Ozone effect on the inflammatory and proteomic profile of human macrophages and airway epithelial cells.

Ozone (O3) is one of the most harmful urban pollutants, but its biological mechanisms have not been fully elucidated yet. Human bronchial epithelial cells (HBEpC) and human macrophage cells (differentiated human monocytic cell line) were exposed to O3 at the concentration of 240 µg/m3 (120 ppb), corresponding to the European Union alert threshold. Cell viability, reactive oxygen species (ROS) production, and pro-inflammatory cytokines release (IL-8 and TNF-α) were evaluated. Results indicated that O3 exposure increases ROS production in both cell types and enhances cytokines release in macrophages. O3 stimulated IL-8 and TNF-α in HBEpC when the cells were pretreated with Lipopolysaccharide, used to mimic a pre-existing inflammatory condition. Proteomics analysis revealed that, in HBEpC, O3 caused the up-regulation of aldo-keto reductase family 1 member B10, a recognized critical protein in lung carcinogenesis. In conclusion, our results show that 120 ppb O3 can lead to potential damage to human health suggesting the need for a revision of the actual alert levels.

Skin cancer and welding.

Many workers in several different occupations can be exposed to ultraviolet radiation (UVR), which may increase their risk of developing skin cancer. Welding, an occupation employing an estimated 11 million people worldwide, is one such occupation. Welders are known to be exposed to the full spectrum of UVR from the welding arc and often experience burns and localized cutaneous erythema. In 2017, UVR from welding was classified as carcinogenic to humans based on sufficient evidence of ocular melanoma in humans. It has been hypothesized that exposure to UVR from the welding arc also may increase the risk of skin cancer among workers in this occupation. This review summarizes the current literature on skin cancer risk in welders.

Inhalation of iron-abundant gas metal arc welding-mild steel fume promotes lung tumors in mice.

Welding fumes were reclassified as a Group 1 carcinogen by the International Agency for Research on Cancer in 2017. Gas metal arc welding (GMAW) is a process widely used in industry. Fume generated from GMAW-mild steel (MS) is abundant in iron with some manganese, while GMAW-stainless steel (SS) fume also contains significant amounts of chromium and nickel, known carcinogenic metals. It has been shown that exposure to GMAW-SS fume in A/J mice promotes lung tumors. The objective was to determine if GMAW-MS fume, which lacks known carcinogenic metals, also promotes lung tumors in mice. Male A/J mice received a single intraperitoneal injection of corn oil or the initiator 3-methylcholanthrene (MCA; 10 µg/g) and, one week later, were exposed by whole-body inhalation to GMAW-MS aerosols for 4 hours/day x 4 days/week x 8 weeks at a mean concentration of 34.5 mg/m3. Lung nodules were enumerated by gross examination at 30 weeks post-initiation. GMAW-MS fume significantly increased lung tumor multiplicity in mice initiated with MCA (21.86 ± 1.50) compared to MCA/air-exposed mice (8.34 ± 0.59). Histopathological analysis confirmed these findings and also revealed an absence of inflammation. Bronchoalveolar lavage analysis also indicated a lack of lung inflammation and toxicity after short-term inhalation exposure to GMAW-MS fume. In conclusion, this study demonstrates that inhalation of GMAW-MS fume promotes lung tumors in vivo and aligns with epidemiologic evidence that shows MS welders, despite less exposure to carcinogenic metals, are at an increased risk for lung cancer.

Genetic engineering of T cells for the immunotherapy of haematological malignancies.

Accumulating experimental and clinical evidence has been obtained over recent years in support of the notion that the immune system has the potential to cure cancer. The most convincing example is the graft versus leukaemia effect observed after allogeneic haematopoietic stem cell transplantation. In the autologous setting, however, the isolation and expansion of naturally occurring tumour-specific T cells is a challenging task. Cancer antigens are often self-antigens and cancer-specific T cells are thus subject to selective mechanisms of central and peripheral tolerance. The significant advances in gene-transfer technologies developed over the last decade have offered new tools to overcome these limitations. Natural T cells can be genetically modified to generate high numbers of 'supernatural' tumour-reactive T cells from virtually every cancer patient. Supernatural T cells may express clonal receptors providing new specificities, factors increasing T-cell performance or safety factors enabling their elimination in case of toxicity. Technological improvements applied to novel concepts of T-cell biology and oncogenesis will allow to simultaneously equip T cells with different transgenes and expand a real 'army' of lymphocytes trained to selectively eradicate cancer cells.

Effect of enzymatic debridement with collagenase on acute and chronic hard-to-heal wounds.

OBJECTIVE: A large retrospective analysis was performed to assess the outcomes of burns and chronic ulcers treated with collagenase in an outpatient setting. No cost comparison was performed. METHOD: Of 979 patients entered into the analysis, 647 had burns affecting < or = 15% body surface area (BSA) and 332 had chronic ulcers of various aetiologies. AII were treated with collagenase-based ointments once daily (Noruxol or Iruxol, Smith and Nephew). Treatment was continued until complete healing was achieved. RESULTS: In burns patients the overall average healing time was 17.9 days in the paediatric population and 23.6 days in adults. Burn depth and presence of eschar were the main factors affecting healing probability. The chronic ulcers were predominantly of diabetic, venous and mixed aetiology. Average healing time was 15.4 weeks, with ulcers of mixed aetiology showing the shortest average healing time (9.2 weeks). There was a positive correlation between wound area and healing time. The topical application of collagenase-based ointments was well tolerated by patients and caregivers. CONCLUSION: This large retrospective analysis shows that collagenase treatments in outpatient clinics are effective and well accepted in patients with burns affecting < or = 15% BSA or with chronic ulcers of various aetiologies. Implementation of collagenase treatments in outpatient clinics has the potential to improve wound healing and may also decrease the cost of wound care.

IGF-I stimulates proliferation of spontaneously immortalized human keratinocytes (HACAT) by autocrine/paracrine mechanisms.

HaCaT keratinocytes are derived from adult human skin and although spontaneously immortalized, remain highly related to their normal counterparts. We observed that HaCaT cells can proliferate in serum-free medium (SFM), in contrast to normal human keratinocytes whose growth in vitro requires a feeder layer and/or the supplementation with hormones and growth factors. Since autocrine production of growth factors has been proposed as the pathway that cells may exploit to escape growth regulation, we have investigated whether this is occurring in HaCaT cultured in SFM. Either epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-I) was effective and dose-dependently stimulated HaCaT replication. The ability of these keratinocytes to express EGF and IGF-I and their receptors was investigated by northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). We report that HaCaT cells synthesize mRNAs for IGF-I, IGF-II, IGF-IR and EGF-R but not EGF mRNA. Immunoneutralization of IGF-I with specific monoclonal antibodies blocked spontaneous HaCaT proliferation in SFM, as did incubation with antibodies against IGF-IR. These data demonstrate that an autocrine/paracrine loop based on IGF-I may allow HaCaT keratinocytes to proliferate autonomously in culture in contrast to keratinocytes in primary culture. A similar mechanism may be involved in the development of hyperproliferative diseases of human skin and its functional disruption may represent the target for therapeutic approaches.

A comparative evaluation of chondrocyte/scaffold constructs for cartilage tissue engineering.

This study aimed to evaluate three biodegradable scaffolds as cell carriers for in vitro cartilage regeneration using mature human chondrocyte cells. We compared cell distribution, viability and morphology and we evaluated the mechanical properties of the constructs after 2 weeks of in vitro culture. The materials used as scaffolds were fibrin glue, a collagen sponge and a polyurethane foam (DegraPol(R)). Fibrin glue was found unsuitable as a chondrocyte carrier vehicle after culture times longer than a few days, probably due to significant barriers to nutrients and oxygen diffusion, and the material weakened rapidly. The collagen-based sponge was found to be unsuitable to support chondrocyte survival in vitro, although the presence of newly synthesized collagen was observed in these constructs. The synthetic biodegradable scaffold was more adequate in supporting cell survival and mechanical properties. After 2 weeks of static culture, the storage modulus obtained by dynamic shear testing was in the order of 0.7 kPa in fibrin constructs, 3.7 kPa in collagen constructs and 105 kPa in DegraPol(R) constructs. The better mechanical stability of the synthetic foam supports further investigation in the possible use of synthetic biomaterials as biodegradable scaffolds for in vitro cartilage regeneration. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 55-64).

Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment.

Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis on animal cells. A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems. We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure. A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described. The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 x 10(-3) Pa (0.03 dyn/cm(2)) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 mum/s. Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure.

Regioselective enzymatic glycosylation of natural polyhydroxylated compounds: galactosylation and glucosylation of protopanaxatriol ginsenosides.

Ginsenoside Rg1 (1), the most representative Ginsenoside from Panax Ginseng C.A. Meyer belonging to the protopanaxatriol family, has been galactosylated by action of the beta-(1,4)-galactosyltransferase (GalT) from bovine colostrum, using UDP-galactose as an activated sugar donor. The enzyme showed the well-known specificity for the formation of a beta-linkage with the C-4 OH of the glucose acceptor, but it was not able to discriminate between the two glucose moieties of 1, giving a mixture of mono- and digalactosylated derivatives. Other natural Rg1-analogues such as F1, Rh1, Re, as well as the synthetic derivative 6'-O-acetyl-Rg1 have been also galactosylated, giving monolactosyl derivatives. GalT was also able to accept UDP-glucose as an activated sugar donor, giving rise to cellobiosyl derivatives of Rg1.

Comparison of different membranes with cultures of keratinocytes from man for percutaneous absorption of nitroglycerine.

The permeability barrier function of cell-culture membranes to the permeation of nitroglycerine was evaluated to find an alternative to skin from man for ex-vivo skin-permeation tests. The membranes were prepared, under submerged conditions, by inducing the growth of keratinocytes, from different donors, on a film of esterified jaluronic acid for different times (10, 20 and 30 days). Their permeability barrier functions were compared with those of some of the most widely used artificial membranes, silicone rubber (Silastic), cellulosic material (Cuprophan, Millipore HAWP), polysulphone membrane (Supor) and polytetrafluoroethylene membrane (TF-PTFE), and with those of biological membranes such as fresh and frozen skin, stratum corneum and epidermis from man, and hairless mouse skin. For each membrane the permeation profile was obtained and the flux was calculated. The permeation profiles for nitroglycerine were similar and linear in the first 2-3 h for all the synthetic membranes tested except TF-PTFE. For this membrane the profile was linear throughout the period considered and the amount permeating in 24 h (1603 microg cm(-2)) was significantly lower than those obtained for the other artificial membranes (between 1926 and 2508 microg cm(-2)). The amounts permeating through all the biological membranes in 24 h were in the range 520 to 781 microg cm(-2), except those for the keratinocyte-culture membranes, which were in the range 1730 to 2553 microg cm(-2). Prolonging the growth period of cultured keratinocytes did not affect nitroglycerine permeation. The findings suggest that these keratinocyte-culture membranes have some advantages--good reproducibility if obtained from the same donor; many membranes can be obtained from the same donor; the preparation is simple; they can be handled more easily than traditional cell-culture membranes; and they afford constant penetration rates for a longer period than synthetic membranes. The membranes could be used for preliminary in-vitro permeation studies.

Interaction of liposomes composed of phospholipids, GM1 ganglioside and cholesterol with human keratinocytes in culture.

We studied the possibility of supplementing human keratinocytes with exogenous lipids (phospholipids, sphingolipids and cholesterol) and evaluated their influence on cell proliferation, using cells cultured in vitro. Experiments carried out with liposomes composed of cholesterol/GM1 ganglioside and different phospholipids (5:1.5:10, M/M/M), showed that liposomes associated with cells more efficiently when they contained soya lecithin. The treatment with liposomes made of the ternary mixture did not modify the rate of cell proliferation, as assessed by the incorporation of [3H]-thymidine. In contrast, the proliferation rate strongly decreased (65% with respect to the control) using the same liposomes without GM1. Experiments carried out with GM1 alone showed a strong stimulation of the proliferation rate (144% with respect to the control). Fluorescence dequenching experiments, carried out with the probe octadecyl rhodamine B chloride, showed that fusion was the main mechanism of liposome-cell interaction. Metabolic studies established that exogenously administered GM1--either embedded in liposomes or as a pure glycolipid dispersion--led to the production of several products, including ceramide. Altogether, these results show that different, opposing effects can be exerted on cell proliferation by the administration of lipids, separately or in mixtures, to human keratinocytes, and indicate the importance of a correct formulation for supplementing human keratinocytes with exogenous lipids.

Ten-year experience with craniofacial implants: clinical and experimental results.

The choice of bone substitute to be used in the cranio-maxillofacial districts raises several problems relating to the biocompatibility and mechanical resistance of the material. Over the past 10 years a total of 245 cranio-maxillofacial skeletal implants have been performed by the Department of Plastic Surgery at Niguarda Ca' Granda Hospital in Milan. The choice of material varied depending on the market availability of products and the experience acquired of both positive results and the onset of short-term and long-term complications. All patients were monitored with a minimum follow-up of 2 years and a maximum of 10 (mean 36 months). The following parameters were taken into account: complications linked to graft contamination, graft extrusion and decubitus, resorption times, degradable materials, resorption of underlying bone tissue and reactivity of the surrounding tissues. Studies on the cell cytohistotoxicity of materials used were performed in parallel by the cell culture laboratory at the Department of Plastic Surgery of Niguarda Hospital, using human keratinocyte and fibroblast cultures.

[Cytochemical study of human sperm metabolism during in vitro capacitation after cryopreservation]. / Studio citochimico sul metabolismo degli spermatozoi umani nel corso della capacitazione in vitro anche dopo crioconservazione.

To study the effects of deep freezing on the energy metabolism of human spermatozoa, we investigated, by cytochemical quantitative methods, cytochrome oxidase and lactate dehydrogenase activities of fresh and frozen human spermatozoa during in vitro capacitation. Fresh and frozen human spermatozoa were incubated in Biggers, Whitten and Wittingham's medium supplemented with 15% heat-inactivated human serum. Both histoenzymological reactions can be quantitated and have been evaluated by microdensitometric method. The results indicate that human spermatozoa depend almost entirely on anaerobic glycolysis during in vitro capacitation and suggest that both aerobic and anaerobic metabolism in spermatozoa are only slightly impaired by freezing-thawing and storage.

Etiology of severe asthenozoospermia and fertility prognosis. A screening of 5216 semen analyses.

A review of n = 5216 semen analyses performed in our two Clinics from January 1986 to December 1989 allowed to identify n = 35 patients whose sperm had constantly very low motility (less than 5% progressive motile gametes in three subsequent analyses; necrozoospermia cases were excluded from this study). This apparently rare but severe anomaly was found to be associated not only with ultrastructural anomalies (n = 18), but also with positive seminal bacteriology (n = 8) or the presence of antisperm antibodies (n = 2). In eight cases the cause(s) for this constant asthenozoospermia remained obscure. The fertility potential of the men affected was followed-up and is discussed in relation to their anamnesis, physical exam and seminal characteristics.

Follicular fluid enhances sperm motility and velocity in vitro.

Forty-three follicular fluids (FFs) obtained during laparoscopy were tested in vitro for their effect(s) on sperm motility using gametes obtained by the swim-up procedure. Both the proportion of motile sperm and the velocity distribution patterns were evaluated as function of time by multiple-exposure photography technique. At the various incubation periods considered, all FFs maintained or then enhanced sperm motility as compared with the paired control suspension incubated with a sperm survival medium. The results of the sperm contact test for FFs from women who achieved pregnancy versus FFs from women who remained infertile were not significantly different for both parameters measured. Comparing these with our previously reported results, we may hypothesize that FF released at ovulation into the peritoneal cavity may counteract some sperm-immobilizing effect of peritoneal fluid, thereby increasing the fertility potential of the male gametes.