude that the distribution of breast milk leukocyte populations is different from blood leukocytes and may contribute to the decrease of breast milk viral load in the late phase of HCMV reactivation in the mammary gland

The Effect of Mindfulness-based Programs on Cognitive Function in Adults: A Systematic Review and Meta-analysis.

peer reviewedMindfulness-based programs (MBPs) are increasingly utilized to improve mental health. Interest in the putative effects of MBPs on cognitive function is also growing. This is the first meta-analysis of objective cognitive outcomes across multiple domains from randomized MBP studies of adults. Seven databases were systematically searched to January 2020. Fifty-six unique studies (n = 2,931) were included, of which 45 (n = 2,238) were synthesized using robust variance estimation meta-analysis. Meta-regression and subgroup analyses evaluated moderators. Pooling data across cognitive domains, the summary effect size for all studies favored MBPs over comparators and was small in magnitude (g = 0.15; [0.05, 0.24]). Across subgroup analyses of individual cognitive domains/subdomains, MBPs outperformed comparators for executive function (g = 0.15; [0.02, 0.27]) and working memory outcomes (g = 0.23; [0.11, 0.36]) only. Subgroup analyses identified significant effects for studies of non-clinical samples, as well as for adults aged over 60. Across all studies, MBPs outperformed inactive, but not active comparators. Limitations include the primarily unclear within-study risk of bias (only a minority of studies were considered low risk), and that statistical constraints rendered some p-values unreliable. Together, results partially corroborate the hypothesized link between mindfulness practices and cognitive performance. This review was registered with PROSPERO [CRD42018100904]

Monitoring of systemic and local immune signatures in response to HCMV reactivation during lactation

The human herpesvirus cytomegalovirus (HCMV) persists in the host in latent form for a lifetime and may periodically reactivate. During lactation, reactivation of HCMV occurs locally in the mammary gland without detectable systemic DNAemia. Preterm infants are at risk of infection through ingesting breast milk and may develop life-threatening disease with sepsis-like symptoms. In this thesis, the modulation of the immune signature in response to HCMV reactivation in the mammary gland, investigated non-invasively by analyzing breast milk, was compared to corresponding simultaneously drawn blood samples of HCMV-seropositive, as well as HCMV-seronegative mothers. This “BlooMil” study was performed on longitudinal samples taken from birth through 60 days postpartum over four time ranges. The viral load in breast milk whey of HCMV-seropositive mothers showed a unimodal course and revealed high differences in peak viral loads between individuals (104 – 106 copies/ml). Breast milk whey of only one of 18 HCMV-seropositive mothers did not contain reactivated virus during the observation period. The humoral immune response against HCMV assessed as IgG in breast milk was very low. However, six of 18 mothers showed an increase of HCMV-specific IgGs after peak viral load. Five of 18 mothers had no measurable HCMV-IgG concentrations in breast milk using an electro-chemiluminescence immunoassay. Nevertheless, recomLine blots for six prominent HCMV antigens (IE1; CM2, fusion protein of pUL44 and UL57; p150; p65 and gB glycoprotein 1 and 2) detected anti-p150-IgG reactivity with intensities at least around the cut-off level in all breast milk samples. Anti-gB1 IgG was detected in all plasma samples, but only in 5 of 18 milk samples. Plasma HCMV-IgG titers increased over the analyzed time range. Immunomonitoring of mothers revealed a significant increase of CD3+ T cells in breast milk of HCMV-seropositive but not seronegative mothers over time. The CD4+/CD8+ T cell ratio was significantly lower in breast milk of HCMV-seropositive than seronegative mothers. Additionally, most T cells were activated (HLA-DR+) memory T cells, which increased over time. Naïve T cells were present only in very limited numbers in breast milk. HCMV-specific T cells were measured by quantifying CD8+ T cells binding to pp65 and IE1 peptides using MHC class I tetramers. Most breast milk samples displayed slightly higher frequencies of HCMV-specific T cells than the corresponding blood samples. The analysis of 92 inflammatory cytokines revealed elevated levels of CC- and CXC- chemokines in HCMV-seropositive compared to -seronegative mothers’ breast milk. The humoral IgG immune response in breast milk of most HCMV-seropositive mothers resulted in no evidence of interrelation to the observed decreasing viral load in the milk during the unimodal course of reactivation. However, a compartmentalization in the mammary gland regarding immune cells, such as CD3+ T cells and activated (HLA-DR+) CD8+ T cells, was observed. These immune cells showed increasing frequencies in breast milk over time compared to corresponding blood samples or seronegative mothers’ breast milk and might therefore contribute to the decrease of HCMV loads in breast milk after the peak

Human cytomegalovirus reactivation during lactation: impact of antibody kinetics and neutralization in blood and breast milk

Human cytomegalovirus (HCMV) is shed into breast milk in nearly every seropositive woman during lactation. This reactivation shows mostly a self-limited, unimodal course. The dynamics and functional role of HCMV-specific-IgG in breast milk and in plasma during reactivation are unknown. Milk whey viral loads were monitored with real-time PCR in 18 HCMV-seropositive mothers over two months postpartum. HCMV-antibody binding assays (ECLIA) and antigen-specific immunoblotting were performed from plasma and corresponding milk samples. Epithelial-cell-specific neutralization was used to analyze functional antibodies in plasma- and whey-pools. Viral loads in milk whey showed unimodal courses in 15 of 18 mothers with peak viral loads around one month postpartum. HCMV-specific-IgG-antibodies increased significantly in plasma and milk whey during reactivation. The mean levels of plasma IgG were about 275-fold higher than in whey. Only antibodies against tegument protein p150 were continuously expressed in both compartments. Anti-glycoprotein-B1 IgG-antibodies were variably expressed in whey, but continuously in plasma. Neutralization assays showed 40-fold higher NT-50 values in plasma compared to whey at two months postpartum. During reactivation, HCMV-specific-IgG reactivities and neutralizing capacities are much lower in whey than in plasma. Therefore, their specific role in the decrease and discontinuation of virus-shedding in milk remains unclear

Human Cytomegalovirus (HCMV) Reactivation in the Mammary Gland Induces a Proinflammatory Cytokine Shift in Breast Milk

A locally restricted human cytomegalovirus (HCMV) reactivation in the mammary gland commonly occurs in nearly every IgG-seropositive breastfeeding mother. This unique phenomenon can therefore be used to study the reactivation process in an immunocompetent healthy host. Breast milk contains a variety of immunoactive compounds, including immune cells, antibodies, growth factors, and cytokines supporting the newborn’s immature immune system. To characterize the impact of HCMV reactivation on breast milk cytokines, we analyzed longitudinal breast milk samples of four IgG-seropositive and three IgG-seronegative mothers of preterm infants using Proximity Extension Assay (PEA) technology (Olink Proteomics, Uppsala, Sweden). Cytokine profiling revealed elevated cytokine levels in IgG-seropositive mothers’ milk whey. Reactivating mothers showed higher levels of CC-chemokines (MCP-2, CCL19, and CCL20) and CXC-chemokines (IL-8, CXCL9, CXCL10, and CXCL11), such as the proinflammatory cytokine IL-17C, glycoprotein CD5, and TNFSF14. HCMV reactivation seems to influence the cytokine profile in human breast milk. This work could open the door for further studies analyzing distinct relations of the cytokine network as well as phenotypical and functional T cell properties in background of HCMV DNA dynamics in early lactation

Immunomonitoring of Human Breast Milk Cells During HCMV-Reactivation

BACKGROUND: Breast milk leukocytes may play a role in protecting the infant from pathogens. The dynamics and the role of lymphocytes in human cytomegalovirus (HCMV)-seropositive mothers shedding HCMV into breast milk during the first months postpartum (p.p.) are mostly unclear. METHODS: Breast milk cells were analyzed by Pappenheim panoptic and alpha-naphthyl acetate esterase staining as well as by imaging and polychromatic flow cytometry to simultaneously establish their morphological and phenotypic properties. The latter were characterized in HCMV-seropositive and seronegative mothers´ breast milk cells at different time points p.p. RESULTS: Panoptic staining of breast milk cells revealed the presence of monocytes/macrophages, granulocytes and lymphocytes. Imaging flow cytometry data combining phenotypic and morphological analysis identified NKT-like cells, NK cells, epithelial cells, T cells and monocytes/macrophages. HCMV-seropositive but not -seronegative mothers had significantly higher T cell frequencies in mature milk. CONCLUSIONS: The presence of lymphocyte subsets in breast milk may be more influenced by the HCMV-seropositivity of the mother than previously recognized