Transcriptome organization of white blood cells through gene co-expression network analysis in a large RNA-seq dataset.

Gene co-expression network analysis enables identification of biologically meaningful clusters of co-regulated genes (modules) in an unsupervised manner. We present here the largest study conducted thus far of co-expression networks in white blood cells (WBC) based on RNA-seq data from 624 individuals. We identify 41 modules, 13 of them related to specific immune-related functions and cell types (e.g. neutrophils, B and T cells, NK cells, and plasmacytoid dendritic cells); we highlight biologically relevant lncRNAs for each annotated module of co-expressed genes. We further characterize with unprecedented resolution the modules in T cell sub-types, through the availability of 95 immune phenotypes obtained by flow cytometry in the same individuals. This study provides novel insights into the transcriptional architecture of human leukocytes, showing how network analysis can advance our understanding of coding and non-coding gene interactions in immune system cells.

Immune and glial cell alterations in the rat brain after repeated exposure to the synthetic cannabinoid JWH-018.

The use of synthetic cannabinoid receptor agonists (SCRAs) poses major psychiatric risks. We previously showed that repeated exposure to the prototypical SCRA JWH-018 induces alterations in dopamine (DA) transmission, abnormalities in the emotional state, and glial cell activation in the mesocorticolimbic DA circuits of rats. Despite growing evidence suggesting the relationship between substance use disorders (SUD) and neuroinflammation, little is known about the impact of SCRAs on the neuroimmune system. Here, we investigated whether repeated JWH-018 exposure altered neuroimmune signaling, which could be linked with previously reported central effects. Adult male Sprague-Dawley (SD) rats were exposed to JWH-018 (0.25 mg/kg, i.p.) for fourteen consecutive days, and the expression of cytokines, chemokines, and growth factors was measured seven days after treatment discontinuation in the striatum, cortex, and hippocampus. Moreover, microglial (ionized calcium-binding adaptor molecule 1, IBA-1) and astrocyte (glial fibrillary acidic protein, GFAP) activation markers were evaluated in the caudate-putamen (CPu). Repeated JWH-018 exposure induces a perturbation of neuroimmune signaling specifically in the striatum, as shown by increased levels of cytokines [interleukins (IL) -2, -4, -12p70, -13, interferon (IFN) γ], chemokines [macrophage inflammatory protein (MIP) -1α, -3α], and growth factors [macrophage colony-stimulating factor (M-CSF), vascular endothelial growth factor (VEGF)], together with increased IBA-1 and GFAP expression in the CPu. JWH-018 exposure induces persistant brain region-specific immune alterations up to seven days after drug discontinuation, which may contribute to the behavioral and neurochemical dysregulations in striatal areas that play a role in the reward-related processes that are frequently impaired in SUD.

The PPARα agonist fenofibrate reduces the cytokine imbalance in a maternal immune activation model of schizophrenia.

Maternal infections during pregnancy may increase the risk of psychiatric disorders in offspring. We recently demonstrated that activation of peroxisome proliferator-activate receptor-α (PPARα), with the clinically available agonist fenofibrate (FEN), attenuates the neurodevelopmental disturbances induced by maternal immune activation (MIA) in rat offspring. We hypothesized that fenofibrate might reduce MIA-induced cytokine imbalance using a MIA model based on the viral mimetic polyriboinosinic-polyribocytidilic acid [poly (I:C)]. By using the Bio-Plex Multiplex-Immunoassay-System, we measured cytokine/chemokine/growth factor levels in maternal serum and in the fetal brain of rats treated with fenofibrate, at 6 and 24 h after poly (I:C). We found that MIA induced time-dependent changes in the levels of several cytokines/chemokines/colony-stimulating factors (CSFs). Specifically, the maternal serum of the poly (I:C)/control (CTRL) group showed increased levels of (i) proinflammatory chemokine macrophage inflammatory protein 1-alpha (MIP-1α), (ii) tumor necrosis factor-alpha (TNF-α), the monocyte chemoattractant protein-1 (MCP-1), the macrophage (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Conversely, in the fetal brain of the poly (I:C)/CTRL group, interleukin 12p70 and MIP-1α levels were lower than in vehicle (veh)/CTRL group. Notably, MIP-1α, TNF-α, keratinocyte derived chemokine (GRO/KC), GM-CSF, and M-CSF levels were lower in the poly (I:C)/FEN than in poly (I:C)/CTRL rats, suggesting the protective role of the PPARα agonist. PPARα might represent a therapeutic target to attenuate MIA-induced inflammation.

Immune resilience despite inflammatory stress promotes longevity and favorable health outcomes including resistance to infection.

Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8+ and CD4+ T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes.

Effect of Genetic Factors, Age and Sex on Levels of Circulating Extracellular Vesicles and Platelets.

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8. These add to a signal in the 3'UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

GWAS of genetic factors affecting white blood cell morphological parameters in Sardinians uncovers influence of chromosome 11 innate immunity gene cluster on eosinophil morphology.

Few genome-wide association studies (GWAS) analyzing genetic regulation of morphological traits of white blood cells have been reported. We carried out a GWAS of 12 morphological traits in 869 individuals from the general population of Sardinia, Italy. These traits, included measures of cell volume, conductivity and light scatter in four white-cell populations (eosinophils, lymphocytes, monocytes, neutrophils). This analysis yielded seven statistically significant signals, four of which were novel (four novel, PRG2, P2RX3, two of CDK6). Five signals were replicated in the independent INTERVAL cohort of 11 822 individuals. The most interesting signal with large effect size on eosinophil scatter (P-value = 8.33 x 10-32, beta = -1.651, se = 0.1351) falls within the innate immunity cluster on chromosome 11, and is located in the PRG2 gene. Computational analyses revealed that a rare, Sardinian-specific PRG2:p.Ser148Pro mutation modifies PRG2 amino acid contacts and protein dynamics in a manner that could possibly explain the changes observed in eosinophil morphology. Our discoveries shed light on genetics of morphological traits. For the first time, we describe such large effect size on eosinophils morphology that is relatively frequent in Sardinian population.

Adolescent self-administration of the synthetic cannabinoid receptor agonist JWH-018 induces neurobiological and behavioral alterations in adult male mice.

RATIONALE: The use of synthetic cannabinoid receptor agonists (SCRAs) is growing among adolescents, posing major medical and psychiatric risks. JWH-018 represents the reference compound of SCRA-containing products. OBJECTIVES: This study was performed to evaluate the enduring consequences of adolescent voluntary consumption of JWH-018. METHODS: The reinforcing properties of JWH-018 were characterized in male CD1 adolescent mice by intravenous self-administration (IVSA). Afterwards, behavioral, neurochemical, and molecular evaluations were performed at adulthood. RESULTS: Adolescent mice acquired operant behavior (lever pressing, Fixed Ratio 1-3; 7.5 µg/kg/inf); this behavior was specifically directed at obtaining JWH-018 since it increased under Progressive Ratio schedule of reinforcement, and was absent in vehicle mice. JWH-018 IVSA was reduced by pretreatment of the CB1-antagonist/inverse agonist AM251. Adolescent exposure to JWH-018 by IVSA increased, at adulthood, both nestlet shredding and marble burying phenotypes, suggesting long-lasting repetitive/compulsive-like behavioral effects. JWH-018 did not affect risk proclivity in the wire-beam bridge task. In adult brains, there was an increase of ionized calcium binding adaptor molecule 1 (IBA-1) positive cells in the caudate-putamen (CPu) and nucleus accumbens (NAc), along with a decrease of glial fibrillary acidic protein (GFAP) immunoreactivity in the CPu. These glial alterations in adult brains were coupled with an increase of the chemokine RANTES and a decrease of the cytokines IL2 and IL13 in the cortex, and an increase of the chemokine MPC1 in the striatum. CONCLUSIONS: This study suggests for the first time that male mice self-administer the prototypical SCRA JWH-018 during adolescence. The adolescent voluntary consumption of JWH-018 leads to long-lasting behavioral and neurochemical aberrations along with glia-mediated inflammatory responses in adult brains.

The longitudinal dynamics and natural history of clonal haematopoiesis.

Clonal expansions driven by somatic mutations become pervasive across human tissues with age, including in the haematopoietic system, where the phenomenon is termed clonal haematopoiesis1-4. The understanding of how and when clonal haematopoiesis develops, the factors that govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression remains limited5,6. Here we track 697 clonal haematopoiesis clones from 385 individuals 55 years of age or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A and TP53) to more than 50% per year (SRSF2P95H). Growth rates of clones with the same mutation differed by approximately ±5% per year, proportionately affecting slow drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole-genome sequences of haematopoietic colonies from 7 individuals from an older age group, we reveal distinct patterns of lifelong clonal behaviour. DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations drove expansion only later in life, whereas TET2-mutant clones emerged across all ages. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterize the lifelong natural history of clonal haematopoiesis and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection.

Comparison of Whole Blood Cryopreservation Methods for Extensive Flow Cytometry Immunophenotyping.

Fresh blood immunophenotyping by flow cytometry, based on the reliable simultaneous detection of several markers in a cell, is the method of choice to study the circulating human immune system. Especially in large and multicenter studies, high sample quality is difficult to achieve, and adequate collection and storage of samples with fine-tuned whole blood cryopreservation is mandatory. Here, we compared the quality of immunophenotypic data obtained from fresh blood with those obtained after five cryopreservation methods by quantifying the levels of 41 immune cell populations. They comprised B and T lymphocyte subsets and their maturation stages, as well as monocytes and granulocytes. Three methods used fixative solutions and two other methods used dimethyl sulfoxide solutions to preserve cell viability. The fixative methods prevented detection of markers critical for identification of B and T cell subsets, including CD27, CXCR3, and CCR6. The other two methods permitted reliable discrimination of most immune-cell populations in thawed samples, though some cell frequencies varied compared to the corresponding fresh sample. Of those two methods, the one preserving blood in media containing dimethyl sulfoxide produced results that were most similar to those with fresh samples.

Autoimmunity Is a Significant Feature of Idiopathic Pulmonary Arterial Hypertension.

Rationale: Autoimmunity is believed to play a role in idiopathic pulmonary arterial hypertension (IPAH). It is not clear whether this is causative or a bystander of disease and if it carries any prognostic or treatment significance. Objectives: To study autoimmunity in IPAH using a large cross-sectional cohort. Methods: Assessment of the circulating immune cell phenotype was undertaken using flow cytometry, and the profile of serum immunoglobulins was generated using a standardized multiplex array of 19 clinically validated autoantibodies in 473 cases and 946 control subjects. Additional glutathione S-transferase fusion array and ELISA data were used to identify a serum autoantibody to BMPR2 (bone morphogenetic protein receptor type 2). Clustering analyses and clinical correlations were used to determine associations between immunogenicity and clinical outcomes. Measurements and Main Results: Flow cytometric immune profiling demonstrates that IPAH is associated with an altered humoral immune response in addition to raised IgG3. Multiplexed autoantibodies were significantly raised in IPAH, and clustering demonstrated three distinct clusters: "high autoantibody," "low autoantibody," and a small "intermediate" cluster exhibiting high concentrations of ribonucleic protein complex. The high-autoantibody cluster had worse hemodynamics but improved survival. A small subset of patients demonstrated immunoglobulin reactivity to BMPR2. Conclusions: This study establishes aberrant immune regulation and presence of autoantibodies as key features in the profile of a significant proportion of patients with IPAH and is associated with clinical outcomes.

Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function.

The use of cryopreserved peripheral blood mononuclear cells is common in biological research. It is widely accepted that primary cells are rendered unusable by several freezing cycles, although this practice might be very helpful when the biological material is valuable and its re-collection is impractical. To determine the extent to which primary cells undergoing repeated freezing cycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability, their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in ten human peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected, we observed a progressive increase in cell death percentages on three rounds of thawing, but the frequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, we observed a significant reduction of B cell frequency in frozen samples compared to fresh ones. On repeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantly decreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.

Genetic variant within CDK6 regulates immune response to palbociclib treatment.

Everyone carries a set of genetic variants that contribute to regulation of the levels of blood cells, with unknown clinical impact. One of them, rs445 within the cell-cycle checkpoint gene CDK6, reduces the levels of myeloid cell types including granulocytes. We treated CD3+ T cells and whole blood with palbociclib in 41 individuals, who were stratified by genotype for analyses. In T cells we assessed cell cycle and apoptosis, whereas in whole blood, apoptosis in activated (CD11b+), unactivated (CD11b-) granulocytes, cytotoxic (CD8 + CD4-), and helper (CD8-CD4+) T cells. We find that rs445 modulates the immune response of CD8+ T cells. It also increases the level of apoptotic CD11b + activated granulocytes after palbociclib treatment, which, in synergy with neutropenia, may affect drug related adverse events. These results suggest that the effect of palbociclib treatment may depend on underlying genetically encoded individual immune response as well as the direct response to the drug.

Effect of Different Disease-Modifying Therapies on Humoral Response to BNT162b2 Vaccine in Sardinian Multiple Sclerosis Patients.

Objectives: Vaccination against COVID-19 is highly recommended to patients affected by multiple sclerosis (MS); however, the impact of MS disease-modifying therapies (DMTs) on the immune response following vaccination has been only partially investigated. Here, we aimed to elucidate the effect of DMTs on the humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines in MS patients. Methods: We obtained sera from 912 Sardinian MS patients and 63 healthy controls 30 days after the second dose of BNT162b2 vaccine and tested them for SARS-CoV-2 response using anti-Spike (S) protein-based serology. Previous SARS-CoV-2 infection was assessed by anti-Nucleocapsid (N) serology. Patients were either untreated or undergoing treatment with a total of 13 different DMTs. Differences between treatment groups comprised of at least 10 patients were assessed by generalized linear mixed-effects model. Demographic and clinical data and smoking status were analyzed as additional factors potentially influencing humoral immunity from COVID-19 vaccine. Results: MS patients treated with natalizumab, teriflunomide, azathioprine, fingolimod, ocrelizumab, and rituximab showed significantly lower humoral responses compared to untreated patients. We did not observe a statistically significant difference in response between patients treated with the other drugs (dimethyl fumarate, interferon, alemtuzumab and glatiramer acetate) and untreated patients. In addition, older age, male sex and active smoking were significantly associated with lower antibody titers against SARS-CoV-2. MS patients previously infected with SARS-CoV-2 had significantly higher humoral responses to vaccine than uninfected patients. Conclusion: Humoral response to BNT162b2 is significantly influenced by the specific DMTs followed by patients, as well as by other factors such as previous SARS-CoV-2 infection, age, sex, and smoking status. These results are important to inform targeted strategies to prevent clinically relevant COVID-19 in MS patients.

Immunologic resilience and COVID-19 survival advantage.

BACKGROUND: The risk of severe coronavirus disease 2019 (COVID-19) varies significantly among persons of similar age and is higher in males. Age-independent, sex-biased differences in susceptibility to severe COVID-19 may be ascribable to deficits in a sexually dimorphic protective attribute that we termed immunologic resilience (IR). OBJECTIVE: We sought to examine whether deficits in IR that antedate or are induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection independently predict COVID-19 mortality. METHODS: IR levels were quantified with 2 novel metrics: immune health grades (IHG-I [best] to IHG-IV) to gauge CD8+ and CD4+ T-cell count equilibrium, and blood gene expression signatures. IR metrics were examined in a prospective COVID-19 cohort (n = 522); primary outcome was 30-day mortality. Associations of IR metrics with outcomes in non-COVID-19 cohorts (n = 13,461) provided the framework for linking pre-COVID-19 IR status to IR during COVID-19, as well as to COVID-19 outcomes. RESULTS: IHG-I, tracking high-grade equilibrium between CD8+ and CD4+ T-cell counts, was the most common grade (73%) among healthy adults, particularly in females. SARS-CoV-2 infection was associated with underrepresentation of IHG-I (21%) versus overrepresentation (77%) of IHG-II or IHG-IV, especially in males versus females (P < .01). Presentation with IHG-I was associated with 88% lower mortality, after controlling for age and sex; reduced risk of hospitalization and respiratory failure; lower plasma IL-6 levels; rapid clearance of nasopharyngeal SARS-CoV-2 burden; and gene expression signatures correlating with survival that signify immunocompetence and controlled inflammation. In non-COVID-19 cohorts, IR-preserving metrics were associated with resistance to progressive influenza or HIV infection, as well as lower 9-year mortality in the Framingham Heart Study, especially in females. CONCLUSIONS: Preservation of immunocompetence with controlled inflammation during antigenic challenges is a hallmark of IR and associates with longevity and AIDS resistance. Independent of age, a male-biased proclivity to degrade IR before and/or during SARS-CoV-2 infection predisposes to severe COVID-19.

Application of Genetic Studies to Flow Cytometry Data and Its Impact on Therapeutic Intervention for Autoimmune Disease.

In recent years, systematic genome-wide association studies of quantitative immune cell traits, represented by circulating levels of cell subtypes established by flow cytometry, have revealed numerous association signals, a large fraction of which overlap perfectly with genetic signals associated with autoimmune diseases. By identifying further overlaps with association signals influencing gene expression and cell surface protein levels, it has also been possible, in several cases, to identify causal genes and infer candidate proteins affecting immune cell traits linked to autoimmune disease risk. Overall, these results provide a more detailed picture of how genetic variation affects the human immune system and autoimmune disease risk. They also highlight druggable proteins in the pathogenesis of autoimmune diseases; predict the efficacy and side effects of existing therapies; provide new indications for use for some of them; and optimize the research and development of new, more effective and safer treatments for autoimmune diseases. Here we review the genetic-driven approach that couples systematic multi-parametric flow cytometry with high-resolution genetics and transcriptomics to identify endophenotypes of autoimmune diseases for the development of new therapies.

Prolonged B-cell depletion after rituximab in AQP4-IgG-positive neuromyelitis optica spectrum disorder.

Rituximab (a B-cell depleting monoclonal antibody) is increasingly utilized for treatment of different immune-mediated neurologic disorders, including aquaporin-4-IgG-positive neuromyelitis optica spectrum disorder (AQP4-IgG-NMOSD). After an initial treatment course, the drug is generally reinfused when peripheral blood B-cells levels re-increase >1% (usually after 6-12 months), or at fixed pre-planned 6-month intervals. We describe the unusual case of a 40-year-old woman with AQP4-IgG-NMOSD who showed a prolonged B-cell depletion for nearly five years after a single rituximab reinfusion. In similar rare patients with exceptionally long-lasting B-cell depletion, rituximab reinfusions at fixed pre-planned intervals would result in unnecessary treatment-related risks and health-care expenses.

PRF1 mutation alters immune system activation, inflammation, and risk of autoimmunity.

BACKGROUND: Defective alleles within the PRF1 gene, encoding the pore-forming protein perforin, in combination with environmental factors, cause familial type 2 hemophagocytic lymphohistiocytosis (FHL2), a rare, severe autosomal recessive childhood disorder characterized by massive release of cytokines-cytokine storm. OBJECTIVE: The aim of this study was to determine the function of hypomorph PRF1:p.A91V g.72360387 G > A on multiple sclerosis (MS) and type 1 diabetes (T1D). METHODS: We cross-compare the association data for PRF1:p.A91V mutation derived from GWAS on adult MS and pediatric T1D in Sardinians. The novel association with T1D was replicated in metanalysis in 12,584 cases and 17,692 controls from Sardinia, the United Kingdom, and Scotland. To dissect this mutation function, we searched through the coincident association immunophenotypes in additional set of general population Sardinians. RESULTS: We report that PRF1:p.A91V, is associated with increase of lymphocyte levels, especially within the cytotoxic memory T-cells, at general population level with reduced interleukin 7 receptor expression on these cells. The minor allele increased risk of MS, in 2903 cases and 2880 controls from Sardinia p = 2.06 × 10-4, odds ratio OR = 1.29, replicating a previous finding, whereas it protects from T1D p = 1.04 × 10-5, OR = 0.82. CONCLUSION: Our results indicate opposing contributions of the cytotoxic T-cell compartment to MS and T1D pathogenesis.

Carotid Beta Stiffness Association with Thyroid Function.

BACKGROUND: Thyroid hormone modulation of cardiovascular function has been associated with cardiovascular disease. Recent evidence suggests that free thyroxine (FT4) levels are associated with an increase in systemic arterial stiffness, but little is known about the effects of FT4 at the local level of the common carotid artery. ß-stiffness index is a local elastic parameter usually determined by carotid ultrasound imaging. METHODS: We conducted a cross-sectional analysis in the ProgeNIA cohort, including 4846 subjects across a broad age range. For the purpose of this study, we excluded subjects with increased thyrotropin (TSH) levels and those treated with levothyroxine or thyrostatic. We assessed ß stiffness, strain, wall-lumen ratio, carotid cross-sectional area (CSA), and stress and flow in the right common carotid artery. We tested whether FT4, heart rate, and their interactions were associated with carotid parameters. RESULTS: FT4 was positively and independently associated with ß stiffness index (ß = 0.026, p = 0.041), and had a negative association with strain (ß = -0.025, p = 0.009). After adding heart rate and the interaction between FT4 and heart rate to the model, FT4 was still associated with the ß stiffness index (ß = 0.186, p = 0.06), heart rate was positively associated with the stiffness index (ß = 0.389, p < 0.001) as well as their interaction (ß = 0.271, p = 0.007). CONCLUSION: This study suggests that higher FT4 levels increase arterial stiffness at the common carotid level, consistent with a detrimental effect on elastic arteries. The effect of FT4 is likely to be primarily attributable to its effect on heart rate.