Lymphocytes detect the antigens in the environment

by means of antibodies on the surface of B cells and T-cell receptors (TCR) on the T cells. With the diverse and expanding array of antigens, the generation of antibody/TCR diversity using limited genetic resources remained a question that baffled scientists for decades. An almost limitless number of antigens exist in the environment and recent research suggests that among the millions of lymphocytes, each one expresses a structurally different antigen receptor to combat this plethora of antigens. How is the genetic information for all of these antigen receptors encoded in the DNA? Do cells carry enough DNA to encode all the antibody specificities? Or is it that random mutations generate this enormously diverse repertoire of antibodies? Two theories arose initially to answer these questions. Somatic STA-9090 mouse mutation/variation theory BAY 80-6946 order suggested that a few inherited genes, with time, encountered mutations or recombinations to encode each antibody.[1] In contrast, germline theory proposed that the genome contains a large repertoire of antigen receptor genes and each of them encodes for separate, specific antibody.[2] Arguments supporting

and opposing these theories were put forward and remained unresolved for several years. In this review, we summarize the basic principles that presently govern the generation of diversity of antibody and TCR with special emphasis on V(D)J recombination. We also discuss the role of recombination activating genes (RAGs) in the generation of antibody diversity and chromosomal translocations. In the early 1990s, it was shown that isothipendyl two tightly linked genes, RAG1 and RAG2, which were unique to vertebrates, were responsible for the generation of antigen receptor diversity.[3, 4] An elegant series of experiments involving genomic DNA transfections into mouse

3T3 fibroblasts lacking V(D)J recombination activity, showed that the transfer of a single genomic locus could make these cells proficient for V(D)J recombination.[5] Following this, using the technique of ‘genome walking’, the RAG1 gene was discovered. Comparative sequence analysis of RAG1 genes from various species indicated that they were evolutionarily conserved.[3] Further studies demonstrated that the locus contained two closely linked genes, RAG1 and RAG2 on chromosome 11p in humans and chromosome 2p in mice.[4, 6] The coding and 3′ untranslated sequences of RAG1 and RAG2 were contained in a single exon.[6] The proteins encoded by the RAG genes play a crucial role in the generation of antigen receptor diversity as discussed below. There are two major antigen receptors for the lymphoid system, antibodies and TCR in B and T cells, respectively. Antibodies or immunoglobulins are glycoproteins that are either secreted out from B cells or remain bound to their membrane.

Inclusion bodies were collected by centrifugation at 10,000 g for 10 min, and pellets selleck chemical were washed twice with TE buffer, twice with 0.5 m

NaCl, once with 0.5 m NaCl–1% Triton X-100, once with 0.5 m NaCl and once with cold distilled water and finally solubilized in CBP buffer (0.1 m Na2CO3 1% 2-mercaptoethanol [pH 9.6]). Particulate material was discarded by centrifugation at 10,000 g for 10 min, and the purified solubilized protoxin was stored at 4 °C and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein concentration was determined by the Bradford method [16], and purity was examined by SDS-PAGE. Endotoxin contamination in Cry1Ac protoxin preparations was tested using the E-toxate Part

1 kit (Sigma-Aldrich, St Louis, MO, USA ) with a limit of sensitivity of 0.05–0.1 endotoxin units (EU)/ml following manufacturer’s instructions. Endotoxin levels in the purified Cry1Ac protoxin preparations were below 0.1 EU/ml, but they were further treated with an excess of a polymixyn B resin (BioRad, Hercules, CA, USA) to remove any possible remnants of endotoxin BioRad. Immunization.  Nine groups of animals were i.n. immunized with Cry1Ac or administered with the vehicle to carry out three independent experiments for each assay type tested: (1) phenotypic and activation analysis, (2) cytokine assays and iii) Enzyme-linked immunospot (ELISPOT) assay. As a positive mafosfamide control for the ELISPOT assay was included a group of animals that were intranasally immunized with cholera find more toxin (CT; Sigma–Aldrich), which is considered the most potent mucosal immunogen. Each group (control and experimental) contained seven animals. For i.n. immunization, mice were lightly anesthetized with ethyl ether, and the antigen (in 30 μl of PBS) was delivered into the nostrils. For experimental group, 50-μg Cry1Ac doses were applied on days 1, 7, 14 and 21 by the i.n. route. For CT group, 10-μg doses were applied on same days. Control mice received 30 μl of PBS. Mice from each group were killed on day 28, and pooled lymphocyte suspensions from

the NALT and NP were obtained as described previously [8]. ELISPOT.  Specific anti-Cry1Ac or anti-CT Ab-secreting cells were enumerated by ELISPOT assay. Briefly, a 24-well plate with a nitrocellulose base (Millipore Corp., Bedford, MA, USA) was coated overnight at 4 °C with 10 μg of Cry1Ac or 10 μg of CT in PBS (500 μl per well). All wells were then blocked with 1% BSA in PBS for 120 min at room temperature. Lymphocytes (1 × 106 cells) were suspended in RPMI-1640 medium containing 5% FCS and added to each well (500 μl per well) and incubated for 4 h at 37 °C in 5% CO2 in air. The plates were thoroughly washed with PBS ± Tween and then incubated for 2 h at room temperature with 500-μl goat anti-mouse IgA α chain specific, peroxidase conjugated (Zymed Laboratories, Inc.

v. Extremely useful (A) Moderately useful (B) Mildly useful (C) Not useful at all (D) Agammaglobulinaemia XLA Ataxia telangiectasia Chronic granulomatous disease Chronic mucocutanous candidiasis CVIDs Complement deficiency DiGeorge syndrome Hyper-IgM syndromes Hyper-IgE syndrome IgG subclass deficiencies Selective IgA deficiency SCID Severe congenital neutropenia Specific antibody deficiency IFN-γ/IL-12 cytokine axis

defect Wiskott–Aldrich syndrome XLP ____________________________ at a dose of ________mg/kg every ______• Selleck Talazoparib hours • days ____________________________ at a dose of ________mg every ______• hours and for • days MARK AS MANY AS APPLY MARK AS MANY AS APPLY MARK AS MANY AS APPLY _____________________________ _____ YEAR Please try to answer all questions to the best of your ability based upon your average approach to the ‘typical’ patient with PID. If you have specific additional concerns or comments regarding a particular question you may list them below (or separately). Question concern ____________________________ ____________________________ ____________________________ ____________________________ Geographic distribution of ESID respondents “
“For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific

target proteins are known www.selleckchem.com/products/azd9291.html and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived

peptide sequences. Using three separate peptide sequences from prostate-specific Methocarbamol membrane antigen (PSMA) (peptides PSMA27, PSMA663, and PSMA711), this vaccine design induced high levels of CD8+ T cells against each peptide in a HLA-A*0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA27 or PSMA663 peptides, and not PSMA711, were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8+ T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials. Prostate cancer (PCa) is the second most common cause of male cancer death in the UK and USA. Although current treatment can cure localized disease, many patients will have occult micrometastases that lead to subsequent relapse and development of detectable metastatic disease 1. Patient groups at risk could benefit from activating immune attack early against undetected, residual cancer cells using specific vaccines.

The first is clonal deletion. Although it can be very effective, when actually studied in the periphery it seems to take a very long time to eliminate the autoreactive population [5]. In cases where

the antigen is chronic, this presents a problem since the animal continues to suffer a risk of autoimmunity while the cells are being “slowly deleted.” Therefore, two other processes are thought to operate to keep the cells in check — a functional inactivation, originally termed anergy and the action of Treg cells [6, 7]. However, a clear separation between the three processes in vivo and an understanding of the principles that CP-690550 datasheet lead to the choice of any one or a combination of them is still lacking. We have previously reported that adoptively transferring antigen specific T cells to mice expressing their target antigen resulted in the induction of anergy and “slow deletion”, but not of Treg cells [5]. Typically, these studies involved the infusion of 1–3 million TCR transgenic T cells to BGB324 concentration congenic hosts. About 10% of the injected cells effectively incorporate into the secondary lymphoid organs. Nevertheless, work from several labs (using acute immunization, not chronic or self-antigens)

subsequently suggested that at such high frequencies, the T-cell responses were severely constrained by interference between the transferred T cells themselves [8-14]. This phenomenon, termed clonal competition, affects the robustness of the initial T-cell response, the subsequent survival of the activated T cells (memory) and even the extent of differentiation into different subsets [13, 15]. We therefore wondered if such a “precursor frequency effect” could also influence the behavior of self-reactive T cells. Interestingly, we find that chronic antigen stimulation elicits a precursor frequency independent response pattern, compared to an acute challenge. In the latter case the expansion phase and to a much lesser extent, the

onset of contraction was influenced by how many T cells participated in the original response. However, the self-reactive T cells were only minimally affected by precursor frequency during the initial expansion phase. MYO10 Furthermore, in the later phase, recipients seeded with about a 100 self-reactive T cells showed no evidence of clonal deletion for over 4 months. But, even at lower frequency, the self-reactive T cells entered an anergic state marked by reduced recall cytokine production and no conversion to Foxp3 positivity. These data suggest that in the normal repertoire, T cells reactive to chronic self-antigens that escape thymic deletion can respond and persist in the periphery, albeit in an anergic state. The impact of initial precursor frequency on the magnitude of the subsequent T-cell response was modeled using an adoptive transfer strategy wherein log dilutions of congenically marked naïve T cells were injected intravenously into recipient mice and challenged in vivo.

[37, 40, 42] Superficial infections can occur in patients suffering from an immunosuppressive disorder, such as leukaemia or HIV, but also in premature infants and apparently healthy adult persons.[42, 45-52] They are characterised by rapidly developing extensive tissue necrosis leading to purple to black discolouration of the skin.[45, 53] In individual cases Selinexor cell line involvement

of deeper tissue, leading to necrotising fasciitis and cellulitis, has also been reported.[40, 46, 54] In the most severe cases, cutaneous infections can progress to disseminated disease, especially in immunocompromised patients and premature infants.[47, 55] In premature infants, Lichtheimia infections furthermore commonly affect the gastrointestinal tract,[56] often resembling necrotising enterocolitis.[57] Since most studies on mucormycosis do not examine the type of infection on a species-specific level, it is hard to assess the incidence of

different types of infections for Lichtheimia. Only two studies include more detailed information about infections with Lichtheimia species. The study of Alvarez et al. included seven cases of Lichtheimia infections with pulmonary infection and infections of the sinuses as the most important presentations (6 of 7 cases).[22] Only one additional study focused on species-specific analysis of healthcare-associated mucormycosis. Cutaneous and pulmonary infections learn more were the most common types of infection representing 70% and 20% respectively.[83] However, due to the limitations of the currently available studies, e.g. low numbers of cases aminophylline or restriction to a special patient group, no clear conclusions can be drawn about the incidence of the different types of infections and underlying conditions for the development of Lichtheimia infections. In addition to causing infections, Lichtheimia species have been implicated in the form of occupational hypersensitivity pneumonitis termed Farmer’s lung disease (FLD). Farmer’s lung disease is caused by recurrent exposure to certain microorganisms, especially

in farming personnel. The acute form is characterised by influenza-like symptoms like sweating, chills, fever, nausea and headache. The (sub)chronic form is associated with coughing and dyspnoea for up to several weeks.[58] As mentioned above, Lichtheimia species represent a major contaminant of farming material like hay and straw. The occurrence of FLD has been associated with increased numbers of L. corymbifera in the farm environment and L. corymbifera-specific antibodies in affected patients.[59] Furthermore, in vitro experiments with lung epithelial cells revealed high expression of pro-inflammatory and allergic mediators (IL-8, IL-13) after exposure to extracts of L. corymbifera.[60] These results support the role of Lichtheimia in the development of hypersensitivity pneumonia.

The first was the one induced with multiple low doses of streptozotocin (MLD–STZ). STZ is a chemical substance with alkylation properties that interferes with glucose transportation. A single high-dose strategy results in severe toxicity and acute diabetes. Conversely, the multiple low-dose regimen, characterized by minimal β cell toxicity, Selleckchem Metformin results in autoantigen release and a possible break in self-tolerance [3]. The T cell dependence of this model is a debated topic, and needs

further evaluation. What is well established is that diabetes in this model cannot be transferred reliably to syngenic recipients by transfer of splenocytes [4]. Non-obese diabetic (NOD) mice are an inbred strain derived from Jcl:ICR mice [5], which develop type 1 diabetes spontaneously. The infiltration in the islets starts around 4–5 weeks, when pockets of lymphocytes are first observed juxtaposed to the pancreatic islets of young NOD mice. As the animals grow older, these mononuclear cells migrate into the islets, and by the time hyperglycaemia occurs destructive insulitis is present. This model is very similar to the human disease. Disease onset, for example, is preceded by infiltration of pancreatic islets by mononuclear cells and is controlled by many quantitative trait loci, particularly major histocompatibility

complex (MHC) class II genes. Diabetes in NOD mice is the most extensively studied model of autoimmune

disease [6, 7]. The discovery of regulatory T cells Proteasome inhibitor (Tregs) disclosed a new field to be explored in the control of autoimmune pathologies [8]. Heat shock proteins (hsps) are molecules up-regulated in conditions of stress that are highly conserved throughout evolution [9]. Although recent research implicates hsp60 as an autoantigen involved in type 1 diabetes pathogenesis [10], this protein also contributes to protection against autoimmune diseases. It has been described that microbial homologues of mammalian hsps could induce the recruitment of Tregs to inflamed tissues [9]. In this study, we investigated the possible protection against type 1 diabetes through a prime-boost vaccination strategy. This strategy consists in priming the system with the antigen administered in one vector and then boosting it with the same antigen, but through another Amino acid vector [11]. Thus, we made use of two different vaccines containing mycobacterial hsp65: bacille Calmette–Guérin (BCG) and pVAXhsp65, a DNA vaccine. This association could, theoretically, be interesting because both vaccines have been already tested separately against diabetes and other autoimmune diseases and showed positive results [12-15]. We hypothesized that the prime-boost strategy could expand these beneficial effects. Female NOD mice and male C57BL/6 mice were obtained from the animal facility of State University of Campinas (UNICAMP, Campinas, São Paulo, Brazil).

Data entry

and management were performed on Microsoft Office Excel 2007. All analyses and calculations were performed using statistical analysis software SAS 9.3 (SAS Institute, Cary, NC, USA). Data are presented as the mean ± standard deviation (SD) for continuous variables and as proportions for categorical variables. Based on the mean, GalNAc exposure rate was 0.4 ± 0.2, the prevalence and mean values of selected IgAN parameters were compared between GalNAc exposure levels (<0.4 and ≥0.4) by using χ2 statistics for categorical variables and the Student t-test for continuous values. The unadjusted odds ratio (OR) between IgAN traits and GalNAc exposure level (≥0.4) was determined by univariate logistic regress models and then adjustments were made for age and gender, as this website well as other IgAN traits by multivariate logistic regression models. All statistical tests were two-sided, and P < 0.05 was considered statistically significant. Table 1 summarizes patient demographics and the clinical characteristics of 199 IgAN patients. There were 90 males and 109 females Dorsomorphin in the study. The mean age was 34.5 ± 11.0 years. The proteinuria of 24 h was 1.77 ± 1.84 g/24 h and about 53.8% of patients had proteinuria excretion more than 1 g/24 h. Serum creatinines of these patients were about 159.9 ± 184.8 μmol/L. The mean GalNAc

exposure rate was 0.4 ± 0.2. It was shown that, the proteinuria excretion was a light negative correlation with GalNAc exposure rate (R = −0.184, P = 0.011; see Fig. 1). In the patients with elevated serum creatinine, the GalNAc exposure rate was comparable to G protein-coupled receptor kinase that in patients with normal serum creatinine (0.44 ± 0.19 vs. 0.43 ± 0.15). There is no relation between the GalNAc exposure rate and serum creatinine. It was also demonstrated that the serum IgA concentration (R = 0.297, P < 0.001; Fig. 2)

and the GalNAc exposure rate (R = 0.24, P = 0.001; Fig. 3) were positively correlated with serum IgG concentration. Patients were divided into two groups according the GalNAc exposure rate more or less than 0.4. The mean ages for the low and high exposure groups were 34.3 ± 11.5 and 34.6 ± 10.6 years, respectively. There were no significant differences in age or gender. The serum creatinine, uric acid, and serum IgA concentration were comparable for the two groups. However, the 24 h urine protein excretion was significantly heavier in the low exposure group than that in the high exposure group (2.14 ± 1.91 g/24 h vs. 1.47 ± 1.73 g/24 h, P = 0.01). Simultaneously, the total cholesterol, low density lipoproteins and complement C3 level was significantly higher in the low GalNAc exposure group (P < 0.05 for all parameters). However, the IgG concentration had the same trend with GalNAc exposure rate, 10.0 ± 3.0 mg/L in the low exposure group and 11.3 ± 2.

The increased TREC levels in the intestinal mucosa could, theoretically, represent T lymphocytes that have matured in situ in the intestinal mucosa, as the intestinal mucosa

can act as a site for extrathymic maturation of both IEL and LPL T lymphocytes in human infants [17], and developing T cells that are rearranging their TCR genes are found in the small intestine in human adults [18]. In addition, immunocompromized mice, i.e. major histocompatibility complex (MHC) class I-deficient and TCR-αβ-deficient mice, of which the latter spontaneously develop colitis [5,29], also have evidence of extrathymic maturation. Thus, it is possible that T cell progenitors in the bone marrow receive signals from the inflamed intestine to go directly to the intestinal mucosa for further maturation. However, we employed flow Akt inhibitor cytometric analysis using previously established phenotypic characteristics LY2606368 clinical trial of T cell progenitors in the gut, identified as CD19-CD16-CD3-CD2+CD5+CD7+ lymphocytes [17,18][30], and found no differences in frequencies of this

population between IBD patients and non-inflamed controls. As only the LPL population was investigated, due to limited amounts of IEL, it could be argued that extrathymic maturation could be increased, specifically in the IEL compartment. However, as quantitative RT–PCR analysis of pre-TCR-α and RAG1 mRNA expression [18,30,31] was performed in mucosal biopsies containing both IEL and LPL, and revealed no increased expression in IBD patients compared to controls, this is highly unlikely. Corroborating our findings of significantly increased frequencies of mucosal T cells expressing

CD62L/L-selectin in UC but not CD patients is a report that HEV-like vessels expressing PNAd, one of the ligands for CD62L, were induced preferentially in active UC [32]. In addition, serum concentrations of soluble L-selectin have been shown to Elongation factor 2 kinase be correlated positively to disease activity in UC but not CD [33]. In mice, CD62L+ expressing CD4+ T cells [34], as well as CD4+CD45RBhi[1,2,35], can induce colitis upon transfer into immunodeficient recipient mice. However, in humans CD62L is expressed by both CD45RA+ and CD45RA- T lymphocytes, of which naive T cells express both, while the CD62L+CD45RA- T lymphocytes have been shown previously to be central memory T cells [36]. Although we did not analyse this population for expression of the chemokine receptor CCR7, this suggests that the increased frequency of CD4+CD62L+CD45RA- lymphocytes found in the intestinal mucosa of UC patients represents CD62L+CD45RA-CCR7+ central memory T lymphocytes, found predominantly in lymphoid tissue [37]. Although the present study investigated a limited number of patients, we demonstrate that UC patients, and not CD patients, display an increased recruitment of RTE to the colonic mucosa, possibly before acquiring immunoregulatory properties in the periphery.

hmpdacc.org/reference_genomes.php) and the assembled and annotated genomic sequences of this bacterium have been submitted to the GenBank/EMBL/DDBJ Forskolin nmr database (http://www.ncbi.nlm.nih.gov/genome?Db=genome&Cmd=ShowDetailView&TermToSearch=7229; accession number AEVO01000000, and consists of sequences AEVO01000001-AEVO01000169, submitted (31-JAN-2011) by Genome Sequencing Center, Washington University School of Medicine). Based on blast analysis and inspection of the annotation of the draft sequence, it is indicated that there is a lack of the genes for CA in this bacterium. However, the sequence data whole genome shotgun draft generated by illumina reads that it consists of 169 contigs with gaps. Therefore,

we speculate that, as in S. thermophilum, the requirement for CO2 in S. hippei YIT 12066T is due to a CA deficiency. To the best of our knowledge, this is the first report of the isolation of a strictly CO2-requiring bacterium from human GI microbiota. The CO2 concentrations selleck compound required for the growth of S. hippei in the human intestinal tract may be achieved by the metabolic activities of other microbiota. Alternatively, the growth of S. hippei may be supported by bicarbonate secreted into the GI tract from the pancreas (19). The loss of the carbonic anhydrase gene in S. hippei may have occurred as a result of its adapting to its niche, the GI tract, which is rich in CO2/bicarbonate, although it is also possible that the ancestor

of this bacterium did not retain the corresponding gene from the beginning.

No potential conflicts of interest were disclosed. “
“Various studies have shown that dietary glutamine can modify the course of an immune response, through altering the release of cytokines. Nutritional supplementation of glutamine may therefore be of advantage to patients, particularly those with compromised immunity. Given that polymorphisms in cytokine genes can also affect cytokine levels, we have undertaken a study to identify whether there was a differential click here effect of glutamine supplementation in the context of different IL-2 -330 (T/G) and TNF-α -308 (A/G) genotypes. Overall, there was no significant impact of glutamine supplementation on IL2 release. However, analysing low, medium and high expressors independently, there was an effect of high glutamine levels on cytokine release from the low and medium expressors. Likewise, there was no effect of glutamine supplementation on the TNF-α release, although a tendency to lower cytokine release at high levels of glutamine. Irrespective of the glutamine concentrations, there was no difference in IL2 release between the IL2 -330 genotypes; there was an effect of the TNF-α genotypes, with the AG and GG genotypes showing greater cytokine release than from the AA genotype. The nutritional status is a very important criterion of assessment for patients’ immunocompetence. In certain situations, patients require the reasonable substitution of different dietary components.

The observed end points were CVD

death, myocardial infarction, unstable angina or ischaemic stroke during a follow-up period of the average of 519 days (range 138–924 days) [14]. The study design was approved by the Ethics Committee of Helsinki University Central Hospital, Helsinki, Finland, and an informed consent was obtained from each subject enroled in the study. Radiographic dental status.  The dental status of the patients was acquired by panoramic tomography taken after the admission to the hospital, as previously described [15]. The presence or absence of erupted teeth and periodontal breakdown was recorded. Patients were divided into three groups: edentulous, without periodontitis (later in the text referred to as non-periodontitis) NVP-AUY922 cell line and with periodontitis. Periodontal breakdown was established when the distance from the cementoenamel junction to the alveolar bone margin was more than 4 mm. The non-periodontitis patients had no radiographic evidence of periodontal breakdown [15, 16]. Serum analysis and sampling time points.  Baseline serum samples were

taken within 48 h of the arrival to the hospital. Follow-up samples were taken after 1 week, 3 months and 1 year of hospitalization owing to the ACS event. IgG and IgA antibody levels to A. actinomycetemcomitans and P. gingivalis were measured by multiserotype ELISA [17]. The inter-assay coefficient of variation was 6.6% and 6.2% for A. actinomycetemcomitans IgG and IgA assays, ABC294640 concentration and 5.3% and 5.6% for P. gingivalis

IgG and IgA assays, respectively. The cut-off limits for seronegatives and seropositives were 2.0 EU and 5.0 EU for IgA- and IgG-class antibody levels, respectively, corresponding to the mean + 1.5 × SD Oxymatrine of periodontally healthy individuals [17]. Serum IgG and IgA levels to human HSP60 were determined by ELISA [18]. The inter-assay coefficient of variation was 4.6 for IgA and 5.2% for IgG assays, respectively. In all antibody assays, the intra-assay coefficient of variation was 2.0–2.5%. High-sensitive C-reactive protein concentrations were quantified by immunoturbidometry [19]. All serum samples were taken after overnight fasting, and the laboratory analyses were performed in a blind fashion. Salivary bacterial analysis.  Salivary samples were taken at baseline within 48 h of arrival to the hospital. Paraffin-stimulated samples were collected and processed as previously described [14]. Aggregatibacter actinomycetemcomitans and P. gingivalis were PCR-amplified using species-specific primers as previously described [20]. Chromosomal DNA isolated from A. actinomycetemcomitans ATCC 43718 and P. gingivalis W50 strains were used as positive controls and sterile water as negative controls in each series of PCR reactions, which were performed blinded for the study groups. Statistics.  The significance of differences was analysed by Mann–Whitney U-test, Chi-square test and Wilcoxon signed ranks test.