Bifidobacteria-mediated immune system imprinting early in life.

Immune-microbe interactions early in life influence the risk of allergies, asthma, and other inflammatory diseases. Breastfeeding guides healthier immune-microbe relationships by providing nutrients to specialized microbes that in turn benefit the host's immune system. Such bacteria have co-evolved with humans but are now increasingly rare in modern societies. Here we show that a lack of bifidobacteria, and in particular depletion of genes required for human milk oligosaccharide (HMO) utilization from the metagenome, is associated with systemic inflammation and immune dysregulation early in life. In breastfed infants given Bifidobacterium infantis EVC001, which expresses all HMO-utilization genes, intestinal T helper 2 (Th2) and Th17 cytokines were silenced and interferon ß (IFNß) was induced. Fecal water from EVC001-supplemented infants contains abundant indolelactate and B. infantis-derived indole-3-lactic acid (ILA) upregulated immunoregulatory galectin-1 in Th2 and Th17 cells during polarization, providing a functional link between beneficial microbes and immunoregulation during the first months of life.

Stereotypic Immune System Development in Newborn Children.

Epidemiological data suggest that early life exposures are key determinants of immune-mediated disease later in life. Young children are also particularly susceptible to infections, warranting more analyses of immune system development early in life. Such analyses mostly have been performed in mouse models or human cord blood samples, but these cannot account for the complex environmental exposures influencing human newborns after birth. Here, we performed longitudinal analyses in 100 newborn children, sampled up to 4 times during their first 3 months of life. From 100 µL of blood, we analyze the development of 58 immune cell populations by mass cytometry and 267 plasma proteins by immunoassays, uncovering drastic changes not predictable from cord blood measurements but following a stereotypic pattern. Preterm and term children differ at birth but converge onto a shared trajectory, seemingly driven by microbial interactions and hampered by early gut bacterial dysbiosis.

Effect of CTLA4-Ig (abatacept) treatment on T cells and B cells in peripheral blood of patients with polymyositis and dermatomyositis.

We aimed to evaluate in vivo effects of abatacept on phenotypes of T and B cells in the circulation of myositis patients in a sub-study of the ARTEMIS trial. Twelve patients with paired frozen PBMCs before and after 6-month abatacept treatment were included in this sub-study where mass cytometry (CyTOF) was chosen as a technology to be tested for its utility in a real-life clinical immune monitoring setting. Using CyTOF, the peripheral T cell phenotypes demonstrated considerable variation over time and between individuals precluding the identification of treatment-specific changes. We therefore conclude that studies of patient cohorts displaying wide clinical heterogeneity using mass cytometry must be relatively large in order to be suited for discovery research and immune monitoring. Still, we did find some correlations with functional muscle outcome, namely positive correlations between the ratio of CD4+ T cells and CD8+ T cells (CD4/CD8) in peripheral blood samples both at baseline and after treatment with muscle endurance improvement as assessed by the functional index-2 (FI-2) test. Our data suggest that the CD4/CD8 ratio in circulation at time of active disease may be a predictor of treatment efficacy in myositis patients.

YM155, a small molecule inhibitor of survivin expression, sensitizes cancer cells to hypericin-mediated photodynamic therapy.

Photodynamic therapy (PDT) represents a rapidly developing alternative treatment for various types of cancers. Although considered highly effective, cancer cells can exploit various mechanisms, including the upregulation of apoptosis inhibitors, to overcome the cytotoxic effect of PDT. Survivin, a member of the inhibitor of apoptosis protein family, is known to play a critical role in cancer progression and therapeutic resistance and therefore represents a potential therapeutic target. The aim of this study was to investigate whether YM155, a small molecule inhibitor of survivin expression, can potentiate the cytotoxic effect of hypericin-mediated PDT (HY-PDT). Accordingly, two cell lines resistant to HY-PDT, HT-29 (colorectal adenocarcinoma) and A549 (lung adenocarcinoma), were treated either with HY-PDT alone or in combination with YM155. The efficacy of different treatment regimens was assessed by MTT assay, flow cytometry analysis of metabolic activity, viability, phosphatidylserine externalisation, mitochondrial membrane potential and caspase-3 activity and immunoblotting for the cleavage of poly (ADP-ribose) polymerase (PARP). Here we show for the first time that the repression of survivin expression by YM155 is effective in sensitizing HT-29 and A549 cells to HY-PDT, as measured by the decrease in cell viability and induction of apoptosis. Combined treatment with hypericin and YM155 led to a more severe dissipation of the mitochondrial membrane potential and caused an increase in caspase-3 activation and subsequent PARP cleavage. Our results demonstrate that the repression of survivin expression by YM155 potentially represents a novel alternative strategy to increase the efficacy of HY-PDT in cancer cells that are otherwise weakly responsive or non-responsive to treatment.

Tacrine derivatives as dual topoisomerase I and II catalytic inhibitors.

This study examines the binding properties of a series of newly synthetized tacrine derivatives 1-4 and their anticancer effects. Spectroscopic techniques (UV-Vis, fluorescence spectroscopy, thermal denaturation, and linear spectropolarimetry) and viscometry were used to study DNA binding properties and to determine the types of DNA interaction with the studied derivatives. The binding constants for the complexes with DNA were obtained using UV-Vis spectroscopic titrations (K = 1.6 × 10(4)-4.0 × 10(5) M(-1)) and electrophoretic methods were used to determine the effect of the derivatives on topoisomerase I and II activity. Monotacrine derivative 1 showed evidence of topoisomerase Irelaxation activity at a concentration of 30 × 10(-6) M, while bistacrine derivatives 2-4 produced a complete inhibition of topoisomerase Iat a concentration of 5 × 10(-6) M. The biological activities of the derivatives were studied using MTT-assay and flow cytometric methods (detection of mitochondrial membrane potential and measurement of cell viability) following incubation of 24 and 48 h with human leukemic cancer cell line HL60. The ability of the derivatives to impair cell proliferation was also tested through the analysis of cell cycle distribution.

Flow cytometric determination of 5-bromo-2'-deoxyuridine pharmacokinetics in blood serum after intraperitoneal administration to rats and mice.

5-Bromo-2'-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.

Novel trisubstituted acridines as human telomeric quadruplex binding ligands.

A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV-vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the "braco"-like derivatives was to arrest the cells in the S- and G2M-phases of the cell cycle within the first 72h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.

[Separation of sperm by micromanipulator from unusual forensic sample - case report]. / Separace spermií pomocí mikromanipulátoru z neobvyklého forenzního vzorku - kazuistika.

The aim of this study is to provide an information about the method of processing of unusual forensic sample that was submitted for the sexual assault case. We analyzed microscopic sample of vaginal swab stained using the hematoxylin-eosin mixture. After removing the covering glass we failed to collect the sperm cells to the micromanipulator capillary. The cells even started, due to the mechanical stress, to fall apart. We think that the main reason of the microdissection failure is the advanced cell lysis. Due to the negative results of the DNA analysis we defined a set of preventive and corrective actions that would (in case of application) increase the chance of positive identification in similar sexual assault cases.

Achieving symptom relief in patients with myalgic encephalomyelitis by targeting the neuro-immune interface and optimizing disease tolerance.

Myalgic encephalomyelitis (ME) previously also known as chronic fatigue syndrome is a heterogeneous, debilitating syndrome of unknown etiology responsible for long-lasting disability in millions of patients worldwide. The most well-known symptom of ME is post-exertional malaise, but many patients also experience autonomic dysregulation, cranial nerve dysfunction and signs of immune system activation. Many patients also report a sudden onset of disease following an infection. The brainstem is a suspected focal point in ME pathogenesis and patients with structural impairment to the brainstem often show ME-like symptoms. The brainstem is also where the vagus nerve originates, a critical neuro-immune interface and mediator of the inflammatory reflex which regulate systemic inflammation. Here, we report the results of a randomized, placebo-controlled trial using intranasal mechanical stimulation targeting nerve endings in the nasal cavity, likely from the trigeminal nerve, possibly activating additional centers in the brainstem of ME patients and correlating with a ∼30% reduction in overall symptom scores after 8 weeks of treatment. By performing longitudinal, systems-level monitoring of the blood immune system in these patients, we uncover signs of chronic immune activation in ME, as well as immunological correlates of improvement that center around gut-homing immune cells and reduced inflammation. The mechanisms of symptom relief remain to be determined, but transcriptional analyses suggest an upregulation of disease tolerance mechanisms. We believe that these results are suggestive of ME as a condition explained by a maladaptive disease tolerance response following infection.

Lower sensitivity of FHC fetal colon epithelial cells to photodynamic therapy compared to HT-29 colon adenocarcinoma cells despite higher intracellular accumulation of hypericin.

Preferential uptake of photosensitizer by tumour tissue is an elementary prerequisite of effective and successful photodynamic therapy (PDT). Therefore intracellular concentration of photosensitizer is one of the limiting factors affecting PDT efficiency. Hypericin (HY) has found applications in photodynamic diagnostics solely due to its high specificity for tumour cells and tissues. However, here we suggest that not only HY uptake, but importantly also the cell ability to manage oxidative stress induced by HY-PDT can be important decisive factors finally affecting the cell death response. We showed that despite the higher accumulation of HY in FHC human fetal colon epithelial cells compared to HT-29 colon adenocarcinoma cells, the cytotoxic effects of this photosensitizer were more pronounced in the latter cell line, and this was associated with enhanced accumulation of HY-PDT-induced reactive oxygen species (ROS).

PUFAs enhance oxidative stress and apoptosis in tumour cells exposed to hypericin-mediated PDT.

Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin.

Human Immune System Variation during 1 Year.

The human immune system varies extensively between individuals, but variation within individuals over time has not been well characterized. Systems-level analyses allow for simultaneous quantification of many interacting immune system components and the inference of global regulatory principles. Here, we present a longitudinal, systems-level analysis in 99 healthy adults 50 to 65 years of age and sampled every third month for 1 year. We describe the structure of interindividual variation and characterize extreme phenotypes along a principal curve. From coordinated measurement fluctuations, we infer relationships between 115 immune cell populations and 750 plasma proteins constituting the blood immune system. While most individuals have stable immune systems, the degree of longitudinal variability is an individual feature. The most variable individuals, in the absence of overt infections, exhibited differences in markers of metabolic health suggestive of a possible link between metabolic and immunologic homeostatic regulation.

Systems-Level Immunomonitoring from Acute to Recovery Phase of Severe COVID-19.

Severe disease of SARS-CoV-2 is characterized by vigorous inflammatory responses in the lung, often with a sudden onset after 5-7 days of stable disease. Efforts to modulate this hyperinflammation and the associated acute respiratory distress syndrome rely on the unraveling of the immune cell interactions and cytokines that drive such responses. Given that every patient is captured at different stages of infection, longitudinal monitoring of the immune response is critical and systems-level analyses are required to capture cellular interactions. Here, we report on a systems-level blood immunomonitoring study of 37 adult patients diagnosed with COVID-19 and followed with up to 14 blood samples from acute to recovery phases of the disease. We describe an IFNγ-eosinophil axis activated before lung hyperinflammation and changes in cell-cell co-regulation during different stages of the disease. We also map an immune trajectory during recovery that is shared among patients with severe COVID-19.

Integration of molecular profiles in a longitudinal wellness profiling cohort.

An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies and immune cell profiling, complemented with gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine.

The role of p53 in the efficiency of photodynamic therapy with hypericin and subsequent long-term survival of colon cancer cells.

Photodynamic therapy with hypericin (HY-PDT) is known as an efficient modality for treatment of various cancerous and non-cancerous diseases. Although the role of p53 protein in cell death signaling is well established, relatively little is known of its impact on the efficiency of HY-PDT. Comparison of sensitivity and long-term survival of p53-null versus wt-p53-expressing HCT-116 cells is reported here. The lack of p53 function did not affect cell proliferation or attenuate the initial phases of programmed cell death. However, analyses of apoptosis in the final stages revealed suppression of its incidence and delayed activation of caspase-3 in p53-null cells. Significantly higher clonogenic ability, especially in hypoxia, was identified in the case of p53-null cells. Induction of Mcl-1 and Bax levels were more prominent in wt-pt53 cells. Interestingly, the level of Bcl-2 did not react to HY-PDT at all, in both cell lines. Bringing the evidence together, we prove that despite insignificant impact on overall toxicity, expression of p53 affects the clonogenic efficiency of HCT-116 cells. Since destruction of tumor tissue and its vascular system by PDT tends to lead to hypoxia, superior survival of p53-deficient tumor cells under given conditions might result in recurrence of cancer diseases.

Drug efflux transporters, MRP1 and BCRP, affect the outcome of hypericin-mediated photodynamic therapy in HT-29 adenocarcinoma cells.

Photodynamic therapy (PDT) is a flexible multi-target therapeutic approach. One of the main requirements of successful PDT is sufficient intracellular concentration of an applicable photosensitizer. Mechanisms of anticancer drug elimination by tumour cells are mostly linked to the elevated expression and activity of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP) and P450 monooxygenases. The interaction of hypericin with this cell drug-defence system is still unclear. We report here for the first time increased activity of MRP1 and BCRP in HT-29 colon cancer cells treated with hypericin per se. On the contrary, pre-treatment with proadifen (SKF525A) affected the function of MRP1 and BCRP leading to increased hypericin content, which might indicate a possible link between proadifen and these ABC transporter proteins. Subsequent enhanced intracellular oxidative stress was accompanied by loss of mitochondrial membrane potential, activation of caspase-9 and -3, PARP cleavage and onset of apoptosis. In conclusion, our study suggests that drug efflux transporters MRP1 and BCRP affect the pharmacokinetics of hypericin in HT-29 colon adenocarcinoma cells, and the action of hypericin-mediated PDT (HY-PDT) should be modulated by pre-treatment with their specific inhibitors.

Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells.

Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin.

Automated Cell Processing for Mass Cytometry Experiments.

Mass cytometry is a powerful technology for high-dimensional single-cell measurements in millions of individual cells. Antibodies and other detection probes are coupled to elemental tags, each with a unique mass and detectable at single-cell resolution using an ICP-MS type of instrument. Given the sensitivity of the detection system, any free metal ions must be carefully removed through multiple rounds of washing in order to prevent background signal. This results in significant loss of cells. Together with cells lost during acquisition, the final data can represent as little as 10% of the starting material, seriously limiting the amount of information that can be extracted from small samples. Furthermore, complex staining protocols introduce experimental variations that limit comparisons across experiments. Here we present a cell processing and staining procedure for mass cytometry fully automated using a liquid handling robotic system and we present measures taken to optimize all steps of the protocol. These advances are applicable to both manual and automated protocols and provide a six-fold higher cell yield as compared to a standard protocol. With this increased yield and improved reproducibility this protocol now allows us to perform mass cytometry analysis using as little as 100 µL of whole blood as starting material.

Mass cytometry identifies distinct CD4+ T cell clusters distinguishing HIV-1-infected patients according to antiretroviral therapy initiation.

Recent guidelines recommend antiretroviral therapy (ART) to be administered as early as possible during HIV-1 infection. Few studies addressed the immunological benefit of commencing ART during the acute phase of infection. We used mass cytometry to characterize blood CD4+ T cells from HIV-1-infected patients who initiated ART during acute or chronic phase of infection. Using this method, we analyzed a large number of markers on millions of individual immune cells. The results revealed that CD4+ T cell clusters with high expression of CD27, CD28, CD127, and CD44, whose function involves T cell migration to inflamed tissues and survival, are more abundant in healthy controls and patients initiating ART during the acute phase; on the contrary, CD4+ T cell clusters in patients initiating ART during the chronic phase had reduced expression of these markers. The results are suggestive of a better preserved immune function in HIV-1-infected patients initiating ART during acute infection.

The repertoire of maternal anti-viral antibodies in human newborns.

All circulating immunoglobulin G (IgG) antibodies in human newborns are of maternal origin1 and transferred across the placenta to provide passive immunity until newborn IgG production takes over 15 weeks after birth2. However, maternal IgG can also negatively interfere with newborn vaccine responses3. The concentration of IgG increases sharply during the third trimester of gestation and children delivered extremely preterm are believed to largely lack this passive immunity1,2,4. Antibodies to individual viruses have been reported5-12, but the global repertoire of maternal IgG, its variation in children, and the epitopes targeted are poorly understood. Here, we assess antibodies against 93,904 epitopes from 206 viruses in 32 preterm and 46 term mother-child dyads. We find that extremely preterm children receive comparable repertoires of IgG as term children, albeit at lower absolute concentrations and consequent shorter half-life. Neutralization of the clinically important respiratory syncytial virus (RS-virus) was also comparable until three months of age. These findings have implications for understanding infectious disease susceptibility, vaccine development, and vaccine scheduling in newborn children.