High-dose Infection Research Articles

Emergence of antibiotic-specific Mycobacterium tuberculosis phenotypes during prolonged treatment of mice.

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiologic state of M. tuberculosis (Mtb) which may enable the pathogen to withstand treatment. While antibiotic-treated Mtb have been evaluated in short-term in vitro experiments, it is unclear if and how long-term in vivo treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affect Mtb physiologic states differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the Mtb transcriptome in the BALB/c high-dose aerosol infection mouse model following 4-week treatment with three sterilizing and three non-sterilizing antibiotics. Certain transcriptional changes were concordant among most antibiotics, including decreased expression of genes associated with protein synthesis and metabolism, and the induction of certain genes associated with stress responses. However, the magnitude of this concordant response differed between antibiotics. Sterilizing antibiotics rifampin, pyrazinamide, and bedaquiline generated a more quiescent Mtb state than did non-sterilizing antibiotics isoniazid, ethambutol, and streptomycin, as indicated by decreased expression of genes associated with translation, transcription, secretion of immunogenic proteins, metabolism, and cell wall synthesis. Additionally, we identified distinguishing transcriptional effects specific to each antibiotic, indicating that different mechanisms of action induce distinct patterns of cellular injury. In addition to elucidating Mtb physiologic changes associated with antibiotic stress, this study demonstrates the value of SEARCH-TB as a highly granular pharmacodynamic assay that reveals antibiotic effects that are not apparent based on culture alone.

Context-dependent role of group 3 innate lymphoid cells in mucosal protection.

How group 3 innate lymphoid cells (ILC3s) regulate mucosal protection in the presence of T cells remains poorly understood. Here, we examined ILC3 function in intestinal immunity using ILC3-deficient mice that maintain endogenous T cells, T helper 17 (TH17) cells, and secondary lymphoid organs. ILC3s were dispensable for generation of TH17 and TH22 cell responses to commensal and pathogenic bacteria, and absence of ILC3s did not affect IL-22 production by CD4 T cells before or during infection. However, despite the presence of IL-22-producing T cells, ILC3s and ILC3-derived IL-22 were required for maintaining homeostatic functions of the intestinal epithelium. T cell-sufficient, ILC3-deficient mice were capable of pathogen clearance and survived infection with a low dose of Citrobacter rodentium. However, ILC3s promoted pathogen tolerance at early time points of infection by activating tissue-protective immune pathways. Consequently, ILC3s were indispensable for survival after high-dose infection. Our results demonstrate a context-dependent role for ILC3s in immune-sufficient animals and provide a blueprint for uncoupling of ILC3 and TH17 cell functions.

Interactions between migration and immunity among oriental armyworm populations infected with the insect pathogenic fungus, Beauveria bassiana.

Migration and immunity are behavioral and physiological traits that protect organisms from environmental stressors or pathogen infection. Shifting from migration to residency has become more common in some wildlife populations owing to environmental changes. However, other biological shifts, such as interactions between migration and immunity among populations within a species are largely unexplored for many agricultural migratory pests. In the field, entomopathogenic fungi infection and transmission, particularly Beauveria bassiana, can cause reduced fitness and population declines across a broad range of insect species. Here, we investigated migration-immunity interactions between migrant and resident populations of the oriental armyworm, Mythimna separata, infected with B. bassiana (the sole fungus used in this work). We found that migratory M. separata exerted stronger pathogen resistance, faster development and lower pupal weight than residents. High-dose infections (5.0 × 105 and 5.0 × 106 conidia mL-1) led to seriously decreased reproductive capacity in migrants and residents. Low-dose infections (1.0 × 104 and 5.0 × 104 conidia mL-1) led to significantly increased host flight capacities. Consecutive flight tests showed that five flight nights inhibited the reproduction of paternal infected M. separata populations. The flights also led to far-reaching transgenerational impairment of larval development and immune defense among offspring populations. By contrast, two flight nights enhanced the reproductive capacities of both M. separata populations and did not exert negative transgenerational effects on offspring populations, which may facilitate migration. This study provides insights into interactions between migration and immunity among M. separata populations. These insights will guide development of future monitoring and management technologies of this pest. © 2024 Society of Chemical Industry.

Characterization of a Molecular Clone of Deformed Wing Virus B.

Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments.

Influenza A virus infection disrupts the function of syncytiotrophoblast cells and contributes to adverse pregnancy outcomes.

Pregnancy heightens susceptibility to influenza A virus (IAV) infection, thereby increasing the risk of severe pneumonia and maternal mortality. It also raises the chances of adverse outcomes in offspring, such as fetal growth restriction, preterm birth, miscarriage, and stillbirth in offsprings. However, the underlying mechanisms behind these effects remain largely unknown. Syncytiotrophoblast cells, crucial in forming the placental barrier, nutrient exchange and hormone secretion, have not been extensively studied for their responses to IAV. In our experiment, we used Forskolin-treated BeWo cells to mimic syncytiotrophoblast cells in vitro, and infected them with H1N1, H5N1 and H7N9 virus stains. Our results showed that syncytiotrophoblast cells, with their higher intensity of sialic acid receptors, strongly support IAV infection and replication. Notably, high-dose viral infection and prolonged exposure resulted in a significant decrease in fusion index, as well as gene and protein expression levels associated with trophoblast differentiation, β-human chorionic gonadotropin secretion, estrogen and progesterone biosynthesis, and nutrient transport. In pregnant BALB/c mice infected with the H1N1 virus, we observed significant decreases in trophoblast differentiation and hormone secretion gene expression levels. IAV infection also resulted in preterm labor, fetal growth restriction, and increased maternal and fetal morbidity and mortality. Our findings indicate that IAV infection in syncytiotrophoblastic cells can result in adverse pregnancy outcomes by altering trophoblast differentiation, suppressing of β-hCG secretion, and disrupting placental barrier function.

Susceptibility and cecal microbiota alteration to Eimeria-infection in Yellow-feathered broilers, Arbor Acres broilers and Lohmann pink layers

Coccidiosis, which is caused by Eimeria species, results in huge economic losses to the poultry industry. Arbor Acres (AA) broilers and yellow-feathered broilers are the dominant broilers in northern and southern China, respectively. However, their susceptibility to coccidiosis has not been fully compared. In this study, the susceptibility of yellow-feathered broilers, AA broilers and Lohmann pink layers to E. tenella was evaluated based on mortality rate, relative body weight gain rate, intestinal lesion score, oocyst output, anticoccidial index (ACI), and cecum weight and length. The yellow-feathered broilers were shown to produce significantly fewer oocysts with higher intestinal lesion score compared to AA broilers, which had the highest growth rates and ACI scores. Subsequently, changes in the cecal microbiota of the 3 chicken lines before and after high-dose infection (1 × 104 oocysts) with E. tenella were determined by 16S rRNA sequencing. The results showed that composition of the microbiota changed dramatically after infection. The abundance of Firmicutes and Bacteroidetes in the infected chickens decreased, and Proteobacteria increased significantly among the different chicken lines. At the genus level, Escherichia increased significantly in all 3 groups of infected chickens, but Lactobacillus decreased to 0% in the infected yellow-feathered broilers. The results of the study indicate that the susceptibility to E. tenella varies among the 3 chicken lines, and that changes in intestinal microbiota by E. tenella-infection among the different chicken lines had a similar trend, but to different degrees. This study provides basic knowledge of the susceptibility in the 3 chicken lines, which can be helpful for the control and prevention of coccidiosis.

Quantification and Potential Viability of Human Noroviruses in Final Effluent from Wastewater Treatment Works in Pretoria, South Africa

Growing global concerns over water scarcity, worsened by climate change, drive wastewater reclamation efforts. Inadequately treated wastewater presents significant public health risks. Previous studies in South Africa (SA) have reported high norovirus levels in final effluent and sewage-polluted surface water, indicating pathogen removal inefficiency. However, the viability of these virions was not explored. This study assessed human norovirus viability in final effluent from wastewater treatment works (WWTWs) in Pretoria, SA. Between June 2018 and August 2020, 200 samples were collected from two WWTWs, including raw sewage and final effluent. Norovirus concentrations were determined using in-house RNA standards. Viability of noroviruses in final effluent was assessed using viability RT-qPCR (vPCR) with PMAxx™-Triton X-100. There was no significant difference in GI concentrations between raw sewage (p = 0.5663) and final effluent (p = 0.4035) samples at WWTW1 and WWTW2. WWTW1 had significantly higher GII concentrations in raw sewage (p < 0.001) compared to WWTW2. No clear seasonal pattern was observed in norovirus concentrations. At WWTW1, 50% (7/14) of GI- and 64.9% (24/37) of GII-positive final effluent samples had no quantifiable RNA after vPCR. At WWTW2, the majority (92.6%, 25/27) of GII-positive final effluent samples showed a 100% RNA reduction post vPCR. PMAxx™-Triton X-100 vPCR provides a more accurate reflection of discharge of potentially viable noroviruses in the environment than standard RT-qPCR. Despite significant reductions in potentially viable noroviruses after wastewater treatment, the levels of potentially viable viruses in final effluent are still of concern due to the high initial load and low infectious dose of noroviruses.

VapC12 ribonuclease toxin modulates host immune response during Mycobacterium tuberculosis infection.

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with ΔvapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with ΔvapC12 succumbed early to the disease. Finally, we reported that the above phenotype of ΔvapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the ΔvapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host.

Comparison of the oncolytic activity of a replication-competent and a replication-deficient herpes simplex virus 1.

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-β mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.

Autophagy promotes efficient T cell responses to restrict high-dose Mycobacterium tuberculosis infection in mice.

Although autophagy sequesters Mycobacterium tuberculosis (Mtb) in in vitro cultured macrophages, loss of autophagy in macrophages in vivo does not result in susceptibility to a standard low-dose Mtb infection until late during infection, leaving open questions regarding the protective role of autophagy during Mtb infection. Here we report that loss of autophagy in lung macrophages and dendritic cells results in acute susceptibility of mice to high-dose Mtb infection, a model mimicking active tuberculosis. Rather than observing a role for autophagy in controlling Mtb replication in macrophages, we find that autophagy suppresses macrophage responses to Mtb that otherwise result in accumulation of myeloid-derived suppressor cells and subsequent defects in T cell responses. Our finding that the pathogen-plus-susceptibility gene interaction is dependent on dose has important implications both for understanding how Mtb infections in humans lead to a spectrum of outcomes and for the potential use of autophagy modulators in clinical medicine.

Purinergic P2X7R expressed on regulatory T cells potentially links molecular mimicry to autoimmune responses

&lt;abstract&gt; &lt;p&gt;The immune system is meant to protect against invading microbes. Although this system is effective against many microbes, some can use molecular mimicry to turn the immune system against the host and activate autoimmune responses. The resulting autoimmunity has significant implications for public health and healthcare costs. It is well known that regulatory T cells (Tregs) are crucial for self-tolerance and that their function is impaired after exposure to self-antigens or antigens with molecular mimicry, leading to the activation of autoimmune responses. How molecular mimicry disrupts Tregs in this manner remains under debate. This review contributes to the field of the pathogenesis of autoimmunity by proposing that purinergic signaling in the lymph nodes, with extracellular ATP, ADP, and adenosine as ligands, plays a pivotal role in this process. Repeated or high-dose microbial infection causes the release of large amounts of extracellular ATP sufficient to reach the threshold of extracellular ATP levels for activating P2X7 purinergic receptors (P2X7Rs) on dendritic cells and Tregs. This hampers the ability of Tregs to suppress autoimmune responses. Crucially, P2X7Rs are activated at very high extracellular ATP levels, thus only after repeated or high-dose infection with microbes. Arguably, in contrast to the rapid elimination of microbes with foreign antigens, the clearance of invading microbes that employ molecular mimicry requires the activation of P2X7Rs at the expense of self-tolerance. Because all processes required to activate autoimmune responses occur in secondary lymphoid organs, this article hypothesizes that, contrary to current convention, microbes do not need to enter organs to initiate autoimmunity. However, some types of microbes can prevent P2X7R-induced Treg disruption by converting extracellular ATP to adenosine, mitigating autoimmune responses resulting in chronic diseases with less severe inflammation. The proposed hypothesized mechanism has potential implications for the understanding and treatment of autoimmune disorders.&lt;/p&gt; &lt;/abstract&gt;

The detection of Toxoplasma gondii ME49 infections in BALB/c mice using various techniques.

Toxoplasma gondii infections are primarily diagnosed by serological assays, whereas molecular and fluorescence-based techniques are garnering attention for their high sensitivity in detecting these infections. Nevertheless, each detection method has its limitations. The toxoplasmosis detection capabilities of most of the currently available methods have not been evaluated under identical experimental conditions. This study aimed to assess the diagnostic potential of enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and immunofluorescence (IF) in BALB/c mice experimentally infected with various doses of T. gondii ME49. The detection of toxoplasmosis from sera and brain tissues was markedly enhanced in mice subjected to high infection doses (200 and 300 cysts) compared to those subjected to lower doses (10 and 50 cysts) for all the detection methods. Additionally, increased B1 gene expression levels and cyst sizes were observed in the brain tissues of the mice. Importantly, IHC, IF, and ELISA, but not RT-PCR, successfully detected T. gondii infections at the lowest infection dose (10 cysts) in the brain. These findings may prove beneficial while designing experimental methodologies for detecting T. gondii infections in mice.

Investigation of the Detectability of Corn Smut Fungus (Ustilago maydis DC. Corda) Infection Based on UAV Multispectral Technology

Corn smut fungus (Ustilago maydis [DC.] Corda) is a globally widespread pathogen affecting both forage and sweet maize hybrids, with higher significance in sweet maize. Remote sensing technologies demonstrated favorable results for disease monitoring on the field scale. The study focused on the changes in vegetation index (VI) values influenced by the pathogen. In this study, four hybrids, two forage maize and two sweet maize hybrids were examined. Artificial infection was carried out at three different doses: a low (2500 sporidium number/mL), medium (5000 sporidium number/mL) and high dose (10,000 sporidium number/mL) with a non-infected control plot for each hybrid. The experimental plots were monitored using a multispectral UAV sensor of five monochrome channels on three different dates, i.e., 7, 14 and 21 days after infection. Five different indices (NDVI, GNDVI, ENDVI, LCI, and NDRE) were determined in Quantum GIS 3.20. The obtained results demonstrated that the infection had a significant effect on the VI values in sweet maize hybrids. A high-dose infection in the Dessert R 73 hybrid resulted in significantly lower values compared to the non-infected hybrids in three indices (NDVI, LCI and GNDVI). In the case of the NOA hybrids, GNDVI and ENDVI were able to show significant differences between the values of the infection levels.

STAT3 operates as a rheostat regulating sensitivity of NK cells to inflammatory cytokines

Abstract NK cells are innate lymphocytes that respond to a variety of cytokines during viral infection. Several homeostatic and inflammatory cytokines bind to receptors that signal via STAT3. STAT3 can form homodimers or heterodimers. To understand the role of STAT3 homodimers and heterodimers, we made use of knockout models in which NK cells lack STAT3 or specific cytokine receptors. We found that NK cells deficient in STAT3 expand less than wildtype NK cells during mouse cytomegalovirus (MCMV) infection. IL-21R and IL-10R are the only receptors expressed on NK cells that signal through STAT3 homodimers. However, IL-21R or IL-10R-deficient NK cells showed no or little expansion differences, suggesting additional cytokine receptors may be signaling through STAT3. To evaluate whether STAT3 regulates the sensitivity of NK cells to cytokines that signal through heterodimerization partners STAT1 and STAT5, we performed cytokine titrations. We found that sensitivity towards IFN-a and IL-15 was reduced in STAT3-deficient NK cells. NK cells require inflammatory cytokines for antiviral expansion but proliferate poorly in hyper-inflammatory environments. Because STAT3 appears to regulate the sensitivity of NK cells to inflammatory cytokines, we hypothesized that STAT3-deficient NK cells would be shielded in settings of hyper-inflammation. Indeed, using high-dose MCMV infection, we observed that STAT3-deficient NK cells expanded better than WT NK cells. RNA-seq analysis further corroborated that in high-dose infection STAT3-deficient NK cells are nearly indistinguishable from WT NK cells during regular-dose infection. Thus, we believe STAT3 operates as a rheostat, modulating the sensitivity of NK cells to their inflammatory environment. Supported by the Cancer Research Institute, Donald J. Gogel Postdoctoral Fellowship, CRI393

Visualizing the early immune response to Mycobacterium tuberculosisinfection in macaques immunized with intravenous BCG

Abstract Tuberculosis (TB) causes 1.6 million deaths annually and the standard human vaccine, intradermal (ID) Bacille-Calmette Guérin (BCG), does not prevent pulmonary TB. Previously, we showed that intravenous (IV) BCG immunization elicited robust TB-specific lung-resident T cell responses compared to ID BCG in rhesus macaques (RMs). Following Mycobacterium tuberculosis (Mtb) challenge, 6 out of 10 RMs had no detectable disease as demonstrated by bacterial burden, granuloma formation, and primary Mtb-specific immune responses. Together, these data suggest that Mtb is cleared rapidly from the lungs of IV BCG-vaccinated RMs. To investigate the vaccine-induced lung environment and anamnestic response that might mediate protection against Mtb early after challenge, we developed a high-dose Mtb infection model in RMs that allowed us to locate and live-image areas of Mtb infection in the lungs using confocal microscopy. RMs immunized for 5 months with either IV or ID BCG were challenged with Mtb Erdman mCherry and euthanized 4 or 11 days later to characterize the lung microenvironment. We observed reductions in bacterial burdens in the lungs of RMs that received IV BCG compared to ID BCG-vaccinated and unvaccinated controls by CFU plating, flow cytometry, and confocal microscopy despite using a high challenge dose. Increased T cells were quantified in the lungs of both IV and ID BCG-vaccinated RMs compared to unvaccinated controls by flow cytometry and confocal microscopy. This research provides an opportunity to mine immune correlates of protection in a preclinical model, at the site of infection, and at a post-challenge time point that has been minimally characterized. NIH Intramural Program

An effective live-attenuated Zika vaccine candidate with a modified 5′ untranslated region

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that has caused devastating congenital Zika syndrome (CZS), including microcephaly, congenital malformation, and fetal demise in human newborns in recent epidemics. ZIKV infection can also cause Guillain-Barré syndrome (GBS) and meningoencephalitis in adults. Despite intensive research in recent years, there are no approved vaccines or antiviral therapeutics against CZS and adult Zika diseases. In this report, we developed a novel live-attenuated ZIKV strain (named Z7) by inserting 50 RNA nucleotides (nt) into the 5′ untranslated region (UTR) of a pre-epidemic ZIKV Cambodian strain, FSS13025. We used this particular ZIKV strain as it is attenuated in neurovirulence, immune antagonism, and mosquito infectivity compared with the American epidemic isolates. Our data demonstrate that Z7 replicates efficiently and produces high titers without causing apparent cytopathic effects (CPE) in Vero cells or losing the insert sequence, even after ten passages. Significantly, Z7 induces robust humoral and cellular immune responses that completely prevent viremia after a challenge with a high dose of an American epidemic ZIKV strain PRVABC59 infection in type I interferon (IFN) receptor A deficient (Ifnar1−/−) mice. Moreover, adoptive transfer of plasma collected from Z7 immunized mice protects Ifnar1−/− mice from ZIKV (strain PRVABC59) infection. These results suggest that modifying the ZIKV 5′ UTR is a novel strategy to develop live-attenuated vaccine candidates for ZIKV and potentially for other flaviviruses.

Assessment of esxA, hsp18, and 16S transcript expression as a measure of Mycobacterium leprae viability: A comparison with the mouse footpad assay

Objectives Shepard’s mouse footpad (MFP) assay is the definitive method for ascertaining Mycobacterium leprae viability; however, this technique is laborious and prolonged. Alternate methods that alleviate burden and time while maintaining sensitivity would benefit clinical and experimental studies. We compared a molecular viability assay (MVA), which measures normalized hsp18 and esxA transcript expression, with growth in the MFP as a determinant of M. leprae viability. Methods Receiver operating characteristic curve analyses were performed to determine viability cut-off values for M. leprae expression of hsp18, esxA, and 16S transcripts. To verify these values, immunocompetent BALB/c mice were inoculated in the footpads with a high dose of viable M. leprae. Footpads were harvested on Day 1 and at various timepoints up to 12 months post infection. M. leprae viability was determined by MVA or by sub-inoculating bacilli into passage mice per Shepard’s MFP assay. Results Cut-off levels for expression of each transcript for M. leprae viability were established with high confidence (CI = 99%, P &lt; 0.0001). In the high-dose infection assay, M. leprae exhibited a significant decrease in transcript detection by three months post-infection. Expression of hsp18 and esxA transcripts by M. leprae correlated with bacterial growth in the MFP assay. In contrast, 16S transcripts were detected in M. leprae populations confirmed dead by the MFP assay. Conclusion The MVA, which is a sensitive, specific, and rapid viability indicator, was directly validated by the MFP assay. This study confirms the accuracy of the MVA for determining M. leprae viability.

Characterization of an air-liquid interface primary human vaginal epithelium to study Ebola virus infection and testing of antivirals

Ebola virus (EBOV) is the causative agent of the often-fatal Ebola virus disease (EVD) characterized by hemorrhagic fever in humans and non-human primates. Sexual transmission from male survivors has been at the origin of multiple outbreak flare-ups between 2015 and 2021. However, this route is still poorly understood and the resulting EVD from it is also understudied. To support epidemiological studies documenting sexual transmission to women, and as a transition from previously using monolayer vaginal epithelial cells (VK2/E6E7), we first determined the biological relevance of two similar air-liquid interface models of the human vaginal epithelium (VEC and VLC Epivaginal™) and then characterized their susceptibility to EBOV and virus-induced inflammation. Finally, we evaluated toxicity of Polyphenylene Carboxymethylene (PPCM) microbicide in VLC and reassessed its antiviral effect.As expected, the VEC, but also VLC model showed stratified layers including a lamina propria under an epithelial structure similar to the full thickness of the human vaginal epithelium. However, we could not detect the immune cells featured in the most relevant model (VLC) of the vaginal epithelium using the dendritic cell CD1a and CD11c markers. Consistent with our previous work using the VK2/E6E7 cell line, infectious virus was detected from the apical side of both primary human cell systems, but only when using a high infective dose, with titers remaining at a constant level of 103−4 pfu/ml over 7 days suggesting lasting infectious virus shedding. In addition, infection caused disruption of the epithelium of both models and virus antigen was found from the apical superficial layers down to the lamina propria suggesting full virus penetration and overall confirming the susceptibility of the human vaginal tissue for EBOV. Just like previously seen in VK2/E6E7 cells, VLC infection also caused significant increase in inflammatory markers including IL-6, IL-8, and IP-10 suggesting vaginitis which is again consistent with tissue lesions seen in non-human primates. Finally, both virus infection and virus-induced inflammatory response in VLC could be prevented by a single 5-min PPCM microbicide treatment prior infection.

Pneumocystis jirovecii Infections Among COVID-19 Patients: A Case Series and Literature Review.

Pneumocystis jirovecii pneumonia (PCP) is a serious, emerging complication of coronavirus disease 2019 (COVID-19). We performed a systematic review of published cases. We describe 6 new cases of PCP/COVID-19 coinfection. Among our cases (n = 6) and those in the literature (n = 69) with available data, the median age (interquartile range [IQR]) was 59 (44-77) years (n = 38), 72% (47/65) were male, and the mortality rate was 30.9% (21/68). Long-term corticosteroid use was noted in 45.1% (23/51), advanced HIV infection (defined as a CD4 count <200 cells/μL) in 17.6% (9/51), and antineoplastic chemotherapy in 13.7% (7/51), consistent with known PCP risk factors. Notably, 56.7% (38/47) had verifiable risk factors for PCP (high-dose corticosteroids, immunosuppressive therapy, and HIV infection) before COVID-19 infection. A median absolute lymphocyte count (IQR) of 0.61 (0.28-0.92) ×103 cells/mm3 (n = 23) and CD4 count (IQR) of 66 (33-291.5) cells/mm3 (n = 20) were also discovered among the study population. These findings suggest a need for greater attention to PCP risk factors among COVID-19 patients and consideration of PCP prophylaxis in these high-risk populations.

Severe persistent mycobacteria antigen stimulation causes lymphopenia through impairing hematopoiesis.

Miliary tubersculosis (TB), an acute systemic blood disseminated tuberculosis mainly caused by Mycobacterium tuberculosis (M. tuberculosis), can cause signs of lymphopenia in clinical patients. To investigate whether/how persistent mycobacteria antigen stimulation impairs hematopoiesis and the therapeutic effect of interleukin-7 (IL-7), a mouse model of Mycobacterium Bovis Bacillus Calmette-Guérin (BCG) intravenous infection with/without an additional stimulation with M. tuberculosis multi-antigen cocktail containing ESAT6-CFP10 (EC) and Mtb10.4-HspX (MH) was established. Consistent with what happened in miliary TB, high dose of BCG intravenous infection with/without additional antigen stimulation caused lymphopenia in peripheral blood. In which, the levels of cytokines IFN-γ and TNF-α in serum increased, and consequently the expression levels of transcription factors Batf2 and IRF8 involved in myeloid differentiation were up-regulated, while the expression levels of transcription factors GATA2 and NOTCH1 involved in lymphoid commitment were down-regulated, and the proliferating activity of bone marrow (BM) lineage- c-Kit+ (LK) cells decreased. Furthermore, recombinant Adeno-Associated Virus 2-mediated IL-7 (rAAV2-IL-7) treatment could significantly promote the elevation of BM lymphoid progenitors. It suggests that persistent mycobacteria antigen stimulation impaired lymphopoiesis of BM hematopoiesis, which could be restored by complement of IL-7.