Staphylococcus aureus. Previously, we conducted a transcriptomics study for identification of SaeRS regulon and found that inactivation of SaeRS dramatically enhances the transcription of a novel transcriptional regulator (SA1804). This led us to question whether SA1804 is involved in bacterial pathogenicity by regulating the expression of virulence factors. To address this question, we created sa1804, saeRS, and sa1804/saeRS double deletion mutants in a USA300 community-acquired MRSA strain, 923, and determined their impact on the pathogenicity. The deletion of sa1804 dramatically increased the cytotoxicity and enhanced the capacity of bacteria to invade into the epithelial cells (A549), whereas the deletion of saeRS eliminated the cytotoxicity and abolished the bacterial ability to invade into the epithelial cells. Moreover, the double deletions of sa1804 and saeRS appeared a similar phenotype with the saeRS null mutation. Furthermore, we determined the regulatory mechanism of SA1804 using qPCR and gel-shift approaches. Our data indicate that the novel virulence repressor SA1804 is dependent on the regulation of SaeRS. This study sheds light on the regulatory mechanism of virulence factors and allows for us further elucidate the molecular pathogenesis of S. aureus.The two-component regulatory system, SaeRS, controls expression of important virulence factors, including toxins and invasins, which contribute to the pathogenicity of  Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is a major community and hospital acquired pathogen that can cause superficial infections, including skin and soft tissue lesions  production in vitro  have been implicated together with other regulators to mediate the pathogenicity of . aureus . The exp. aureus , ArlRS  and USA300 MRSA isolate 923 (Escherichia coli DH10B (Invitrogen) served as the host for all in vitro recombinant DNA. Bacteria were grown in Tryptic Soy broth  and on TSA agar at 37°C with appropriate antibiotics. Bacterial cell cultures were incubated at 37°C with shaking at 200 rpm.late 923 , as secosaeRS and/or sa1804 was carried out following the pKOR1 allelic exchange protocol as described  into the AscI and PmeI sites of pYH4 , and cloned into same enzyme sites of pCY1006. The reformed Psa1804-lux reporter fusion was confirmed by diagnostic PCR and electroporated into the saeRSko and parental control. The Psa1804-lux reporter fusion strains were grown in TSB at 37°C with shaking overnight. Bioluminescence intensity and optical density of the cultures were measured at different times of the experiment in duplicate. The relative light units (RLU) were calculated by dividing the average bioluminescence reading by the average OD600nm reading (lum/OD600nm) at each time point. The experiment was repeated three times with separate colonies of each strain.In order to examine whether the expression of  of pYH4 . The resescribed . The upsS. aureus were inoculated in 5% in TSB medium and grown to the mid-exponential (3 h) phase of growth. Cells were harvested by centrifugation; RNA was isolated using the RNAPrep Kit  as described (free Kit (Ambion). The first strand cDNA was synthesized using reverse transcriptase with the SuperScript III Platinum Two-Step qRT-PCR Kit (Life Science Technology). For each RNA sample, duplicate reactions of reverse transcription were performed, as well as a control without reverse transcriptase, in order to determine the levels of DNA contamination. PCR reactions were set up in triplicate by using the SYBR Green PCR Master Mix (Bio-Rad). Real-time sequence-specific detection and relative quantization were performed with the Stratagene Mx3000P Real Time PCR System. Gene-specific primers were designed to yield ∼100 bp of specific products (Table 1). Relative quantification of the product will be calculated using the Comparative CT method, as described for the Stratagene Mx3000P system. The housekeeping gene gyrB was used as an endogenous control. All samples were analyzed in triplicate and normalized against gyrB gene expression. The experiments were at least repeated twice and analyzed for correlation to the microarray results.Overnight cultures of escribed . Contami5 cells were seeded in each well of 24-well plates, and incubated overnight at 37°C in a CO2 incubator. Monolayers of A549 cells (2 × 105 cells/well) were infected by adding 0.5 ml RPMI containing approximately 5 × 105 cfu of bacteria followed a centrifugation at 100 g for 5 min, and incubated for 2 h at 37°C in 5% CO2. The bacteria outside of the monolayer cells were killed by adding1 ml of RPMI-10% FCS containing 100 μg/ml gentamicin and 5 μg/ml lysostaphin to invasion wells. We used 0.025% TritonX-100 for better cell lysis. The numbers of bacterial cfu released from the lysed epithelial cells were determined by plating of diluted lysates on TSA-agar plates. Each experiment was repeated three times and statistically analyzed by TTEST, using Microsoft Excel software. P-values of < 0.05 were considered significant.A549 human lung epithelial cells (ATCC CCL 185) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum . Cultures of A549 cells were maintained in a medium containing penicillin (5 μg/ml) and streptomycin . Bacterial invasion assyas were performed in RPMI 1640 medium with supplement of 10% FBS (RPMI-FBS) as described . Briefly2 overnight for 18 h. At the end of the experiment, cell viability was determined using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega) as manufacturer’s instructions. Each experiment was repeated three times and statistically analyzed by TTEST, using Microsoft Excel software. P-values of < 0.05 were considered significant.All cells were grown in 96-well plates to 70% confluence. Inhibitors were diluted serially in complete medium and applied to the cells. The supernatants were collected from the overnight cultures, sterilized by filtration, and applied to the epithelial cells, and the treated cells were incubated at 37°C with 5% COsa1804 gene was obtained by PCR amplification using the premiers listed in Table 1, cloned into pET28a, and resulted in pSA1804-28a. Plasmid pSA1804-28a were introduced into a BL21(DE3) strain. The resulting strains were grown in LB medium at room temperature; the expression of SA1804 was induced when the culture media reached OD600nm equal to 0.6 by addition of 1 mM IPTG  and incubation pursued for 4 h. The SA1804-his tagged protein was purified using Ni-NTA agarose column (Novagen) as described . The purified biotin-labeled probes were utilized for the electrophoretic mobility shift assay (EMSA) using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). All samples contained 1X LightShift Binding Buffer, 50 ng/μl Poly (dI∙dC), and 2.5% glycerol. The labeled probe, SA1804-His, non-labeled specific probe, BSA, and non-specific non-labeled probe were all added to concentrations as outlined in the figure and ultrapure water was added so that all reaction volumes totaled 20 μl. The reactions were incubated at room temperature for 20 min followed by addition of 5 μl of 5X loading buffer to each reaction. 20 μl of each reaction were loaded into the wells of a pre-run 8% TBE native polyacrylamide gel and electrophoresed at 75 V for three hours at 4°C. The samples were transferred to nylon membrane and processed as outlined in the manufacturer’s protocol. BioMax Light Film (Kodak) was used to detect the chemiluminescent reaction.To determining whether SA1804 directly regulates escribed . The duasaeS significantly induced the transcription of a putative regulator gene (sa1804), suggesting that SaeR might be a repressor of SA1804 . This suggests that SaeRS system transcriptionally modulates the expression of SA1804.Our previous microarray analysis showed that the mutation of f SA1804 . To furtsa1804 transcription. This led us to question the impact of SA1804 on the capacity of bacteria to adhere to and invade into the host cells. To address this question, first we constructed a sa1804 null mutant using a USA 300 human clinical S. aureus isolate 923. Then, we examined the impact of the null mutation of SA1804 on invasion using a human lung epithelial cell line (A549). The results showed that the mutation of sa1804 significantly elevated the capacity of bacteria to invade into the epithelial cells . To confirm the role of Sa1804, we constructed sa1804 complementary strain, and determined whether the expression of Sa1804 in trans complements the effect of depletion of SA1804. The results showed that the complementation of Sa1804 alleviated the bacterial ability to invade epithelial cells .Our previous studies demonstrated that the SaeRS is a major regulator of bacterial adhesion and invasion and SaeRS represses saeRS and saeRS/sa1804 double knockout mutants and examined their impact on bacterial invasion. Consistent with our previous findings the deletion of saeRS loci totally eliminated the bacterial capacity of invading of epithelial cells . The deletion mutation of saeRS eliminated the effect of SA1804 on the invasion ability of in the sa1804 null mutant . The above data indicate that the involvement of SA1804 in regulation of bacterial invasion is dependent on SaeRS.To elucidate whether SA1804 functions through the regulation of SaeRS, we generated S. aureus, we performed hemolysis and cytotoxicity assays by measuring LDH as described . We found that the supernatant of the wild type strain, 923, caused 54% of A549 cells died 16 h after exposure, whereas 75% of the cells died after exposing to the supernatant from the sa1804 knockout mutant culture . Deletion of saeRS abolished the toxicity of the supernatant of the culture, whereas knockout of saeRS had no impact of the deleted sa1804 on cytotoxicity. This further indicates that the regulator SA1804 plays a role in the regulation of virulence factors in a saeRS dependent manner.To further determine the role of SA1804 in pathogenicity of escribed . The delsa1804 null mutant, saeRS null mutant, saeRS/sa1804 double knockout mutant, and parental control. The transcription levels of selected genes were detected by qPCR. The housekeeping gene gyrB was used as an internal control. No difference of gyrB gene transcription level was revealed between the sa1804 and/or saeRS null mutant and it parental control. However, the transcriptional levels of hla, efb, and sa1000 increased approximately threefold in the sa1804 null mutant than that parental control (Table 2). In contrast, the deletion of saeRS remarkably decreased these genes transcription. Taken together, the above data demonstrated that the putative regulator SA1804 transcriptionally represses the expression of hla, efb, and sa1000 in S. aureus 923 isolate.To elucidate potential molecular mechanism behind the impact of SA1804 on bacterial invasion and cytotoxicity, we examined the impact of SA1804 on transcriptions of several potential target genes using qPCR approach. Total RNA was purified from the mid-log phase of cultures of the hla, efb, and sa1000 genes using gel-shift assays. We cloned, expressed, and purified SA1804-his tag protein as described . BSA protein was used as a control. The promoter regions of hla, efb, and sa1000 genes were obtained by PCR, labeled, and purified using PCR cleanup kits. Unlabeled probe was utilized as specific competitor (SC) accordingly. Apparent hla probe-SA1804 complex formed in a dose-dependent manner . However, no hla probe-SA1804 complex was observed for the efb and sa1000 promoters (data not shown). These suggest that SA1804 may directly mediate hla transcription by binding to hla promoter region, but indirectly regulate efb and sa1000 transcription.To elucidate the potential mechanism of regulation, we examined whether SA1804 directly or indirectly regulates the transcription of the hla, efb, and sa1000 in a CA-MRSA USA300 isolate 923. Moreover, our data suggest that SA1804 indirectly mediates the transcription of efb and sa1000, whereas directly controls the transcription of hla through binding to the promoter region of hla. Furthermore, our data clearly indicate the role of SA1804 in manipulation of the pathogenicity of S. aureus, as the null mutation of sa1804 enhanced the capacity of bacteria internalizing into the epithelial cells and the cytotoxicity. In addition, we further confirmed that SaeRS negatively regulates the transcription of the novel transcriptional regulator SA1804. Our data suggest that the regulatory function of SA1804 is dependent on the activation of SaeRS, as the disruption of SaeRS eliminated the influence of SA1804 on the transcription of selected virulence factors, including hla, efb, and sa1000, as well as the bacterial invasion ability and cytotoxicity.In this study, we are the first demonstrating that SA1804 is a repressor of several virulence factors, including sa1804 promoter-lux reporter fusion experiment showed that the inactivation of SaeRS remarkably elevated the transcription of sa1804, which is consistent with our microarray analysis result (S. aureus isolates (hla, efb, fnbB, and sa1000 in CA-MRSA USA300 isolate 923. Moreover, our invasion and cytotoxicity assays demonstrated that SaeRS plays a key role in the pathogenesis of S. aureus, which is highly supportive for the importance of SaeRS in pathogenicity in animal models of infection (fnb (fnbA and fnbB was detectable in the saeS mutant strain (saeRS had a weak effect on the transcription of fnbB. This discrepancy is likely due to the difference of genetic background among different S. aureus isolates.Our s result . Togetheisolates , our qPCnfection . Previouion (fnb  since not strain . In thishla expression in S. aureus (hla transcription by binding to its promoter region (hla promoter region, our data clearly demonstrated that SaeRS plays a predominant role in the regulation of the hla transcription in S. aureus.It was well established that SaeRS is a major regulator of . aureus ; SaeR car region . Althoug


How do single mothers who have conceived a child via anonymous or identity-release sperm donation represent the donor?While the majority of mothers described their anonymous and identity-release donors as symbolically significant to their families, others were more likely to emphasize that their lack of information limited their thoughts about him.There is limited understanding of the factors that impact upon how single mothers represent the donor, and whether or not they are determined by specific donor programmes (anonymous or identity-release).Qualitative interviews were conducted with 46 women who had treatment at a UK licensed fertility clinic during the years 2003–2009. Twenty mothers (43%) had used an anonymous donor, and 26 (57%) had used an identity-release donor.Among the 46 mothers interviewed, all had at least one child conceived via donor insemination who was between the ages of 4 and 9 years. Mothers were heterosexual and were currently without a live-in and/or long-term partner. Interview data were analysed qualitatively according to the principles of thematic analysis.n = 27) mothers talked about the donor as symbolically significant to family life and were likely to describe the donor as (i) a gift-giver, (ii) a gene-giver and (iii) a potential partner. Others (n = 16) talked about the donor as (i) unknown, (ii) part of a process and (iii) out of sight and out of mind. There were mothers with anonymous and identity-release donors in each group. Several mothers explained that their feelings about the donor had changed over time.Findings indicated marked diversity in single mothers' representations of the donor. Most (All mothers conceived at a licensed fertility clinic in the UK. Findings are limited to individuals willing and able to take part in research on donor conception.The study offers greater insight into the factors influencing the donor narratives produced in single-mother families. It has implications for the counselling and treatment of single women seeking fertility treatment with donor gametes in both anonymous and identity-release programmes. Given that the number of clinics offering identity-release programmes worldwide seems to be increasing, the finding that single women may have varying preferences with regard to donor type, and varying interest levels with regard to donor information, is important. It is recommended that clinicians and other fertility clinic staff guard against making assumptions about such preferences and any thoughts and feelings about the donor or donor information on the basis of marital status.This study was funded by the Wellcome Trust [097857/Z/11/Z]. The authors have no conflicts of interest to declare. Donor insemination is a form of third-party assisted reproduction that enables individuals and couples to conceive using donor sperm. Although initially established in order to assist heterosexual couples to become parents, a significant proportion of those who now seek fertility treatment with donated gametes are women without a male partner . CoupledStudies measuring the preference rates for known, identity-release or anonymous donation have shown that heterosexual couples may be less likely than single women and lesbian couples to actively choose an identity-release donor, and less likely to attribute importance to identifying information about him , and thaIn keeping with this literature, research of a qualitative nature has also indicated that heterosexual couples may minimize the role of the donor in their family narratives , and thaBy way of explaining these findings, it has been suggested that interest in donor information may be guided by recipients' intentions to tell their children about their conception .Participants were recruited through the London Women's Clinic, the largest fertility clinic in the UK that offers donor insemination to single women. Mothers had at least one child aged between 4 and 9 years conceived by sperm donation, were heterosexual and were currently without a live-in and/or long-term partner. These inclusion criteria were determined by mothers' involvement in a larger study of family functioning and child development in single-mother families  and had never been married . Twenty mothers (43%) had used an anonymous donor and 26 (57%) had used an identity-release donor. The mean age of children conceived using an anonymous donor was 6.9 years (SD = 1.30). The mean age of children conceived using an identity-release donor was 4.5 years (SD = 0.76).Of a total of 63 women contacted by the research team, 46 agreed to take part, giving a response rate of 73%. Mothers' ages ranged from 32 to 51 years . The majority had one child  mothers talked about the donor as an ‘absent presence’ who was in one or more ways symbolically significant to family life, and were likely to describe the donor as (i) having given them a gift (the ‘gift giver’), (ii) having provided their child with genetic material (the ‘gene-giver’), and (iii) as in some sense part of ‘traditional’ family life . Others (n = 16) talked about the donor as an ‘absence’ and described him as (i) a figure about whom little or nothing was known (the ‘unknown’), (ii) donor sperm, rather than a sperm donor (‘part of a process'), and a person whose contribution was intangible . Within this second subgroup, mothers expressed either positive or negative feelings about the donor's ‘absence’.Findings indicated that participants' thoughts and feelings about the donor could be understood in relation to two overarching themes of ‘presence’ and ‘absence’. Most (n = 3) of mothers appeared to currently represent the donor as both an ‘absent presence’ and an ‘absence’ in equal measure, often vacillating between the two in the course of the interview.The themes of ‘presence’ and ‘absence’ were found to characterize participants' narratives both in terms of how they specifically described the donor, and in terms of the strategies they described as facilitating their representation of the donor as either a ‘presence’ or an ‘absence’. A minority The majority of mothers described the donor as having given them a ‘gift’, for which their gratitude was unquantifiable. Some mothers suggested that they had specifically chosen a donor on the basis that he too saw his donation in such terms:I only think of him as a very kind and generous soul. That's almost the deal. He's done his bit … done and dusted.Other mothers explained that although they were not given information from the clinic about the donor's reason for donating, they were assured that the donor was a ‘kind man’, since he had chosen to donate his sperm in the first place. However, although several mothers stated that they would like to meet the donor to personally express their gratitude, others appeared to conceptualize the donor's gift as marking the end of a specific—albeit extremely generous—exchange:What's the point of knowing that you're gonna get knocks on your door in 18 years' time, to say ‘I don't want to know you?’ I know these men, who are becoming the donor dads, are gonna be happy to have someone who's related to them come and say hello to them.Responding to questions about the donor's possible future involvement in their families, some mothers expressed a belief that donors who specifically made a donation after 2005 would, as ‘kind men’, be willing to have contact with donor-conceived children:I have to be careful that I don't embellish him with stuff that I don't know he has.We don't really go into—we don't build up a fantasy. We just [talk about] some man who was really kind, and allowed mummy to use the special stuff.Many mothers who had begun the process of telling their children about their donor conception explained that they had described the donor as a ‘kind man’, although some expressed some discomfort about doing so. This was the case irrespective of whether mothers had used an anonymous or identity-release donor:Other mothers indicated that although they had described the donor to their child as a ‘kind man’ who had given them a gift, they took great care to describe him in terms that distinguished him from a father figure. Several mothers, for example, made a distinction between a ‘daddy’ and a ‘father’; others clarified that the ‘donor daddy’ was not a ‘day-to-day daddy’ or a ‘normal dad’.I wasn't bothered about IQ in the slightest. I'm sure that's more social than genes. I was bothered about character, and … very vainly, what he looked like.Being not terribly tall myself, I just thought that if I had a boy then … at least I wanted to give him some extra genes for height.Alongside describing the donor as ‘gift-giver’, the majority of mothers explicitly designated the donor as ‘present’ in their family narratives as part of the story of where their child ‘came from’. For most, the donor was seen to be the carrier of specific genetic traits, and as somebody who had therefore provided their child with some of their genetic material. Some mothers described having chosen their specific donor on the basis of this belief:I'm really intrigued to know what he's like … whether there's similarities and whether there are things about her that I'm not like … so that must come from someone else … There must be mannerisms and stuff that's similar.At the same time, several mothers reflected upon how their thoughts and feelings about the donor had changed post-birth, and especially after acknowledging that their children possessed particular characteristics and mannerisms that they could not identify in themselves:Sometimes when I look in [my child's] eyes, cos his eyes aren't anything like any of our family, that's the link I feel with the dad, his eyes.One mother specifically suggested that the identification of these characteristics in her child made the donor continually ‘present’ for her:Now I realise for them how important it will be … because it's part of them.I think before … I would have thought it would be better to be anonymous, but then I thought oh well it doesn't matter, and then when I thought about it, I think it's really important because … if she ever wants to know, which, you know, she may do, I think that's a really key part for her.Alongside envisioning the donor as a ‘giver’ of genes, several mothers specifically suggested that the donor's genetic contribution meant that he represented the ‘root’ of their child's identity, often likening the experience of donor-conceived children to adopted children who search for information about their origins. A minority of mothers stressed that this was not how they had thought about the donor during treatment:That was my overriding focus, the best way and the quickest way of getting a baby … It's awful isn't it? Like, worst, I spend more time choosing, I don't know, things in the supermarket, isn't that strange? Or a pair of shoes or something. Now of course I'm thinking … there's all these half brothers and sisters out there in the world, which could cause issues as they get older.I feel very protective … We have our own family, and I want to protect them. The whole sibling [thing] … I could go on and contact all these people, but I have no control about them.Some mothers also remarked that further information from the HFEA had been especially helpful, not only in allowing them to think about the donor in greater detail, but also in facilitating their knowledge of their child's ‘genetic background’ in general. Several mentioned the significance of information provided about ‘donor siblings'—other children who had also been created through the use of their donor's sperm. For some participants, however, knowing more about these children served to provoke a great deal of anxiety:I think it was important to me that they were educated, just because that reflects my own kind of beliefs and what I would look for in a partner, I guess, so that's the only way I could judge it really. And yeah, someone tall, because I like tall men.Several mothers explained that the donor was a figure who had ‘presented’ himself as a ‘potential partner’, stating that they had chosen a donor on the basis of characteristics that they were attracted to:I remember saying to [the nurse], if you had to date one of them, who would you go out with? And we had a very funny conversation around that … I mean every now and then she'll say a little bit, she'll put a little meat on the bones, nothing much, but that makes the person come alive a bit.In so doing, mothers described how fertility clinic staff had been instructive in their decision-making and had provided them with additional information about the donor:Although it was rare for mothers to discuss the donor as a potential future partner, one mother did describe wondering whether ‘there might even be a spark’ , referring to the romantic potential of future contact with the donor. Some also suggested that it was regrettable that the donor was ‘missing out on this wonderful thing’  he had helped to create, thereby alluding to ‘traditional’ family circumstances.His age, no I don't think his age was on it, no. Um, what else was on it, oh I can't remember now! No, there were some other things, but I can't remember what they were.I was [given information] but I never wrote any of it down, and I never thought to … ask them to write it down … After so many tries, they did so many different donors and I think I'm probably getting muddled up.Several mothers commented upon the donor as an ‘unknown’ entity; some responded to questions about the information they had about the donor in ways that clearly indicated that they had not incorporated information about him into their family narratives, but had rather rendered him ‘absent’:I don't really know much about the donor … No I don't know anything. I know they, they offered to give me a [pen] sketch, and I said no.Mothers describing the donor as ‘unknown’ varied in their thoughts and feelings about their lack of information about him. Although it is possible that the participants quoted above were in fact not comfortable to share information about the donor with researchers, others explicitly stated that they had refused information:I didn't know if I had another child that the reasons could be disclosed, or there could be some claim or something, just something that would come back and haunt me in the future.One mother indicated that in order to maintain her representation of the donor as ‘unknown’ she had chosen not to have more children after legislative changes to donor anonymity:I thought he was a known donor, and he actually isn't … The donor probably gives me the creeps, actually. He's this strange person that she will never, you know … a white blank page.Relatedly, although several mothers expressed indifference about the fact that they could not contact the donor, others reflected upon the practice of anonymous donation—and their resultant representation of the donor as ‘unknown’—in highly negative terms. This was particularly true of mothers who wanted more information about their identity-release donor, but were currently unable to access it, and for those who at the time of treatment had believed their anonymous donor to be identity-release:It doesn't matter how much stuff you read about someone on a piece of paper, you can never know what they're like, really, unless you meet them, and you're not gonna meet them.Some mothers who had received further information about their identity-release donor nevertheless also described him as an ‘absence’ who was difficult to think of as anything other than a ‘ghost’ or ‘stranger’:I think maybe as she gets older it will get a bit more difficult because, you know, she'll want to ask more questions and want more answers and it may get to the stage where I can't answer some of her questions … about her donor …I'm hoping it won't be such a big deal that she'll go, ‘I can't really be bothered [to contact him]’.Mothers describing the donor as ‘unknown’ also differed in their thoughts and feelings about how this might impact upon the future conversations they may have with their children about him. While some mothers expressed some anxiety around this issue, others described how they hoped that their children would follow suit in ‘absenting’ the donor from their families in the future:The clinic is under an obligation to make sure that there were no [problems in the] medical history involved, so I'm quite happy that they did their job properly.The sperm's tested so much they, like any defect or anything just doesn't get through.Mothers who mostly described the donor as an ‘absence’ were also likely to stress that they had used a fertility clinic to create their family, and to emphasize the use of ‘donor sperm’—rather than a ‘sperm donor’—in their path to parenthood:So I just told her that um, a nurse … identifying a person, rather than just a strange, um, helped mummy to have you in a very very special way.Some mothers specifically described how they had chosen to undergo donor insemination in a clinical setting because of the potential risks involved in using a known donor or a donor outside of a licensed fertility clinic who may later become ‘present’ in their child's life. Those who conceptualized the donor in this way also tended to describe him to their children as ‘part of a process' that led to them becoming a mother, instead emphasizing the contribution others had made to their child's conception, such as fertility clinic staff:The clinic was additionally described as significant by participants who labelled the donor as an ‘absent’ figure who was also ‘out of sight’. Several mothers explained that they were advised by clinic staff to use a donor with similar physical attributes to themselves, so that there would be ‘no obvious signs of someone else being involved’ . Indeed, mothers mostly described choosing a donor based on the fact that his characteristics aligned with their own, or those of their family members.I didn't want [my child] to look different to us. I mean it probably doesn't matter anyway, because at the end of the day he's my dad's double, isn't he? … I don't think any other genes in that sense got a look in.As it happens, my daughter's the spitting image of me as a child … and [my son] is very like the men in our family … so it's obvious my genes were the strongest anyway!Although some of the mothers who chose to use a physically similar donor described being able to identify his characteristics in their children, others suggested that they had been successful in physically—and therefore genetically—‘absenting’ the donor from their families:I don't really talk about him as a person, cos he's not really a person, is he, in our lives? He's not, we don't see him on a personal basis.I forget that there is a donor … it's not a big part of our lives.In addition to mothers suggesting that the donor's ‘absence’ was reinforced by him being ‘out of sight’, some participants explained that the donor was not relevant to their everyday experience of family life. Portraying the donor as ‘out of mind’, these mothers described an overarching lack of interest, and a general lack of wondering about him or his life:The findings of this study reveal marked diversity in single mothers' representations of the donors involved in their paths to parenthood. While most mothers conceptualized the donor as an ‘absent presence’, that is, as a figure important to—although not physically present in—their families, others were more likely to describe the donor as an ‘absence’. The analysis revealed that mothers in the latter group differed in their feelings about the donor's ‘absence’, such that for some, the perceived inability to make the donor ‘present’ was a source of difficulty and anxiety, and for others, it was not. These findings incite reflection upon the existing literature on representations of the donor in single-mother families. In contrast to the idea that single women may be congruent in their views of the donor , the preIndeed, the experiences of the participants in this study show that complex and changeable feelings about the donor may be experienced by mothers in both one and two-parent families. As in previous research that has indicated that mothers' feelings about the donor are likely to change over time, and especially post-birth , the analysis also complicates the idea that donor information may be especially desired by recipients who intend to tell their children about their donor conception , and/or specific donor information, is made available. The study makes clear that marital status cannot be assumed to determine recipients' thoughts and feelings about the donor or donor information. Nor can these thoughts and feelings be assumed to be static. Given that the children in the families studied were still of a relatively young age, longitudinal research on single-mother families formed through donor insemination is now recommended.All authors were involved in study design, data acquisition, analysis and interpretation. This manuscript was drafted by S.Z. and has been approved by all authors.Wellcome Trust [097857/Z/11/Z]. Funding to pay the Open Access publication charges for this article was provided by the University of Cambridge.This study was supported by the The authors have no conflicts of interest to declare.


The scope of functional heterogeneity in macrophages has been defined by two polarized end states known as M1 and M2, which exhibit the proinflammatory activities necessary for host defense and the tissue repair activities required for restoration of homeostasis, respectively. Macrophage populations in different tissue locations exist in distinct phenotypic states across this M1/M2 spectrum and the development and abundance of individual subsets result from the local and systemic action of myeloid colony-stimulating factors (CSFs) including M-CSF and GM-CSF. These factors have relatively non-overlapping roles in the differentiation and maintenance of specific macrophage subsets. Furthermore, there is now evidence that CSFs may also regulate macrophage phenotype during challenge. Cell culture studies from multiple laboratories demonstrate that macrophages developed in the presence of GM-CSF exhibit amplified response to M1 polarizing stimuli while M-CSF potentiates responses to M2 stimuli. As a consequence, these factors can be important determinants of the magnitude and duration of both acute and chronic inflammatory pathology and may, therefore, be potential targets for therapeutic manipulation in specific human disease settings. The mononuclear phagocyte system is known to exhibit substantial functional heterogeneity –3. Thoughi and Ly6Clo/CX3CR1hi. The former is likely the source of most infiltrating inflammatory macrophages or dendritic cells, while the latter is a patrolling and longer lived cell population that provides maintenance of the vascular endothelium  induced by M1 polarizing stimuli. Correlates of the M2 phenotype include IL-10, TGFβ, arginase, YM1, FIZZ1 (in the mouse), the mannose receptor (CD206), and scavenger receptors, such as CD163 induced following exposure to IL-4, IL-13, TGFβ, IL-10, and other agents.Classically and alternatively activated (M1 or M2) macrophage phenotypes are defined by the specific molecular characteristics induced in response to prototypic pro- and anti-inflammatory stimuli  , 15, 18.in vivo and the variability in markers of polarization.In this context, M1 and M2 phenotypes are often presented as relating to the actions of Th1 and Th2 cell subsets, respectively, because of the Th1 and Th2 lymphokine-based modulation of their defining features , 21. It Macrophage populations may also exhibit a predisposition for polarization toward the M1 or M2 phenotype. For example, different strains of mice and rats have been shown to have skewed patterns of activation potential that ultimately correlate with their innate and adaptive immune functionality , 24, 25.The myeloid colony-stimulating factors (CSFs) M-CSF and GM-CSF are known to modulate macrophage phenotype , 27. WhiWhile receptor signaling mechanisms mediating responses to M-CSF and GM-CSF are appreciated in basic detail , 6, 26, in vivo will always occur in a complex stimulus environment, these additional agents are likely to co-operate with or antagonize the actions of the CSFs. PPARγ, in particular, has been reported to be required for development of alternatively activated macrophages in the context of insulin resistance and metabolic inflammatory disease . Second, resident macrophage populations are generally found to exhibit an M2-like phenotype under resting conditions, consistent with the need to minimize tissue damaging inflammatory reaction  What mechanisms are involved in skewing responses to polarizing stimuli? Can we identify specific CSF-induced patterns of gene expression that are requisite to generating macrophages predisposed for more potent responses to cytokines and pattern recognition receptors? Can we correlate outcomes obtained in vitro with those obtained in vivo? (2) What are the sources and timing of M-CSF and GM-CSF expression within specific tissues during different forms of inflammatory response? Which myeloid cell populations are the targets of the CSFs and how is this co-ordinated with the need to enhance or diminish specific aspects of function over the full course of inflammatory response? (3) Finally, we must begin to consider not only the mechanisms through which M-CSF and GM-CSF operate but also how these stimuli are integrated with the host of other agents encountered within inflammatory settings that also have marked influence on M1/M2 skewing?Macrophage heterogeneity or phenotype polarization is an area of high current interest and the impact of myeloid CSFs on this process The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most offered pancreases are not transplanted. This study investigates the factors that inform and influence the transplant surgeon’s decision to select an offered pancreas.Semi-standardized interviews were conducted with 14 highly qualified transplant surgeons from all 14 German transplant centers performing > 5 pancreas transplantations per year. The interviews focused on medical and non-medical criteria on which the individual accept/refuse decision depends. Interviews were recorded, transcribed and underwent content analysis.The interviewees agreed upon certain main selection criteria, e.g. donor age, lab results, ICU stay. However, there was no consistency in judging these parameters, and clear cut-offs did not exist. The pancreas macroscopy is a pivotal factor, as well as knowing (and trusting) the donor surgeon. 3/14 surgeons reported that they had occasionally refused a pancreas because of staff shortage. Some interviewees followed a restrictive acceptance policy, whereas others preferred to accept almost any pancreas and inspect it personally before deciding.The assessment of medical donor characteristics is highly inconsistent. Both very cautious as well as very permissive acceptance policies may render the allocation process less efficient. A more standardized policy should be discussed. Finally, better training for donor surgeons seems advisable, in order to increase trust and thus pancreas utilization. Whole organ pancreas-kidney transplantation has become the standard treatment for type 1 diabetes mellitus patients with renal failure . HoweverWhen an organ is consented to be allocated for transplantation, the physician or surgeon from the transplant center with the patient ranked highest on the waiting list is contacted by phone . In paraThe objective of this study was to analyze(1) Which medical donor characteristics have an impact on a transplant surgeon’s decision to accept or refuse an organ offer;(2) Whether there are differences in evaluating medical donor characteristics between transplant surgeons of different centers with regard to acceptance of an offered pancreas;(3) To what extent non-medical reasons, e.g. staff shortage, may play a role in the accept/refuse decision in pancreas transplantation.In Germany, 163 pancreases from donors after brain death were transplanted in the year 2010  . The interview partners (n = 14) met the following inclusion criteria:employment at a transplant center performing >5 pancreas transplantations per year (according to 2010 data)minimum of three years of experience in pancreatic transplant surgeryroutine responsibility for decisions on accepting or refusing offered donor pancreasesPurposive sampling was used to recruit the sample. Two transplant surgeons from transplant centers with less than five pancreas transplants per year were interviewed first in order to pre-test the interview guide; they provided comments on the questions  conducted all the interviews, each of which was audio-taped and lasted for 20–70 minutes.Interview tapes were transcribed verbatim. The transcripts were de-identified and continuously numbered (Interview Partner = IP 01-IP 14), so none of the researchers, except the interviewer, could link the answers back to the interviewed transplant surgeon or the respective transplant center. The transcripts were examined using thematic content analysis . Key theNo authorized ethical approval was needed as per the ethics committee of the University of Regensburg (reference number 11-160-0192).The 14 transplant surgeons reported to be performing transplant surgery in authoritative positions for 3–15 years. All interview partners with the exception of one reported to be routinely involved in donor surgery of the pancreas as well, being a member of a regional recovery team. According to the interview partners, transplant surgeons have the sole responsibility of the accept/refuse decision for offered pancreases in 11 transplant centers, whereas a joint decision between transplant surgeons and nephrologists is the usual procedure in three transplant centers.As illustrated schematically in Figure 1. Donor medical historyAll interview partners mentioned at least 3–6 donor characteristics that are considered decisive for evaluating a pancreas offer. Some of these characteristics were named by almost every interview partner: age, length of ICU stay, cause of death, lab results, and co-morbidities. Other characteristics, such as BMI, history of alcohol abuse, or administration of blood transfusions were named less frequently. When asked about resuscitation of the donor, the majority of interview partners denied this criterion to be of importance.While there was a broad consensus on the donor characteristics that are relevant to evaluate a pancreas offer, the cut-offs used for some of these characteristics varied substantially between interview partners.Donor age. Donor age plays a prominent role for the evaluation of the pancreas. The upper age limit which was considered acceptable ranged from 40 to 55 years of age. The majority of interviewed surgeons pointed out that the donor age needed to be judged in relation to other donor characteristics: the more risk factors were present, the less tolerable is an age above 45 or 50 years.With a 50-year-old, you need to check what other factors there might be…How is his/her glucose level? Does co-morbidity play a role? Does the offered organ appear to be from a biologically younger donor? (IP11).Two surgeons, however, reported to have fix cut-offs for donor age.We have relatively strict guidelines… It starts already with age. As a rule, we do not accept organs that are more than 40 years old. (IP 06)Length of ICU stay. Most interview partners explained that they appraised the length of ICU stay in relation to other donor characteristics; however, there was a wide range of rough cut-offs mentioned (from 5 days to 2–3 weeks up to 20–30 days).[The length of ICU stay] should not exceed a week. If it’s two and half to three weeks, I would be skeptical about the organ. (IP 10)[I don’t draw] a line there. There are patients who have stayed in the ICU for three or four weeks. With this deadline of one week and then rolling their eyes, saying: ‘Oh, 10 days already!’ For us, this is nonsense; it’s no reason to turn down an organ. (IP 05)Lab results. The majority of interviewed surgeons reported to routinely check the laboratory values of the donor, but none named concrete limits. The donor’s lab results are only one of many aspects that influence the accept/refuse decision. Some surgeons stated that lab results play a minor role compared to the organ macroscopy.Interdependence of donor characteristics. Most surgeons explained that as a rule, none of the mentioned donor criteria alone justified an organ refusal; different factors need to be weighed against each other. Organs may be declined for a number of relative risk factors. It became clear, throughout the interviews, that different factors were not assessed in relation to each other in a systematic process, but very individually and intuitively for every donor.The decision is cumulative: old age, pancreatitis, poor circulation, and then - on top of that - maybe overweight. Then you add it all up. But refusing it [the organ] just because of a single criterion, we wouldn’t do that. (IP 05)The refusal reasons that are given to the ET, these are just an accumulation of bad gut feelings. (IP08)P-PASS: The P-PASS, a score system combining several risk factors of the donor, does not serve as a decision aid; four out of 14 interview partners mentioned that they use it, but explained that its significance was either limited or decreasing in importance.Matching between donor and potential recipient. The majority of interview partners stated that it is not crucial that the donated organ matches well with the potential recipient, e.g. in terms of size – other than e.g. in liver transplantation.Evidence base. Although not explicitly asked in the interview, the surgeons expressed that their criteria and cut-offs are based on various grounds, as presented in Table 2. Organ macroscopyThe surgeons stated unanimously that the macroscopy of the pancreas is the key parameter for judging its quality. Many surgeons described it as superior to information about the donor’s general medical condition for the assessment of the pancreas quality.If the CRP is a little high, it doesn’t mean that you can’t transplant [the pancreas]. It’s the same with lipase: as long as it isn’t above 1000 [U/l], it doesn’t bother me. If it [the pancreas] is macroscopically fine and recovered well, you would certainly transplant it. (IP 09)For me, the surgical parameters are more important, but the nephrologists primarily look at age, lab results, sodium, amylase, … creatinine, glucose (…). I rather look at the parenchyma (…), vascularization [and the] cold ischemic time. (IP 07)3. Confidence in recovery teamPancreas macroscopy as viewed by recovery team. Normally, the transplant surgeons who need to decide to accept an organ have to rely on the donor surgeon’s judgment of the organ macroscopy, often communicated via phone. If the recovery team describes the pancreas quality as poor, transplant surgeons react differently, according to the interviews, as shown in Table The policy of eagerly accepting offered pancreases with the aim to inspect the organs personally Technical quality of pancreas recovery. Almost all interviewed consultants agreed that the success of the transplantation is dependent upon a skilled organ recovery. In principal, this factor cannot influence the accept/refuse decision prior to actually receiving the accepted organ in the transplant center; however, there were hints that a lack of confidence in the donor surgeon’s competence might influence the decision to accept an organ.For me, it’s essential to have a good donor and an excellent recovery. That‘s why I‘m convinced […] that you can say that your reason for refusal was that the recovery surgeon had no clue. And I‘m one of the few people who gives this as the reason. (IP04)4. Capacity of transplant centerThe vast majority of the interviewed transplant surgeons was convinced that transplant centers in Germany occasionally turn down pancreas offers due to staff shortage. However, nine out of 14 clearly ruled out that option for their own respective hospitals, declaring that refusals on the grounds of staff shortage were ‘nonsense’ or ‘a non-issue’. According to the interview partners, the main reason for capacity problems is a limited number of experienced transplant surgeons in a hospital, coinciding with sickness, holidays or scientific conferences. Three interviewed surgeons admitted that they had already refused organs for capacity reasons.I’m with a center now where this [capacity] problem is not really the issue. Because we’ve spread ourselves out pretty well…So there’s always someone there who is available and who is able to transplant a pancreas. I could certainly imagine though, that it’s different in smaller centers, because I already know how difficult it is for us to organize ourselves this way. (IP 11)I have already refused organs because it simply wasn’t possible, logistically. On Easter Sunday, an organ offer arrives, and the surgeon on call is sick. Then I have to refuse the organ for organizational reasons. (IP 07)We inform all potential recipients on the waiting list about the possibility of a capacity shortage – for whatever reasons: ICU bed, surgeon, other things… I have already cancelled organs due to capacity problems. We communicate this to our patients in a transparent way. (IP 08)Whereas two interview partners emphasized that it was the obligation of a transplant center to have the necessary resources for transplantation available at any time and under any circumstances, others (6/14) described capacity problems as unavoidable or even legitimate.If you provide a transplant program in your hospital, you have to make sure that the organs that come in can be transplanted. […] You also have the responsibility for the patient, who has been on the waiting list for years. And then say, ‘Oh, there were three others in parallel and there was only one surgeon who had to take care of all of them” – the patient on the waiting list will not understand that.’ (IP05)[A transplant surgeon] is allowed to get sick, to go on holiday…, meaning that he can‘t [operate]. And I can very well imagine that someone says, due to logistical reasons [they can‘t do it]. And this shouldn‘t be seen as a reason to stigmatize these people. (IP04)It transpired in the interviews that transplant surgeons might be led into using medical reasons as a pretext when an organ needs to be refused for organizational reasons.5. Previous turn-downs by other centersIf the pancreas had previously been offered to other transplant centers by Eurotransplant and had been refused, it was of little to no relevance for most interview partners.We always look at the organ individually. You can’t imagine what we have experienced – pancreases are refused for the most diverse reasons. We always want to see for ourselves what’s there. (IP 12)In four out of 14 interviewed surgeons, however, there was a tendency to consider or to at least be influenced by the decisions of other transplant centers.I ask what was the reason [for turning down the organ]. This is for me, above all, a time-saving factor. When I get a phone call at 3 a.m., and they tell me there had already been 3 centers. And these 3 centers are certainly not that stupid, they must have had some reason. (IP 08)As a rule, a good organ is accepted by the first center. If it isn’t accepted, this is already sort of suspicious. (IP 07)6. Patient’s prognosisThe interviewed surgeons pointed out that a pancreas transplant differs from a liver, heart or lung transplant in that the indication is, as a rule, not vital. Therefore, the organ needs to be selected more carefully, because the willingness to compromise is smaller than in cases of life-threatening conditions. This may lead to a cautious acceptance policy.If I am in doubt, I’d rather refuse. Simply because the recipients are so selected. You can hardly expect them to put up with the possibility that it doesn’t work well afterwards […] If you don’t transplant the recipient, the chance that he or she dies is small. If you give him or her a bad organ, the chance that he or she dies is actually given. You simply have to consider that. (IP 08).The waiting time …for pancreas-kidney-transplants in Germany is… not so extreme that you need to rush someone into transplant surgery…I cannot endanger a human being in order to perform a transplant when I know that an organ from a good pool will arrive within the next 18 months anyway. (IP 13)This flexibility in accepting or turning down an offered pancreas is accentuated by the lack of clear standards and cut-offs; therefore almost any pancreas can be refused.And most of the donors do have some flaw…. And then this one flaw can be made into a big deal, and that’s why we only utilize only [so few] of the offered organs. And then there is the question: how many flaws do you want to accept? If you don’t want to run any risks, then you don’t accept any flaw. (IP 03)7 Strategic aspectsSome further factors which relate to expectations of the hospital administration or aspects of competition (with other transplant centers) might also influence the decision making process, although this was only hinted at in few interviews.Here, we need to justify ourselves very well, in the clinic and to our boss, and need to have good reasons if we turn down such an offer. (IP 05)In addition, younger surgeons who are inexperienced or surgeons from a center with a higher rate of complications might decline an organ offer in order not to risk a surgical failure, as two interviewees surmised.A lot of this is about experience. A center that, for example, has had many complications will certainly be more restrictive. And when there’s a tiny little thing [about the pancreas], they say, for safety reasons, that they refuse it. A center with more experience and fewer complications will surely loosen its criteria…Experience plays a major role there. (IP 09)From the interview results we could gain insight into the decision-making process of transplant surgeons who are offered a pancreas: The main factors that influence the decision  are relatively consistent throughout the interviews. However, when analyzed in depth, there are extreme differences between the surgeons’ assessment and general handling of pancreas offers. Firstly, while nearly all of interviewed surgeons list the same medical donor factors as being relevant for pancreas selection , the assessment of these factors varies substantially between the surgeons; no standardized or consistent cut offs exist. For example, some surgeons told the interviewer that they had no doubts about accepting the pancreas of a 55-year old if the other parameters were favorable, whereas others claimed not to accept the pancreas of a donor over 40.Secondly, the macroscopic appearance of the donor organ and the technical quality of the pancreas recovery are the predominant factors which influence the decision. In Germany, the transplanting surgeons usually do not perform the pancreas recovery as well, so the macroscopy is described to them on the telephone. The transplant surgeons differed significantly as to whether or not they relied on the donor surgeon’s assessment of the macroscopic pancreas quality. Some prefer to have the organ shipped to their center even if it has been described as having poor quality. Others trust the donor surgeon’s judgment – some surgeons do so in every case and others only if they know and have confidence in the respective donor surgeon.Non-medical factors play a minor role, but certainly exist; above all, capacity problems can lead to the refusal of pancreases in some centers. Interestingly, the sample was split on the legitimacy of occasional staff shortages.It became clear in the interviews that the relatively benign prognosis of the patients on the pancreas waiting list may induce surgeons to wait for excellent (‘flawless’) organs. This restrictive policy is in contrast to some surgeons’ preference to accept pancreases in a very permissive way – even if turned down frequently before and having been described as macroscopically poor – so that they can inspect and evaluate the organ personally.Although the phenomenon of pancreas under-utilization is well known in European and North American transplantation networks and has been described as a ‘major issue in pancreas transplantation’ , the anaIn order to explain the phenomenon of pancreas under-utilization, few quantitative studies analyzed donor characteristics of transplanted and/or discarded pancreases. Wullstein et al. compared donor profiles of accepted pancreas grafts versus grafts declined due to ‘medical reasons’. They found significant differences between both groups for cause of death, age, BMI, serum Lipase, alcohol abuse and history of smoking. They concluded that these aspects might be the most important reasons to refuse a pancreas . These mThe interviews shed light on some aspects that help explain the under-utilization of donor pancreases.It became clear that transplant surgeons who decide to accept or refuse a pancreas offer always act on conflicting priorities: on one hand, the expectations of patients and hospital administration not to turn down a rare and precious donor organ; and on the other hand, the fear of endangering the patient’s health by accepting an organ that is not flawless. The latter is especially important in pancreas transplantation because unlike liver or heart transplantation, the patient’s condition is usually not life threatening, so there is less willingness to compromise. If many centers dictate a cautious acceptance policy, organs might be refused repeatedly. Consequently a ‘cascade effect’  can ensuThe interview results suggested that the assessment of medical donor characteristics is subjective, inconsistent and hardly standardized. This observation can also be explained by the fact that the interviewed surgeons base their cut-offs on varying evidence or customs. A more standardized approach in terms of cut-offs seems to be difficult, however, because one parameter needs to be assessed in conjunction with other risk factors. One has to discuss whether the allocation process would profit from a better standardization or an evidence-based approach. Particularly younger surgeons who have less experience might benefit from recommendations or guidelines, which can be developed by experts. However, one needs to consider that there is still incomplete evidence on whether or not a pancreas is suitable for transplantation, as recently pointed out by Magione et al. .Within the relatively small community of surgeons who are experienced in pancreas transplantation, donor surgery and pancreas surgery, the aspect of trust seems to be an influential factor in the decision-making process: confidence in the donor surgeon and his or her capacity to a) assess the pancreas quality and b) to recover a pancreas without damaging it, and maybe even trust in the transplant center that had turned down the organ offer prior to one’s own decision. Better and more standardized training or perhaps more rigorous selection of donor surgeons could improve the expertise and thus boost confidence.The assessment of medical donor characteristics is highly subjective and inconsistent. Both very cautious as well as very permissive acceptance policies may render the allocation process less efficient. A more standardized policy should be discussed. Finally, better training for donor surgeons seems advisable, in order to increase trust and thus pancreas utilization.aP-PASS = pre-procurement pancreas allocation suitability score, combining nine donor parameters, e.g. age, body mass index, ICU stay, and (nor)adrenaline use. The DRI (donor risk index), an alternative prognostic score for pancreas transplantation, is not commonly used in Germany and was therefore not included explicitly in the interview guide.The following authors of this manuscript have conflicts of interest to disclose: ML has received consulting fees and lecture fees from Novartis, Astellas, Genzyme, Roche, and Biotest. HJS has received honorarium for lectures, advisory boards, data monitoring committees (DMCs) and research support from Novartis and Roche, honorarium for advisory boards from Bristol-Myers Squibb, and research support from Pfizer. JL has received support to her institution from Novartis in order to cover the travel expenses for KPD.ML and JL had the idea for the study, developed the research design and methods, participated in data analysis and wrote the manuscript. KPD performed the data collection and analyzed the data. HJS contributed to developing the research design, was involved in the discussion of the analysis, and contributed to writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/13/47/prepub


Background. Histoplasmosis is a common fungal infection in the southeastern, mid-Atlantic, and central states; however, its presentation can be atypical. Case Presentation. We report a case of Histoplasma capsulatum infection presenting as slowly progressive weakness in the lower extremities, followed by the development of numbness below the midthoracic area, urinary incontinence, and slurred speech. Brain MRI showed leptomeningeal enhancement, predominantly linear, involving the basal cisterns, the brainstem, and spinal cord. Cerebrospinal fluid analysis showed lymphocytic pleocytosis. Discussion. CNS histoplasmosis is usually seen in patients with disseminated histoplasmosis. Isolated CNS histoplasmosis is rarely seen, especially in immunocompetent patients. Conclusions. Histoplasmosis should be considered in the differential diagnosis of patients experiencing slowly progressive neurological deficit. Histoplasma capsulatum throughout the world. In the United States, it is commonly seen in the southeastern, mid-Atlantic, and central states. Typically, patients become infected through inhalation of fungal spores causing an infection in the lungs. The infection might be asymptomatic but it could lead to flu-like syndrome, infection localized to the lungs, or disseminated infection. Herein, we report of case of Histoplasma capsulatum infection localized to the central nervous system (CNS).Histoplasmosis is a fungal infection caused by a dimorphic fungus calledA 53-year-old woman with no significant past medical history presented with slowly progressive weakness in the lower extremities, new-onset numbness below the midthoracic area, urinary incontinence, and slurred speech. One year prior to presentation, she experienced left-sided transitory weakness involving the face and extremities. She was evaluated by multiple neurologists and her brain imaging studies were unrevealing. She later developed tremor in the upper extremities and she was suspected to have central tremor or Parkinson's disease. The patient never had fever, chills, or sweating; however, she had lost 30 pounds during the three-month period preceding her presentation.The patient grew up on a dairy farm and played in silos. She is married; she lives and works in a farm in rural Kansas, where they grow corn and soy. She has no significant travel history. She denied having sick contacts or significant animal exposure. And she had no history of drug use, incarceration, or exposure to tuberculosis.3, 157 wbc/mm3 , protein 362 mg/dL, and glucose 24 mg/dL. CSF Gram stain showed no organisms and AFB smear was negative. Bacterial culture was negative. CSF studies including cryptococcal antigen, human herpes simplex virus PCR, West Nile virus PCR, Lyme antibodies, M. tuberculosis (TB) PCR, and cytology were all negative. HIV screen, RPR, and QuantiFERON-TB Gold were negative as well. A CT of the chest, abdomen, and pelvis was unremarkable. The patient was treated with antibiotics and steroids and transferred to our hospital 8 days after her initial admission for further evaluation and treatment. At that time, her CSF fungal and mycobacterial cultures were pending.The patient first presented to an outside hospital. MRI of the brain and spine, as well as lumbar puncture (LP), was obtained. The MRI images showed leptomeningeal enhancement, predominantly linear, involving the basal cisterns, the brainstem, and spinal cord. Cerebrospinal fluid (CSF) analysis showed 4 rbc/mm3 , protein 340 mg/dL, and glucose 51 mg/dL. Routine bacterial culture was again negative. C-reactive protein and procalcitonin levels were normal. Repeat brain and spine MRI at our facility showed mild improvement of the meningeal enhancement , glucose 14 mg/dL, and protein 587 mg/dL. The shift from lymphocytes to neutrophils was felt to be due to steroid therapy. The CD4 lymphocyte count was 372 (58.5%), despite treatment with steroids. The work-up at this point focused on ruling out a fungal infection since TB seemed unlikely.After transfer to our facility, a second LP was performed, three days after the initial one. CSF analysis revealed 248 wbc/mm Histoplasma antibody was positive by EIA; however, complement fixation and immunodiffusion assays were negative. Histoplasma urine antigen was 0.68 ng/mL. Blastomyces antibody was negative (immunodiffusion assay). Blastomyces urine antigen was 0.55 ng/dL. Sporothrix antibody was negative. CSF Histoplasma and Blastomyces antibodies were both negative. At this time, fluconazole, which was given initially for empiric antifungal therapy, was changed to liposomal amphotericin B. Repeat Histoplasma urine antigen was 0.71 ng/mL and Blastomyces urine antigen was 0.56 ng/dL. CSF Histoplasma and Blastomyces antigens were both positive, above the limits of quantification.Fungitell was negative. Serum Histoplasma and Blastomyces antibodies were again negative, while repeat CSF Histoplasma and Blastomyces antigens were both positive, above limits of quantification. Serum Histoplasma antigen was measured as well and came back positive, below limit of quantification (<0.4 ng/dL). Two CSF fungal and mycobacterial cultures done at the outside hospital remained negative. One of two CSF fungal cultures, obtained two days after admission to our facility, eventually grew Histoplasma capsulatum one month later. Four weeks after admission to our facility, the patient was discharged to a long-term acute care hospital to continue treatment. She received 6 weeks of treatment with liposomal amphotericin B followed by oral voriconazole with minimal improvement of her weakness during the first two months of treatment. Patient was lost to follow-up afterwards.Last LP, two weeks later, showed further improvement of pleocytosis and protein level . Repeat CSF Histoplasma endocarditis, and cerebral vasculitis with focal ischemia [CNS histoplasmosis is usually seen as a manifestation of disseminated histoplasmosis, at the time of presentation or during disease relapse after discontinuation of antifungal therapy . Isolateischemia . Even thischemia . Histoplasma capsulatum can take up to 6 weeks to grow in culture, which significantly delays the diagnosis and possibly the treatment. In early reports, the sensitivity of Histoplasma antigen detection in the CSF was 38% in all patients and 67% in patients with AIDS. However, the current antigen assay is likely more sensitive than the older one [ Penicillium marneffei. False-positive results can be caused by interfering substances such as human anti-rabbit antibodies, rheumatoid factor, or heterophil antibodies. However, the specificity of the CSF Histoplasma antigen remains high (96%). Anti-Histoplasma antibodies in the CSF have good sensitivity (80–89%) and specificity (83%) and can be a helpful adjunct to establish the diagnosis. Serum anti-Histoplasma antibodies are positive in 86% of immunocompetent patients, 80% of non-AIDS immunosuppressed patients, and 67% of patients with AIDS. Interestingly enough, in our immunocompetent patient, both CSF and serum anti-Histoplasma antibodies were repeatedly negative. Urine Histoplasma antigen has 71% sensitivity and 99% specificity in patients with Histoplasma meningitis, while serum antigen has only 38% sensitivity but ~98% specificity [The diagnosis of CNS histoplasmosis can be challenging. None of the diagnostic tests has high sensitivity, which mandates the concomitant use of multiple tests in order to establish the diagnosis. Even though CSF fungal culture is the most specific test, its sensitivity can be as low as 25%. Large volume of CSF is required to increase the yield. Therefore, at least 10 mL of CSF should be cultured, and cultures should be repeated when additional CSF samples are obtained. Histoplasma antigen [The optimal treatment for CNS histoplasmosis is not known. However, the recommended treatment is liposomal amphotericin B  followed by itraconazole (200 mg 2 or 3 times daily) for at least 1 year and until resolution of CSF abnormalities, including negative


These studies are the first to indicate a crucial role for G-CSF inhibition in promoting protective anti-tumor immunity, and suggest that anti-G-CSF treatment is a potential therapeutic approach for CRC.Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is highly expressed in human and mouse colorectal cancers (CRC). We previously reported that G-CSF stimulated human CRC cell growth and migration, therefore in this study we sought to examine the therapeutic potential of anti-G-CSF treatment for CRC. G-CSF is known to mobilize neutrophils, however its impact on other immune cells has not been well examined. Here, we investigated the effects of therapeutic anti-G-CSF treatment on CRC growth and anti-tumor immune responses. C57BL/6 mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS) to induce neoplasms were administered anti-G-CSF or isotype control antibodies three times a week for three weeks. Animals treated with anti-G-CSF antibodies had a marked decrease in neoplasm number and size compared to the isotype control group. Colon neutrophil and macrophage frequency were unchanged, but the number of macrophages producing IL-10 were decreased while IL-12 producing macrophages were increased. NK cells were substantially increased in colons of anti-G-CSF treated mice, along with IFNγ producing CD4 Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States, suggesting that new treatment approaches are needed . ChronicG-CSF is a pro-inflammatory cytokine with the well established function of inducing mobilization of neutrophils from bone marrow to the periphery . HoweverDespite the well-known actions of G-CSF for neutrophil mobilization, the influence of tumor-produced G-CSF on other immune cells is not clear. There is emerging evidence that both neutrophils and macrophages play a role in solid tumor growth, including CRC , 10, yet+ T cell numbers were markedly enhanced in colons of anti-GCSF treated mice along with evidence of cytolytic activity. This is the first study to indicate the potent tumor immune promoting mechanisms of G-CSF and decreased neoplasm size after G-CSF blockade, suggesting that G-CSF is a potential therapeutic target for CRC. Since the activity of G-CSF has only been examined on neutrophil mobilization in cancer, these previously unknown findings that G-CSF has potent effects on other immune cells is critical for evaluation of new immunotherapies for tumors that produce G-CSF.For this study, we hypothesized that G-CSF supports tumor-promoting immune responses and as such is a potential therapeutic target for CRC. To test this hypothesis, we administered anti-G-CSF or isotype control to AOM/DSS treated mice. Upon completion of the treatment regimen, anti-G-CSF treatment led to an 88% decrease in neoplasm number and an 93% decrease in neoplasm size compared to isotype control. Unexpectedly, colon macrophages and T cell phenotypes were transformed to anti-tumorigenic phenotypes. NK cell and CD8To examine the role of G-CSF in a mouse model of CRC, C57BL/6 mice were administered an AOM injection followed by three rounds of DSS treatments, which is an established model to induce multiple neoplasms between days 40–80 after AOM injection , 15. Her2) compared to isotype control (9.9 mm2)  Figure . Histolo) Figure  compared) Figure . These d+ cells (granulocyte marker indicative of neutrophils) and F4/80+ cells (macrophage marker) were found to be increased in AOM/DSS treated mouse colons compared to control mice. Surprisingly, treatment with anti-G-CSF did not affect the influx of neutrophils into mouse colons  in anti-G-CSF treated mouse colons compared to isotype control treated mouse colons  , 13. Thep Figure . To furthenotype . For bots Figure  and 5C. p Figure . Since te Figure . To furte Figure . Total Ie Figure . Taken tIn vitro, we further found that recombinant G-CSF treatments of colon cancer and gastric cancer cells induced proliferation and migration. In addition to our human study, several others have noted that G-CSF expression in solid tumors promotes tumor growth. In particular, in one study of human skin cancer, G-CSF promoted malignant progression of tumor cells [G-CSF is a pro-inflammatory cytokine that is well documented to mature and mobilize neutrophils from bone marrow. Despite its known effects on neutrophils, G-CSF has been largely overlooked for a role in tumors and the tumor microenvironment. Our studies indicate that G-CSF may have unrecognized, critical tumor-promoting functions in gastrointestinal cancers. We first examined this in human tumors where we found that G-CSF and G-CSFR expression were highly associated with lymph node metastasis, suggesting a link with poor disease outcome . In vitror cells . Furtheror cells . There aor cells , 25–27. or cells , G-CSF min vitro and suggested that G-CSF may induce IL-10 production in cells of the monocyte lineage. In the AOM/DSS model, macrophages expressing markers of both M1 and M2 phenotypes were recently shown to be present [Given the well-known role of G-CSF in mobilizing bone marrow neutrophils, most of the attention in G-CSF research has been focused on this function. Thus, we predicted that G-CSF may affect the immune response in the AOM/DSS model. No change in neutrophil influx was detected so it is possible that neutrophils are recruited before anti-G-CSF treatment or by other chemokines that are induced by DSS treatment such as MIP-2, KC, and CXCL2 –31. Macr present , but G-CGiven that IL-12 also promotes NK function and protective immunity in CRC , we furt+ and CD8+ T cells and found increased numbers in the colons of mice that had been treated with anti-G-CSF compared to isotype control. Both CD4+ and CD8+ T cells were increased in number, IFNγ producing cells were increased, and IL-10 producing cells were decreased concurrently. These results represent a shift from a regulatory phenotype to an anti-tumorigenic phenotype and suggest that G-CSF plays a role in promoting IL-10 producing immune cells that are likely inhibitory in the tumor microenvironment. This concept is supported by the work of others in diseases where inhibitory immune responses are helpful, such as graft vs host disease and diabetes. In several studies, G-CSF induced IL-10 production by T cells [Finally, we examined CD4 T cells , 12, 41. T cells , 43 also+ cells numbers were heightened along with the presence of perforin and granzyme B gene expression suggesting enhanced cytotoxic function. Furthermore, an increase in potentially anti-tumorigenic IFNγ producing CD4+ and CD8+ T cells also indicate improved anti-tumor immunity. Taken together, these results reveal that anti-G-CSF treatment has potent immune-modulating effects that demonstrate the potential for anti-G-CSF as a therapeutic approach for human cancers expressing this cytokine.Our findings provide evidence that anti-G-CSF treatment induces protective anti-tumor immunity in the colons of AOM/DSS treated mice. The decrease in IL-10 with concurrent increase in IL-12 producing macrophages in mouse colons with anti-G-CSF treatment suggests a shift from a pro-tumorigenic to an anti-tumorigenic phenotype. NK and CD82. Neoplasms were counted and measured L x W (mm) under a dissecting microscope. Colon tissues were divided for flow cytometry, quantitative real time PCR (qRT-PCR) and histological examination.Female C57Bl/6 (B6) mice from Harlan Laboratories  were housed under pathogen free conditions. Under approval of the UNM IACUC, at 6 weeks of age the mice received one 12.5mg/kg intraperitoneal (IP) injection of AOM. During days 5–10 and 26–31 after injection mice received 2.5% DSS in drinking water and on days 47–51 they received 2% DSS. At day 54, mice were administered either 25 μg of IgG1 or anti-G-CSF antibody (R&D Systems MAB005 and MAB414) via IP injection 3 times a week for 3 weeks and sacrificed on Day 80 using CORNA was isolated using Ribozol  according to the manufacturer's instructions. RNA concentrations were measured using a Nanodrop . qRT-PCR was performed according to Applied Biosystems'  two-step protocol as we have previously published .2, MultiScribe Reverse Transcriptase (3.125 U/μL) and its buffer, and 1 μg of cellular RNA. The RT step was performed according to the following protocol: 10 min at 25°C, 60 min at 37°C, 5 min at 95°C. Obtained cDNA samples were stored at −80°C and used for the PCR reaction step. The PCR reaction mix was prepared using the Assays-on-Demand™ gene expression assay mix (Applied Biosystems) for mouse 18S, G-CSF, G-CSFR, perforin, and granzyme B  and 2 μL of cDNA were added to the PCR reaction mix. The reaction was carried out according to the following protocol: 2 min at 50°C, 10 min at 95°C (1 cycle), and 15 sec at 95°C and one min at 60°C (45 cycles) on Applied Biosystem's StepOnePlus instrument. The endpoint used in real-time PCR quantification, CT, was defined as the PCR cycle number that crossed the signal threshold. Quantification of cytokine gene expression was performed using the comparative CT method  and reported as the fold difference relative to the human housekeeping gene, 18S mRNA.The RT reaction mixture includes random 2.5 μM hexamers, 500 μM dNTPs, 0.4 U/μL of the RNase inhibitors, 5.5 mM MgClMouse colons and lymph nodes were processed for single cell suspension as previously published  and multColon tissue pieces were cut to 8mg and put into complete RPMI for 12 hours. Supernatants were collected and cytokines were measured by multiplex bead array  using a Luminex™200 machine.t-test for multiple comparisons. Values of P < 0.05 were considered statistically significant.Results were expressed as the mean ± SE of data. Differences between means were evaluated by ANOVA using Student's


Intestinal monocytes/macrophages sustain the intestinal immune homeostasis and might be an attractive therapeutic target for the management of inflammatory bowel disease (IBD). Granulocyte macrophage colony-stimulating factor (GM-CSF) exerts beneficial effects on intestinal inflammation and promotes signal transducer and activator of transcription 3 (STAT3)-mediated expansion of myeloid-derived suppressor cells (MDSCs). However, the full action mechanism of GM-CSF, and especially whether monocytes mediate its therapeutic effects in vivo, had not been previously elucidated.  This review article summarizes recent developments in the immunology of mucosal diseases and describes new aspects of the role of myeloid regulatory cells in IBD and the function of GM-CSF in maintaining the intestinal immune homeostasis in Crohn’s disease (CD). This review article highlights the exploration of stimulating in addition to suppressive therapies for patients with IBD and underpins that myeloid regulatory cells might become a promising novel cell-based therapeutic option. Crohn’s disease (CD) is a chronic relapsing disease and is a manifestation of a dysregulated immune response against the microorganisms of the intestinal flora in genetically susceptible individuals . VariousMonocytes and their derivative cells play an important role in the pathophysiology of IBD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine . IntestiGM-CSF also triggers several different signaling pathways in myeloid-derived suppressor cells (MDSCs) that mainly involve the signal transducer and activator of transcription (STAT) family of transcription factors. MDSCs are a heterogeneous group of immature cells that includes precursors of macrophages, granulocytes, DCs, and myeloid cells at earlier stages of differentiation. MDSCs expand rapidly during inflammation, infection, and cancer and suppress T cell responses . Althougin vivo, had not been previously elucidated.The potential for future cell-based therapies for IBD is enhanced by the advances being made in the understanding of the innate immune system in the intestine. Based on this, any therapeutic approach aimed at modulating monocytes/macrophages as a key player in innate immunity could be a potent way to modulate/manage intestinal inflammation in patients with chronic intestinal inflammation. However, the full action mechanism of GM-CSF, and especially whether monocytes mediate its therapeutic effects in vitro using microarray technology and functional assays taking all potential functions of human GM-CSF-activated monocytes into account . Our findings suggest that the early imprinting of monocytes after activation with GM-CSF is of crucial importance, because monocytes play an important role during the recruitment phase of the innate immune response and have the potential to regulate adaptive immune mechanisms.We hypothesized that GM-CSF might activate monocytes in a way that modulates their function during intestinal inflammation. We analyzed the immunoregulatory potential of GM-CSF on human monocytes + T cells, we observed that GM-CSF-activated monocytes attract T cells and shape their differentiation toward Th2 cells by upregulating T cell-derived IL-4, IL-10, and IL-13 and downregulating T cell-derived IFNγ in vitro. In addition, cocultures of GM-CSF-activated monocytes and naïve T cells led to an induction of Treg, which produce anti-inflammatory IL-10 and TGFβ and have important anti-inflammatory functions [We showed that GM-CSF-activated monocytes combine (i) an anti-inflammatory phenotype, (ii) features of augmented innate immune functions , (iii) the ability to facilitate epithelial healing, and (iv) the regulatory potential on adaptive immunity. GM-CSF-activated monocytes represent an intermediate cell type, combining cell-surface expression characteristics and functional features of different M2-type macrophage subsets . In contunctions .Finally, we showed that GM-CSF may regulate the homing molecules CCR2 and CCR6 on human monocytes , which ain vitro, we next aimed to address the functionality of these cells in vivo in the context of CD. Because systematic analyses in the human system are not feasible, we went on analyzing the effects of GM-CSF-activated monocytes on the murine system. In the first step, we activated bone-marrow-derived murine monocytes with GM-CSF in vitro and analyzed the phenotype and functional properties of murine GM-CSF-activated monocytes in vitro.After having analyzed the specific activation pattern of human monocytes in response to GM-CSF in vitro that this population has a lower capacity for phagocytosis and adhesion but triggers increased generation of reactive oxygen species, which are required for pathogen clearance. Moreover, after lipopolysaccharide treatment, proinflammatory cytokine production was found to be higher in GM-CSF-activated monocytes compared with untreated monocytes. However, despite their phenotypical and functional diversity, murine GM-CSF-activated monocytes mainly seem to represent monocytes in a developmental stage passing toward M2-polarized macrophages with an upregulation of M2 markers and important anti-inflammatory functions [Our gene expression analyses showed that murine GM-CSF-activated monocytes have a similar gene regulation profile when compared with the GM-CSF-dependent gene expression in human monocytes . Like huunctions .In addition, we performed flow cytometry-based staining of various gut homing molecules on monocytes and found, in agreement with our studies on human GM-CSF-activated monocytes, CCR2, necessary for migration toward inflammatory sites, and CCR6, used for the migration toward lymph follicles such as Peyer patches, to be upregulated on murine GM-CSF-activated monocytes .+CD25− T cell-induced experimental colitis when they received GM-CSF-activated monocytes but not untreated monocytes. In agreement with earlier work, we found that Rag1-deficient mice that did not receive GM-CSF-activated monocytes but GM-CSF intraperitoneally after the transfer of T cells were not completely protected from colitis but showed reduced disease severity. Our data demonstrate that the protective effect of monocytes depends on their GM-CSF pre-activation in a T cell-dependent model of colitis [In the next step, we tested whether GM-CSF-activated monocytes have a protective and therapeutic effect against intestinal inflammation in vivo. Since none of the currently available animal models of IBD reflects all aspects of IBD in humans, we used various mouse models of chronic colitis. Firstly, we showed that Rag1-deficient mice  were protected from disease progression during CD4 colitis . Secondl colitis . Thirdly colitis .+ Tregs in the lymph follicles of mice with experimental colitis when compared with untreated mice or those treated with nonactivated monocytes. In an in vitro model in which GM-CSF-activated monocytes were cocultured with naïve T cells, we observed increased proliferation and differentiation of Foxp3+ Treg cells and found that this occurred through a mechanism that involves the CD39-mediated conversion of ATP into adenosine—a mechanism that was previously reported to induce Treg cell differentiation [We next sought to decipher the mechanism by which GM-CSF-activated monocytes confer their therapeutic effects on intestinal inflammation. We found that, compared with control monocytes, the GM-CSF-activated monocytes were taken up faster by the inflamed intestine and stayed there longer, especially in Peyer patches , 16. In + Treg cells. This is in agreement with the work of others, who showed that GM-CSF can influence the interactions between DCs and T cells and in this manner determine a pro- versus anti-inflammatory adaptive immune response via Th1 and Th17 cell responses or the induction of Treg cells, respectively [Taken together, these important data show that GM-CSF-activated monocytes are a specific population of monocytes that has a protective effect against intestinal inflammation Fig. . The benectively .Fig. 2MeGM-CSF bioactivity varies inversely with increasing serum levels of endogenous neutralizing GM-CSF autoantibodies (Ab). GM-CSF Ab as well as defective GM-CSF receptor expression have been described in IBD –25. IncrOur phenotypic and functional studies showed that peripheral blood monocytes from CD patients in clinical remission are not impaired compared to healthy controls . We next757F/F mice is associated with protection from DSS-induced colitis. We hypothesized that the protective role of gp130-dependent STAT3 activation in experimental IBD involves the expansion and activation of MDSCs, in addition to the previously reported proliferative, regenerative, and survival effects on intestinal epithelial cells. We therefore induced acute and also chronic colitis with DSS and analyzed the effects in mice with systemic hyperactivation of STAT3 (gp130757F/F) ± myeloid-specific STAT3-deficiency (LysMcre/STAT3flox). We showed that the resistance to DSS-induced acute colitis in gp130757F/F mice is via myeloid cell-specific STAT3 activation, expansion of granulocytic MDSCs in the colon, and increased production of suppressive and protective mediators. Interestingly, gp130757F/F mice were not resistant to chronic DSS-induced colitis [Several studies have investigated the mechanisms of MDSC T cell suppressive activity in health and disease including experimental colitis models and human IBD , 9. MDSC colitis .Thus, our study identified new immunoregulatory mechanisms of STAT3 during intestinal inflammation. Importantly, the adoptive transfer of MDSCs in different animal models of IBD ameliorates colitis and suggests that MDSCs may be used as the basis for a novel adoptive cellular cell therapy in IBD (–36. In aThe work of others and our in vitro and in vivo data show that GM-CSF-activated monocytes are a specific population of monocytes that has a protective effect against intestinal inflammation as shown in various independent mouse models of experimental colitis. The beneficial effects of GM-CSF-activated monocytes are T cell dependent and involve the proliferation and differentiation of Th2 and Treg cells in the intestine (Fig.


Macrodontia is a rare dental anomaly that refers to teeth that appear larger than normal. Generalised macrodontia can be associated with certain medical conditions and syndromes. This case report presents clinical and radiographic findings of isolated bilateral macrodontia in a 14-year-old child. The patient was referred to the clinic with local crowding of maxillary and mandibular teeth. Radiographic findings revealed the presence of impacted macrodont mandibular second premolar on one side and erupted macrodontic premolar on the other side and their distinct morphological appearance, characterized by large, multitubercular, and molariformcrowns and tapering, single roots. Macrodontia is a morphoanatomical change that can affect any tooth in a way that the body of the tooth is enlarged and roots are smaller. The affected teeth have proportionately shortened roots, and their pulp chambers are enlarged as a result of apical prolongation. Teeth may occur in varying sizes and shapes that do not always correspond to the accepted descriptions. When dental size and anatomy present characteristics that deviate from what is supposed to be accepted range of normality, they are termed anomalies . MandibuIdentification of specific patterns of associated dental anomalies could be related to certain genetic and environmental factors contributing to different dental anomaly subphenotypes. Macrodontia (or megadontia) is a rare dental anomaly characterized by an excessive enlargement of all tooth structures and, in few cases, may be associated with morphological anomalies. Tooth anomaly can be categorized as follows: true generalized (large percentage of dentition), relative generalized (entire dentition), and isolated macrodontia of single tooth . MultiplA 14-year-old female child visited the outpatient department of pediatric dentistry with a chief complaint of irregular teeth in oral cavity. The patient was nonsyndromic and all her vital signs were well within normal range. There was no familial history of any anomaly. On intraoral examination the patient had crowding in the mandibular arch and there was an unusually bulbous second premolar on the right side which had molar-like appearance. Intraoral periapical radiograph of the tooth showed that it had an enlarged pulp chamber and short roots, suggestive of macrodontia . On the Nonsyndromic bilateral macrodontia of mandibular second premolars is an extremely rare dental anomaly with very few cases reported to date. Mandibular second premolars show many variations in their morphology; that is to say, the anatomy of this tooth is particularly hard to predict. There can be variations in the cusp wherein we can see one, two, or three cusps on the buccal and lingual side of the tooth . Apart fBeing an extremely rare condition , macrodoThe mesiodistal size of tooth 35 13 mm) was higher than the 7.3 mm for a normal size of second mandibular premolar as reported by others but lower to range between 10.6 and 13.1 mm for macrodontic premolars reported by Dugmore on this way  mm was h, 15. TheIt is very important for a dental practitioner to be familiar with macrodontia with regard to not only clinical complications but also its management. Macrodontia also can provide valuable clues in detecting its association with many syndromes and other systemic conditions.