Tocilizumab in COVID-19 therapy: who benefits, and how?

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Novel decay dynamics revealed for virus-mediated drug activation in cytomegalovirus infection

Human cytomegalovirus (CMV) infection is a substantial cause of morbidity and mortality in immunocompromised hosts and globally is one of the most important congenital infections. The nucleoside analogue ganciclovir (GCV), which requires initial phosphorylation by the viral UL97 kinase, is the mainstay for treatment. To date, CMV decay kinetics during GCV therapy have not been extensively investigated and its clinical implications not fully appreciated. We measured CMV DNA levels in the blood of 92 solid organ transplant recipients with CMV disease over the initial 21 days of ganciclovir therapy and identified four distinct decay patterns, including a new pattern exhibiting a transient viral rebound (Hump) following initial decline. Since current viral dynamics models were unable to account for this Hump profile, we developed a novel multi-level model, which includes the intracellular role of UL97 in the continued activation of ganciclovir, that successfully described all the decline patterns observed. Fitting the data allowed us to estimate ganciclovir effectiveness in vivo (mean 92%), infected cell half-life (mean 0.7 days), and other viral dynamics parameters that determine which of the four kinetic patterns will ensue. An important clinical implication of our results is that the virological efficacy of GCV operates over a broad dose range. The model also raises the possibility that GCV can drive replication to a new lower steady state but ultimately cannot fully eradicate it. This model is likely to be generalizable to other anti-CMV nucleoside analogs that require activation by viral enzymes such as UL97 or its homologues

In Vivo Dynamical Interactions between CD4 Tregs, CD8 Tregs and CD4+CD25− Cells in Mice

BACKGROUND: Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1--a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4(+)CD25(-) cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity. CONCLUSIONS/SIGNIFICANCE: In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work

Changes in anti-viral effectiveness of interferon after dose reduction in chronic hepatitis C patients: a case control study

BACKGROUND: High dose interferon induction treatment of hepatitis C viral infection blocks viral production over 95%. Since dose reduction is often performed due to clinical considerations, the effect of dose reduction on hepatitis C virus kinetics was studied. METHODS: A new model that allowed longitudinal changes in the parameters of viral dynamics was used in a group of genotype-1 patients (N = 15) with dose reduction from 10 to 3 million units of interferon daily in combination with ribavirin, in comparison to a control group (N = 9) with no dose reduction. RESULTS: Dose reduction gave rise to a complex viral kinetic pattern, which could be only explained by a decrease in interferon effectiveness in blocking virion production. The benefit of the rapid initial viral decline following the high induction dose is lost after dose reduction. In addition, in some patients also the second phase viral decline slope, which is highly predictive of success of treatment, was impaired by the dose reduction resulting in smaller percentage of viral clearance in the dose reduction group. CONCLUSIONS: These findings, while explaining the failure of many induction schedules, suggest that for genotype-1 patients induction therapy should be continued till HCVRNA negativity in serum in order to increase the sustained response rate for chronic hepatitis C

Combining 16S sequencing and qPCR quantification reveals Staphylococcus aureus driven bacterial overgrowth in the skin of severe atopic dermatitis patients

Atopic dermatitis (AD) is an inflammatory skin disease with a microbiome dysbiosis towards a high relative abundance of Staphylococcus aureus. However, information is missing on the actual bacterial load on AD skin, which may affect the cell number driven release of pathogenic factors. Here, we combined the relative abundance results obtained by next-generation sequencing (NGS, 16S V1-V3) with bacterial quantification by targeted qPCR (total bacterial load = 16S, S. aureus = nuc gene). Skin swabs were sampled cross-sectionally (n = 135 AD patients; n = 20 healthy) and longitudinally (n = 6 AD patients; n = 6 healthy). NGS and qPCR yielded highly inter-correlated S. aureus relative abundances and S. aureus cell numbers. Additionally, intra-individual differences between body sides, skin status, and consecutive timepoints were also observed. Interestingly, a significantly higher total bacterial load, in addition to higher S. aureus relative abundance and cell numbers, was observed in AD patients in both lesional and non-lesional skin, as compared to healthy controls. Moreover, in the lesional skin of AD patients, higher S. aureus cell numbers significantly correlated with the higher total bacterial load. Furthermore, significantly more severe AD patients presented with higher S. aureus cell number and total bacterial load compared to patients with mild or moderate AD. Our results indicate that severe AD patients exhibit S. aureus driven increased bacterial skin colonization. Overall, bacterial quantification gives important insights in addition to microbiome composition by sequencing

Benchmarking MicrobIEM – a user-friendly tool for decontamination of microbiome sequencing data

Background Microbiome analysis is becoming a standard component in many scientific studies, but also requires extensive quality control of the 16S rRNA gene sequencing data prior to analysis. In particular, when investigating low-biomass microbial environments such as human skin, contaminants distort the true microbiome sample composition and need to be removed bioinformatically. We introduce MicrobIEM, a novel tool to bioinformatically remove contaminants using negative controls. Results We benchmarked MicrobIEM against five established decontamination approaches in four 16S rRNA amplicon sequencing datasets: three serially diluted mock communities (108–103 cells, 0.4–80% contamination) with even or staggered taxon compositions and a skin microbiome dataset. Results depended strongly on user-selected algorithm parameters. Overall, sample-based algorithms separated mock and contaminant sequences best in the even mock, whereas control-based algorithms performed better in the two staggered mocks, particularly in low-biomass samples (≤ 106 cells). We show that a correct decontamination benchmarking requires realistic staggered mock communities and unbiased evaluation measures such as Youden’s index. In the skin dataset, the Decontam prevalence filter and MicrobIEM’s ratio filter effectively reduced common contaminants while keeping skin-associated genera. Conclusions MicrobIEM’s ratio filter for decontamination performs better or as good as established bioinformatic decontamination tools. In contrast to established tools, MicrobIEM additionally provides interactive plots and supports selecting appropriate filtering parameters via a user-friendly graphical user interface. Therefore, MicrobIEM is the first quality control tool for microbiome experts without coding experience

Seroma after simple mastectomy in breast cancer — the role of CD4+ T helper cells and the evidence as a oossible specific immune process

Seroma development after breast cancer surgery is the most common postoperative complication seen after mastectomy but neither its origin nor its cellular composition is known. To investigate the assumption of immunological significance, one of the first aims of this pilot study is to describe the cellular content of collected seroma fluids and its corresponding serum in patients with simple mastectomy after needle aspiration, as well as the serum of healthy controls. The content of red blood cells (RBC) was measured by haemato-counter analyses, and the lymphocyte identification/quantification was conducted by flow cytometry analyses in seroma fluid (SFl) and the sera of patients (PBp) as well as controls (PBc). Significantly lower numbers of RBCs were measured in SFl. Cytotoxic T cells are significantly reduced in SFl, whereas T helper (Th) cells are significantly enriched compared to PBp. Significantly higher numbers of Th2 cells were found in SFl and PBp compared to PBc. The exact same pattern is seen when analyzing the Th17 subgroup. In conclusion, in contrast to healthy controls, significantly higher Th2 and Th17 cell subgroup-mediated immune responses were measured in seroma formations and were further confirmed in the peripheral blood of breast cancer (including DCIS) patients after simple mastectomy. This could lead to the assumption of a possible immunological cause for the origin of a seroma

Interferon (IFN)-γ-Inducible Protein-10: Association with Histological Results, Viral Kinetics, and Outcome during Treatment with Pegylated IFN-α2a and Ribavirin for Chronic Hepatitis C Virus Infection

BackgroundWe investigated associations between interferon (IFN)-γ-inducible protein (IP)-10 and liver histological results, viral kinetic response, and treatment outcome in patients infected with hepatitis C virus (HCV) genotypes 1-4 MethodsPlasma IP-10 was monitored before, during, and after treatment with pegylated IFN-α2a and ribavirin in 265 HCV-infected patients ResultsIn univariate analyses, a low baseline IP-10 level was significantly associated with low baseline viral load, rapid viral response (RVR), a sustained viral response (SVR), body mass index <25 kg/m2, and less-pronounced fibrosis, inflammation, and steatosis (for HCV genotypes other than 3). When the results of the univariate analyses were included in multivariate analyses, a low plasma IP-10 level, low baseline viral load, and genotype 2 or 3 infection were independent predictors of an RVR and SVR. IP-10 levels decreased 6 weeks into treatment and remained low in patients with an SVR. By contrast, plasma levels of IP-10 rebounded in patients who had detectable HCV RNA after the completion of treatment. Using cutoff IP-10 levels of 150 and 600 pg/mL for predicting an SVR in patients infected with HCV genotype 1 yielded a specificity and sensitivity of 81% and 95%, respectively ConclusionBaseline IP-10 levels are predictive of the response to HCV treatmen

Changes in anti-viral effectiveness of interferon after dose reduction in chronic hepatitis c patients: a case control study

BACKGROUND: High dose interferon induction treatment of hepatitis C viral infection blocks viral production over 95%. Since dose reduction is often performed due to clinical considerations, the effect of dose reduction on hepatitis C virus kinetics was studied. METHODS: A new model that allowed longitudinal changes in the parameters of viral dynamics was used in a group of genotype-1 patients (N = 15) with dose reduction from 10 to 3 million units of interferon daily in combination with ribavirin, in comparison to a control group (N = 9) with no dose reduction. RESULTS: Dose reduction gave rise to a complex viral kinetic pattern, which could be only explained by a decrease in interferon effectiveness in blocking virion production. The benefit of the rapid initial viral decline following the high induction dose is lost after dose reduction. In addition, in some patients also the second phase viral decline slope, which is highly predictive of success of treatment, was impaired by the dose reduction resulting in smaller percentage of viral clearance in the dose reduction group. CONCLUSIONS: These findings, while explaining the failure of many induction schedules, suggest that for genotype-1 patients induction therapy should be continued till HCVRNA negativity in serum in order to increase the sustained response rate for chronic hepatitis C