Preservation of vaccine-induced long-term B cell memory and the effects of immunosuppressive treatment

Immune memory after vaccination is largely dependent on the combination of antibody production from long-lived plasma cells, and a supporting pool of antigen-primed memory B cells. It has been observed that individuals with certain immunosuppressive conditions or treatments have a weakened B cell memory, but the mechanisms behind remain elusive. The aim of this thesis was to evaluate B cell immunity in healthy children, and how HIV-1 infection, antineoplastic therapy, and rheumatic disease and treatment can impact on various features of B cell memory induction and maintenance. In paper I, we explored the hyperactivation of B cells observed in patients carrying HIV-1 infection, and showed that it can be partly induced by ligation of soluble cleaved CD27 to CD70 on the surface of memory B cells. In paper II, we aimed at comparing the establishment of serum antibody titers and memory B cells after vaccination against measles and rubella in healthy children. We found that the memory B cell pool remained stable also early after vaccination, whereas the corresponding serum IgG titers decayed with time. In contrast, both the serum IgG levels and frequency of blood memory B cells in healthy young adults appeared stable. This implied that the antibody production and memory B cell compartment are two separate entities with independent regulation, and that it takes longer time to establish a stable pool of circulating antibodies. How these two parts of B cell memory are affected by immunosuppressive disease and treatment was addressed in papers III and IV. In paper III, we used a rhesus macaque model for high-dose Doxorubicin treatment, and concluded that the established vaccine-induced memory B cell pool was depleted, contrary to long-lived plasma cells and the resulting serum IgG titers. These observations supported the finding of independent regulation of the two B cell memory compartments, and revealed different sensitivity to chemotherapy. The bone marrow plasma cell niche was additionally studied in an in vitro model for plasma cell – stromal cell cross talk, where we discovered that in vivo relevant concentrations of Doxorubicin could hamper the output of pivotal survival factors from stromal cells. In paper IV, we examined memory B cells and circulating IgG titers in children with rheumatic disease, treated with low-dose Methotrexate and TNF-α inhibition. We noted that serum IgG titers against tetanus were lower in rheumatic patients than in healthy controls, and that patients who had only received one measles vaccine dose had lower levels of measles-specific memory B cells. This stresses the importance for children with rheumatic disease and treatment to follow the full vaccine schedule. To summarize, this thesis has contributed to enhanced knowledge on how B cell memory is induced, preserved and at risk of disruption by common immune disorders and treatment. Hopefully, our findings can aid future improvement of functional vaccine regimes for immunocompromised children

Assessing Cumulative Health Risks from Exposure to Environmental Mixtures—Three Fundamental Questions

Differential exposure to mixtures of environmental agents, including biological, chemical, physical, and psychosocial stressors, can contribute to increased vulnerability of human populations and ecologic systems. Cumulative risk assessment is a tool for organizing and analyzing information to evaluate the probability and seriousness of harmful effects caused by either simultaneous and/or sequential exposure to multiple environmental stressors. In this article we focus on elucidating key challenges that must be addressed to determine whether and to what degree differential exposure to environmental mixtures contributes to increased vulnerability of exposed populations. In particular, the emphasis is on examining three fundamental and interrelated questions that must be addressed as part of the process to assess cumulative risk: a) Which mixtures are most important from a public health perspective? and b) What is the nature (i.e., duration, frequency, timing) and magnitude (i.e., exposure concentration and dose) of relevant cumulative exposures for the population of interest? c) What is the mechanism (e.g., toxicokinetic or toxicodynamic) and consequence (e.g., additive, less than additive, more than additive) of the mixture’s interactive effects on exposed populations? The focus is primarily on human health effects from chemical mixtures, and the goal is to reinforce the need for improved assessment of cumulative exposure and better understanding of the biological mechanisms that determine toxicologic interactions among mixture constituents

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.Peer reviewe

MOVING TOWARD GENETIC PROFILING IN PATIENT CARE: THE SCOPE AND RATIONALE OF PHARMACOGENETIC/ECOGENETIC INVESTIGATION

This paper is available online at http://dmd.aspetjournals.org The topic of genetic profiling in patient care was recently reviewed in Nature Biotechnology under the title "Laying the foundations for personalised medicines" Genetic variability and selection directed toward the characters of the individuals are considered to be the fundament of the evolution and adaptation of living organisms to all environments where they are able to exist. The interplay between genetic constitution and other factors has consistently been emphasized. The true meaning of "heritage" is sometimes misunderstood. The fact that a character is inherited or has a genetic predisposition does not mean that it has to penetrate into the phenotype of the next generation or that parents necessarily need to share a quality that is obvious in their children. Many characters may be conceived as continuous variables, and among these are diverse physical and intellectual capacities, talents for art, etc. However, they all go back to the genetic code and will only appear as continuous or even gaussian variables if a large enough number of factors is involved in their control and a large enough number of individuals is studied. When investigated in detail, however, most characters will show skewness or separation into different modes. This can be explained by the influence of particularly strong factors such as monogenic or oligogenic coding systems or the influence of singular environmental factors. Many examples of characteristics such as eye color, blood groups, tissue antigens, etc. show discrete variation into separate groups. This is also true for certain drug-metabolizing enzymes. An early observation was that isoniazid might be slowly or rapidly acetylated The most important drug-metabolizing enzyme family is the cytochrome P450 system. It comprises several enzymes that show distinct but partially overlapping substrate specificity Tricyclic antidepressants were early found to display vast interindividual variability in steady-state plasma concentrations. The Debrisoquine/Sparteine Hydroxylation Polymorphism (CYP2D6) Debrisoquine was launched as an antihypertensive agent but is no longer on the market. It was found to induce orthostatic hypotension in a small percentage of healthy volunteers who took the drug for investigational purposes. The reason for the exaggerated effect in these subjects was found to be the lack of an enzyme almost exclusively responsible for the metabolic elimination of debrisoquine, and the affected subjects were classified as poor metabolizers of debrisoquine The character of being a poor (PM) or an extensive metabolizer (EM) of debrisoquine is controlled as an autosomal, recessive monogenic trait with the PM phenotype being the recessive alternative. The genetic heritability of the debrisoquine hydroxylation phenotype is very high (79%), while only 6% of all variability of the debrisoquine metabolic ratio could be ascribed to environmental or cultural factors (Steiner et al.

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.2 +/- 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH- deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH- deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH- deer mice were 2.0 +/- 0.1 and 2.3 +/- 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH- deer mice in addition to the dehydrogenase systems

Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation

Hepatocytes are dynamic cells that, upon injury, can alternate between nondividing differentiated and dedifferentiated proliferating states in vivo . However, in two‐dimensional cultures, primary human hepatocytes (PHHs) rapidly dedifferentiate, resulting in loss of hepatic functions that significantly limits their usefulness as an in vitro model of liver biology, liver diseases, as well as drug metabolism and toxicity. Thus, understanding the underlying mechanisms and stalling of the dedifferentiation process would be highly beneficial to establish more‐accurate and relevant long‐term in vitro hepatocyte models. Here, we present comprehensive analyses of whole proteome and transcriptome dynamics during the initiation of dedifferentiation during the first 24 hours of culture. We report that early major rearrangements of the noncoding transcriptome, hallmarked by increased expression of small nucleolar RNAs, long noncoding RNAs, microRNAs (miRNAs), and ribosomal genes, precede most changes in coding genes during dedifferentiation of PHHs, and we speculated that these modulations could drive the hepatic dedifferentiation process. To functionally test this hypothesis, we globally inhibited the miRNA machinery using two established chemically distinct compounds, acriflavine and poly‐l ‐lysine. These inhibition experiments resulted in a significantly impaired miRNA response and, most important, in a pronounced reduction in the down‐regulation of hepatic genes with importance for liver function. Thus, we provide strong evidence for the importance of noncoding RNAs, in particular, miRNAs, in hepatic dedifferentiation, which can aid the development of more‐efficient differentiation protocols for stem‐cell‐derived hepatocytes and broaden our understanding of the dynamic properties of hepatocytes with respect to liver regeneration. Conclusion: miRNAs are important drivers of hepatic dedifferentiation, and our results provide valuable information regarding the mechanisms behind liver regeneration and possibilities to inhibit dedifferentiation in vitro

The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids

There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human in vitro disease model to understand mechanisms linked to genetic risk variants associated with NAFLD. The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD