Cesare Saltini

Immunogenetic basis of environmental lung disease: lessons from the berylliosis model

The role of genetic factors has been hypothesized in the pathogenesis of a number of chronic inflammatory lung diseases. The genes of the major histocompatibility complex (MHC) locus on human chromosome 6 have been identified as important determinants in diseases caused both by inorganic and organic compounds such as beryllium, gold, acid anhydrides, isocyanates and grass pollens. Since many environmental factors are the determinants of the immunopathogenesis of asthma, pulmonary granulomatous disorders, hypersensitivity pneumonitis and fibrotic lung disorders, an understanding of the interaction between environmental factors is crucial to epidemiology, prevention and treatment of these disorders. Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts. A human leukocyte antigen class II marker (HLA-DP Glu69) has been found to be strongly associated with the disease. In in vitro studies, the gene has been shown to play a direct role in the immunopathogenesis of the disease. In human studies, the gene has been shown to confer increased susceptibility to beryllium in exposed workers, thus suggesting that HLA gene markers may be used as epidemiological probes to identify population groups at higher risk of environmental lung diseases, to identify environmental levels of lung immunotoxicants that would be safe for the entire population and to prevent disease risk associated with occupation, manufactured products and the environment. Studies on the associations between human leukocyte antigens and chronic inflammatory lung disorders are reviewed in the context of the berylliosis model.

HLA-DP-unrestricted TNF-α release in beryllium-stimulated peripheral blood mononuclear cells

Berylliosis is a granulomatous disorder of the lung caused by inhalation of beryllium (Be) and dominated by the accumulation of CD4+ T-helper (Th)1 memory T-cells proliferating in response to Be in the lower respiratory tract. Two gene markers have been associated with susceptibility to berylliosis: 1) the human leucocyte antigen (HLA)-DP gene whose allelic variants, carrying glutamate in position 69 of the β-chain (HLA-DPGlu69), can bind Be directly and present it to interferon (IFN)-γ releasing Th1 T-cell clones from patients with berylliosis; and 2) the cytokine gene tumour necrosis factor (TNF)-α which has been shown to increase berylliosis risk independent of HLA-DPGlu69. In order to determine whether TNF-α release was triggered by Th1 T-cell activation by Be stimulation in the context of HLA-DPGlu69 molecules, the proliferation of BeSO4-stimulated blood mononuclear cells and the release of IFN-γ, TNF-α, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 by BeSO4-stimulated blood mononuclear cells was quantified in 11 individuals with berylliosis using an anti-HLA-DP antibody as a probe for HLA-DP restricted T-cell activation. While proliferation and IFN-γ release were completely abrogated by HLA-DP inhibition (inhibition with anti-HLA-DP monoclonal antibody (mAb): 88±16 and 77±16%, respectively; anti-HLA-DR: 29±38 and 14±10%, respectively), the release of TNF-α was not (inhibition with anti-HLA-DP mAb: 8.9±7.8%). No other cytokine was detected at significant levels. Moreover, Be was able to induce TNF-α production in healthy control subjects not exposed to Be in the absence of T-cell proliferation and IFN-γ production. In conclusion, these data suggest that the tumour necrosis factor-α response of mononuclear cells is independent of the activation of beryllium-specific human leucocyte anitgen-DP restricted T-cells, which is consistent with the finding that the tumour necrosis factorA2 and the human leucocyte anitgen-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk.

Evaluation of a western blot serum test for the diagnosis of Mycobacterium tuberculosis infection

This study was designed to evaluate the possibility of monitoring Mycobacterium tuberculosis infection using a serological assay. A discriminant score comprising antigen fractions of 38, 28, 24 and 19 kDa, identified in western blots using the Mycobacterium bovis bacille Calmette-Guérin (BCG) A60 antigen complex was established in a sample of 57 purified protein derivative (PPD)-negative and 47 PPD-positive individuals. It was then tested in a group of 140 subjects undergoing BCG vaccination as a model of tuberculosis complex infection and in a group of human immunodeficiency virus (HIV)-infected individuals as a model of cell-mediated immunodeficiency-related risk of tuberculosis. The discriminant score identified 57 out of 57 (100%) PPD-positives and none (0%) of the 47 PPD-negatives. In the BCG vaccinated subjects, 1.4% tested positive before vaccination and 90% after vaccination. In the HIV-positive subjects, 90% of the PPD-positive and 5% of the PPD-negative subjects had a positive score. This study suggests that the western blot discriminant score is an accurate test to survey M. tuberculosis infection in serum samples.

Evaluation of a western blot test as a potential screening tool for occupational exposure to Mycobacterium tuberculosis in health care workers

Health care workers (HCWs) have a higher than average risk for contracting Mycobacterium tuberculosis (MTB) infection and tuberculosis (TB). No markers of MTB-exposure are available, and TB risk assessment is performed by tuberculin screening, identifying individuals with acquired MTB infection. This study evaluated a western blot (WB) anti-M. bovis A60 complex antibody as a MTB-exposure marker. WB reactivity was evaluated on 127 exposed and 28 non-exposed HCWs from four divisions of the Policlinico Hospital of Modena, and 140 non-exposed bacille Calmette-Guérin-vaccinated controls. Excess of occupational TB risk according to the Occupational Safety and Health Administration (OSHA) was calculated in each division. WB-positivity (%) was: (1) significantly higher in exposed HCWs compared with non-exposed (72% vs 25%, P < 0.00001), (2) highly related (r = 0.99) to OSHA risk excess in all divisions, (3) higher than non-exposed in HCWs with short (< 5 years) MTB-exposure (purified protein derivative [PPD], P > 0.18; WB, P < 0.04). PPD-positivity (%) was higher than controls only in HCWs with longer (> 5 years) MTB-exposure. The study suggests that the WB antibody might represent a more sensitive biological marker of MTB contact among exposed HCWs, related to the level of TB risk and detectable earlier than the PPD skin test, thus providing new tools for TB risk assessment in health care facilities.

Role of the HLA-DP Glu 69 and the TNF-alpha TNF-alpha 2 gene markers in susceptibility to beryllium hypersensitivity.

Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts, characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. Beryllium presentation to CD4(+) T cells from patients with berylliosis results in T cell activation, and these Be-specific CD4(+) T cells undergo clonal proliferation and Th1-type cytokine production such as interleukin-2, interferon-gamma and tumor necrosis factor-alpha. In exposed workers, genetic susceptibility to this granulomatous disorder is associated with major histocompatibility gene and the TNF-α gene. The HLA-DP glutamic 69 residue was shown to be the MHC genetic marker associated with disease susceptibility; furthermore the TNF-α TNFA-308*2 allele was found to be independently associated with HLA-DP GIu69 in the determination of berylliosis risk.