Interleukin-1 family cytokines and mast cells: activation and inhibition.

Activated mast cells (MCs) secrete a number of compounds including pro-inflammatory and anti-inflammatory cytokines. MCs are a potential source of cytokines and chemokines which participate in allergic reactions and inflammation. MCs can be activated by IgE through its receptor FceRI, but also by Toll-like receptors and/or interleukin (IL)-1. MCs can be a target for both pro-inflammatory and anti-inflammatory cytokines. IL-1 activates MCs to release inflammatory chemical mediators, and cytokines/chemokines, an effect which can be potentially inhibited by IL-37. In addition, IL-36 is also a powerful cytokine with a pro-inflammatory activity. IL-38 binds IL-36R and inhibits the pro-inflammatory activity of IL-36, thus performing a therapeutic action. In this article we review the role of MCs in relation to pro-inflammatory and anti-inflammatory IL-1 family member cytokines and a possible therapeutic effect in inflammatory disorders.

Alternating block copolymer-based nanoparticles as tools to modulate the loading of multiple chemotherapeutics and imaging probes.

Cancer therapy often relies on the combined action of different molecules to overcome drug resistance and enhance patient outcome. Combined strategies relying on molecules with different pharmacokinetics often fail due to the lack of concomitant tumor accumulation and, thus, to the loss of synergistic effect. Due to their ability to enhance treatment efficiency, improve drug pharmacokinetics, and reduce adverse effects, polymer nanoparticles (PNPs) have been widely investigated as co-delivery vehicles for cancer therapies. However, co-encapsulation of different drugs and probes in PNPs requires a flexible polymer platform and a tailored particle design, in which both the bulk and surface properties of the carriers are carefully controlled. In this work, we propose a core-shell PNP design based on a polyurethane (PUR) core and a phospholipid external surface. The modulation of the hydrophilic/hydrophobic balance of the PUR core enhanced the encapsulation of two chemotherapeutics with dramatically different water solubility (Doxorubicin hydrochloride, DOXO and Docetaxel, DCTXL) and of Iron Oxide Nanoparticles for MRI imaging. The outer shell remained unchanged among the platforms, resulting in un-modified cellular uptake and in vivo biodistribution. We demonstrate that the choice of PUR core allowed a high entrapment efficiency of all drugs, superior or comparable to previously reported results, and that higher core hydrophilicity enhances the loading efficiency of the hydrophilic DOXO and the MRI contrast effect. Moreover, we show that changing the PUR core did not alter the surface properties of the carriers, since all particles showed a similar behavior in terms of cell internalization and in vivo biodistribution. We also show that PUR PNPs have high passive tumor accumulation and that they can efficient co-deliver the two drugs to the tumor, reaching an 11-fold higher DOXO/DCTXL ratio in tumor as compared to free drugs. STATEMENT OF SIGNIFICANCE: Exploiting the synergistic action of multiple chemotherapeutics is a promising strategy to improve the outcome of cancer patients, as different agents can simultaneously engage different features of tumor cells and/or their microenvironment. Unfortunately, the choice is limited to drugs with similar pharmacokinetics that can concomitantly accumulate in tumors. To expand the spectrum of agents that can be delivered in combination, we propose a multi-compartmental core-shell nanoparticles approach, in which the core is made of biomaterials with high affinity for drugs of different physical properties. We successfully co-encapsulated Doxorubicin Hydrochloride, Docetaxel, and contrast agents and achieved a significantly higher concomitant accumulation in tumor versus free drugs, demonstrating that nanoparticles can improve synergistic cancer chemotherapy.

Antifungal activity of essential oils against azole-resistant and azole-susceptible vaginal Candida glabrata strains.

Candida glabrata is an opportunistic pathogen, associated with endocarditis, meningitis, and disseminated disease, and also with complicated vaginitis. Essential oils derived from aromatic plants are known in traditional medicine as antimicrobial agents and have antifungal properties. The aim of this work was to evaluate whether 12 tested essential oils (tea tree, laurel, anise, basil, bergamot, lavender, mint, oregano, grapefruit, rosemary, winter savory, and ginger) could have a transverse effect on C. glabrata sensitive strains but above all on strains resistant to the three main azole antifungals used (clotrimazole, fluconazole, itraconazole). For this reason, different strains of C. glabrata, vaginal isolated, were characterized (disk diffusion assay, minimal inhibitory concentration) with respect to their response to such antifungals. Electron microscopy analyses were performed to examine cellular damages in depth. Subsequently, we wanted to evaluate the effect of the oils on human cells to estimate their potential cytotoxicity. Oregano and winter savory were the two most effective essential oils, inducing growth inhibition, cell damage of C. glabrata strains (both sensitive and resistant to azole antifungal drugs), and medium-high level of toxicity against human keratinocytes. The results of this work support the research for new alternatives or complementary therapies against vaginal candidiasis.

Cytokine expression profile and blood parameter evaluation of patients undergoing cardiac surgery.

Cardiac surgery is accompanied by an important immune response that is poorly understood. This inflammatory response is caused by several stimuli: surgical trauma, cardiopulmonary bypass apparatus, aortic-cross clamping, reperfusion injury and hypothermia. The aim of the present study is to investigate the cytokine level profile involved in the inflammatory pathway of patients undergoing cardiac surgery. One hundred and two patients undergoing elective cardiac surgery utilizing cardiopulmonary bypass (CPB) apparatus were enrolled in the study. In the hematological and biochemical profiles investigated, we observed a significant increase of WBC and blood glucose concentration and a strong decrease of RBC, HB, HCT and PLT 24 h post-surgery compared to baseline and immediately after surgery groups. Furthermore, we found a modulation of cytokine levels mostly for IL-10 and an increase of IL-6, detected at 6 h post-surgery, IL-8 at 6 and 24 h, and TNFα only at 24 h post-surgery. In conclusion, these findings evidence a time course profile on cytokine levels and a balance between pro- and anti-inflammatory cytokine activation during and after cardiac surgery. In fact, IL-6 and IL-10, a pro- and an anti-inflammatory cytokine, respectively, increased immediately after surgery. The plasma level of TNF-α could be inhibited by the high concentration of IL-10 up to 6 h post-surgery. An IL-10 reduction at baseline level, after 24 h post-surgery, could explain a rise of TNF-α plasma concentration. On the other hand, considering the dual role of IL-6 on inflammation acting both as an activator of inflammatory cascade or an anti-inflammatory agent, the increased IL-6 levels 24 h after surgery could be related to the negative feedback action on TNFα activity.

Effects of central fibroblast growth factor 21 and irisin in anxiety-like behavior.

Adipose tissue and skeletal muscle are organs capable of secreting many bioactive molecules, such as adipomiokines that could be possibly involved in mood disorders. In the present work, we investigated the possible behavioral effects of a single intracerebroventricular (i.c.v.) injection of two adipomiokines, fibrobroblast growth factor (FGF)-21 (0.5-5.0 µg) and irisin (0.4-0.6 µg), in male rats tested in the open field and elevated plus maze tests. Prefrontal cortex levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and the gene expression of catechol-O-methyltransferase (COMT), dopamine transport (DAT) and tyrosine hydroxylase (TH), were measured by high performance liquid chromatography (HPLC) analysis and real-time reverse transcription polymerase chain reaction (RT-PCR). Both FGF-21 and irisin administration induced anxiogenic behavior, increased DA levels in prefrontal cortex, decreased COMT, DAT and increased TH gene expression. In conclusion, in the present study we demonstrated behavioral effects induced by central FGF-21 and irisin injections that could involve increased DA signaling in the prefrontal cortex.

Effects of central fibroblast growth factor 21 (FGF21) in energy balance.

Fibroblast growth factor 21 (FGF21) is known as a major metabolic regulator of glucose and lipid homeostasis. Continuous intracerebroventricular (i.c.v.) administration of FGF21 was found to modulate feeding and energy expenditure in rats with diet-induced obesity, suggesting a central effect by the peptide. In this context, in the present work, we studied the effects of a single central FGF21 administration (0.5-5 µg) on feeding and energy expenditure by evaluating locomotor activity, interscapular brown adipose tissue (BAT) weight, gene expression of uncoupling protein-1 (UCP-1) in BAT and plasma norepinephrine (NE) levels in Sprague-Dawley fed rats. In addition, we evaluated the effects of FGF21 on orexigenic [agouti-related peptide (AgRP) and neuropeptide Y (NPY)] and anorexigenic [cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)] peptides, in the hypothalamus, and dopamine (DA) and serotonin (5-hydroxytriptamine, 5-HT) levels in nucleus accumbens (NAc). We confirmed that central FGF21 administration induced a significant increase in food intake, possibly mediated by increased NPY and AgRP, and decreased POMC and CART gene expression. Moreover, FGF21 could modulate the motivational aspects of feeding, possibly through stimulated NAc DA levels. On the other hand, our findings of decreased locomotor activity, BAT weight, UCP-1 gene expression and plasma NE levels support a role for FGF21 in decreasing energy expenditure.

Crocus sativus, Serenoa repens and Pinus massoniana extracts modulate inflammatory response in isolated rat prostate challenged with LPS.

Prostatitis is a common prostate disease that could be promoted by bacterial or non-bacterial infectious agents. In addition, inflammatory pathways involved in prostatitis have been increasingly studied, and herbal extracts endowed with anti-inflammatory effects are under investigation, individually or in combination, for their efficacy in alleviating the burden of inflammation, with possible improvements in symptoms. Serenoa repens (Serenoa), in combination with Crocus sativus (Crocus) and Pinus massoniana (Pinus), has previously shown to improve sexual function and limit urinary symptoms in patients suffering from concomitant erectile dysfunction and lower urinary tract symptoms. In this context, the aim of the present study is to evaluate the efficacy of Serenoa, Crocus and Pinus extracts, either alone or in combination, on immortalized prostate cells (PC3) and in an experimental model of bacterial prostatitis constituted by ex vivo prostate specimens challenged with lipopolysaccharide (LPS). We found that the tested extracts were able to reduce ROS production by PC3 cells and NFkB and PGE2 activity in prostate specimens challenged with LPS. In addition, the pharmacological association of the extracts displayed synergistic effects indicating a rational use of the mixture of the tested extracts as a novel anti-oxidant and anti-inflammatory formulation in bacterial prostatitis. Finally, we performed analytical and in vitro evaluation to better characterize the phytochemical profile and the mechanism of action of selected secondary metabolites.

Cytokine modulation in patients with idiopathic pulmonary fibrosis undergoing treatment with steroids, immunosuppressants, and IFN-γ 1b.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology and pathogenic mechanisms. From an etiopathogenic point of view, alveolar macrophages play a key role in accumulation of fibroblasts and deposition of collagen and extracellular matrix by releasing specific cytokines and inflammatory mediators. IPF seems to be also associated with circulating fibrocytes, which might be involved with an abnormal pulmonary vascular repair and remodeling. Based on its hypothesized pathologic mechanisms, anti-inflammatory, anti-fibrotic and immunosuppressive therapies are often used. For these reasons, Interferon-g (IFN-g) has been used to exploit its activity on macrophages and fibroblasts. The aim of this study was to investigate the response to corticosteroids and/or IFN-g 1b treatments based on pulmonary function tests and on inflammatory cytokine patterns of expression on bronchoalveolar lavage (BAL), at baseline and during and after the therapies. Unlike previous studies, we analyzed a period of therapy longer than 1 year. Our results demonstrated the effectiveness of IFN-γ in a group of IPF patients in whom the treatment was prolonged for over a year. These data suggest a positive role of IFN-γ; treatment in patients in the initial stage of the disease.

miR-2861 is involved in osteogenic commitment of human periodontal ligament stem cells grown onto 3D scaffold.

miR-2861 endorsing osteoblast differentiation through the overexpression of Runt-related transcription factor 2 (RUNX2) protein has been recently described. In this study we evaluated: the performance of living construct, composed by human Periodontal Ligament Stem Cells (hPDLSCs) and 3D scaffold (EXg), and the behaviour of miR-2861/RUNX2 expression pathway on the osteogenic commitment. Human PDLSCs were seeded with and without EXg scaffold and cultured under basal and osteogenic conditions. Morphological features, adhesiveness and differentiation abilities were analysed using scanning electron and confocal laser scanning microscopy. Time-course of RUNX2, ALP, OPN and miR-2861 were evaluated through RT-PCR analysis. Our results highlighted that the osteogenic differentiation was mostly obvious in the hPDLSCs, grown onto 3D scaffold in presence of osteoinductive medium. Moreover, the overexpression of miR-2861 and RUNX2 in hPDLSCs cultured in presence of EXg under osteogenic and standard conditions was demonstrated. In synthesis, the increased expression of miR-2861/RUNX2 provides new insights regarding miRNA signaling network in the presence of scaffold providing an additional method to evaluate the performance of biomaterial in bone regeneration.

Effect of low energy light irradiation by light emitting diode on U937 cells.

Photobiomodulation (PBM) can induce a set of different biological modulators either in vitro or in vivo. Experimental evidence has highlighted the role of light effects on the mechanisms related to inflammation, apoptosis and autophagy. The goal of this project was the evaluation of PBM on U937, an established cell line of histiocytic lymphoma origin. Several aspects of modulation of proinflammatory pathways were analyzed and autophagic and proapoptotic mechanisms related to low laser light exposure of cells were studied. As a source of low energy light emission, we used an NIR-LED device, characterized by an 880 nm-wavelength as light source. Flow cytometry analysis was performed on supernatants of controls and treated U937 cells to detect inflammatory cytokine levels. In order to evaluate NF-kB and caspase3 expressions, Western blot analysis was performed according to standard procedures. In this report, we show the effect of PBM on a monocyte/macrophage established tumor cell line (U-937). We demonstrate that LED exposure, in the presence or absence of lipopolysaccharide (LPS), activates cell degranulation, increased expression of Interleukin-8 (IL-8) and modulation of beta galactosidase activity. Evidence shows that the well-known pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the apoptotic marker (caspase3/cleaved-caspase3 ratio) are up-regulated in response to a proinflammatory biochemical pathway.

Comparative molecular analysis of bacterial species associated with periodontal disease.

Periodontal disease is an inflammatory disorder affecting the supporting teeth structures, including gingiva, periodontal ligament and alveolar bone, causing loss of connective tissue, reabsorption of alveolar bone and formation of periodontal pockets. The aim of this study is to find a correlation between bacterial growth and periodontal disease. Fifty-seven patients aged between 21 and 65 years, median age 46 years, were enrolled. According to gingival pocket depth, ranging from 3 to 7 mm, patients were divided into two groups: the first (30 patients, 53%) with deep pockets ³ 5 mm and the second (27 patients, 47%) less than 5 mm. The samples taken were processed for microbiological analysis by absolute quantitative real-time Taq-Man technique. Patients affected by periodontal disease were 32 (56%) and patients with gingival bleeding were 35 (61%). This data showed that the presence, the type and the bacterial load in gingival pockets were strongly correlated with gingival depth, periodontal disease and gingival bleeding. Quantitative microbiological analysis is a key point to improve patient compliance, allowing to choose the specific antibiotic treatment. avoiding antibiotic resistance and ensuring the successful outcome of therapy for periodontal disease.

Enigmatic question of early reactive arthritis disclosed after researches of mycoplasmas, Chlamydia trachomatis and enteropathogens following the holistic vision of human being.

An HLA-B27 genetic profile patient is fully investigated by molecular analyses after an anamnestic assessment of multi-site ecosystems, following the holistic vision of human being.VDRL and Widal-Wright (WWR) resulted positive, showing at Wright’s reaction a title of 1:40. Of all the enzymatic activities measured, only the ALP enzymatic pool activities showed a low increasing value of 297 U/L. Of all later acute phase proteins, Only C3 c protein value (127 mg/dL) and fibrinogen (376 mg/dL) were altered. Cultural and molecular oropharyngeal ecosystem investigation resulted significantly positive to Mycoplasmas(Mhand Uu) and Chlamydia trachomatis(Ct) together with a spread of saprophytic flora. From an accurate anamnesis, several and severe uro-genital clinical symptomatology emerged from birth until the beginning of rheumatologic symptomatologies that were confirmed by oldest Mh, Uu and Ctsilent chronic infections between these ecosystems. The molecular HPV research was negative, while the Thin prep pap-test was indicative of vaginosis and cellular reactive changes associated with inflammation. Parasitological research resulted positive for presence of 5-7 newly-formed G. lambliacysts for microscopic field, while digestibility test was positive for presence of several free fatty acid crystals. The remarkable presence of indigested meat fibre and several mucous dense filaments were observed. The pH value was 6.5, while blood faecal test was positive. The values observed were: ferritin 12 microg/L (10-120), total iron-binding capacity (TIBC) 310 &mgr;g/dL (300+-20), unsaturated iron-binding capacity (UIBC) 286 microg/dL (200-220) and iron seric level 24 microg/dL (60-130). Faecal research highlighted a very scarce presence of E. coli, resulting in 102 UFC/g of stool. Of all enteroinvasive pathogens, researched by molecular analyses, only Yersinia spp. was positive. After several specific cycles of antibiotic and antinflammatory therapies, the patient improved its general health condition considerably and showed almost complete regression of aching inguinal lymph node inflammation. In a picture of a worsening inflammatory process, produced by pathogens like Mycoplasmas, chronic silent or low grade inflammation atypical agents, in young HLA-B27 positive patient, VDRL test resulted positive. This value represents the first non-specific unique spy to reveal the precocious immunological signal in order to register the beginning of early innate immune system decay, keeping in mind that mycoplasmal and chlamydial infections are the triggering of cancer in patients genetically susceptible.

Can latent synergism of intestinal pathogens be responsible for inflammaging process causing Reiter's syndrome in a young patient HLA-B27 infected by atypical pathogens? A holistic view and clinical biochemical reinterpretation.

A case of a genetically HLA-B27 patient fully investigated by molecular analyses, following a holistic vision and an anamnestic assessment of multi-site ecosystems is repeated. VDRL, Lupus anti-coagulant (LAC) and Widal-Wright (WWR), resulted positive. The antibodies (IgG/IgA anti-Ct) against chronic Chlamydia trachomatis inflammation were positive. In the context of all the enzymatic activities in reference range, the AMS and the ALP enzymatic activities showed an increasing trend and a time course augment depending respectively. Cultures, parasitological, digestibility tests and molecular analyses were then performed to investigate the different human ecosystems. Parasitological research and digestibility test were performed, resulting a latent chronic bowel inflammation, including certain enteroinvasive pathogens, such as, Salmonella, Shigella, Yersinia and Campylobacter (Enteric Pathogens Group, EPG) and Escherichia Coli pathogens (Escherichia Coli Pathogens Group, ECPG). The Salmonella typhi-DNA resulted positive, while 90% of the total microbic charge (TMC) was represented by C. freundi in culture analyses. Interpreting the VDRL positive test as early triggering of autoimmune disease, a few acute phase proteins as a pauci-symptomatic chronic phlogistic process, the amylase and alkaline phosphatase alterations as tissue markers of early intestinal inflammation, the Widal's reaction positivity together with the precocious clinical and faecal manifestations, this study suggests the prime triggering role of these atypical pathogens to cause a chronic low grade autoimmune response against the tissue/organ susceptible target, causing inflammaging phenomenon in young patient with chronic latent infection by Salmonella typhi, leading to Reiter's syndrome, in HLA-B27 positive patient.

A novel biotechnology product for the degradation of biofilm-associated polysaccharides produced by Streptococcus mutans.

In this study we evaluated the activity of ABR preparation, a first-in-class agent obtained through fermentation process by genetically unmodified Bacillus spp., in breaking down polysaccharide produced by Streptococcus mutans, primary coloniser of tooth surface and abundant in dental biofilms. Our results showed that ABR preparation is able in degrading sugars formed by S. mutans, both in broth culture and onto teeth surface. Its activity is not influenced by the presence of saliva, commercial mouthwashes or oral disinfectants. ABR preparation has the potential to remove preformed plaque and counteract its development, thus offering conservative control of gingival and periodontal disease.

Cyanidin reduces preadipocyte differentiation and relative ChREBP expression.

Adipogenesis is a continuous process even in adult adipose tissue for the presence of preadipocytes that, when subjected to appropriate stimuli can proliferate and differentiate. ChREBP, the essential transcription factor for lipogenesis, is expressed in all tissues, but mainly in lipogenic organs. In this study, we focused on ChREBP expression during preadipocytes differentiation. Since it was found that cyanidin-3 reduces body weight in mice even in the presence of a high-fat diet, by decreasing levels of blood glucose and by improving insulin sensitivity, we studied the effect of this substance on adipogenic differentiation. For this purpose we used preadipocytes obtained from subcutaneous and visceral human adipose explant tissue, characterized and stimulated to differentiate in selective media. On cytofluorimetric analysis these cells showed mesenchymal markers (CD29, CD90, CD44), whereas they were negative for hematopoietic markers (CD45, CD10, CD117,CD31). ChREBP expression levels were quantified by immunoelectron-microscopy and western blotting analysis. In this report we show that ChREBP is expressed in preadipocytes (both nuclear and cytoplasmic compartments); the cytoplasmic level of ChREBP increased by 50 percent on day seven of differentiation into mature adipocytes. Cyanidin reduced differentiation by 20 percent (as evaluated by red oil O staining) and the expression of ChREBP. In addition, cyanidin-treated cells showed abnormal morphology, a square shape with irregular size, probably due to the fact that cyanidin may interfere with the extracellular matrix. These findings suggest that dietary cyanidin, may have inhibitory effects on adipogenesis.

Jugulodigastric lymph node inflammation derived from chronic atypical oropharyngeal phlogosis recurring annually after flu virus vaccination: a holistic vision of a clinical case solved after chlamydicidal antibiotic therapy.

In this report, we evaluated the case history of a patient with longstanding chronic pharyngitis who had periodic clinical manifestation for three years after a flu vaccine administration, and after various treatments tried to resolve the chronic pharyngitis with unsuccessful antibiotic and anti-inflammatory therapies. The patient occasionally presented a slight ocular inflammation, while dysuria occurred after sexual activity. The search for common pathogens by use of pharyngeal swabs resulted only in Corynebacterium ulcerans growth. After this first result, we focused our investigations on ocular and uro-genital infections of Chlamydiaceae (Ct and Cp) and Mycoplasmataceae (Mh and Uu) families. We examined the patient?s pharynx using molecular and culture techniques from three different sites. Although several infectious agents, including viruses and bacteria, causing chronic pharyngitis are reported in the literature, these ocular and uro-genital pathogens are seldomly routinely investigated in the same patient in ORL. Furthermore, while episodes of chronic pharyngitis is one of the most common clinical manifestation in ENT patients, these atypical pharyngitis represent ever-increasing infections which must always be considered and researched by suitable instruments such as PCR. Only from the collection of detailed medical history and careful observations of clinical manifestation, indicative of an oral chronic pathologic phenomenon of low intensity initiated several years previously, starting with sudden outbreak and relapse like a bout of flu, we suggest to study these atypical infecting agents frequently localized in the urogenital human area, awhich would allow to highlight and to recognize these clinical cases that manifest themselves as chronic inflammation of jugulodigastric lymph nodes, remaining still unrecognized and rarely associated to chlamydial infection, confused with the response to flu vaccination. After several specific cycles of antibiotic therapy, the patient's health improved considerably and showed almost complete regression of jugulodigastric lymph node inflammation.

Ocular clinical pictures disclosed by PCR molecular diagnosis of Chlamydia trachomatis infection performed following the appropriate sampling modality in ocular ecosystem.

Four clinical cases regarding the correct diagnosis of early ocular Chlamydia trachomatis (Ct) inflammation, performed by two different modalities on the ocular ecosystem, are discussed. The present study was carried out in parallel using a cotton flock ocular swab and the scraping of upper lid conjunctiva. The ocular samplings were carried out by a first ocular swab from inner canthus and fornix, while the second by a conjunctival scraping from upper the conjunctiva of four patients. In the first case, by ocular swab, all samples resulted negative to Ct-DNA research by PCR, while the cultural analyses showed a growth of saprophytic and opportunist germs in all patients. No growth micetes resulted. On the contrary, in the second case, by conjunctival scraping, three of four samples were positive to Ct-DNA research. No fungal growth was observed, while only the 3rd patient, negative to Ct-DNA research, showed microbial growth. Our study, carried out with two different modalities of sampling on different areas of the same ecosystem, showed different results, demonstrating the importance of sampling accuracy for chlamydial research by molecular analysis in PCR, during the slight phase of inflammation. These initial data indicate that laboratory diagnosis by PCR for precocious Ct infection, not revealed clinically, could represent the first step for a correct diagnostic procedure, eliminating one of the critical points, allowing an accurate, effective and precocious antibiotic therapy. We hypothesize that only by following these correct procedures of sampling during the early phase of chlamydial inflammation, in the future, will it be possible to reduce a pejorative evolution of this worsening disease in people genetically susceptible, building a more efficacious Public Health program of prevention against chronic conjunctivitis and to favour a major prevention of trachoma in endemic areas.

Molecular approach by PCR is the best method to detect the presence of Chlamydia trachomatis and to define the true agent of ocular bacterial inflammation.

Chlamydia trachomatis (Ct) is an atypical agent for acute, subclinical and chronic conjunctivitis in developed countries, as stated by the International League against Trachoma. In order to evaluate the presence of Ct, from a total of 3,520 patients visiting the consulting room of the Eye Clinic of the University of Chieti, Italy from 2006-2008, we enrolled 171 patients affected by occasional mild, moderate or severe conjunctivitis in a three-arm prospective open study, using traditional analysis such as Immune Fluorescent Assay and Enzyme–Linked Fluorescent Assay (IFA and ELFA) and molecular analysis with Polymerase Chain Reaction (PCR) procedure for Ct DNA research (Ct DNA). At the same time, microbiological culture was carried out for common germs and mycetes. These patients were analyzed at different subsequent times. In the first arm (Group A) of 82 patients with IFA and ELFA only 10 people (12.2%) resulted positive to Ct infection with both methods. The presence of Ct was never alone, but always overlapped with contaminants, like corynebacteria, staphylococci, streptococci and colonbacteria, randomly distributed, while no growth of mycetes was observed. Of these positive patients, only one 47-year-old female, suffering from a moderate form of ocular chlamydial infection, showed serological conversion against this infection; furthermore, this female had also been suffering from reactive arthritis for sometime. In the second arm (Group B) of 89 patients, we carried out PCR for Ct detection: 82 (94.25%) were found positive to Ct – DNA research, with common germ growth randomly associated, without sex or age prevalence, as in group A; no mycetes were found. The third arm (Group C) included 37 negative patients from Group A with severe or moderate chronic conjunctivitis, randomly recruited between relapsing cases, with the addition of the single previously positive seroconversion case, for a total of 38 patients, who were re-evaluated by PCR Ct-DNA analysis. All these patients, negative to IFA and ELFA, were positive to Ct-DNA analysis. These data indicate a higher rate of Ct infection in patients with severe or moderate chronic conjunctivitis, resistant to usual therapies even after eradication of common germs, thus showing the advantage of introducing this molecular technique of analysis in mild to severe chronic or recurrent conjunctivitis.

Upgraded diagnostic value of Gen-Probe PACE 2 assay for detection of Chlamydia trachomatis infection.

In this study, we evaluate the performance of a nucleic acid amplification assay, COBAS AMPLICOR (Roche Molecular systems) (PCR), compared to non-amplified DNA probe assay PACE2 (Gen-Probe Inc.) for the detection of C. trachomatis in a total of 2,916 samples (2,114 females and 802 males) consecutively collected in two different clinical pathology laboratories, over a period of three years. In the females, the endocervical swabs showed a similar range of detection when using the two different methods: out of 1,581 females processed with PACE 2, 1.4% (2005), 0.9% (2006), 0.5% (2007), resulted positive for C. trachomatis; out of 533 females processed with PCR, 1.3% (2005), 1.5% (2006) and 1.2% (2007), resulted positive. However, in the male subjects we found an increased positivity of Chlamydia detection on urethral swabs by using PACE 2: 4.8% (2005), 1.9% (2006) and 2.9% (2007), compared to urine specimen processed by PCR: 1% (2005), 1.4% (2006) and 0% (2007). Even if PCR should be considered a most promising tool for routine diagnosis of Chlamydia infection, Gen Probe allowed us to better identify Chlamydia trachomatis (in 4.8% of urethral swabs compared to urine) leading to a hypothesis that extracellular EB forms of Chlamydia could be absent in urine in persistent infectious.