Club cell protein 16 (CC16) is the most abundant protein in broncho-alveolar secretions. CC16 inherits an anti-inflammatory property in the lungs. Chronic Obstructive Pulmonary disease (COPD) caused due to exposure of lungs to smoke or any other particulate pollutants1. CC16 has a molecular mass of 16 kilo Dalton, and belongs to the secretory globin sub-family of proteins. CC16 is a homodimeric protein with identical 70-amino acid subunits linked in an antiparallel orientation by two disulfide bonds2.
The main source of CC16 is the Club cell (formerly known as Clara cell) which is a non-ciliated, non-mucous secreting club-shaped cell present mainly in distal bronchioles as well as basal cells found in large airways. The density of Club cells throughout the respiratory tract varies substantially between species. In humans, Club cells represent 22% in the respiratory bronchioles3. Other organs that contain few CC16-secreting cells are the prostate, ovaries, pancreas, mammary glands, and uterine endometrium4,5.
As per the literature data, many chronic pulmonary inflammatory diseases such as anthraco-silicosis, chronic obstructive pulmonary disease (COPD), asthma etc. cause depletion of CC16. In all types of cases, Club cells are degenerated and reduced in number resulting in decreased levels of CC16 in Broncho-alveolar lavage fluid (BALF) and serum. The anti-inflammatory and protective effect of CC16 on the airway epithelium is gone resulting in inflammation of lungs. In case of acute inflammatory conditions, the CC16 level increases temporarily and attempts in the repair of the airway epithelium. But in cases of chronic exposures, the CC16 levels reduce gradually resulting in the chronic inflammation which ultimately leads towards fibrosis of lungs.
Chronic silicosis, the commonest and widely prevalent form of silicosis, is an irreversible occupational ailment of the respiratory system caused by the invasion of lung tissue (parenchyma) due to continuous or intermittent inhalation of dusts consisting of crystalline silica or silicon dioxide of respirable size (less than 10µ in diameter) while working in the relevant industries. Individuals with various durations of exposure ranging from 2 to 15 years or more in the above-said industries like mines, stone quarry, agate, construction sites and non-metallic product manufacturing units for example refractory (articles with heat resistant ability), ceramic, glass, mica, and structural clay are more prone to silicosis6. These micro-particles get trapped in the interstitial lung collagen tissue resulting in fibrosis of the lung. Therefore, it becomes one of the major occupational health hazards for the silica dust exposed workers working in relevant industries. Regrettably, most of the silicosis cases remain undiagnosed or misdiagnosed at an early stage due to asymptomatic or mild symptomatic nature of the initial stage of the disease, lack of suitable biomarker for early detection, poor health-seeking behavior of the workers and poor occupational health care delivery service at the workplaces, particularly in unorganized sectors7,8,9.
Patients with silicosis are prone to pulmonary tuberculosis also called silico-tuberculosis, probably due to destruction of alveolar macrophages (declined lung immunity). Differential diagnosis is difficult unless the physician is aware of the occupational history of silica exposure, which is very subjective in nature. Also, it becomes very difficult to differentiate between silicotic nodules and tuberculous infiltration in radiography. There is a lifelong risk of tuberculosis in silicotic patients even after stopping silica dust exposure. Secondly anti-tubercular treatment outcome is known to be uncertain with higher chance of reinfection in silicotic patients10. Additionally, isolation of Mycobacterium bacilli from the sputum of silico-tuberculosis is difficult as moderate to advanced stage silicotic fibrosis prevents discharge of the Mycobacterium in sputum, making the situation more difficult11. Hence, a suitable biomarker is urgently required for early detection of silicosis among silica dust exposed workers. Early detection using a suitable bio-marker will prevent generating moderate and advanced silicosis and its associated co-morbidities (silico-tuberculosis, renal disease, stroke, heart disease, lung cancer etc.) by minimizing further dust exposure12,13. Hence, countries with high burden of silicosis are in need of a suitable bio-marker at an affordable cost as silicosis is a neglected occupational disease with high disease burden and victims are often from the under-privileged communities.
As per rule, the diagnosis of silicosis needs to be confirmed by chest radiology following clinical examination along with a history of occupational exposure to silica dust for a varying period. X-ray of chest shows bi-lateral pathognomonic patchy nodular opacities in silicosis. Diagnosis is invariably made at a moderate or an advanced stage when nothing could be done to save or prolong the patients’ lives. Considering all, a suitable biomarker for early suspicion of silicosis may be a useful screening tool for secondary prevention of silicosis and silico-tuberculosis to protect these vulnerable workers.
A number of anti-inflammatory biomarkers for early diagnosis of silicosis have been attempted, but most of these were found to be non-specific and hence unsuitable for detection of lung related pathologies with certainty14,15. Club cell protein (CC16) is secreted by Club cells of broncho-alveolar epithelial tissue of the lung16 (Bernard et al., 1994). CC16 is proposed to be a peripheral marker of respiratory epithelial injury that protects the respiratory tract against oxidative stress-induced inflammation17,18,19 and passively diffuses in broncho-alveolar-blood barrier to plasma20. The serum concentration of CC16 can be used to decipher the degree of chronic respiratory tract injury at an early stage. Though the exact physiological mechanism of CC16 remains unknown, but evidences suggest significant reduction of CC16 in chronically silica dust-exposed workers with no change in respiratory symptoms, normal chest radiology and lung function tests indicates that serum CC16 could be an early asymptomatic detection tool for silicosis among silica-exposed population at risk21. Above principle has been kept in mind while developing a point of care, semi-quantitative CC16 kit as a screening tool for early detection of silicosis.
At present-day, the CC16 detection is performed with commercially available enzyme linked immunosorbent (ELISA) assays of clinical field application22 (Biovendor–Laboratorni) that requires to be imported from the foreign countries. The available commercial assays are very expensive and the cost cannot be afforded by the daily wage workers and/or by the health authority for mass use. Moreover, it requires expensive instrumentation that is available only in the large cities. Thus, there is a need for economical, user friendly and rapid detection devices and methods which do not require expensive instrumentation or specialized skills for testing and analysis. The purpose of developing this kit is to facilitate even for the remote rural health care workers for use of it in need with little training.
In this current study, we describe a Point of Care assay that can be particularly employed for semi-quantitative estimation of CC16 in human serum samples. This assay can be used periodically at regular intervals to assess the serum CC16 levels among workers with the history of silica dust exposure. This assay would give an idea about an estimated lung injury caused by silica dust exposure before advising for their radiological confirmation to arrive at a confirmed diagnosis. So, it may be considered as a proxy bio-marker as well as a screening tool for early detection of silicosis among silica dust exposed workers exclusively. It should not be used for assessing other lung diseases without further disease specific validation.
