Combined evaluation of prolactin-induced peptide (PIP) and extracellular signal-regulated kinase (ERK) as new sperm biomarkers of FSH treatment efficacy in normogonadotropic idiopathic infertile men.

PURPOSE: Nearly, 40% of the causes of male infertility remain idiopathic. The only suggested treatment in idiopathic oligo- and/or asthenozoospermia in normogonadotropic patients is the FSH. In the current clinical practice, efficacy is exclusively assessable through semen analysis after 3 months of treatment. No molecular markers of treatment efficacy are appliable in clinical practice. The aim of the present work is to evaluate the combination of extracellular signal regulated kinase (ERK) 1 and 2 and prolactin inducible peptide (PIP) as potential markers of idiopathic infertility and FSH treatment efficacy. METHODS: Western blot and confocal microscopy were performed to analyze the modulation of PIP and ERK1/2 in idiopathic infertile patients (IIP) sperm cells. Taking advantage of mass spectrometry analysis, we identified these proteins unequivocally in sperm cells. RESULTS:  We demonstrated a significant decrease of both PIP protein and of ERK1/2 levels in spermatozoa obtained from IIP in comparison to healthy fertile patients (HFP). Conversely, we reported a significant increase of these markers comparing infertile patients before and after 3 months of FSH treatment. Importantly, this correlated with an increase in total number of sperm and sperm motility after FSH treatment. Finally, we identified of PIP and ERK2 proteins in sperm samples by proteomic analysis. CONCLUSIONS: The combined evaluation of ERK1/2 and PIP proteins might represent a useful molecular marker to tailor FSH treatment in the management of male normogonadotropic idiopathic infertility.

Proteomic analysis of salivary inflammatory biomarkers of developmental gingival enlargements in patients with West and Noonan syndromes: a preliminary pilot single-center retrospective study.

OBJECTIVE: The aim of the preliminary pilot single-center retrospective cross-sectional study was to analyze and compare the presence of non-secretory salivary inflammatory biomarkers in pediatric patients with West syndrome, Noonan syndrome, and a healthy control group. PATIENTS AND METHODS: A total of 60 saliva samples were collected during dental check-ups. The saliva samples collected were analyzed by liquid chromatography. The results were analyzed with a t-test, and the statistical significance was given by a p-value lower than 0.05. RESULTS: We found statistical significance for defensin α1 (p=0.006) and thymosin ß4 (p=0.025) in the Noonan syndrome. In the West syndrome, only the defensin α1 had a statistically significant difference with the other groups (p=0.022). Proteomic analysis revealed an overexpression of peptides related to the innate (thymosin ß4) and acquired (defensin α1, α3) immunity. CONCLUSIONS: West and Noonan's syndromes showed the overexpression of molecular biomarkers involved in the pathogenesis of chronic periodontitis. The inflammatory status is triggered and amplified by the abnormal overgrowth of gingival tissues, the amplified release of proinflammatory cytokines from the immune cells, and the poor cooperation in maintaining adequate oral hygiene.

The prevention of obesity starting from the kindergarten by means of peer educator mothers and WhatsApp messages.

BACKGROUND: Early-life diet plays a crucial role in the development of body weight issues. We therefore investigated the effectiveness of a peer-education intervention, aiming at encouraging the intake of fruits and vegetables and of a healthy mid-morning snack, targeting the mothers of 3-8 years old children attending kindergarten and primary school. STUDY DESIGN: Experimental approach. METHODS: The study involved 924 children (intervention group) and 450 children (control group). The intervention was carried out by organizing three meetings at school with the participation of healthcare professionals, mothers and teachers in a maieutic way. The meetings focused on i) discussing the issues that mothers face with their children's nutrition; ii) possible solutions; iii) identification of "leading mothers" playing as peer-educators. As control a similar intervention targeting only the teachers, without involving the mothers, was also carried out. The effectiveness of such interventions was evaluated by external evaluators, before, the interventions and 3 and 9 months after the interventions. RESULTS: The check, carried out 3 months after the intervention, showed a significant improvement with respect to the mid-morning snack, and the consumption of the main dishes as well as fruits; the check carried out 9 months after the intervention, showed that such improvements were maintained in the case of the mid-morning snack and the fruit intake. No improvement was observed in relation to the control group. CONCLUSIONS: This is the first example of an intervention promoting a healthy diet involving mothers of children attending kindergarten and primary school as peer educators and it actually improved the dietary habits.

Thymosin ß4 and ß10 are highly expressed at the deep infiltrative margins of colorectal cancer - A mass spectrometry analysis.

OBJECTIVE: Colorectal cancer (CRC) is a complicated tumor, involving several oncogenic signaling pathways, and with a molecular mechanism not fully understood yet. The implication of thymosin ß4 (Tß4) with tumor insurgence and in migration of CRC cells was evidenced in the past with different methodologies, while Tß10 connection with CRC has been sporadically investigated. This study focused on the implication of both types of thymosin in CRC progression and invasion by analyzing the changes in their levels according to different zones of the tumor, and to Dukes stage and budding index. PATIENTS AND METHODS: Tß4 and Tß10 were analyzed in deep and superficial tumor samples, and normal mucosa from 18 patients. Concentrations of Tß4 and Tß10 have been measured by high-pressure liquid chromatography (HPLC) coupled to electrospray-ion trap mass spectrometry (ESI-IT-MS). MS data were compared by t-test and ANOVA statistical analysis. Identification of thymosin and their proteoforms has been performed by HPLC-high resolution-ESI-IT-MSMS. RESULTS: Both Tß4 and Tß10, exhibited intra-tumoral quantitative differences, being upregulated in the deep part of the CRC. They exhibited, moreover, strong association with the Dukes stage and the budding grade, being more concentrated in patients at Dukes stage B and with budding index "2". CONCLUSIONS: The results obtained in the present investigation encouraged the hypothesis that the two thymosin are involved in colorectal cancer progression, and in promoting cancer invasion. Thus, they are good candidates to be diagnostic/prognostic biomarkers and therapy targets.

Proteomic evaluation of GCF in the development of pregnancy related periodontal disease: a pilot clinical study.

OBJECTIVE: This pilot study analyzed the possible changes of periodontal disease status in female patients during the period following pregnancy. Both clinical and laboratory data were collected and analyzed. PATIENTS AND METHODS: A non-randomized controlled clinical trial was conducted by the Periodontal Department of the Dental Clinic in collaboration with the Pediatrics Department, at Fondazione Policlinico Universitario A. Gemelli, Rome, Italy. Ten female patients, who completed the pregnancy without complications, were enrolled in this research protocol forming the experimental group. During the first post-partum days, gingival crevicular fluid (GCF) samples were collected and analyzed with high-performance liquid chromatography associated with high-resolution mass spectrometry (HPLC ESI MS); periodontal parameters as pocket depth (PD), full mouth plaque score (FMPS) and full mouth bleeding score (FMBS) were recorded, and a professional oral hygiene session was performed. The same protocol was applied after three months with the same patients forming the recall group. A control group was created in order to compare the results with GCF samples from 10 not pregnant fertile women. RESULTS: Student's t-test has been used to evaluate the statistical significance of the collected data. Mean levels of PD decreased from 3.75 mm ± 1.2 mm after pregnancy to 2.88 mm ± 0.85 mm at three months post-partum (p<0.01). Mean value of FMPS and FMBS decreased from 21.8% ± 1.35% and 34.27% ± 1.5% after pregnancy to 13% ± 2.81% and 17.55% ± 2.84% at three months post-partum, respectively (p<0.05). The concentration of each analyzed peptide has changed in relation to the general improvement of the periodontal status at three months post-partum. CONCLUSIONS: Pregnancy may be associated with an increased risk of periodontal disease. Both clinical and laboratory data have demonstrated that a professional oral hygiene session can affect the course of pregnancy inducing periodontal diseases allowing a faster healing and restitutio ab integrum.

Salivary biomarkers and proteomics: future diagnostic and clinical utilities.

Saliva testing is a non-invasive and inexpensive test that can serve as a source of information useful for diagnosis of disease. As we enter the era of genomic technologies and -omic research, collection of saliva has increased. Recent proteomic platforms have analysed the human salivary proteome and characterised about 3000 differentially expressed proteins and peptides: in saliva, more than 90% of proteins in weight are derived from the secretion of three couples of "major" glands; all the other components are derived from minor glands, gingival crevicular fluid, mucosal exudates and oral microflora. The most common aim of proteomic analysis is to discriminate between physiological and pathological conditions. A proteomic protocol to analyze the whole saliva proteome is not currently available. It is possible distinguish two type of proteomic platforms: top-down proteomics investigates intact naturally-occurring structure of a protein under examination; bottom-up proteomics analyses peptide fragments after pre-digestion (typically with trypsin). Because of this heterogeneity, many different biomarkers may be proposed for the same pathology. The salivary proteome has been characterised in several diseases: oral squamous cell carcinoma and oral leukoplakia, chronic graft-versus-host disease Sjögren's syndrome and other autoimmune disorders such as SAPHO, schizophrenia and bipolar disorder, and genetic diseases like Down's Syndrome and Wilson disease. The results of research reported herein suggest that in the near future human saliva will be a relevant diagnostic fluid for clinical diagnosis and prognosis.

Available influenza vaccines: immunization strategies, history and new tools for fighting the disease.

The main public health strategy for containing influenza-related disease is annual vaccination, which is recommended for the elderly and others belonging to risk-factor categories, who present the highest morbidity and mortality, as reported by the World Health Organization (WHO) Recommendations. The availability of different influenza vaccine formulations makes the choice of the best immunization strategy a challenge for stakeholders and public health experts. Heterogeneity in at-risk categories included in national influenza vaccine recommendations still exists, in particular among European countries. Broader consensus is expected, which should positively impact on influenza vaccination coverage. The availability of quadrivalent vaccines, containing both influenza B lineages, offers the potential to improve protection by overcoming the drawbacks of wrongly predicting which B lineage will predominate in a given year.

Modifications of the acidic soluble salivary proteome in human children from birth to the age of 48months investigated by a top-down HPLC-ESI-MS platform.

During the first year of life the infant oral environment undergoes dramatic changes. To investigate how the salivary proteome of human children evolves during infant development we have analyzed whole saliva of 88 children aged between 0 and 48months by a top-down platform based on RP-HPLC-ESI-MS. Children were divided according to their age into five groups (A, 0-6months, N=17; B, 7-12months, N=14; C, 13-24months, N=32; D, 25-36months, N=16; E, 37-48months, N=9). The proteins and peptides analyzed were histatins (histatin-1, histatin-3 1/24), acidic proline-rich proteins, statherin, P-B peptide, and salivary cystatins. Protein and peptide quantification based on the area of the RP-HPLC-ESI-MS extracted ion current peak evidenced that: (i) concentrations of the major salivary proteins/peptides showed a minimum in the 0-6-month-old group and increased with age; (ii) the level of histatin-1 reached a maximum in the 7-12-month-old group, a minimum in the 13-24-month-aged babies and it increased again in the 25-36-month-old group; (iii) S-type cystatins were almost undetectable in the 0-6-month-old group; (iv) P-B peptide concentration greatly increased with age; (v) histatin-3 1/24 and statherin concentrations did not show any age-related variation. BIOLOGICAL SIGNIFICANCE: The top-down proteomic approach undertaken in this work reveals that the salivary proteome of human children from birth to 48months of age shows important quantitative modifications. The concentrations of the major salivary proteins, with the exception of statherin and histatin-3 1/24, showed a minimum in the 0-6-month-old group when the expression in salivary glands is probably not fully activated. Concentrations of the salivary proteins slowly increased with age, with different trends. Only histatin-1 showed the highest concentration in the 7-12-month-old group, followed by a decrease in the 13-24-month-aged children. This particular trend could be related to the phenomenon of eruption of primary dentition. This study gives a contribution to the knowledge on the physiological variability occurring in human saliva during the early childhood. It could represent a strong and reliable basis for further investigation of saliva to develop diagnostic and prognostic biomarkers.

Potential applications of human saliva as diagnostic fluid.

The use of human saliva as a diagnostic and prognostic fluid has until recently been somewhat disregarded. Although sample collection is non-invasive, physiological and genetic variations were largely responsible for its infrequent application in the past. Recently, several proteomic studies contributed to partial elucidation of the salivary proteome (more than 2400 protein components have been characterized), both in terms of composition, contributions to whole saliva and genetic/physiological variability. On this basis, is not too optimistic to believe that in the near future human saliva could become a relevant diagnostic fluid. In this review, the characterization by proteomic approaches of new salivary markers in oncology, head and neck carcinoma (oral cavity, oropharynx, larynx, and salivary glands), breast and gastric cancers, salivary gland function and disease, Sjögren syndrome, systemic sclerosis, dental and gingival pathology, systemic, psychiatric and neurological diseases, is described.

Morpho-functional modifications of human neutrophils induced by aqueous cigarette smoke extract: comparison with chemiluminescence activity.

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke-related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles.