Netherton syndrome and its multifaceted defective protein LEKTI.

Netherton syndrome (NS, OMIM 256500) is a rare autosomal recessive disorder manifesting with congenital ichthyosis, a specific hair shaft abnormality named trichorrhexis invaginata, and atopic manifestations. Because of severe complications frequently occurring in the neonatal period, NS prognosis can be poor in infancy. NS is due to loss-of-function mutations in the SPINK5 gene and to the consequent lack of expression of its encoded protein LEKTI in the skin and all stratified epithelial tissues. Following the identification of the NS causative gene and protein, specific diagnostic tools have been developed, thus breaking up the challenge of distinguishing NS from other congenital ichthyoses with overlapping features, and from severe, early-onset forms of atopic dermatitis or psoriasis. Intensive efforts to extend the knowledge into the pathomechanisms of NS have also been made. However, NS management is still problematic due to the lack of specific treatment and unmet needs. This overview summarizes the current state of the art in NS research with an emphasis on the progress made toward disease-specific innovative therapy development.

Continuity and discontinuity of attachment patterns: a short-term longitudinal pilot study using a sample of late-adopted children and their adoptive mothers.

This study analysed the attachment patterns of 28 late-adopted children (placed when they were between four and seven years of age) and their adoptive mothers. The change in the children's internal working models (IWMs) within seven to eight months of their placement was evaluated. In addition, we wanted to observe the influence of a secure-autonomous maternal state of mind in facilitating the change in the children's IWMs and the possible associations between the maternal IWMs and the children's IWMs in the adoptive dyads. The separation-reunion procedure (SRP) was used for the late-adopted children in order to assess their attachment behavioural patterns, and the Manchester Child Attachment Story Task (MCAST) was used to evaluate their attachment narrative patterns. The adoptive mothers completed the Adult Attachment Interview (AAI) in order to classify their state of mind with regard to attachment. The results showed a significant change in the attachment behavioural patterns of late-adopted children, from insecure to secure (p = .002). Furthermore, the children who presented this change were predominantly placed with secure-autonomous adoptive mothers (p = .047), although the link between the adoptive mothers' representations of their attachment history and their adopted children's completed narratives was not significant. In conclusion, it seems possible to revise the attachment behaviour of late-adopted children but, for about one-third of children, the adverse history will persist at a narrative/representational level.

Decreased expression of heparin-binding epidermal growth factor-like growth factor as a newly identified pathogenic mechanism of antiphospholipid-mediated defective placentation.

OBJECTIVE: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL)-mediated defective placentation. METHODS: HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal beta(2)-glycoprotein I-dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF. RESULTS: Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels. CONCLUSION: These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding.

Oxidation-dependent maturation and survival of explanted blood monocytes via Bcl-2 up-regulation.

Monocytes isolated and cultured according to standard procedures from the blood of 22 healthy donors display an activation process, monitored as adhesion and increased exposure of CD11. Starting from very early time points, monocytes undergo a deep redox modulation, i.e., they increase reactive oxygen species (ROS) formation and decrease glutathione content; at the same time, the anti-apoptotic protein Bcl-2 is substantially up-regulated. The cause-effect relationship between these parameters was investigated. On the one side, pharmacological glutathione depletion with BSO further increases ROS formation and Bcl-2 levels. On the other side, scavenging of ROS by Trolox prevents Bcl-2 up-regulation. Two lipoxygenase (LOX) inhibitors (CAPE or AA861) prevent ROS increase and, accordingly, also prevent Bcl-2 up-regulation. All this evidence supports the redox-sensitivity of Bcl-2 regulation. Trolox, CAPE and AA861, i.e., all treatments that abolish ROS increase and prevent Bcl-2 up-regulation, increase the rate of cell loss, whereas BSO, increasing Bcl-2, reduces cell loss and induces chemo-resistance. Thus, explanted healthy monocytes seem to undergo an oxidation-dependent maturation implying increased survival via Bcl-2 up-regulation, perhaps mimicking physiological activation.

Low-molecular weight heparin induces in vitro trophoblast invasiveness: role of matrix metalloproteinases and tissue inhibitors.

Heparin is used widely for the prevention of pregnancy loss in pregnant women with thrombophilia. However, it is still unknown if heparin may be able to affect trophoblast functions. Therefore, we investigated the hypothesis that low-molecular weight heparin (LMWH) might regulate in vitro trophoblast invasiveness and placental production of matrix metalloproteinases (MMPs) and tissue inhibitors (TIMPs). In the first-trimester placental tissue, the MMP-9 expression was observed in both villous and extravillous cytotrophoblast cells, and MMP-2 mainly in villous cytotrophoblast. In human choriocarcinoma cells (JAR), MMP-2 was the dominant form. Heparin significantly enhanced both pro-MMPs and the active forms, and increased Matrigel invasiveness of extravillous trophoblast and choriocarcinoma cells. In choriocarcinoma cells the heparin effect was also indirect, inducing a significant decrease in TIMP-1 and TIMP-2 protein expressions and mRNAs. The present data suggest that the increase in trophoblast invasion by heparin is due to a specific protein playing a role in placental invasion. These observations may help in understanding the effects of heparin treatment during pregnancy.

Pathogenic role of anti-beta2-glycoprotein I antibodies on human placenta: functional effects related to implantation and roles of heparin.

Most of the clinical manifestations of the antiphospholipid syndrome (APS) can be related to thrombotic events; however, placental thrombosis cannot explain all of the pregnancy complications that occur in women with this syndrome. In this regard, it has been hypothesized that antiphospholipid (aPL) antibodies can directly attack trophoblasts, but it is still unclear what pathogenetic mechanisms play a role and which aPL antibodies subpopulations are involved. Although it has been assumed that aPL antibodies are directed against anionic phospholipids (PLs), current advances in the field suggest that antibodies to PL-binding plasma protein such as beta2-glycoprotein-I (beta2-GPI) are the clinically relevant aPL antibodies. It appears that following the attachment of beta2-GPI to PLs, both molecules undergo conformational changes that result in the exposure of cryptic epitopes within the structure of beta2-GPI allowing the subsequent binding of antibodies. aPL antibodies detected by anti-beta2-GPI assays are associated with fetal loss. However, there is still debate on how the antibodies might induce the obstetrical manifestations. The significantly improved outcome of pregnancies treated with heparin has stimulated interest in the drug's mechanisms of action. Several mechanisms could explain its beneficial effects, because in addition to a direct effect of heparin on the coagulation cascade, it might protect pregnancies by reducing the binding of aPL antibodies, reducing inflammation, facilitating implantation and/or inhibiting complement activation. Further investigations are needed to better understand how aPL antibodies induce obstetric complications and to better clarify the functional role of heparin in the human placenta leading to more successful therapeutic options.

NMR exposure sensitizes tumor cells to apoptosis.

NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3-66 mT) static magnetic fields deeply affect apoptosis in a Ca2+ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95-102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8-2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca2+ overload consequent to NMR-promoted Ca2+ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca2+ chelation or by inhibition of plasma membrane L-type Ca2+ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies.

Magnetic fields protect from apoptosis via redox alteration.

Magnetic fields (MFs) are receiving much attention in basic research due to their emerging ability to alter intracellular signaling. We show here that static MFs with intensity of 6 mT significantly alter the intracellular redox balance of U937 cells. A strong increase of reactive oxygen species (ROS) and a decrease of glutathione (GSH) intracellular levels were found after 2 h of MF exposure and maintained thereafter. We found that also other types of MFs, such as extremely-low-frequency (ELF) MFs affect intracellular GSH starting from a threshold at 0.09 mT. We previously reported that static MFs in the intensity range of 0.3-60 mT reduce apoptosis induced by damaging agents (Fanelli et al., 1998). Here, we show that ELF-MFs are also able to protect U937 from apoptosis. Interestingly, this ability is limited to the ELF intensities able to alter redox equilibrium, indicating a link between MF's antiapoptotic effect and the MF alteration of intracellular redox balance. This suggests that MF-produced redox alterations may be part of the signaling pathway leading to apoptosis antagonism. Thus, we tested whether MFs may still exert an antiapoptotic action in cells where the redox state was artificially altered in both directions, that is, by creating an oxidative (via GSH depletion with BSO) or a reducing (with DTT) cellular environment. In both instances, MFs fail to affect apoptosis. Thus, a correct intracellular redox state is required in order for MFs to exert their antiapoptotic effect.

The cleavage mode of apoptotic nuclear vesiculation is related to plasma membrane blebbing and depends on actin reorganization.

In U937 monocytic cells induced to apoptosis, plasma membrane blebbing of different intensities appears, before the development of nuclear alterations; this latter phenomenon can occur through two major pathways, namely the cleavage and the budding mode (Dini et al., 1996). Strongly blebbing cells develop deep nuclear constrictions leading to nuclear fragmentation according to the cleavage mode, while cells with milder forms of blebbing, or no blebbing at all, undergo nuclear fragmentation along the budding mode. Compounds interfering with different cytoskeletal components affect blebbing, which is completely inhibited by the actin polymerization inhibitors, cytochalasins, while disturbance of tubulin network with taxol limits blebbing to milder forms. At the same time, the cytoskeletal poisons affect the type of nuclear fragmentation, abolishing the cleavage mode, shifting all events into the budding pathway. Adherent cells, which possess a more structured cytoskeleton, do not develop strong blebs and undergo nuclear fragmentation via budding. These observations suggest that the deep cytoskeletal movements that cause the strongest forms of plasma membrane blebbing strangle the nucleus, leading to the constrictions that later evolve into nuclear fragmentation by cleavage. The trigger for the cytoskeletal movements, known to be redox-sensitive, is probably the apoptotic GSH extrusion.

Hyperpolarization of plasma membrane of tumor cells sensitive to antiapoptotic effects of magnetic fields.

Chemical/physical agents able to prevent apoptosis are receiving much attention for their potential health hazard as tumor promoters. Magnetic fields (MFs), which have been shown to increase the occurrence of some tumors, reduce damage-induced apoptosis by a mechanism involving Ca2+ entry into cells. In order to discover the mechanism of such effect of MFs, we investigated the interference of MFs on cell metabolism and analyzed cell parameters that are involved in apoptotic signaling and regulation of Ca2+ fluxes. Here we show that different types (static and extremely low-frequency, ELF pulsating) of MFs of different intensities alter plasma membrane potential. Interestingly, MFs induce plasma membrane hyperpolarization in cells sensitive to the antiapoptotic effect of MFs, whereas cells that are insensitive showed a plasma membrane depolarization. These opposite effects suggest that protection against apoptosis and membrane potential modulation are correlated, plasma membrane hyperpolarization possibly being part of the signal transduction chain determining MFs' antiapoptotic effect.

Medicolegal aspects of an unusual uterine perforation with multiload-Cu 375R.

Perforation of the uterus is one of the most serious complications associated with insertion of intrauterine contraceptive devices (IUD). According to recent studies, this lesion occurs in 0.87 per 1000 cases, but statistics generally fluctuate between 0.05 and 13 per 1000 insertions. "Primary" perforations occur at the time of IUD insertion, and "secondary," or delayed, perforations are usually assumed to be caused by reactive uterine contractions. We report an unusual case of uterine perforation, which occurred 9 days after the insertion of a Multiload-Cu 375 IUD. The criteria for differential diagnosis between primary and secondary complications are discussed, particularly from the perspective of medical malpractice cases.

[Lack of direct cytotoxic effect of intracellular nanotubes]. / Impatto occupazionale ed ambientale dei nanotubi di carbonio, analisi della citotossicità su linee cellulari.

Nanotubes have a great therapeutic potential due to their astounding physico-chemical features, the possibility to be funtionalised for ad hoc uses, and the specific interaction of nanotubes as such with life molecules (DNA and proteins). These features recommend a thorough toxicological study before widespread pharmaceutic use. We provide evidence that culture cells with phagocytic potential internalise multi wall nanotubes (10-50 nm average size). This is not accompanied by cytotoxicity in terms of induction of &apoptosis or necrosis at the doses used (up to 125 microg/mI).

Oxidative Bax dimerization promotes its translocation to mitochondria independently of apoptosis.

Bax is a cytosolic protein, which in response to stressing apoptotic stimuli, is activated and translocates to mitochondria, thus initiating the intrinsic apoptotic pathway. In spite of many studies and the importance of the issue, the molecular mechanisms that trigger Bax translocation are still obscure. We show by computer simulation that the two cysteine residues of Bax may form disulfide bridges, producing conformational changes that favor Bax translocation. Oxidative, nonapoptogenic treatments produce an up-shift of Bax migration compatible with homodimerization, which is reverted by reducing agents; this is accompanied by translocation to mitochondria. Dimers also appear in pure cytosolic fractions of cell lysates treated with H2O2, showing that Bax dimerization may take place in the cytosol. Bax dimer-enriched lysates support Bax translocation to isolated mitochondria much more efficiently than untreated lysates, indicating that dimerization may promote Bax translocation. The absence of apoptosis in our system allows the demonstration that Bax moves because of oxidations, even in the absence of apoptosis. This provides the first evidence that Bax dimerization and translocation respond to oxidative stimuli, suggesting a novel role for Bax as a sensor of redox imbalance.

Intracellular degradation of beta4 integrin in lethal junctional epidermolysis bullosa with pyloric atresia.

BACKGROUND: Junctional epidermolysis bullosa with pyloric atresia (PA-JEB) is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous blistering and gastric outlet obstruction. The disease, which is caused by mutations in the alpha6beta4 integrin genes (ITGA6, ITGB4), is usually lethal. However, nonlethal cases have also been reported. Mutation database analysis has suggested that premature termination codons predominantly result in lethal forms while missense mutations frequently associate with nonlethal variants. Nevertheless, it is becoming more and more evident that the disease phenotype is also influenced by the position of the mutation in the protein functional domains. OBJECTIVE: To investigate the molecular basis of a novel PA-JEB lethal case. METHODS: Reverse transcriptase-polymerase chain reaction and direct sequencing-based mutation screening were performed. Mutation consequences in the patient's keratinocytes were then analysed by Northern blot and immunoprecipitation. Immunofluorescence analysis of cultured keratinocytes treated with protein intracellular degradation pathway inhibitors was also carried out. RESULTS: The phenotype was caused by the presence, in the homozygous state, of a novel 33 bp in-frame deletion (nucleotides 175-207) in the ITGB4 coding sequence. Despite the normal steady-state level of integrin beta4 mRNA, the mutation, designated DeltaR59-A69, results in the almost complete absence of alpha6beta4 integrin in the patient's skin and cultured keratinocytes. Exposure of the patient's keratinocytes to the proteasomal inhibitor clasto-lactacystin beta-lactone increased the expression of the mutated beta4 integrin chains indicating that the proteasome complex is involved in the degradation of the internally deleted beta4 polypeptides. CONCLUSIONS: We report for the first time a homozygous in-frame deletion in the ITGB4 gene. Our results suggest that the deletion of amino acids R59-A69 interferes with the biosynthetic folding of the protein, leading to a rapid degradation of the mutated beta4 chains. These findings provide new insight into the pathogenic effects of mutations affecting different functional domains of the beta4 integrin molecule and their prognostic implications in PA-JEB patients.

Hypoxic stress stably alters apoptotic parameters on U937 cells.

Tumor promonocytic U937 cells cultured under a low O(2)/high CO(2) atmosphere display altered characteristics after restoration of normal atmosphere: increased resistance to apoptosis induced by different treatments; apoptotic morphology; lack of glutathione (GSH) extrusion in apoptosis; lack of protection by antioxidants; and lack of Ca(2+) mobilization with thapsigargin. These alterations were stably maintained for many months of culture in normal conditions, originating the stable U937-HX variant. Since the hypoxic treatment did not produce a great selective pressure, the alterations are conceivably the result of stable adaptative response.

Apoptotic GSH extrusion is associated with free radical generation.

Reactive oxygen species (ROS) are involved in many forms of apoptosis and mediate apoptosis in a number of cell types. In this paper, we use a variant of U937 monocytic cells (U937 HX) that show different biochemical features with respect to standard U937. Apoptotic standard U937 extrude reduced glutathione (GSH) and generate free radicals concomitantly with loss of mitochondria transmembrane potential (mt Deltapsi). These events are correlated with the extrusion of intracellular GSH. Conversely, apoptotic U937 HX cells retain GSH, and the loss of mt Deltapsi is not accompanied by generation of free radicals. The perfect inverse correlation between (a) ROS generation and (b) the presence of intracellular GSH during apoptosis suggests novel mechanisms to finely tune ROS generation in apoptosis.

Novel and recurrent mutations in the integrin beta 4 subunit gene causing lethal junctional epidermolysis bullosa with pyloric atresia.

In this study we examined two unrelated patients affected with the lethal variant of junctional epidermolysis bullosa with pyloric atresia (PA-JEB) who were found to carry mutations in the integrin beta4 subunit gene (ITGB4). Although in both patients Northern blot analysis showed only a 50% reduction in the level of ITGB4 transcript, a complete lack (patient 1) or a strong reduction (patient 2) of beta4 immunoreactivity was observed in the skin. Using immunoprecipitation analysis, integrin beta4 could not be visualized in patient 1 cells while a markedly reduced amount (approximately 20%) of normal sized beta4 chains was detected in patient 2. These data suggested the presence of ITGB4 mutations that interfere with both mRNA and protein stability. Using molecular analysis, patient 1 was shown to be a compound heterozygous for a single amino acid deletion (deltaN318) and a not yet identified mutation that induces a very rapid decay of the encoded mRNA transcript. Patient 2 was, instead, a compound heterozygous for a novel 4-bp tandem duplication (4298-4299ins4) and a previously described missense mutation (R252C). Our data support the notion that PA-JEB lethal phenotypes associated with a markedly decreased/absent alpha6beta4 expression can be due not only to the presence of null alleles, but also to specific mutations leading to protein instability and/or altered function.

A homozygous nonsense mutation in type XVII collagen gene (COL17A1) uncovers an alternatively spliced mRNA accounting for an unusually mild form of non-Herlitz junctional epidermolysis bullosa.

In this study we describe six Italian patients presenting an unusually mild variant of non-Herlitz junctional epidermolysis bullosa associated with a reduced expression of type XVII collagen. All patients are homozygous for a novel nonsense mutation (R795X) within exon 33 of COL17A1 and show a common haplotype, attesting propagation of an ancestral allele within the Italian population. Analysis of patients' COL17A1 transcripts showed the presence of two mRNA species: a normal-sized mRNA carrying mutation R795X that undergoes rapid decay, and a transcript generated by in-frame skipping of exon 33. Patients keratinocytes were shown to synthesize minute amounts of type XVII collagen, which appeared correctly localized along the cutaneous basement membrane. We therefore suggest that the exon 33-deleted COL17A1 splice variant encodes for type XVII collagen molecules that maintain a functional role and account for the mild phenotype of our patients.