There is a theoretical risk of adverse events following immunization with a preservative-free, 2-dose vial formulation of 10-valent-pneumococcal conjugate vaccine (PCV10). We set out to measure this risk. Four population-based surveillance sites in Kenya  were used to conduct a 2-year post-introduction vaccine safety study of PCV10. Injection-site abscesses occurring within 7 days following vaccine administration were clinically diagnosed in all study sites (passive facility-based surveillance) and, also, detected by caregiver-reported symptoms of swelling plus discharge in two sites (active household-based surveillance). Abscess risk was expressed as the number of abscesses per 100,000 injections and was compared for the second vs first vial dose of PCV10 and for PCV10 vs pentavalent vaccine (comparator). A total of 58,288 PCV10 injections were recorded, including 24,054 and 19,702 identified as first and second vial doses, respectively . The risk ratio for abscess following injection with the second  vs first  vial dose of PCV10 was 1.22 . The comparator vaccine was changed from a 2-dose to 10-dose presentation midway through the study. The matched odds ratios for abscess following PCV10 were 1.00 (95% CI 0.12–8.56) and 0.27 (95% CI 0.14–0.54) when compared to the 2-dose and 10-dose pentavalent vaccine presentations, respectively. In Kenya immunization with PCV10 was not associated with an increased risk of injection site abscess, providing confidence that the vaccine may be safely used in Africa. The relatively higher risk of abscess following the 10-dose presentation of pentavalent vaccine merits further study. Streptococcus pneumoniae causes an estimated 14.5 million episodes of serious disease  and over 800,000 deaths annually in children aged <5 years, with most pneumococcal-associated serious morbidity and mortality occurring in low- and middle-income countries , induration, fluctuance, erythema, discharge, local hotness, blisters, red streaks, or swollen regional lymph nodes), circumference of right and left thighs (if there was history of thigh vaccination), interventions/treatments provided , results of laboratory evaluations, if any, outcome of clinical encounter, and clinical impression, such as injection site abscess. Depending on symptom severity, MCH clinics could refer patients with suspected AEFI to a hospital for further evaluation.In the Kilifi HDSS site, data were recorded on facility-based questionnaires by medical staff at any one of 26 health centers within the Kilifi HDSS. Data were checked on-site by the Kilifi VAEIK study project manager and scanned copies of completed facility-based questionnaires were stored. In study health facilities in the KEMRI/CDC HDSS , Rarieda PBIDS  and Kibera PBIDS (Tabitha Clinic) surveillance sites, trained study clinicians entered data on suspected AEFI onto scannable paper forms.Household-based detection of abscesses occurred in the Rarieda and Kibera PBIDS surveillance sites. Trained CIs visited the homes of all PBIDS participants weekly (through June 2011) or once every two weeks (beginning July 2011), and used personal digital assistants or smart phones to administer standard questions about vaccine history and morbidity . If a caAn injection site abscess is a localized soft tissue collection of material, occurring at the site of immunization . For purHealth workers  in all study sites received training on intramuscular abscess identification and management conducted by a pediatric surgeon from the University of Nairobi School of Medicine. In all study sites, health workers and data entry staff were trained on how to complete the VAEIK AEFI case report form used in the health facility-based surveillance. Monthly (KEMRI/CDC HDSS and PBIDS sites) or quarterly (Kilifi HDSS site) refresher trainings, specific to each site, were conducted, and in all sites, health workers were encouraged to complete both the Kenya MoPHS AEFI form and the VAEIK study case report form for all adverse events reported.The primary objective was to evaluate whether the site of injection of a second aliquot of PCV10 was more likely to develop an abscess or related symptoms within 7 days than the site of injection of a first aliquot of PCV10. The 7-day time lag was chosen to optimize the specificity of the AEFI endpoint by restricting to events occurring within a short period of time following immunization. Specificity also was enhanced by limiting surveillance of abscess events to the appropriate thigh that received the vaccine injection. For the primary objective, we computed risk ratios (RRs) using log binomial regression. Ninety-five percent confidence intervals (CIs) and the corresponding p-values were calculated with generalized estimating equations using an exchangeable correlation structure to account for intra-child correlation in injection site abscesses or related symptoms. An additional objective was to evaluate whether an injection site in the leg that received PCV10 was more likely to develop an abscess or related symptoms within 7 days than an injection site in the leg that received pentavalent vaccine. The Mother Child Booklet contained pre-printed instructions directing vaccinators to place PCV10 only into the right thigh, and training provided by the MoPHS at the time of PCV10 introduction instructed vaccinators to place pentavalent vaccine only into the left thigh; for VAEIK analyses, correct vaccine placement was assumed. Exact conditional logistic regression was used to calculate matched odds ratios (mORs) and p-values to evaluate abscess risk among matched pairs of PCV10 and pentavalent vaccine injections received at the same immunization encounter; 95% CIs were obtained using the asymptotic method. Fisher’s exact test was used for risk comparisons with small cell sizes (<5). All analyses were performed in SAS version 9.2 .Pentavalent vaccine was selected as the comparator because it was given according to the same 3-dose schedule as PCV10 and both vaccines were administered in a thigh. In addition, the GSK pentavalent vaccine Tritanrix™-HepB/Hiberix™  already was established in Kenya’s routine immunization program at the time of PCV10 introduction and was considered to have an acceptable safety profile. However, in July 2011, the Kenya Expanded Programme on Immunization initiated a switch from the GSK pentavalent vaccine to a Serum Institute of India (SII) pentavalent vaccine product . Within 3 months of introduction, all pentavalent immunizations delivered through Kenya’s routine immunization program used the new SII formulation.ad hoc analysis to compare observed risks of abscess or related symptoms between the two pentavalent vaccine formulations using data from the Kilifi HDSS, Rarieda PBIDS, and KEMRI/CDC HDSS sites. Data from the Kibera PBIDS site were not included in this analysis because Kibera PBIDS records did not distinguish whether observed abscesses or related symptoms occurred at a PCV10 injection site or a pentavalent vaccine injection site from February 2011 through July 2011 (i.e. whether the abscess was located on the left or right leg), limiting data available for analysis from the GSK pentavalent vaccine period. We also compared risks of abscess and related symptoms for PCV10 separately to each pentavalent vaccine formulation.Following the introduction of the new presentation of pentavalent vaccine, we noticed an increase in the frequency of reporting of abscesses in the control group and decided therefore to do an The VAEIK protocol was reviewed and approved by KEMRI’s Ethical Review Committee (KEMRI #1873) and the Oxford Tropical Research Ethics Committee (#48–10). CDC’s Institutional Review Board approved the VAEIK protocol as non-research; the home-visit assessment of abscesses in Kibera and Rarieda was approved within an amendment to the PBIDS protocol . Data collected in the KEMRI/CDC HDSS were approved through the HDSS protocol . Parents or guardians of all participants provided written informed consent to participate in surveillance of abscess-related health outcomes following immunization, as approved under the respective site protocols . Written informed consent for recording of vaccination history also was provided by parents or guardians in the KEMRI/CDC HDSS site and both PBIDS sites under the same approved protocols used for abscess surveillance. In the Kilifi HDSS, vaccine history monitoring was conducted by staff of the Kenya MOPHS under the Pneumococcal Conjugate Vaccine Impact Study (KEMRI #1433); the KEMRI Ethical Review Committee determined this vaccine history monitoring to be public health surveillance which did not require individual informed consent.A total of 58,288 doses of PCV10 and 46,265 doses of pentavalent vaccine were administered to enrolled infants at all four sites during the VAEIK study period . The larAmong all study sites, a total of 16 injection-site abscesses were identified within 7 days following injections of PCV10 for which aliquot number was recorded . OverallAmong a total of 38,958 matched vaccination encounters in which the infant received both PCV10 and pentavalent vaccine, 17 injection site abscesses were identified within 7 days following PCV10 injection  and 43 abscesses were identified within 7 days following pentavalent vaccine injection  . Across The risks of abscess per 100,000 injections for the SII versus GSK pentavalent vaccines were, respectively, 44 and 0 in the Kilifi HDSS site , 1,304 and 363 in the Rarieda PBIDS site , and 108 and 22 across all study sites except the Kibera PBIDS site  . In matcIn this 2-year, multisite Phase IV study, we did not find evidence of increased risk of injection site abscess following infant immunization with the second compared to the first aliquot of a 2-aliquot, preservative-free formulation of 10-valent pneumococcal conjugate vaccine introduced into Kenya’s routine immunization program in 2011. Further, no increased risk of abscesses was seen following PCV10 compared to pentavalent vaccine (either formulation) administered according to the same 3-dose schedule as PCV10. To our knowledge, this was the first and to-date is the longest Phase IV study of a multi-aliquot, preservative-free vaccine formulation used routinely in a developing country, and our findings suggest that 2-aliquot vial formulations of such vaccines have the potential for safe use in developing country settings, assuming adherence to policies regarding storage and use. We made the incidental observation that the risk of abscess following the 10-dose formulation of pentavalent vaccine was higher than the risk following the 2-dose formulation. This difference was large but it was not statistically significant. Our finding that the 2-aliquot, preservative-free formulation of PCV10 has been safely introduced in Kenya is important because it supports introduction of this vaccine in other developing countries where pneumococcal immunization may substantially reduce morbidity and mortality from pneumococcal disease among young children [For our primary analysis, evaluating the abscess risk of second compared to first aliquot injections of PCV10, the study had sufficient power to detect only large effect sizes. We calculate that all study sites combined had 86% power to detect a risk ratio of 3.5 but only 56% power to detect a risk ratio of 2.5 for abscesses associated with aliquot 2 when compared with aliquot 1. Smaller differences in abscess risk between PCV10 aliquots 1 and 2 might not have been detected because of insufficient sample size. To detect a 2-fold increased risk—which would be of considerable public health importance—the study would need to have been 2.4 times larger overall. Encouragingly, a one-year study of PCV10 AEFI in Ethiopia that was patterned on VAEIK methods also found no statistically significant increased risk of injection site abscess following immunization with the second compared to the first aliquot of PCV10 .The comparison of abscess and related symptom risks of PCV10 and pentavalent vaccine was complicated by the change in the formulation of pentavalent vaccine used in Kenya’s routine immunization program during the study period, from a 2-aliquot presentation manufactured by GSK to a 10-aliquot presentation manufactured by the Serum Institute of India. Because of the potential difference in abscess risks related to the two pentavalent vaccine formulations, we performed stratified analyses comparing PCV10 abscess risk separately to the GSK and SII pentavalent vaccine products, in addition to the planned comparison of abscess risk between PCV10 and pentavalent vaccine over the entire 2-year study period. None of these analyses showed increased risk of abscess following immunization with PCV10 compared to the pentavalent vaccine products, providing reassurance that the preservative-free 2-aliquot vial formulation of PCV10 may be used safely in Kenya. Nevertheless, the power of the study to detect differences in vaccine safety between PCV10 and the originally intended comparator, the GSK pentavalent vaccine, was reduced in the stratified analysis.Given the heterogeneity in our surveillance methods (active versus passive surveillance), differences in risk estimates across sites, and a lack of external validation of our abscess case definitions, a concern might exist that our failure to find an increased abscess risk for the second compared to first aliquot of PCV10 could reflect bias toward the null resulting from nondifferential misclassification errors related to exposures and/or outcomes. We do not have information on the frequency of potential misclassification of the first versus second aliquot of PCV10, which could have reduced the observed effect sizes. However, the incidental observation of an increased abscess risk of the SII compared to GSK pentavalent vaccine confirms the potential of the surveillance methodology to detect systematic differences in abscess risk for products within Kenya’s routine immunization program. Thus, as the VAEIK study did have the capacity to identify programmatic associations between specific vaccine exposures and risks of injection site abscess, it is likely that the study would have identified similar excess risk related to the second aliquot of PCV10, if one had existed.Our estimates of abscess risk should be interpreted in light of the different surveillance practices used across sites. Active surveillance, which was used only in the two PBIDS sites, is normally associated with higher ascertainment of AEFIs, especially for mild symptoms. This is likely to account for our findings that the estimates of abscess risk following immunization were 10–100 times greater in the PBIDS sites compared to the Kilifi HDSS site. Despite standard training in the recognition and case-definition of abscess at each study site, inherent differences between active and passive surveillance processes raise the possibility that the clinical entities classified as abscess may also have differed across sites.We incidentally observed that the risk of abscess was 4.8-fold higher across study sites following immunization with the 10-aliquot vial SII pentavalent vaccine formulation compared to the 2-aliquot vial GSK pentavalent vaccine formulation. This association was not statistically significant and could have resulted from sampling error. The association most closely approached statistical significance in the Kilifi HDSS site (p = 0.088), which contributed the largest share of vaccine observations among the four study sites. It also is possible that some proportion of the observed difference in abscess risk for the SII versus GSK pentavalent vaccine could have resulted from increased sensitivity of VAEIK surveillance procedures over time, or from other aspects of the VAEIK study design that were not tailored to properly evaluate the safety of the change in pentavalent vaccine formulation. There may have been misclassification of exposure regarding the formulation of pentavalent vaccine used during the period of transition from GSK to SII pentavalent vaccine in Kenya’s routine immunization program, although such misclassification would have biased our comparison of abscess risk between the pentavalent vaccine formulations toward the null. If the observed association is not attributable to sampling error or ascertainment bias, then it would have significant implications for vaccine policy in Kenya and elsewhere in the developing world. The risk difference between pentavalent vaccine formulations is most interpretable in the Rarieda PBIDS site, where non-zero abscess incidences were observed for both pentavalent vaccine products—there, the risk difference between the 10-aliquot vial and the 2-aliquot vial pentavalent vaccine formulations was roughly 1% , suggesting that 1% of children receiving the 10-aliquot vial pentavalent vaccine may develop abscesses attributable to the new formulation. Abscesses in the Rarieda PBIDS site were identified through active household surveillance which, as noted above, could identify abscess events not severe enough to prompt a health facility visit. Nevertheless, Kenya administers approximately 5 million doses of pentavalent vaccine per year, which, with the 10-aliquot vial product, could be responsible for 50,000 unnecessary abscesses, some proportion of which may be associated with more severe clinical manifestations, like bacteremia and sepsis. The magnitude of the risk ratio across two sites suggests that the potential of contamination of doses of the 10-aliquot vial pentavalent vaccine in Kenya should be further evaluated and additional training on proper handling and administration of this pentavalent vaccine should be considered. Inclusion of a preservative may not wholly counter the risk of contamination in 10-aliquot vial vaccine presentations used in developing countries.This study provides important lessons for vaccine safety monitoring in developing countries. The infrastructure for vaccine safety surveillance is often limited in the very settings that are most in need of expanded access to immunizations. In the current study, even the combination of four field study sites in Kenya with existing surveillance capacity provided a study power of only ~80% to detect a three-fold increased risk of injection site abscess, which is a relatively common potential AEFI. Less frequent but more serious AEFI would be even more difficult to detect with existing surveillance systems. As new vaccines are introduced into GAVI countries, the potential for undetected adverse events is considerable. In addition, the capacity of national immunization programs and global vaccine policy bodies to refute spurious associations  is determined by the robustness of AEFI surveillance systems. HDSS sites and similar health surveillance platforms in developing countries provide useful frameworks for AEFI surveillance, but more such platforms are needed and specific effort and resources should be allocated to develop effective AEFI surveillance programs in multiple sites. The potential for breaks in sterile technique and vaccine-handling practices likely differs from site to site and in different countries in Africa, so substantial additional experience and more post-licensure observational studies will be needed to fully characterize risks associated with use of preservative-free, multi-dose vial vaccine formulations such as PCV10 in developing countries.In summary, this Phase IV multisite study found no evidence to suggest that use of a 2-aliquot vial, preservative-free formulation of PCV10 in Kenya’s routine immunization program was associated with an increased risk of injection site abscesses, related to the second aliquot or overall. The incidental observation of an increased risk of abscess associated with a change in the formulation of the comparator vaccine  during the study period, while complicated, confirms the capacity of the VAEIK surveillance methods to detect relevant, clinically important changes in the safety profile of vaccines administered in Kenya’s routine immunization program and provides context for the findings regarding PCV10.


In cell therapy and regenerative medicine, a reliable source of stem cells together with cytokine growth factors and biomaterial scaffolds seem necessary. As adipose tissue is easy accessible and is abundant source of adult stem cells and can differentiate along multiple lineages, it can be considered as a good candidate in aesthetic medicine. The clinical application of adipose-derived stem cells (ASCs) is reviewed in this article. They could be provided in abundant quantities (millions to billions of stem cells); (ii). They could be differentiated into many cell lineages in a regulatable and reproducible way; (iii). They could be harvested by minimally invasive techniques; (iv). They could safely and effectively be used in either an autologous or allogeneic host; and (v). They could be produced according to current Good Manufacturing Practice guidelines.1 iPSCs as the other group of stem cells are derived from differentiated cells including skin fibroblasts and keratinocytes by transduction with a combination of many transcription factors involved in reprogramming. These cells are phenotypically and functionally indistinguishable from ES cells. However, their production is not dependent on a source of embryos.3Among stem cells, ESCs have the potential of extensive self-renewal, expansion and differentiatiation into any type of somatic tissues making them for future use in regenerative medicine.1,4iPSCs could potentially be used in construction of tissue-engineered skin even several limitations were seen in practical use of iPSCs and ESCs including cellular regulation of teratoma formation, immune concerns and ethical considerations for them and problems in genetic manipulation of iPSCs.4 They are called multipotent mesenchymal stem cells (MSCs) and are non-hematopoietic adult stem cells of mesodermal derivation seen in many postnatal organs and connective tissues.4 MSCs either derived from bone marrow or adipose tissue were shown to be suitable candidates for cell therapy. Among them, bone marrow MSCs (BM-MSCs) are a heterogeneous group of multipotent progenitor cells with the potential of self-renewal and differentiation into cells with ectodermal, mesodermal and endodermal characteristics.5 They have the intrinsic potential to leave the bone marrow, circulate in the blood and home to injured tissues.6 BM-MSCs are a standard population in regenerative medicine due to their high differentiation capacity and low morbidity during harvesting. But harvesting in bone marrow aspiration is still considered as a painful procedure and the quantity of cells acquired is usually low.7The third group of stem cells are postnatal adult stem cells which by their nature are immuno-compatible, and there are no ethical concerns for their use.8 trabecular bone,9 synovial membrane,10 pericytes,11 peripheral blood,12 skeletal muscle,13 skin,14 periodontal ligament,15 deciduous teeth,16 and umbilical cord.17,18MSCs, with identical characteristics to BM-MSCs were isolated from various tissues such as periosteum,19 Therefore, adipose tissue was considered an attractive alternative source which can be provided in large quantities from adipose tissue fragments .20Based on the limitations with these BM-MSCs including low number of harvested cells, limited amount of harvested tissues and donor site morbidity or patient discomfort in providing a sample, there was a need for ex vivo expansion or further manipulation of these cells before their preclinical or clinical used to satisfy the safety and efficacy requirements.21 So adipose tissue has successfully been used as autologous fat grafts for structural fat grafting in lip, facial, and hand rejuvenation and body contour improvement, but a relatively lower level of attention was directed to their application in cosmetic, plastic, and reconstructive surgery.22-26Based on the reports of the American Society for Aesthetic Plastic Surgery, about five hundred thousand elective liposuction surgeries were undertaken each year in the United States.19 making the use of autologous ASCs an appropriate research tool and cellular therapy.27 Lipoaspirates provide an easily obtainable source of ASCs at a frequency of 1:100 to 1:1500 cells exceeding the frequency of MSCs from bone marrow 500-fold, while 1 g of adipose tissue was shown to yield nearly 5,000 ASCs.28Adipose tissue is considered as a source of MSCs, termed adipose-derived stem cells (ASCs). They are ubiquitous and easily obtained in large quantities with little donor site morbidity or patient discomfort29 Several researches were undertaken on tissue remodeling and differentiation of ASCs into specialized somatic cell types to replace damaged organs and tissues.30 They were demonstrated to be immunoprivileged31 and seem to be more genetically stable in long-term culture32 in comparison with BM-MSCs.33ASCs have also the potential for banking as an alternative or complement to cord blood banking for many therapeutic applications in which MSCs could be used.34 ASCs have mesodermal origin, but they have the potential to differentiate into several lineages of osteogenic, chondrogenic, adipogenic, cardiomyogenic, myogenic, and neurogenic cells.28 They can differentate into tissues of endo- and ectodermal lineages such as hepatocytes, pancreatic islet cells, endothelial cells, neural cells, and epithelial cells too.28Adipose tissue is composed of mature adipocytes constituting about 90% of the tissue volume, and a stromal vascular fraction (SVF) including fibroblasts, endothelial cells, preadipocytes, vascular smooth muscle cells, lymphocytes, resident monocytes/macrophages and ASCs.35 ASCs are available within the brown adipose tissue.36 Fresh SVF cells were shown to be heterogeneous with putative ASCs , endothelial (progenitor) cells , vascular smooth muscle cells or pericytes , and hematopoietic cells (CD45þ) in uncultured conditions..37 Also, freshly SVF cells and early passage of ASCs were demonstrated to present higher levels of human leukocyte antigen-DR (HLA-DR), CD117 (c-kit), and stem cell-associated markers such as CD34, along with lower levels of stromal cell markers such as CD13, CD29 (b1 integrin), CD44, CD63, CD73, CD90, CD105, and CD166.37 Pericyte markers are also expressed by ASCs such as platelet-derived growth factor (PDGF) receptor-b, smooth muscle b-actin, and neuroglial proteoglycan 2,38 while the markers shared by ASCs and MSCs include CD13, CD29, CD44, CD58, and CD166. ASCs like other MSCs can show telomerase activity even lower than the cancer cell lines revealing the capacity of ASCs for self-renewal and proliferation.39 Puissant et al.40 reported the absence of HLA-DR expression and the immunosuppressive properties of ASCs. Fang et al. showed that severe steroid-refractory acute graft-versus-host disease (GVHD) could be treated with ASCs from HLA-mismatched donors.41It was shown that ASCs harvested from superficial abdominal regions are significantly more resistant to apoptosis than other parts.42 Surgeons have tried to market ‘‘stem cell face-lifts’’ when in fact the procedure involving merely facial fat injections.43Therefore, cell therapy and regenerative medicine have opened a window as an interdisciplinary field of clinical use of stem cells focussing on the repair, replacement or regeneration of cells, tissue or organs to restore impaired function due to disease, congenital deformities, trauma and ageing. In regenerative medicine, many technological approaches are combined such as stem cell transplantation, gene therapy, the use of soluble molecules, tissue engineering and the reprogramming of cell and tissue types.44In regenerative medicine, tissue engineering combines stem cells, growth factors, and biomaterials for repair of failing organs using fabricate biocompatible scaffolds which would promote cell infiltration and angiogenesis together with production of highly purified, bioactive cytokines in large quantity.ASC therapy in regenerative laboratories and clinical settings was used in treatment of wound beds with a poor blood supply and for healing of radiation injuries. The safety and efficacy of ASCs in reconstructive medicine were evaluated in many clinical trials. The number of these trials had an increasing trend from a total of nine in December 2009 to 18 by May 2010. These trials investigated the efficacy of ASCs in treatment of many diseases and disorders .45 bone tissue repair,46 graft-versus-host disease,47 immunosuppression48 and multiple sclerosis.49 Augmentation of soft-tissue deformities using scaffolds seeded with ASCs were previously reported.50 ASCs or preadipocytes were used as seeded ones on hyaluronic acid-based scaffolds in 12 volunteers resulting into matrix deposition and cell infiltration. But the hyaluronic acid-based scaffolds could not support pre-adipocyte survival and were not inductive towards adipose tissue formation.50 ASCs were also evaluated in clinical case studies for soft tissue augmentation,et al. used adipose-derived SVF cells in augmentation of soft tissues by cell-assisted lipotransfer (CAL)51 in treatment of breast augmentation and facial lipoatrophy. In facial lipoatrophy, no complications or adverse side effects were noticed. Yoshimura In augmentation of the breast, the augmentation of breast was successful and had satisfactory clinical results without any major complications too.52In regenerative medicine, skin is an attractive model organ for the use of stem cells. Recent breakthroughs in understanding the role of ASCs in wound healing and tissue regeneration have lead to new options for treating difficult wounds. It was shown in a diabetic animal that the topical administration of autologous ASCs together with a type I collagen sponge matrix could accelerate the healing of diabetic ulcers.53Another clinical study on treatment of radiation-induced tissue damages by administration of human ASCs resulted into progressive improvement in tissue hydration and new vessel formation due to the release of growth factors such as VEGF and HGF leading to the subsequent angiogenesis and proliferation of keratinocytes or dermal fibroblasts.54 including a basal keratinocyte population found in the interfollicular epithelium and stem cells residing in the bulge region of the hair follicle.55 The other reservoir of skin stem cells is the bulge region of the ORS of hair follicles. Stem cells provided from hair follicles could be differentiated into the ORS of the hair follicle.56It was shown that epidermal stem cells might provide a prominent source of multipotent stem cells to replace damaged tissues and lead to wound healing56,57 Also as ASCs have gained popularity in regenerative medicine, improvement in methods to assess their reproducibility, safety and quality of the vitro expanded stem cells is important. In addition, producing cells that are genetically stable is a step towards getting insurance that the cells would not transform, leading to genetically aberrated progeny when transplanted into the recipient.57The application of ASCs and other stem cells in treatment of many medical conditions has been reflected in several mass media. But it is of great importance in scientific and medical communities to balance the hope from the hype and just time will clarify whether, someday, healthy subjects will voluntarily undergo liposuction to donate fat identically when they donate blood.Based on the available data in literature, further studies are needed in both basic and clinical sciences on application of ASCs to clarify if ASC-supplemented fat transplants are really safe and effective and also on the costs, graft survival, predictability of findings, ease of procedures and aesthetic outcomes. The authors declare no conflict of interest.


Mitogen- and stress-activated kinases (MSK) 1 and 2 are nuclear proteins activated downstream of the ERK1/2 or p38 MAPK pathways. MSKs phosphorylate multiple substrates, including CREB and Histone H3, and their major role is the regulation of specific subsets of Immediate Early genes (IEG). While MSKs are expressed in multiple tissues, their levels are high in immune and neuronal cells and it is in these systems most is known about their function. In immunity, MSKs have predominantly anti-inflammatory roles and help regulate production of the anti-inflammatory cytokine IL-10. In the CNS they are implicated in neuronal proliferation and synaptic plasticity. In this review we will focus on recent advances in understanding the roles of MSKs in the innate immune system and neuronal function. Ciona intestinalis, Ciona savignyi, and Branchiostoma floridae possess a single MSK homolog indicating that MSKs diverged from RSK before the onset of vertebrate evolution. MSK1 and 2 probably derive from a duplication event early in vertebrate evolution; orthologs of both MSK1 and 2 exist in the elephant shark, Callorhinchus milii  activates PKA in macrophages and acts synergistically with TLR agonists to induce IL-10. This effect is dependent on CREB, but does not require PKA-mediated CREB phosphorylation , CCL3 and CCL4 were reduced by Ro318220, an inhibitor that targets MSKs amongst other AGC kinases. In support of a role for CREB, transfection of PLB-985 cells, a neutrophil-like cell line, with a dominant negative CREB reduced IL-8 and TNF induction  and human cytomegalovirus (HCMV), cause human disease. Upon infection these viruses can either enter a lytic replication cycle to produce further virions or enter a latent phase , a condition resulting from the expansion of a polyglutamine tract in ataxin-1, results in neurodegeneration in the cerebellum and brain stem Manto, . Ataxin-in vivo (Brami-Cherrier et al., The striatum is also involved in addiction. MSK1 regulates glutamate-stimulated Histone H3 phosphorylation in cultured striatal neurons and cocaine administration activated MSK1 in the striatum The hippocampus plays important roles in encoding memory and is involved in rodent models of spatial memory and contextual fear conditioning. CREB is implicated in synaptic plasticity and memory (Shaywitz and Greenberg, In primary cortical neuronal cultures stimulated with the neurotrophin BDNF, MSK1 was the critical isoform for both CREB phosphorylation and the induction of CREB-dependent IEGs (Arthur et al., In contextual fear conditioning mice learn to associate a specific context with an aversive stimulus. MSK1 activation has been shown to occur during this process (Sindreu et al., in vivo (Baroncelli et al., Although the role of MSKs has not been reported in LTP, their role has been investigated in homeostatic scaling. This is a form of non-Hebbian plasticity that allows a neuron to co-ordinately regulate the strengths of all its synaptic inputs in order to maintain its firing rate within physiological boundaries Davis, , thus alin vivo in health and disease.This review highlights the critical importance of MSKs in limiting inflammation in innate immunity and their role in inflammatory disease, however their role in the adaptive immune responses remain uncharacterized. MSKs are also implicated in neurodegeneration and synaptic plasticity. Currently, much more work remains to uncover their precise roles in these processes of inflammation and neurodegenerative disease. Outside these systems, work has recently suggested further roles for MSKs. For example MSKs may be involved in skin tumor formation (Chang et al., All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.SA is funded by the Medical Research Council and Arthritis Research UK.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.


It is a transmembrane, highly glycosylated protein with several isoforms resulting from alternative gene splicing. The CD44 molecule consists of several domains serving different functions: the N-terminal extracellular domain, the stem region, the transmembrane domain and the C-terminal tail. In the nervous system, CD44 expression occurs in both glial and neuronal cells. The role of CD44 in the physiology and pathology of the nervous system is not entirely understood, however, there exists evidence suggesting it might be involved in the axon guidance, cytoplasmic Ca CD44 is a transmembrane glycoprotein mediating cell responses to the extracellular microenvironment. Also known as the hermes antigen, Pgp1, MDU3 and Inlu-related p80 glycoprotein, it was first described as a surface molecule present in lymphocytes, thymocytes and granulocytes , but also extracellular matrix (ECM) glycoproteins and proteoglycans, growth factors, cytokines and matrix metalloproteinases Figure . The amiThe human gene for CD44 consists of 20 exons and is located in the 11p13 locus , epileptogenesis, response to injury, neurodegeneration and brain tumour invasion , was decreased upon CD44 knockdown.Recently, a novel signaling pathway has been described in pyramidal neurons of the hippocampus and cerebral cortex, involving CD44 and Src tyrosine kinase-induced cascade, regulating Golgi apparatus morphology and dendritic tree arborization , has been described in sensory neurons  and FAK cascade. Since Ca2+ clearance from the neuronal cytoplasm regulates a number of Ca2+-dependent processes in neurons, including excitability, plasticity and neurotransmitter release, it appears that CD44 might be involved in all the above processes. Moreover, hyaluronic acid has been implicated in hippocampal synaptic plasticity by modulating postsynaptic L-type voltage-dependent Ca2+ channels  and serve as a platform for signaling molecule assembling. For instance, in the peripheral nervous system, CD44 enhances the neuregulin signaling by mediating the ErbB receptor heterodimerization in Schwann cells of the developing peripheral nerves  activity which, in turn up-regulates the phosphorylation of the cytoskeletal protein, cortactin. This HA/CD44 interaction with Rac1-PKNγ leads to cytoskeleton activation and enhanced astrocyte migration  treatment or use of blocking antibodies was associated with increased MFS  rather than with CD44 expressed by OPCs , as compared with the normal brain tissue , convert to reactive cells that lost their bushy-like morphology and become multinucleated and hypertrophic (Sosunov et al., Chronically elevated CD44 expression and HA accumulation occur in non-human primate CNS with normal aging as a result of age-related astrogliosis and are linked to the aberrant accumulation of OPCs (Cargill et al., in vitro and in vivo (Kuppner et al., cis-acting elements contribute to the downregulation of CD44 gene expression in neuroblastoma cells (Shtivelman and Bishop, Several studies have demonstrated the correlation between high CD44 expression and poor prognosis in patients with brain tumour. CD44s and CD44v are frequently expressed in primary brain tumours and seem to be essential for the invasive growth of various CNS-derived tumour cell types Overall, it appears that both the overexpression and lack of expression of CD44 might be involved in the development and invasiveness of various brain tumour types. The results of the performed studies are consistent with the hypothesis put forward by Herrlich and colleagues, stating that CD44 might play a dual role in cancer pathogenesis, acting as either an oncogene or a tumour-suppressing factor (Herrlich et al., In conclusion, there is growing evidence that the previously underappreciated CD44 molecule might be a key signal transducer in neurons, where it can act as a c-Met co-receptor to regulate synaptic transmission or through the activation of Src-dependent signaling pathways to influence dendritic arborization and calcium ions clearance. Additionally, CD44 can also modulate many other processes important for neuronal functions summarized in Figure 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.


Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential—impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism.Cells of the green alga  Chlamydomonas reinhardtii has been widely used as a model organism to study photosynthesis  of this organism were presented and used for prediction of metabolic fluxes in different growth conditions  and Flux Variability Analysis (FVA) for the prediction of metabolite flux through a metabolic network reconstruction  were subtracted from maximal fluxes under phototrophic conditions (b). Conversely, minimal fluxes under phototrophic conditions (c) were subtracted from maximal fluxes of mixotrophic conditions (d).FVA is an FBA-based method for characterizing all feasible states of genome-scale metabolic models that satisfy a set of given constraints and objective function  environment, used in conjunction with Gurobi optimizer solver (version 5.6) employing a linear programming based optimization algorithm , flux substitutable (where the range of possible fluxes span zero) and flux blocked (reactions with minimum and maximum flux of zero). This method uses FVA results to determine whether a flux is always required for optimal biomass production, is potentially used for optimal biomass production or is never used for optimal biomass production.To determine which reactions and metabolic pathways are active or dormant in a particular condition, a reaction essentiality analysis was performed from the non-overlapping reactions derived from FVA  were used in this study. Cells were cultured in 250 ml conical flasks with 200 ml medium, which were placed in a shaker at 180 rpm and illuminated on a 16 h light/8 h dark cycle with light provided by warm-white LED lamps (color temperature 3000–3200 K), at 140 μmol m−2 s−1, 22°/16°C day/night. Mixotrophic cultures were grown in a Tris-Acetate-Phosphate (TAP), with added potassium phosphate (pH 7.0) and Hutner trace elements  was measured using an Ocean Optics USB4000 spectrophotometer . Specific growth rates (μ) were derived from cells in an exponential phase of growth, from two time points  with associated OD readings used in the equation below.For inoculation, 250 μl phototrophic cultures in exponential growth phase were axenically transferred to 250 ml conical flasks containing 200 ml of the appropriate medium. Following introduction of algae, cell growth was monitored at intervals for 12 days, until a stationary or death phase was reached. The optical density of cultures at 680 nm . Intact C. reinhardtii cells in culture medium were placed into the chamber, maintained at 20°C and continuously stirred. Saturating light was provided by a Led Engin LZ4 warm white LED  driven by a laboratory built constant current power supply. Rates of photosynthesis were normalized to cell number for each sample. Oxygen evolution was determined every 24 h from Day 4 until cells reached a stationary phase, expressed as oxygen evolution on a cellular basis.Measurements of O2 evolution steps of the TCA cycle are bypassed, resulting in retention of organic carbon by the cell. Model simulations are consistent with published experimental data, which have shown that ICL activity is essential for efficient mixotrophic growth  whilst mixotrophic growth was simulated using the phototrophic model as a base model and allowing the uptake of acetate at an experimentally defined rate Hoober, . All othIn phototrophic simulations, pyruvate, originating from carbon fixation is converted into acetyl-CoA and fed into the TCA cycle. ICL activity was redundant and the model chose to export isocitrate to the cytosol in exchange for malate. Cytosolic isocitrate was ultimately converted into biomass and the newly imported malate was converted into oxaloacetate and exported into the cytosol which fed back into pyruvate metabolism.In addition to increasing fluxes through enzymes involved in the TCA cycle and localized in the mitochondria, acetate also increased fluxes associated with the glycolytic pathway. Oxaloacetate, generated in TCA cycle reactions is converted into glycerate 2-phosphate (2PG) and exported into the cytosol, where a flux partition occurs, resulting in the majority of flux continuing through gluconeogenesis leading to the formation of both starch and sucrose, accumulating within biomass. Both starch and sucrose are the main products of photosynthesis , the primary reaction of photosynthetic carbon fixation. The reactions of gluconeogenesis in the mitochondrion are otherwise inactive. The predominant flux from 3PG was branched toward the Calvin-Benson cycle in the chloroplast. A further branching of flux toward erythrose 4-phosphate (E-4P) and sedoheptulose 7-phosphate (S7P) and their immediate removal from gluconeogenesis toward carbon fixation is observed. The resulting flux toward glucose 1-phosphate (G1P) production was reduced due to branching in the network and when compared to mixotrophic simulations, was reduced by a factor of 2.4, and as such, only starch was accumulated within biomass.Phototrophic metabolism Figure  on the o2 into organic compounds. In mixotrophic simulations  and fructose 6-phosphate (F6P) via the reversible action of the enzymes ribulose 5-phosphate epimerase (RPEh) and transketolase (TKT2h) respectively. It is with these reactions that the OPPP combines with the Calvin-Benson cycle and gluconeogenesis and ultimately demonstrates the interconnectivity of photosynthesis with central carbon metabolism.In addition to altering fluxes in central carbon metabolism, acetate is also predicted to modulate fluxes in photosynthesis and the Calvin-Benson cycle. The Calvin-Benson cycle uses the products of photosynthetic electron transport, ATP and NADPH, to convert COs Figure  the Calvb6f from ferredoxin. As a result, ATP production resulting from CEF increased without production of NADPH, as described previously. The activity of CEF was otherwise non-evident with phototrophic simulations  and 1546 (70%) respectively]. Flux essential reactions for phototrophic growth and mixotrophic growth comprised the lowest subset of reactions with 16 essential reactions in each condition, representing less than 1% of reactions, whilst 626 reactions (28.6%) were blocked for phototrophic simulations verses 629 (28.7%) for mixotrophic growth.2 transport into the cytosol (CO2t), the conversion of ribulose 5-phosphate and Xu5P via ribulose-5-Phosphate 3-Epimerase (RPEh), cytochrome b6/f complex (CBFC), ferredoxin-NADP+ reductase (FNORh) and the two photosystems (PSI and PSII). Here, it seems that the activity of CSm, allowing for the entry of acetyl-CoA into the TCA cycle, and PEP carboxykinase suggest that TCA cycle itself is fundamentally important to provide a heightened biomass for mixotrophic growth, as these reactions were all substituble for phototrophic growth. The finding that CEF was substitutable for both growth regimes is important indicating its non-essential role in both mixotrophic and phototrophic metabolism.Out of the 16 essential reactions identified under mixotrophic growth, 10 were also deemed essential for phototrophic growth. These reactions all consisted of photon exchange and uptake. The remaining six reactions were classified as substitutable  whilst acetate uptake (EX_ac(e)) and transport into the cytoplasm ACt were blocked. There were no blocked reactions observed for mixotrophic growth, however, there were seven substitutable reactions that were classed as essential for a phototrophic simulation. These included COiCR1080 to predict primary metabolism with respect to phototrophic and mixotrophic growth. To further asses the reliability of model predictions, growth rate, and oxygen evolution, were measured to compare predicted growth and photosynthetic oxygen evolution to address the initial question of the reported down regulation of photosynthesis with mixotrophic growth.We used the modified C. reinhardtii cell growth occurred following a logistic curve for both phototrophic and mixotrophic cultures  to evaluate the down regulation of photosynthesis associated with a mixotrophic growth regime, in the presence of acetate. Furthermore, model predictions have been validated using experimental results presented here and can be compared to results in the literature (Merchant et al., It is widely accepted that growth in the presence of acetate induces a down regulation of photosynthesis in the model organism Mixotrophic simulations included the uptake of acetate set at an experimentally determined level and allowed for the free exchange of gasses. It has been previously reported that the incorporation of acetate into acetyl CoA might occur via two possible pathways. The first pathway involves a two-step reaction involving ACK, prior to phosphate-acetyltransferase whilst the second pathway involves a single step conversion catalyzed by ACS (Johnson and Alric, C. reinhardtii has the capacity to convert acetate to acetyl-CoA in the cytosol, into the mitochondria through the TCA cycle, into the glyoxylate cycle and in the chloroplast due to localization of the enzyme ACS in these organelles. Our analysis suggests that acetate taken up into the cytoplasm is converted to acetyl-CoA by the enzyme ACS. However, our essentiality analysis did show this reaction to be substitutable for both conditions. Therefore, we are not able to conclusively say which reaction is responsible for acetate assimilation.Different models contain different structures, constraints and different optimization criteria, hence used to address different questions. Based on the annotated genome and metabolic reconstruction, Regardless of the primary acetate assimilatory reaction, we predict that acetyl-CoA feeds directly into a simplified TCA cycle, using the mitochondrial ICL with reducing power being consumed through oxidative phosphorylation. Operation of mitochondrial ICL was deemed essential for mixotrophic growth, but substitutable for phototrophic growth. Our model simulations are thus consistent with the experimental observation that acetate induces the expression of ICL and that ICL is required for growth on acetate (Plancke et al., 2 fixation within the chloroplast, yet still exhibiting a greater biomass than phototrophically growing cells. The suppression of CO2 fixation by acetate has been predicted under a mixotrophic growth regime at low light (Boyle and Morgan, C. reinhardtii mutants devoid of active Rubisco are still able to grow in the presence of acetate, albeit sub-optimally, compared to wild type (Pinto et al., 2 fixation indicating an overestimation of the inhibitory effect of acetate.Acetyl-CoA was then fed into the mitochondria with the export of succinate toward mitochondrial respiration and increasing flux toward ubiquinol. In addition, acetate increased fluxes along the pathway of gluconeogenesis whilst suppressing CO6fb complex. Previously, De Oliveira Dal'Molin et al. (6fb complex and feredoxin NADPH oxidoreductase has been characterized (Iwai et al., 2. As such, our mixotrophic simulations suggest C. reinhardtii prefers to assimilate acetate rather than CO2 by way of suppressing the CO2 fixation step, thereby alleviating the requirement for NADPH. Acetate assimilation via ACS however still requires ATP (Morales-Sanchez et al., We observe an increased flux through the CEF reactions, consistent with the suggestion that CEF around PSI explains the down-regulation of photosynthesis (Johnson and Alric, n et al.  reportedn et al. . Consistn et al. . The genn et al. . The wayn et al. . CEF alsn et al. . HoweverHere we have presented a GEM which is able to predict and explain the role of CEF in acetate metabolism while demonstrating the non-essentiality of CEF. It is well known that acetate metabolism leads to an increased growth rate and biomass. Model simulations are able to reproduce this and suggest that an enhanced biomass is achieved in mixotrophic cells by increased fluxes into starch and sucrose metabolism resulting from increased gluconeogenesis. In addition, the OPPP was active resulting in the incorporation of 6PGC into R5P. R5P is used in the synthesis of nucleotides and nucleic acids, supporting greater cell profilation and increased biomass (Bar-Peled and O'Neill, As a tool, flux balance models are becoming increasingly popular for explaining biological phenomena. As we have shown here, FBA is a powerful tool for data qualification. Of course, assumptions are made in constraint based modeling approaches and, as such, the questions investigated need to be applicable to the methodology being used. To accurately predict the fate of acetate using constraint based modeling techniques presents a challenge due to the presence of isozymes. To fully constrain the solution space requires integration of targeted omics data.We have modeled acetate metabolism in the light and as such, in a photosynthetic organism, metabolic pathways that are specifically active in the night are ignored. For example, starch degradation was constrained to zero to reflect the suppression of this reaction in the light. As a result, although starch contributed to the biomass, its accumulation was not specifically optimized account for low synthesis of starch. Experimental studies have shown that phototrophic growth favors starch accumulation when compared to mixotrophic cultures (Singh et al., Just as the ratio of ATP and NADPH have been shown to modulate the reactions of photosynthesis, elemental ratios of carbon and nitrogen have also been shown to affect cellular metabolism of photosynthetic microalgae (Talmy et al., Chlamydomonas reinhardtii, with an accurate representation of CEF, which is now able to explain the down regulation of photosynthesis observed under mixotrophic growth conditions in the presence of acetate. The predictive capabilities of the model were used to highlight changes in metabolism that were essential for this trait. We can conclude that acetate incorporation into metabolism is achieved via reactions of the TCA cycle before entering central metabolism requiring CEF. CEF serves to support the production of a proton motive force that allows ATP generation without the net formation of NADPH, serving as a balance to meet metabolic demands imposed on the system. The functioning of CEF is however non-essential. In doing so, we have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the cell-wide scale.In conclusion, we have produced a curated version of a GEM of 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.


The last Brazilian guidelines on melanoma were published in 2002. Developmentin diagnosis and treatment made updating necessary. The coordinatorselaborated ten clinical questions, based on PICO system. A Medline search,according to specific MeSH terms for each of the 10 questions was performedand articles selected were classified from A to D according to level ofscientific evidence. Based on the results, recommendations were defined andclassified according to scientific strength. The present Guidelines weredivided in two parts for editorial and publication reasons. In the firstpart, the following clinical questions were answered: 1) The use ofdermoscopy for diagnosis of primary cutaneous melanoma brings benefits forpatients when compared with clinical examination? 2) Does dermoscopy favordiagnosis of nail apparatus melanoma? 3) Is there a prognostic differencewhen incisional or excisional biopsies are used? 4) Does revision by apathologist trained in melanoma contribute to diagnosis and treatment ofprimary cutaneous melanoma? What margins should be used to treat lentigomaligna melanoma and melanoma in situ? Although some concepts have not changed, it is necessary to updatethe standards of practice on this important health problem.Cutaneous melanoma (CM) is one of the most potentially dangerous forms of skincancers, accounting for approximately 90% of deaths. The dermatologist is in theforefront of diagnosis and treatment of CM. It is his duty to keep updated onbest practices in diagnosis, treatment and disease monitoring to be able todiagnose, treat and council patients in the best way. The last Brazilianguidelines on CM were published in 2002.These guidelines are intended for diagnostic and therapeutic approach andfollow-up of patients with suspected or confirmed diagnoses of primary CM (PCM)with no clinical nor histological evidence of metastatic disease . They do not include ocular nor mucosal melanoma.To introduce the most advanced evidences in diagnosis, therapeutic management andmonitoring of PCM clinical stages 0, I and II patients, describing diagnosticpeculiarities that allow identification of these tumors in early stages, as wellas measures most widely accepted for treatment and follow-up in the context ofBrazilian dermatology.PICO , according to regulations of the National Health Agency,the Brazilian Medical Association and Federal Council of Medicine, described in"The process of development, validation and implementation of clinicalguidelines in the private healthcare system in Brazil".2 To answer these questions, aliterature review of scientific articles was conducted in MEDLINE database.Search for evidence was limited primarily to articles published between01/01/2009 and 06/30/2014 and keywords (MeSH terms) present in the title and/orsummary were used, grouped into specific syntaxes for each of the 10 questionsalone, as described in The coordinators have defined 10 questions that reflect issues of clinicalrelevance on the subject. The questions were structured according to the acronymAfter reading the abstracts, articles containing information relevant to thesubject were selected. When appropriate, references present in these papers wereselected, without limit for publication period, and were analyzed using the samemethodology as that for the primarily selected studies.Articles that presented the expected contribution were analyzed regarding thelevel of evidence (Table 2). Recommendations were written in response to thequestions elaborated. Recommendations were also graduated according to level ofevidence .Discussion and recommendations for the first 5 questions appear below. The other 5questions will be presented in a new publication.1) Is the use of dermoscopy for diagnosis of PCM beneficial for patients withsuspicious lesions when compared to clinical examination?For many years dermatologists could only rely on clinical diagnosis when facing asuspicious melanoma lesion. Implications of such diagnosis justified extrememeasures such as removal of large amounts of suspicious lesions, whichsubsequently revealed to be benign (unnecessary resections).3 Althoughincreasing diagnosis sensitivity and specificity, the number of nevus excisedfor diagnosing a melanoma was still high. A few years later the letter "E" wasincluded in the acronym to represent "Evolution" or changes inthe appearance of a suspicious lesion.5A major step in improving accuracy of diagnosis of primary cutaneous melanoma(PCM) occurred with the introduction of "Melanoma ABCD" rule, acronym introducedin 1985, using the initials of four clinical features of skin lesions that maybe indicative of malignancy .Dermoscopy (DMCP) enables the examination of skin lesions, melanocytic or not, andthe observation of structures and colors of the epidermis, dermo-epidermaljunction and superficial dermis, not visible to the naked eye. These findingscorrelate with histological features and are used to determine whether thelesion is malignant or benign and if surgical removal is indicated. Whenintegrated with information obtained from the clinical history and macroscopicexamination, DMCP allows obtaining such a large amount of information, that ithas gradually became an irreplaceable instrument for the dermatologist, such asthe stethoscope is for the clinician.6There was no decrease in specificity, suggesting that the DMCP increasesdiagnostic accuracy without increasing the number of undiagnosed melanomas(level of evidence A).8Naked eye diagnostic sensitivity for PCM (percentage of correctly diagnosedmelanomas) is around 71% whereas with the use of dermatoscope is 90%. Ameta-analysis of 22 studies showed that diagnostic accuracy was higher thannaked eye examination when experts used DMCP (sensitivity 89% and specificity79%) (level of evidence A).9 At a consensus DMCP expertsmeeting, standard analysis algorithm reached the highest specificity,sensitivity and accuracy for melanoma detection when compared with the other 3algorithms (level of evidence B).10 Any of these algorithms presents higher effectiveness thanexamination with naked eye (level of evidence A).13 In summary, when using DMCPto evaluate any suspicious lesion, compared with evaluation by the naked eye,the probability of classifying an actual malignant lesion as malignant ishigher, as well as classifying as benign a truly benign lesion. At this point itis important to remember that even with the use of DMCP accuracy of diagnosismelanoma does not reach 100%. This is due to the lack of dermoscopic featuressuggestive of the diagnosis in very early lesions or in some cases ofuncharacteristic melanomas (featureless).14To achieve these higher efficiency levels it is necessary to use an algorithm foranalyzing suspicious skin lesions. The 4 best known are: Menzies' scoringmethod; pattern analysis; 7-points check list; and ABCD of DMCP, which - it'simportant to note - is different from the clinical ABCD.18 Arandomized study showed 42% decrease in the number of unnecessarybiopsies.16 Thisfinding is similar to a retrospective study of excisions of pigmented lesionsperformed before and after training in DMCP. The rate of benign/malignantlesions decreased from 18.0: 1 to 4.3:1 (level of evidence A).16In addition to increased PCM diagnostic accuracy, use of DMCP presents 2 moreclear benefits: reduction of unnecessary resections and increased diagnosis ofnon-melanoma skin cancer (level of evidence A).19 This learning process is time consuming and in theearly stages of the learning curve worsening of accuracy can occur.20 Dermatologists, physiciansfrom other specialties, paramedical staff and even medical students haveconsistently improved diagnosis accuracy after being trained in DMCP use (levelof evidence A). A considerable number of dermatologists believe that the timerequired for training in DMCP is excessive.21 At the same time, some studies show a low level ofsatisfaction among chief-residents of dermatology in relation to training inDMCP. Many want more intense instruction in the area. There are also complaintsabout the limited availability of courses (level of evidence C).22Importantly, the effectiveness DMCP use depends on formal training and experience.It was demonstrated that dermatologists with formal training and at least3-years experience in DMCP reach higher CM detection rates than non-traineddermatologists (level of evidence B).DMCP increases diagnostic accuracy of PCM compared to clinicalexamination. It should be used routinely in the evaluation ofmelanocytic lesions (grade of recommendation A);The use of DMCP for this purpose requires specific training inorder to achieve the desired accuracy (grade of recommendationA).In addition to being useful in diagnosing PCM, regular use of DMCPalso has the advantage of reducing the number of unnecessaryexcisions (grade of recommendation A);In addition to being useful in diagnosing PCM and reducing thenumber of unnecessary excisions, regular use of DMCP leads to anincrease in diagnosis of non-melanoma skin cancer (grade ofrecommendation A).2) Does dermoscopy favor diagnosis of primary cutaneous melanoma of the nailapparatus (NA)?24Although deemed little frequent when considering that the palm is equivalent to1% of the body surface area and the total area of the 20 nails is much smallerthan the area of the palm, it becomes clear the incidence of NA-PCM isproportionally greater than in the rest of the body.25 In Caucasians NA-PCM responds from 1,4 to2,0% of all melanomas.27 In Afro-american populationit is more frequente, reaching 20% of all melanoma. In American indiansfrequency may be even higher (31%).30 In noethnic group the frequency of NA-PCM is as high as in japanese population. Inthis population, acral melanoma  responds for 50 to77% of all melanomas.32.NA-PCM represents 0.7% to 3.5% of all melanomas.Early diagnosis of NA-PCM improves prognosis. However, biopsies, essential forcorrect diagnosis and staging, carry the possibility of permanent dystrophies ofthe NA. The inherent complexity of surgical procedures in the NA delays greatpart of melanoma diagnosis in this location, closing the opportunity window forcurative treatment. Lesions arousing much suspicion, such as large lesions orlesions causing nail dystrophy, are usually biopsied. However, these are usuallyassociated with more advanced stages and worse prognosis.33 Over the years, signs and methods to assistthe physician in early diagnosis of NA melanoma of have been sought. Among themare:Clinically, subungual melanoma (SUM) at initial stages is often characterized asstriated melanonychia (SM), defined as dark bands growing in the longitudinallyon the nail. Since it is not a pathognomonic NAPCM clinical finding, melanomaassociated SM should be differentiated from those of non-malignant etiology.Early differential diagnosis of SM still remains a challenge for dermatologists,even for nails experts (level of evidence B).a) Clinical examination:35 However, itis not a pathognomonic sign since it is not present in amelanotic melanoma andcan be seen in benign conditions such as melanocyte activation and even inbenign melanocytic nevi (level of evidence A).36 HS should be differentiated from pseudo-HS that may be present in Bowen's disease and in benign conditionsthat cause subungual hyperpigmentation.38 Topical orsystemic drugs can also cause SM, which may or may not be associated with theperi-ungual pigmentation.42Hutchinson's signal (HS), periungual pigmentation with or without SM, ishistorically considered as the most important clinical sign to differentiate SUMfrom benign melanocytic nevi.44 Each letter refers to a characteristicassociated with increased risk for SUM, as it is shown in Clinical examination with naked eye was used for decades as the sole diagnosticmethod. To increase accuracy, ABCDEF rule for SUM was created, like the ABCDrule for CM.b) Nail plate dermoscopy:42DMCP plays a key role in PCM early diagnosis, however, this role is less impactingon the evaluation of SM and SUM. It's important to remember that DMCP helps todifferentiate melanin and blood . There is no evidence of the superiority of polarizedin relation to non-polarized light in the examination of SM.c) Intraoperative dermoscopy:The nail plate is translucent and allows dermoscopic visualization of the pigmentpresent in the nail bed, but visual characteristics are altered, making itdifficult to reach a correct interpretation. If the nail plate is surgicallyremoved, it is possible to perform direct DMCP on the nail bed using a polarizedlight dermatoscope.43 It isimportant to note that, intraoperative DMCP does not replace histologicexamination, which remains the gold standard for diagnosis of SUM and providescrucial data for evaluation of surgical margins, therapy and prognosis (level ofevidence B).Studies correlating nail bed DMCP with histological evaluation of SM allowedidentifying patterns with high diagnostic accuracy (level of evidenceA).44Tangential excision, also known as "nail matrix shave biopsy" allows the removalof lesions larger than 3 mm with minimal risk of scarring the NA, as well asenabling an assessment of safety margin (level of evidence B).in situ PCM.Recognizing and biopsying SUM in early stages is key to achieving cure ratessimilar to those of ABCDEF rule provides a methodology for clinical evaluation of SMand increases clinical diagnosis sensitivity of SUM (grade ofrecommendation A).nail plate DMCP increases accuracy for indicating SM biopsies(grade of recommendation A).Intraoperative DMCP has high level of accuracy in the correlationbetween visualized and histological pattern (grade ofrecommendation A).Histologic examination still remains the gold standard fordefinitive diagnosis of NA melanoma (grade of recommendationA).3) Is there prognostic difference when performing incisional or excisional biopsyof suspected melanoma lesions?45 There are situations where EB is difficult to perform,such as, but not only extensive pigmented lesions on the face, mucous membranesor acral lesions or extensive lesions with low suspicion of melanoma. In suchcases, incisional biopsy (IB) would be less invasive than EB.46Excisional biopsy (EB) with 1 to 3 mm margins is recommended as first choicetherapy for pigmented lesion suspected of melanoma. EB enables betterhistological evaluation, which includes: tumor thickness (Breslow depth), MIandpresence or absence of ulceration. These 3 items are essential to definestaging, prognosis and treatment.46 Theconcern that mechanical manipulation (such as punch biopsy or needle biopsy)could implant neoplastic cells and increase the risk of metastases was great formany years and IB was contraindicated at that time. This potential risk has beendiscussed not only for melanoma, but also for various types of tumors.47Presently it is believed this risk has been overestimated and there areconsiderations that tumor cells molecular characteristics, such as adhesionmolecules, matrix metalloproteinase and vascular growth factors, may influencethe ability to migrate and to induce vascularization, which would be moreimportant in disseminating tumor cells than just mechanicalmanipulation.48Although IB is technically simpler to perform and has lower cost, a point thatshould be taken into consideration since healthcare resources are scarce in manycountries, it allows only a partial histological evaluation of thelesion.57Most of them, especially those with larger numbers of patients, showed that thetype of biopsy does not play a role in the evolution of melanoma patients (levelof evidence A).To assess whether IB resulted in worse prognosis compared to EB, severalretrospective and prospective studies were conducted.58IB can be performed by shave, punch or incisional techniques. A study comparinghistological evaluation of EB, shave biopsy, punch biopsy and IB, showed thattreatment recommendations changed in 6% of the cases: . Thus, it was found thatEB allows better histological evaluation and should be preferred wheneverpossible (level of evidence A).59A prospective study designed to evaluate the impact on staging, area of marginsexpansion, sentinel node positivity, tumor recurrence and survival evaluated 709patients who underwent punch biopsy (23%), shave biopsy (34%) and EB (43%).There was no significant difference in accuracy of sentinel lymph node results,tumor recurrence nor in patient survival. The study concluded that biopsy typedid not impact SLNB accuracy or results, tumor recurrence, or prognosis. Punchand shave biopsies, when used appropriately, should not be discouraged for thediagnosis of melanoma (level of evidence A).46IB may be used for lesions suspected of melanoma located in areas where EB isdifficult to perform, such as - but not limited to - large lesions, lesionslocated on the face, mucous membranes and acral regions or lesions with lowprobability of melanoma (level of evidence B).60When deciding on IB, it must be performed in locations that allow histologicalevaluation of the greater depth of the lesion . DMCP can be a useful tool for selecting the best place for biopsy, butthere are still no studies with large number of patients that support thisrecommendation (level of evidence D).EB should be the preferred technique whenever possible, because itallows better tumor histological evaluation, which directlyimpacts in the conduct and prognosis (grade of recommendationA).IB does not affect prognosis of melanoma patients and can be usedwhen necessary (grade of recommendation A).IB is suggested in the following cases, but is not limited to(grade of recommendation B):Extensive pigmented lesions with low suspicion of melanoma;Suspected extensive lentigo maligna on the face;Suspicious pigmented lesions, extensive or in an acral region;Mucosal lesions.The major axis of the biopsy should be oriented so as to facilitatepossible margins expansion. In general, when performed in themembers it means along the major axis (grade of recommendationC).4) Does the review of histological findings by a pathologist trained in melanomacontribute to the diagnosis and treatment of PCM?61. Experienceand interest of general pathologists on this issue are heterogeneous, as is thefrequency such a neoplasm is found in diagnostic routine in general pathologylaboratories.The gold-standard for PCM diagnosis is histopathology. Roughly, there exist about20 histological items to be evaluated in each case before a final diagnosis isrendered. This is a challenging task, which can be influenced by interpretationbiases62. Since appropriate local treatment and the decisionof performing sentinel lymphnode biopsy rely on MST items, histologic reportmust include at least the following data68:In addition to correct diagnosis, it is essential that histologic reports onmelanoma address microstaging (MST) items, which will be essential fordetermining treatment and prognosisMargin involvement and margin distances from the neoplasm;Presence or not of dermal invasion;In invasive neoplasms:Tumor thickness (Breslow depth);Mitotic index (MI);Ulceration: present or not;Microsatellitosis /satellitosis.71 In order toreduce such frequencies, some of the most influential pathology societiesworldwide created a consensus protocol on mandatory and wishful items to beincluded in melanoma histologic reports.66Studies have shown that information on MI, ulceration, microsatellitosis and tumorthickness are not originally included in melanoma histologic reports in 47-50%,13-72%, 50%, and 29% of cases, respectively.66Correct histological criteria to determine dermal invasion, ulceration, andmicrosatellitosis, as well as proper methodology for measuring Breslow thicknessand mitotic counting are of equal importance.Physicians reading melanoma histologic reports cannot directly check if propermethodology for each item was used correctly in a given case. The only practicalway to achieve this goal is to request a second opinion to a pathologist withexpertise in melanoma or to send the slides to a melanoma referral hospital forhistopathological review.69 Similarly, a retrospective large series showed thatmargin status was modified in 11% of cases after expert review.72Indeed, a recent study has shown that review of thin and in situ melanomas by anexpert pathologist has led to changes in diagnosis and microstaging withsubsequent modification of original proposed local treatment and SLNB in 12% and16%, respectively. (Level of evidence B).73-7)6 On the other hand, microsatellitosis remains a microstaging itemof low reproducibility.75 This data seems very important because the detectionof microsatellitosis in primary melanoma resection (or biopsy) specimen modifiespathologic staging from pN0 (or pNx) to pN2c.69It has been shown that reporting of Breslow thickness, MI, and ulceration has goodinter-observer reproducibility among pathologists.78 It has also been known for a long time that ambiguousmelanocytic lesions can lead to diagnostic disagreement even amongexperts.79 Examplesof ambiguous lesions include atypical nevus with severe cytological andarchitectural atypia, atypical Spitz tumors, etc. Advances in genetics knowledgehave brought profound changes in the understanding of these ambiguous lesions.As a result, the polarization "melanocytic nevus (benign) versus melanoma" has progressively given way to the recognition of a range ofintermediate lesions categorized as low-grade malignant or of uncertainbiological behavior. Although such lesions do not constitute aclinicopathological entity, they form a spectrum with intermediate morphologicaland genetic aberrations lying between classical nevi and melanomas. As a group,these lesions have the potential to evolve with microscopic regional lymphnodemetastases, but they rarely lead patients to death 80. Examples of such lesionsinclude atypical Spitz tumors, atypical blue nevi, and melanocytoma 82.With regard to differential diagnoses of melanocytic nevus versus melanoma,studies report that reproducibility ranges from 73% to 97.3% between general andexpert pathologists.83.Immunohistochemistry, FISH, and other molecular pathology techniques have beenused in order to reduce subjectivity of morphological examination of ambiguouscases (level of evidence B)87.Great effort of the scientific community has been made to stratify thisintermediary group of lesions by fluorescent in situ hybridization (FISH) sothat lesions with higher probability of aggressive behavior may be detected andmanaged accordingly, particularly atypical Spitz tumors (level of evidenceB)Definite diagnosis of melanoma is based on histopathology ofexcision or biopsy specimens (level of recommendation A);Melanoma histologic report must include histological items of MST(level of recommendation A).Histological review by an expert pathologist/melanoma referralcenter should be sought whenever possible before definitivetreatment (level of recommendation B).Immunohistochemistry and molecular studies can be used to aid inthe diagnosis of selected cases of ambiguous melanocytic lesions(level of recommendation B).in situ be excised with?5) What margin should lentigo maligna melanoma and melanoma 89Historically, through the 20th century, resection with circumferential margin of3-5 mm was indicated for PCM of any thickness, followed by grafting. Rationalewas based on the occurrence of "centrifugal lymphatic spread", seen inhistological preparations of excised melanomas.90The primary treatment for PCM is surgical resection.103 These studies demonstratedthat histological findings will determine the appropriate surgical margins, andthe Breslow thickness is the most important data (level of evidence A).Current recommendation is that smaller margins are used, reducing surgerymorbidity. This is based on prospective studies comparing more conservative withmore radical surgeries, on randomized studies and on international consensusconferences.in situ and invasivemelanomas, histological examination with paraffin embedded sections is the goldstandard for evaluation of surgical margins and that margins expansion surgeryshould be performed preferably between 4 to 6 weeks from the initial biopsy(level of evidence C).100It is important to remember that for both Margins recommended for PCM surgical excision appear below .There is no consensus regarding deep margins resection as it occurs for lateralmargins. Historically, the recommendation is that invasive PCM is resected untilthe muscle fascia. There is, however, no evidence confirming that such action isnecessary in all cases. A group of experts recommends that, whenever possible,resection is made to the fascia or to the deep subcutaneous tissue, depending ontumor location (level of evidence B).in situmelanoma (isM) and for LM, scientific evidences regarding the surgical marginsto be used are not as clear as for invasive melanomas. This is because it isknown that large isM, LM and lentigo maligna melanoma (LMM) often presentsubclinical atypical junctional hyperplasia in peripheral and peri-adnexal areasof the lesion, which can extend for several millimeters beyond the visiblemargins.103Although surgical resection is the standard treatment for 104According to Kunishige et al, 9 mm margins would be sufficient in 99% of cases(level of evidence B).105Several authors agree that 5 mm margins may often be insufficient. Many studieshave shown that up to 20% of LM require margins larger than 5 mm, while 12% ofLMM with Breslow thickness up to 1.0 mm require margins with more than 10 mm(level of evidence B).It is also known that recurrent LM and LMM tend to require wider margins (level ofevidence B). The lack of prospective studies suggests that, in the cases oflarge isM, LM and LMM, the determination of the margins is defined by consensusof expert groups or by retrospective studies (level of evidence B).103 The most disseminated is Mohs' method, but severalother methods of margins micrographic control appear in the literature, withdifferent nomenclatures such as 3D histology method, Tubingen pie chart, stagedsurgical excision, etc. Micrographic surgery is a very useful method to treatlesions in areas where healthy tissue preservation is important, such as theface, as well as to allow greater surgical margins control. It also diminishesthe risk of recurrence and may reduce the final size of surgical wounds (levelof evidence C).106The usefulness of the various methods of micrographic control of margins is verydiscussed.111At this point it is important to remember that histological examination byconventional frozen sections are not suitable for determining surgical marginsof melanocytic lesions (level of evidence A). The ideal is to examine paraffinsections. Advances in IHC techniques towards melanocytes identification willallow frozes sections to achieve higher reliability rates.112Some authors recommend the use of Wood's light to assist in assessing the realextent of the tumor (level of evidence D).isM, (imiquimod and radiation) are justified in caseswhere surgery can cause great aesthetic/functional damage or in patients unableto undergo surgery.104 Literature presentsreports of complete response of up to 88% of cases, but it is important toconsider that it is not possible to obtain a complete histological analysis ofthe lesion, thus eventual invasive melanoma foci would not be identified.Histological examination after treatment showed persistence of disease in up to25% of treated patients and there were cases of LM progression to invasivemelanoma. Moreover, diversity of therapeutic regimens and short monitoringperiod limit the comparative studies (level of evidence C).101Use of alternative non-surgical therapeutic methods for treating LM or101Cryosurgery with liquid nitrogen has not been adequately studied, but it's onemore option. There are no comparative long-term studies on the subject (level ofevidence D).RecommendationFor patients with isM, surgical margins of 0.5 cm are sufficient(grade of recommendation B).Treatment margins for large isM / LM are at least 0.5 cm, and itmay be necessary to increase them (grade of recommendationA).The use of micrographic surgery techniques for large isM, LM andLMM can assist in more accurate determination of surgicalmargins, but paraffin sections should be used and, whenavailable, IHC techniques (grade of recommendation B).Use of imiquimod to treat LM or isM, although off-label, isjustified in cases where surgery can cause greataesthetic/functional impairment or in patients unable to undergosurgery. Because data for these situations are limited, thesurgeon must make the decision with the patient (grade ofrecommendation C).At the end of this first part of the Brazilian guidelines on melanoma, it isextremely important to point out that they are not intended to stifle medicalpractice, but to make it more homogeneous, reducing uncertainty/disagreement ongood practice standards. Stablishing standards, besides reducing the differencesin patient care, can also make possible to provide options based on evidence,allowing the physician to make decisions about treatment or diagnostic methods,thus reducing the strain on patients, doctors and on the healthcare system.These guidelines reflect the best scientific information published about thesubject to the date of its preparation. Nevertheless, we must be careful wheninterpreting these data, since the outcome of future studies can lead to changesin recommendations. In some cases, it may be necessary not to follow theseguidelines, always keeping in mind patients' well-being , as well as otherspecial circumstances.