T-cell heterogeneity, progenitor-progeny relationships, and function during latent and chronic viral infections.

Upon resolution of an acute viral infection, during latent-reactivating infection and during chronic active infections virus-specific T-cells differentiate into distinct subsets that differ in phenotype, longevity, transcriptional, metabolic, and epigenetic profiles, and effector functions. With recent advances in single-cell profiling, this substantial heterogeneity has become apparent and new subsets of virus-specific T cells, either of stable or transitory nature, are being identified. A unifying principle of T cells emerging in these different conditions is their precursor-progeny relationship. For acute and resolved viral infections, this relationship becomes apparent during re-challenge, whereas a constant differentiation of progenitor T cells into more differentiated cells occurs during latent-reactivating and active chronic viral infections. In this review, we summarize and discuss current knowledge about T-cell heterogeneity and progenitor-progeny relationships in the setting of persistent viral infections.

The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells.

Expression levels of the chemokine receptor CX3CR1 serve as high-resolution marker delineating functionally distinct antigen-experienced T-cell states. The factors that influence CX3CR1 expression in T cells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8+ T cells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T-cell differentiation. Nevertheless, in vivo generated memory CD8+ T cells maintained CX3CR1 expression during culture. This allowed us to investigate whether T-cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T-cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1+ CD8+ T cells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose-dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T-cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T-cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8+ T cells.

Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Profiling the specificity of clonally expanded plasma cells during chronic viral infection by single-cell analysis.

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy- and light-chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to nonviral and autoantigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies.

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity.

Adaptive immune repertoires are composed by the ensemble of B and T-cell receptors within an individual, reflecting both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Here, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following lymphocytic choriomeningitis virus (LCMV) infection, CD8+ T cells with binding specificity restricted to two distinct LCMV peptides, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T-cell inflation, and regulation. Together, this dataset provides a resource for immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

Non-neutralizing antibodies protect against chronic LCMV infection by promoting infection of inflammatory monocytes in mice.

Antibodies play an important role in host defense against microorganisms. Besides direct microbicidal activities, antibodies can also provide indirect protection via crosstalk to constituents of the adaptive immune system. Similar to many human chronic viral infections, persistence of Lymphocytic choriomeningitis virus (LCMV) is associated with compromised T- and B-cell responses. The administration of virus-specific non-neutralizing antibodies (nnAbs) prior to LCMV infection protects against the establishment of chronic infection. Here, we show that LCMV-specific nnAbs bind preferentially Ly6Chi inflammatory monocytes (IMs), promote their infection in an Fc-receptor independent way, and support acquisition of APC properties. By constituting additional T-cell priming opportunities, IMs promote early activation of virus-specific CD8 T cells, eventually tipping the balance between T-cell exhaustion and effector cell differentiation, preventing establishment of viral persistence without causing lethal immunopathology. These results document a beneficial role of IMs in avoiding T-cell exhaustion and an Fc-receptor independent protective mechanism provided by LCMV-specific nnAbs against the establishment of chronic infection.

Memory CD8 T cell inflation vs tissue-resident memory T cells: Same patrollers, same controllers?

The induction of long-lived populations of memory T cells residing in peripheral tissues is of considerable interest for T cell-based vaccines, as they can execute immediate effector functions and thus provide protection in case of pathogen encounter at mucosal and barrier sites. Cytomegalovirus (CMV)-based vaccines support the induction and accumulation of a large population of effector memory CD8 T cells in peripheral tissues, in a process called memory inflation. Tissue-resident memory (TRM ) T cells, induced by various infections and vaccination regimens, constitute another subset of memory cells that take long-term residence in peripheral tissues. Both memory T cell subsets have evoked substantial interest in exploitation for vaccine purposes. However, a direct comparison between these two peripheral tissue-localizing memory T cell subsets with respect to their short- and long-term ability to provide protection against heterologous challenge is pending. Here, we discuss communalities and differences between TRM and inflationary CD8 T cells with respect to their development, maintenance, function, and protective capacity. In addition, we discuss differences and similarities between the transcriptional profiles of TRM and inflationary T cells, supporting the notion that they are distinct memory T cell populations.

Aquimarins, Peptide Antibiotics with Amino-Modified C-Termini from a Sponge-Derived Aquimarina sp. Bacterium.

Genome mining and bioactivity studies suggested the sponge-derived bacterium Aquimarina sp. Aq135 as a producer of new antibiotics. Activity-guided isolation identified antibacterial peptides, named aquimarins, featuring a new scaffold with an unusual C-terminal amino group and chlorine moieties. Responsible for the halogenation is the FeII /α-ketoglutarate-dependent chlorinase AqmA that halogenates up to two isoleucine residues in a carrier protein-dependent fashion. Total syntheses of two natural aquimarins and eight non-natural variants were developed. Structure-activity relationship (SAR) studies with these compounds showed that the synthetically more laborious chlorinations are not required for antibacterial activity but enhance cytotoxicity. In contrast, variants lacking the C-terminal amine were virtually inactive, suggesting diamines similar to the terminal aquimarin residue as candidate building blocks for new peptidomimetic antibiotics.

Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice.

Neuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimer's disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system. Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor and T cell receptor repertoires and accompanying gene expression profiles in young and old mouse brains. We observed the presence of clonally expanded B and T cells in the central nervous system of aged male mice. Furthermore, many of these B cells were of the IgM and IgD isotypes, and had low levels of somatic hypermutation. Integrating gene expression information additionally revealed distinct transcriptional profiles of these clonally expanded lymphocytes. Our findings implicate that clonally related T and B cells in the CNS of elderly mice may contribute to neuroinflammation accompanying homeostatic ageing.

Evaluation of frailty and its impact on geriatric assessment.

AIM: Frailty has become an increasingly important topic, being directly correlated with ageing, presence of comorbidities, and also with other factors. It is a state of high vulnerability and is a consequence of ageing-related decline in whole body functioning. In order to initiate preventive and therapeutic measures, we need to identify the characteristics of current frail older adults. To identify the characteristics of frailty depending on age, area of residence and to assess the relationship between frailty and disability, cognitive impairment, malnutrition, depression and comorbidities. METHODS: Included in the study were 663 consecutively admitted patients over the age of 65. Frailty was evaluated using the Fried criteria and a comprehensive geriatric assessment. The mean age of the subjects enrolled in this study was 76.58 ± 6.5 years, most of the patients residing in rural areas (62.4%). RESULTS: A diagnosis of frailty was made in 73% of the study patients. The presence of frailty was found to be negatively correlated, statistically significant with cognitive function assessed by Mini-Mental State Examination (P = 0.039, r = -0.094) and malnutrition assessed by Mini Nutritional Assessment (P = 0.001, r = -0.151). Also, the presence of frailty was positively correlated, statistically significant with depression evaluated by Geriatric Depression Scale (P = 0.046, r = 0.093). CONCLUSIONS: Frailty is a common condition in the surveyed elderly population. It is associated with the presence of other geriatric syndromes such as malnutrition, cognitive impairment, depression, and is influenced by age.

Elder abuse and associated factors in eastern romania.

BACKGROUND: This article explores elder abuse in a hospitalised population. We wanted to identify details related to psychological and emotional abuse in the older population in our region and to determine the importance of the Elderly Abuse Suspicion Index (EASI© ) in comprehensive geriatric assessments. METHODS: This cross-sectional study conducted between March 2015 and May 2016 included 386 consecutive hospitalised patients over 65 years of age. All patients underwent a geriatric assessment, data were collected about their medical history, and the EASI© was administered to each. The main outcome was identifying the presence, the type of abuse and the factors associated with abuse. RESULTS: There were 21.5% of patients who suffered any form of abuse. Women were more frequently abused than men. Emotional abuse was the most common (60.2%) followed by neglect (53%) and physical abuse (22.91%); sexual abuse was absent in our study group. The abused patients had an impaired cognitive function (P = 0.034). They were also malnourished (P ≤ 0.001) and depressed (P = 0.001). The presence of peripheral artery disease, stroke, pneumonia, chronic kidney disease, musculoskeletal diseases and anxiety correlated with the presence of abuse. No statistically significant correlation was found between the degree of independence in instrumental activities of daily living and the presence of abuse (r = 0.105, P = 0.051). CONCLUSIONS: EASI is a tool for detecting elder abuse and should be included in the standard geriatric assessment to prevent ageism. The number of abused elderly patients is significant, and the multiple factors associated with abuse are diverse.

Therapeutic challenges in epidermal inclusion cysts with periocular localization: case reports.

Epidermal inclusion cysts in the periocular region are distinctive pathologies exhibiting varied clinical and radiological features, and they should be taken into consideration in the differential diagnosis of cystic lesions near the orbit. This article discusses the clinical and radiological details, along with the surgical results, of two individual cases of epidermal inclusion cysts, with different localization and without any preceding trauma, surgical history, or eyelid inflammation. In the first case, a substantial spherical structure closely connected to the tarsal plate was identified via excisional biopsy, whereas the second case involved a soft, oval tumor located at the outer right orbital corner, as determined clinically and validated through computed tomography. The histological examination showed cysts lined with a keratinized squamous layer, confirming an epidermoid cyst. The surgical removal of the cysts led to esthetically satisfactory outcomes in both cases. The particularity of the presented cases lies in the locations and considerable sizes of the tumors, which have complicated their surgical management. Such instances of epidermal inclusion cysts attached to the tarsus are rarely reported in the literature.

The Impact of Heart Rate Variability Monitoring on Preventing Severe Cardiovascular Events.

The increase in the incidence of cardiovascular diseases worldwide raises concerns about the urgent need to increase definite measures for the self-determination of different parameters, especially those defining cardiac function. Heart rate variability (HRV) is a non-invasive method used to evaluate autonomic nervous system modulation on the cardiac sinus node, thus describing the oscillations between consecutive electrocardiogram R-R intervals. These fluctuations are undetectable except when using specialized devices, with ECG Holter monitoring considered the gold standard. HRV is considered an independent biomarker for measuring cardiovascular risk and for screening the occurrence of both acute and chronic heart diseases. Also, it can be an important predictive factor of frailty or neurocognitive disorders, like anxiety and depression. An increased HRV is correlated with rest, exercise, and good recovery, while a decreased HRV is an effect of stress or illness. Until now, ECG Holter monitoring has been considered the gold standard for determining HRV, but the recent decade has led to an accelerated development of technology using numerous devices that were created specifically for the pre-hospital self-monitoring of health statuses. The new generation of devices is based on the use of photoplethysmography, which involves the determination of blood changes at the level of blood vessels. These devices provide additional information about heart rate (HR), blood pressure (BP), peripheral oxygen saturation (SpO2), step counting, physical activity, and sleep monitoring. The most common devices that have this technique are smartwatches (used on a large scale) and chest strap monitors. Therefore, the use of technology and the self-monitoring of heart rate and heart rate variability can be an important first step in screening cardiovascular pathology and reducing the pressure on medical services in a hospital. The use of telemedicine can be an alternative, especially among elderly patients who are associated with walking disorders, frailty, or neurocognitive disorders.

Is the Angiostrongylus vasorum infection in domestic dogs underestimated or misdiagnosed? A comprehensive presentation of four lethal cases.

Introduction: Angiostrongylus vasorum (A. vasorum) is a widely distributed gastropod-borne nematode, causing severe cardio-pulmonary disorders in dogs. In Romania, A. vasorum was detected in foxes and serologically confirmed in domestic dogs, but no clinical cases are currently diagnosed. Methods: Four dogs with clinical history of respiratory distress, originating from different geographical regions of Romania, were included in the study. One dog (case 1) was clinically evaluated using thoracic radiology and cardiac ultrasound; examination of feces and PCR were also performed for the etiologic diagnosis. The postmortem exam was performed in the other three cases, followed by parasitological and molecular analyses. Results: In the first case, parasitic pneumonia was suspected based on the radiographic examination of the thorax and the infection with A. vasorum was confirmed by L1 morphological identification and PCR. The main postmortem changes included large, coalescing, dark red areas of pulmonary consolidation (n = 3) and numerous adult nematodes in the pulmonary arteries (n = 2). The histopathological examination of the lungs showed necrotizing and granulomatous pneumonia with severe hemorrhages and chronic pulmonary arterial changes. Intralesional nematodes were seen in all necropsied cases. Additional inflammatory changes related to A. vasorum infection were identified in the brain and tracheobronchial and mediastinal lymph nodes (n = 2). Identification of larvae, histopathology and PCR confirmed the infection with A. vasorum. Conclusions: This study describes the first cases of canine cardiopulmonary angiostrongylosis in domestic dogs in Romania, and focuses on clinical presentation, pathological findings and molecular analysis. Angiostrongylosis should be included on the list of differential diagnoses of canine cardiopulmonary distress and/or haemorrhagic diathesis in Romania and awareness should be raised among clinicians to avoid post-mortem diagnosis in the future.

Factors Associated with Burnout in Medical Staff: A Look Back at the Role of the COVID-19 Pandemic.

Despite the significant consequences for medical practice and public health, burnout in healthcare workers remains underestimated. Pandemic periods have increased the reactivity to stress by favoring some changes whose influence are still felt. PURPOSE: This study aims to identify opportune factors during pandemic periods that predispose medical personnel to burnout and the differences between medical staff which worked with COVID-19 patients and those who did not work with COVID-19 patients. MATERIAL AND METHODS: This is a prospective study on 199 subjects, medical staff and auxiliary staff from national health units, COVID-19 and non-COVID-19, who answered questions using the Google Forms platform about the level of stress related to the workplace and the changes produced there. All statistical analyses were conducted using IBM SPSS Statistics (Version 28). RESULTS: The limited equipment and disinfectant solutions from the lack of medical resources category, the fear of contracting or transmitting the infection from the fears in relation to the COVID-19 pandemic category and the lack of personal and system-level experience in combating the infection due to the lack of information on and experience with COVID-19 were the most predisposing factors for burnout. No significant differences were recorded between those on the front line and the other healthcare representatives. CONCLUSIONS: The results of this study identify the stressors generated in the pandemic context with prognostic value in the development of burnout among medical personnel. At the same time, our data draw attention to the cynicism or false-optimism stage of burnout, which can mask a real decline.

Preventing Dementia-A Cross-Sectional Study of Outpatients in a Tertiary Internal Medicine Department.

Dementia is a significant health problem worldwide, being the seventh leading cause of death (2,382,000 deaths worldwide in 2016). Recent data suggest there are several modifiable risk factors that, if addressed, can decrease dementia risk. Several national dementia screening programs exist; however, limited-income countries do not have the means to implement such measures. We performed a prospective cross-sectional study in an outpatient department to identify individuals at risk for dementia. Patients with no known cognitive dysfunction seeking a medical consult were screened for dementia risk by means of the cardiovascular risk factors, ageing, and dementia (CAIDE) and modified CAIDE tests. Additionally, we collected demographic and clinical data and assessed each participant for depression, mental state, and ability to perform daily activities. Of the 169 patients enrolled, 63.3% were identified as being in the intermediate-risk or high-risk group, scoring more than seven points on the mCAIDE test. Over 40% of the elderly individuals in the study were assessed as "somewhat depressed" or "depressed" on the geriatric depression scale. Almost 10% of the study population was diagnosed de novo with cognitive dysfunction. In conclusion, using a simple questionnaire such as the mCAIDE in a predefined high-risk population is easy and does not represent a major financial burden. At-risk individuals can subsequently benefit from personalized interventions that are more likely to be successful. Limited-resource countries can implement such screening tools in outpatient clinics.

Generation of a single-cell B cell atlas of antibody repertoires and transcriptomes to identify signatures associated with antigen specificity.

Although new genomics-based pipelines have potential to augment antibody discovery, these methods remain in their infancy due to an incomplete understanding of the selection process that governs B cell clonal selection, expansion, and antigen specificity. Furthermore, it remains unknown how factors such as aging and reduction of tolerance influence B cell selection. Here we perform single-cell sequencing of antibody repertoires and transcriptomes of murine B cells following immunizations with a model therapeutic antigen target. We determine the relationship between antibody repertoires, gene expression signatures, and antigen specificity across 100,000 B cells. Recombinant expression and characterization of 227 monoclonal antibodies revealed the existence of clonally expanded and class-switched antigen-specific B cells that were more frequent in young mice. Although integrating multiple repertoire features such as germline gene usage and transcriptional signatures failed to distinguish antigen-specific from nonspecific B cells, other features such as immunoglobulin G (IgG) subtype and sequence composition correlated with antigen specificity.

Clonally Expanded Virus-Specific CD8 T Cells Acquire Diverse Transcriptional Phenotypes During Acute, Chronic, and Latent Infections.

CD8+ T cells play a crucial role in the control and resolution of viral infections and can adopt a wide range of phenotypes and effector functions depending on the inflammatory context and the duration and extent of antigen exposure. Similarly, viral infections can exert diverse selective pressures on populations of clonally related T cells. Technical limitations have nevertheless made it challenging to investigate the relationship between clonal selection and transcriptional phenotypes of virus-specific T cells. We therefore performed single-cell T cell receptor (TCR) repertoire and transcriptome sequencing of virus-specific CD8 T cells in murine models of acute, chronic and latent infection. We observed clear infection-specific populations corresponding to memory, effector, exhausted, and inflationary phenotypes. We further uncovered a mouse-specific and polyclonal T cell response, despite all T cells sharing specificity to a single viral epitope, which was accompanied by stereotypic TCR germline gene usage in all three infection types. Persistent antigen exposure during chronic and latent viral infections resulted in a higher proportion of clonally expanded T cells relative to acute infection. We furthermore observed a relationship between transcriptional heterogeneity and clonal expansion for all three infections, with highly expanded clones having distinct transcriptional phenotypes relative to less expanded clones. Together our work relates clonal selection to gene expression in the context of viral infection and further provides a dataset and accompanying software for the immunological community.

Chronic Coronary Syndrome in Frail Old Population.

The demographic trend of aging is associated with an increased prevalence of comorbidities among the elderly. Physical, immunological, emotional and cognitive impairment, in the context of the advanced biological age segment, leads to the maintenance and precipitation of cardiovascular diseases. Thus, more and more data are focused on understanding the pathophysiological mechanisms underlying each fragility phenotype and how they potentiate each other. The implications of inflammation, sarcopenia, vitamin D deficiency and albumin, as dimensions inherent in fragility, in the development and setting of chronic coronary syndromes (CCSs) have proven their patent significance but are still open to research. At the same time, the literature speculates on the interdependent relationship between frailty and CCSs, revealing the role of the first one in the development of the second. In this sense, depression, disabilities, polypharmacy and even cognitive disorders in the elderly with ischemic cardiovascular disease mean a gradual and complex progression of frailty. The battery of tests necessary for the evaluation of the elderly with CCSs requires a permanent update, according to the latest guidelines, but also an individualized approach related to the degree of frailty and the conditions imposed by it. By summation, the knowledge of frailty screening methods, through the use of sensitive and individualized tools, is the foundation of secondary prevention and prognosis in the elderly with CCSs. Moreover, a comprehensive geriatric assessment remains the gold standard of the medical approach of these patients. The management of the frail elderly, with CCSs, brings new challenges, also from the perspective of the treatment particularities. Sometimes the risk-benefit balance is difficult to achieve. Therefore, the holistic, individualized and updated approach of these patients remains a desired objective, by understanding and permanently acquiring knowledge on the complexity of the frailty syndrome.

Relationship between Frailty and Depression in a Population from North-Eastern Romania.

(1) Background: In the older population, depression often affects people with chronic medical illnesses, cognitive impairment, or disability. Frailty is another important issue affecting older adults, being difficult to clinically distinguish from frailty in advanced old age. Well-designed interventional studies and clinical strategies targeting both frailty and depression are rare or nonexistent. (2) Methods: We realized a retrospective study in which we included a total of 411 patients that were admitted to the Geriatric Clinic from "Dr. C. I. Parhon" Hospital from Iasi for a period of 13 months. The aim of our study was to investigate the relationship between depression and frailty in a geriatric population due to the fact that the quality of life is negatively influenced by both frailty and depression. (3) Results: The prevalence of the depressive symptoms screened by the GDS-15 was 66.7%, with women being more depressed than men. Furthermore, an obvious relationship between depression and the dependence degree in performing daily activities has been observed. In addition, the mean MMSE score decreased with an increasing degree of depression. (4) Conclusions: Our study demonstrates the association between frailty and depression, one of them being a risk factor in the development of the other. A poor acknowledgment of the problem and an underdiagnosis of these conditions are important public health concerns due to the high healthcare costs. Thus, an active primary prevention would be imperiously needed in order to diagnose frailty and depression at an early stage, increasing the quality of life of the elderly and also their successful aging.