Layer-by-layer assembly for biomedical applications in the last decade.

In the past two decades, the design and manufacture of nanostructured materials has been of tremendous interest to the scientific community for their application in the biomedical field. Among the available techniques, layer-by-layer (LBL) assembly has attracted considerable attention as a convenient method to fabricate functional coatings. Nowadays, more than 1000 scientific papers are published every year, tens of patents have been deposited and some commercial products based on LBL technology have become commercially available. LBL presents several advantages, such as (1): a precise control of the coating properties; (2) environmentally friendly, mild conditions and low-cost manufacturing; (3) versatility for coating all available surfaces; (4) obtainment of homogeneous film with controlled thickness; and (5) incorporation and controlled release of biomolecules/drugs. This paper critically reviews the scientific challenge of the last 10 years--functionalizing biomaterials by LBL to obtain appropriate properties for biomedical applications, in particular in tissue engineering (TE). The analysis of the state-of-the-art highlights the current techniques and the innovative materials for scaffold and medical device preparation that are opening the way for the preparation of LBL-functionalized substrates capable of modifying their surface properties for modulating cell interaction to improve substitution, repair or enhancement of tissue function.

Clinicopathologic spectrum of cutaneous diseases in patients with hematologic malignancies with or without allogeneic bone marrow transplantation: an observational cohort study in 101 patients.

AIM: Objective of the study was to determine the most common cutaneous lesions in patients with haematologic malignancies observed at dermatologic consultation and to identify the impact parameters related to the haematologic condition, like disease type/duration, remission, chemotherapy and transplantation, have on skin manifestations. METHODS: A total of 101 consecutive patients with onco-haematological malignancies referred for dermatological consultation over a two-year period were included in this prospective single-centre observational cohort study. RESULTS: The most common finding was infection (19.8%), followed by drug adverse reactions (16.8%) and malignant neoplasia (11.9%). Elderly patients and those with a longer disease duration had a higher frequency of cutaneous neoplasia. Squamous cell carcinoma was the most frequent cutaneous neoplasia; three cases of melanoma were diagnosed and had a high Breslow thickness. Cutaneous involvement due to the haematological malignancies was observed in 5 patients. Common chronic dermatoses (psoriasis and eczema) were found in 10% of patients. Transplant had no effect on the percentage of infections or tumours. CONCLUSION: Patients with haematological malignancies have a higher incidence of adverse drug reactions with peculiar morphologic features and a lower incidence of common chronic dermatoses than patients referred for dermatological consultation by their general practitioner or other hospital services. Infectious dermatoses were less frequent than in solid organ transplanted patients. The complex variety of cutaneous lesions, the differential diagnostic pitfalls and the prognostic relevance of early skin tumour diagnosis, evidence the importance of a correct dermatological approach.

Brief executive-function assessment tool: A new cognitive impairment screening tool for alcohol and other drug services.

Accurate screening for cognitive impairment in alcohol and other drug (AOD) services would help to identify individuals who may need supports to obtain the greatest benefit from substance use disorder (SUD) treatment. At present there is no screening measure that has been developed specifically to detect cognitive impairment in a SUD population. This study examines the psychometric properties of the Brief Executive-function Assessment Tool (BEAT), which was specifically designed for this purpose. This study involving 501 individuals with SUD and 145 normal control participants established internal consistency (n = 646; 0.734), interrater (n = 60; 0.994), and test-retest reliability (n = 177; 0.845), and construct (all correlations p ≤ 0.05), and criterion (n = 467; ANCOVA p < 0.001) validity. Test operating characteristics (n = 500; 87% sensitivity, 71% specificity, 21% PPP, and 99% NPP) were also established relative to an independent criterion variable made up of three established performance-based neuropsychological tests. Findings support the reliability and validity of the BEAT as a screening measure of executive function impairment with high sensitivity and a low rate of false negatives.

[Comparison of plasma matrix metalloproteases 2, 3, 9 in breast carcinomas and fibroadenomas]. / Confronto dei valori plasmatici delle metalloproteasi della matrice 2, 3, 9 in pazienti con carcinomi e fibroadenomi mammari.

Tumor cell infiltration causes the remodelling of peritumoral tissues, determined by an increased lytic activity of extracellular matrix exerted by the neoplastic invasive phenotype. Among the principal lytic enzymes produced by tumor cells and mainly involved in invasion process there are the matrix metalloproteases (MMPs). The Authors compared the plasmatic values of MMPs 2, 3, 9 from patients with breast carcinomas and fibroadenomas in order to evaluate whether there was a significant difference between the two groups of patients. MMPs 2, 3, 9 values were quantified by ELISA test from plasma collected 24 hours before surgery in 50 breast carcinomas and 30 fibroadenomas. MMP2 mean value from the patients with carcinomas resulted significantly higher as compared to that from the patients with fibroadenomas; while for MMP 3 and 9 mean values was not possible to find a significant difference between the two groups of malignant and benign breast tumors. These data confirm the main role played by MMPs during the tumor invasion process. Therefore, it is possible to propose the future inclusion of MMP2 test, together to other biological and clinical data, for prognostic evaluation of neoplastic breast lesions.

Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.

Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant.

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.

Mutations in the XPC gene in families with xeroderma pigmentosum and consequences at the cell, protein, and transcript levels.

Xeroderma pigmentosum (XP)-C is one of the more common complementation groups of XP, but causative mutations have thus far been reported for only six cases (S. G. Khan et al., J. Investig. Dermatol., 115: 791-796, 1998; L. Li et al., Nat. Genet., 5: 413-417, 1993). We have now extended this analysis by investigating the genomic and coding sequence of the XPC gene, the level of expression of the XPC transcript and the status of the XPC protein in 12 unrelated patients, including all of the 8 Italian XP-C cases identified thus far and in 13 of their parents. Eighteen mutations were detected in the open reading frame of the XPC gene, 13 of which are relevant for the pathological phenotype. The mutations are distributed across the gene, with no indication of any hotspots or founder effects. Only 1 of the 13 relevant changes is a missense mutation, the remainder causing protein truncations as a result of nonsense mutations (3), frameshifts (6), deletion (1) or splicing abnormalities (2). These findings indicate that the XPC gene is not essential for cell proliferation and viability and that mutations causing minor structural alterations may not give an XP phenotype and may not, therefore, be identified clinically. XP13PV was the only patient carrying a missense mutation (Trp690Ser on the paternal allele). This was also the only patient in which the XPC transcript was present at a normal level and the XPC protein was detectable, although at a lower than normal level. No quantitative alterations in the transcript or protein levels were detected in the XP-C heterozygous parents. However, the expression of the normal allele predominated in all of them, except the father of XP13PV, which suggests the existence of a possible mechanism for monitoring the amount of the XPC protein.

Proneness to UV-induced apoptosis in human fibroblasts defective in transcription coupled repair is associated with the lack of Mdm2 transactivation.

The apoptotic response and the level of expression of p53 and of three genes transcriptionally activated by p53 (Mdm2, p21 and bax) were investigated in UV-sensitive cells from patients with xeroderma pigmentosum (XP) or Cockayne syndrome (CS). These disorders are due to different genetic defects affecting transcription-coupled repair (TCR) and/or global genome repair (GGR), the nucleotide excision repair subpathways which remove UV-induced lesions from the transcribed strand of active genes or from the rest of the genome, respectively. After 20 J/m2 UV light, normal and GGR-defective XP-C fibroblasts showed rapid increase in p53, late induction of Mdm2 and no evidence of apoptosis even 96 h after irradiation. In contrast, in XP-A (defective in GGR and TCR), CS-A and CS-B (defective only in TCR) fibroblasts, the p53 increase was not followed by Mdm2 induction and the persistence of high levels of p53, due to the lack of its degradation by Mdm2, was associated with the appearance of apoptosis. Besides indicating that the persistence of DNA damage in the transcribed strand of active genes leads to apoptosis, these findings provide the first evidence that the lack of activation of Mdm2 plays a key role in the cascade of events leading to apoptosis. Oncogene (2000).

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients.

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38-50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Tyrosinase mRNA RT-PCR analysis as an additional diagnostic tool for the identification of melanoma cells in biological fluid samples other than blood: a preliminary report.

Reverse transcription polymerase chain reaction (RT-PCR) of tyrosinase mRNA has been applied for the detection of melanoma cells in the peripheral blood, lymph nodes and bone marrow of melanoma patients. We evaluated the diagnostic accuracy of RT-PCR in comparison to standard cytology and immunocytochemistry (ICC) for the identification of melanoma cells in biological fluids other than blood. Tyrosinase expression was evaluated together with standard cytology and ICC (anti-S100, HMB-45 and Melan-A antibodies) in biological fluid samples collected from 17 melanoma patients according to the site of metastatic involvement or clinical suspicion (eight cerebrospinal fluid (CSF) samples; three pleural effusions; four ascites; one bile sample, one pericardial effusion); 17 samples collected from patients with non-melanoma metastatic cancer were used as controls. Tyrosinase expression in the biological fluid sample was compared with the expression determined at the same time in peripheral blood. Positive tyrosinase expression was found in 12/17 melanoma and 3/17 non-melanoma cancer patients. Cytology/ICC showed the presence of neoplastic cells in only 7/12 melanoma samples with positive tyrosinase expression: radiological evidence of disease involvement was found in all these patients (three meningeal, two pleural, two peritoneal). Clear-cut radiological evidence of disease involvement at the sampling site was found in the five patients with negative cytology/ICC and positive RT-PCR (one CSF; four serous membrane effusions); all patients died of disease progression within four months of sampling. The five patients who were negative for both cytology/ICC and RT-PCR did not show any clinical evidence of disease recurrence at the sampling site. Only five of the 12 metastatic patients with positive tyrosinase expression in biological fluid showed positivity for tyrosinase in the peripheral blood. These preliminary results suggest that the analysis of biological fluids other than blood could be considered as a new potential clinical field of application for the tyrosinase mRNA assay.

Telomeric fusions in cultured human fibroblasts as a source of genomic instability.

In a human fibroblast clone we studied the evolution, during culture propagation, of a dicentric chromosome consisting of the end-to-end association of the short arm of chromosome 5 and the long arm of chromosome 16. Dual-color fluorescence in situ hybridization (FISH) with painting probes allowed us to define the structure of a variety of derivative chromosomes and to identify the mechanisms by which they originated. Asymmetric interchanges involving the intercentromeric region of the dicentric, bridge-breakage-fusion events, or breaks followed by sister chromatid fusion, originate unstable hetero- or homodicentric chromosomes with deletion or duplication; breakages not followed by reunion, or intradicentric recombination, presumably originate stable rearranged monocentric chromosomes. The variety of the derivatives is extremely large because the observed events may involve any site of the intercentromeric region, although the majority of them occurs after a break in 16qh. The results of this investigation document the evolution through successive steps of a telomeric fusion, a chromosome anomaly frequently observed in tumor and senescent cells. They also demonstrate that in cultured cells of normal origin, starting with this anomaly, various chromosomal mechanisms may produce translocations, duplications, and deletions. The karyotype instability produced by a telomeric fusion can be relevant for carcinogenesis because it may generate genetic changes critical in the multistep process of transformation.

DNA repair investigations in nine Italian patients affected by trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair, mental and growth retardation, peculiar face, ichthyosis, and in 20% of the reported cases photosensitivity. Cellular photosensitivity due to the same genetic defect present in xeroderma pigmentosum group D (XP-D) has been described in several patients. Nine patients with clinical symptoms diagnostic for TTD have been identified in Italy to date. We report the results of DNA repair investigations performed in cultured fibroblasts from these patients and 8 TTD parents. Survival, DNA repair synthesis and RNA synthesis following UV irradiation were all normal in the 8 TTD heterozygous cell strains. Among the 9 TTD-affected individuals, normal cellular UV sensitivity was observed in the 2 patients without signs of clinical photosensitivity. In contrast, the other 7 TTD cell strains showed a notable reduction in UV-induced DNA repair synthesis (UDS) levels, ranging between 40% and 5-15% of normal values. Complementation analysis indicated that in the repair-deficient TTD cell strains the genetic defect is the same as that present in XP-D cells. The biochemical heterogeneity of the XP-D defect in TTD patients characterized by different degrees of defective UDS results in different patterns of response to the killing effect of UV light in non-proliferating cells.

Molecular analysis of the XP-D gene in Italian families with patients affected by trichothiodystrophy and xeroderma pigmentosum group D.

In several patients with the rare hereditary disorder trichothiodystrophy (TTD), a DNA repair defect has been shown to be in the same gene as in xeroderma pigmentosum complementation group D (XP-D). The ERCC-2 gene (excision repair cross-complementing rodent repair deficiency of group 2) has recently been identified as a strong candidate gene for XP-D, since it restores normal UV sensitivity to XP-D cells after transfection. Using Southern blotting, we have analysed the ERCC-2 gene in DNA samples from 28 members of nine Italian families with individuals affected by XP-D (three patients) or by TTD with photosensitivity due to the XP-D defect (eight patients). No major modifications of the ERCC-2 gene were detected with two cDNA probes in either XP-D or TTD patients indicating that the association between TTD and XP-D is not likely to result from a large deletion or rearrangement involving this gene. We found two RFLPs after digestion of the DNA samples with TaqI or MspI, but neither of them could be related to the molecular alteration determining the pathological phenotype. We also analysed a human homologue detected with the hamster sequence isolated by Arrand et al. (1989), which specifically, but partially, complements the DNA repair deficiency in XP-D cells. Our analysis demonstrated that this gene is not the primary gene defective in XP-D. In fact two RFLPs detected with a genomic probe do not co-segregate with the disease in an XP-D family.

Circulating CD4+ CD25brightFOXP3+ regulatory T-cells are significantly reduced in bullous pemphigoid patients.

Bullous pemphigoid (BP) is the most frequent autoimmune bullous skin disease, characterised by auto-antibodies against the hemidesmosome complex. Recently, regulatory T cells (Tregs) have been implicated in the development of several autoimmune diseases; few data are available in BP, failing to demonstrate a role of this subset in disease pathogenesis. The aim of this study was to investigate the expression and phenotypes of different Tregs (CD4+ CD25brightFOXP3+ and CD8+ CD28- cells) in BP to clarify whether the depletion of this subset constitutes one mechanism of tolerance loss. The CD4+ CD25brightFOXP3 and CD8+ CD28- circulating subsets were determined by flow-cytometry in 26 untreated BP patients and compared with a group of age- and sex-matched healthy controls (HC, n = 30). Absolute and percentage values of the CD4+ CD25brightFOXP3+ cells were significantly reduced in BP compared with HC (median CD25brightFOXP3+ expression within CD4+ cells: 1.8 vs. 3.5%, p = 0.002); conversely, BP patients were characterised by a significant expansion of the CD25brightFOXP3- "activated" T-cell subset. CCR4 and CD62L were expressed on the majority of CD4+ CD25brightFOXP3+ cells (75.2 and 82.3%, respectively). No differences in the CD8+ CD28- subset were found between BP and HC. This is the first report showing a significant reduction of circulating CD4+ CD25brightFOXP3+ Treg frequency in BP patients.

Traditional urinary cytology and tyrosinase RT-PCR in metastatic melanoma patients: correlation with clinical status.

BACKGROUND: The finding of a suspicious urinary cytology is not uncommon in melanoma patients, in as much as morphology alone is often unable to distinguish the variable cytological features of melanoma cells. To date, although tyrosinase reverse transcription (RT)-PCR assay has been used to identify melanoma cells in peripheral blood and tissues, this method has not been applied to the analysis of urine samples. METHODS: RT-PCR mRNA tyrosinase expression was analysed in 79 urine samples from patients with metastatic melanoma and correlated with standard morphology/immunocytology. The results were compared with the disease course and presence of genito-urinary involvement. RESULTS: A positive RT-PCR expression was found in 18/79 urine samples from patients with metastases; four of the 18 patients had positive cytology, nine had atypical cytology, and five had negative cytology. Genito-urinary metastases were demonstrated in 27.8% tyrosinase-positive patients but in only 9.8% of the negative patients. The majority of tyrosinase-positive patients had a progressive disease unresponsive to chemotherapy. Urine samples from 20 patients with non-melanoma cancer and 20 healthy subjects were all negative. CONCLUSIONS: Our data demonstrate the higher sensitivity of RT-PCR compared with standard cytology in detection of urinary melanoma cells, and suggest that this assay could be used as an additional tool in the presence of negative or suspicious cytology.

A novel mutation in the XPA gene associated with unusually mild clinical features in a patient who developed a spindle cell melanoma.

BACKGROUND: Xeroderma pigmentosum (XP) is an autosomal recessive disorder of, in most cases, defective nucleotide excision repair (NER) of ultraviolet radiation (UV)- and chemical-induced DNA damage. The condition is characterized by an increased sensitivity of the skin to UV radiation, with early development of pigmentary changes and premalignant lesions in sun-exposed areas of the skin, signs of photoageing and a greatly increased incidence from a young age of skin tumours including melanoma. Approximately 20% of patients with XP show neurological abnormalities of varying severity due to primary neuronal degeneration. Genetic analysis by somatic cell hybridization has led to the identification in the NER-defective form of XP of seven complementation groups, designated XP-A to XP-G. These complementation groups correspond to different proteins involved in the NER process. XP-A classically includes some of the most severely affected patients. OBJECTIVES: We describe a 61-year-old Punjabi woman with XP. Remarkably she had only mild cutaneous abnormalities, minimal neurological features and unusual longevity, and developed a malignant spindle cell melanoma. There are few previous reports of spindle cell melanoma associated with XP. To gain insight into the aetiology of these unusual features, we sought to analyse the DNA repair properties of the patient and identify the complementation group and the causative mutation in the defective gene. METHODS: Unscheduled DNA synthesis and the inhibition of RNA synthesis were measured. The complementation group was assigned by fusing the cells of our patient with XP cells of known complementation groups and determining the ability to carry out unscheduled DNA repair. Molecular analysis of the cDNA was carried out by polymerase chain reaction and DNA sequencing. RESULTS: Levels of DNA repair were extremely low and complementation analysis assigned the defect to the XP-A group. Sequencing of the XPA gene revealed a novel homozygous mutation of A-->G at the eighth nucleotide of intron 4 causing aberrant splicing and a nonfunctional truncated XP-A protein. However, a small amount of normally spliced mRNA was detected at <5% the level in normal cells. CONCLUSIONS: The small amount of normally spliced mRNA detected may be sufficient to explain the relatively mild clinical features in our patient.

Analysis of mutations in the XPD gene in Italian patients with trichothiodystrophy: site of mutation correlates with repair deficiency, but gene dosage appears to determine clinical severity.

Xeroderma pigmentosum (XP) complementation group D is a heterogeneous group, containing patients with XP alone, rare cases with both XP and Cockayne syndrome, and patients with trichothiodystrophy (TTD). TTD is a rare autosomal recessive multisystem disorder associated, in many patients, with a defect in nucleotide-excision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repair-deficient TTD patients, the defect has been assigned to the XPD gene. The XPD gene product is a subunit of transcription factor TFIIH, which is involved in both DNA repair and transcription. We have determined the mutations and the pattern of inheritance of the XPD alleles in the 11 cases identified in Italy so far, in which the hair abnormalities diagnostic for TTD are associated with different disease severity but similar cellular photosensitivity. We have identified eight causative mutations, of which four have not been described before, either in TTD or XP cases, supporting the hypothesis that the mutations responsible for TTD are different from those found in other pathological phenotypes. Arg112his was the most common alteration in the Italian patients, of whom five were homozygotes and two were heterozygotes, for this mutation. The presence of a specifically mutated XPD allele, irrespective of its homozygous, hemizygous, or heterozygous condition, was always associated with the same degree of cellular UV hypersensitivity. Surprisingly, however, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect. The most severe clinical features were found in patients who appear to be functionally hemizygous for the mutated allele.

Genetic analysis of twenty-two patients with Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder with dwarfism, mental retardation, sun sensitivity and a variety of other features. Cultured CS cells are hypersensitive to ultraviolet (UV) light, and following UV irradiation, CS cells are unable to restore RNA synthesis rates to normal levels. This has been attributed to a specific deficiency in CS cells in the ability to repair damage in actively transcribed regions of DNA at the rapid rate seen in normal cells. We have used the failure of recovery of RNA synthesis, following UV irradiation of CS cells, in a complementation test. Cells of different CS donors are fused. Restoration of normal RNA synthesis rates in UV-irradiated heterodikaryons indicates that the donors are in different complementation groups, whereas a failure to effect this recovery implies that they are in the same group. In an analysis of cell strains from 22 CS donors from several countries and different racial groups, we have assigned five cell strains to the CS-A group and the remaining 17 to CS-B. No obvious racial, clinical or cellular distinctions could be made between individuals in the two groups. Our analysis will assist the identification of mutations in the recently cloned CSA and CSB genes and the study of structure-function relationships.