Because of this close association between chemotherapy and cell-mediated immunity, treatment for L. donovani infection has been thought to be more amenable to combined therapy, that is, immunochemotherapy [16]. Therefore, we tested immunochemotherapy to determine the safety, immunogenicity and probable curative potential of 78 kDa antigen in combination with a newly tested drug cisplatin in mice infected with L. donovani. The current Opaganib cell line study is expected to assist in the evaluation of immunochemotherapy as a better alternative antileishmanial therapy. Promastigotes of L. donovani, strain MHOM/IN/80/Dd8, were grown at 22°C in NNN medium

supplemented with MEM (pH 7·2), 200U of streptomycin, 200U of benzyl penicillin and 40 μg of gentamycin per mL and subcultured in

the same medium after every 48–72 h. Inbred BALB/c mice of either sex weighing 20–25 g were used for the present study. During the start of the experiment, the mice weigh around 20–25 g, but by the time, infection was given and treatment was completed weight increased to 25–30 g. These animals were obtained from Institute of Microbial Technology, Chandigarh, India, and then maintained in the Central Animal House, Panjab University, Chandigarh. All the mice were kept in appropriate cages and fed with water and food ad libitum throughout the study period. The ethical clearance for conducting various experiments on BALB/c mice was taken from Institutional Animal Ethics Committee (IAEC) of the Panjab PI3K inhibitor University, Chandigarh. Cis-diamminedichloroplatinum (II) dichloride (CP) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) in the pure form, and then it was dissolved in distilled water to get the

requisite concentration of 0·5 mg/kg body wt [14]. The 78 kDa antigen of L. donovani was identified and eluted as described by Nagill and Kaur [6]. The 78 kDa antigen alone (without any adjuvant) was also used as a vaccine candidate for immunization. 78 kDa + MPL-A vaccine was prepared by the addition Quisqualic acid of 144 μL solution of MPL-A (conc. 10 mg/mL) to 360 μg of 78 kDa antigen. Subcutaneous route was used for immunization of mice in all the groups [6]. Mice were infected intracardially with 107 promastigotes/0·1 mL [14]. Animals were divided into different groups, and each group consisted of eighteen mice. Animals of Group 1 (Chemotherapy) received intraperitoneal injection of cisplatin at a dose of 0·5 mg/kg body wt. continuously for 5 days in two cycles with an interval of 14 days between each cycle, while Group 2 (cisplatin + 78 kDa) and Group 3 (cisplatin + 78 kDa + MPL-A) received immunochemotherapy, respectively.

These results suggest that the EBNA1-derived HPV epitope may be a relevant target of EBV-specific CTL responses. To investigate the presentation of the HPV CTL epitope in EBV-positive cells, HLA-B35 or HLA-B53 positive LCLs and BL cells were used as targets of HPV-specific CTL learn more cultures obtained from donors 5 and 7. We found, in the 5-hr 51Cr-release assay that unmanipulated HLA-B35- and HLA-B53-matched LCLs were lysed by HPV-specific CTL cultures whereas BL cells were not recognized, suggesting that the HPV epitope is poorly presented at the surface of BL cells (Fig. 2a,b). To exclude poor sensitivity

to lysis of BL lines, we evaluated the killing of BLs loaded with the synthetic HPV epitope by cytotoxic assay. We found that HPV-pulsed BL cells were recognized by HPV-specific CTLs, indicating that BL cells are sensitive to lysis and able to present the HPV T-cell epitope when exogenously added (Fig. 2b). The IFN-γ production assays have been mainly used in studies documenting the presentation of EBNA1-derived MHC-I-presented CTL epitopes because it is considered a more sensitive indicator

of target cell recognition.10–12 Therefore, we tested whether recognition of EBNA1-expressing BL cells could be revealed by monitoring IFN-γ release in ELISPOT assays. To this end, HPV-specific CTLs and matched LCLs and BL cells were seeded at an effector : target STA-9090 ic50 ratio of 10 : 1, and the number of HPV-specific IFN-γ-producing cells was evaluated after 24 hr. As shown in Fig. 2(c), eltoprazine release of IFN-γ was specifically induced by HLA-B35-matched LCLs while HLA-B35-matched and HLA-B53-matched BL cells did not stimulate IFN-γ release, thereby confirming the poor presentation of this epitope in BL cell lines. As a whole, these results demonstrate that the EBNA1-derived HPV epitope is generated and presented in LCLs but not in BL cells. This suggests

that HPV generation does not exclusively depend on the presence of the GAr domain. Loss or down-regulation of HLA class I is one of the routes of immune escape in a variety of human tumours, including BL cell lines.25–28 Therefore, the surface expression of class I molecules in BL cells and LCLs was tested by indirect immunofluorescence. As shown in Fig. 3 and supplementary material, see Table S1, Jijoye cells expressed lower amounts of class I molecules whereas BJAB B95.8 cell lines showed similar levels of total HLA class I molecules, compared with LCLs. However, significant levels of lysis were achieved by the addition of HPV peptide to BL cells, thereby suggesting that sufficient levels of class I molecules were expressed at the cell surface (Fig. 2b).

For instance, IL-7 is essential for the generation of murine pre-B cells and the IL-7 receptor synergizes with the pre-BCR to activate pre-B cell cycling 28, 29. This dual regulation of early B-cell generation might be important to prevent an uncontrolled proliferation of pre-B or

autoreactive B cells, while allowing a certain magnitude of cell cycling, which is followed by the rearrangement Poziotinib in vivo of the LC genes. Thus, regulating the concentration of growth factors in the microenvironment or altering the responsiveness of developing B cells to these factors seems to control the switch from proliferation to differentiation (i.e. LC gene rearrangement) in response to pre-BCR or autoreactive BCR signaling. Conversely, combining autoreactive BCRs with elevated expression of growth factors

such as IL-7 might lead to lymphoproliferative and/or autoimmune diseases as suggested by transgenic over-expression of IL-7 30. It would be interesting to test whether the BCRs of these immature B-cell lymphomas possess increased autoreactivity and whether selleck this is involved in the increased lymphoproliferation. Altogether, understanding the positive role of autoreactivity in precursor B-cell proliferation not only highlights the importance of pre-BCR expression for early B-cell selection but might also help to explain the molecular mechanisms that underlie the development of autoimmune and lymphoproliferative diseases. Our study demonstrates the importance of autoreactivity for proper B-cell development with the pre-BCR

being an invariantly autoreactive Fossariinae receptor. In the presence of a strongly recognized antigen the self-reactivity of the pre-BCR can be substituted by an autoreactive BCR to allow efficient generation of B cells. Thus, it is conceivable that at the early immature B-cell stage, cells bearing an autoreactive BCR may continue to proliferate and to recombine their LCs just as their pre-B cell predecessors do. After having changed their autoreactive specificity by receptor editing, such BCRs may get stably expressed on the surface of immature B cells, which then proceed in development. Our results are reminiscent of a hypothesis published by Niels Jerne in the very first issue of this journal 40 years ago, in which he proposed the selection of escape mutants through the initial expansion and subsequent negative selection of progenitor cells expressing germ line encoded autoreactive receptors as a mechanism of somatic antibody diversification 31. Mb1-lox-GFP mice 18, λ5−/− mice 10, 3-83Igi mice carrying the pre-rearranged 3-83Hi/33-83κi Ig gene segments 14 and mice carrying the B1-8Hi/3-83κi Ig gene segments 15 were used in this study. All mice used for the generation of HSCs were backcrossed on H-2d background. Rag-2/λC−/− mice 17, either on Balb/C or BL/6 background, were used as recipient mice for adoptive transfer experiments.

In a number of species (e.g., rats, guinea pigs, rabbits, and rhesus monkeys [13, 1, 49-52]), the blood pressure entering the placenta is quite low (8–15 mmHg under anesthetized conditions), highlighting the contribution of maternal vessels upstream of the spiral arteries, Lumacaftor in vitro particularly radial and arcuate arteries, to uterine vascular resistance. The increase in uterine artery diameter can also be modified by environmental conditions. For example, pregnant

guinea pigs exposed chronically to high altitude show only half the low-altitude rise in DNA synthesis, with the proliferative response of uterine artery vascular smooth muscle cells in vitro being blunted as well [68, 67]. Chronic hypoxia also Decitabine decreases uterine artery flow-mediated vasodilation in the guinea pig and eliminates the normal pregnancy-associated reduction in myogenic tone seen in ovine resistance-sized uterine vessels [43, 10]. Colorado women residing at high altitude only show about half the pregnancy-associated increase in uterine artery diameter and a lesser increase in uterine artery blood flow than seen in well-controlled

studies at low altitude, a difference that does not appear to reflect changes in downstream vessels insofar as the high-altitude women had normal, “low resistance” uterine artery waveforms [29]. That the vascular changes are present before the marked pregnancy rise in uterine blood flow in the PAK6 guinea pig or the onset of reduced fetal growth in humans supports the likelihood that chronic hypoxia interferes with normal uterine artery remodeling during pregnancy. Such a causal role for hypoxia is further suggested by recent studies in resistance-sized ovine uterine vessels in which 48 hours of hypoxia (10.5% O2) ex vivo reproduced the inhibitory effects of chronic hypoxia on pregnancy-

(or steroid hormone-) associated reductions in myogenic tone [11]. Although they are part of the same hemodynamic network, upstream changes (large artery) differ from those occurring in downstream (smaller/pre-placental) uterine vessels, a fact that is often overlooked. Their time course is distinctive insofar as the upstream changes in blood flow begin during the first few weeks of pregnancy well before placentation is complete (as reviewed above). In addition, changes in main uterine artery blood flow can occur in the absence of a placenta as demonstrated by a recent report from an abdominal pregnancy in which both uterine arteries displayed normal, “low resistance” waveforms despite the fact that only one was supplying the placenta (implanted on the pelvic wall) and the uterus was of pre-pregnancy size [14].

The human cathelicidin LL-37 is expressed in neutrophils, epithelial cells, mast cells, B cells, NK cells, and γδ T cells (reviewed in 1,

28), while the detailed expression of mCRAMP is less well described. To determine whether splenic B and T cells express mCRAMP, splenocytes from C57BL/6 mice were sort-purified to obtain MZ (B220+, CD21hi, CD23low) B cells, FO (B220+, CD21int, CD23+) B cells, CD4+ and CD8+ T cells. In addition, total peritoneal lavage cells were sort-purified to obtain B1a (CD5+ Mac-1+ B220int), B1b (CD5− Mac-1+ B220int), B2 (CD5− Mac-1− B220high), and T cells (CD5+ B220−). Post-sort analysis Bioactive Compound Library manufacturer revealed greater than 95% purity for each B- and T-cell population (data not shown). Total RNA was isolated from each sort-purified cell population and RT-PCR was performed to detect Camp, CD19, CD3e, and actin mRNA. All B and T cells tested expressed Camp mRNA directly ex vivo (Fig. 1A). To determine whether B and T cells express the mCRAMP protein, total

protein was isolated from purified B and T cells and analyzed using Western blot. Figure 1B confirms the expression of the immature mCRAMP protein in the total resting B and T cells. To determine whether B and T cells regulate the expression of Camp following cell activation, total CD43− splenic B cells were sort-purified and activated with CD40L and IL-4 or IFN-γ, while selleck purified CD4+ T cells were cultured in either Th1- or Th2-inducing conditions. Real-time PCR analysis for the relative expression level of Camp, normalized to actin expression, revealed that both B and T cells increased Camp expression following activation (Fig. 1C). Interestingly, B and T cells express less Camp mRNA and mCRAMP protein Morin Hydrate relative to purified neutrophils (Fig. 1B and C). In addition, total numbers of B- and T-cell subsets as well as serum antibody levels were equivalent between

C57BL/6 and Camp−/− mice (data not shown). These data show that all B and T cells tested express Camp mRNA and mCRAMP protein, suggesting that mCRAMP has the potential to regulate B- and T-cell functions. The ability of mCRAMP to directly regulate mouse T-cell cytokine production has not been fully investigated. WT and Camp−/− naïve CD4+ T cells were sort-purified and cultured in either Th1 (anti-CD3, -CD28, and rIFN-γ) or Th2 (anti-CD3, -CD28, -IL-12, and rIL-4) inducing conditions. Under Th1-inducing conditions, WT and Camp−/− T cells expressed equivalent amounts of IFN-γ mRNA (Fig. 2A), equivalent numbers of IFN-γ+ cells (Fig. 2B), and equivalent IFN-γ mean fluorescent intensity (MFI) (Fig. 2C). In contrast, Camp−/− T cells cultured under Th2-inducing conditions expressed more IL-4 mRNA (Fig. 2D), more IL-4+ cells (Fig. 2E), and equivalent IL-4 MFI (Fig. 2F).

The

three failed cases were found in patients with hyperfibrinogenemia and needed further reconstruction with another flap. The overall success rate was 88.5% (23/26). Hematologic disorder is not a predicted factor of free flap failure. The key factors for success flap survival in patients with hematologic disorders include Selleckchem Daporinad preoperative knowledge of the medical condition and monitoring potential post-operative complications, aggressive hematologist consultations, and meticulous non-traumatic surgical anastomosis. © 2014 Wiley Periodicals, Inc. Microsurgery 34:505–510, 2014. “
“The acellular nerve graft that can provide internal structure and extracellular matrix components of the nerve is an alternative for repair of peripheral nerve defects. However, results of the acellular nerve grafting for nerve repair still remain inconsistent. This study aimed to investigate if supplementing bone marrow mesenchymal stromal cells (MSCs) could improve the results of nerve repair with the acellular nerve graft in a 10-mm sciatic nerve defect model in mice. Eighteen mice were divided into three groups (n = 6 for each group) for nerve repairs with the nerve autograft, the acellular nerve

graft, and the acellular nerve graft by supplemented with MSCs (5 × 105) fibrin glue around the graft. The mouse static sciatic Cabozantinib index was evaluated by walking-track testing every 2 weeks. The weight preservation of the triceps surae muscles and histomorphometric assessment of triceps surae muscles and repaired nerves were examined at week 8. The results showed that the nerve www.selleck.co.jp/products/Temsirolimus.html repair by the nerve autografting obtained the best functional recovery of limb. The nerve repair with the acellular nerve graft supplemented with MSCs achieved better functional

recovery and higher axon number than that with the acellular nerve graft alone at week 8 postoperatively. The results indicated that supplementing MSCs might help to improve nerve regeneration and functional recovery in repair of the nerve defect with the acellular nerve graft. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“This study aims to compare the major anatomical aspects among anterolateral thigh, parascapular and lateral arm flaps. Sixty flaps were dissected in 20 human cadavers, comparing their vascular pedicle length, flap thickness and arterial/venous pedicle diameters. The vascular pedicle length (from the origin of the vascular pedicle to its entry into the skin flap) of anterolateral thigh flap (13.43 ± 3.92 cm, lateral circumflex femoral artery) was longer than parascapular (9.07 ± 1.20 cm, circumflex scapular artery) and lateral arm flap (8.90 ± 1.65 cm, posterior collateral radial artery) (P < 0.001). The thickness of lateral arm flap (6.32 ± 2.33 mm) was lesser than parascapular (8.59 ± 2.93 mm) and anterolateral thigh flap (9.30 ± 3.54 mm) (P < 0.001). The arterial/venous pedicle diameters of lateral arm flap (2.

However, c-Rel−/− mice contained a significantly lower percentage of CD4+Foxp3+ nTreg compared with WT mice (Fig. 2A and B). Further, we examined Treg populations in peripheral lymphoid tissues. Consistent with the phenotype in the thymus, percentages of CD4+Foxp3+ cells in c-Rel−/− mice were also greatly reduced in the spleen and LN as compared with WT mice (Fig. 2A and B). These data, together Selleckchem GS 1101 with our in vitro studies on c-Rel-deficient

iTreg, demonstrate that c-Rel is a critical molecule required for the development of both nTreg and iTreg. Previous studies using IL-2-deficient and IL-2Rα-deficient mice have shown that IL-2 is dispensable for the generation of nTreg in the thymus 26. The absence of IL-2 in the thymus of IL-2-deficient mice is likely to be compensated by IL-15 and IL-7. Interestingly, a profound

reduction in nTreg development was reported in IL-2 and IL-15 double-deficient mice 27. Therefore, we assume that, besides the c-Rel-mediated transcriptional control of IL-2, other mechanisms that regulate the expansion of nTreg may also be defective in c-Rel-deficient mice. Recently, it has been shown that differentiation of TH17 and Treg is interrelated 25. To examine the function of c-Rel during TH17 differentiation, c-Rel−/− CD4+ cells were stimulated via their TCR and CD28 for 3 days in a cytokine milieu optimal for TH17 differentiating conditions or in media alone. Similar IL-17 production and thus TH17 differentiation were observed in the presence Ensartinib cell line and absence of exogenous IL-2 in both c-Rel−/− and WT TH cells (Fig. 3A), as determined by intracellular cytokine staining. Confirming previous reports 24, we observed that addition of exogenous IL-2 resulted in somewhat reduced TH17 development. In the absence of exogenous IL-2, the proportion of c-Rel-deficient IL-17-producing cells was in the same order of magnitude as in WT cells (Fig. 3A). Previously, we have shown that the development of inflammatory TH17 cells is crucially dependent on the transcription Amobarbital factor IRF-4: IRF-4-deficient CD4+ TH were incapable to differentiate into TH17 cells in vitro and in vivo28, 29.

Intriguingly, it was previously reported that in activated lymphocytes, expression of IRF-4 at the RNA level is induced by c-Rel 30. This finding is difficult to be reconciled with normal c-Rel−/− TH17 cell differentiation, as shown in the current publication. However, experiments testing control of IRF-4 expression by c-Rel at the protein level are still missing. Therefore, we examined the protein expression of IRF-4 in c-Rel-deficient splenocytes as well as purified CD4+ TH by western blot analysis. Surprisingly, we found strong expression of IRF-4 in c-Rel−/− splenocytes, probably due to its constitutive expression in B cells (Fig. 3B). Moreover, activation of both WT and c-Rel-deficient CD4+ cells by PMA/ionomycin revealed similarly strong induction of IRF-4 protein after 16 h of culture (Fig. 1C).

The primers amplify a 432 bp DNA fragment. To specifically amplify T. rubrum and T. mentagrophytes, we aligned the two reference Panobinostat concentration sequences (T. rubrum: Z97993, T. mentagrophytes: Z98000) of the internal transcribed spacer ITS and we chose two sets of specific primers in the site where the sequences were divergent. The selected primers and their PCR product size are shown in Table 2. The primers consisted of the following: Derm primers that amplify all dermatophyte species, TR primer and TM primer that specifically amplify T. rubrum and T. mentagrophytes respectively. Before the MX assays

were set up and to optimise the specificity of the primers, 23 T. rubrum and 35 T. mentagrophytes strains were tested in a species-specific PCR by using separately the TR and TM primers amplifying 214 and 132 bp fragments respectively. After verification of the specificity of each set, we performed a MX PCR using the three primers in the same reaction. Multiplex PCR was performed on DNA extracts from all fungal isolates under the following conditions: the amplification reaction was performed in a total volume of 50 μl; the PCR mixture contained 10 μl of 5× reaction

buffer (GoTaq DNA buffer; Promega, Madison, WI, USA), 0.5 μl of 25 mmol l−1 desoxynucleoside triphosphates containing an equimolar mixture of dATP, dCTP, dGTP and dTTP (Promega), selleck compound 1 μl (30 μmol l−1) of each primer, 1.25 unit of GoTaq DNA polymerase (Promega) and 50 ng of template DNA. Samples were amplified through 30 cycles in a thermocycler (Thermolyne Amplitron II Series 1091, Barnstead Thermolyne Corporation, Dubuque, IA, USA) as follows: initial denaturation for 5 min at 95 °C, denaturation for 30 s at 94 °C, annealing for 30 s at 60 °C and extension for 30 s at 72 °C. This was followed by a final extension step for 10 min Docetaxel mw at 72 °C.

PCR products were separated on 2% agarose gel, stained with ethidium bromide and visualised under an UV illumination. Appropriate positive and negative controls were included in every amplification. Analytical sensitivity was determined using serial dilutions (starting from 5 pg up to 50 pg per reaction) of purified DNA extracted from the two reference targets: T. rubrum CBS 494.62 and T. interdigitale CBS 165.66. DNA was extracted from pure cultures as described by Liu et al. [15]. Common dermatophytes, reference strains, non-dermatophytic moulds, yeast and human DNA were used to determine the specificity of the MX PCR (Table 1). Data from mycological test and MX PCR were compared using analysis of chi-squared test as appropriate. The level of statistical significance was set at P < 0.05. Figure 1 shows PCR results with Derm, TR and TM primers by using serial dilution of extracted DNA; starting from 5 pg up to 50 pg per reaction. The lowest concentration of DNA that gave a positive MX PCR result for all the investigated dermatophyte species was 50 pg in a PCR volume of 50 μl.

By contrast, when IFNAR−/− bone marrow cells were cultured with influenza viruses, the proportion of CD11c+/MHCII+ BMDCs generated was similar to that observed in untreated cultures, suggesting that the IFNAR was required to mediate these effects. To further investigate the role of type 1 IFN, BALB/c bone marrow was cultured in the presence of GM-CSF, with or without Jap or recombinant IFN-α. The data (Fig. 5b) demonstrated that cultures treated with IFN-α showed a reduction

in BMDC production similar to that observed in cultures stimulated with Jap virus. We next examined the effects of neutralizing IFN. Cultures were treated with IFN-α in the presence or absence of neutralizing antibody to IFN-α. Palbociclib cost The results (Fig. 5c) showed that in the presence of neutralizing antibody the effects of IFN-α were negated and CD11c+/MHCII+ BMDC production was restored to levels corresponding to those observed in unstimulated cultures. To investigate whether the effects of influenza virus were mediated by IFN-α, cultures were treated with the Jap virus in the presence or absence of neutralizing anti-IFN-α (Fig. 5d). The addition of antibody clearly reversed the effects induced by the virus. Taken together, this evidence clearly demonstrates a role for type 1 IFN, signalling through the IFNAR, in mediating

Metformin solubility dmso responses to influenza viruses that lead to the observed changes in BMDC generation. As described above, ligands for TLRs 3, 4 and 9 were shown to initiate changes in haematopoiesis, inducing a marked reduction in BMDC production. In many cells the cytokine

TNF-α is produced in response to MyD88-dependent TLR signalling and this cytokine has also been shown to inhibit haematopoiesis19. To examine a possible role Pyruvate dehydrogenase lipoamide kinase isozyme 1 for TNF in mediating the observed effects, recombinant TNF-α was added to bone marrow cultures containing GM-CSF. The results (Fig. 6a) show that the addition of TNF-α led to a reduction in the production of CD11c+/MHCII+ BMDC similar to that observed in cultures stimulated with influenza viruses or TLR ligands. The addition of a neutralizing antibody, anti-TNF-α (Fig. 6b), restored the production of CD11c+/MHCII+ BMDCs, confirming that TNF-α was responsible and that the antibody could abolish its effects. To assess whether TNF-α was mediating the effects of LPS and CpG ODN, bone marrow cells were cultured with GM-CSF and these stimuli in the presence or absence of the neutralizing antibody, anti-TNF-α. The resulting data (Fig. 6c) showed that anti-TNF-α had no effect on the modulation of BMDC production by LPS or CpG ODN. Data compiled from cell numbers (Fig. 6d) revealed that although there was little change in the proportion of cells displaying a CD11c+/MHCII+ phenotype, anti-TNF-α did appear to suppress the increase in cell number usually observed to occur in response to LPS and CpG ODN.

OS is invariably fatal within the first months of life unless immune restoration is performed by haematopoietic stem cell transplantation (HSCT). Abnormal autoreactive T cells may infiltrate and expand R788 research buy into different organs (e.g. skin, gut, liver and spleen) and cause significant tissue damage [3]. Poor clinical status before the HSCT results in high transplantation-related mortality [4]. In the past, interferon (IFN) gamma was used to counteract the predominance of T cell activation and proliferation,

to down-regulate interleukin (IL)-4 and IL-5 production, to modulate the inflammatory reaction by enhancing phagocytic functions and to improve clinical status [5]. Today, topical/systemic steroids or cyclosporin A (CsA) are the widely used medications to control the skin manifestations [6]. CsA, a known calcineurin inhibitor, seems to act on the IL-2 by inhibiting its production and

repressing the activity of various transcription factors, thus leading to a decrease in the proliferation of the activated lymphocyte [7,8]. Moreover, it may interfere with specific signal transduction pathways which are important to the hypertrophic response [9]. Little is known about the immune modifications induced by CsA in OS patients. Such information will further improve our understanding the pathophysiology underlying OS and mechanisms of potential treatment modalities. Here we describe two OS patients anti-PD-1 monoclonal antibody and their clinical and immune response to CsA. Two patients with recombinase activating gene (RAG)2 deficiency SCID and clinical and immunological features suggestive of the diagnosis of OS phenotype were reported. Significant transplacentally acquired maternal T lymphocyte was excluded in both patients by fluorescence in-situ hybridization (FISH). The study was approved by the Institutional Review Board and informed consent was obtained from all participants’ Adenylyl cyclase parents. Cell surface markers of peripheral blood mononuclear cells (PBMCs) and lymphocyte proliferative

responses to mitogens were performed as described previously. The amount of signal joint (sj) T cell receptor excision circles (Trecs) were determined by quantitative real-time reverse transcriptase – polymerase chain reaction (qRT–PCR). Reactions were performed using 0·25–0·5 µg genomic DNA extracted from the patients’ PBMCs. The standard curve was constructed by using serial dilutions of a known Trec plasmid (generously provided by Dr Daniel Douek, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA). The number of Trecs in a given sample was calculated automatically by comparing the obtained Ct value of a patient’s sample to the standard curve using an absolute quantification algorithm.