Plasma Levels of Secreted Cytokines in Virologically Controlled HIV-Infected Aging Adult Individuals on Long-Term Antiretroviral Therapy.

HIV-infected (HIV+) aging adult individuals who have achieved undetectable viral load and improved CD4 T cell counts due to long-term antiretroviral therapy (ART) may continue to experience inflammation and immunosenescence. Therefore, we evaluated the plasma levels of proinflammatory and anti-inflammatory cytokines in 173 HIV+ aging adult individuals with age ranging from 22 to 81 years on long-term ART with viral load mostly <20 HIV RNA copies/mL and compared with 92 HIV-uninfected (HIV- or healthy controls) aging individuals. We found that the median levels of TNF-α, IFN-γ, IL-1ß, IL-6, and IL-10 were higher (p < 0.001 to <0.0001) and IL-17 trended lower in HIV+ individuals than healthy controls. Increasing CD4 T cell counts in the HIV+ cohort did not significantly change the circulating cytokine levels, although levels of IL-1ß increased. However, IL-17 levels significantly decreased with increasing CD4 counts in the healthy controls and yet unchanged in the HIV+ cohort. Of note, the levels of circulating IL-17 were significantly reduced comparatively in the healthy controls where the CD4 count was below 500, yet once above 500 the levels of CD4, IL-17 levels were comparable with the HIV+ cohort. With increasing CD8 T cell counts, the levels of these cytokines were not significantly altered, although levels of TNF-α, IFN-γ, and IL-6 declined, whereas IL-1ß and IL-17 were slightly elevated. Furthermore, increasing age of the HIV+ cohort did not significantly impact the cytokine levels although a slight increase in TNF-α, IL-6, IL-10, and IL-17 was observed. Similarly, these cytokines were not significantly modulated with increasing levels of undetectable viral loads, whereas some of the HIV+ individuals had higher levels of TNF-α, IFN-γ, and IL-1ß. In summary, our findings show that HIV+ aging adult individuals with undetectable viral load and restored CD4 T cell counts due to long-term ART still produce higher levels of both proinflammatory and anti-inflammatory cytokines compared with healthy controls, suggesting some level of inflammation.

Genetic Analysis of HIV-1 vpr Sequences from HIV-Infected Older Patients on Long-Term Antiretroviral Therapy.

BACKGROUND: Many HIV-infected individuals have achieved undetectable viral load and increased CD4 T cell counts due to the success of Antiretroviral Therapy (ART). However, HIV persists in resting T cells, monocytes/macrophages and other quiescent cells. Furthermore, the HIV- 1 vpr accessory gene may play an important role in the persistence of HIV in these infected patients. OBJECTIVES: Therefore, we characterized the HIV-1 vpr gene from PBMC DNA of 14 HIV-infected older patients on long-term ART with mostly undetectable viral load and increased CD4 T cell counts. METHODS: Peripheral Blood Mononuclear Cells (PBMC) were isolated from 14 HIV-infected individuals, followed by extraction of genomic DNA, amplification of HIV-1 vpr gene by polymerase chain reaction (PCR), cloning of vpr gene in TOPO vector and characterization of correct size recombinant inserts containing vpr genes. An average of 13 clones were sequenced from each patient, followed by sequence analysis by bioinformatic tools. RESULTS: Phylogenetic analysis of 182 vpr sequences demonstrated that the vpr sequences of each patient were well separated and discriminated from other patients' sequences and formed distinct clusters. The vpr sequences showed a low degree of viral heterogeneity, lower estimates of genetic diversity and about half of the patients' sequences were under positive selection pressure. While the majority of the vpr deduced amino acid sequences from most patients contained intact open reading frames, several sequences, mostly from two patients, had stop codons. Numerous patient-specific and common amino acid motifs were found in deduced vpr sequences. The functional domains required for vpr activity, including virion incorporation, nuclear import of pre-integration complex and cell cycle arrest, were generally conserved in most vpr sequences. Several of the known Cytotoxic T-lymphocytes (CTL) epitopes in vpr showed variation in our patients' sequences. CONCLUSION: In summary, a low degree of genetic variability, conservation of functional domains and variations in CTL epitopes were the features of vpr sequences from the 14 HIV-infected older patients with controlled viremia on long-term ART.

Phenotypic frailty in people living with HIV is not correlated with age or immunosenescence.

BACKGROUND: It has been hypothesized that HIV-1 infection prematurely "ages" individuals phenotypically and immunologically. We measured phenotypic frailty and immune "aging" markers on T-cells of people living with HIV on long term, suppressive anti-retroviral therapy (ART) to determine if there is an association between frailty and immunosenescence. METHODS: Thirty-seven (37) community-dwelling people living with HIV were measured for frailty using a sensor-based frailty meter that quantifies weakness, slowness, rigidity, and exhaustion. An immunological profile of the patients' CD4+ and CD8+ T-cell expression of cell surface proteins and cytokines was performed (n = 20). RESULTS: Phenotypic frailty prevalence was 19% (7/37) and correlated weakly with the number of past medical events accrued by the patient (r = 0.34, p = .04). There was no correlation of frailty with age, sex, prior AIDS diagnosis or HIV-1 viral load, or IFN-γ expression by CD4+ or CD8+ T-cells. There were more immune competent (CD28+ CD57-) cells than exhausted/senescent (CD28- CD57+) T cells. CONCLUSION: Frailty in people living with HIV on long term, suppressive ART did not correlate with aging or T cell markers of exhaustion or immunosenescence.

Fibrotic interstitial lung disease - palliative care needs: a World-Café qualitative study.

OBJECTIVES: The importance of palliative care in those with advanced fibrotic interstitial lung diseases (F-ILD) is recognised, but the palliative care requirements of patients and caregivers affected by F-ILD regardless of disease course are not established. We set out to explore this and identify optimal solutions in meeting the needs of a F-ILD population in Ireland. METHODS: Implementing a World-Café qualitative research approach, we captured insights evolving, iteratively in interactive small group discussions in response to six predefined topics on palliative care and planning for the future. Thirty-nine stakeholders participated in the World-Café including 12 patients, 13 caregivers, 9 healthcare professionals, 4 industry representatives and 1 representative of the clergy. RESULTS: Palliative care emerged as fundamental to the care and treatment of F-ILDs, regardless of disease progression. Unmet palliative care needs were identified as psychological and social support, disease education, inclusion of caregivers and practical/legal advice for disease progression and end-of-life planning. Participants identified diagnosis as a particularly distressing time for patients and families. They called for the introduction of palliative care discussions at this early-stage alongside improvements in integrated care, specifically increasing the involvement of primary care practitioners in referrals to palliative services. CONCLUSION: Patients and caregivers need discussions on palliative care associated with F-ILD to be included at the point of diagnosis. This approach may address persisting inadequacies in service provision previously identified over the course of the last decade in the UK, Ireland and European F-ILD patient charters.

Characterization of HIV-1 Envelope V3 Region Sequences from Virologically Controlled HIV-Infected Older Patients on Long Term Antiretroviral Therapy.

Although many HIV-infected patients have attained older age owing to the success of antiretroviral therapy (ART) in controlling viremia and increasing CD4 T cell counts, HIV continues to persist in several target cells. We have characterized 514 HIV-1 envelope V3 region sequences (94-96 amino acids [aa]) from 25 HIV-infected older patients' peripheral blood mononuclear cell DNA on long-term ART with controlled viremia (undetectable viral load) and improved CD4 T cell counts. Phylogenetic analysis revealed that the V3 region sequences of each patient formed distinct clusters that were well separated and discriminated from other patients' sequences. The coding potential of the V3 region, including several patient-specific amino acid motifs and functional domains, including the two cysteines sandwiching the V3 loop, the central GPGR motif with variation at one position in some sequences, the base GDIR motif, and the N-glycosylation sites were generally conserved. The patients' V3 region sequences contained amino acid motifs conferring affinity mostly for CCR5 coreceptor, suggesting R5 phenotype. There was a low degree of heterogeneity and lower estimates of genetic diversity in all 25 patients' V3 region sequences. Twelve of 25 patients' V3 region sequences were found to be under positive selection pressure. Analysis of the several cytotoxic T lymphocytes (CTL) epitopes showed variation, whereas some of known neutralizing antibodies (nAbs) epitopes showed conservation in patients' V3 region sequences. In conclusion, a low degree of genetic variability and maintenance of functional domains with R5 phenotypes, and variation in CTL and conservation of nAb epitopes were the hallmarks of V3 region sequences from our 25 virologically controlled HIV-infected older patients on long-term ART.

Evaluation of HIV-specific T-cell responses in HIV-infected older patients with controlled viremia on long-term antiretroviral therapy.

HIV-infected older individuals may have a diminished immune response because of exhaustion/immune aging of T-cells. Therefore, we have investigated HIV-specific CD4 and CD8 T-cell responses in 100 HIV-infected patients (HIV+) who have aged on long-term antiretroviral therapy (ART) and achieved controlled viremia (mostly undetectable viral load; 92 patients with <20 to <40 HIV RNA copies/mL and 8 <60 to <100) and improved CD4 T-cell counts. We show that the median frequencies of HIV-specific CD4+ and CD8+ IFN-γ T-cells were higher in HIV+ than uninfected individuals (HIV-), including increasing levels of IFN-γproduced by CD4+ T-cells and decreasing levels by CD8+ T-cells with increasing CD4 T-cell counts in HIV+. No correlation was found between T-cell responses and varying levels of undetectable viremia. HIV-specific TNF-α made by CD8+ T-cells was higher in HIV+ than HIV-, including decreasing levels with increasing CD4 T-cell counts in HIV+. Furthermore, the CD8+ T-cell mediators, CD107a and Granzyme-B, were higher in HIV+ than HIV-, and decreased with increasing CD4 T-cell counts in HIV+. Remarkably, HIV-specific CD8 T-cells produced decreasing levels of IFN-γwith increasing age of HIV+, including decreased levels of CD107a and Granzyme-B in older HIV+. However, HIV-specific CD8+ T-cells produced increasing levels of TNF-α with increasing age of the HIV+, suggesting continued inflammation. In conclusion, HIV+ with controlled viremia on long-term ART and with higher CD4 T-cell counts showed reduced HIV-specific CD8 T-cell responses as compared to those with lower CD4 T-cell counts, and older HIV+ exhibited decreasing levels of CD8 T-cell responses with increasing age.

Kissing Bugs Harboring Trypanosoma cruzi, Frequently Bite Residents of the US Southwest But Do Not Cause Chagas Disease.

BACKGROUND: Kissing bugs are common household pests in the Desert Southwest of the United States. These hematophagous bugs enter homes and suck blood from resident humans and pets. They are vectors of Trypanosoma cruzi, an enzootic parasite in small mammals and the cause of Chagas disease in humans. Autochthonous cases of Chagas disease are rare in the United States despite the presence of the vector and parasite. Environmental and biological factors accounting for this phenomenon need studying. METHODS: Homeowners in Bisbee and Tucson, Arizona captured kissing bugs inside homes during 2017-2018. Bugs were tested for presence of T. cruzi by polymerase chain reaction. Residents bitten by kissing bugs were tested for Chagas disease by serology. We evaluated invaded homes in the 2 cities. RESULTS: Three species of kissing bugs (n = 521) were collected in or near homes. Triatoma rubida was the most common triatomine in Tucson; T. recurva in Bisbee. T. protracta was uncommon. Seventeen percent of bugs captured in Bisbee and 51.1% in Tucson harbored T. cruzi. Bite victims (n = 105) recalled more than 2200 bites. Reactions to bites were common, including 32 episodes of anaphylaxis in 11 people (10.5%). Tests for Chagas disease (n = 116) were negative. Median age of houses was 91 years in Bisbee and 7 years in Tucson. Bisbee houses had pier and beam foundations. Tucson houses were built on concrete slabs. CONCLUSIONS: Kissing bugs harboring T. cruzi readily entered new and old homes. Bites of humans caused severe, life-threatening reactions. There was no serological evidence of Chagas disease among those bitten.

Iron and Ferritin Deposition in the Ovarian Tissues of the Yellow Fever Mosquito (Diptera: Culicidae).

Dengue, yellow fever, and Zika are viruses transmitted by yellow fever mosquito, Aedes aegypti [Linnaeus (Diptera: Culicidae)], to thousands of people each year. Mosquitoes transmit these viruses while consuming a blood meal that is required for oogenesis. Iron, an essential nutrient from the blood meal, is required for egg development. Mosquitoes receive a high iron load in the meal; although iron can be toxic, these animals have developed mechanisms for dealing with this load. Our previous research has shown iron from the blood meal is absorbed in the gut and transported by ferritin, the main iron transport and storage protein, to the ovaries. We now report the distribution of iron and ferritin in ovarian tissues before blood feeding and 24 and 72 h post-blood meal. Ovarian iron is observed in specific locations. Timing post-blood feeding influences the location and distribution of the ferritin heavy-chain homolog, light-chain homolog 1, and light-chain homolog 2 in ovaries. Understanding iron deposition in ovarian tissues is important to the potential use of interference in iron metabolism as a vector control strategy for reducing mosquito fecundity, decreasing mosquito populations, and thereby reducing transmission rates of vector-borne diseases.

Rapid detection of human blood in triatomines (kissing bugs) utilizing a lateral flow immunochromatographic assay - A pilot study.

OBJECTIVES: We tested a rapid and specific immunochromatographic assay (that detects human blood in forensic samples) to determine if human blood was present in triatomines and their fecal excreta. METHODS: We fed Triatoma rubida human blood (positive control) or mouse blood (negative control) and performed the assay on the abdominal contents and fecal excreta. Triatomine field specimens collected in and around human habitations and excreta were also tested. FINDINGS: The assay was positive in triatomines fed human blood (N = 5/5) and fecal excreta from bugs known to have ingested human blood (N = 5/5). Bugs feeding on mice (N = 15/15) and their fecal excreta (N = 8/8) were negative for human blood. Human blood was detected in 47% (N = 23/49) triatomines, representing six different species, collected in the field. MAIN CONCLUSIONS: The pilot study shows that this rapid and specific test may have applications in triatomine research. Further study is needed to determine the sensitivity of this assay compared to other well-established techniques, such as DNA- and proteomics-based methodologies and the assay's application in the field.

Rapid detection of human blood in triatomines (kissing bugs) utilizing a lateral flow immunochromatographic assay - A pilot study

BACKGROUND DNA- and proteomics-based techniques are currently used to identify a triatomine human blood meal. These methods are time consuming, require access to laboratories with sophisticated equipment, and trained personnel. OBJECTIVES We tested a rapid and specific immunochromatographic assay (that detects human blood in forensic samples) to determine if human blood was present in triatomines and their fecal excreta. METHODS We fed Triatoma rubida human blood (positive control) or mouse blood (negative control) and performed the assay on the abdominal contents and fecal excreta. Triatomine field specimens collected in and around human habitations and excreta were also tested. FINDINGS The assay was positive in triatomines fed human blood (N = 5/5) and fecal excreta from bugs known to have ingested human blood (N = 5/5). Bugs feeding on mice (N = 15/15) and their fecal excreta (N = 8/8) were negative for human blood. Human blood was detected in 47% (N = 23/49) triatomines, representing six different species, collected in the field. MAIN CONCLUSIONS The pilot study shows that this rapid and specific test may have applications in triatomine research. Further study is needed to determine the sensitivity of this assay compared to other well-established techniques, such as DNA- and proteomics-based methodologies and the assay's application in the field.

Effects of biphasic and monophasic electrical stimulation on mitochondrial dynamics, cell apoptosis, and cell proliferation.

Currently, electrical stimulation (ES) is used to induce changes in various tissues and cellular processes, but its effects on mitochondrial dynamics and mechanisms are unknown. The aim of this study was to compare the effects of monophasic and biphasic, anodal, and cathodal ES on apoptosis, proliferation, and mitochondrial dynamics in neuroblastoma SH-SY5Y cells. Cells were cultured and treated with ES. Alamar blue assay was performed to measure cell proliferation. The proteins expression of apoptotic-related proteins Bcl-2 associated X (Bax), B cell lymphoma 2 (Bcl-2), optic-atrophy-1 (OPA1), mitofusin2 (Mfn2), phosphorylated dynamin-related protein 1 at serine 616 (p-DRP1), and total dynamin-related protein 1 (Total-DRP1) were also determined. The results showed that monophasic anodal and biphasic anodal/cathodal (Bi Anod) ES for 1 hr at 125 pulses per minute (2.0 Hz) produced the most significant increase in cell proliferation. In addition, monophasic anodal and Bi Anod ES treated cells displayed a significant increase in the levels of anti-apoptotic protein Bcl-2, whereas the Bax levels were not changed. Moreover, the levels of Mfn2 were increased in the cells treated by Bi Anod, and OPA1 was increased by monophasic anodal and Bi Anod ES, indicating increased mitochondrial fusion in these ES-treated cells. However, the levels of mitochondrial fission indicated by DRP1 remained unchanged compared with non-stimulated cells. These findings were confirmed through visualization of mitochondria using Mitotracker Deep Red, demonstrating that monophasic anodal and Bi Anod ES could induce pro-survival effects in SH-SY5Y cells through increasing cell proliferation and mitochondrial fusion. Future research is needed to validate these findings for the clinical application of monophasic anodal and Bi Anod ES.

Effects of electrical stimulation on cell proliferation and apoptosis.

The application of exogenous electrical stimulation (ES) to cells in order to manipulate cell apoptosis and proliferation has been widely investigated as a possible method of treatment in a number of diseases. Alteration of the transmembrane potential of cells via ES can affect various intracellular signaling pathways which are involved in the regulation of cellular function. Controversially, several types of ES have proved to be effective in both inhibiting or inducing apoptosis, as well as increasing proliferation. However, the mechanisms through which ES achieves this remain fairly unclear. The aim of this review was to comprehensively summarize current findings from in vitro and in vivo studies on the effects of different types of ES on cell apoptosis and proliferation, highlighting the possible mechanisms through which ES induced these effects and define the optimum parameters at which ES can be used. Through this we hope to provide a greater insight into how future studies can most effectively use ES at the clinical trial stage.