Immunofluorescence mapping, electron microscopy and genetics in the diagnosis and sub-classification of inherited epidermolysis bullosa: a single-centre retrospective comparative study of 87 cases with long-term follow-up.

BACKGROUND: Epidermolysis bullosa (EB) comprises a heterogeneous group of skin fragility disorders, classified in four major types based on skin cleavage level, i.e. EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), Kindler EB, and in more than 30 subtypes defined by the combination of laboratory and clinical data, including disease course. OBJECTIVES: Our aims were to address whether, in the age of genomics, electron microscopy (TEM) has still a role in diagnosing EB, and whether the genotype per se may be sufficient to sub-classify EB. METHODS: A thoroughly characterized single-centre EB case series was retrospectively evaluated to compare the power of TEM with immunofluorescence mapping (IFM) in establishing the EB type, and the ability of TEM, IFM and genetics to predict selected EB subtypes, i.e. severe dominant EBS (DEBS), severe JEB, severe recessive DEB (RDEB) and DEB self-improving, using genetic and final diagnosis, respectively, as gold standard. RESULTS: The series consisted of 87 patients, including 44 newborns, with a median follow-up of 54 months. Ninety-five mutations were identified in EB-associated genes, including 25 novel variants. Both IFM and TEM were diagnostic in about all cases of JEB (21/21 for both) and DEB (43/44 for IFM, 44/44 for TEM). TEM sensitivity was superior to IFM for EBS (19/20 vs. 16/19). As to EB subtyping, IFM performed better than genetics in identifying severe JEB cases due to laminin-332 defect (14/14 vs. 10/14) and severe RDEB (eight/nine vs. seven/nine). Genetics had no role in self-improving DEB diagnosis; it almost equalled TEM in predicting severe DEBS (eight/nine vs. nine/nine) and enabled to discriminate dominant from recessive non-severe DEB phenotypes and to identify special subtypes, e.g. DEBS with KLHL24 mutations. CONCLUSIONS: Transmission electron microscopy remains relevant to the diagnosis of EBS. IFM and genetics are essential and complementary tools in the vast majority of EB cases.

Linguistic analysis of autobiographical narratives in unipolar and bipolar mood disorders in light of multiple code theory.

BACKGROUND: Discriminating bipolar disorder (BD) from unipolar disorder (UD) is crucial in diagnosing mood disorders. Neurophysiological studies have identified different correlates of emotional regulation in BD and UD. According to the Multiple Code Theory, bodily modifications relate to linguistic styles, as highlighted by studies on the language of depression. Our purpose is to verify the existence in the Italian language of linguistic features of depression differentiating BD from UD to provide tools for clinicians to use beyond self-report measures. METHODS: The sample included 20 BD, 20 UD (all diagnosed using DSM-5), and 20 Control Group (CG) participants. Participants completed the Profile of Mood States (POMS) and an audio-recorded Relationship Anecdotes Paradigm Interview, transcribed and analyzed by the Discourse Attributes Analysis Program for Referential Process Linguistic Measures. RESULTS: One-way ANOVAs confirmed that specific linguistic features characterized BD, UD and CG. The use of Sensory-Somatic words was significantly different in the groups: higher in BD, intermediate in UD, and lower in CG. Individuals with BD produced higher scores on the Referential Activity Intensity Index and the use of singular pronoun "I". Negative Affect, as well as several POMS subscales, distinguished UD and BD from CG. LIMITATIONS: Narrow sample size, use of a single self-report instrument and treatment effects on measures in the clinical groups are limitations of the study. CONCLUSION: Individuals with UD and BD appear to use sensory-somatic language in predictably different patterns from each other and from the non-clinical population. Observation and assessment of linguistic features could improve diagnostic accuracy.

Aggressive Traits in People with Multiple Sclerosis-A Case-Control Study.

OBJECTIVE: Multiple sclerosis (MS) is a chronic neuroimmunological disease that mainly affects young adults and leads to neurological disabilities. Depression, anxiety, cognitive dysfunction, and other psychiatric conditions have often been reported in patients with MS. Other, subtler aspects of psychosocial conditions in MS have been studied, but there is a paucity of papers on the subject. Remarkable degrees of aggression have been described in up to a quarter of patients with MS, but few studies have targeted this outcome in the psychopathological assessment on patients. The objective of the present study was to assess aggressiveness in patients with MS and compare with matched control subjects. METHOD: The present study included a group of 24 patients and 24 healthy controls matched for gender, age, and socioeconomic level. Patients with moderate or severe disability, anxiety, or depression were excluded. A validated tool was used for assessment of aggressive trait. RESULTS: Aggressive traits were studied in patients and matched controls, and the results point to a very low level of aggressive tendency in patients with MS, in comparison with controls. CONCLUSION: The results from the present study do not confirm findings from other authors who had observed high levels of aggressive behavior in patients with MS. The authors are aware that exclusion of patients with moderate or severe disability, anxiety, or depression might have influenced the results.

A recent epidemiological cluster of acute hepatitis B genotype F1b infection in a restricted geographical area of Italy.

In this study, by phylogenetic analysis, we identified an epidemiological cluster involving eight individuals diagnosed with acute hepatitis B virus (HBV) infection related to unprotected sexual intercourse in a restricted area of central Italy (time period: 2011-2014). Notably, these patients (six of eight Italians) were infected by subgenotype F1b, which is not commonly found in western countries. Ultra-deep pyrosequencing confirmed a superimposable composition of HBV quasi-species in these patients. Despite the availability of effective vaccination, this study highlights the importance of not underestimating the risk of HBV infection, of continuing to set up surveillance programmes for HBV infection, and of investigating the pathogenetic potential of these atypical genotypes.

Entomofauna of a buried body: study of the exhumation of a human cadaver in Buenos Aires, Argentina.

This study focuses on insects and other arthropods sampled on the exhumation of an infant skeleton belonging to 'Prof. Dr. Rómulo Lambre' skeletal collection. The body was buried in soil inside a wooden coffin in a grave 40cm deep, in autumn, and stored in the cemetery deposit after exhumation. Death records were obtained from the cemetery archive. Samples of faunal remains were recovered from wrappings, clothes, bones and soil samples, and were identified at different taxonomic levels depending on the stage of conservation. The dominant taxon was the muscid fly Ophyra aenescens (Wiedemann). The relationships among the identified taxa and the moving of the corpse, from the burial context to the cemetery deposit, are discussed and used to create a hypothetical colonization sequence after death. The application of entomological data to anthropological research can provide valuable information for the interpretation of taphonomic processes and burial contexts.

Genetic and metabolic factors are associated with increased hepatic iron stores in a selected population of p.Cys282Tyr heterozygotes.

Heterozygosity for p.Cys282YTyr is not ordinarily associated with a hemochromatosis phenotype, unless associated in the compound heterozygous state with other HFE mutations. The aims of the study were to identify factors responsible for iron overload in patients who were only heterozygous for p.Cys282Tyr at first genetic testing. Since 2001, twelve p.Cys282Tyr heterozygous patients with iron overload, defined by increased transferrin saturation, serum ferritin and hepatic iron stores, were identified. Four patients showed rare nonsense or missense HFE mutations in the compound heterozygous state with p.Cys282Tyr. One mutation (p.Gln233X) was never described before. The other 8 patients did not carry any other causal mutations in iron-related genes, but showed a very high prevalence of hepatic steatosis and steato-hepatitis, and metabolic alterations. Serum ferritin levels did not differ between the two groups, but transferrin saturation, hepatic iron amount and distribution significantly did. These last indices should be then strongly considered to decide for additional genetic characterization in p.Cys282Tyr heterozygotes. Our results also highlights the influence of metabolic alterations on serum iron indices and pattern of hepatic iron accumulation.

Bacterial RNA polymerase inhibitors: an organized overview of their structure, derivatives, biological activity and current clinical development status.

RNA polymerase (RNAP) currently represents an important target for the development of new antibacterial agents. RNAP is a nucleotidyl transferase enzyme able to generate an RNA copy of a DNA or RNA template chain, controlling initiation and termination of transcription. RNAP is found in nature in all eukaryotes, prokaryotes and archaea, as well as in many viruses. Bacterial RNAP is a large molecule (about 400 kDa) and its core structure is composed of four polypeptide subunits: alpha (alpha) required for assembly of the enzyme, beta (beta) involved in chain initiation and elongation, beta' (beta') which binds to the DNA template, and omega (omega) which constrains the beta' subunit and aids its assembly into RNAP, in the stoichiometry alpha2betabeta'omega. The bacterial enzyme differs both from eukaryotic RNAP, which is composed of different subunits and is present in several variants, and from archaeal or viral RNAP. These differences allow to selectively target the bacterial enzyme with appropriately designed inhibitors, excluding interactions with eukaryotic RNAP, accounting for the deep interest developed around these compounds as selective antibacterial agents. In this review the known natural and synthetic inhibitors of RNAP will be described considering their mechanism of action, biological activity, availability of analogues, Structure Activity Relationship (SAR) information and clinical use when already approved or recently entered into clinical trials.

Cardiovascular exercise intervention improves the primary antibody response to keyhole limpet hemocyanin (KLH) in previously sedentary older adults.

Based upon a prior cross-sectional study, we hypothesized that an aerobic exercise intervention in sedentary older adults would improve a primary T cell-dependent immune response. Participants were a subset of older subjects from a large, ongoing exercise intervention study who were randomly assigned to either an aerobic exercise (Cardio, n=30, 68.9+0.8 years) or flexibility/balance (Flex, n=20, 69.9+1.2 years) intervention. The intervention consisted of either three aerobic sessions for 30-60 min at 55-70% VO(2 max) or two 60 min flexibility/balance sessions weekly for 10 months. Eight months into the intervention, samples were collected before intramuscular administration of KLH (125 microg), followed by sampling at 2, 3, and 6 weeks post-KLH. Serum anti-KLH IgM, IgG1, and IgG2 was measured by ELISA. Physiological and psychosocial measures were also assessed pre- and post-intervention. While there was no difference in the anti-KLH IgG2 response between groups, Cardio displayed significantly (p<0.05) higher anti-KLH IgG1 (at weeks 2, 3, and 6 post) and IgM responses when compared to Flex. Despite cardiovascular intervention-induced improvement in physical fitness (approximately 11% vs. 1% change in VO(2 peak) in Cardio vs. Flex, respectively), we found no relationship between improved fitness and enhanced anti-KLH antibody responses. Optimism, perceived stress, and affect were all associated with enhanced immune response. We have shown for the first time that cardiovascular training in previously sedentary elderly results in significantly higher primary IgG1 and IgM antibody responses, while having no effect on IgG2 production.

Novel mutations of the ferroportin gene (SLC40A1): analysis of 56 consecutive patients with unexplained iron overload.

The aim of this study was to search for SLC40A1 mutations in iron overloaded patients, which tested negative for HFE mutations and other iron-related genes. After a careful differential diagnosis, we selected 56 patients with unexplained iron overload whose phenotype could suggest the ferroportin disease. Iron overload was assessed by liver biopsy or by superconducting quantum interference device. SLC40A1 exons and intron-exon boundaries were amplified by polymerase chain reaction and sequenced. We also evaluated the presence of the insulin-resistance hepatic iron overload and of non-alcoholic fatty liver disease. Iron status was assessed in 44 families. We identified two novel mutations (D157N and V72F) at the heterozygous state in two probands. Phenotype heterogeneity was observed in both families, suggesting variable penetrance and expression. Including the two affected ones, 25 of the 44 families (57%) available for the iron study had one or more relatives with increased serum iron indices. Our findings not only suggest that the presence of major alterations of serum iron parameters in probands' relatives is a main criteria to improve the power of the genetic testing for ferroportin disease but also indicate that a number of patients exists in which the etiology of iron overload remains still undefined.

Manipulating dendritic cells to induce regulatory T cells.

Dendritic cells (DCs) induce and regulate T-cell responses, and tolerogenic DCs can promote the development of regulatory T cells with suppressive activity. The possibility of manipulating DCs using different pharmacological or biological agents, enabling them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection.

Iron chelation therapy in aceruloplasminaemia: study of a patient with a novel missense mutation.

We describe a novel missense mutation of ceruloplasmin in a patient with aceruloplasminaemia causing the replacement of a neutral amino acid (phenylalanine) with a polar one (serine) at position 198, probably leading to abnormal folding and secretion of the protein. The patient showed mild microcytic anaemia, mild hepatic iron overload, and marked brain iron overload. Six months of therapy with deferiprone was ineffective in removing iron from the tissues. Deferoxamine was more efficient in removing excess iron from the liver but aggravated the disease related anaemia. After more than one year of chelation treatment, the brain magnetic resonance imaging signal did not change. Overall, these findings indicate that treatment of iron overload in aceruloplasminaemia is a difficult challenge and that new iron chelators, more efficient in crossing the blood-brain barrier, are needed.

Prevalence of HFE mutations in upper Northern Italy: study of 1132 unrelated blood donors.

BACKGROUND: In the Italian general population, prevalence of C282Y is lower than in Northern European countries. We hypothesised a higher prevalence of C282Y in Northern than in Central and Southern Italy. We previously identified a nonsense mutation (W169X) in haemochromatosis probands originating from a Northern Italian region (Brianza). AIM: To define the prevalence of HFE mutations in that region. Subjects and methods. A total of 1132 unrelated blood donors from the Blood Banks of Monza and Merate were investigated for C282Y, H63D, S65C and W169X mutations by PCR-restriction assays. A total of 300 were also tested for rare HFE and TFR2 mutations by reverse-hybridization test strips. RESULTS: Two C282Y homozygotes, eight C282Y/H63D compound heterozygotes, 27 H63D homozygotes and one W169X heterozygote were found. The allele frequencies of C282Y, H63D, S65C, and W169X were 3.2, 13.4, 1.3, and 0.04%, respectively. CONCLUSIONS: Our results confirm the existence of a decreasing frequency of C282Y allele from upper to lower Northern Italy. This difference is probably related to the larger Celtic component of upper Northern Italian populations in which screening studies for haemochromatosis may even be cost effective. W169X, due to its severity, should be looked for in all haemochromatosis patients of Northern ancestry with an incomplete HFE genotype.

Inhibition of spontaneous and androgen-induced prostate growth by a nonhypercalcemic calcitriol analog.

We have recently found that analog V (BXL-353, a calcitriol analog) inhibits growth factor (GF)-stimulated human benign prostate hyperplasia (BPH) cell proliferation by disrupting signal transduction, reducing Bcl-2 expression, and inducing apoptosis. We now report that BXL-353 blocks in vitro and in vivo testosterone (T) activity. BPH cells responded to T and dihydrotestosterone (DHT) with dose-dependent growth and reduced apoptosis. Exposure of BPH cells to BXL-353 significantly antagonized both T- and DHT-induced proliferation and induced apoptosis, even in the presence of T. To verify whether BXL-353 reduced prostate growth in vivo, we administered it orally to either intact or castrated rats, supplemented with T enanthate. Nonhypercalcemic doses of BXL-353 time- and dose-dependently reduced the androgen effect on ventral prostate weight, similarly to finasteride. Comparable results were obtained after chronic administration of BXL-353 to intact rats. Clusterin (an atrophy marker) gene and protein were up-regulated by BXL-353 in rat prostate, and nuclear fragmentation was widely present. The antiandrogenic properties of BXL-353 did not interfere with pituitary and testis function, as assessed by serum determination of rat LH and T. BXL-353 did not compete for androgen binding to BPH homogenates and failed to inhibit 5alpha-reductase type 1 and type 2 activities. In conclusion, BXL-353 blocks in vitro and in vivo androgen-stimulated prostate cell growth, probably acting downstream from the androgen receptor, without affecting calcemia or sex hormone secretion. BXL-353 and other vitamin D(3) analogs might thus represent an interesting class of compounds for treating patients with BPH.

Immune-endocrine status and coeliac disease in children with Down's syndrome: relationships with zinc and cognitive efficiency.

Immune defects, thyroid abnormalities, plasma zinc levels, and the presence of gastrointestinal disease were investigated in 43 children with Down's syndrome (DS). Peripheral T lymphocytes with the phenotype of helper cells or cluster of differentiation 4 (CD4) were decreased. Circulating activated T cells (CD3/HLA-DR-positive cells) and large granular lymphocytes (CD16/CD56 positive cells) were increased. Plasma levels of interleukin-6 were higher in DS children than in controls. Serum levels of thyroid-stimulating hormone were increased in DS. Coeliac disease was over-represented in the group of DS children and many of these children also showed increased serum levels of immunoglobulin-G (IgG) specific for gliadin antigen. The increment of serum interleukin-6 was age-related and correlated with anti-gliadin IgG levels in DS. Plasma zinc levels were lower in DS children with coeliac disease and in those with anti-gliadin IgG than in DS without detectable anti-gliadin IgG. Dietary antigens may represent a continuous stimulus for the immune system in this syndrome and interfere with normal immune responses. Altered intestinal absorption of nutrients may in turn affect endocrine functions, brain development, and cognitive performances.

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

Haemochromatosis in patients with beta-thalassaemia trait.

Severe iron overload has been reported in patients with the beta-thalassaemia trait. Studies performed before the discovery of the haemochromatosis gene (HFE) have yielded conflicting results: some suggest that iron overload might arise from the interaction of the beta-thalassaemia trait with heterozygosity for haemochromatosis, some with homozygosity for haemochromatosis and others that it was unrelated to haemochromatosis. We have studied the clinical phenotype, iron indices and HFE genotypes of 22 unrelated patients with the beta-thalassaemia trait and haemochromatosis, the inheritance of chromosome 6p and 1q haplotypes in families of non-homozygous C282Y probands and serum measures of iron status in relatives heterozygous for C282Y with or without the beta-thalassaemia trait. We demonstrate that the beta-thalassaemia trait aggravates the clinical picture of C282Y homozygotes, favouring higher rates of iron accumulation and the development of severe iron-related complications. We suggest that the coexistence of the beta-thalassaemia trait might also increase the risk of iron overload in patients with HFE genotypes at a mild risk of haemochromatosis. Our findings do not support the hypothesis that the association of the beta-thalassaemia trait with a single C282Y or H63D allele might lead to iron overload and suggest that other non-HFE-related inherited factors are present in haemochromatosis patients with incomplete HFE genotypes.

Soluble CD40 ligand induces beta-chemokine production by macrophages and resistance to HIV-1 entry.

CD40 ligand (CD40L) is a cell surface molecule of CD4(+)T cells that interacts with its receptor CD40 on antigen presenting cells to mediate thymus-dependent humoral immunity and inflammatory reactions. We report here that treating monocyte-derived macrophages (MDM) with a trimeric soluble form of CD40L (CD40LT) induced them to secrete high levels of the beta-chemokines RANTES, MIP-1alpha and MIP-1beta that are ligands for CCR5 and able to inhibit HIV-1 entry. CD40LT inhibited the entry of M-tropic HIV-1 reporter viruses. Furthermore, supernatants obtained from CD40LT-stimulated macrophages protected CEMx174-CCR5 cells from infection by HIV-1(JRFL)reporter virus. The inhibitory activity appeared to be due to beta-chemokines present in the supernatant, since pretreating them with a cocktail of antibodies to RANTES, MIP-1alpha and MIP-1beta neutralized the inhibitory activity of the supernatants. In addition, treating monocytes with CD40LT caused CCR5 and CD4 to be downregulated from the cell surface. In vivo, macrophages activated through CD40 could interfere with HIV replication.

[Sweet syndrome in an infant]. / Syndrome de Sweet chez un nourrisson.

BACKGROUND: Acute febrile neutrophilic dermatosis (Sweet's syndrome) is very uncommon in infancy. Systemic corticosteroid treatment is the standard therapy, usually leading to dramatic improvement within a few days. CASE REPORT: A seven-month-old female infant was admitted for investigation of a rash developing over ten days with fever. Physical examination and skin biopsy led to a diagnosis of Sweet's syndrome. The relative inefficiency of systemic corticotherapy induced the parents to stop all treatment. CONCLUSION: This case report allows us: 1) to consider the clinical and biological features of Sweet's syndrome in infancy; 2) to describe a corticosteroid resistant disease; and 3) to observe the course of a spontaneous evolution of Sweet's syndrome.

A block to human immunodeficiency virus type 1 assembly in murine cells.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.