Mathematical modelling of contact dermatitis from nickel and chromium.

Dermal exposure to metal allergens can lead to irritant and allergic contact dermatitis (ACD). In this paper we present a mathematical model of the absorption of metal ions, hexavalent chromium and nickel, into the viable epidermis and compare the localised irritant and T-lymphocyte (T-cell) mediated immune responses. The model accounts for the spatial-temporal variation of skin health, extra and intracellular allergen concentrations, innate immune cells, T-cells, cytokine signalling and lymph node activity up to about 6 days after contact with these metals; repair processes associated with withdrawal of exposure to both metals is not considered in the current model, being assumed secondary during the initial phases of exposure. Simulations of the resulting system of PDEs are studied in one-dimension, i.e. across skin depth, and three-dimensional scenarios with the aim of comparing the responses to the two ions in the cases of first contact (no T-cells initially present) and second contact (T-cells initially present). The results show that on continuous contact, chromium ions elicit stronger skin inflammation, but for nickel, subsequent re-exposure stimulates stronger responses due to an accumulation of cytotoxic T-cell mediated responses which characterise ACD. Furthermore, the surface area of contact to these metals has little effect on the speed of response, whilst sensitivity is predicted to increase with the thickness of skin. The modelling approach is generic and should be applicable to describe contact dermatitis from a wide range of allergens.

Systematic review of respiratory case definitions in metalworking fluid outbreaks.

BACKGROUND: Since the mid-1990s, outbreaks of asthma and extrinsic allergic alveolitis (EAA) have been identified in workers exposed to metalworking fluids (MWFs). The cause of these outbreaks remains to be determined. AIMS: To identify and review all previously published occupational lung disease case definitions and diagnostic criteria that have been utilized during MWF outbreak investigations. METHODS: Respiratory outbreaks due to MWFs were identified by a systematic literature search for articles published between 1990 and October 2011. Investigations reporting the usage of disease case definitions or diagnostic criteria for respiratory disease were reviewed and summarized. RESULTS: The literature search identified 35 papers relating to 27 outbreaks of respiratory disease in MWF-exposed workers. Fourteen case definitions for MWF-related respiratory disease were identified: seven for EAA, five for occupational asthma and one each for humidifier fever and industrial bronchitis. A single paper was identified where any comparison of different disease case definitions (for EAA) had been performed. CONCLUSIONS: A range of case definitions and diagnostic criteria for MWF respiratory disease have been utilized in outbreak investigations, but the majority have been produced for individual outbreak investigations without previous validation. It may be difficult to compare the findings of future workplace studies without a more standardized approach to case identification and diagnosis.

A mathematical model of the in vitro keratinocyte response to chromium and nickel exposure.

A mathematical model describing the main mechanistic processes involved in keratinocyte response to chromium and nickel has been developed and compared to experimental in vitro data. Accounting for the interactions between the metal ions and the keratinocytes, the law of mass action was used to generate ordinary differential equations which predict the time evolution and ion concentration dependency of keratinocyte viability, the amount of metal associated with the keratinocytes and the release of cytokines by the keratinocytes. Good agreement between model predictions and existing experimental data of these endpoints was observed, supporting the use of this model to explore physiochemical parameters that influence the toxicological response of keratinocytes to these two metals.

Basic fibroblast growth factor increases the multiplication and migration of a serum-free derivative of CACO-2 but does not affect differentiation.

Basic fibroblast growth factor (bFGF) is found in the extracellular matrix and around the endothelial and epithelial cells of some human colon carcinomas. It is believed to play a role in angiogenesis, but in addition, recent data suggest that it can directly stimulate mitogenesis in some colon carcinoma cell lines. To clarify the role of bFGF in human colon carcinoma, we developed a model of Caco-2 which grew in serum-free conditions so that the effect of bFGF on multiplication, migration, and differentiation could be studied in defined conditions. Through morphological and biochemical studies in serum-free conditions, we demonstrated that this subline of Caco-2 differentiated spontaneously on reaching confluence. Using this model, we found that bFGF did not affect differentiation but that multiplication and migration were increased. The implication of these findings is that bFGF, released from the extracellular matrix by invading cells or produced by neovascular endothelial cells, can increase the mitogenic rate and migratory potential of colon carcinoma cells. In addition, the dual role of bFGF in stimulating colon carcinoma cells directly and promoting angiogenesis suggests that anti-bFGF strategies could form the basis of a novel approach to the treatment of colon carcinoma.

Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.

A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.

Changes in the hormone dependency of epithelial cell proliferation in the genital tract of mice following neonatal oestrogen treatment.

The genital tract epithelium of the female laboratory mouse has been widely studied as a model of oestrogen-dependent growth and proliferation. Perturbation of the hormonal imprinting of these tissues during neonatal development has also been used to study the development of pathological abnormalities, particularly in the cervical epithelium. This study demonstrates that mice treated neonatally from days 1-5 with supraphysiological concentrations of oestrogen are able to maintain high levels of proliferation following the removal of the ovaries later in adult life. This high level of proliferation was shown to be independent of the ovarian oestrogens and of oestrogens produced peripherally by aromatisation. These results suggest conversion of the genital tract in these mice to a fully hormonal "independent" state. However, neonatal treatment with oestrogen was not found to produce a uniform change to hormonal independence. Further challenge of the adult ovariectomised mice with oestrogen, demonstrated that a population of cells still retained the ability to respond to the mitogenic influence of this hormone.

Progressive morphogenesis in vivo after transplantation of cultured small bowel epithelium.

An experimental model for the primary culture and transplantation of late foetal rat small intestinal epithelium is described. Multicellular aggregates of mucosal epithelium containing pre-crypt proliferative cells were isolated from 20-day foetal rat intestine by enzymatic disaggregation. Cellular aggregates, which we refer to as "epithelial organoids," attached readily in culture, proliferated, and spread to produce coalescing colonies within 10 days. Enterocytes were maintained in culture for 3 days, removed as cell sheets, and incubated overnight with foetal mesenchyme. Fourteen recombinant preparations were then grafted to the renal subcapsular space of adult nude mice. Four of six grafts retrieved after 1 wk had developed. Histology demonstrated the formation of simple tubular structures lined by a polarized columnar epithelium. At 14 days, two of eight grafts had developed and demonstrated temporal progression of morphogenesis. Histology showed rudimentary crypts and villi lined by different epithelial cell types, including enterocytes and goblet cells. Small bowel proliferative cells within "epithelial organoids" from 20-day foetal intestine, may be maintained in primary culture for up to four days. After short term primary culture, these proliferative cells retain the capacity for progressive organotypic morphogenesis and pluripotent cytodifferentiation, after transplantation to adult recipients.

Colonic mucosal replacement by syngeneic small intestinal stem cell transplantation.

A novel method of colonic mucosal replacement by transplantation of disaggregated small intestinal epithelium is described. Thirty-one inbred rats had the ascending colon isolated, and surgical mucosectomy was performed on the "free" loop. Epithelial cell aggregates were isolated from postnatal small intestine using collagenase and dispase digestion, then 20 microL of the cell suspension was "seeded" over the denuded colonic muscle of 25 recipient rats. Six control rats had surgical mucosectomy only. All loops were retrieved after 14 days for histologic examination. Stem cell lineage studies were used with selective staining protocols to identify enterocytes, goblet cells, entero-endocrine cells, and Paneth cells. A neomucosa with typical small bowel morphology including crypts and villi and all four stem cell lineages was regenerated by transplanted cells on the colonic muscle in 19 of 25 (76%) recipients. Control loops showed no epithelial regrowth confirming total mucosectomy. With appropriate stromal support, transplanted small intestinal stem cells have the capacity to re-epithelialize denuded colonic muscle with small bowel neomucosa.

Growth factor regulation of proliferation in primary cultures of small intestinal epithelium.

Although the intestinal epithelium is one of the most rapidly renewing tissues, little is known about the major growth factors that control the rate of cell replacement and migration. Recently, a primary culture model has been described for the developing rat small intestinal epithelium, which permits epithelial growth while maintaining interactions with associated stromal cells, thereby possessing several contextual advantages over established cell lines (Evans et al., 1992). We have used this model to begin to determine the factors that may be involved in controlling intestinal epithelial cell proliferation. Under the conditions examined, no single growth factor promoted exclusive proliferation of epithelial cells; stromal cell proliferation was also apparent. The most potent stimulators of epithelial proliferation were insulin and insulin-like growth factor 1 (IGF-1). These factors also appeared to inhibit migration of the epithelial cells. 5-10 ng/ml EGF, 5-20 ng/ml TGF alpha, and 10-20 ng/ml PDGF also slightly increased epithelial cell numbers. Cell proliferation was inhibited by 0.1 ng/ml TGF beta-1. In Dulbecco's modified Eagle's medium (DMEM) containing 0.25 IU/ml insulin, glucose levels of 2-3 g/liter permitted epithelial growth with limited expansion of the stromal cell population. Higher levels of glucose further stimulated the nonepithelial cell types. Transferrin was also a potent stimulator of both cell types.

Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line).

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability of the HepG2 cells they were cultured in 4-32 microM copper(II) sulphate (CuSO4)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 microM Cu2+ but significantly declined at 64 microM compared to the controls. Lysosomal acid phosphatase showed a concentration dependent decline in activity at 72 h. Cellls exposed to 64 microM Cu2+ had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 microM Cu2+, increases of necrosis up to 18% were seen whereas comparable levels of apoptotic and necrotic cells (<5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming efficiency at concentrations of 16 microM Cu2+ and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.

Clinical investigation of an outbreak of alveolitis and asthma in a car engine manufacturing plant.

BACKGROUND: Exposure to metal working fluid (MWF) has been associated with outbreaks of extrinsic allergic alveolitis (EAA) in the USA, with bacterial contamination of MWF being a possible cause, but is uncommon in the UK. Twelve workers developed EAA in a car engine manufacturing plant in the UK, presenting clinically between December 2003 and May 2004. This paper reports the subsequent epidemiological investigation of the whole workforce. The study had three aims: (1) to measure the extent of the outbreak by identifying other workers who may have developed EAA or other work-related respiratory diseases; (2) to provide case detection so that those affected could be treated; and (3) to provide epidemiological data to identify the cause of the outbreak. METHODS: The outbreak was investigated in a three-phase cross-sectional survey of the workforce. In phase I a respiratory screening questionnaire was completed by 808/836 workers (96.7%) in May 2004. In phase II 481 employees with at least one respiratory symptom on screening and 50 asymptomatic controls were invited for investigation at the factory in June 2004. This included a questionnaire, spirometry and clinical opinion. 454/481 (94.4%) responded and 48/50 (96%) controls. Workers were identified who needed further investigation and serial measurements of peak expiratory flow (PEF). In phase III 162 employees were seen at the Birmingham Occupational Lung Disease clinic. 198 employees returned PEF records, including 141 of the 162 who attended for clinical investigation. Case definitions for diagnoses were agreed. RESULTS: 87 workers (10.4% of the workforce) met case definitions for occupational lung disease, comprising EAA (n = 19), occupational asthma (n = 74) and humidifier fever (n = 7). 12 workers had more than one diagnosis. The peak onset of work-related breathlessness was Spring 2003. The proportion of workers affected was higher for those using MWF from a large sump (27.3%) than for those working all over the manufacturing area (7.9%) (OR = 4.39, p<0.001). Two workers had positive specific provocation tests to the used but not the unused MWF solution. CONCLUSIONS: Extensive investigation of the outbreak of EAA detected a large number of affected workers, not only with EAA but also occupational asthma. This is the largest reported outbreak in Europe. Mist from used MWF is the likely cause. In workplaces using MWF there is a need to carry out risk assessments, to monitor and maintain fluid quality, to control mist and to carry out respiratory health surveillance.

Differentiation and immune function of human dendritic cells following infection by respiratory syncytial virus.

RSV causes annual epidemics of bronchiolitis in winter months resulting in the hospitalization of many infants and the elderly. Dendritic cells (DCs) play a pivotal role in coordinating immune responses to infection and some viruses skew, or subvert, the immune functions of DCs. RSV infection of DCs could alter their function and this could explain why protection after natural RSV infection is incomplete and of short duration. In this study, this interaction between DCs and RSV was investigated using a human primary culture model. DCs were generated from purified healthy adult volunteer peripheral blood monocytes. Effects of RSV upon DC phenotype with RSV primed DCs was measured using flow cytometry. Changes to viability and proliferation of cocultured DCs and T-cells were determined using microscopy with fluorescent dyes (Hoechst 33342 and propidium iodide). DC maturation was not prevented by the RSV challenge. RSV infected a fraction of DCs (10-30%) but the virus replicated slowly in these cells with only small reduction to cell viability. DCs challenged with RSV stimulated T-cell proliferation less well than lipopolysaccharide. This is the first study to demonstrate RSV infection of human monocyte derived DCs and suggests that the virus does not significantly interfere with the function of these cells and potentially may promote cellular rather than humoral responses.

Enzyme exposure in the British baking industry.

OBJECTIVES: Enzymes are commonly used in the baking industry, as they can improve dough quality and texture and lengthen the shelf life of the final product. There is little published information highlighting exposure to enzymes (other than fungal alpha-amylase) in the baking industry, therefore the purpose of this study was to identify antibodies and develop assays for the measurement of a variety of such enzymes in samples of airborne flour dust. METHODS: Polyclonal antibodies to bacterial amylase, glucose oxidase and amyloglucosidase were identified and developed into ELISA assays. The assays showed limited cross-reactivity with other enzymes commonly used in the baking industry. RESULTS: We measured levels of airborne enzymes in 195 personal air samples taken from a sample of 55 craft baking establishments. We were able to detect amyloglucosidase in 9% (16/184) of the samples, fungal alpha-amylase in 6% (11/171), bacterial alpha-amylase in 7% (13/195). However, we were unable to detect glucose oxidase in any of the samples. Measurements for protease enzymes were not carried out. Median levels in detectable samples of amyloglucosidase, fungal alpha-amylase and bacterial amylase were similar at 10.3, 5.3 and 5.9 ng/m(3), respectively. These figures represent the total enzyme protein (active and inactive) measured. CONCLUSIONS: There are few data in the literature regarding sensitization and exposure-response relationships to these enzymes, and indeed there is often a lack of information within the industry as to the precise enzyme content of particular baking ingredients. As a precautionary measure, all enzymes are regarded as having the potential to cause respiratory sensitization. Consequently, exposures need to be controlled to as low a level as reasonably practicable, and future investigation may highlight the importance of measuring a variety of enzyme exposures and standardizing these methodologies to inform approaches to adequate control.

Respiratory syncytial virus and neutrophil activation.

Respiratory syncytial virus infects almost all children by 2 years of age. Neutrophils are the predominant airway leucocytes in RSV bronchiolitis and they are activated in the presence of infection. However it is not clear whether RSV can directly signal to activate neutrophil cytotoxic function. To investigate this we have used a preparation of RSV washed using a new centrifugal diafiltration method to rapidly remove inflammatory molecules produced by the epithelial cells used to propagate the RSV stock. Human neutrophils were isolated from peripheral blood and activated with either the unwashed crude RSV preparations or the purified intact RSV. Neutrophils were also challenged with purified RSV G-glycoprotein. The effect of challenging human neutrophils with these preparations of intact RSV, or the RSV G-glycoprotein, was assessed by measuring the cell surface expression of CD11b and CD18b, the phagocytic oxidative burst, and intracellular release of calcium pools. Neutrophils challenged with the washed RSV exhibited significantly lower activation of surface marker expression (P < 0.001) and oxidative burst (P < 0.001) than those challenged with unwashed virus or with virus free supernatant. There was no increase in intracellular calcium release on exposure to the washed RSV. Purified G glycoprotein did not stimulate neutrophils, whilst the use of a blocking antibody to the F protein did not prevent unwashed RSV from activating cytotoxic responses. These results suggest that neutrophils have no innate signalling system that recognizes RSV but they are activated at sites of RSV infection as a result of the cytokines and inflammatory molecules released by virally infected cells.

The distribution of endocrine cells along the mouse small intestine. Bombesin and somatostatin producing cells.

The topographical distribution and incidence of endocrine cells in the crypt and villus epithelium and along the length of the mouse intestine was studied. Cells containing somatostatin and bombesin like reactivity were stained by immunocytochemical techniques using polyclonal antiserum. Most of the somatostatin cells were found in the duodenum, jejunum and ileum, and these cells were generally more frequent on the villus compared to the crypts. This may indicate that the somatostatin cells develop late in the endocrine cell lineage. Bombesin like cells were rare in occurrence, and were only present in measureable numbers in the ileum, where they were observed in the crypt and villi. The application of ELISA assays to determine the specificity of the antisera for these peptides is also discussed.

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study.

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.