Maintaining oral health is a continuous and dynamic process that also involves the immune system. Polymorphonuclear neutrophils (PMNs) migrate from blood circulation and become apparent in the oral fluid. Controversies exist regarding the specific role of the oral PMNs (oPMNs) in the presence of chronic oral inflammation, such as periodontitis. In this study we characterized cell counts, activation status, apoptosis, and reactive oxygen species (ROS) generation by oPMNs and circulatory (cPMNs), and the salivary protease activity, in subjects with and without periodontitis. Venous blood and oral rinse samples were obtained from 19 patients with untreated periodontitis and 16 control subjects for PMN isolation. Apoptosis and expression of cell activation markers CD11b, CD63, and CD66b were analyzed using flow cytometry. Constitutive ROS generation was detected using dihydrorhodamine123. Additionally, ROS production in response to stimulation was evaluated in samples incubated with 10 μM phorbol myristate acetate (PMA) or Fusobacterium nucleatum. Total protease activity was measured using substrate PEK-054. Periodontitis patients presented with over 4 times higher oPMN counts compared to controls (p = 0.007), which was a predictor for the total protease activity (r² = 0.399, P = 0.007). More oPMNs were apoptotic in periodontitis patients compared to the controls (P = 0.004). All three activation markers were more expressed on the oPMNs compared to the cPMNs (p < 0.05), and a higher expression of CD11b on the oPMNs from periodontitis patients was observed compared to the control subjects (P = 0.024). Constitutive ROS production per oPMN was higher compared to the cPMN (P < 0.001). Additional analysis showed that the oPMNs retained their ability to respond to stimulation, with no apparent differences between the periodontitis and control subjects. Higher numbers of oral PMNs, being more apoptotic and having increased levels of degranulation markers were found in periodontitis compared to periodontal health. However, since the oPMNs in periodontitis were responsive to ex vivo stimulation, we conclude that the oPMNs are active in the oral ecosystem. It is currently unknown whether the oPMN counts, which correlated with the detected protease levels, are detrimental in the long term for the oral mucosa integrity. This study was retrospectively registered at the ISRCTN registry (trial ID ISRCTN15252886 ). Registration date August 11, 2017.

Since periodontitis is bi-directionally associated with several systemic diseases, such as diabetes mellitus and cardiovascular diseases, it is important for medical professionals in a non-dental setting to be able examine their patients for symptoms of periodontitis, and urge them to visit a dentist if necessary. However, they often lack the time, knowledge and resources to do so. We aim to develop and assess “quick and easy” screening tools for periodontitis, based on self-reported oral health (SROH), demographics and/or salivary biomarkers, intended for use by medical professionals in a non-dental setting. Consecutive, new patients from our outpatient clinic were recruited. A SROH questionnaire (8 questions) was conducted, followed by a 30 s oral rinse sampling protocol. A complete clinical periodontal examination provided the golden standard periodontitis classification: no/mild, moderate or severe periodontitis. Total periodontitis was defined as having either moderate or severe. Albumin and matrix metalloproteinase-8 concentrations, and chitinase and protease activities were measured in the oral rinses. Binary logistic regression analyses with backward elimination were used to create prediction models for both total and severe periodontitis. Model 1 included SROH, demographics and biomarkers. The biomarkers were omitted in the analysis for model 2, while model 3 only included the SROH questionnaire. The area under the receiver operating characteristic curves (AUROCC) provided the accuracy of each model. The regression equations were used to create scoring algorithms, composed of the remaining predictors, each with its own weight. Of the 156 patients participating in this study, 67% were classified with total periodontitis and 33% had severe periodontitis. The models for total periodontitis achieved an AUROCC of 0.91 for model 1, 0.88 for model 2 and 0.81 for model 3. For severe periodontitis, this was 0.89 for model 1, 0.82 for model 2 and 0.78 for model 3. The algorithm for total periodontitis (model 2), which we consider valid for the Dutch population, was applied to create a freely accessible, web-based screening tool. The prediction models for total and severe periodontitis proved to be feasible and accurate, resulting in easily applicable screening tools, intended for a non-dental setting.

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control—scrambled F-Hst1 (F-Hst^scr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotracker^TM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1^scr co-localized with the ER-tracker^TM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1^scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

Cervical, uterine, and ovarian cancers are the most common malignancies of the female genital tract worldwide. Despite advances in prevention, early diagnosis, effective screening, and treatment programs, mortality remains high. Consequently, it is important to search for new treatments. The activity of bovine lactoferrin (bLF) and LF peptides against several types of cancer has been studied; however, only a few studies report the effect of bLF and LF peptides against cervical and endometrial cancers. In this study, we explored the effect of bLF as well as LF chimera and its constituent peptides LFcin17–30 and LFampin265–284 on the viability of cervical (HeLa, SiHa) and endometrial (KLE, HEC-1A) cancer cell lines. Cell proliferation was quantified with an MTT assay, cell morphological changes and damage were determined by Giemsa and phalloidin-TRITC and DAPI staining, and apoptotic and necrotic cells were identified by Alexa Fluor® 488 Annexin V and propidium iodide staining. Additionally, the effect of combinations of bLF and LF peptides with cisplatin was assessed. bLF and LF peptides inhibited the proliferation of uterine cancer cells and caused cellular morphological changes and damage to cell monolayers. bLF induced apoptosis, LFcin17–30 and LFampin265–284 induced apoptosis and necrosis, and LF chimera induced necrosis. Additionally, bLF and LF chimera showed an additive interaction with cisplatin against uterine cancer cells.

Aim was to profile salivary total protease, Porphyromonas gingivalis gingipain, and neutrophil elastase activities in relation to the resolution of periodontal inflammation, salivary macrophage-derived chemokine (MDC), and macrophage inflammatory protein-1α concentrations. Nonsurgical periodontal treatment was performed in 24 periodontitis patients in a prospective interventional study design. Periodontal clinical parameters were recorded, and stimulated saliva samples were collected at baseline and 2, 6, and 12 weeks after treatment. Salivary total protease and gingipain activities were determined using fluorogenic substrates, elastase activity by chromogenic substrates, and cytokine concentrations by Luminex immunoassay. For statistical analyses, generalized linear mixed models for repeated measures were used. Salivary total protease activity elevated, while gingival inflammation and plaque accumulation decreased 2 and 6 weeks after periodontal therapy. Salivary MDC concentration was elevated 12 weeks after periodontal treatment. Patients with elevated protease activities at baseline in comparison to patients with low baseline total protease activities, had higher levels of gingival inflammation before and after periodontal treatment. In conclusion, elevations in salivary total protease activity seem to be part of periodontal healing at its early phases. Higher levels of salivary total protease activities before periodontal treatment may predict the severity and steadiness of unresolved gingival inflammation.

Oral microbiota have been implicated in pancreatic ductal adenocarcinoma (PDAC) and may contribute to chemotherapy resistance. While previous studies attributed bacteria-induced resistance to indirect host modulation, recent findings suggest a direct mechanism. Escherichia coli expressing long-form cytidine deaminase (CDD_L) can degrade gemcitabine, a chemotherapeutic agent, into a non-toxic form, leading to resistance. In contrast, bacteria carrying short form (CDD_S) or lacking CDD did not induce resistance. This study investigates whether oral bacteria can cause gemcitabine resistance in PDAC cells through CDD-mediated degradation. Oral microbes associated with PDAC were selected based on CDD isoforms: Aggregatibacter actinomycetemcomitans carrying CDD_L, Enterococcus faecalis, Streptococcus mutans, Porphyromonas gingivalis, all carrying CDD_S, and Fusobacterium nucleatum lacking CDD. The selected microbes, along with wild-type and CDD-deficient E. coli, were co-incubated with gemcitabine to assess its degradation and PDAC cell proliferation. A. actinomycetemcomitans fully degraded gemcitabine and induced resistance. Surprisingly, CDD_S-expressing oral bacteria partially degraded gemcitabine in a strain-dependent manner. Expressing either CDD_L or CDD_S in CDD-deficient E. coli resulted in equivalent gemcitabine degradation and resistance, indicating that CDD function is independent of isoform length. These findings highlight the role of oral bacteria in gemcitabine resistance and the need for strategies to mitigate microbial-driven resistance in PDAC treatment.