The latter was achieved Ipatasertib cell line by generation of mixed BM chimeras through reconstitution of lethally irradiated WT recipient mice

with an equal mixture of B7-deficient (CD80−/−CD86−/−) BM 18 and CD11c:DTA (CD45.1) BM 15. For controls, we included mice reconstituted with a mixture of B7− and WT (CD45.1) BM or CD11c:DTA, B7− and WT BM only (Fig. 2A). In the resulting mixed [B7−/CD11c:DTA>WT ] BM chimeras, wt cDC are constantly ablated due to DTA expression. The cDC compartment of these animals thus consists exclusively of CD80−/−CD86−/− cDC. On the contrary, B cells and other hematopoetic cells in these animals are composed of both B7-proficient and -deficient cells, whereas nonhematopoetic cells, including the radio-resistant thymic epithelium, are exclusively of WT recipient genotype. Notably, the specific absence of CD80−/−CD86−/− from cDC in [B7−/CD11c:DTA>wt] BM chimeras had no effect on the percentages

of thymic Foxp3+ Treg out of single-positive CD4+ thymocytes (Fig. 2B). This corroborates earlier notions that mTEC and other, BM-derived APC can mediate the generation of nTreg in the thymus via B7 interactions 7, 19 and that thymic DC are dispensable for the generation of nTreg 14, 15. On the contrary, EGFR inhibitor peripheral Foxp3+ Treg in [CD11c:DTA>WT] chimeras, constitutively lacking cDC, and [B7−>WT] chimeras lacking CD80/CD86

expression on all BM-derived cells displayed markedly reduced Treg frequencies, when compared with [WT>WT] control chimeras (Fig. 2C and D). Moreover, importantly, the specific absence of CD80/CD86 on cDC, in the mixed [B7−/CD11c:DTA>wt] BM chimeras, also resulted in more than twofold reduction of peripheral Foxp3+ Treg. In contrast, mixed [B7−/WT>WT] BM chimeras retaining both B7-proficient and -deficient cDC displayed PIK3C2G elevated percentages of Foxp3+ Treg, as compared with [B7−/CD11c:DTA>wt] chimeras (Fig. 2C and D). It is worth noting that the only difference between these two groups of mixed BM chimeras is the absence of CD80/CD86-proficient cDC in [B7−/CD11c:DTA>wt] chimeras. To substantiate our findings, we next generated mixed chimeras using BM of B7− mice (CD45.2) and CD11c-DTR mice (CD45.1) that allow for the conditional ablation of cDC 20. The resulting chimeras harbor a mixed DC-compartment consisting of DTx-sensitive WT DC and DTx-resistant B7− DC. DTx injection which leaves the chimeras only with CD80/CD86-deficient cDC resulted in a reduction of peripheral Treg (Fig. 2E).

[89] The pathogenesis and mechanisms AG-014699 cost involved in vertical transmission are still not completely understood. HCMV spreads from the infected mother’s decidual cells to the fetus. Sites

of viral replication include cytotrophoblast progenitor cells in chorionic villi and differentiating/invading cytotrophoblasts.[90] Until recently, the role of dNK cells in controlling viral infection was not known. However, epidemiological studies indicate that the rate of congenital HCMV infection is often low in the first trimester of pregnancy, which coincides with high numbers of dNK cells within the decidua, which suggests that dNK cells might be involved in protection against congenital HCMV infection. Decidual NK cells express all the receptors involved selleck compound in the response to HCMV and they also contain the necessary arsenal for cell cytotoxicity (Fig. 2). In a recent work, we provided the first evidence for the involvement of dNK cells in the response against congenital HCMV infection (see Fig. 3 for visual summary).

Interestingly, dNK cells can be found in the vicinity of infected cells within floating chorionic villi, suggesting that the functional plasticity of dNK cells in response to invading pathogens is associated with modulation of their migratory phenotype.[91] Deciual NK cells respond to congenital HCMV infection by lowering the secretion of several soluble factors (CCL2, CCL4, CCL5, CXCL10, granulocyte–macrophage colony-stimulating factor and CXCL8) that are involved in trophoblast invasion. By interfering with trophoblast

invasion, dNK cells can participate actively in limiting viral spreading and congenital infection. Along the same lines, such changes within the microenvironment itself will not only limit trophoblast invasion but also induce inappropriate activation of other immune cells namely dendritic cells and T cells. The ability to cross the placental barrier is one key determinant of invasive viruses and pathogens (hepatitis viruses, HIV, Plasmodium). Isoconazole Yet little is known about mechanisms underlying the fetal placenta tropism and the ability of dNK cells in the defence against these agents. Recent studies demonstrated that under certain conditions NK cells isolated from non-pregnant uterine mucosa and soluble factors secreted by decidual cells can control X4-tropic HIV-1 infection.[92, 93] Hence, it is conceivable that uterine NK and decidual NK cells act as local guardians against infection and their immune modulation might ensure efficient anti-viral protection. During the first trimester of pregnancy dNK cells display unique phenotypic and functional properties that distinguish them from other peripheral blood or tissue NK cells. They orchestrate fetal trophoblast invasion and placental vasculature remodelling, which are necessary for the maintenance of a healthy pregnancy.

However, here we concentrate on evidence for differential sensitivity as measured by T cell effector functions. Thornton and Shevach described

a co-culture system to measure Treg-mediated suppression that not only provided important mechanistic data on the requirements for suppression, but also laid down a template for demonstrating the functional activity of Tregs. The classical suppression assay involves the co-culture of CD25+ Tregs and CD25– responder T cells over a range of suppressor : responder ratios and measurement of the extent to which Tregs restrain the proliferation of CD25– T cells [40]. There is almost no area of Treg selleck screening library biology which has not been assessed by some modification of this basic technique. This assay has been used to compare the regulatory function of different subsets of Tregs[64], of in vitro-activated versus freshly explanted Tregs[65–68], of Tregs from sites of inflammation [69], of nTregs and iTregs[26] and of Tregs in infected versus healthy mice [70] and humans [71]. The findings of

many of these studies informed further in-vivo experiments and they have greatly enhanced our knowledge of Treg function. However, the specificity and activation status of regulatory and effector T cell populations as well as the cytokines present in the microenvironment and the activation status of antigen-presenting cells (APCs) will influence the capacity of Tregs to suppress in vivo. These conditions are often not well modelled in vitro and this caveat represents the greatest limitation of this type of assay. Particularly in mice, Torin 1 concentration most often the responder population used for in vitro suppression assays are CD4+CD25– T cells from naive mice, and such cells are highly susceptible to Treg-mediated suppression.

Indeed, it has been suggested that the window of susceptibility to Treg-induced suppression in vitro is regulated tightly and restricted to the first 12 h of stimulation [72]. Limiting the Carbohydrate proliferation or cytokine production of highly activated polarized T cells is a much more demanding task, and this may be why a clear comparison of the capacity of Tregs to limit the activity of polarized Th1, Th2 and Th17 cells is missing from the literature. It has been shown, however, that while Tregs can suppress the priming of Th2 responses, they are unable to suppress the proliferation or cytokine production of established Th2 effectors unless they themselves are pre-activated in vitro[73]. The importance of the comparative activation status of effectors and Tregs has been well illustrated. Tregs at sites of inflammation, for example, are typically more highly activated than peripheral Tregs[74,75], and this draws into question the extrapolation of functional assays carried out using mismatched responder : suppressor co-cultures and argues in favour of sampling both Tregs and effector T cells from the tissue of interest wherever possible [44,69,76].

(p. 287) I can think of no better term than “awesome” to describe the excitement and vibrancy of our field. This article is a revised version of a presidential address delivered on June 8, 2012 at the biennial meeting of the International Society on Infant Studies, held in Minneapolis, MN. I am indebted to the many faculty mentors, collaborators, postdoctoral fellows, AZD2014 datasheet and graduate students who have filled my head with ideas and implemented

those ideas in ways that I never dreamed possible. Grant support was provided by NIH research grants HD-037086 to RNA and Elissa Newport, HD-073890 to Michael Tanenhaus and RNA, and HD-067250 to Daniel Weiss and RNA. “
“We conducted two experiments to address questions over whether 9-month-old find more infants believe that objects depicted in realistic photographs can be picked up. In Experiment

1, we presented 9-month-old infants with realistic color photographs of objects, colored outlines of objects, abstract colored “blobs,” and blank pages. Infants most commonly rubbed or patted depictions of all types. They also showed significantly more grasps toward the realistic photographs than toward the colored outlines, blobs, and blank pages, but only 24% of infants directed grasping exclusively at the photographs. In Experiment 2, we further explored

infants’ actions toward objects and pictures while controlling for tactile information. We presented 9-month-old infants with objects and pictures of objects under a glass cover in a false-bottom table. Although there were no significant differences between the proportion of rubs and pats infants directed toward the objects versus the photographs, infants exhibited significantly more grasping toward the objects than the photographs. Together, these findings show that 9-month-old infants largely direct appropriate actions toward realistic photographs and real objects, indicating that they perceive different affordances for pictures and objects. “
“This very study explores the relationship between tonal synchrony and maternal-infant social engagement based on free-play recordings of 15 mothers and their 3-month-old infants in a laboratory setting. Moment-by-moment analyses on a microlevel were used to study social engagement and vocal interaction. We analysed and categorized 854 vocalization periods (mother-only vocalizations, tonal interaction periods, nontonal interaction periods, and mutual silence). Tonal synchrony was analysed in terms of harmonic and pentatonic series based on pitch frequency analyses. Social engagement was microanalyzed in terms of matched and mismatched engagement states.

Our study is the first to evaluate the percentage of blood monocytes in CRPS patients. Although the percentage of total monocytes selleck products (CD14+ peripheral blood mononuclear cells) remained unchanged in CRPS, the percentage of the CD14+CD16+ monocyte subgroup was elevated significantly (P < 0·01) in individuals afflicted with CRPS compared to healthy controls. Previous studies have

determined that these cells represent a potent antigen-presenting and proinflammatory subpopulation of monocytes [28] that has been shown to be expanded in inflammatory conditions [34]. Although there was no correlation between the increased number of CD14+CD16+ monocytes in the CRPS group and the patients’ overall pain level, there was a correlation between increased numbers of CD14+CD16+ monocytes in CRPS patients demonstrating cold allodynia. This finding

suggests that the increased percentage of CD14+CD16+ monocytes may be associated with central sensitization. As reported previously, there was no difference in plasma levels of TNF-α, IL-10, IL-8, IL-6 and IL-1β between CRPS patients and controls [35,36]. However, individuals with high levels of CD14+CD16+ monocytes demonstrated a significantly https://www.selleckchem.com/products/bay-57-1293.html lower (P < 0·05) plasma level of IL-10 compared to individuals with low levels of CD14+CD16+. This is consistent with a study showing that CD14+CD16+ monocytes produce similar levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β and lower levels of the anti-inflammatory cytokine IL-10 [26]. This study also showed that the percentage of lymphocytes (T helper cells, T cytotoxic cells, NK cells or B cells) did not differ between CRPS patients and healthy control individuals. These results are in agreement with the study of Ribbers and colleagues that reported no association between lymphocyte subpopulations and patients with reflex sympathetic dystrophy (currently referred to as CRPS-type 1) [37]. A subsequent study by Kaufmann and colleagues also found no changes in the percentage of T cytotoxic cells, NK cells and B cells in CRPS patients [38]. However, they reported a reduction

of T helper cells (CD8+ lymphocytes) as well as an increase in the CD4/CD8 ratio [38] in CRPS patients compared to healthy controls. Although our study also Low-density-lipoprotein receptor kinase found a small reduction of CD8+ lymphocytes and an increase in the CD4/CD8 ratio, these changes were not statistically significant (P > 0·05). The elevation in the percentage of CD14+CD16+ monocytes seen in CRPS patients in this study could be due to the syndrome itself or may result from other factors. Factors such as physical inactivity, morbid obesity and sleep have been shown to alter the percentage of CD14+CD16+ monocytes [39–41]. Morbidly obese individuals have been reported to show elevated levels of the CD14+CD16+ monocyte subset [39]. The percentage of obese individuals (BMI > 30) in both the CRPS and control groups was approximately 20%.

[24] However, this population does not account for all the stromal cells lying in the double-negative gate, and suggests further stromal subset heterogeneity within lymphoid selleckchem tissue. Once SLOs are formed,

a major functional role of stromal cells is undoubtedly the maintenance of SLO structural integrity, and many subsets secrete large amounts of extracellular matrix (Table 1). The FRCs form collagen-rich reticular fibres, which they then surround to form conduits for afferent lymph.[25] These function by allowing for the transport of low-molecular-weight antigen and so facilitate antigen presentation by antigen-presenting cells in the T-cell zone.[26] Similar conduits have been found in the subcapsular sinus of the lymph node that are specialized for transport of antigen to the B-cell zone[27] and may be formed by marginal reticular cells that are present at this distinct location.[28] Stromal BMS-907351 concentration cells also play a vital role in lymphocyte trafficking by maintaining a functional separation of B-cell and T-cell zones via specific chemokine expression.

The FRCs in the T-cell zone express CCL19 and CCL21, which act to recruit CCR7+ naive T cells.[29] The importance of the stromal chemokine gradient induced is shown by aberrant SLO structure and T-cell distribution in the plt/plt mutant mouse,[30] which lacks CCL19 and CCL21 expression. In contrast, FDCs and marginal reticular cells express CXCL13,[31, 32] which acts on CXCR5 to Chlormezanone attract B cells to the B-cell zone of SLOs. As naive

T cells and B cells do not express CXCR5 and CCR7, respectively (except for T-follicular helper cells, which express enough CXCR5 to enter the B-cell zone[33]), the stromal chemokine gradients restrict lymphocytes to their respective zones during steady-state conditions. Moreover, stromal chemokine production can even play a role in the further differentiation of lymphocytes. Recently, a key role for stromal cells in the functional activation of T helper cells in the LN has been revealed, whereby stromal cell production of CXCL9 optimizes the polarization of CXCR3+ T cells toward an interferon-γ+ T helper type 1 phenotype in vivo.[34] Multiple stromal subsets also provide vital survival signals to peripheral lymphocytes, e.g. FRC and lymphatic endothelial cell-derived IL-7 for T cells[23, 35] and FDC-derived BAFF for B cells.[36] Stromal cells control the influx and retention of naive lymphocytes to SLOs via chemokines, yet they may also control the egress of lymphocytes via sphingosine-1-phosphate (S1P) signalling.[37] Levels of S1P are much lower in SLOs than in the circulation because of increased SLO expression of S1P-lyase.[38] Cyclic expression of the S1P receptor on lymphocytes competes with CCR7 or CXCR5 signalling to determine lymphocyte retention versus egress.

This study for Dasatinib in vivo recurrent hyponatremic episodes following the first admission with severe hyponatremia (<125 mEq/L) on thiazide aimed to investigate whether other coexistent factors than thiazide are responsible.

Methods: In the retrospective chart review over 5 yrs, out of 1,625 pts admitted with severe hyponatremia on hydrochlorothiazide (HCTZ), 24 pts (M : F, 7:17; age 71 ± 11 yrs, mean ± SD) were re-admitted for the recurrent hyponatremia (up to 4 times). Results: Among the 1st (n, 24), the 2nd (n, 24), the 3rd (n, 6), and the 4th admission (n, 2), serum sodium levels on admission were not significantly different (122 ± 3.8 vs 120 ± 6.7 vs 119 ± 5.4 vs 115 ± 19.1 mEq/L, p = ns). Successful managements of the 1st admission (n, 24) included discontinuing HCTZ with intravenous salt (n, 17), withdrawing HCTZ only (n, 1), and inadvertent continuation of HCTZ with intravenous salt (n, 6). As the causes of hyponatremia on

the 2nd admission (n, 24), 14 10 pts (42%) with no further exposure to HCTZ revealed volume depletion (n, 6), SIADH (n, 3), and adrenal insufficiency (n, 1), respectively. Four pts on the second admission died of malignancy, not from hyponatremia. Moreover, 4 pts on HCTZ in the 1st (17%) and the 2nd admission (17%), and also 2 pts on the 3rd admission (33%) were simultaneously taking neuropsychiatric medications and other diuretics with either furosemide or spironolactone. The latter 2 pts of the 3rd admission experienced the 4th admission of recurrent hyponatremia. Conclusion: In summary, hyponatremia Casein kinase 1 on thiazide Opaganib supplier does not mean necessarily thiazide alone as the sole cause of its frequent occurrence. Therefore, thiazide-associated hyponatremia warrants prudent exploration for other coexistent causes of hypontremia. SOHARA

EISEI, SUSA KOICHIRO, RAI TATEMITSU, ZENIYA MOKO, MORI YUTARO, SASAKI SEI, UCHIDA SHINICHI Department of Nephrology, Tokyo Medical and Dental University Introduction: Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding the WNK1 and WNK4 kinases were known to be responsible. Recently, two genes (KLHL3 and Cullin3) were newly identified as responsible for PHAII. KLHL was identified as substrate adaptors in the Cullin3-based ubiquitin E3 ligase. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and NCC were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is true substrate(s) of KLHL3-Cullin3 E3 ligase, in other words, what is the true pathogenesis of PHAII caused by KLHL3 mutation. Methods: To investigate the pathogenesis of PHAII by KLHL3 mutation, we generated and analyzed KLHL3R528H/+ knock-in mice.

Helicobacter pylori is able to transform to a coccoid form, which is able to access the intestinal lumen and is subsequently captured by DC in Peyer’s patches (PP) (Nagai et al., 2007). The CD4-positive T cells that are activated MLN2238 in vitro in PP subsequently migrate into the gastric mucosa, resulting in the development of gastritis (Kiriya et al., 2007; Nagai et al., 2007). In the inflammatory response, T helper (Th) 1 and Th2 lymphocyte-derived cytokines

control the clearance of intracellular and extracellular pathogens, respectively. According to this Th paradigm, the T cells in the H. pylori-infected gastric mucosa are predominantly Th1 cells, which secrete interferon (IFN)-γ (D’Elios et al., 1997; Bamford et al., 1998; Itoh et al., 1999), and H. pylori infection leads to the upregulation of IFN-γ production and the downregulation of interleukin (IL)-4 and IL-10 production in the gastric mucosa, resulting in the enhancement of the Th1 pathway and the subsequent development of chronic gastritis (Smythies et al., 2000). Th17 cells, which produce IL-17, modulate the Th1 response in gastric inflammation induced by H. pylori (Kabir, 2011). In addition, the role of regulatory T cells in H. pylori infection is now being investigated regarding escape from the host immune response (Kandulski

et al., 2010). Thus, the role of CD4-positive T cells has been widely studied in the context of adaptive immunity against H. pylori. On the contrary, immune responses against H. suis and the relationship between H. suis and gastric disease have been less Ferroptosis inhibitor understood. Recently, Nakamura et al. (2007) reported the animal model of gastric Meloxicam MALT lymphoma using H. suis, previously named ‘H. heilmannii’ type 1, obtained from a Cynomolgus monkey. In this model, the formation of MALT lymphoma-like lesions was observed in 100% of mice at 6 months after infection. In our previous study using the same animal model, the mRNA expression level

of IFN-γ was upregulated in the gastric mucosa of C57BL/6J mice at 3 months after H. suis inoculation, suggesting the occurrence of a local Th1 response (Nobutani et al., 2010). However, regarding H. suis infection, no detailed analysis of cytokine profiles or experiments using cytokine-deficient mice have been performed. In the present study, we aimed to assess the role of helper T cells in the development of gastric lymphoid follicles in H. suis-infected animals. C57BL/6J wild type (WT), IFN-γ−/−, and IL-4−/− mice were infected with H. suis, and then histological and immunohistological examinations were carried out. In addition, the expression levels of Th cytokines in the gastric mucosa of C57BL/6 WT mice were examined. All animal experiments were performed according to the ‘Guidelines for Animal Experimentation at Kobe University (Permission No. P-090707)’. C57BL/6J WT mice were purchased from CLEA Japan Inc. (Shizuoka, Japan). IFN-γ−/− mice (Tagawa et al.

Immunohistochemical investigation demonstrated an increased cytokine production, including interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-6, and tumour necrosis factor (TNF)-α in senile plaques in the hippocampus and cortex of Alzheimer’s brain [3]. Microglia and astrocytes can produce cytotoxic molecules and these pro-inflammatory cytokines [5]. The presence of peripheral monocytes/macrophages within the central nervous system (CNS) can reduce the extension of β-amyloid plaques

Selleckchem GSK1120212 via multiple mechanisms regulated by immune system [5]. Although the attempts for clarifying the environmental aetiology of AD have been hopeless, however, many researchers have demonstrated an increased risk among those people Selinexor supplier with a family history of AD [6]. Diversity of risk factors for sporadic AD has shown that it is a multifactorial

disease [2]. Natural killer (NK) cells are granular lymphocytes and play an important role in the immune system [7]. Involvement of NK cells in some neurodegenerative diseases such as multiple sclerosis (MS) has been well studied [8]. A decreased NK cell activity has been reported in AD patients [9], which may suggest that NK cells may also contribute in AD immunopathogenesis. However, the role of NK cells in AD patients is not well studied and requires to more investigation. In this paper, we tried to review the data resulting from different studies regarding the role of NK cells in AD. Natural killer (NK) cells were defined by their ability to spontaneously kill tumour cells and virally infected cells [10, 11]. These cells are derived from hematopoietic stem cells in the bone marrow (BM). Moreover, the development of NK cells in other organs such as liver and thymus have also been reported [12]. Peripheral activation of NK cells may lead to phenotype modification and modulation of NK cell functions [13]. In humans, NK cells have been phenotypically defined as CD3−CD56+ lymphocytes that may be further subdivided into CD56dimCD16bright Protein kinase N1 (90% of all NK) and CD56brightCD16− cells. These subpopulations differ based on cytotoxic capacity

and cytokine production [14]. NK cells main functions are destroying a wide variety of target cells or production of cytokines [15] (Fig. 1). NK cells destroy the target cells by perforin and granzymes, which are stored in cytoplasmic granules and released upon activation [16]. NK cells also express TNF-related apoptosis-inducing ligand (TRAIL) and FasL, which are important mediators of apoptosis. Notably, cytokine production by NK cells can be regulated through both activating and inhibitory receptors. Hence, NK cells may have both immunostimulatory and immunomodulatory effects through production of cytokines such as interferon (IFN)-γ, TNF-α, granulocyte monocyte colony-stimulating factor (GM-CSF), IL-5, IL-13, IL-10 and transforming growth factor (TGF)-β.

All samples included junctional and sulcular epitheliums and connective gingival tissue. The gingival biopsies were divided into two portions. One portion was immediately placed in microcentrifuge tubes containing 250 μl phosphate-buffered saline and protease inhibitor cocktail (Sigma-Aldrich), and homogenized (Kinematica Polytron PT3100, Littau-Luzern, Switzerland), and then centrifuged at 13,000 g for 5 min at 4 °C. The resulting supernatants, devoid of debris, were stored at −70 °C until subjected to cytokine measurements by ELISA. The additional portion was stored in a tube containing RNA later (Ambion Inc., Austin, TX, USA) and stored at −20 °C for subsequent assays. Enzyme linked

immunosorbent assay (ELISA).  Target Selective Inhibitor Library nmr Total levels of IgA were determined by ELISA using microtiter plates (Costar click here 3590, Corning, NY, USA)

coated for 24 h at 4 °C with 2 μg/ml of goat IgG anti-human IgA (Southern Biotech, Birmingham, AL, USA) in carbonate-bicarbonate buffer, pH 9.6. After being coated, plates were washed and blocked for 1 h at room temperature with bovine serum albumin (0.1%) in phosphate-buffered saline (PBS), pH 7.5. Diluted saliva samples (1:200 in PBS) were applied in triplicate, and plates were incubated for 2 h at room temperature. All experiments included serial dilutions (1.0, 0.5, 0.25, and 0.125 μg/ml) of a standard sample of human IgA antibody purified from serum (Southern Biotech). The secondary antibody was biotin-conjugated goat IgG anti-human IgA (Southern Biotech) at a dilution of 1:14,500. After incubation with a solution of streptavidin, conjugated with alkaline phosphatase (Southern Biotech) (1:500 in PBS, pH 7.5), antibody reactions were revealed by incubation with the substrate p-nitrophenyl phosphate disodium. In order to obtain the A405 units, plates were read in an ELISA plate reader (Epoch, Biotek, Winooski, VT, USA). Negative controls included the uncoated wells without saliva and primary antibody. For determination of IgA concentrations, absorbance values were plotted

Carnitine palmitoyltransferase II against the standard curve obtained for the serial dilutions of the purified human IgA within a linear range. IgA levels were expressed as pg/ml of saliva. The gingival biopsies were analyzed by ELISA for IL-4 and IL-10 using commercially available ELISA kits (Quantikine; R&D Systems Inc., MN, USA). Assays were carried out according to the manufacturer’s recommendations using human recombinant standards. The optical density was measured at 450 nm according to recommendation. Results are reported as total amount (pg/mg) of each cytokine. Sites with cytokine levels below the detection limit of assay were scored as 0 pg. RNA extraction.  The gingival biopsies stored in RNA later (Ambion) were evaluated for mRNA levels of IL-4, IL-10, IL-21, IL-21R, CD40L and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).