The role of allergoids in allergen immunotherapy: from injective to sublingual route.

Summary: Summary Allergen immunotherapy (AIT) is aimed at inducing tolerance to allergens, such as pollens, dust mites or moulds, by administering increasing amounts of the causative allergen through subcutaneous or sublingual route. The evidence of efficacy of AIT is high, but the issue of safety, especially for the subcutaneous route, must be taken into account. The search for safer AIT products aimed at reducing the allergenicity, and thus adverse reactions, while maintaining the immunogenicity, that is essential for effectiveness, gave rise to the introduction of allergoids, which were conceived to fulfill these requirements. In the first allergoids glutaraldehyde or formaldehyde were used as cross-linking agent to polymerize allergens, this resulting in high molecular weight molecules (200,000 to 20,000,000 daltons) which were significantly less allergenic due to a decreased capacity to bridge IgE on its specific receptor, while maintaining the immunogenicity and thus the therapeutic efficacy. In recent years further agents, acting as adjuvants, such as L-tyrosine, monophosphoryl lipid A, aluminium hydroxide, were added to polymerized extracts. Moreover, a carbamylated monomeric allergoid was developed and, once adsorbed on calcium phosphate matrix, used by subcutaneous route. At the same time, in virtue of its peculiarities, such allergoid revealed particularly suitable for sublingual administration. A lot of clinical evidences show that it is well tolerated, largely safer and effective. Importantly, the higher safety of allergoids allows faster treatment schedules that favor patient compliance and, according to pharmaco-economic studies, they might be more cost-effective than other AIT options.

Occurrence of enteropathogenic bacteria in birds of prey in Italy.

The importance of wild birds as potential vectors of disease has received recent renewed empirical interest, especially regarding human health although information regarding the enteropathogenic bacteria in birds of prey continue to be scant. This study was performed with the aim to evaluate the occurrence of enteropathogenic bacteria (i.e. Campylobacter spp. Escherichia coli, Salmonella spp.) in birds of prey carcasses in Southern Italy. The results of the present study showed a prevalence of 33·1% (49/148) for Campylobacter spp. where all positive isolates (49/49) were identified as Campylobacter jejuni, and among these positive 12/49 were also identified as Campylobacter coli. Thus, 12/49 birds of prey showed mixed infections for both Campylobacter species. Differences in Campylobacter spp. prevalence between diurnal and nocturnal birds were statistically significant (P = 0·016). Escherichia coli showed a prevalence of 6·8% (10/148) and were serogrouped as O26 (n = 3), O55 (n = 2), O145 (n = 5). Salmonella spp. showed a prevalence of 6·8% (10/148) and were serotyped as S. Napoli (n = 4), Salmonella salamae (n = 3) and S. Typhimurium (n = 3). Although wildlife disease outbreaks have often been underreported in the broader context of global epidemiology, results of the present study suggest that birds of prey may serve as a reservoir of pathogens for livestock and human health, acting at the animal-human-ecosystem interface. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the role of birds of prey as a reservoir of enteropathogenic bacteria (i.e. Campylobacter spp., Escherichia coli, Salmonella spp.). Wild birds can contaminate environment with their faeces and play a crucial role in the transmission of pathogens to poultry and livestock farms and aquifers supplying water to humans. Furthermore, wild birds could disseminate pathogens within rescue and rehabilitation centres where they are admitted.

Dilute versus concentrated vasopressin administration during laparoscopic myomectomy: a randomised controlled trial.

OBJECTIVE: To determine if higher-volume, fixed-dose administration of vasopressin further reduces blood loss at the time of minimally invasive myomectomy. DESIGN: Randomised multicentre clinical trial. SETTING: Tertiary-care academic centres in the USA. POPULATION: Women undergoing conventional laparoscopic or robot-assisted laparoscopic myomectomy. METHODS: All participants received the same 10-unit (U) dose of vasopressin, but were randomly assigned to one of two groups: (i) received 200 ml of diluted vasopressin solution (20 U in 400 ml normal saline), and (ii) received 30 ml of concentrated vasopressin solution (20 U in 60 ml normal saline). MAIN OUTCOME MEASURES: The primary study outcome was estimated blood loss; the study was powered to detect a 100-ml difference. RESULTS: A total of 152 women were randomised; 76 patients in each group. Baseline demographics were similar between groups. The primary outcome of intraoperative blood loss was not significantly different, as measured by three parameters: surgeon estimate (mean estimated blood loss 178 ± 265 ml and 198 ± 232 ml, dilute and concentrated groups respectively, P = 0.65), suction canister-calculated blood loss, or change in haematocrit levels. There were no vasopressin-related adverse events. CONCLUSION: Both dilute and concentrated vasopressin solutions that use the same drug dosing demonstrate comparable safety and tolerability when administered for minimally invasive myomectomy; however, higher volume administration of vasopressin does not reduce blood loss. TWEETABLE ABSTRACT: This randomised trial failed to show benefit of high-volume dilute vasopression.

End-colostomy parastomal hernia repair: a systematic review on laparoscopic and robotic approaches.

INTRODUCTION: Parastomal hernia (PSH) is the most common and challenging complication after stoma creation, with an estimated 50% incidence 2 years after the index surgery. Mesh repair is the treatment of choice. Laparoscopic and/or robotic approaches allow acceptable outcomes. MATERIALS AND METHODS: A systematic literature review from January 2012 to November 2023 was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Embase, PubMed, and Scopus search were performed to select articles dealing with minimally invasive surgical treatment for PSH after end colostomy. RESULTS: 603 studies were found, and 24 were chosen. When compared to open surgery, laparoscopy showed decreased postoperative complications and recurrence. The main laparoscopic approaches are the keyhole (KH), the Sugarbaker (SB), and the sandwich technique. Continuous improvement in surgery, mesh technology, and surgeons' expertise led to an amelioration of surgical outcome and recurrence rate after repair. Recent studies showed comparable outcomes for SB and KH techniques, while novel surgical approaches have been proposed in an attempt to further increase the operative and long-term results. Reports on PSH robotic repairs are scarce and describe small series results, suggesting a role of the initial learning curve as a risk factor for complications. CONCLUSION: End-colostomy PSH surgical repair still represents a challenge for surgeons. Recent evidence has not shown a significant advantage in postoperative complications and recurrence with a specific repair among SB, KH, and sandwich technique. The paucity of data on robotic surgery does not allow to draw definitive conclusion. Further primary, multicentric, and larger cohort studies are needed.

Prevalence of thermotolerant Campylobacter in partridges (Perdix perdix).

AIM: To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges (Perdix perdix) in southern Italy. METHODS AND RESULTS: Cloacal swabs of partridges (n = 240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49.2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12.7%) out of the 118 positive samples were also positive for Campylobacter jejuni. In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence (P < 0.05) than younger ones. CONCLUSION: These results reinforce the assumption that game birds may be considered as potential carriers of Campylobacter spp. for human being and other animal species. SIGNIFICANCE AND IMPACT OF THE STUDY: Although an earlier 1986 publication described the prevalence of Campylobacter coli in commercially reared partridges, this is the first report to confirm the species of Campylobacter using a PCR test.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Survey of Campylobacter jejuni in stray cats in southern Italy.

AIM: To evaluate the presence of thermotolerant Campylobacter spp. in stray cats in southern Italy. METHODS AND RESULTS: One hundred and thirteen animals were trapped in two different environments (urban area, harbour area) of the city of Naples. From each cat, rectal swabs were collected. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Campylobacter jejuni was isolated from 19/113 (16.8%) stray cats, whereas Campylobacter coli was not isolated. The cat data (age, environment and sex) were analysed by two statistical analyses using the C. jejuni status (positive/negative) as a dependent variable. As regards statistical regression model results, age and environment were risk factors for C. jejuni positivity. In particular, cats older than 1 year had a significantly higher risk of being positive for C. jejuni than cats aged up to 1 year (OR = 10.440; P = 0.000). Moreover, cats living in the harbour area had a significantly higher risk to be a carrier of C. jejuni than cats living in urban area (OR = 17.911; P = 0.008). CONCLUSION: The findings of the present survey confirm stray cats as potential carriers of C. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the prevalence of C. jejuni in stray cats in Europe.

Nicotine induces tissue factor expression in cultured endothelial and smooth muscle cells.

BACKGROUND AND OBJECTIVES: Cigarette smoking is associated with an increased risk to develop myocardial infarction and ischemic stroke. However, the mechanisms responsible for these effects are still poorly understood. AIM: To investigate whether nicotine, the major component of cigarette smoking, and its main metabolite, cotinine, might induce a pro-thrombotic state via stimulation of tissue factor (TF) expression in two cell population widely represented in the arterial wall such as endothelial cells (ECs), and smooth muscle cells (SMCs). METHODS AND RESULTS: Incubation of ECs and SMCs with nicotine and cotinine induced TF expression in both cell types in a dose-dependent fashion, exerting its effect at the transcriptional level, as demonstrated by semiquantitative and by real-time PCR. Nicotine- and cotinine-induced TF expression was mediated by the activation of the transcription factor, nuclear factor-kappa B (NF-kappaB), as demonstrated by electrophoretic mobility shift assay and by the suppression of TF expression by the NF-kappaB inhibitor, pyrrolidine dithio carbamate ammonium. CONCLUSIONS: These data indicate that nicotine and cotinine exert direct effects on ECs and SMCs, shifting them toward a pro-thrombotic state via induction of TF expression. These effects on cells of the vessel wall might explain, at least in part, the deleterious cardiovascular consequences of cigarette smoking.

Neopterin induces pro-atherothrombotic phenotype in human coronary endothelial cells.

BACKGROUND: Inflammation plays a pivotal role in atherothrombosis. Recent data indicate that serum levels of neopterin, a marker of inflammation and immune modulator secreted by monocytes/macrophages, are elevated in patients with acute coronary syndromes and seem to be a prognostic marker for major cardiovascular events. The aim of the present study was to determine whether neopterin might affect the thrombotic and atherosclerotic characteristics of human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: In HCAECs, neopterin induced TF-mRNA transcription as demonstrated by real time polymerase chain reaction and expression of functionally active tissue factor (TF) as demonstrated by procoagulant activity assay, and of cellular adhesion molecules (CAMs) as demonstrated by FACS analysis, in a dose-dependent fashion. These neopterin effects were prevented by lovastatin, a HMG-CoA reductase inhibitor. Neopterin-induced TF and CAMs expression was mediated by oxygen free radicals through the activation of the transcription factor, nuclear factor-kappa B (NF-kappaB), as demonstrated by electrophoretic mobility shift assay and by suppression of CAMs and TF expression by superoxide dismutase and by NF-kappaB inhibitor, pyrrolidine-dithio-carbamate ammonium. CONCLUSIONS: These data indicate that neopterin exerts direct effects on HCAECs by promoting CAMs and TF expression and support the hypothesis that neopterin, besides representing a marker of inflammation, might be an effector molecule able to induce a pro-atherothrombotic phenotype in cells of the coronary circulation.

Apoptotic cell death in canine hair follicle.

Apoptotic cell death is an essential homeostatic mechanism involved in the control of cellular turnover in a variety of adult tissues. Cytoplasmic and nuclear condensation morphologically define this process whose biochemical hallmark is extensive DNA fragmentation into discrete oligonucleosomic units. Hair follicle growth and regression has been shown to be correlated with apoptosis in humans, mice, rats and guinea pigs. The present study was carried out to evaluate its implication in canine hair biology in order to define the spatio-temporal relationship between apoptosis and the hair cycle in dogs. As assessed by terminal deoxy-nucleotidyl transferase-mediated d-UTP nick-end-labelling (TUNEL) and by basic histological and ultrastructural assays, apoptotic cells appeared both in the growing and in the regressing follicle epithelium showing the well characterized morphological features described in the previous relevant literature.

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis.

In the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasma membrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida.

Pregnancy outcomes in in vitro fertilization cycles with serum estradiol drop prior to human chorionic gonadotropin.

OBJECTIVE: To study the effect of an unpredictable drop in serum estradiol prior to hCG administration on pregnancy outcomes in in vitro fertilization cycles. METHODS: 3653 consecutive IVF cycles from January 1, 1998 to December 31, 2000 at Brigham and Women's Hospital were reviewed, and 65 cycles in which oocyte retrieval (ER) was performed following a drop in serum estradiol (E(2)) not associated with intentional withdrawal of gonadotropins were identified. Daily gonadotropin dose was decreased at some time in 25 of these cycles, while the remaining 40 cycles did not have a reduction in gonadotropin dose. A retrospective case-control study of the respective live birth rates and pregnancy loss rates of patients with unpredictable E(2) drops in the 65 study cycles were compared to 65 age matched controls. RESULTS: Live birth rates (32% vs. 35%, p=0.72) and pregnancy loss rates (28% vs. 30%, p=0.76) were similar for all study and control groups respectively. There were no differences in live birth and pregnancy loss rates in cycles undergoing gonadotropin dose reduction (40% vs. 44%, p=0.78 and 29% vs. 39%, p=0.70) and cycles without gonadotropin dose reduction (28% vs. 30%, p=0.81 and 27% vs. 20%, p=0.72). CONCLUSIONS: In the absence of coasting, a drop in serum estradiol levels during GnRH-agonist downregulated controlled ovarian hyperstimulation for IVF prior to hCG is not associated with a decrease in live birth rates or pregnancy loss rates.

miR-23a, miR-24 and miR-27a protect differentiating ESCs from BMP4-induced apoptosis.

Numerous studies have indicated that BMP4 signaling is involved in the regulation of the early steps of development. In mouse embryonic stem cells (ESCs), BMP4 is crucial to sustain pluripotency and blocks differentiation towards neural fate. Here, through a systematic analysis of miRNAs in ESCs, we establish that BMP4 signaling regulates miR-23a, 27a and 24-2, through the recruitment of phospho-Smads at the promoter of the gene encoding this miRNA cluster. Suppression of miR-23a/b, 27a/b and 24 does not affect self-renewal or pluripotency, but induces an evident change of ESC differentiation, with a significant increase of the cells undergoing apoptosis after the transition from ESCs to epiblast stem cells (EpiSCs). BMP4 has been previously reported to cause apoptosis during ESC differentiation. By blocking BMP4 signaling, we completely prevent the apoptosis induced by suppression of the miRs. This suggests that the effects of miR suppression are the result of enhanced BMP4 signaling. This hypothesis is further supported by the observation that Smad5, the transcription factor downstream of the BMP4 receptor, is targeted by the miRNAs of the 23a and 23b clusters. Altogether, our results highlight the existence of a regulatory loop, involving Smad5 and the miR-23a clusters, that modulates the apoptotic response of ESCs to BMP4.

Epidermal growth factor receptors in uteroplacental tissues in term pregnancy before and after the onset of labor.

Using saturation binding assays and Scatchard analyses, we determined the concentrations and binding affinities of epidermal growth factor (EGF) receptors in human myometrium (n = 13) and decidua (n = 10) before and during labor and in placenta (n = 15), chorion (n = 17), and amnion (n = 17) before labor, during labor, and after vaginal delivery. Each tissue was individually assayed. In myometrium and chorion, EGF receptors increased significantly from 5.6 +/- 0.8 and 13.5 +/- 1.7 fmol/mg protein (mean +/- SEM) before labor to 11.1 +/- 2.8 and 26.7 +/- 3.0 fmol/mg protein, respectively, after the onset of labor (P < 0.05). In amnion, EGF receptors increased from 12.8 +/- 2.7 fmol/mg protein before labor to 33.0 +/- 2.3 fmol/mg protein during labor, but decreased significantly (5.9 +/- 1.2 fmol/mg protein) with vaginal delivery (P < 0.05). Decidual and placental concentrations of EGF receptors did not change significantly with labor. The binding affinity of EGF receptors in all tissues studied did not change significantly with labor, as reflected by their respective association and dissociation constants. Up-regulation of EGF receptors in myometrium, chorion, and amnion with spontaneous labor may enhance stimulation of prostanoid production and stimulate uterine activity.

Baboon corpus luteum: autonomous pulsatile progesterone secretion and evidence for an intraluteal oscillator demonstrated by in vitro microretrodialysis.

Pulsatility of serum progesterone (P) is usually ascribed to stimulation of the corpus luteum (CL) by pulsatile release of pituitary LH. We investigated P secretion by the primate CL by performing microretrodialysis on 6 fresh CL obtained at laparotomy from baboons (Papio anubis) with well defined menstrual cycles. Individually microdialyzed for 24-26 h with Dulbecco's Modified Eagle's Medium and Ham's F-12 enriched with HEPES buffer in a perifusion chamber, the retrodialyzed fluid was collected every 10 min and measured for P, estradiol, and 17 alpha-hydroxyprogesterone by specific and sensitive RIAs. The chronodynamics of hormone secretion were analyzed for pulse detection by PC-Pulsar 3.0. All 6 CL (2 each from early, LH +1 to +5; mid, LH +6 to +10; and late luteal phases, LH +11 to +15) demonstrated pulsatile secretion of P in vitro, with distinct and detectable peaks over the 24-26 h studied. The CL secreted 23-27 pulses of P in 24 h in early luteal, 8-20 pulses in midluteal, and 6-19 pulses in late luteal phases. Peak lengths were 23.8 +/- 18.5 to 35.7 +/- 17.1 min. Four CL gave interpeak intervals of 46-55 min, whereas two gave intervals of 136-137 min. Analysis of distribution of pulses against different interpulse intervals in individual CL and all CL together revealed a bell-shaped distribution, with the largest number of pulses seen at an interpulse interval of 21-40 min. Because of the low concentrations of estradiol and 17 alpha-hydroxyprogesterone retrodialyzed, a similar analysis of these data was not possible. Histological examination of the tissue at the termination of the experiment using hematoxylin and eosin and localization of 3 beta-hydroxysteroid dehydrogenase activity indicates that the steroidogenic potential of the tissue is minimally affected, although some morphological changes do occur. Our findings suggest autonomous pulsatile P secretion by the primate CL, indicating local control by and the presence of an intraluteal oscillator or pulse generator for P secretion.

The Amount of MUC5B mucin in cervical mucus peaks at midcycle.

The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.