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C Castagnoli

Publications and source records attributed to C Castagnoli.

17 recordsLinked to original sources
PubMed2002

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider linear range, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4 degrees C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative, whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.

Journal Article↗
PubMed1999

Functional analysis of T lymphocytes infiltrating the dermis and epidermis of post-burn hypertrophic scar tissues.

The cytokine profile of T cell clones (TCC) from the dermis and epidermis of burn patients with hypertrophic scars (HS) in active (AHS) and remission phases (RHS) was determined in this study. We found that AHS tissues are heavily infiltrated by Type 0-Type 1 polarized CD3+ lymphocytes producing high IFN-gamma and low IL-4 levels. Analysis of their surface marker phenotype showed that the high IFN-gamma production was shared equally between the CD4+ TCRalpha/beta and CD8+ TCRalpha/beta clones. The profile of TCC from RHS tissues revealed pronounced infiltration of Type 0-Type 1 polarized lymphocytes with an even more evident Type 1 profile. However, the levels of IFN-gamma produced by RHS-derived TCC were 4-6 times lower than those produced by AHS-derived TCC. These data show that high levels of IFN-gamma produced by Type 0-Type 1 lymphocytes infiltrating HS are a feature of AHS, whereas reduction of this ability to produce high levels of IFN-gamma, though without a shift towards a Type 0-Type 2 phenotype through an increase in IL-4, is characteristic of RHS.

Burns↗
PubMed1999

Expression and role of IL-15 in post-burn hypertrophic scars.

Hypertrophic scarring is a skin disorder that occurs after wounding and thermal injury. There is accumulating evidence that immunologic processes such as infiltration of activated T lymphocytes and altered cytokine production may play a role in the formation of hypertrophic scars. Interleukin-15, a cytokine identified as a T cell growth factor, also acts as a chemoattractant for T cells and has pro-inflammatory properties. We investigated the expression and the role of this cytokine in hypertrophic scarring. IL-15 expression was compared in skin biopsies of hypertrophic scars (HS) both in active (AHS) and in remission (RHS) phases, in normotrophic scars (NTS) and in normal skin using reverse transcriptase-polymerase chain reaction and immunohistochemistry. IL-15 expression in HS was significantly higher than in NTS or normal skin. Furthermore, AHS expressed higher levels of IL-15 than RHS. Immunohistologic analysis of AHS samples showed strong IL-15 immunoreactivity in keratinocytes and Langerhans cells in the epidermis and in macrophages, fibroblasts, and dermal dendritic cells in the dermis. High levels of IL-15 expression in AHS correlated with abundant infiltration of activated CD3+ cells. Ex vivo experiments indicate that IL-15 can sustain the proliferative response of T cells derived from AHS but not from RHS and NTS. In addition, IL-15 prevents both cytokine deprivation and activation-induced apoptosis of T cells derived from AHS. Taken together, these results suggest that IL-15 can be involved in the recruitment, proliferation, and apoptosis inhibition of T cells in AHS. The findings that the evolution from an AHS to a RHS is associated with a decrease in IL15 expression, and with a loss of IL-15 responsiveness in ex vivo-cultured T cells, indicate that this cytokine plays an important role in the biology of pathologic scar formation.

Adolescent↗
PubMed1999

RR interval variation, sympathetic skin reflex and QT dispersion in the assessment of autonomic function in peripheral neuropathy.

In recent years, two simple non-invasive tests, sympathetic skin response (SSR) and RR interval variation (RRIV) have been used to assess autonomic function (Wang et al., 1994; Drory et al., 1995; Chassande et al., 1996; Bordet et al., 1996; Spitzer et al., 1997). Their easy performance by an electromyographic (EMG) machine and successful clinical correlation (Shahani et al., 1990) have made them routine in autonomic assessment in several EMG laboratories. More recently QT dispersion on electrocardiogram (ECG) has been shown to have increased in patients with diabetic neuropathy and several autonomic dysfunctions (Wei et al., 1995; Lengyel et al., 1997; Langen et al., 1997; Axelrod et al., 1997). In order to evaluate the diagnostic value of the RR interval variation, sympathetic skin response and QT dispersion combined, we performed the three tests on a group of 37 patients with several peripheral neuropathies with and without clinical signs of dysautonomia. All patients were studied using an electromyograph (EMG). The results of the SSR, RRIV and QT dispersion combined have shown abnormal values in all patients with neuropathy and clinical dysautonomia and in some patients without clinical signs of dysautonomia, suggesting a subclinical autonomic dysfunction. The principal finding of this study is that the evaluation of the RRIV, SSR and QT dispersion combined may contribute to the assessment of dysautonomia in patients with somatic neuropathy and that they may be currently performed by an electromyograph.

Adult↗
PubMed1998

Primary ex vivo culture of keratinocytes isolated from hypertrophic scars as a means of biochemical characterization of CD36.

The CD36 antigen is a molecule which is ectopically expressed on epidermal keratinocytes of hypertrophic scars and is a good candidate for a marker for a broad range of skin pathologies. Most marker studies have been performed using immunohistochemical techniques on fixed skin sections. Our aim was to investigate the biochemical features of the CD36 expressed in pathological keratinocytes and to find an in vitro model for the study of the regulation of its expression. Here we show how keratinocytes isolated from hypertrophic scars can be cultivated in vitro and employed as a model for the study of these cells. We demonstrated that the antigenic features of the CD36 expressed on keratinocytes of hypertrophic scars are identical to those described for the CD36 expressed by other cell types. The molecule was expressed on the surface of keratinocytes which were non-adherent in vitro. Adherent and proliferating keratinocytes, as well as normal keratinocytes, were CD36 negative both at the surface and intracellularly. The in vitro proliferating cells from hypertrophic scars, but not the normal keratinocytes, showed intracellular expression of CD36 after long-term culture and cell stratification, suggesting a regulated expression of CD36 in pathological keratinocyte differentiation.

Adult↗
PubMed1996

The influence of progress in the treatment of severe burns on the quality of life.

The problems related to burns treatment can be considered among the oldest and most passionating in history of medicine. Since the early forties amazing progresses have been done in the comprehension of the physiopathology of burns. The fast development of resuscitating techniques determined a remarkable reduction of mortality in the first phase; in a similar way through new concepts in the project and construction of intensive care facilities dedicated to burns, where patients can be isolated and a high standard of environmental control can be guaranteed, together with new topical and systemic antibacterial treatment protocols, a significant reduction of infectious complications has been achieved. Concerning surgical treatment early tangential excision and cultured epidermal grafts can be considered the cornerstones of burn therapy. Quality of life of burnt patients have been greatly ameliorated by these technical advances. Burn sequelae however remain the main concern of survivors because of the many controversial aspects of burn scar physiopathology and treatment. Along my career many endeavours I dedicated in this important research field. I will then report the results of most interesting clinical and experimental studies carried out in the last 30 years by our group in collaboration with basic researchers. All the work done in this domain enhance our hope that good results can really improve quality of life in burns: this is the goal for those who dedicated the whole life to relieve the suffering of these badly injured patients.

Anti-Bacterial Agents↗
PubMed1994

Similar ectopic expression of ICAM-1 and HLA class II molecules in hypertrophic scars following thermal injury.

In previous studies we have shown that HLA Class II antigens are expressed by keratinocytes and fibroblasts in hypertrophic scars. Because of the potential role of immunological events in the pathogenesis of hypertrophic scars, in the present study we have tested hypertrophic scars for the expression of intercellular adhesion molecule-1 (ICAM-1), a molecule which plays an important role in immunological phenomena. Immunoperoxidase staining with anti-ICAM-1 MoAb of 10 hypertrophic scar samples detected this molecule on epidermal keratinocytes and on about 30 per cent of fibroblasts at the site of lymphoid infiltration. The expression of ICAM-1 in hypertrophic scars was similar to that of HLA Class II antigens. Since the concomitant expression of ICAM-1 and HLA Class II by keratinocytes is known to enhance their antigen-presenting properties, the present results support the possibility that immunological events play a role in the disruption of the normal processes of wound healing and tissue remodelling which result in hypertrophic scars.

Adult↗
PubMed1994

Altered biosynthesis of tumour necrosis factor (TNF) alpha is involved in postburn hypertrophic scars.

The present study shows that the decrease of TNF alpha in postburn hypertrophic scars is due to a decrease in the steady-state level of TNF alpha mRNA and thus to an altered biosynthesis of the cytokine. Thirteen scars, including seven hypertrophic and six normotrophic scars, were tested for TNF alpha mRNA production by a semiquantitative reverse polymerase chain reaction (PCR) method. TNF beta and beta actin were tested as a control. Six out of six normotrophic scar samples amplified with primers for TNF alpha showed a positive PCR signal up to the 1:32 dilution. On the contrary all the hypertrophic tested samples (7/7) had a positive PCR signal only at the 1:1 or 1:2 dilution. All samples, both normotrophic and hypertrophic, were homogeneous as to TNF beta production.

Actins↗
PubMed1993

TNF production and hypertrophic scarring.

The percentage of TNF alpha- and beta-positive cells was analyzed in hypertrophic scar (N = 13), normotrophic scar (N = 7), and normal skin (N = 6) biopsies using monoclonal antibodies and immunoperoxidase staining of cryostat tissue sections. Samples were first characterized for infiltrating cells. In hypertrophic samples there was a significant increase in activated infiltrating cells, capable of producing TNF beta and IL-1 beta. In contrast, the percentage of TNF alpha-positive cells was significantly lower than that detected in normotrophic scars. In fact, in hypertrophic scar samples a positive staining with anti-TNF alpha mAb was restricted to 8% of tissue-infiltrating cells compared to 35.4% of the cells present in normotrophic scars; 12% of infiltrating cells were stained in normal skin sections. These results suggest that TNF alpha may be important for normal wound healing and that hypertrophic scarring might be partially a consequence of a low amount of TNF alpha.

Adolescent↗
PubMed1990

The HLA-DR beta 16 allogenotype constitutes a risk factor for hypertrophic scarring.

Nineteen patients that had developed hypertrophic scars subsequent to thermal injury were typed for HLA class II allogenotypes with the restriction fragment length polymorphism technique. A significant association was found with DR beta 16 (pc = 1.45 x 10(-4); relative risk = 12.25). This finding adds evidence to other data suggesting that immunologic phenomena are involved in pathologic scarring. Moreover, the results presented here have allowed an identification of a genetically determined risk factor for hypertrophic scar formation located in the HLA region.

Cicatrix↗
PubMed1990

Anomalous expression of HLA class II molecules on keratinocytes and fibroblasts in hypertrophic scars consequent to thermal injury.

Immunoperoxidase staining of skin sections obtained from 11 hypertrophic scars, six normotrophic scars and three samples of normal skin were performed using anti-HLA monoclonal antibodies (HLA-DR, -DQ, class I), anti-interleukin-2 receptor (IL-2R) and anti-CD1. Sections from all hypertrophic scars showed an anomalous expression of HLA-DR molecules on keratinocytes and fibroblasts. Moreover hypertrophic scars were characterized by dense infiltrates of IL-2R-positive cells and by the presence of abundant Langerhans (CD1+) cells in the epidermis and dermis. These results support the hypothesis that immunologic mechanisms play an important role in hypertrophic scarring and point to an involvement of cell-mediated immune phenomena.

Adolescent↗
PubMed1987

HLA class I- like antigen expression on human leukemic cells.

The expression of HLA class I- like molecules was analyzed on human acute and chronic leukemic cells. The presence on leukemic cells of class I- like molecules, absent on the patient's normal lymphocytes, was examined by complement- dependent lymphocytotoxicity using platelet absorbed alloantisera that recognize HLA-linked, 45-12 kd, beta-2-microglobulin associated molecules, selectively expressed on PHA-activated cells. A positive reactivity of the anti- class I- like alloantisera was found in 50% of the acute leukemias (cALL, T-ALL and AML), independently of the lineage of differentiation, while chronic lymphocytic leukemias (B-CLL) were constantly negative. It is suggested that beta-2-microglobulin associated HLA molecules may represent markers of leukemic blast activation and/or maturation state.

Adult↗
PubMedDate not supplied

Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissues.

In this study, skin-infiltrating cells were characterized in both the active and remission phases of post-burn hypertrophic scar biopsies. Immunohistochemistry examination of active phase samples showed an abundant presence of Langerhans cells, T cells, macrophages, a low presence of natural killer cells and the lack of B lymphocytes. In active hypertrophic scars T lymphocytes infiltrate deep into the superficial dermis and are also observed in the epidermis: CD3+ cells were present at about 222 +/- 107 per 0.25 mm2. In particular the analysis of lymphocyte subpopulations showed that CD4+ T cells predominate in the dermis as well as in the epidermis of active hypertrophic scars whereas CD8+ cells were less well represented (CD4/CD8 ratio is 2.06). This distribution was also shown in remission phase samples and in normotrophic scar specimens, although the lymphocyte number was significantly lower. Approximately 70 per cent of T lymphocytes present in the tissue involved in active phase hypertrophic scar samples were activated (positive with anti-HLA-DR and IL-2 receptor antibodies) which is significantly higher than remission phase hypertrophic and normotrophic scars, in which positivity was 40 and 38 per cent, respectively. Upon activation, the lesional lymphocytes release several cytokines, locally and transiently, that interact with specific receptors in response to different stimulation. Central to the immune hypothesis of hypertrophic scars is that some of the T-cell lymphokines act on keratinocytes, fibroblasts and other cell types to induce changes characteristic of these scars. The presence and close proximity of activated T lymphocytes and antigen-presenting cells of various phenotypes in both the epidermis and dermis of hypertrophic tissues provides strong circumstantial evidence of a local immune response. However, the manner in which T cells achieve and maintain their activated state in hypertrophic tissues is not yet known, and both antigen-dependent and independent mechanisms may contribute.

Adolescent↗