Production of immunoglobulins was lower in ST subjects as a resul

Production of immunoglobulins was lower in ST subjects as a result of reduced survival and not lower proliferation selleck inhibitor of B cells. Increased apoptosis of B cells in the MB0 group can result in fewer cells developing into antibody-secreting cells upon stimulation, hypogammaglobulinaemia and poor humoral response to antigens. For CVID MB1 patients a different mechanism should be responsible, because their B cells behave like control B cells in their sensitivity

to apoptosis. This holds true for the two evaluated CVID MB2 patients. Their B cell apoptosis rescue was similar to CVID MB1 patients and controls (data not shown). In a recent paper, Borte et al. [35] suggested that IL-21 restores immunoglobulin production in patients with CVID. Using purified B cells, they found that IL-21 reduced apoptosis from naive and memory B cells from 14 CVID patients. However, no CVID group distinction was made; stimulation with anti-CD40 and IL-21 also included IL-4, and they considered only the CD27– naive and CD27+ IgD– memory B cell populations (excluding CD27+IgD+). The proportion of MB1/MB2 to MB0 patients in their studied cohort

might have influenced the final result and explain the apparently distinct conclusions. We cannot exclude the possibility that the peripheral blood B cells with increased apoptosis found in CVID MB0 could be the result of incomplete activation by follicular CD4+ T cells. In keeping Deforolimus with this, Hagn et al. [36] have demonstrated that human B cells co-cultured with incompletely activated CD4 T cells that secrete IL-21, but do not express CD40L, differentiate into granzyme B (GzmB)-secreting and potentially cytotoxic BCKDHB cells, able to induce slowly developing apoptosis of several cell lines. Activation of human B cells by IL-21 and BCR engagement in the absence of CD40 ligation results in their differentiation into GzmB-secreting

cytotoxic cells rather than into plasma cells. In summary, our findings reinforce the fact that (in humans) the net effect of different stimuli on B cells depends upon both the B cell subpopulation studied and the activation status of the B cell and underscore the relevance of these features in CVID physiopathology. We suggest that higher levels of apoptosis of CVID MB0 CD27+ B cells during an immune response can result in lower levels of immunoglobulin production, irrespective of their proliferation. The results highlight the heterogeneity among CVID patients, where distinct molecular mechanisms underlie common clinical symptoms, and highlight the need to classify and study CVID patients separately when evaluating B cell responses. A.C., J.P., N.L. and J.M.F. designed and performed the experiments and analysed the data. N.M. and J.P. contributed to patient selection. All authors contributed to writing the manuscript.

Genome-wide studies in human T cells have also characterized patt

Genome-wide studies in human T cells have also characterized patterns associated with promoters, enhancers and other well-conserved genomic regulatory regions.[34-38] For example, at promoter regions, H3K4me3 exists as a double peak immediately upstream of transcriptional start sites because of nucleosome depletion or Pol II binding.[34, 37, 39-42] In contrast, enhancers are characterized by the three H3K4 methylation states as well as the histone variant, H2A.Z in human T cells.[34, 38, 41] Bioinformatics analysis on 21 histone modifications in CD4+

T cells Selleck BMS-777607 was used to classify genomic regions based on their regulatory functions. The study identified 14 distinct clusters of chromatin signatures for promoters.[43] A similar bioinformatics approach buy AZD1208 separated 51 functionally distinct chromatin states

by using 38 histone modifications, Pol II and the insulator binding protein, CTCF (CCCTC-binding Factor). These chromatin states could be further categorized into five broad classes, namely promoter-associated states, transcription-associated states, active intergenic states, large-scale repressed states and repetitive states.[44] In addition, CpG islands have been linked with active marks like histone acetylation and H3K4me3 both in human T cells and embryonic stem cells.[35, 36, 45] Collectively, these distinct histone modifications specific to regional domains contribute to functional differences in gene regulation. Given the distinct chromatin states that govern specific regions of the genome, it is likely that genes with comparable transcription profiles

possess similar epigenetic landscapes. Genome-wide studies in human Liothyronine Sodium T cells have extensively characterized a large number of histone modifications using chromatin immunoprecipitation assays (ChIP) combined with massively parallel sequencing (ChIP-Seq) and have been particularly informative in identifying modification patterns associated with active and inactive genes.[34-38, 46, 47] In general, promoters with an active chromatin signature have intermediate to high gene expression levels but genes with low expression levels are associated with promoters with repressed chromatin signatures.[43] A major study focusing on 37 histone acetylation and methylation marks in human CD4+ T cells has shown that genes with different basal expression levels are associated with specific combinations of histone modifications.[38] A common backbone of histone modifications consisting of: histone variant H2A.Z, H2BK5ac, H2BK12ac, H2BK20ac, H2BK120ac, H3K4ac, H3K4me1, H3K4me2, H3K4me3, H3K9me1, H3K18ac, H3K27ac, H3K36ac, H4K5ac, H4K8ac, H4K91ac and H3K9ac was identified at a large number of promoters and tended to correlate with higher expression levels.

For analysis of intracellular

IL-17A, Brefeldin A (GolgiP

For analysis of intracellular

IL-17A, Brefeldin A (GolgiPlug® 1 μL/mL, BD Biosciences) was added to cultures for 8 h prior to analysis and, following surface staining, intracellular staining was carried out using Cytofix/Cytoperm® reagents. For FACS, magnetic column-enriched CD4+ T cells were incubated for 20 min in FACS sorting buffer at 4°C with combinations of fluorochrome-labelled antibodies then sorted using a BD FACSAriaII®sorter. In some experiments, MSCs were re-purified from co-cultures by FACS based on CD45 surface expression and then subjected to Western Blotting, quantitative RT-PCR or re-cultured to generate conditioned media. Representative examples of gating strategies used for MSC re-purification experiments are Selleckchem PD-332991 provided in Supplementary Fig. S6. Representative gating strategies for additional flow cytometry and FACS experiments are

Enzalutamide mw provided in Supplementary Fig. S9. Sorted cells were re-analysed to ensure high purity. FACS-purified MSCs were incubated for 1 h on ice in complete lysis buffer. The protein concentration was determined using a BCA Protein Assay Kit (Fisher Scientific) and proteins were separated on 4–20% Precise™ Protein Gels (Fisher Scientific) in a Mini-Protean® Tetra Cell (Bio-Rad, Hercules, CA, USA). Electro-transfer to Immobilion P PVDF membranes (Millipore, Billerica, MA, USA) was performed prior to blocking for 1 h at room temperature in 5% w/v skimmed milk powder. Membranes were incubated with anti-mouse COX-1 (1:200), anti-mouse COX-2 (1:200) or anti-β-actin (1:50 000) overnight at 4°C followed by washing in TBST, incubation for 1 h at room temperature with goat anti-rabbit IgG-HRP (1:5000), development using Immobilon® Western Chemiluminescent HRP Substrate (Millipore) and imaging on a Kodak® Image Station 4000MM Pro (Eastman Kodak, Rochester, NY, USA). Total RNA was extracted from FACS-purified MSCs using RNeasy Micro kits (Qiagen, Hilden, Germany). Reverse transcription

Phospholipase D1 was carried out using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative (Real Time) RT-PCR was performed for murine COX-1 and COX-2 (see Supplemental Methods for primer sequences) using SYBR® Green primer pairs and SYBR® Green PCR Master Mix with 18S rRNA as a normalisation control. Samples were amplified on a Prism 7900HT Real-time PCR System (Applied Biosystems). Relative quantification was performed using the comparative CT method with results expressed as fold difference relative to the MSCs-alone sample. UUO with preparation of cell suspensions by collagenase/DNase digestion was conducted as previously described 22, 43 (see also Supplemental Methods). Leukocyte-enriched fractions were prepared from kidney cell suspensions by positive magnetic selection using anti-CD45 microbeads (Miltenyi Biotec).

The fusion protein, but not rS450–650 or rCRT/39–27, successfully

The fusion protein, but not rS450–650 or rCRT/39–27, successfully induced S450–650-specific IgG production in nude mice (Fig. 4). The potent adjuvanticity of rCRT/39–272 can be partially explained by its direct activating effect on B lymphocytes (12). However, there are other possible ways for it to enhance target Ag-specific humoral lresponses in vivo. After all, adjuvants are typically characterized by their ability to activate professional APCs, Erlotinib such as DCs and macrophages, rather than B or T cells. Bone marrow-derived mouse DCs were stimulated

with rCRT/39–272, or rS450–650-CRT, or LPS, or rEGFP for 24 hrs and then analyzed by flow cytometry for CD40 expression, which is regarded as a marker for DC maturation (17). As illustrated in Figure 5, the percentage of CD40+ cells of the groups treated with rCRT/39–272 (24.5%) or rS450–650-CRT (18.6%) was considerably higher than that of the rEGFP control group (6.8%), thus confirming rCRT/39–272 and rS450–650-CRT as potent activators of murine DCs. This is further supported by the fact that rS450–650-CRT as well as rCRT/39–272, but not rEGFP, were able to induce production of IL-12 and IL-1β by DCs in vitro (data not shown). The ability of rCRT/39–272 and rS450–650-CRT

to activate DC is not due to endotoxin contamination because the recombinant proteins used in this study PS-341 in vitro were passed through polymyxin B agarose to remove endogenous LPS that could have come from the E. coli system. Newly emerging pathogens such as SRAS-CoV and avian influenza viruses are of major concern for public health today. The development of more effective vaccines (and adjuvants) against such infectious agents is urgently needed. Our results reported herein show that fusion protein rS450–650-CRT exhibits much more potent immunogenicity than rS450–650 alone in terms of

eliciting rS450–650-specific IgG responses in vivo. It should be noted, however, that whether such Abs exhibit any neutralizing effect against SARS-CoV infection remains to be tested by using either live virus or pseudo-virus systems. Physical linkage between rS450–650 and CRT/39–272 is necessary for the improved immunogenicity, because a mixture of rS450–650 and of rCRT/39–272 was no more immunogenic than rS450–650 alone (Fig. 2). Another advantage of rS450–650-CRT over rS450–650 as an immunogen is its better hydrophilicity. When preparing rS450–650, renaturation steps were necessary after Ni-column purification and the resultant product had to be maintained at a relatively low concentration in order to avoid protein aggregation and precipitation. By contrast, no renaturation steps are necessary for preparation of rS450–650-CRT and the final product is less likely to form aggregates in PBS.

[141] Moreover, several studies have described higher circulating

[141] Moreover, several studies have described higher circulating IL-18

in SLE patients than in control subjects, and the levels correlates with the anti-dsDNA titres and the SLEDAI score.[138, 140, 142, 143] Apart from the kidneys, IL-18 was also highly relevant in other organ manifestations of lupus. IL-18 was abundantly expressed in biopsy samples of lesional skin from patients with cutaneous lupus.[144] These patients also expressed higher levels of IL-18 receptor on their keratinocyte surface in response to TNF-α and IFN-γ buy Pexidartinib stimulation. Kahlenberg et al. have recently demonstrated that inflammasome activation of IL-18 would result in endothelial progenitor cell (EPC) dysfunction in SLE patients, which might explain premature atherosclerosis in SLE. In these selleck chemical experiments, neutralization of IL-18 in SLE EPC cultures restores their capacity to differentiate into mature endothelial cells, supporting a deleterious effect of IL-18 on vascular repair in vivo.[145] Nold et al. demonstrated that the use of a IL-18 binding protein would significantly inhibit the release of IFN-α and matrix metalloproteinase-9 (MMP-9) from whole blood samples obtained from SLE patients, and anti-IL18 might confer additional inhibitory

effect on the pro-inflammatory cytokines when compared with samples incubated with corticosteroids or mycophenolic acid alone.[146] Although IL-18 blockade appeared to a potential therapeutic concept in SLE, the clinical data regarding this approach are still lacking. In this review, we have highlighted the cytokines which have crucial pathogenic significance in SLE (Fig. 1). The growing knowledge in these cytokines has introduced opportunities for the design of innovative diagnostics and therapeutic approaches (Table 1). Currently, these novel therapies which involve the attenuation of the cytokine system are often used as add-on treatment or for recalcitrant cases. However, one should expand the use of these biologics such as minimization of other immunosuppressive drugs which CYTH4 have more significant toxicities.

While some of these agents have proven efficacy and tolerability in the initial studies, the long-term safety remains undefined. Both upcoming randomized trials and long-term follow-up studies are needed to adequately address these concerns. Taken together, data regarding the manipulation of the cytokine systems are encouraging and it is worthwhile to invest resources for the development of therapy in this promising direction. “
“The Cochrane Collaboration is a global network whose aim is to improve health-care decision making through systematic reviews of the effects of health-care interventions. Cochrane systematic reviews are published in the Cochrane Database of Systematic Reviews within The Cochrane Library ( http://www.thecochranelibrary.

One to three per cent of inspired molecular oxygen is converted t

One to three per cent of inspired molecular oxygen is converted to O2-,

which is the most common of the ROS and a powerful precursor of H2O2.5 Although cellular H2O2 is stable, it has the potential to interact with a variety of substrates to cause damage, especially in the presence of the reduced metal ion C59 wnt Fe2+. This leads to H2O2 to break down and form the most reactive and damaging of the ROS, OH-. In healthy cells, the production of the potentially harmful H2O2 is countered by the catalysing actions of mitochondrial or cytosolic catalase (CAT) or thiol peroxidases into H2O and O2. Figure 1 demonstrates pathways to, and natural anti-oxidant neutralization of, common ROS. Given that ROS are likely to be highly damaging molecules to cells, why have the mitochondria not evolved more efficient systems that limit mitochondrial oxidants? One possible answer is that ROS have an essential

role in oxidant metabolism where they are involved in highly conserved basic physiological processes as effectors of downstream pathways. Thus, to some, oxidative stress theories of disease pathogenesis must be intrinsically flawed.6 Nonetheless, ROS are damaging molecules. Even when they are produced during normal respiration, they could cause cumulative damage that would eventually lead to loss of cell and tissue function and, ultimately, disease. Their production is known to increase, over natural anti-oxidant levels, CT99021 during progressive disease and during ageing.4 The kidney is highly energetic and therefore relies heavily on aerobic metabolism for the production of ATP by oxidative phosphorylation. The reduction of molecular O2 along the electron transport chain (ETC) within mitochondria is vital for renal cellular function, yet potentially devastating long-term. The ETC consists of five multi-enzyme complexes responsible for maintaining mitochondrial membrane potential Phosphatidylinositol diacylglycerol-lyase and ATP generation.

Each of these complexes presents a site of ROS generation; however, complexes I and III have been identified as primary sites of O2- generation.7 Complex I, also known as nicotinamide adenine dinucleotide (NADH) dehydrogenase, or NADH-CoenzymeQ (NADH-CoQ) reductase, facilitates the transfer of electrons between NADH and CoQ10 (sometimes known as ubiquinone). Defects in oxidative phosphorylation may be due to the use of substrates in the respiratory chain, such as the reduced NADH and NADH oxidase, and not due to alterations in the proteins of the respiratory complexes. Thus, it is likely that altered respiratory complexes and substrates lead to an inefficiency of electron transport, and subsequent increased ROS, decreased ATP and a loss of the mitochondrial membrane potential. Oxidatively damaged proteins of the mitochondrial complexes increase with age in mice.8 In CKD patients (stages 2–3) and haemodialysis patients, impaired mitochondrial respiration was recorded.

There were no major complications, and very satisfactory results

There were no major complications, and very satisfactory results have been obtained.

This retrospective study showed that both options of raising a large DIEAP flap for unilateral breast reconstruction, namely unipedicled flap based on large medial perforator/s plus additional venous discharge or double-pedicle flap, are safe. Preoperative examination of the dominant perforator/s with CDS and/or MDCT is mandatory in both cases. © 2010 Wiley-Liss, Inc. Microsurgery 2010. “
“Breast conservation surgery in the treatment of early stage breast cancer has become increasingly utilized as a means to avoiding mastectomy. While partial mastectomy defects (PMDs) may often be cosmetically acceptable, some cases warrant consideration of reconstructive options, and while several reconstructive options have been described in this role, a series of deep selleck chemicals inferior epigastric perforator (DIEP) flaps R788 nmr has not been reported to date. A cohort of 18 patients undergoing PMD reconstruction with a DIEP flap were included. Patient-specific data, operation details, cosmetic results, and complication rates were assessed. Oncologic outcomes, in particular recurrence rates, were also evaluated. In our series there were no cases of partial or total flap necrosis, and overall complications were

low. There were two cases of wound infection (both had undergone radiotherapy), managed conservatively, and one case of reoperation due to hematoma. There were no cancer recurrences or effect on oncologic management. Cosmetic outcome was rated as high by both patients and 3-oxoacyl-(acyl-carrier-protein) reductase surgeon. The results were thus comparable with other reconstructive options. Although autologous reconstruction has an established complication rate, our results suggest that the DIEP flap may be of considerable value for delayed reconstruction of selected larger partial mastectomy defects. © 2010 Wiley-Liss, Inc. Microsurgery, 2011. “
“The latissimus dorsi (LD) muscle flap is one of the most versatile flaps used for reconstruction of soft tissue defects. With knowledge of its anatomy, harvest of the segmental LD muscle has been introduced as a reliable technique with

the advantage of muscle preservation. We devised a new harvest technique for the segmental LD flap using a limited transverse incision to elevate a less bulky distal segment of the muscle with a sufficient pedicle length obtained by intramuscular dissection of the vascular pedicle. Two cases, in which this technique was effectively applied to reconstruct plantar defects after wide excision of malignant melanoma with a maximally efficient use of donor and recipient tissues, are presented. Satisfactory results were gained with stability in walking. When the defect size permits use of a segmental muscle and the long pedicle is needed, this pedicle-lengthened segmental LD muscle harvest technique would be a valuable method. © 2013 Wiley Periodicals, Inc. Microsurgery 33:491–495, 2013.

Right panel: Similarity analysis between Hoechst 33258 and IRF-7

Right panel: Similarity analysis between Hoechst 33258 and IRF-7 in untreated or CpG-stimulated CAL-1 cell variants. Values depicted in the histograms represent the percentage of cells with similarity values above an arbitrary value of 1.7 over a total of approximately 20.000 cells. Supporting Information Figure 3. NAB2 knowdown by siRNA reduces TRAIL induction in CpG treated CAL1 cells but does not affect CD40 expression. CAL-1 cells were transfected with siGLO transfection indicator

together with Ctrl siRNA or siRNA targeting NAB2 in a ratio of 1:3. (A) 48h post-transfection TRAIL expression of unstimulated, or CpG-stimulated CAL-1 cells was measured by flow cytrometry in the siGLO+ and total transfected cell populations. Numbers in the upper right corner represent TRAIL GeoMFI of CpG stimulated cells. (B) The knock-down of NAB2 protein of the total transfected cell population was assessed selleck screening library by Western Lapatinib price blot analysis. (C) CD40 expression was measured by flow cytometry in siGLO+ (left panel) or in the total cell population (right panel). Numbers depict the percentage of CD40+ cells. Data are representative of 2 independent experiments. Supporting Information Figure 4. Activated CAL-1 NAB2E51K cells are less potent in inducing

apoptosis in Jurkat cells. (A) DDAO-labeled Jurkat cells were co-cultured for 20h with unstimulated or CpG stimulated CAL-1-EV, -NAB2, or -NAB2E51K cells. Active Caspase-3 was measured in Jurkat cells by CaspGLOW Red Active Caspase-3 Staining Kit. Data are representative of 2 independent experiments. Supporting Information Figure 5. Analysis of the specificity of inhibition of PI3K [7], p38MAPK, NF-kB and effects of

mTOR and PI3K pathways. (A-B) CAL-1 cells were pre-incubated for 30 min with PI-103 (PI), SB203580 (SB), and BAY11–7082 (Bay), DMSO (Ctrl) or left untreated (-), before being activated with CpG for 30min (A) or 1h (B). Protein expression of Akt, p38MAPK, NF-kB p65 and the respective phosphorylated forms (p-) were assessed by Western blot analysis. NAB2 induction is independent on mTOR. (C) CAL-1 cells were incubated for 30 min with PI-103 (PI) HSP90 or Rapamycin (Rap) followed by 4h activation with CpG. NAB2 mRNA levels were measured by RT-PCR. (D) CAL-1 cells were stimulated for 4h with CpG in the absence or presence of PI-103, and IFNβ mRNA levels were measured. Supporting Information Figure 6. Differential TRAIL levels in CAL-1-NAB2E51K cells are not correlated with NAB2E51K expression levels, but rather a consequence of not fully activated CAL-1 cells. (A) CAL-1- NAB2E51K cells were activated for 6h with CpG, and TRAIL expression levels were assessed by flow cytometry of the top GFP-expressing cells (GFP high) the bottom GFP-expressing cells (GFP low). Shaded plots represent unstimulated CAL-1-NAB2E51K cells.

Although anti-inflammatory therapies have attenuated cystogenesis

Although anti-inflammatory therapies have attenuated cystogenesis in animal models, inflammatory cells may also have reparative actions. Thus, in developing therapies for PKD, it is prudent to consider the potential negative outcomes of ablating inflammation, and whether it is more viable to target certain inflammatory pathways over others. Polycystic kidney diseases (PKD) are a group of genetically inheritable disorders that are characterized by the formation of bilateral renal cysts.[1] Autosomal dominant PKD (ADPKD) involves

mutation of the genes Pkd1 and/or Pkd2, which encode the ciliary cystoproteins, polycystin 1 and 2 (PC1 and PC2) respectively.[2, 3] Autosomal recessive PKD (ARPKD) is characterized by genetic mutation of Pkhd1, leading to defects in the cystoprotein, fibrocystin.[4] In both forms of PKD, dilation of renal tubules gives learn more rise to the cystic morphology.[5, 6] Cyst growth is propagated by cystic epithelial cell (CEC) proliferation and dedifferentiation,[7] Trametinib fluid secretion[8] and basement membrane abnormalities.[9] This cystic expansion compresses the surrounding renal parenchyma and microvasculature, obstructing nephrons and thus impairing their function, resulting in renal failure.[7] Although research in PKD has focussed on preventing cyst growth and expansion, another key pathological feature of cystic renal disease is the development of interstitial

inflammation and fibrosis, typically associated with inflammatory cell infiltration.[7, 10, 11] Generally speaking,

PKD is not a primary inflammatory disorder. However, for many years it has been unclear whether interstitial inflammation is merely associated with disease progression in PKD, or whether it essentially plays a role in pathogenesis.[7] Recent studies in animal models suggest that the chronic interstitial inflammation in PKD possibly contributes to cyst development and renal impairment, but the precise roles of macrophages and other infiltrating inflammatory cells have not been defined. This review aims to analyse the potential mechanisms leading to renal interstitial inflammation in PKD, including the roles of soluble mediators, intracellular signalling pathways, and the interplay between these pathways and cystoprotein dysregulation. There is substantial heterogeneity among peripheral and tissue monocytes, in humans, as well PAK5 as mice.[12] Resident monocytes are characterized by CD16+ and Ly6Clow expression in humans and mice, respectively (see Table 1).[12] These cells ‘crawl’ across endothelial vessels, and are therefore thought to monitor surrounding cells for injury.[12] In contrast, inflammatory monocytes display a CD16− and Ly6Chigh profile in humans and mice, respectively,[12] and infiltrate renal tissue in inflammatory states such as ischemia reperfusion injury (IRI).[13] Once they have migrated to the injured region, these monocytes differentiate into inflammatory macrophages.

Most of the current devices use a wavelength of 780 nm,

Most of the current devices use a wavelength of 780 nm, EGFR cancer which provides good skin penetration independently of skin color and oxygen saturation [151]. The first laser Doppler technique developed is called

flowmetry (LDF), also referred to as laser Doppler perfusion monitoring (LDPM). Single point LDF assesses blood flow over a small volume (1 mm3 or smaller) with a high sampling frequency (often 32 Hz) and is accurate at detecting and quantifying relative changes in skin blood flow in response to a given stimulus [25]. However, the regional heterogeneity of skin perfusion [11] leads to spatial variability, which contributes to the relatively poor reproducibility of the technique [114]. In contrast, the more recently developed laser Doppler imaging (LDI), or laser Doppler perfusion imaging (LDPI), provides 2D images using the same physical principle as LDF [25]. In LDI, the laser beam is reflected by a computer-driven mirror to progressively scan the area of interest. A fraction of the backscattered light is detected and used to map tissue blood flux, each pixel representing a perfusion value. LDI decreases spatial variability, but it is much slower than LDF, making rapid changes in skin blood flow over the larger areas more difficult to record. Nevertheless, more recent imagers use a multi channel laser Doppler

line permitting faster scanning. A linear relationship between the laser Doppler signal and microvascular selleck chemical flow has been demonstrated

in the range from GDC0449 0 to 300 mL/min per 100 g tissue [3]. However, it does not provide an exact measure of flow (i.e., mL/min) as can be extrapolated when using strain gauge plethysmography. Therefore, laser Doppler is mostly used to assess microvascular reactivity, by challenging microvessels with various tests. Among the different tests used in combination with laser Doppler, the most common are iontophoresis of vasoactive drugs, PORH, and thermal challenges. Results are often expressed as arbitrary PU (1 PU = 10 mV) or as CVC (i.e., flux divided by arterial pressure [in mV/mmHg]) [25]. Microdialysis is a technique consisting of the intradermal insertion of small fibers with semipermeable membranes and is mostly used for the continuous sampling of small water-soluble molecules within the extracellular fluid space in vivo [22]. Nonetheless, it can also be used to deliver drugs to a small area of tissue, avoiding confounding systemic effects [25]. Although minimally invasive, microdialysis offers the advantage of a controlled drug infusion rate and the absence of current-induced vasodilation, compared with iontophoresis. However, it is painful and justifies the use of local anesthesia. Both local inflammation and anesthetic drugs may interfere with the response. This approach coupled with LDF has been used to assess the role of NO in skin post-occlusive and thermal hyperemia [101,145].