Dermatofibrosarcoma protuberans in childhood: two case reports and review of the literature.

Dermatofibrosarcoma protuberans (DFSP) is a rare skin cancer with intermediate malignancy, characterized by a progressive local growth and a propensity for local recurrence. DFSP is most frequent in adults; however, in recent years, DFSP in childhood emerged to be more common than previously believed. Unfortunately DFSP in children may be misdiagnosed, leading to a delay in the treatment. The authors report two cases of childhood DFSP with unusual clinical presentation: a congenital nodular variant and an atrophic variant developed at 2 years of age, both with acral localization. They highlight the importance of an early diagnosis by pediatricians and dermatologists to ensure an appropriate complete excision and reduce the risks of recurrences.

T regulatory cells mediate immunosuppresion by adenosine in peripheral blood, sentinel lymph node and TILs from melanoma patients.

T regulatory cells (Tregs), involved in tumour tolerance, can generate Adenosine by CD39/CD73 surface enzymes, which identify four Tregs subsets: CD39+CD73- nTregs, CD39+CD73+ iTregs, CD39-CD73+ oTregs and CD39-CD73- xTregs. In melanoma patients, increased Tregs levels are detected in peripheral blood (PB), sentinel lymph node (SLN) and tumour infiltrating lymphocytes (TILs), but Adenosine role was not investigated yet. We examined total Tregs and Adenosine subsets in PB, SLN and TILs from melanoma patients (n = 32) and PB from healthy donors (HD; n = 10) by flow cytometry. Total Tregs significantly increased in stage III-IV patients PB, in SLN and TILs, as compared to HD/stage I-II patients. Tregs subsets analyses showed that: 1) PB nTregs significantly increased in SLN and decreased in TILs; 2) iTregs significantly increased in stage III-IV patients PB and further significantly increased in SLN and TILs; 3) PB oTregs and xTregs significantly decreased in SLN and TILs. Patients clinical features did not significantly influence total Tregs, except SLN excision order. Results confirmed Tregs role in melanoma progression and indicate Adenosine generation as a novel escape mechanism, being nTregs and iTregs increased in PB/SLN/TILs.

CD4+FOXP3+ T regulatory cells decrease and CD3+CD8+ T cells recruitment in TILs from melanoma metastases after electrochemotherapy.

Electrochemotherapy (ECT) represents an effective local treatment for skin unresectable melanoma metastases with high overall objective response rate. ECT is based on the combination of anti-neoplastic drugs administration and cancer cells electroporation. Whether ECT can also activate the immune system is a matter of debate, however a significant recruitment of dendritic cells in melanoma treated metastases has been described. Herein we investigated immediate and late effects of ECT treatment on T cell subsets in ECT-treated lesions by fluorescent immunohistochemistry. Biopsies from melanoma patients (n = 10) were taken before ECT (t0), at d1 and d14 from treatment. At t0, CD3+CD4+ T cells were the most represented T cells, well detected in the perilesional dermis, particularly at tumour margin, while CD3+CD8+ T cells were less represented. CD4+FOXP3+ T regulatory (Treg) cells were present in the perilesional dermis and within the lesion. ECT induced a significant decrease of CD4+FOXP3+ Treg cells percentage in the perilesional dermis, observed at d1 and at d14 (p < 0.001). CD3+CD8+ T cells frequency significantly increased at d14 from treatment in the perilesional dermis (p < 0.001). Furthermore calreticulin translocation to the plasma membrane, a hallmark of immunogenic cell death, was observed in metastatic cells after ECT. The data reported here confirm that ECT induces a local response, with a lymphoid infiltrate characterized by CD4+FOXP3+ Treg cells decrease and CD3+CD8+ T cells recruitment in the treated lesions. These results might contribute to design novel combinational therapeutic approaches with ECT and immunotherapy in order to generate a systemic long-lasting anti-melanoma immunity.

Cd1d is expressed on dermal dendritic cells and monocyte-derived dendritic cells.

CD1 proteins are a family of cell surface molecules that present lipid antigens to T cells. We investigated skin dendritic cells and monocyte-derived dendritic cells for expression of CD1 molecules using a panel of 10 different monoclonal antibodies focusing on the recently described CD1d molecule. By immunohistochemical analysis, CD1d expression in normal human skin was restricted to dendritic appearing cells in the papillary dermis mainly located in a perivascular localization. Langerhans cells did not show detectable CD1d expression in situ. Epidermal/dermal cell suspensions analyzed by flow cytometry demonstrated distinct subpopulations of HLA-DR positive dermal dendritic cells expressing CD1a, CD1b, and CD1c. CD1d was expressed on HLA-DRbright dermal antigen-presenting cells in dermal suspensions (16% +/- 3.6%), as well as on highly enriched dermal dendritic cells migrating out of skin explants (60.5% +/- 8.0%). Migrated mature dermal dendritic cells coexpressed CD83 and CD1d. Western blot analysis on microdissected skin sections revealed the presence of a 50-55 kDa CD1d molecule in dermis, suggesting that CD1d is highly glycosylated in skin. Both immature and mature monocyte-derived dendritic cells cultured in autologous plasma expressed CD1d molecules. In contrast, culture in fetal bovine serum downregulated CD1d expression. In conclusion, antigen-presenting cells in skin express different sets of CD1 molecules including CD1d and might play a role in lipid antigen presentation in various skin diseases. Differential expression of CD1 molecules depending on culture conditions might have an impact on clinical applications of dendritic cells for immunotherapy.

Cyclosporin-A affects the organization of cytoskeleton of normal human keratinocytes in culture.

Cyclosporin-A (CsA) is a potent immunoregulatory molecule which has been widely used in many immunomediated and inflammatory skin diseases. It inhibits the proliferation of keratinocytes, but its possible effects(s) on cell differentiation are poorly known. To address this issue, we have studied the influence of CsA on the assembly of intermediate filaments by normal human keratinocytes in culture. Control keratinocytes were flat; the cells which had not reached confluence stained intensely for vimentin and weakly for cytokeratins; confluent cells stained with intermediate intensity for both types of proteins and the cells adhering on the top of others, interpreted as the best differentiated ones, stained for cytokeratins but not for vimentin. CsA (1.6 micrograms/ml for 10 days) inhibited the growth of keratinocytes, which never reached confluence; most cells appeared small and roundish, only some stained for cytokeratins and few for vimentin. By electron microscopy, a well organized meshwork of tonofibrils was recognized in many control keratinocytes, but never in CsA-treated keratinocytes. We propose that the cytoskeleton could be a target of CsA and that its alteration mediates other effects of CsA on keratinocytes, including those on cell growth.

Human keratinocytes cultured without a feeder layer undergo progressive loss of differentiation markers.

Culture of keratinocytes in conventional medium without a mesenchyme-derived feeder layer leads to poor growth and impaired differentiation; however, the exact pathway and degree of differentiation achieved in such conditions is unclear. We have cultured normal human keratinocytes in Rheinwald and Green's medium, on plastic without a feeder layer, in order to investigate the degree of differentiation that they achieve in these conditions. Intermediate filament proteins, tonofibrils and desmosomes were assumed as markers of differentiation and their expression was analyzed by immunohistochemistry and electron microscopy. Before reaching confluence, keratinocytes expressed keratin molecules, as well as vimentin, and formed tonofibrils and desmosomes. The expression of these markers was progressively reduced until confluence and was totally lost thereafter, while cultures could be propagated for at least six passages. On the contrary, reseeding on a feeder layer after the first passage led to rapid cell death. It could be concluded that signals from a feeder layer are relevant to support continuous synthesis of intermediate filaments proteins and formation of tonofibils and desmosomes, and that the derangement of the cytoskeleton in these conditions leads to altered, not simply defective, response to delayed stimulation by a feeder layer.

Influence of maternal metabolic control and insulin antibodies on neonatal complications and B cell function in infants of diabetic mothers.

Circulating insulin antibodies at birth and the degree of maternal metabolic control were measured in 68 infants of insulin-treated diabetic mothers. Their correlation with neonatal B cell function and with the clinical features of the infants was evaluated in order to better understand their influence on fetal outcome. Maternal metabolic control was assessed on the basis of blood glucose levels, glycosuria and the occurrence of hypoglycemia and/or ketonuria. All infants were clinically evaluated for gestational age, macrosomia, hypoglycemia, hyperbilirubinemia, hypocalcemia, and respiratory distress syndrome. Cord blood plasma glucose, C peptide, and IgG insulin antibodies were also measured. It was shown that poor maternal metabolic control was associated with a higher prevalence of fetal morbidity as well as with signs of B cell hyperfunction. Also the presence of circulating insulin antibodies correlated well with higher C peptide levels and with several neonatal complications. B cell hyperfunction, indicated by high C peptide levels in the infants of diabetic mothers, may possibly play a causal role in the pathogenesis of fetal morbidity. In conclusion, a good fetal outcome in insulin-treated diabetic pregnancies was associated with and may have depended upon: (1) good maternal metabolic control, and (2) absence or low levels of circulating insulin antibodies.

Foetal macrosomia and erythrocyte acid phosphatase (ACP1) polymorphism in diabetic and normal pregnancy.

Both in diabetic and in normal pregnancy the proportion of macrosomic fetuses is much lower among newborns carrying Pc allele of erythrocyte acid phosphatase (ACP1) than among other ACP1 genotypes. In diabetic pregnancy the well known increased incidence of fetal macrosomia has been observed only among fetuses which do not carry this allele. ACP1 probably functions as a flavin-mononucleotide phosphatase. Since Pc allele is associated with the highest enzymatic activity it is likely that subjects carrying this gene may have a relatively lower concentration of flavin-mononucleotide cofactors and in turn a reduced rate of metabolic activities controlled by flavoenzymes. It is possible that in fetuses carrying Pc, flavo-enzyme activities are regulated at a level that does not allow a full response to stimuli (both genetic and/or environmental) aimed to maximize fetal growth.

'Animal-type' melanoma of the scalp with satellitosis and positive sentinel nodes in a 4-year-old child: case report and review of the literature.

BACKGROUND: Although showing a rapidly rising incidence, paediatric melanoma is relatively rare, accounting for 1-4% of all cases of melanoma and for 1-3% of all paediatric malignancies. The overall survival rate in paediatric patients seems to be similar to that recorded in adults. 'Animal-type' melanoma (ATM) is a rare melanoma subtype, occurring both in childhood and in adults, that shows a close histological resemblance to the heavily pigmented melanocytic tumours observed in grey and white horses. CASE PRESENTATION: We present a case of ATM of the scalp with satellitosis and two positive sentinel nodes in a 4-year-old male child. No other tumour deposits were found in the subsequent regional lymphadenectomy; the patient has been tumour free for 30 months. CONCLUSIONS: We treated our case of ATM in a child as the other types of paediatric melanoma, therefore as an adult melanoma. ATM is generally considered a neoplasm with an indolent course, that occasionally shows an aggressive behaviour, and patient deaths of ATM have been reported. Due to the rarity of ATM, further studies are needed to better define the biological behaviour of this particular melanoma subtype and the therapeutic and follow-up strategies.

Specific immune therapy-related cutaneous B-cell pseudolymphoma with following dissemination.

We report two cases of cutaneous B-cell pseudolymphoma (PCBCL) induced by intradermal antigen injections for specific immune therapy (SIT). In both cases, the lesions had first developed on the area of injection; years later, new lesions appeared far from the original site. The histological, immunohistochemical, and molecular findings of the lesions showed features consistent with the diagnosis of PCBCL in both cases. In particular, the staining for sIg light chains showed a polyclonal pattern, and the molecular analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot showed a germ-line configuration of the Ig heavy chain genes. While the development of PCBCL related to a specific stimulus is well known and widely reported, the development of histologically and immunohistologically identical lesions far from the injection site is definitely worthy of note. This behaviour might be due to the presence of retained antigens in the injection site. This chronic antigenic stimulation could induce the progressive selection--and subsequent dissemination--of antigen-specific B cell clones.

Relationship between ultrasound findings in pregnancy and neonatal morbidity.

In 142 diabetic pregnancies fetal biparietal diameter (BPD), transverse abdominal diameter (TAD) and abdominal circumference (AC) were estimated by means of serial ultrasound examinations. An accurate evaluation of fetal growth was obtained in 85% of the cases. AC was found to have the highest degree of reliability in predicting macrosomia (71%) followed by TAD (60%) and BPD (17%). Placental morphology was estimated at the last scanning before delivery in 85 of the patients. In pregnancies resulting in macrosomic infants the placental maturation seemed to be retarded. In 26 patients a third trimester amniocentesis was performed. A mature (grade III) placenta was always associated with a lecithin-sphingomyelin (L/S) ratio greater than 2, indicating fetal lung maturity but there was no constant relation between placental maturity and the L/S ratio. In conclusion, ultrasound examination during diabetic pregnancy offers valuable information concerning the prenatal diagnosis of macrosomia and could serve as a guidance in the determination of the optimal time and route of delivery. The grading of placental maturity might help to identify fetuses at risk, but further studies in this area are needed before final conclusions can be drawn.

Evidence of selective interaction between adenosine deaminase and acid phosphatase polymorphisms in fetuses carried by diabetic women.

Possible selective interaction between genetic polymorphisms of acid phosphatase locus 1 (ACP1) and adenosine deaminase (ADA) has been investigated in a sample of 211 infants from diabetic women, and in 350 consecutive infants from normal women. Newborns from diabetic pregnancies carrying the ADA2 allele show a lower proportion of BA and CB phenotypes (heterozygotes for the main allele of ACP1 system), compared with both their mothers and normal infants. The observation suggests that, in a diabetic environment, intrauterine selection may act against double heterozygotes for the ACP1 and ADA systems.

Haptoglobin development in newborn infants from diabetic mothers.

Haptoglobin (Hp) development during the neonatal period has been studied in 325 newborn infants from normal pregnancies and in 242 infants from diabetic mothers. In infants from diabetic mothers Hp development is delayed as compared to infants from normal pregnancies. This delay is associated with a change in the pattern of relationship between Hp development and the polymorphism of acid phosphatase (ACP1) (an enzyme which shows phosphotyrosine phosphatase (PTPase) activity). In infants from normal pregnancies who show ACP1 phenotypes with the highest activity, the appearance of Hp is accelerated as compared to other infants. In contrast, infants from diabetic pregnancies who have ACP1 phenotypes with the highest activity, show delayed Hp development.

Genetic and non genetic factors in the outcome of diabetic pregnancy.

In insulin-dependent diabetes mellitus, maternal Phosphoglucomutase genotype is a predictor of fetal macrosomia much more important than quality of metabolic control during pregnancy. In gestational diabetes and in non insulin-dependent diabetes, on the contrary, the most important predictor is the metabolic control of diabetes.

Enzyme polymorphism and clinical variability of diseases: study of acid phosphatase locus 1 (ACP1) in obese subjects.

The ACP1*A allele of erythrocyte acid phosphatase (ACP1) has a lower enzymatic activity when compared to other ACP1 alleles and is associated with maximal rate of body growth during intrauterine life. In three different samples of obese subjects (total number = 218). ACP1*A was associated with extreme body mass deviations. No difference in ACP1 allele distribution was observed between obese and nonobese subjects. These data suggest that a genetically determined variability of ACP1 influences the degree of obesity, but only when obesity itself has been triggered by some other factors.

Diabetic pregnancy: evidence of selection on the Rh blood group system.

The blood glucose levels of pregnant women with insulin-dependent diabetes mellitus and the blood glucose levels of newborns during the first few hours of life show an association with maternal Rh genotype. Distortions of joint maternal-fetal Rh phenotype distribution have also been observed. Because a cluster of genes involved in glycide metabolism is located on the short arm of chromosome 1, the present observations may reflect the action of these genes.

Immunophenotypic analysis of normal human dendritic cells isolated from epidermis and dermis.

BACKGROUND: The skin immune system comprises two types of dendritic cells, i.e. CD1a-positive Langerhans cells in the epidermis and CD36-positive dendritic macrophages in the dermis. Dendritic cells can migrate from skin explants into a culture medium. METHODS: We have examined the morphology and immunophenotype of the dendritic cells migrating from epidermal and dermal sheets in vitro. The epidermis and dermis of keratomes of normal human skin were separated with dispase and cultured for 72 h. At this time, the non-adherent cells in the medium were removed, enriched on a metrizamide or Lymphoprep gradient, counted, prepared by cytospin, and labeled for CD1a, CD36, and HLADr. RESULTS: Cells migrating from the epidermis and dermis show many thin projections or a few veils from the cell surface. Approximately four times more cells migrate from epidermal than dermal sheets from the same keratome. CONCLUSIONS: Using methods to separate the epidermis from the dermis, both CD1a-positive Langerhans cells and CD36-positive dendritic macrophages can be obtained from both tissues, although in different numbers.

Amniotic fluid insulin and glucagon in normal pregnancy and pregnancy complicated by rhesus isoimmunization.

Immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) were determined in the amniotic fluid from 28 rhesus isoimmunized pregnancies with moderately affected fetuses and from 15 normal pregnancies; in umbilical arterial plasma from nine newborn infants with rhesus haemolytic disease of moderate degree and from 19 normal infants; in plasma from their respective mothers at delivery; and in the urine of 13 normal infants at birth. Levels of IRI and IRG in amniotic fluid from rhesus cases were not different from those of normal pregnancies. IRG was detected in the first voided neonatal urine.

[Idiopathic neonatal jaundice. 1. Influence of use of oral contraceptives before pregnancy and of smoking during pregnancy].

PIP: The influence of former oral contraceptive (OC) use and smoking during pregnancy on neonatal bilirubinemia was evaluated prospectively in 371 newborn infants. No significant influence was observed upon bilirubin levels in cord blood, bilirubin increase during the first 24 hours, and incidence of values higher than 12 mg/dl. Correction of the effect of OCs for maternal smoke demonstrated a reduced incidence of values higher than 12 mg/dl when both factors were present together. Correction of the effect of smoke for gestational age demonstrated an increased incidence of values higher than 12 mg/dl in infants of smokers with gestational ages lower than 37 weeks. The correlations, however, were not significant. (author's)^ieng

Enzyme polymorphism and clinical variability of diseases: a study of diabetes mellitus.

We investigated possible relations among four common neonatal manifestations of diabetic pregnancy (macrosomia, hypoglycemia, hypocalcemia, jaundice) and four enzyme polymorphisms (PGM1, ADA, AK1, ACP1 in a sample of infants born of diabetic mothers. The pattern of associations observed between the two sets of variables is consistent with known differences in enzymatic activity within phenotypes of each system, suggesting that low enzymatic activity may have unfavorable effects on fetal development and on adaptability of the neonate to the extrauterine environment, Some of the polymorphic enzymes studied influence fetal growth in normal pregnancy as well. Analysis of relations between genetic polymorphisms and the clinical pattern of common diseases may provide a better understanding of the genetic basis of the clinical variability of diseases within and between human populations.