Effect of initial periodontal therapy on metallothionein levels in smokers and non-smokers with periodontitis.

The aim of this study was to determine the effect of non-surgical periodontal therapy (NSPT) on mRNA expression of metallothionein (MT) and its levels in serum, saliva and gingival crevicular fluid (GCF) of smokers (S) and non-smokers (NS) with periodontitis (P).A total of 100 participants were included: 48 periodontally healthy (PH) subjects (24 S [PH + S] and 24 NS [PH + NS]) and 52 patients with P (27 S [P + S] and 25 NS [P + NS]). Clinical parameters were recorded, and biofluids (serum, saliva and GCF) and gingival tissue samples were obtained at baseline in all groups and 3 months after NSPT in P groups. MT levels in biofluids were determined by ELISA. In gingival tissues, MT-mRNA expression was quantified using real-time PCR. mRNA expression of MT and its levels in biofluids were significantly higher in P + S compared to other groups, and the differences between P + NS and PH + S were non-significant. A significant decrease was observed for MT levels in biofluids, and MT-mRNA expression in periodontitis patients after NSPT. In conclusion, smoking and periodontitis are associated with higher MT expression which decreases after NSPT. MT as an oxidative stress biomarker and its therapeutic role in periodontitis should be investigated in future studies.Clinical trial registration: The study was prospectively registered at Clinical Trials Registry-India (ctri.nic.in) as CTRI/2018/08/015427 on August 23, 2018.

Cotreatment With Clofazimine and Rapamycin Eliminates Drug-Resistant Tuberculosis by Inducing Polyfunctional Central Memory T-Cell Responses.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is acquiring drug resistance at a faster rate than the discovery of new antibiotics. Therefore, alternate therapies that can limit the drug resistance and disease recurrence are urgently needed. Emerging evidence indicates that combined treatment with antibiotics and an immunomodulator provides superior treatment efficacy. Clofazimine (CFZ) enhances the generation of T central memory (TCM) cells by blocking the Kv1.3+ potassium channels. Rapamycin (RAPA) facilitates M. tuberculosis clearance by inducing autophagy. In this study, we observed that cotreatment with CFZ and RAPA potently eliminates both multiple and extensively drug-resistant (MDR and XDR) clinical isolates of M. tuberculosis in a mouse model by inducing robust T-cell memory and polyfunctional TCM responses. Furthermore, cotreatment reduces the expression of latency-associated genes of M. tuberculosis in human macrophages. Therefore, CFZ and RAPA cotherapy holds promise for treating patients infected with MDR and XDR strains of M. tuberculosis.

Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples.

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

Assay design for unambiguous identification and quantification of circulating pathogen-derived peptide biomarkers.

Rationale: Circulating pathogen-derived proteins can serve as useful biomarkers for infections but may be detected with poor sensitivity and specificity by standard immunoassays due to masking effects and cross-reactivity. Mass spectrometry (MS)-read immunoassays for biomarker-derived peptides can resolve these issues, but lack standard workflows to select species-specific peptides with strong MS signal that are suitable for antibody generation. Methods:Using a Mycobacterium tuberculosis (Mtb) protein as an example, candidate peptides were selected by length, species-specificity, MS intensity, and antigenicity score. MS data from spiked healthy serum was employed to define MS feature thresholds, including a novel measure of internal MS data correlation, to produce a peak detection algorithm. Results: This algorithm performed better in rejecting false positive signal than each of its criteria, including those currently employed for this purpose. Analysis of an Mtb peptide biomarker (CFP-10pep) by this approach identified tuberculosis cases not detected by microbiologic assays, including extrapulmonary tuberculosis and tuberculosis cases in children infected with HIV-1. Circulating CFP-10pep levels measured in a non-human primate model of tuberculosis distinguished disease from asymptomatic infection and tended to correspond with Mtb granuloma size, suggesting that it could also serve as a surrogate marker for Mtb burden and possibly treatment response. Conclusions: These biomarker selection and analysis approach appears to have strong potential utility for infectious disease diagnosis, including cryptic infections, and possibly to monitor changes in Mtb burden that may reflect disease progression or a response to treatment, which are critical needs for more effective disease control.

Corrigendum: Testosterone Acts Within the Medial Amygdala of Rats to Reduce Innate Fear to Predator Odor Akin to the Effects of Toxoplasma gondii Infection.

[This corrects the article DOI: 10.3389/fpsyt.2020.00630.].

Characterizing Early T Cell Responses in Nonhuman Primate Model of Tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease killer worldwide with 1.4 million TB deaths in 2019. While the majority of infected population maintain an active control of the bacteria, a subset develops active disease leading to mortality. Effective T cell responses are critical to TB immunity with CD4+ and CD8+ T cells being key players of defense. These early cellular responses to TB infection have not yet been studied in-depth in either humans or preclinical animal models. Characterizing early T cell responses in a physiologically relevant preclinical model can provide valuable understanding of the factors that control disease development. We studied Mtb-specific T cell responses in the lung compartment of rhesus macaques infected with either a low- or a high-dose of Mtb CDC1551 via aerosol. Relative to baseline, significantly higher Mtb-specific CD4+IFN-γ+ and TNF-α+ T cell responses were observed in the BAL of low dose infected macaques as early as week 1 post TB infection. The IFN-γ and TNF-a response was delayed to week 3 post infection in Mtb-specific CD4+ and CD8+T cells in the high dose group. The manifestation of earlier T cell responses in the group exposed to the lower Mtb dose suggested a critical role of these cytokines in the antimycobacterial immune cascade, and specifically in the granuloma formation to contain the bacteria. However, a similar increase was not reflected in the CD4+ and CD8+IL-17+ T cells at week 1 post infection in the low dose group. This could be attributed to either a suppression of the IL-17 response or a lack of induction at this early stage of infection. On the contrary, there was a significantly higher IL-17+ response in Mtb-specific CD4+ and CD8+T cells at week 3 in the high dose group. The results clearly demonstrate an early differentiation in the immunity following low dose and high dose infection, largely represented by differences in the IFN-γ and TNF-α response by Mtb-specific T cells in the BAL. This early response to antigen expression by the bacteria could be critical for both bacterial growth control and bacterial containment.

Correction: Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008887.].

Luteolin as a potential host-directed immunotherapy adjunct to isoniazid treatment of tuberculosis.

Tuberculosis (TB) remains a major health problem throughout the world with one third of the population latently infected and ~1.74 million deaths annually. Current therapy consists of multiple antibiotics and a lengthy treatment regimen, which is associated with risk for the generation of drug-resistant Mycobacterium tuberculosis variants. Therefore, alternate host directed strategies that can shorten treatment length and enhance anti-TB immunity during the treatment phase are urgently needed. Here, we show that Luteolin, a plant-derived hepatoprotective immunomodulator, when administered along with isoniazid as potential host directed therapy promotes anti-TB immunity, reduces the length of TB treatment and prevents disease relapse. Luteolin also enhances long-term anti-TB immunity by promoting central memory T cell responses. Furthermore, we found that Luteolin enhances the activities of natural killer and natural killer T cells, both of which exhibit antitubercular attributes. Therefore, the addition of Luteolin to conventional antibiotic therapy may provide a means to avoid the development of drug-resistance and to improve disease outcome.

Xenodiagnosis to evaluate the infectiousness of humans to sandflies in an area endemic for visceral leishmaniasis in Bihar, India: a transmission-dynamics study.

BACKGROUND: Visceral leishmaniasis, also known on the Indian subcontinent as kala-azar, is a fatal form of leishmaniasis caused by the protozoan parasite Leishmania donovani and transmitted by the bites of the vector sandfly Phlebotomus argentipes. To achieve and sustain elimination of visceral leishmaniasis, the transmission potential of individuals exposed to L donovani from across the infection spectrum needs to be elucidated. The aim of this study was to evaluate the relative infectiousness to the sandfly vector of patients with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, and individuals with asymptomatic infection. METHODS: In this prospective xenodiagnosis study done in Muzaffarpur district of Bihar, India, we included patients with clinically confirmed active visceral leishmaniasis or post-kala-azar dermal leishmaniasis who presented to the Kala-Azar Medical Research Center. These participants received treatment for L donovani infection. We also included asymptomatic individuals identified through a serosurvey of 17 254 people living in 26 high-transmission clusters. Eligible participants were aged 12-64 years, were HIV negative, and had clinically or serologically confirmed L donovani infection. During xenodiagnosis, the forearms or lower legs of participants were exposed to 30-35 female P argentipes sandflies for 30 min. Blood-engorged flies were held in an environmental cabinet at 28°C and 85% humidity for 60-72 h, after which flies were dissected and evaluated for L donovani infection by microscopy and quantitative PCR (qPCR). The primary endpoint was the proportion of participants with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, as well as asymptomatic individuals, who were infectious to sandflies, with a participant considered infectious if promastigotes were observed in one or more individual flies by microscopy, or if one or more of the pools of flies tested positive by qPCR. FINDINGS: Between July 12, 2016, and March 19, 2019, we recruited 287 individuals, including 77 with active visceral leishmaniasis, 26 with post-kala-azar dermal leishmaniasis, and 184 with asymptomatic infection. Of the patients with active visceral leishmaniasis, 42 (55%) were deemed infectious to sandflies by microscopy and 60 (78%) by qPCR before treatment. No patient with visceral leishmaniasis was found to be infectious by microscopy at 30 days after treatment, although six (8%) were still positive by qPCR. Before treatment, 11 (42%) of 26 patients with post-kala-azar dermal leishmaniasis were deemed infectious to sandflies by microscopy and 23 (88%) by qPCR. Of 23 patients who were available for xenodiagnosis after treatment, one remained infectious to flies by qPCR on the pooled flies, but none remained positive by microscopy. None of the 184 asymptomatic participants were infectious to sandflies. INTERPRETATION: These findings confirm that patients with active visceral leishmaniasis and patients with post-kala-azar dermal leishmaniasis can transmit L donovani to the sandfly vector and suggest that early diagnosis and treatment could effectively remove these individuals as infection reservoirs. An important role for asymptomatic individuals in the maintenance of the transmission cycle is not supported by these data. FUNDING: Bill & Melinda Gates Foundation.

Intranasal immunization with peptide-based immunogenic complex enhances BCG vaccine efficacy in a murine model of tuberculosis.

Prime-boost immunization strategies are required to control the global tuberculosis (TB) pandemic, which claims approximately 3 lives every minute. Here, we have generated an immunogenic complex against Mycobacterium tuberculosis (M.tb), consisting of promiscuous T cell epitopes (M.tb peptides) and TLR ligands assembled in liposomes. Interestingly, this complex (peptide-TLR agonist-liposomes; PTL) induced significant activation of CD4+ T cells and IFN-γ production in the PBMCs derived from PPD+ healthy individuals as compared with PPD- controls. Furthermore, intranasal delivery of PTL significantly reduced the bacterial burden in the infected mice by inducing M.tb-specific polyfunctional (IFN-γ+IL-17+TNF-α+IL-2+) immune responses and long-lasting central memory responses, thereby reducing the risk of TB recurrence in DOTS-treated infected animals. The transcriptome analysis of peptide-stimulated immune cells unveiled the molecular basis of enhanced protection. Furthermore, PTL immunization significantly boosted the Bacillus Calmette-Guerin-primed (BCG-primed) immune responses against TB. The greatly enhanced efficacy of the BCG-PTL vaccine model in controlling pulmonary TB projects PTL as an adjunct vaccine against TB.

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

The immune landscape in tuberculosis reveals populations linked to disease and latency.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) latently infects approximately one-fourth of the world's population. The immune mechanisms that govern progression from latent (LTBI) to active pulmonary TB (PTB) remain poorly defined. Experimentally Mtb-infected non-human primates (NHP) mirror the disease observed in humans and recapitulate both PTB and LTBI. We characterized the lung immune landscape in NHPs with LTBI and PTB using high-throughput technologies. Three defining features of PTB in macaque lungs include the influx of plasmacytoid dendritic cells (pDCs), an Interferon (IFN)-responsive macrophage population, and activated T cell responses. In contrast, a CD27+ Natural killer (NK) cell subset accumulated in the lungs of LTBI macaques. This NK cell population was also detected in the circulation of LTBI individuals. This comprehensive analysis of the lung immune landscape will improve the understanding of TB immunopathogenesis, providing potential targets for therapies and vaccines for TB control.

High glucose alters the DNA methylation pattern of neurodevelopment associated genes in human neural progenitor cells in vitro.

Maternal diabetes alters the global epigenetic mechanisms and expression of genes involved in neural tube development in mouse embryos. Since DNA methylation is a critical epigenetic mechanism that regulates gene functions, gene-specific DNA methylation alterations were estimated in human neural progenitor cells (hNPCs) exposed to high glucose (HG) in the present study. The DNA methylation pattern of genes involved in several signalling pathways including axon guidance (SLIT1-ROBO2 pathway), and Hippo pathway (YAP and TAZ) was altered in hNPCs exposed to HG. The expression levels of SLIT1-ROBO2 pathways genes (including its effectors, SRGAP1 and CDC42) which mediates diverse cellular processes such as proliferation, neurogenesis and axon guidance, and Hippo pathway genes (YAP and TAZ) which regulates proliferation, stemness, differentiation and organ size were downregulated in hNPCs exposed to HG. A recent report suggests a possible cross-talk between SLIT1-ROBO2 and TAZ via CDC42, a mediator of actin dynamics. Consistent with this, SLIT1 knockdown downregulated the expression of its effectors and TAZ in hNPCs, suggesting that HG perturbs the cross-talk between SLIT1-ROBO2 and TAZ in hNPCs. Overall, this study demonstrates that HG epigenetically alters the SLIT1-ROBO2 and Hippo signalling pathways in hNPCs, forming the basis for neurodevelopmental disorders in offspring of diabetic pregnancy.

Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice.

Despite the availability of multiple antibiotics, tuberculosis (TB) remains a major health problem worldwide, with one third of the population latently infected and ~2 million deaths annually. The only available vaccine for TB, Bacillus Calmette Guérin (BCG), is ineffective against adult pulmonary TB. Therefore, alternate strategies that enhance vaccine efficacy are urgently needed. Vaccine efficacy and long-term immune memory are critically dependent on central memory T (TCM) cells, whereas effector memory T (TEM) cells are important for clearing acute infections. Recently, it has been shown that inhibition of the Kv1.3 K+ ion channel, which is predominantly expressed on TEM but not TCM cells, profoundly enhances TCM cell differentiation. We exploited this phenomenon to improve TCM:TEM cell ratios and protective immunity against Mycobacterium tuberculosis infection in response to BCG vaccination of mice. We demonstrate that luteolin, a plant-derived Kv1.3 K+ channel inhibitor, profoundly promotes TCM cells by selectively inhibiting TEM cells, and significantly enhances BCG vaccine efficacy. Thus, addition of luteolin to BCG vaccination may provide a sustainable means to improve vaccine efficacy by boosting host immunity via modulation of memory T cell differentiation.

Testosterone Acts Within the Medial Amygdala of Rats to Reduce Innate Fear to Predator Odor Akin to the Effects of Toxoplasma gondii Infection.

Rats infected with the protozoan Toxoplasma gondii exhibit a reduced aversion to cat odor. This behavioral change is thought to increase trophic transmission of the parasite. Infected male rats also show a greater testicular synthesis of testosterone and epigenetic change in arginine vasopressin within the medial amygdala. Here, we show that exogenous supply of testosterone within MeA of uninfected castrates recapitulates reduction in innate fear akin to behavioral change attributed to the parasite. We also show that castration post establishment of chronic infection precludes changes in fear and medial amygdala arginine vasopressin in the infected male rats. These observations support the role of gonadal hormones and pursuant neuroendocrine changes in mediating the loss of fear in the infected rats. This work also demonstrates that testosterone acting specifically within the medial amygdala sufficiently explains reduced defensive behaviors often observed during the appetitive component of reproductive behaviors.

Hypoxia and myelination deficits in the developing brain.

Myelination is a complex and orderly process during brain development that is essential for normal motor, cognitive and sensory functions. Cellular and molecular interactions between myelin-forming oligodendrocytes and axons are required for normal myelination in the developing brain. Oligodendrocyte progenitor cells (OPCs) proliferate and differentiate into mature myelin-forming oligodendrocytes. In this connection, astrocytes and microglia are also involved in survival and proliferation of OPCs. Hypoxic insults during the perinatal period affect the normal development, differentiation and maturation of the OPCs or cause their death resulting in impaired myelination. Several factors such as augmented release of proinflammatory cytokines by activated microglia and astrocytes, extracellular accumulation of excess glutamate and increased levels of nitric oxide are some of the underlying factors for hypoxia induced damage to the OPCs. Additionally, hypoxia also leads to down-regulation of several genes involved in oligodendrocyte differentiation encoding proteolipid protein, platelet-derived growth factor receptor and myelin-associated glycoprotein in the developing brain. Furthermore, oligodendrocytes may also accumulate increased amounts of iron in hypoxic conditions that triggers endoplasmic reticulum stress, misfolding of proteins and generation of reactive oxygen species that ultimately would lead to myelination deficits. More in-depth studies to elucidate the pathophysiological mechanisms underlying the inability of oligodendrocytes to myelinate the developing brain in hypoxic insults are desirable to develop new therapeutic options or strategies for myelination deficits.

Nanoparticle-Formulated Curcumin Prevents Posttherapeutic Disease Reactivation and Reinfection with Mycobacterium tuberculosis following Isoniazid Therapy.

Curcumin, the bioactive component of turmeric also known as "Indian Yellow Gold," exhibits therapeutic efficacy against several chronic inflammatory and infectious diseases. Even though considered as a wonder drug pertaining to a myriad of reported benefits, the translational potential of curcumin is limited by its low systemic bioavailability due to its poor intestinal absorption, rapid metabolism, and rapid systemic elimination. Therefore, the translational potential of this compound is specifically challenged by bioavailability issues, and several laboratories are making efforts to improve its bioavailability. We developed a simple one-step process to generate curcumin nanoparticles of ~200 nm in size, which yielded a fivefold enhanced bioavailability in mice over regular curcumin. Curcumin nanoparticles drastically reduced hepatotoxicity induced by antitubercular antibiotics during treatment in mice. Most interestingly, co-treatment of nanoparticle-formulated curcumin along with antitubercular antibiotics dramatically reduced the risk for disease reactivation and reinfection, which is the major shortfall of current antibiotic treatment adopted by Directly Observed Treatment Short-course. Furthermore, nanoparticle-formulated curcumin significantly reduced the time needed for antibiotic therapy to obtain sterile immunity, thereby reducing the possibility of generating drug-resistant variants of the organisms. Therefore, adjunct therapy of nano-formulated curcumin with enhanced bioavailability may be beneficial to treatment of tuberculosis and possibly other diseases.

Blockade of the Kv1.3 K+ Channel Enhances BCG Vaccine Efficacy by Expanding Central Memory T Lymphocytes.

Tuberculosis is the oldest known infectious disease, yet there is no effective vaccine against adult pulmonary tuberculosis. Emerging evidence indicates that T-helper 1 and T-helper 17 cells play important roles in host protection against tuberculosis. However, tuberculosis vaccine efficacy in mice is critically dependent on the balance between antigen-specific central memory T (Tcm) and effector memory T (Tem) cells. Specifically, a high Tcm/Tem cell ratio is essential for optimal vaccine efficacy. Here, we show that inhibition of Kv1.3, a potassium channel preferentially expressed by Tem cells, by Clofazimine selectively expands Tcm cells during BCG vaccination. Furthermore, mice that received clofazimine after BCG vaccination exhibited significantly enhanced resistance against tuberculosis. This superior activity against tuberculosis could be adoptively transferred to naive, syngeneic mice by CD4+ T cells. Therefore, clofazimine enhances Tcm cell expansion, which in turn provides improved vaccine efficacy. Thus, Kv1.3 blockade is a promising approach for enhancing the efficacy of the BCG vaccine in humans.