Study protocol for a zinc intervention in the elderly for prevention of pneumonia, a randomized, placebo-controlled, double-blind clinical pilot trial.

Pneumonia is a major public health problem for older adults, being one of the leading causes of hospitalization and death, particularly for elderly nursing home residents. We previously conducted a clinical trial in which we demonstrated that 29% of nursing home residents had low serum zinc levels coinciding with a two-fold increase in pneumonia incidence and duration in comparison to individuals with adequate serum zinc levels. However, causality could not be inferred and necessitates a double-blind clinical trial. To determine the appropriate supplementation dose for such a trial we are conducting a randomized, placebo-controlled, double-blind clinical pilot trial aimed at delineating the optimal dosage (30 and 60 mg/day elemental Zn) and establishing safety. The results from the pilot study will be leveraged to inform our larger randomized clinical trial designed to study the effect of zinc supplementation in nursing home elderly with low serum zinc levels on respiratory infections, antibiotic use, and duration of sick days with pneumonia. In tandem with dose optimization, we will evaluate the correlation between serum zinc and pan-T cell zinc levels, given that T cells and their zinc levels are important in the response and resolution of respiratory infections but whose correlation has only been extrapolated and not demonstrated. Herein we present the study rationale and protocol, as well as discuss specific challenges we encountered in securing a manufacturer for the study agents and when recruiting from nursing home populations during the COVID-19 pandemic. In light of these experiences, we provide recommendations for future clinical trials under circumstances where supply chains are disrupted, and recruitment pools are constrained or unavailable. Clinical trial registration: https://clinicaltrials.gov/, NCT05527899.

Nutritional Aspects of Healthy Aging.

Proper nutrition and healthy eating are key determinants of healthy aging. In older age, energy requirements decrease, yet micronutrient requirements stay the same or increase, which make older adults susceptible to nutrient deficiencies. Therefore, it is important to encourage older adults to consume nutrient-dense foods. Many older adults do not maintain proper hydration, so adequate water intake should also be encouraged. Most older adults have multiple chronic diseases that may influence their dietary intake and nutritional needs. However, currently, our understanding of how individual chronic diseases and their associated treatments influence dietary requirements is limited.

Short-Chain Fatty Acids Impair Neutrophil Antiviral Function in an Age-Dependent Manner.

Half of the people living with HIV are women. Younger women remain disproportionally affected in endemic areas, but infection rates in older women are rising worldwide. The vaginal microbiome influences genital inflammation and HIV infection risk. Multiple factors, including age, induce vaginal microbial alterations, characterized by high microbial diversity that generate high concentrations of short-chain fatty acids (SCFAs), known to modulate neutrophil function. However, how SCFAs may modulate innate anti-HIV protection by neutrophils is unknown. To investigate SCFA-mediated alterations of neutrophil function, blood neutrophils from younger and older women were treated with SCFAs (acetate, butyrate and propionate) at concentrations within the range reported during bacterial vaginosis, and phenotype, migration and anti-HIV responses were evaluated. SCFA induced phenotypical changes preferentially in neutrophils from older women. Butyrate decreased CD66b and increased CD16 and CD62L expression, indicating low activation and prolonged survival, while propionate increased CD54 and CXCR4 expression, indicating a mature aged phenotype. Furthermore, acetate and butyrate significantly inhibited neutrophil migration in vitro and specifically reduced α-defensin release in older women, molecules with anti-HIV activity. Following HIV stimulation, SCFA treatment delayed NET release and dampened chemokine secretion compared to untreated neutrophils in younger and older women. Our results demonstrate that SCFAs can impair neutrophil-mediated anti-HIV responses.

Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses.

Legionella pneumophila elicits caspase-11-driven macrophage pyroptosis through guanylate-binding proteins (GBPs) encoded on chromosome 3. It has been proposed that microbe-driven IFN upregulates GBPs to facilitate pathogen vacuole rupture and bacteriolysis preceding caspase-11 activation. We show here that macrophage death occurred independently of microbial-induced IFN signaling and that GBPs are dispensable for pathogen vacuole rupture. Instead, the host-intrinsic IFN status sustained sufficient GBP expression levels to drive caspase-1 and caspase-11 activation in response to cytosol-exposed bacteria. In addition, endogenous GBP levels were sufficient for the release of DNA from cytosol-exposed bacteria, preceding the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway for Ifnb induction. Mice deficient for chromosome 3 GBPs were unable to mount a rapid IL-1/chemokine (C-X-C motif) ligand 1 (CXCL1) response during Legionella-induced pneumonia, with defective bacterial clearance. Our results show that rapid GBP activity is controlled by host-intrinsic cytokine signaling and that GBP activities precede immune amplification responses, including IFN induction, inflammasome activation, and cell death.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22-35 years) or elderly (65-69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

Longitudinal expression of Toll-like receptors on dendritic cells in uncomplicated pregnancy and postpartum.

OBJECTIVE: Toll-like receptors (TLRs) are integral parts of the innate immune system and have been implicated in complications of pregnancy. The longitudinal expression of TLRs on dendritic cells in the maternal circulation during uncomplicated pregnancies is unknown. The objective of this study was to prospectively evaluate TLRs 1-9 as expressed on dendritic cells in the maternal circulation at defined intervals throughout pregnancy and postpartum. STUDY DESIGN: This was a prospective cohort of 30 pregnant women with uncomplicated pregnancies and 30 nonpregnant controls. TLRs and cytokine expression was measured in unstimulated dendritic cells at 4 defined intervals during pregnancy and postpartum. Basal expression of TLRs and cytokines was measured by multicolor flow cytometry. The percent-positive dendritic cells for each TLRs were compared with both nonpregnant and postpartum levels with multivariate linear regression. RESULTS: TLRs 1, 7, and 9 were elevated compared with nonpregnant controls with persistent elevation of TLR 1 and interleukin-12 (IL-12) into the postpartum period. Concordantly, levels of IL-6, IL-12, interferon alpha, and tumor necrosis factor alpha increased during pregnancy and returned to levels similar to nonpregnant controls during the postpartum period. The elevated levels of TLR 1 and IL-12 were persistent postpartum, challenging notions that immunologic changes during pregnancy resolve after the prototypical postpartum period. CONCLUSION: Normal pregnancy is associated with time-dependent changes in TLR expression compared with nonpregnant controls; these findings may help elucidate immunologic dysfunction in complicated pregnancies.

Statistical approaches for analyzing immunologic data of repeated observations: a practical guide.

Translational research not only encompasses transitioning from animal to human models but also must address the greater heterogeneity of humans when designing and analyzing experiments. Appropriate study designs can address heterogeneity through a priori data collection, and taking repeated measures can improve the power and efficiency of a study to detect clinically meaningful differences. Although common in other areas of biomedical research, modern statistical methods using repeated measurements on the same subject and accounting for their potential correlations are not widely utilized in immunologic studies. To highlight these analytic issues, we present a practical guide to understanding and applying analytic methods from commonly used T-tests without adjusting for multiple comparisons to mixed models with subject-specific adjustments for correlations using our data on Toll-like receptor-induced cytokine production in monocytes from young and older adults.

Dendritic cells in the circulation of women with preeclampsia demonstrate a pro-inflammatory bias secondary to dysregulation of TLR receptors.

Toll-like receptors (TLRs) are central components of the innate immune system that recognize both microbial ligands and host products released during tissue damage. Data from epidemiologic studies and animal models suggest that inappropriate activation of the immune system plays a critical role in the development of preeclampsia. This study evaluates in a systematic fashion the expression and function of TLRs in the circulation of patients with preeclampsia compared to healthy pregnant controls. We evaluated TLR expression and function in primary dendritic cells (DCs) of 30 patients with preeclampsia and 30 gestational age-matched healthy pregnant controls. DCs were stimulated with the different TLR ligands engaging TLR1/2, TLR2/6, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9. The expression of TLR-induced production of TNF-α, IFN-α, IL-6, and IL-12 were measured by multicolor flow cytometry. Basal expression of TLR3, TLR4 and TLR9 was significantly increased in DCs isolated from women with preeclampsia. Preeclamptic DCs also expressed significantly higher basal levels of cytokines. In contrast, preeclamptic DCs demonstrated a less robust response to stimulation with various TLR ligands as compared with healthy pregnant controls. Under basal conditions, DCs from preeclamptic individuals express higher levels of select TLRs and produce more pro-inflammatory cytokines as compared with healthy controls. As such, the ability of these cells to mount an inflammatory reaction in response to a TLR ligand is limited. These data demonstrate a dysregulated pattern of TLR expression and cytokine production in DCs from PE patients that may limit further activation by TLR engagement.

Influenza vaccination during pregnancy and factors for lacking compliance with current CDC guidelines.

BACKGROUND: The Center for Disease Control and Prevention (CDC) and the American College of Obstetricians and Gynecologists (ACOG) recommend influenza vaccination for all pregnant women during the influenza season. However, the actual rate of vaccination is substantially below the target levels. Given the recent emergence of novel influenza strains, there is an important need to address knowledge gaps in women and their healthcare providers to improve vaccination coverage for pregnant women during inter-pandemic and pandemic periods. This study attempted to identify potentially remediable attitudinal factors among women and their physicians that may present barriers to influenza vaccination and then assess the impact of interventions to increase the influenza vaccination rate in pregnant women. METHODS: This prospective study initially analyzed patient and physician knowledge regarding the influenza vaccine in pregnancy and then examined the impact of several interventions aimed to increase immunization rates implemented over the following year. Influenza vaccination rates were assessed before and after the interventions. RESULTS: Five hundred twenty patients were enrolled in the study during the influenza season 2007/2008. Only 19% of those patients reported receiving the influenza vaccination and only 28% recalled that the vaccine was offered. Following this, in the summer and fall of 2008, we performed a physician education program and distributed posters advertising the influenza vaccine to all offices offering prenatal care in our area in order to increase patient awareness of the need for the vaccine. In the following influenza season, we again reassessed the vaccination rate and patient's knowledge and awareness of the vaccine in 480 postpartum women. Influenza vaccination rates increased from 19% to 31%. After the intervention, 51% of patients recalled that the vaccine was offered to them during the pregnancy as opposed to only 28% the year prior. CONCLUSION: Understanding the specific barriers to vaccination that our population faced was helpful in designing the interventions to improve knowledge and acceptance of influenza vaccination in pregnancy, which led to an increased vaccination rates in women.

Dysregulation of human Toll-like receptor function in aging.

Studies addressing immunosenescence in the immune system have expanded to focus on the innate as well as the adaptive responses. In particular, aging results in alterations in the function of Toll-like receptors (TLRs), the first described pattern recognition receptor family of the innate immune system. Recent studies have begun to elucidate the consequences of aging on TLR function in human cohorts and add to existing findings performed in animal models. In general, these studies show that human TLR function is impaired in the context of aging, and in addition there is evidence for inappropriate persistence of TLR activation in specific systems. These findings are consistent with an overarching theme of age-associated dysregulation of TLR signaling that likely contributes to the increased morbidity and mortality from infectious diseases found in geriatric patients.

Selected viral infections in pregnancy.

This article reviews the impact of seasonal influenza on pregnancy with particular emphasis on the 2009 novel H1N1 pandemic. Antiviral therapy for influenza, as well as recommendations and safety data on vaccination are discussed. In addition, the impact of hepatitis A, B, and C in pregnancy is addressed with a focus on prevention and treatment strategies for hepatitis B and C.

Aging of the innate immune system.

The innate immune system is composed of a network of cells including neutrophils, NK and NKT cells, monocytes/macrophages, and dendritic cells that mediate the earliest interactions with pathogens. Age-associated defects are observed in the activation of all of these cell types, linked to compromised signal transduction pathways including the Toll-like Receptors. However, aging is also characterized by a constitutive pro-inflammatory environment (inflamm-aging) with persistent low-grade innate immune activation that may augment tissue damage caused by infections in elderly individuals. Thus, immunosenescence in the innate immune system appears to reflect dysregulation, rather than exclusively impaired function.

Echocardiographic diagnosis, management and monitoring of pulmonary embolism with right heart thrombus in a patient with myotonic dystrophy: a case report.

Acute pulmonary embolism (PE) is a common disease which frequently results in life-threatening right ventricular (RV) failure. High-risk PE, presenting with hypotension, shock, RV dysfunction or right heart thrombus is associated with a high mortality, particularly during the first few hours. Accordingly, it is important to commence effective therapy as soon as possible. In the case described in this report, a 49-year-old woman with myotonic dystrophy type 1 presented with acute respiratory failure and hypotension. Transthoracic echocardiography showed signs of right heart failure and a mobile right heart mass highly suspicious of a thrombus. Based on echocardiographic findings, acute thrombolysis was performed resulting in hemodynamic stabilization of the patient and complete resolution of the right heart thrombus. This case underscores the important role of transthoracic echocardiography for the diagnosis, management and monitoring of PE and underlines the efficacy and safety of thrombolysis in the treatment of PE associated with right heart thrombus.

To bronch or not to bronch? A recurring challenge in neutropenic patients with pulmonary infiltrates.

BACKGROUND: Though it is generally accepted that both noninvasive and bronchoscopic procedures substantially increase the diagnostic yield ofpulmonaryinfiltrates, few studies address the therapeutic implications of invasive bronchoscopic procedures and their impact on survival. METHODS: We prospectively followed all patients with neutropenic fever and pulmonary infiltrates who were either referred to the inpatient Pulmonary Consult Service of Yale New Haven Hospital or admitted to the Medical Intensive Care Unit between July 2006 andJuly 2008. One hundred forty-four patients with febrile neutropenia and associated pulmonary infiltrates were identified of whom 128 underwent flexible bronchoscopy. RESULTS: A diagnosis was obtained in 91 (71%) of the 128 patients. The diagnostic yield was highest when sputum cultures, bronchoalveolar lavage andtransbronchialbiopsywere combined (70%; 95% CI, 57% to 80%). Survivalwas higher in patients who had an early diagnosis of the underlying cause of the pulmonary infiltrates. The results obtained with the different bronchoscopic techniques led to a change in antibiotic treatment in 70 cases (55%). In 35/128 patients (27%), bronchoscopic techniques led to a definite diagnosis otherwise not detected with nonbronchoscopic techniques. However, in 23% of the cases, where bronchoscopic techniques led to a definite diagnosis, the clinical information was not translated into appropriate changes of the antimicrobial management. CONCLUSION: When noninvasive procedures are not likely to be diagnostic, bronchoscopic procedures should be performed soon after the occurrence of pulmonary infiltrates as early diagnosis improves survival.

Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.

We evaluated TLR function in primary human dendritic cells (DCs) from 104 young (age 21-30 y) and older (> or =65 y) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) and found substantial decreases in older compared with young individuals in TNF-alpha, IL-6, and/or IL-12 (p40) production in mDCs and in TNF-alpha and IFN-alpha production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g., gender, race, body mass index, number of comorbid medical conditions) using mixed-effect statistical modeling. Studies of surface and intracellular expression of TLR proteins and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and posttranscriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in DCs; notably, defects in cytokine production were strongly associated with poor Ab response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.

Human innate immunosenescence: causes and consequences for immunity in old age.

The past decade has seen an explosion in research focusing on innate immunity. Through a wide range of mechanisms including phagocytosis, intracellular killing and activation of proinflammatory or antiviral cytokine production, the cells of the innate immune system initiate and support adaptive immunity. The effects of aging on innate immune responses remain incompletely understood, particularly in humans. Here we review advances in the study of human immunosenescence in the diverse cells of the innate immune system, including neutrophils, monocytes, macrophages, natural killer and natural killer T (NKT) cells and dendritic cells-with a focus on consequences for the response to infection or vaccination in old age.

Bronchogenic cyst of the interatrial septum presenting as atrioventricular block.

Bronchogenic cysts are congenital lesions that are a remnant from abnormal budding of the embryonic foregut. These cysts are usually single; most cases are either asymptomatic or present with respiratory symptoms. A 43-year-old woman presented with intermittent type II atrioventricular block during cholecystectomy. The cardiac evaluation including transthoracic and transesophageal echocardiography and magnetic resonance imaging revealed a cystic homogeneous mass within the interatrial septum. The patient underwent surgical resection of the mass and closure of the septal defect. Histopathology identified ciliated columnar epithelium, consistent with the diagnosis of a bronchogenic cyst.

Antibiotic resistance and penicillin tolerance in ano-vaginal group B streptococci.

OBJECTIVES: We sought to determine the prevalence of group B streptococcus (GBS) colonisation and to characterise antibiotic resistance patterns. METHODS: Vaginal and ano-rectal cultures were evaluated for GBS colonisation, and antibiotic susceptibility profiles were determined to 15 antibiotics according to the guidelines of the National Committee for Clinical Laboratory Standards. RESULTS: Our GBS prevalence was 30%. All isolates were sensitive to amoxicillin/clavulanic acid, ampicillin, ampicillin/sulbactam, cefotaxime, ceftriaxone, cefuroxime-sodium, imipenem, linezolid, penicillin G and vancomycin. Thirty-two percent of the isolates were resistant to azithromycin, 21% to clindamycin, 25% to erythromycin and 23% to tetracycline. CONCLUSIONS: The relatively high rates of resistance to four of the 15 antibiotics tested confirm that for women allergic to penicillin and colonised with GBS, antibiotic sensitivities should be determined. We noticed increasing resistance to clindamycin over a 7-year period. Ongoing surveillance of local antibiotic resistance patterns at the institutional level is important in determining optimal prophylaxis as resistance patterns differ between institutions and are increasing.

Long-term follow-up of a fenestrated Amplatzer atrial septal occluder in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease, with right-heart failure being the main cause of death. In patients refractory to conventional drug therapy, atrial septostomy can serve as palliative treatment or as a bridge to transplantation. A 41-year-old woman with a 15-year history of PAH associated with a corrected atrial septal defect presented with severe deterioration of symptoms. Echocardiography confirmed reocclusion of an atrial septal stoma that had been created several months before. After performing a repeat atrial septostomy, we implanted a custom-made atrial septostomy device, an Amplatzer septal occluder that had been fenestrated to serve as a custom-made atrial septostomy device. This resulted in an improvement in cardiac output and a marked symptomatic relief. During the 6-year follow-up, the patient was clinically stable with limited but constant exercise tolerance, under specific medical therapy. Repeated echocardiography confirmed long-term patency of the device.

Noninvasive monitoring of myocardial function after surgical and cytostatic therapy in a peritoneal metastasis rat model: assessment with tissue Doppler and non-Doppler 2D strain echocardiography.

OBJECTIVE: We sought to evaluate the impact of different antineoplastic treatment methods on systolic and diastolic myocardial function, and the feasibility estimation of regional deformation parameters with non-Doppler 2D echocardiography in rats. BACKGROUND: The optimal method for quantitative assessment of global and regional ventricular function in rats and the impact of complex oncological multimodal therapy on left- and right-ventricular function in rats remains unclear. METHODS: 90 rats after subperitoneal implantation of syngenetic colonic carcinoma cells underwent different onclogical treatment methods and were diveded into one control group and five treatment groups (with 15 rats in each group): group 1 = control group (without operation and without medication), group 2 = operation group without additional therapy, group 3 = combination of operation and photodynamic therapy, group 4 = operation in combination with hyperthermic intraoperative peritoneal chemotherapy with mitomycine, and group 5 = operation in combination with hyperthermic intraoperative peritoneal chemotherapy with gemcitabine, group 6 = operation in combination with taurolidin i.p. instillation. Echocardiographic examination with estimation of wall thickness, diameters, left ventricular fractional shortening, ejection fraction, early and late diastolic transmitral and myocardial velocities, radial and circumferential strain were performed 3-4 days after therapy. RESULTS: There was an increase of LVEDD and LVESD in all groups after the follow-up period (P = 0.0037). Other LV dimensions, FS and EF as well as diastolic mitral filling parameters measured by echocardiography were not significantly affected by the different treatments. Values for right ventricular dimensions and function remained unchanged, whereas circumferential 2D strain of the inferior wall was slightly, but significantly reduced under the treatment (-18.1 +/- 2.5 before and -16.2 +/- 2.9 % after treatment; P = 0.001) without differences between the single treatment groups. CONCLUSION: It is feasible to assess dimensions, global function, and regional contractility with echocardiography in rats under different oncological therapy. The deformation was decreased under overall treatment without influence by one specific therapy. Therefore, deformation assessment with non-Doppler 2D strain echocardiography is more sensitive than conventional echocardiography for assessing myocardial dysfunction in rats under oncological treatment.