Biomedical Journal of Scientific & Technical Research .BJSTR

Monday, July 13, 2026

Comparative Analysis of Salivary Cellular Composition in Patients with Chronic Inflammatory Bowel Disease

Introduction

The oral cavity reflects the overall condition of the body. Signs of systemic disease are commonly manifested in the oral cavity before the systemic disease itself suspected. The various oral tissues (lips, tongue, gingiva, mucous surfaces, teeth) and fluids (saliva and cervical fluid) are involved in the presentation of disease state [1]. Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBD) with multifactorial origin including the immune system (autoimmune response and autoantibodies against organ-specific cellular antigens shared by the gastrointestinal tract and other systems), genetic sensitivity and environmental factors (diet, use of antibiotics or NSAIDs and the presence of enteral infections) [2]. Both the intestinal mucosa and the oral mucosa contain a large number of immune cells that make up the so-called mucosal immune system, functioned to preserve the body’s constancy against antigens that have penetrated the digestive system [3]. The commensal microbial flora promotes immune processes at the level of secretion of antimicrobial peptides, regulatory and effector immune cells [4]. This symbiosis with the microbiome helps maintain homeostasis, while dysbiosis induces altered immune responses with an inflammatory response [5]. CD and UC can engage not only the mucosa of the gut, but also have extra-intestinal manifestations in the oral cavity and other organs (joints, skin, eyes, bile ducts). Oral lesions as a clinical manifestation were first described in 1969 by Dudeney [6]. The incidence ranges from 0.5% to 30% most commonly seen in Crohn’s disease (cheilitis, ulcerations, fissures and glossitis) [7]. Oral manifestations in 5-10% of cases may be the first sign of bowel disease [8] or precede the appearance of intestinal lesions by a year or more [9]. Some of the oral changes are considered disease-specific and others are non-specific (aphthous stomatitis, pyostomatitis), but they can help diagnose and monitor the activity of the process [10,11].

Oral manifestations in IBD patients may be due to other causes, such as drug reactions, infections, malnutrition, adherence to specific diets, complications (inflammatory activity, dyselectrolytemia anemia, and hypovitaminosis) [1]. The relationship between changes in the oral cavity and IBD has not been sufficiently clarified. The inflammatory response, autoimmune genesis, dysbacteriosis, and infection are thought to be factors leading to specific changes in the oral cavity, most pronounced in the activity of inflammatory bowel mucosa [12]. The oral cavity and its oral fluid are an integral part of the gastro-intestinal tract (GIT), have a common phylogenetic and morpho-functional origin and structure, obey general neurohumoral regulation and, in addition to digestive function, have a protective role for the digestive system [13]. Saliva is an aqueous secretion with a slightly acidic to neutral pH of 6.0-7.0 and includes dissolved inorganic ions and various organic substances, including proteins (mucins, immunoglobulins, enzymes), epithelial cells, bacteria, leukocytes and food residues [14]. Oral fluid is a mirror of the metabolic, functional, hormonal and emotional state of the body [15]. Investigation of saliva as a non-invasive biological material may answer the question whether the pathological process and inflammatory activity in IBD can influence and alter its cellular content and functions. These cellular and functional alterations could serve us to monitor patients’ status. The aim of the present study is to investigate the salivary cell content and composition in patients with IBD and evaluate the effect of intestinal inflammatory activity on it.

Material and Methods

Subjects

The study included 54 patients (30 women and 24 men) with IBD (40 with UC and 14 with CDs), mean age 43.9±14.7 (range: 19 to 74 years) admitted to the gastroenterology wards at the University Hospital “St. Marina” and Military Hospital - Varna during September 2017 – May 2019 for the diagnosis of IBD or exacerbation of the disease, as well as for routine control colonoscopies. The diagnoses of UC and CD of the examined patients were made based on the criteria of ECCO Consensus 2019, year (European Crohn’s and Colitis Organization), including a complex of anamnestic, clinical, laboratory and instrumental studies. All participants enrolled in the study have signed informed consent. The local Ethical Committee approved the study (Protocol No 64/13.07.2017). As a control group, 80 healthy subjects (43 women and 37 men) aged between 20-65 years (mean: 43.1±10.8 years, corresponding to that of the patient group; p>0.05) undergoing routine prophylactic examinations, including dental status at the Military hospital-Varna were examined. The exclusion criteria were inflammatory changes in the mouth and dental procedures within 48-72 hours before the examination. The preliminary requirements for the collection and primary treatment of biological material have been clarified and respected. Unstimulated native saliva is collected in the morning by a passive droplet.

Collection Protocol

In all patients, saliva was collected under the same conditions: on an empty stomach (in the morning from 8-10 h) without the use of tonic drinks (coffee) and smoking. Unstimulated saliva is used. The oral fluid is collected in special sterile containers (conical bottom and graduated) by passively repeatedly removing the amount collected in the mouth within 5-10 minutes to a total amount of 2-3 ml. For reliable results, patients were instructed to comply with the following conditions: More than 30 minutes have elapsed since the last meal, drink, chewing gum or the last toothbrush and toothbrush cleaning. Mouth 5 min before the test, rinse twice with grunt for 10 sec. with saline or mineral water.

Material Processing

The saliva is hypotonic, lytic cellular processes are accelerated, so the material is quickly processed within 15-20 minutes. Until then, the samples are refrigerated at 4°C. On the graduated scale of the containers, we count the amount of saliva collected. We spin the containers at 2500 rpm for 5 minutes. The supernatant was separated. Resolve the sediment with dilute (dilution buffer for biological material at FUS 100 cell count) of the same amount to the original volume. We homogenize well. Cell counting is performed by the FUS 100 automated urinary sedimentation system (DIRUI). The method is based on flow cytometric microscopic high-speed images to enumerate and identify the formed elements in the sample. The pictures of the sample cells are compared to a database of images of various morphologies and crystals embedded in the instrument software and classified according to shape, structure and size. The number of images is calculated as count/μL. The morphological characteristics of the different cellular elements obtained by the automatic analysis are also verified visually. Routine laboratory methods were used for the assay of the inflammatory markers: WBC count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). All IBD patients were tested for FC, a specific biomarker for intestinal inflammation, by the quantitative immunochromatographic method Point-of-Care Test (Quantum Blue®fCAL, BÜHLMANN).

Statistical Analysis

Descriptive statistical analysis, non-parametric T-test for comparison of the mean values and Spearman correlation test were used for data processing. Values of p <0.05 were considered significant.

Results

The mean disease duration was 7.44±9.51 (1-31) years. All patients are on therapy (mono- or combination therapy) given in (Table 1). The pharmacological management of IBD aims to reduce inflammation and maintain remission. Therapy includes sulfasalazine, 5-aminosalicylates, glucocorticoids and immunosuppressants, and a series of biological agents, monoclonal antibodies (adalimumab, golimumab, infliximab). The classification of patients with IBD according to disease activity/severity is given in (Table 2). It is based on the indices used to evaluate activity or disease severity: for CD - CDAI (Crohn’s Disease Activity Index), and for UC - Mayo index. According to these indices, we divided the patients included in the study into two groups. The first group was patients with active disease (CDAI> 220 and Mayo index ≥2), and the second group was patients with mild or remission. The number of cellular elements (epithelial cells, erythrocytes, leukocytes and bacteria) are presented in (Table 3). (Figure 1) Leukocytes and epithelial cells, as significant components of the mucosal epithelial barrier, are most affected by changes in physiological and pathological conditions. Physiological factors such as gender and age have been found to influence the values of these parameters in both the control and patient groups. The comparative analysis showed that the number of cells in the norm had a gender variation. Women have slightly higher values than men.

Table 1: Types of drug therapy in the IBD study group.

Table 2: Demographic and clinical characteristics of patients with.

Table 3: Cellular elements in unstimulated saliva in the three study groups.

Note: *Differences in the number of cell elements in the three study groups were evaluated using One way ANOVA Bonferroni correction

**The reference limits were determined for the purposes of our study in a representative group of clinically healthy persons from the Bulgarian population. The selection of persons from the reference group (n =186) was carried out randomly - from persons passing to MMA-Varna, subject to a routine annual preventive examination or expert assessment, without subjective and objective deviations (healthy persons) and normal dental status.

Figure 1: Comparison of salivary cell elements in the three studied groups

A) Erythrocytes,

B) Leukocytes,

C) Epithelial cells and

D) Bacteria (One way ANOVA with Bonferroni correction is attached).

The data are presented in (Table 4). Similar observations are described by Rijkschroeff, et al. [16]. Many studies show that the oral mucosa is sensitive to the effect of sex hormones (estrogens and progesterone). Different periods in a woman’s life, such as puberty, pregnancy, and menopause, are associated with changes in their levels affect oral mucosa. Donald, et al. [17,18] disclose wellexpressed rhythmic changes in the oral cavity cells, coinciding with changes detected in vaginal smears, thus reflecting the hormonal state of the menstrual cycle [17,18]. We also find a moderate positive correlation (r=0.358; p=0.009) between age and increased oral leukocytes, but not with epithelial cells. The number of leukocytes in the mouth decreases in proportion to the reduction of teeth due to their inability to migrate through the gingival crevices. The mean disease duration was 7.44±9.51 (1-31) years. We found no relationship between disease duration and cellular element values. They are influenced by the activity of the inflammatory process.

Table 4: Values of leukocytes and epithelial cells in control and patient groups and their sex dependence.

Discussion

Cells found in the oral fluid have different origins. Leaving the ductus of the parotid gland, the saliva contains no cellular elements [19]. They are added by buccal mucosa, migration of leukocytes and erythrocytes from different areas of the oral mucosa. The largest is the abundance of epithelial cells that are usually excreted in chewing processes. The oral cavity is covered by squamous epithelial cells, which are a barrier against mechanical, chemical damage and pathogenic microbiological invasion [20]. The normal mucosal epithelium is a multilayer flat with high regenerative capacity. Due to its involvement in the digestive process, however, it remains un keratinized or partially corrosive in certain places (such as the intricately arranged mucous membrane of the tongue with the papillae available) and secretes mucus to facilitate the passage of food. It is already known that epithelial cells are not passive bystanders, but are metabolically active and able to respond to external stimuli by synthesizing several cytokines, adhesion molecules, growth factors, chemokines and matrix metalloproteases [21]. Morphologically, squamous cells have a small nucleus and a large polygonal cytoplasm with numerous granulations. Cell sizes are 85-125 μm, while those of the gingiva and periodontium are smaller in size. According to Watanabe, the number of epithelial cells in the saliva is 4x105/ml. [22].

Several studies have tracked the number and change in morphology of epithelial cells in screening and diagnosis of precancerous conditions and oral cancer [23]. Inflammatory lesions and periodontal disease also lead to an increase in the number of squamous cells secreted. Our study found that the control group had higher values of epithelial cells, given by some researchers as the norm. This can be explained by the presence of dental bridges and dental crown, whose finding in the oral cavity increases with age. Smoking is also an essential factor acting irritably on the oral mucosa and a significant risk factor for inflammatory and neoplastic processes. In a previous study, we found a statistically significant difference in the number of epithelial cells and leukocytes in smokers compared to non-smokers. Of the subjects in our control group, smokers were 36, representing 45%, and 23 (43%) from the patient group. We observed an increased number of epithelial cells in patients with IBD, which was statistically significant relative to the control group (p=0.0192). However, no such dependence was observed between the two patient groups. The finding is believed to be due to the chronic inflammatory process, dehydration and poor nutrition, especially during relapse. Little is known about the factors that affect the relative number of epithelial cells and leukocytes in the oral cavity. The buccal mucosa is relatively permeable and has a rich blood supply, with continuous flow and migration of leukocytes from the gingival fluid through the gingival gap in the saliva [24].

Healthy buccal mucosa contains several cells involved in immune function, including lymphocytes (T cells), polymorphonuclear cells (including neutrophils and eosinophils), and mast cells [25,26]. They protect the oral cavity and are an element of the innate immune response. Leukocyte counts in norm and pathology have been the subject of several studies. It is estimated that about 1 – 4x105 cells are normally found in 1 ml of saliva. Their number has inter-individual but also circadian intra-individual variation [19]. Leukocytes are predominantly represented by polymorphonuclear cells (PMNs) - 90% and 10% monomorphonuclear cells (MMNs). The major representatives of MMNs are B lymphocytes (60%), T cells (about 20-30%) and macrophages (~10%), which are integral parts of mucosa-associated lymphoid tissue (MALT). Its role is to induce an immune response to specific antigens from the environment and to develop local immunity. In a healthy state, approximately 30,000 oral PMNs (oPMN) per minute arrive through gingival cervical fluid (GCF), which inflows into the oral cavity from the periodontal sulcus [27]. Eighty percent of oPMNs are viable and functional in the gingival gap and play a phagocytic and antibacterial role. In the hypotonic saliva, oPMNs undergo rapid lytic changes. Functional PMNs are paramount to innate immunological processes, including maintenance of oral health.

In acute and chronic inflammatory processes in the oral cavity, the amount of oPMN increases. Rijkschroeff, et al. [16] report significantly higher expression of CD11b in oPMNs compared to circulating blood PMNs in healthy individuals, suggesting their facilitated migration through oral mucosal tissues. There are insufficient studies on the effects of systemic diseases on salivary cell composition and oral hemostasis. We found a statistically significant difference (p<0.0001) in leukocyte counts between the control group and patients with IBD, but there was no similar relationship between the two patient groups (with activity and mild/remission). The active involvement of oral mucosa in the immunological response and inflammation most likely causes leukocyte growth in the saliva. We did not find a correlation between the total number of circulating blood leukocytes and the number of salivary leukocytes. Although bPMNs reflect the general inflammatory state of an individual, the number of oPMNs varies greatly depending on local influences in the oral cavity [16,28]. A similar weak correlation was observed between oPMN and C-reactive protein as a marker of inflammation. Corticosteroid therapy probably affects the content of cellular elements in the oral fluid, causing increased permeability and migration of erythrocytes and leukocytes through the gingival crevices. Calprotectin is a cytoplasmic protein secreted by the degranulation of neutrophil leukocytes and monocytes locally at the site of inflammation.

Faecal calprotectin (FC) has in recent years become a marker reflecting the activity of the inflammatory mucosal response in IBD. Majster, et al. [28] examined calprotectin in saliva from patients with IBD, finding elevated values in both stimulated and unstimulated saliva. In our patients, we routinely examine FC for disease monitoring. By correlation analysis, we checked whether there was a relationship between oral leukocyte count and FC as a marker of inflamed intestinal mucosa. A weak correlation r=0.2043 was found. For comparability, the correlation between the number of salivary leukocytes and salivary calprotectin should be used. The normal amount of erythrocytes in the oral fluid is negligible, although there is no accurate literature data on their normal count per milliliter. Most studies look at the increase in red blood cells and the presence of salivary blood in fissures, lesions, periodontal or cancerous diseases in the oral cavity [29,30]. Our study found equally higher erythrocyte values in both patient groups, regardless of the inflammatory process activity. This is most likely due to side effects of medicines leading to increased permeability of the vessels in the oral cavity, ecchymoses and bleeding. It is more commonly observed in patients with mono or combination therapy with Infliximab (a TNFα inhibitor) [31]. The oral cavity and gastrointestinal tract are highly colonized sites [16]. Bacteria are the most numerous cells, represented mainly by normal microflora, but pathological species can also be found.

In healthy individuals, the microflora have a symbiotic relationship with the host organism and possess important and unique functions, including metabolic function. The human oral microbiome is regarded as a community of more than 300 species of microorganisms, with a total salivary total of 106-108 CFU/ml (colony-forming unit) [32]. Gram positive bacteria predominate, and anaerobic bacteria are about 10 to 100 times the amount of aerobes [33]. In the study group, especially those with inflammatory process activity, the bacterial count was statistically significantly lower. During the relapse, antibiotic therapy is included as part of the treatment and treatment of the inflammatory process. Most likely, antibiotics lead to microflora suppression by reducing their population or leading to dysbiosis. The thesis about the reducing role of AB against the oral microflora is confirmed by the fact that in the second patient group and without antibiotic therapy, the number of salivary bacteria is almost similar to that of the control group.

Conclusion

Oral health and homeostasis, maintaining oral hygiene is crucial for patients with IBD during their treatment and remission. The concept of enumeration of cellular elements in saliva with the FUS 100 automated system may be proposed as a possible screening method for the indication of inflammatory changes in the mouth. A rapid, non-invasive qualitative and quantitative assessment of cellular composition would be a new approach in laboratory or clinical diagnosis and monitoring and management of patients with IBD.

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Surface Plasmon Resonance: Applications in Detection of Tumour Markers

Introduction

The second most prevalent globally, cancer is a serious issue for human wellbeing [1]. As per World Health Organization (WHO) statistics from 2019, cancer is the third or fourth major cause of mortality before the age of 70 in 23 nations, while it is the top or second main cause in 112 of 183 countries [2]. Cancer is the uncontrollable proliferation of leukemic cells that causes significant modifications in physiological mechanisms [3]. Normal cells, chemicals, and blood arteries that surround and nourish a tumour are affected by cancer cells, which have the ability to elude the immune system [4]. The digestive, neurological, and circulatory systems may be affected by the tumours, which have the ability to spread and expand to other parts of the body. They may also emit hormones that affect how the body works. For anticancer drug development, designing molecules that can selectively inhibit the proliferation of abnormal cells with minimal or no effect on normal cells is critical [5]. Therefore, developing anticancer drugs is of utmost importance worldwide. Developing compounds that may selectively limit the growth of aberrant cells with little to no impact on healthy cells is crucial for the development of anticancer drugs [6]. Therefore, creating anticancer medications is of highest significance everywhere. In 2020, it is anticipated that were 19.3 million cases of cancer globally (18.1 million with the exception of nonmelanoma skin cancer) and over 10 million cancer deaths (9.9 million excluding nonmelanoma skin cancer).

With an expected 2.3 million new cases (11.7%), female breast carcinoma has overtaken lung cancer as the most often detected malignancy. Lung (11.4%), colorectal (10%), prostate (7.3%), and stomach (5.6%) cancers are the next most commonly detected malignancies. With an expected 1.8 million fatalities (18%), lung cancer continued to be the most common kind of cancer. It was then followed by colorectal (9.4%), liver (8.3%), stomach (7.7%), and female breast (6.9%) tumours [7]. Surface plasmon resonance (SPR) has received a lot of interest and offers a wide range of uses in biosensors and chemical sensors [8]. Over than 80 years ago, Wood first identified a phenomenon, and it wasn’t until Liedberg et al. that the first gas sensing plus biosensing principles were developed [9]. The significant aspects of this innovation, such as its sensitivity, real-time identification, and label-free assay, set it apart from prior sensing platform breakthroughs [10]. The core idea of SPR sensor design is the resonance of a strong electromagnetic field oscillation at the interface of a nanometal sheet and a dielectric medium with p-polarized light as the incoming light, which results in a dark band pattern in the light reflectivity at a certain wavelength [11].

Applications of Surface Plasmon Resonance

Various applications of SPR have been outlined in Figure 1.

Figure 1: Applications of Surface plasmon resonance.

SPR-Detection of Tumour Cells

Tumour is a mass of tissue that develops abnormally when cells grow and multiply more often than normal or do not die when they should. Both benign and malignant tumours are possible (cancer). Although benign tumours have the potential to become enormous, they do not penetrate or spread to neighbouring tissues or other body regions [12]. Fathi et al., designed and reported the establishment of a real-time, label-free surface plasmon resonance (SPR)-based biosensor for the identification of cancer stem cells (CSCs) employing the cellular biomarker CD133. A few patients with severe myeloid leukaemia (AML) had this marker detected using the constructed biosensor, and the outcomes were evaluated to those of the flow cytometry (FC) approach. After separating mononuclear cells from the individuals’ bone marrow, CD133 antibody was immobilised on the gold chip surface using the EDC/NHS coupling approach, and binding of the candidate cells to the altered gold sensor surface was observed. The technique was verified using a number of criteria, including cell density and CD133-antibody content. Seven AML patients’ CD133-marked cells were examined.

The findings of the FC technique and all SPR outcomes were compared. The SPR sensing chip’s gold sheets with 25 μg/ml of CD133 antibody put on them exhibited the greatest angle shift, and at a flow rate of 20 ×μl/min, the best grab potential was achieved with 1×105 cells/ml. The association among SPR and FC responses in regard to the densities of CD133-marked cells was extremely strong (R2 = 0.96). In summary, a label-free and real-time SPR cytometry approach was created in this work and effectively used to identify CD133 and track this tumour stem cell biomarker in AML patients [13]. Chiu and co-workers designed a brand-new class of extremely effective biosensing technology is functionalized graphene oxide. Using the cytolerayin 19 (CK19) protein biomarker in spiking human plasma, authors describe a carboxyl-functionalized graphene oxide (GO-COOH)-based surface plasmon resonance (SPR) device for the quick and accurate detection of non-small cell lung cancer (NSCLC). The authors showed that kinetic study of connections between GO-COOH and anti-CK19 and CK19 protein was binding selective. They also determined the relationship of SPR angle and the refractive index of GO-COOH with one another and showed that -COOH modified GO sheets on Au film can increase the field energy transmission intensity of an SPR sensor, leading to a higher sensitivity for the detection of CK19 protein than a traditional Au-based SPR chip. By immobilising CK19 antibody at a trace amount (10 μg/mL) on an SPR chip, the immunosensor was planned and operated.

One fg/mL was the minimal observable concentration. It was possible to generate a boosted 10 percent human plasma CK19 detection limit of 0.05 pg/mL, which is much lower than the physiologically appropriate amount of serum protein (3.3 ng/ mL). For the identification of clinical total plasma biomarkers and potential use in disease diagnosis, a carboxyl-GO based SPR biosensor consequently seems to offer good sensitivity and specificity. In comparison to a traditional SPR chip, this GO-COOH based SPR chip had a quicker reaction time, a better detection limit, and a good linear range (0.001 to 100 pg/mL). We also showed that our carboxyl-GO-based SPR biosensor could detect CK19 at levels as low as 0.05 pg/mL in 10% blood plasma and at levels as low as 0.001 pg/mL in PBS solution. When compared to outcomes in PBS solution, the identification of protein in spiked plasma was demonstrated with an accuracy of R2=0.965 [14].

Surface Plasmon Resonance -Detection of Exosomes

Extracellular vesicles called exosomes comprise the protein, DNA, and RNA of the cells that produce them. They are absorbed by far-off cells, in which they can alter cellular activities and functioning. Exosomes are carrier atoms that cells spontaneously discharge [15]. Cell communication is made possible by these particles. Exosomes provide channels for cell communication and deliver genetic material and proteins to every cell in your body. Currently available techniques for isolation and quantification of exosomes are either requires higher quantities of sample, less specificity and less purity results. To overcome these limitations, researchers have designed some other technologies such as microfluidic device connected with surface plasmon sensors. These sensors exhibited accurate results and isolate exosomes with perfectly sized under the limits [16]. Chen and co-workers designed and reported a label-free, real-time surface plasmon resonance imaging biosensor based on the hydrogel-AuNP supramolecular sphere (H-Au) for the very precise and targeted detection of exosomes produced by prostate carcinoma cells.

The SPR biosensor for exosome identification displayed a vast linear range from 1.00×105 to 1.00×107 particles/mL with a limit of identification of 1.00×105 particles/mL after embedding the signal amplification influence of the mass accumulated hydrogel and the LSPR impact of AuNPs with relatively high aptamer. Most notably, this biosensor demonstrated excellent practical applicability for human serum evaluation, which demonstrates promising applications in diagnosis of the disease and bioanalysis, as demonstrated by a strong correlation between the SPRi signal and the t-PSA value assessed by the clinical chemiluminescence immunoassay. The mass cumulative multilayer hydrogels and the LSPR effect of the AuNPs are primarily responsible for the exceptional sensitivity of this approach. Figure 2 displayed the whole procedure for detection of exosomes. Additionally, the devised test has been successfully used to analyse exosomes in the clinical serum of patients with prostate cancer. Most importantly, there is a substantial association between the t-PSA levels determined by therapeutic chemiluminescence immunoassay and the SPRi signals. Consequently, our technique is capable of differentiating between the sick group and the apparently healthy group, holding tremendous promise for exosome detection-based early cancer diagnosis and therapy monitoring. As a result, we believe that this study may open up a fresh, alternative route for the early detection of prostate cancer [17].

Figure 2: Detection of exosomes – Transmission spectra.

Jabin et al., proposed a biosensor for the prompt identification of various cancer-affected cells, a novel surface plasmon resonance (SPR)-based cancer sensor with an optimal bowl shape. Some significant variations in optical characteristics are seen when the refractive index (RI) of each cancer-contaminated cell is compared to its normal cell counterpart. Additionally, the concentration of malignant cells in liquid is estimated to be 80%, and 2100390 mesh components are used in the finite element model (FEM) for screening. The plasmonic band gap in between silica and cancer cell parts, which are isolated by a thin (35 nm) titanium film coating, is responsible for variation in spectrum shift. The suggested sensor has a maximal coupling length of 66 μM and a high birefringence of 0.04. The suggested structure, however, offers an optimal wavelength sensitivity level in between about 10000 nm/RIU and 17500 nm/ RIU, with a sensor precision between 1.5×10-2 RIU and 9.3×310-3 RIU. Additionally, for carcinoma cells in major polarisation phase with a maximal detection limit of 0.025, the transmittance variance of the malignant cell ranges from around 3300 dB/RIU to 6100 dB/ RIU and the amplitude sensitivity is virtually between -340 RIU-1 and -420 RIU-1. Additionally, the total sensitivity performance is evaluated in relation to their normal cells, which may be superior to any previously presented structures [18].

SPR-Detection of miRNA

Non-coding RNAs known as microRNAs (miRNAs) have significant functions in controlling the expression of genes. Most miRNAs are produced via transcription of DNA sequences into primary miRNAs, precursor miRNAs, and then mature miRNAs [19]. By base-pairing with the target mRNA to inhibit its production, the miRNA acts as a guide. The silencing method is selected by how well the guide and mRNA target complement one other breakage of the target messenger RNA (mRNA), followed by its destruction or suppression of translation [20]. In the presence of single-stranded (ss)DNA/miRNA duplexes, Liyanage et al., (2019) proposed a novel transduction mechanism that incorporates the dispersion of photoexcited conduction electron wave functions of gold triangular nanoprisms (AuTNPs). The plasmoelectronic impact affects the LSPR characteristics of AuTNPs, improving the sensing capabilities. This sensor allowed for the highly specific identification of miR- 10b, miR-182, miR143, and miR145 in serum sample from bladder carcinoma patients with a limit of detection (LOD) as low as 140 zeptomolar (zM). In addition to having the potential to develop into a revolutionary liquid biopsy technology, this ultrasensitive assay may also be used to analyse single malignant cells and identify circulatory miRNA in patient serum for initial-stage, low-volume diagnostic tests for a range of disorders [21].

Recent research on the first LSPR-based sensing method in physiological medium was published by Joshi and group. They created a highly focused plasmonic biosensor to identify miRNA in bloodstream from pancreatic cancer patients with extreme sensitivity. Gold nanoprisms mounted to a glass surface and functionalized with ssDNA (HS-C6-ssDNA) corresponding to the target miRNA are used in this experiment. By observing the LSPR dipole peak (LSPR), the direct hybridization of the target miRNA was discovered. As possible circulatory diagnostic and prognostic indicators for pancreatic ductal adenocarcinoma (PDAC), miR-21 and miR10 levels may be precisely detected with great precision in the sub-femtomolar (fM) range using this biosensor. The LSPRbased observations indicate that, in contrast to conventional qRT-PCR, where a few RNA is lost during sample collection, the concentration of this miRNA is at least 2-fold greater. Any sample collection for the RNA target sequence (such as alteration, amplification, or tagging) has been eliminated in this case, and all issues with the existing sensing methods have been resolved.

Additionally, DNA RNA duplex splitting enzymes might be used to replenish the sensor without reducing its sensing effectiveness. This method transforms the sensor into a straightforward, affordable tool for the early screening of cancer using any miRNA [22]. Research conducted by Ding et al., discloses an SPR biosensor for very rapid screening of miRNA relying on streptavidin signal amplification and DNA super-sandwich complexes, which is intended to solve the drawbacks described earlier. In this test, a corresponding thiolate capturing hairpin probe is mounted on the sensor surface. The structural change is caused by the target miRNA’s hybridization with the thiolate probe. After miRNA hybridization, the capture probe’s hairpin loop reveals interaction sites for the supplementary probe AP1, which has been altered with a biotin tag. On the sensor surface, AP1 partly binds AP2, additional auxiliary probe, to create a super-sandwich. By introducing streptavidin to the surface of sensor, a signal augmentation cascade is further set off. Streptavidin binds to the biotin label of AP1 in the supersandwich to adhere to the chip surface. With higher overexpression in several tumour tissues, miR-21, a possible cancer biomarker was found using this sensing technology. A LOD of 470 pM was obtained with rapid identification, whereas a cascade of signal augmentation reduced the LOD to 9 pM. MiR21 was effectively identified in human breast adenocarcinoma MCF-7 cells following calibration of the sensing platform, exhibiting a non-compromised analysis in complicated components. The test also shown a high level of specificity in detecting complementary target miRNA, single-base mismatch target, double-base mismatch target, and non - specific miRNA sequences. The detection of miRNA with a LOD of 9 p mL-1 is straightforward, quick (30 min), and enzyme-free using this sensing technology. It was also possible to renew the assay, enabling the use of the same chip for at least 20 times [23].

Surface Plasmon Resonance- Protein Marker

Proteins or other compounds that cancer cells produce in greater quantities than healthy cells have historically been used as tumour markers [24]. Some cancer patients may have them in their tumours, blood, urine, faeces, or other body fluids or tissues [25]. Anything appear in or formed by tumour cells, other cells of the body, or certain benign (noncancerous) circumstances is referred to as a tumour marker [26]. Tumour markers can reveal details about a cancer, including its aggressiveness, its ability to be treated with targeted therapies, and its response to therapy [27]. Additional interesting biomarkers towards early detection of cancer are proteins, whose expression, quantity, location, and biochemical changes significantly causes impact on biological process. Only a small subset of those proteins are actually tested as biomarkers in clinical practice for cancer diagnosis and monitoring, despite extensive data showing that hundreds of proteins are differently expressed in human malignancies. Mainly, immunoassays and affinity techniques were used from past times for detection of protein and its quantification in which antibody and targeted protein bound together with another antibody. For this, we have compiled some protein biomarkers generally used clinically for detection of various cancer and their quantification at lower concentration. The whole procedure for detection of protein markers have been depicted in Figure 3. Eletxigerra and coworkers have developed a concise and accurate SPR method for the detection of tyrosine kinase specially (ErbB2) in human serum. the LOD of the sample was found to be 180 pg mL-1, which is under the limits [28].

Figure 3: SPR method for determination of tyrosine kinases.

Conclusion

In this article, we have studied about new applications of surface plasmon resonance for isolation, detection and quantification of various types of tumour markers used in diagnosis of cancer. Most of the SPR methods exhibited better results and minimize the time duration of results than other assays like, ELISA. These methods also detect the markers at low concentration that’s why, the techniques are more reliable and required less amount of sample. These all considerations help SPR techniques for providing cost-effective, robust, accurate and sensitive methods for isolation, detection and quantification of various tumour markers.

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Friday, July 10, 2026

Psychobiotics as Modulators of Gut-Brain Influences

Introduction

Psychobiotics are bacteria beneficial to the body, including probiotic and prebiotic components, capable of influencing gutbrain relationships [1]. This evidence is supported by a pivotal study reporting that mice raised in sterile environments (germfree animals), exhibited excessive physiological reactions to stress compared to normal controls. These abnormal reactions were reversible by recolonization through probiotic-induced bacteria [2]. Thus, the microbiome appears capable of exerting a causal involvement in the maintenance of general homeostasis (body and brain), as well as in the regulation of the hypothalamic-pituitaryadrenal (HPA) axis, a marker of stress. It is now also reported that the vectors of communication between bacteria and brain, to which psychobiotics exert an effect, include the enteric nervous system, the immune system and the vagal nerve. Besides the above considerations, it must be further specified that the nature of psychobiotics, could also be extended to any exogenous influence whose brain effect is bacteria-mediated [1,3].In the present review, we have considered successively: the microbiota of the gut-brain axis; the psychophysiological influences exerted by probiotics and prebiotics; the microbiome-brain-microbiome communications; the psychobiotics different from probiotics and prebiotics; the role of psychobiotics in patient care.

The Microbiota of the Gut-Brain Axis

The gut microbiota includes all microorganisms and their genomes located in the intestinal tract. The bidirectional communications, brain-gut-brain, regulate several important functions (immunity, digestion, metabolism, satiety, and stress) [4,5].

Probiotics

Bacteria beneficial to health, probiotics, when ingested in appropriate amounts, may lead to positive psychiatric effects in some psychopathologies [6]. The bacteria most frequently employed as probiotics are Gram-positive bacteria Bifidobacterium, Lactobacillus [7]. These bacteria do not possess pro-inflammatory lipopolysaccharide chains in the gut and their propagation does not trigger immunological inflammatory reactions. With such bacteria, the immune system learns to distinguish between pro- or antiinflammatory entities and to develop adapted immune responses (Figures 1& 2) [8].

Prebiotics

They can also be included in the definition of psychobiotics since they are compounds that, by fermentation in the gut, produce specific changes in bacterial composition and activity. Moreover, prebiotics also support the growth of commensal bacteria and the majority of them are fructans and oligosaccharides (Figures 1 & 2) [1].

Psychophysiological, Immune and Clinical Influences of Psychobiotics

Probiotics in Rodents

According to the great variety of reports available in literature, to limit redundancy, only two relevant approaches are reported here. In one of them, a maternal separation was achieved to induce an early-life stress. Rat pups were left undisturbed or administered with a neutral vehicle substance or the probiotic Bifidobacterium infantis. Vehicle rats showed psychophysiological patterns corresponding to maternal separation, i.e., poorer performance on the forced swim test; an increased inflammation with heightened peripheral pro-inflammatory cytokines (Interleukin-6, IL-6); decreased presence of brain noradrenaline; elevated concentrations of corticotrophin releasing factor (CRF) mRNA. These indices were normalized in probiotic-fed rats. In this model, probiotics appear thus capable of correcting the imbalance produced by the maternal separation [9]. In the other approach selected, probiotic effects during stressful experiences have been examined [10]. Healthy adult male Sprague-Dawley rats were administered with Lactobacillus helveticus while exposed to chronic-restraint stress or no intervention. Relative to control group, the animals fed with probiotics showed lower levels of post-restraint anxiety as well as an enhanced memory. At the biochemical level, rats supplemented with probiotic displayed lower levels of adrenocorticotropic hormone and corticosterone [11]. The probiotic group also showed increases in anti-inflammatory cytokines (IL-10), noradrenaline and serotonin. Again, probiotics, through influences similar to those reported for maternal separation, appear capable of correcting the imbalance produced by the chronic restraint stress [10].

Probiotics in Humans

While the clinical studies available are much more current than those reported for rodents, they also appear robust [12]. Here, we consider three relevant reports of probiotic effects in healthy medical student athletes [13], in patients with an irritable bowel syndrome and in healthy patients subjected to an emotional situation [1].

Healthy Student Athletes: These subjects fed with Lactobacillus gasseri showed, relative to placebo, elevated mood and reduced natural killer cell activity after strenuous exercise, with some additional alleviation of fatigue when the probiotic α-lactalbumin was consumed [13]. These results suggest that probiotics may have relevant ecological benefits and the potential to improve some life activities.

Patients with an Irritable Bowel Syndrome: Such patients, in addition to the irritable bowel disease [14], also exhibited disturbances in the gut-brain axis [15] and in the composition of their microbiota [16]. The foregoing disturbances were also often associated with anxiety and depressive syndrome [17], and an aberrant ratio of IL-10 and IL-12 suggesting a generalized pro-inflammatory state. Finally, patients who consumed Bifidobacterium infantes had a normalization of the interleukin’s ratio after treatment [1]. These results show that probiotics can induce changes in cytokines and therefore exert immunological effects.

Stressful Situations: Over four weeks, healthy human female participants took either a placebo or a combination of probiotics (Bifidobacterium animalis, Streptococcus thermophiles, Lactobacillus bulgaricus, Lactococcus). They further received a functional Magnetic Resonance Imaging (MRI) to determine how ingestion of probiotics could affect their neurophysiological activity. During the image acquisition phase, faces with emotions (expressions of fear) were presented to the participants. Compared to placebo, participants on probiotics showed a decrease in neuronal activity and emotional response, somatosensory and interoceptive processing in the somatosensory cortex, the insula and the periaqueductal gray [18]. These results may represent a probiotic-induced reduction in the neuronal reactivity to stressful situations. Indeed, the exertion of stress has an important influence on the functional and structural aspects of the microbiome. Glucocorticoids can impair the intestinal barrier function, allowing in this way the migration of bacteria, which in turn generate an inflammatory immune response (Figure 1 & 2) [1, 2].

Figure 1: Psychobiotics actions on commensal bacteria within the gut. Psychobiotics, primarily defined as probiotics, are bacteria involved in the general homeostasis maintenance. They act, at first, through their interactions with normal intestinal flora bacteria (various synergistic effects). These bacteria do not possess the pro-inflammatory lipopolysaccharide chain and their propagation does not trigger immunological inflammatory reactions. Prebiotics can also be included in the definition of psychobiotics since they are compounds that, by fermentation in the gut, produce specific changes in bacterial composition and activity (various pleiotropic effects). Prebiotics are fructans and oligosaccharides that support the growth of normal flora bacteria (double harrows probiotics/probiotics). Psychobiotic communications. Within the gut, they regulate the enteric nervous system either directly or through various neurotransmitters (mainly aminergic). The outputs from the gut include the short-chain fatty acids (SCFAs) capable of influencing the secretions of satiety peptides (PYY, CCK, GLP-1). Psychobiotics also influence the immune system in triggering the release of anti-inflammatory cytokines (IL-10) and in reducing that of proinflammatory (IL-1, IL-6, TNF-a). Finally, the vagus nerve possesses abundant sensory fibers conveying information from body organs to the brain. It is sensitive to nutrition, activity and stress and might be a psychobiotic pathway.

Figure 2: Psychobiotics influences within the gut. They are mainly produced by probiotics and prebiotics. Probiotics introduce beneficial bacteria like lactobacilli and bifidobacteria into the gut. Prebiotics support the growth of such bacteria. Psychobiotics cannot be limited to probiotics and prebiotics only. Any substance that exerts a microbiome effect is potentially also a psychobiotic (other substances). Psychobiotics regulate the enteric nervous system either directly or through various neurotransmitters including, dopamine (DA), noradrenaline (NA), serotonin (5-HT), gamma-aminobutyric acid (GABA) and acetylcholine (Ach). Short-chain fatty acids (SCFAs), issued from the metabolization, in anaerobic conditions, of indigestible polysaccharides are produced by the microbiota in the large intestine. Outside of the gut. Psychobiotics may contribute to eliminating pathogens in decreasing the inflammation (Anti-Infla-Cyt, anti-inflammatory cytokines) that causes the reduction of circulating pro-inflammatory cytokines (Pro-Infla-Cyt). In stressful situations, the hypothalamic-pituitary-adrenal gland (HPA) axis releases glucocorticoids capable of inducing a leaky gut. In this situation, a bacterial migration takes place leading to an inflammatory situation through pro-inflammatory cytokines. Anti-Infla-Cyt counteract this process. SCFAs, in interacting with the gut endocrine cells (EC), catalyze the release of gut hormones such as CCK, PYY and GLP-1. The vagus nerve also plays an important role since it is sensitive to nutrition, exercise and stress. It may exert anti-inflammatory and anxiolytic effects.

Prebiotics in Rodent

A small number of studies have examined the psychophysiological effects of prebiotics. These include investigations of galactooligosaccharides (GOS) and fructo-oligosaccharides (FOS), which are a source of nutrition for microbiota bacteria of Bifidobacterium and Lactobacillus genera and stimulate their activity and propagation in the gut [19]. Proportional to the prebiotic exposure, male rodents (both Sprague-Dawley rats and C57BL/6 mice) showed enhanced learning and working memory, a higher expression in the hippocampal and striatal brain-derived neurotrophic factor (BDNF), and an increased hippocampal longterm potentiation. Moreover, in relationship to the administration, rats fed the prebiotics during lactation showed substantially enhanced maze learning and object recognition one year later [20]. These findings point out the influence of prebiotics on memory processes and have important implications for assessing the longlasting effects of prebiotics.

Prebiotics in Humans

A very small number of studies have examined the effects of prebiotics in humans. These studies include investigations on GOS and FOS (non-digestible oligosaccharides with prebiotic properties), that are sources of nutrition for microbiota bacteria by stimulating their activity and spread within the intestine. The first human study considering the psychophysiological effects of prebiotics [21] was conducted in healthy participants who consumed either GOS or FOS or placebo. Compared to other groups, participants who consumed GOS had a significantly decreased response in “cortisol wakefulness” which is a biomarker of emotional disturbances [1,3]. Further studies are necessary to document the time course of prebiotic effects.

Microbiome-Brain Communications

Mechanisms of the effects exerted by psychobiotics are still poorly understood. The crucial step remaining in this field of research lies in the understanding of the processes through which microbiome and brain communicate.

Bacteria-Enteric Nervous System Inter-Influences

Gut bacteria regulate electrophysiological thresholds in enteric nervous system neurons. Neurons exposed to Bifidobacterium longum showed reduced generation of action potentials when they were electrically stimulated [22]. In the same sense, in germ-free mice, these neurons showed lower levels of excitability compared to their normally colonized counterparts [23]. Thus, the above results provide evidence for a direct bacteria-induced modulation of the enteric nervous system. Gut-derived bacteria, through the metabolism of non-digestible fibbers, also provide various neurotransmitters like dopamine, noradrenaline, serotonin, gamma-aminobutyric acid (GABA) and acetylcholine [1]. Regarding GABA, it is now well known that it is the chief inhibitory neurotransmitter in the central nervous system (CNS). Found in large amounts, it regulates many processes, including anxiety and depression, blood pressure, pain and epilepsy [24,25]. Interest in the role of GABA as an antihypertensive dietary component has recently increased in Japan, due to the high sodium intake in that country. This aspect led to an increase in the research surrounding the development of fermented products containing GABA [24]. This transmitter is produced primarily from the decarboxylation of L-glutamate by glutamate decarboxylase and is found in animals as well as in higher plants and bacteria [26]. One way to increase its concentration in the gut may include the ingestion of probiotic bacteria containing dietary monosodium glutamate (MSG) to generate GABA [27]. In this way, the ingestion of human intestinally derived bifidobacteria and lactobacilli, the most efficient way to produce GABA [24], appears now as an efficient and new pathway to produce GABAbiotic (Figure 1&2).

Short Chain Fatty Acids

The human gut cannot digest macronutrients such as plant polysaccharides since the human genome does not code the enzymes necessary for their digestion [28]. The metabolization, by anaerobic fermentation, of indigestible polysaccharides is produced by the microbiota in the large intestine [28] and produces short-chain fatty acids (SCFAs) [29]. The greater proportion of SCFA is directed into the liver and muscle, through the circulatory system. While it is unclear to what extent the SCFAs enter the CNS, there is some evidence for their psychotropic properties. For instance, systemic sodium butyrate injections in rats produce antidepressant effects, an increase the central serotonin neurotransmission and a brainderived neurotrophic factor (BDNF) expression [30]. SCFAs also stimulate the HPA axis [31] or directly affect the mucosal immune system [32], which may indirectly affect central neurotransmission. SCFAs also influence secretion of satiety peptides, including cholecystokinin (CCK), peptide tyrosine (PYY) and glucagon-like peptide-1 (GLP-1) [33]. They also promote pleiotropic effects including stress and immunity (Figure 1 & 2).

Bacteria and Immune System Interactions

The key function of the immune system is to detect and eliminate pathogens. Psychobiotics may contribute in this process by decreasing the inflammation that conduces to a reduction in circulating pro-inflammatory cytokines. These cytokines can increase the permeability of the blood-brain barrier, permitting, in this way, the brain access to potential pathogenic entities [34]. Then, cytokines alter the neurotransmitters (serotonin, dopamine, and glutamate) involved in brain communication [35]. Finally, cytokines can also enter the brain through active uptake, stimulating secretion of pro-inflammatory substances such as prostaglandins [36], precipitating further inflammation (cytokine storm). In this situation, cytokines produce an excessive host-inflammatory response comprising of the induction of various interleukins (IL- 6, IL-17A, tumor necrosis factor alpha, interferon gamma and free radicals related to nitric oxide (NO) [37]. Here, it must be further documented that when exposed to pro-inflammatory cytokines, the inducible NO-synthase (iNOS) enzyme is expressed and triggers the release of large amounts (Figure 1 & 2) of NO. This compound reacts avidly with oxygen and superoxide radicals to form NO derivatives including peroxynitrite (ONOO-). The latter has a long half-life and is a powerful oxidant that perpetuates acute systemic and CNS damages including inflammation, protein deterioration, cell membrane destruction, DNA/RNA lesions, and cell death [38-41]. Above NO-related mechanism observed with pathogens invasion (bacteria, viruses and parasites), is also present throughout the aging process and neurodegenerative pathologies [39-41].

Vagal Communications

The vagus nerve plays an important role in coordinating parasympathetic activity, including the regulation of heart rate and gut motility. It also possesses abundant sensory fibbers capable of conveying rich information from body organs to the brain [42]. The vagal activity is sensitive to nutrition, exercise, and stress [1]. Stimulating the vagus nerve exerts anti-inflammatory [43] and anxiolytic effects [1] and is used therapeutically for refractory depression and epilepsy [44,45]. Here, it must be recalled that stimulation of the afferent large diameter fibbers of the vagus and aortic nerves (vago-aortic stimulation) can evoke slow wave sleep (SWS) and paradoxical sleep (or rapid eye movement sleep, REM sleep) [46]. Thus, it must be questioned whether the antiinflammatory and anxiolytic effect observed with stimulation of the vagus nerve [44] or the administration of psychobiotics, might run both through the vagus nerve afferents to the brain (Figure 2).

Brain-Microbiome Influences

Stress and Glucocorticoids

While stress cannot be considered per se as a signaling pathway, it may play important influences on structural and functional aspects of the microbiome [47]. During stress, through the HPA axis, a hyper-production of glucocorticoids takes place. Such a production, while contributing substantially to the maintenance of resting and stress-related homeostasis, deregulates gut barrier function, in reducing the epithelium integrity [48]. Such an impairment permits the outward migration of bacteria, triggering inflammatory immune responses. Bacterial translocation from the gut can also modulate inflammation by raising the concentrations of pro-inflammatory cell elements such as lipopolysaccharides, a process associated with human depression [49]. Moreover, probiotic supplementation with the Bifidobacterium or Lactobacillus genera can restore in turn the gut-barrier integrity [50]. These mechanisms illustrate the pleiotropy of the effects that can be exerted by these compounds in healthy or pathological conditions (Figure 1 & 2).

Psychobiotics Different from Probiotics and Prebiotics

Antipsychotics and Antibiotics

Psychobiotics cannot be limited to probiotics and prebiotics only. Any substance that exerts a microbiome-mediated psychological effect is potentially also a psychobiotic. In this sense, the ingestion of an antipsychotic like olanzapine increases the abundance of actinobacteria and proteobacteria. Moreover, a mixture of antibiotics, containing neomycin, metronidazole, and polymyxin, also ameliorate the effects of olanzapine on bacteria [51]. Antibiotic mixtures (bacitracin, neomycin, and pimaricin) have been shown to induce neurochemical and behavioral changes through the microbiome [52]. Therefore, both antibiotics and antipsychotics may also be classified as psychobiotics. Moreover, many other substances may exert secondary psychobiotic effects in addition to their primary intended effect. The study of the above substances (pharmacomicrobiomics) represents a new and open field of research (Figure 1 & 2) [53].

Psychobiotics for Patient Care

Psychobiotics an Alternative to Psychotropic Medications

Through an important volume of scientific approaches, the role of probiotics appears of particular interest since significant evidence indicates that microbiota can maintain or even restore health. In nutritional psychiatry, significant reports also link human intestinal microbiota to mental health giving rise to the concept of psychobiotics. Research conducted with this focus suggests that CNS and gut have bidirectional communications through neuronal, endocrine, and immune pathways. Thus, psychobiotics could offer a natural, low-risk alternative to psychotropic medications. This area needs, however, to be further explored. Clinicians should continue to examine effective strains and doses of probiotics as this information becomes available. The abundance of favorable research conducted to date, with no reported adverse reactions, supports the use and continued investigation of psychobiotics [54,55].

Conclusion

At the completion of this review, it appears that psychobiotics, including probiotics, prebiotics and different other compounds, exert microbiome-mediated psychological effects. Psychobiotics are capable of influencing the microbiota-brain relationships and exert anxiolytic effects characterized by changes in emotional, cognitive and neural parameters. Channels through which psychobiotics exert their effects include the enteric nervous system, the immune system and the vagal nerve. In nutritional psychiatry, experimental evidence suggests that the human intestinal microbiota could be directly linked to mental health, giving rise to the concept of psychobiotics. In the management of patients, psychobiotics may offer a natural, low-risk alternative to psychotropic medications. Finally, regarding the perspectives in the inflammatory conditions of depressive or neurodegenerative pathologies, the use of antioxidants like superoxide dismutase (SOD) and glutathione peroxidase (GPx), or NO-synthase inhibitors (iNOS) should be considered.

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