However, it remains to be clarified whether DCs may participate i

However, it remains to be clarified whether DCs may participate in the pathogenesis of other autoimmune diseases. Previously we have demonstrated that, in primary SS, blood immature myeloid DCs are decreased and mature myeloid DCs are accumulated in salivary glands, suggesting the recruitment of myeloid DCs from blood to inflamed salivary glands. In addition, we demonstrated that numerous IFN-γ-producing CD4+ T cells are also infiltrated into the salivary glands from primary SS patients [2]. Based upon these findings, we proposed a hypothesis that myeloid DCs play a role in pathogenesis of primary SS by initiating Th1 immune response. In this study, we report

that the decrease Pexidartinib solubility dmso of blood myeloid DCs and accumulation of salivary gland-infiltrating DCs is universal in the early phase of not only primary SS but also secondary SS, and this alteration was restored spontaneously during the natural clinical course. As shown in Table 1, patients enrolled into this study comprised 24 patients with secondary SS (two men and 22 women, mean age 55·5 years), 29 with primary SS (two men and 27 women, mean age 58·6 years), 11 with SLE (two men and nine women, mean age 25·3 years),

14 with SSc (one man and 13 women, mean age 54·9 years) and 12 with RA (three men and nine women, mean age 55·9 years). In addition, 32 healthy volunteers (12 men and 20 women, mean age 48·0 years) were also enrolled into this study as normal controls. All patients presented to our hospital between May 1999 and June 2003 and were diagnosed freshly as having autoimmune diseases. No patients or volunteers had evidence of infections at the time of this study. All patients underwent routine laboratory examinations and

were also examined for a variety of autoantibodies. Informed consent was obtained for this study in accordance see more with the provisions of the Declaration of Helsinki. All SS patients met the criteria of the Research Committee on SS of the Ministry of Health and Welfare of Japan [12], as well as the European Community criteria [13]. Patients with SLE or SLE-merged secondary SS fulfilled the diagnostic criteria for SLE of the American College of Rheumatology (ACR) [14,15]. Patients with RA or RA-merged secondary SS fulfilled the diagnostic criteria for RA of the ACR [16]. Patients with SSc or SSc-merged secondary SS fulfilled the diagnostic criteria for SSc of the ACR [17]. We determined the onset of SS by a patient complaint about Sicca syndrome in a medical interview (Table 1). In order to assess whether the number of peripheral blood DCs (PBDCs) changes during the natural course of primary SS, six primary SS patients with long-term follow-up were examined sequentially. All the six primary SS patients’ PBDCs were examined in the chronic phase of the disease, 24 months or after the onset of Sicca syndrome [all women, mean age 56·5 years (range 51–71 years)].

Taken together, these results indicate that induction of CD8+ T-c

Taken together, these results indicate that induction of CD8+ T-cell responses at mucosal sites upon i.m. immunization is independent of a given vaccine platform. Antigen-experienced CD8+ T cells may traffic to the GT with

the help of specific sensors that remain to be identified, or alternatively this process may be random. To gain further insight into the vaccine-induced CD8+ T cells that homed to the GT, we conducted a detailed phenotypical analysis of Gag-specific CD8+ T cells induced by the different immunization protocols, comparing cells isolated from spleen, blood, ILN and the GT at different times after immunization. In some assays, we also tested cells isolated from NALT; Wnt inhibitor the latter were tested for comparison as a population of cells homing to a distinct mucosal site. Phenotypes of Gag-specific

CD8+ T cells isolated from systemic sites and the GT were phenotypically distinct, and this was especially pronounced at 1 year after the i.m./i.m. prime-boost vaccine Akt inhibitor regimen. The phenotypes suggest that most tet+CD8+ T cells present in the GT remain fully activated and would be expected to start target cell lysis immediately upon encounter of infected cells. We evaluated markers that are known to be upregulated on cells derived from the intestinal mucosa. Studies have demonstrated high levels of CD69 expression on intestinal CD8+ cells 22, 30, but expression of CD69 was not increased in the GT at any of the time points analyzed. Although α4β7 has been linked to the genital migration of subsets Obatoclax Mesylate (GX15-070) of CD4+ cells 31, and is a well-known marker for homing of T cells to the intestinal mucosa, our results do not suggest that α4β7 affects homing of CD8+ T cells to the GT. CD103 was slightly increased in tet+CD8+ T cells from the GT at early time points, and by 1 year after immunization became strongly upregulated. In the adoptive transfer experiment, CD103 was low on the Gag-specific CD8+ T cells isolated from the vaccinated donors and upon transfer

remained low on cells isolated from all compartments but the GT, where an increase was observed. Again, these data argue against the notion that CD103 supports mucosal homing but rather suggest that CD103 may contribute to the retention of CD8+ T cells within the GT. The adoptive transfer experiment also showed that Gag-specific CD8+ T cells from the spleen could readily migrate to the GT to a similar extend as observed in vaccinated mice. This argues against the need for a distinct differentiation pathway during activation to allow for migration of CD8+ T cells to the mucosa, as had been described for T-cell homing to GALT 32 or for CD4+ T cells of the female GT 33. On the other hand, the observation that at 2 wk upon i.m. immunization frequencies of Gag-specific CD8+ T cells were ∼10-fold higher in blood but only ∼2-fold higher in the GT than upon i.n.

Among them apolipoprotein B-100, complement component 3, etc decr

Among them apolipoprotein B-100, complement component 3, etc decreased in the last, indicating the association with nephrotic condition. On the other hand, complement component 9, apoprotein E increased probably suggesting of the association with clinical remission. Of interest is that apolipoprotein Selleckchem Y 27632 E and serum amyloid P were high in both the first and last sessions. Moreover, serum apolipoprotein

E was also high in a non-responder group. Conclusion: The present proteomic analysis revealed that the increase in serum apolipoprotein E may predict the responsiveness of LDL-A in steroid-resistant nephrotic syndrome. Further study may clarify the more detailed mechanism of the LDL-A in an intractable setting of steroid-restant nephrotic syndrome. JEONG KYUNGHWAN1,2, ASANUMA KATSUHIKO2,3, LYDIA AIDA4, TAKAKI MIYUKI2, ASAO RIN2, KODAMA PLX4032 research buy FUMIKO2, ASANUMA ETSUKO2, TOMINO YASUHIKO2 1Division of Nephrology, Department of Internal Medicine, Kyung Hee University, Seoul, Korea; 2Division of Nephrology, Department of Internal Medicine,Juntendo University, Faculty of Medicine, Tokyo, Japan; 3Laboratory for Kidney Research, Medical Innovation Center, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 4Division of Nephrology and Hypertension,

Department of Internal Medicine, Cipto Mangun Kusumo Hospital, University of Indonesia, Jakarta, Indonesia Introduction: Blockade of the renin-angiotensin system plays a key role in suppressing the progression of renal diseases. It has been well unknown whether this therapy provides additional effects when combined with vitamin D or its analog in an adriamycin (ADR)-induced nephropathy model. Methods: Here we evaluated the effect of applying the combination of an AT1 receptor blocker, telmisaltan, and a vitamin D analog, oxacalcitriol, in ADR-induced nephropathy mice and immortalized murine podocytes. Podocyte injury was assessed by podocyte apoptosis using the TUNEL assay, podocyte counting, and podocyte-specific expressed protein by immunofluorescence and

western blot analysis. Results: Mice with ADR-induced nephropathy (9.5 mg/kg single intravenous injection) developed progressive albuminuria and glomerulosclerosis within 30 days, accompanied by decreased expression of slit diaphragm-associated proteins (nephrin and podocin), reduced numbers of ID-8 podocytes, and increased systolic blood pressure. Treatment with telmisartan (0.1 mg/kg ip injection, everyday) or oxacalcitriol (0.05 μg/Kg ip injection, three times per week) alone moderately ameliorated the kidney injury; the combined treatment most effectively reduced the albuminuria and glomerulosclerosis. These effects were accompanied by restoration of slit diaphragm-associated proteins (nephrin and podocin) and podocyte apoptosis and podocyte loss in the glomeruli. Cultured podocytes were exposed to 0.25 μg/ml of ADR with telmisartan (10−7 M) or oxacalcitriol (10−8 M) and combination.

These people can be identified by all members of the multi-discip

These people can be identified by all members of the multi-disciplinary team and this identification leads to increased input, e.g. social work, ACPs, greater focus on symptoms. This approach could be considered for institution in Australia and New Zealand as a way of focussing attention on this group, collecting data for a better estimate of the numbers and aiding support and input into these patients’ care as they approach EOL. 3. Conflict Resolution Conflict resolution is a difficult area to deal with and has been a reason

selleck chemicals llc for some patients being initiated on dialysis when it may not have been the most appropriate management choice. NSW Department of Health[9] published a report in 2010 – Conflict Resolution in End of Life Settings (CRELS). This report includes discussion of the problems encountered when clinicians from

other specialities prognosticate on a condition, misconceptions about a ‘Not for Resuscitation’ order and ongoing management, unrealistic expectations of modern medicine as well as ethical and legal issues in EOL decisions. It also includes Ivacaftor nmr a flow chart aimed at resolving EOL conflicts in a patient who has lost decision-making capability as well as guidelines for formulation of and End of Life Care plan. This helpful review can assist in formulating local guidelines which need to take account different legal positions in different countries, states and territories (see section 19). We stress the importance of ‘second’ and other medical and ethical opinions in difficult cases when conflict arises. Many guidelines exist around

the world around RSC but most are Meloxicam based on low level evidence. Analgesic use is probably the best referenced and available but many other areas need ongoing research before guidelines supported by higher level evidence can be formulated. KDIGO No recommendations KDIGO has recently begun work to look at the formulation of guidelines in this area. 1.3 ‘Timing of therapy: When patients reach stage 5 CKD (estimated GFR < 15 mL/min per 1.73 m2), nephrologists should evaluate the benefits, risks, and disadvantages of beginning kidney replacement therapy. Particular clinical considerations and certain characteristic complications of kidney failure may prompt initiation of therapy before stage 5.’ (B) European Best Practice Guidelines Guideline D. ‘Conservative management should be aimed at slowing the progression of renal failure, decreasing proteinuria, strict control of blood pressure, prevention of over-hydration, and treatment of anaemia, renal bone disease and metabolic acidosis.

gondii tachyzoite polyclonal antibody (37°C for 2 h) in a humidif

gondii tachyzoite polyclonal antibody (37°C for 2 h) in a humidified box. After washing the plates three times with PBST, fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG (1 : 2000; Boster, Wuhan, China) was applied to all the cells and incubated at 37°C selleck for 1 h. After washing the cells three more times with PBST, the fluorescence was observed under a fluorescence microscope (Olympus BX-51, Tokyo, Japan). Six- to eight-week-old female BALB/c mice were randomly divided

into three groups (13 mice per group) and immunized by intramuscular injection. pVAX1-TgCyP (100 μg/each in PBS) was used to immunize the mice for the experimental group (a 0·05 mL syringe and a 20G needle were used for the injection); the empty pVAX1 vector (100 μg/each in PBS) and PBS (100 μL/each) were used as negative controls. All groups were vaccinated in the same manner on days 0, 14 and 28. Blood was collected from each group via the venous plexus of the tail before each immunization and stored at −20°C mTOR inhibitor for enzyme-linked immunosorbent assay (ELISA) analysis. An indirect ELISA test was applied to evaluate specific antibodies according

to the procedure described previously [12]. The 96-well microtiter plates were coated overnight at 4°C with crude T. gondii tachyzoite antigens (10 mg/mL). On the second day, the plates were blocked with 5% bovine serum albumin (BSA) in PBS at room temperature for 2 h. Then, the plates were washed three times with PBST, and incubated with mouse sera (1 : 3200 in 1% BSA-PBS) at 37°C for 1·5 h. After washing three times with PBST, the plates were incubated with an HRP-labelled goat anti-mouse IgG antibody (1 : 2000; Boster) at 37°C for 1 h. After washing three times with PBST, a substrate solution containing 15 μL H2O2, 10 ml citrate-phosphae

and 4 mg O-phenylenediamine (OPD) was applied (100 μL/well). The reaction was stopped with 2 m H2SO4, and the optical density values were read at A490. Spleens were removed from five mice per group 14 days after the final vaccination. A splenocyte BCKDHB suspension was obtained by the gentle squeezing of whole spleens in Hank’s balanced salt solution (HBSS, Sigma, St. Louis, MO, USA) and filtration through nylon mesh. The erythrocytes in the spleen cell suspension were removed by lysis and centrifugation. The pellet was washed three times with PBS and resuspended with complete RPMI-1640 medium supplemented with 10% FCS. The cells were cultured in 96-well Costar plates at a density of 103 cells/well. The splenocytes were stimulated with TLA (10 μg/mL), concanavalin A (Con A; 5 μg/mL; Sigma; positive control) or medium alone (negative control). After incubation with Alamar blue (10 μL/well) for 12 h, the plates were read at 570 nm with an ELISA reader. Lymphocyte proliferative responses were represented by a stimulation index (SI), which is the OD570 ration between stimulated cells and nonstimulated cells.

Jean-Luc Cracowski is MD, PhD, professor of Clinical Pharmacology

Jean-Luc Cracowski is MD, PhD, professor of Clinical Pharmacology at Grenoble University, France. He

is in charge of the Clinical Pharmacology Unit at the INSERM Clinical Research Center in Grenoble, France. His main area of research is the pharmacology and physiology of human skin Napabucasin concentration microcirculation. This includes the development of methods to assess skin microvascular function, their use in physiological and pathological conditions such as scleroderma and primary Raynaud’s phenomenon, and the development of new pharmacological approaches. He is coauthor of 139 publications indexed in Medline. “
“Microcirculation (2010) 17, 32–38. doi: 10.1111/j.1549-8719.2009.00004.x Objective:  Fenestrations are pores in the

liver sinusoidal endothelium that facilitate the transfer of particulate substrates between the sinusoidal lumen and hepatocytes. Fenestrations express caveolin-1 and have structural similarities to caveolae, therefore might be a form of caveolae and caveolin-1 may be integral to fenestration structure and function. Therefore, fenestrations were studied in the livers of caveolin-1 knockout mice. Methods:  Scanning, transmission and immunogold electron microscopic techniques were used to study the liver sinusoidal endothelium and other tissues in caveolin-1 knockout and wild-type mice. Results:  Comparison of fenestrations in wild-type and knockout mice did not reveal any differences on either scanning or transmission electron microscopy. The diameter Ribonucleotide reductase of the fenestrations was not significantly different (74 ± 13 nm knockout mice STI571 vs 78 ± 12 nm wild-type mice) nor was the fenestration porosity (6.5 ± 2.1 knockout vs 7.3 ± 2.4% wild-type mice). In contrast, adipocytes and blood vessels in other tissues lacked caveolae in the knockout mice. Caveolin-1 immunogold of livers of wild-type mice indicated sparse expression in sinusoidal endothelial cells. Conclusions:  The normal structure of fenestrations in the liver sinusoidal endothelium is not dependent upon

caveolin-1 and fenestrations are not a form of caveolae. “
“Please cite this paper as: Emmett, Lanati, Dunn, Stone and Bates (2011). CCR7 Mediates Directed Growth of Melanomas Towards Lymphatics. Microcirculation 18(3), 172–182. Objective:  To determine whether chemotactic-metastasis, the preferential growth of melanomas towards areas of high lymphatic density, is CCL21/CCR7 dependent in vivo. Lymphatic endothelial cells (LECs) produce the chemokine CCL21. Metastatic melanoma cells express CCR7, its receptor, and exhibit chemotactic-metastasis, whereby metastatic cells recognise and grow towards areas of higher lymphatic density. Methods:  We used two in vivo models of directional growth towards depots of LECs of melanoma cells over-expressing CCR7.

From each animal, three flat sheets of unstripped ileum free of P

From each animal, three flat sheets of unstripped ileum free of Peyer’s patches were placed in

Teflon holders and mounted in Ussing chambers within 5 min after being cut off from blood supply. Both sides of the sample (exposed area 0·2 cm2) were in contact with 1·6 mL Krebs–Ringer solution, stirred and gassed with humidified 95% O2 + 5% CO2 at 37°C. The transepithelial potential difference Vte click here (mV) was continuously monitored with Calomel electrodes connected to the chambers with Krebs–Ringer-agar bridges. Transepithelial electrical resistance R (Ω/cm2) was calculated from the voltage deflections induced by bipolar current pulses of 10 μA (every 30 s) applied through platinum wires. The potential and resistance data were stored on a PC using custom software (Natural Simstrument, Amsterdam, the Netherlands). During off-line data analysis, corrections were made for resistance of the solution and for potential differences between Calomel electrodes, measured both just before and immediately

after each experiment. The equivalent short-circuit selleckchem current Isc (μA/cm2) was calculated from the continuously monitored values of R and Vte. Reported values for the parameters Vte, R and Isc were obtained at the end of a 15- to 20-min equilibration period. Generally, these values were stable during the subsequent 1- or 2-h experiment. At the end of the experiment, the secretory capacity of the tissue segments was tested by measuring their response (Vte and Isc) to application of the secretagogue carbachol in the serosal compartment (10−4 M). In the Ussing chamber experiments, the measured transepithelial potential

(Vte) and equivalent short-circuit current (Isc) are indicative of the basal epithelial secretion, while the increase in these parameters (dVte and dIsc) in response to the secretagogue carbachol reflects the maximal secretory capacity. Paracellular mucosal-to-serosal permeability was determined using NaFl Thalidomide as a model molecule (25). After the equilibration period, NaFl was added to the mucosal compartment (0·01 g/L) and 200-μL serosal samples were taken every 7·5 min and replaced by Krebs–Ringer. The concentration of NaFl was determined using a fluorimeter (Polarstar Galaxy fluorescence multi-well plate reader; BMG LabTech GmbH, Jena, Germany), with 485 nm and 530 nm as excitation and emission wavelengths respectively. Steady-state NaFl-flux was quantified and expressed as ng/cm2/h. For each animal, average values of electrophysiological parameters and NaFl-flux were calculated from simultaneous measurements of three ileal samples. Statistical analyses were performed using SPSS v.12·0 software (SPSS Inc., Chicago, IL, USA).

Their molecular weights were confirmed by electrospray ionization

Their molecular weights were confirmed by electrospray ionization-mass spectrometry (ESI-MS). The IAb-restricted HBV core antigen-derived T helper epitope (sequence 128–140: TPPAYRPPNAPIL) was used in the in vivo assay. Peptides were dissolved in DMSO at a concentration of 100 mm and stored at −20 °C.

Blood samples and cell line.  Peripheral blood samples were obtained from six HLA-A*02+ healthy donors. The sample collection was approved by the ethics committee of Zhengzhou University. The human TAP-deficient T2 cell line (HLA-A*0201-positive) was a generous gift from professor Yu-zhang Wu (Third Military Medical University, China). The human oesophageal carcinoma cell line EC-9706 (HLA-A2-positive, COX-2-positive [15]) was maintained in our laboratory, human oesophageal carcinoma cell line KYSE-140 (HLA-A2-positive, COX-2-negative) was a gift from professor Qiao-zhen Kang (Zhengzhou University, China), human colon cancer cell line HT-29 (HLA-A2-negative, COX-2-positive) was purchased from American Type Culture Collection (ATCC, check details Rockville, MD, USA). T2 cells and cancer cells were cultured in RPMI 1640 medium (Invitrogen, Grand island, NY, USA) supplemented with 100 units/ml penicillin, 100 units/ml streptomycin, 2 mm L-glutamine, and 10% foetal bovine serum (FBS, Hyclone).

All cells mentioned above were kept at 37 °C in a humidified HSP90 atmosphere containing 5% CO2. Mice.  HLA-A2.1/Kb transgenic mice, 8–12 weeks old, which express a chimeric heavy chain of the MHC-I molecule (α1 and α2 fragments of human HLA-A*0201, and transmembrane and intracytoplasmic domains of mouse H-2Kb), were kindly provided by professor Xue-tao Cao (Second Military Medical University, China). Mice were bred and maintained in a specific pathogen-free (SPF) facility. Peptide-binding assay.  To determine whether the synthetic peptides could bind to HLA-A*0201 molecule, peptide-induced

HLA-A*0201 upregulation on T2 cells was examined according to a protocol described previously [21, 22]. Briefly, T2 cells (1 × 106 cells/ml) were incubated with various concentrations of the candidate peptides and 3 μg/ml human β2-microglobulin (β2-M, Merck, Germany) in serum-free RPMI 1640 medium for 18 h at 37 °C in a 5% CO2 atmosphere. Then, cells were washed twice and incubated with the anti-HLA-A2 mAb, BB7.2 (Santa Cruz, USA), followed by treatment with FITC-labelled goat IgG anti-mouse immunoglobulin (Bioss, China). Cells were harvested and analysed by flow cytometry (FACSCalibur, Becton Dickinson, USA). The fluorescence index (FI) was calculated as follows: FI = [(mean fluorescence intensity (MFI) of the peptide – background) − (MFI of the PBS control group – background)]/[MFI of the PBS control group − background], the MFI value of the cells which were not incubated with peptides or antibodies was used as background.

The number of β cells, determined from β cell mass [17–20], is an

The number of β cells, determined from β cell mass [17–20], is an outcome of developmental turnover and the level of autoimmune destruction [13,16,19,21]. β cell insulin production is regulated by the levels of glucose and inflammatory mediators [22,23]. Autoantigens.  Autoantigens are modelled generically to represent several antigens identified in the literature, including insulin and glutamic acid decarboxylase [24,25]. The autoantigen level is a function of β cell mass, β cell apoptosis and insulin secretion. Autoantigens are acquired and presented on major histocompatibility complex (MHC) class I and II molecules by dendritic cells (DCs), macrophages and B lymphocytes [26–28].

β cells also present autoantigens on MHC class I molecules [29]. Dendritic cells.  DCs are present in each modelled islet, even in the absence of inflammation, and recruitment of DC precursors is amplified by inflammation [30,31]. Both Sunitinib mouse inflammatory and suppressive (tolerogenic) DC phenotypes are represented [32,33]. Each subset influences the developing adaptive immune response, and each has limited phagocytic capabilities [34]. DCs acquire

and present antigens, produce mediators, interact with other cell types and traffic from the islets to the PLN Palbociclib order [26,35–37]. Macrophages.  Macrophages are also present in the islets even in the absence of inflammation, and recruitment of macrophage precursors is amplified by inflammation [38,39]. Macrophages perform phagocytic functions, acquire and present antigens, produce mediators, interact with other cell types and traffic to the PLN [27,37,40,41]. CD4+ T lymphocytes.  Two groups of naive CD4+ T lymphocytes are represented: those specific for islet autoantigens and those specific for other antigens. This same distinction is made for all other T lymphocyte and B lymphocyte populations. In the model, thymic output of naive T MRIP cells is a specified time-dependent

profile representative of what has been observed experimentally [42–44], taking into account the relative proportion of CD4+ and CD8+ T cells [45], but is not regulated dynamically. While the intricate and highly regulated process of thymocyte development has been studied extensively, it was not included in the current model scope based on an initial focus on peripheral mechanisms of autoimmunity and tolerance. The validation protocols used to refine and test virtual mouse behaviours were dependent primarily on peripheral mechanisms. However, the model was designed to accommodate expansion of the represented biology, which could include thymocyte development. During simulations, naive islet-autoantigen-specific (or diabetes-specific) T lymphocytes in the PLN become activated in response to autoantigen presented on MHC class II molecules and differentiate into T helper type 1 (Th1), Th2 or regulatory T cell (adaptive regulatory T cell or aTreg) subsets [46–49].

Louis, MO, USA) in a volume of 100 μL RPMI 1640 (Nissui) without

Louis, MO, USA) in a volume of 100 μL RPMI 1640 (Nissui) without antibiotics for 5 hr. Amounts of CRAMP in the culture supernatant were determined by ELISA as described above. Results expressed as means and SD were compared using one-way analysis of variance. The differences between learn more each group were compared by multiple comparisons (Bonferroni t test). Differences were considered significant at P < 0.05. Cathelin-related antimicrobial peptide was examined for its antimicrobial activity against M. pneumoniae. As shown in Figure 1, CRAMP exerted antimicrobial

activity against M. pneumoniae M129 and FH strains in a dose dependent manner in the range of 10 to 20 μg/mL. At a concentration of 20 μg/mL the number of mycoplasmal colonies was reduced by 100 to 1000-fold as compared with the control. These results show that CRAMP possesses antimicrobial activity against M. pneumoniae. To determine whether M. pneumoniae infection induces CRAMP production, CRAMP concentrations in BALF of M. pneumoniae-infected mice were determined using a sandwich ELISA. As shown in Figure 2, CRAMP concentrations in BALF of M. pneumoniae-infected mice were 20–25 ng/mL, whereas the corresponding concentrations Selleck Ku0059436 for control uninfected mice were 0.7–1.1 ng/mL. To further confirm the presence of CRAMP in the supernatant of

the BALF, Western blotting was performed using a rabbit anti-CRAMP Ab. As shown in Figure 3, the 3.8 kDa band of the mature form of CRAMP and a 18 kDa band corresponding to the CRAMP immature form were detected. Synthetic CRAMP peptide was

Idoxuridine detected at 3.8 kDa in accordance with its molecular weight. The results showed that M. pneumoniae infection induces CRAMP in the BALF of M. pneumoniae-infected mice. It is, however, still unknown which cells are responsible for CRAMP production. Approximately 90% of the cells in the BALF were neutrophils, the rest being monocytic cells. CRAMP expression of the neutrophils in the BALF was also examined. As shown in Figure 4, expression of CRAMP was evident fairly widespread throughout the neutrophils, particularly in the area of the nuclear membranes. The neutrophils were confirmed to have polynuclear morphology by Hoechst 33342 staining. In contrast, CRAMP was not detected within neutrophils by normal serum. These results indicate that neutrophils are a primary source of CRAMP in M. pneumoniae-infected BALF. In the next experiments, we examined whether M. pneumoniae can induce the release of CRAMP from neutrophils. Neutrophils induced by thioglycolate were used in this experiment. Cells that had already been activated by thioglycolate released small amounts of CRAMP, approximately 1.7 ng/mL. Addition of M. pneumoniae induced CRAMP of approximately 20 ng/mL in the supernatant after 5 hr (Fig. 5). The viability of neutrophils after 5 hr incubation was approximately 95% as judged by the trypan blue exclusion test.