Role of NK-Like CD8+ T Cells during Asymptomatic Borrelia burgdorferi Infection.

Lyme disease (LD) due to Borrelia burgdorferi is the most prevalent vector-borne disease in the United States. There is a poor understanding of how immunity contributes to bacterial control, pathology, or both during LD. Dogs in an area of endemicity were screened for B. burgdorferi and Anaplasma exposure and stratified according to seropositivity, presence of LD symptoms, and doxycycline treatment. Significantly elevated serum interleukin-21 (IL-21) and increased circulating CD3+ CD94+ lymphocytes with an NK-like CD8+ T cell phenotype were predominant in asymptomatic dogs exposed to B. burgdorferi. Both CD94+ T cells and CD3- CD94+ lymphocytes, corresponding to NK cells, from symptomatic dogs expressed gamma interferon (IFN-γ) at a 3-fold-higher frequency upon stimulation with B. burgdorferi than the same subset among endemic controls. Surface expression of activating receptor NKp46 was reduced on CD94+ T cells from LD, compared to cells after doxycycline treatment. A higher frequency of NKp46-expressing CD94+ T cells correlated with significantly increased peripheral blood mononuclear cell (PBMC) cytotoxic activity via calcein release assay. PBMCs from dogs with symptomatic LD showed significantly reduced killing ability compared with endemic control PBMCs. An elevated NK-like CD8+ T cell response was associated with protection against development of clinical LD, while excess IFN-γ was associated with clinical disease.

miRNA expression profiling of hereditary breast tumors from BRCA1- and BRCA2-germline mutation carriers in Brazil.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene expression regulation and have been described as key regulators of carcinogenesis. Aberrant miRNA expression has been frequently reported in sporadic breast cancers, but few studies have focused on profiling hereditary breast cancers. In this study, we aimed to identify specific miRNA signatures in hereditary breast tumors and to compare with sporadic breast cancer and normal breast tissues. METHODS: Global miRNA expression profiling using NanoString technology was performed on 43 hereditary breast tumors (15 BRCA1, 14 BRCA2, and 14 BRCAX), 23 sporadic breast tumors and 8 normal breast tissues. These normal breast tissues derived from BRCA1- and BRCA2- mutation carriers (n = 5) and non-mutation carriers (n = 3). Subsequently, we performed receiver operating characteristic (ROC) curve analyses to evaluate the diagnostic performance of differentially expressed miRNAs. Putative target genes of each miRNAs considered as potential biomarkers were identified using miRDIP platform and used for pathway enrichment analysis. RESULTS: miRNA expression analyses identified several profiles that were specific to hereditary breast cancers. A total of 25 miRNAs were found to be differentially expressed (fold change: > 2.0 and p < 0.05) and considered as potential biomarkers (area under the curve > 0.75) in hereditary breast tumors compared to normal breast tissues, with an expressive upregulation among BRCAX cases. Furthermore, bioinformatic analysis revealed that these miRNAs shared target genes involved in ErbB, FoxO, and PI3K-Akt signaling pathways. CONCLUSIONS: Our results showed that miRNA expression profiling can differentiate hereditary from sporadic breast tumors and normal breast tissues. These miRNAs were remarkably deregulated in BRCAX hereditary breast cancers. Therefore, miRNA signatures can be used as potential novel diagnostic biomarkers for the prediction of BRCA1/2- germline mutations and may be useful for future clinical management.

Resident Synovial Macrophages in Synovial Fluid: Implications for Immunoregulation in Infectious and Inflammatory Arthritis.

Objectives: Resident synovial macrophages (RSM) provide immune sequestration of the joint space and are likely involved in initiation and perpetuation of the joint-specific immune response. We sought to identify RSM in synovial fluid (SF) and demonstrate migratory ability, in additional to functional changes that may perpetuate a chronic inflammatory response within joint spaces. Methods: We recruited human patients presenting with undifferentiated arthritis in multiple clinical settings. We used flow cytometry to identify mononuclear cells in peripheral blood and SF. We used a novel transwell migration assay with human ex-vivo synovium obtained intra-operatively to validate flow cytometry findings. We used single cell RNA-sequencing (scRNA-seq) to further identify macrophage/monocyte subsets. ELISA was used to evaluate the bone-resorption potential of SF. Results: We were able to identify a rare population of CD14dim, OPG+, ZO-1+ cells consistent with RSM in SF via flow cytometry. These cells were relatively enriched in the SF during infectious processes, but absolutely decreased compared to healthy controls. Similar putative RSM were identified using ex vivo migration assays when MCP-1 and LPS were used as migratory stimulus. scRNA-seq revealed a population consistent with RSM transcriptionally related to CD56+ cytotoxic dendritic cells and IDO+ M2 macrophages. Conclusion: We identified a rare cell population consistent with RSM, indicating these cells are likely migratory and able to initiate or coordinate both acute (septic) or chronic (autoimmune or inflammatory) arthritis. RSM analysis via scRNA-seq indicated these cells are M2 skewed, capable of antigen presentation, and have consistent functions in both septic and inflammatory arthritis.

EV-miRNAs from breast cancer patients of plasma as potential prognostic biomarkers of disease recurrence.

Background: Extracellular vesicles (EVs), ubiquitously released by blood cells, facilitate intercellular communication. In cancer, tumor-derived EVs profoundly affect the microenvironment, promoting tumor progression and raising the risk of recurrence. These EVs contain miRNAs (EV-miRNAs), promising cancer biomarkers. Characterizing plasma EVs and identifying EV-miRNAs associated with breast cancer recurrence are crucial aspects of cancer research since they allow us to discover new biomarkers that are effective for understanding tumor biology and for being used for early detection, disease monitoring, or approaches to personalized medicine. This study aimed to characterize plasma EVs in breast cancer (BC) patients and identify EV-miRNAs associated with BC recurrence. Methods: This retrospective observational study included 24 BC patients divided into recurrence (n= 11) and non-recurrence (n= 13) groups. Plasma EVs were isolated and characterized. Total RNA from EVs was analyzed for miRNA expression using NanoString's nCounter® miRNA Expression Assays panel. MicroRNA target prediction used mirDIP, and pathway interactions were assessed via Reactome. Results: A stronger presence of circulating EVs was found to be linked with a less favorable prognosis (p = 0.0062). We discovered a distinct signature of EV-miRNAs, notably including miR-19a-3p and miR-130b-3p, which are significantly associated with breast cancer recurrence. Furthermore, miR-19a-3p and miR-130b-3p were implicated in the regulation of PTEN and MDM4, potentially contributing to breast cancer progression.A notable association emerged, indicating a high concentration of circulating EVs predicts poor prognosis (p = 0.0062). Our study found a distinct EV-miRNA signature involving miR-19a-3p and miR-130b-3p, strongly associated with disease recurrence. We also presented compelling evidence for their regulatory roles in PTEN and MDM4 genes, contributing to BC development. Conclusion: This study revealed that increased plasma EV concentration is associated with BC recurrence. The prognostic significance of EVs is closely tied to the unique expression profiles of miR-19a-3p and miR-130b-3p. These findings underscore the potential of EV-associated miRNAs as valuable indicators for BC recurrence, opening new avenues for diagnosis and treatment exploration.

Modulation of Macrophage Redox and Apoptotic Processes to Leishmania infantum during Coinfection with the Tick-Borne Bacteria Borrelia burgdorferi.

Canine leishmaniosis (CanL) is a zoonotic disease caused by protozoan Leishmania infantum. Dogs with CanL are often coinfected with tick-borne bacterial pathogens, including Borrelia burgdorferi in the United States. These coinfections have been causally associated with hastened disease progression and mortality. However, the specific cellular mechanisms of how coinfections affect microbicidal responses against L. infantum are unknown. We hypothesized that B. burgdorferi coinfection impacts host macrophage effector functions, prompting L. infantum intracellular survival. In vitro experiments demonstrated that exposure to B. burgdorferi spirochetes significantly increased L. infantum parasite burden and pro-inflammatory responses in DH82 canine macrophage cells. Induction of cell death and generation of mitochondrial ROS were significantly decreased in coinfected DH82 cells compared to uninfected and L. infantum-infected cells. Ex vivo stimulation of PBMCs from L. infantum-seronegative and -seropositive subclinical dogs with spirochetes and/or total Leishmania antigens promoted limited induction of IFNγ. Coexposure significantly induced expression of pro-inflammatory cytokines and chemokines associated with Th17 differentiation and neutrophilic and monocytic recruitment in PBMCs from L. infantum-seropositive dogs. Excessive pro-inflammatory responses have previously been shown to cause CanL pathology. This work supports effective tick prevention and risk management of coinfections as critical strategies to prevent and control L. infantum progression in dogs.

Epidemiologic, Clinical and Immunological Consequences of Co-Infections during Canine Leishmaniosis.

Canine leishmaniosis (CanL) is a vector-borne, parasitic disease. CanL is endemic in the Mediterranean basin and South America but also found in Northern Africa, Asia, and the U.S. Regions with both competent sand fly vectors and L. infantum parasites are also endemic for additional infectious diseases that could cause co-infections in dogs. Growing evidence indicates that co-infections can impact immunologic responses and thus the clinical course of both CanL and the comorbid disease(s). The aim for this review is to summarize epidemiologic, clinical, and immunologic factors contributing to eight primary co-infections reported with CanL: Ehrlichia spp., Anaplasma spp., Borrelia spp., Babesia spp., Trypanosoma cruzi, Toxoplasma gondii, Dirofilaria immitis, Paracoccidioides braziliensis. Co-infection causes mechanistic differences in immunity which can alter diagnostics, therapeutic management, and prognosis of dogs with CanL. More research is needed to further explore immunomodulation during CanL co-infection(s) and their clinical impact.

MicroRNA Biomarkers of High-Grade Cervical Intraepithelial Neoplasia in Liquid Biopsy.

New prevention strategies are needed to detect cervical intraepithelial neoplasia (CIN). The microRNA expression analysis has already been reported as molecular biomarkers in the early detection of cervical cancer (CC) through minimally invasive samples, such as liquid biopsy, obtained through collection using liquid-based cytology (LBC). In this study, we aimed to identify molecular signatures of microRNAs in cervical precursor lesions from LBC cervical and the molecular pathways potentially associated with the CC progression. We analyzed 31 LBC cervical samples from women who underwent colposcopy. These samples were divided into two groups: the first group was composed of samples without precursor lesions of CC, considering the control group, referred to as healthy female subjects (HFS; n = 11). The second group corresponded to women diagnosed with cervical interepithelial neoplasia grade 3 (CIN 3; n = 20). We performed microRNA and gene expression profiling using the nCounter® miRNA Expression Assays (NanoString Technology) and PanCancer Pathways (NanoString Technology), respectively. A microRNA target prediction was performed by mirDIP, and molecular pathway interaction was constructed using Cytoscape. Bidirectional in silico analyses and Pearson's correlation were performed for associated the relation between genes, and miRNAs differentially expressed related cervical cancer progression were performed. We found that the expression of nine microRNAs was significantly higher, two were downregulated (miR-381-3p and miR-4531), and seven miRNAs were upregulated (miR-205-5p, miR-130a-3p, miR-3136-3p, miR-128-2-5p, let-7f-5p, miR-202-3p, and miR-323a-5p) in CIN 3 (fold change ≥ 2 and p ≤ 0.05). The miRNA expression patterns were independent of hr-HPV infection. We identified four miRNAs (miR-205-5p, miR-130a-3p, miR-4531, and miR-381-3p) that could be used as biomarkers for CIN 3 in LBC samples through multiple logistic regression analyses. We found 16 genes differentially expressed between CIN 3 and HSF samples (fold change ≥ 2 and p ≤ 0.05). We found the correlation between miR-130a-3p and CCND1(R = -0.52; p = 0.0029), miR-205-5p and EGFR (R = 0.53; p = 0.0021), and miR-4531 and SMAD2 (R = -0.54; p = 0.0016). In addition, we demonstrated the most significant pathways of the targets associated with cervical cancer progression (FDR-corrected p < 0.001). This study demonstrated that miRNA biomarkers may distinguish healthy cervix and CIN 3 and regulate important molecular pathways of carcinogenesis.

Identification and performance evaluation of housekeeping genes for microRNA expression normalization by reverse transcription-quantitative PCR using liquid-based cervical cytology samples.

Screening for cervical cancer by cytology has been effective in reducing the worldwide incidence and mortality rates of this disease. However, a number of studies have demonstrated that the sensitivity of conventional cervical cytology may be too low for detection of cervical intraepithelial neoplasias (CIN). Therefore, it is important to incorporate more sensitive molecular diagnostic tests that could substantially improve the detection rates and accuracy for identifying CIN lesions. MicroRNAs (miRNAs) are a class of small non-coding RNAs with the potential to provide robust non-invasive cancer biomarkers for detecting CIN lesions in liquid-based cervical cytology (LBC) samples. At present, there is no consensus on which are the best housekeeping genes for miRNA normalization in LBC. The present study aimed to identify housekeeping genes with consistent and reproducible performance for normalization of reverse transcription-quantitative PCR (RT-qPCR) expression analysis of miRNA using LBC samples. The present study firstly selected six potential candidate housekeeping genes based on a systematic literature evaluation. Subsequently, the expression levels of microRNAs U6, RNU-44, RNU-47, RNU-48, RNU-49 and hsa-miR-16 were measured in 40 LBC samples using RT-qPCR. The stability of each potential housekeeping gene was assessed using the NormFinder algorithm. The results revealed that U6 and RNU-49 were the most stable genes among all candidates requiring fewer amplification cycles and smaller variation across the sample set. However, RNU-44, RNU-47, RNU-48 and hsa-miR-16 stability exceeded the recommended housekeeping value suitable for normalization. The findings revealed that U6 may be a reliable housekeeping gene for normalization of miRNA RT-qPCR expression analysis using LBC samples.

Association between BRAF (V600E) mutation and clinicopathological features of papillary thyroid carcinoma: a Brazilian single-centre case series.

OBJECTIVES: We aimed to investigate the prevalence of the BRAF (V600E) mutation in consecutive cases of papillary thyroid carcinoma (PTC) in patients diagnosed and treated at the Hospital Sao Rafael (Salvador, BA, Brazil) and evaluate its association with clinical and pathological characteristics of PTC. SUBJECTS AND METHODS: We retrospectively enrolled in the study a total of 43 consecutive PTC patients who underwent total thyroidectomy. We performed DNA extraction from formalin-fixed paraffin-embedded (FFPE) tumour tissue samples. Polymerase chain reaction (PCR) and direct sequencing were used to determine BRAF (V600E) mutation status. Univariate and multivariate logistic regression analyses were employed to identify independent associations. RESULTS: The prevalence of BRAF (V600E) mutation was 65.1% (28/43). A high frequency of older patients (p value: 0.004) was observed among the BRAF-mutated PTC group and, in contrast, a low frequency of concurrent Hashimoto's thyroiditis (HT) (p value: 0.011) was noted. Multivariate analysis confirmed that older age (OR: 1.15; 95% CI: 1.00 - 1.33; p value: 0.047) and HT (OR: 0.05; 95% CI: 0.006-0.40; p value: 0.005) were independent factors associated with BRAF (V600E) mutation. CONCLUSION: We found a high prevalence of BRAF (V600E) mutation in PTC cases. Older age and no concurrent HT were independently associated with BRAF (V600E) mutation.

Association between BRAF (V600E) mutation and clinicopathological features of papillary thyroid carcinoma: a Brazilian single-centre case series

ABSTRACT Objectives: We aimed to investigate the prevalence of the BRAF (V600E) mutation in consecutive cases of papillary thyroid carcinoma (PTC) in patients diagnosed and treated at the Hospital Sao Rafael (Salvador, BA, Brazil) and evaluate its association with clinical and pathological characteristics of PTC. Subjects and methods: We retrospectively enrolled in the study a total of 43 consecutive PTC patients who underwent total thyroidectomy. We performed DNA extraction from formalin-fixed paraffin-embedded (FFPE) tumour tissue samples. Polymerase chain reaction (PCR) and direct sequencing were used to determine BRAF (V600E) mutation status. Univariate and multivariate logistic regression analyses were employed to identify independent associations. Results: The prevalence of BRAF (V600E) mutation was 65.1% (28/43). A high frequency of older patients (p value: 0.004) was observed among the BRAF-mutated PTC group and, in contrast, a low frequency of concurrent Hashimoto's thyroiditis (HT) (p value: 0.011) was noted. Multivariate analysis confirmed that older age (OR: 1.15; 95% CI: 1.00 - 1.33; p value: 0.047) and HT (OR: 0.05; 95% CI: 0.006-0.40; p value: 0.005) were independent factors associated with BRAF (V600E) mutation. Conclusion: We found a high prevalence of BRAF (V600E) mutation in PTC cases. Older age and no concurrent HT were independently associated with BRAF (V600E) mutation.