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The Journal of Experimental Medicine  1960;112(6):1173-1194.
In rabbits, the depletion of cartilage matrix which occurs following intravenous administration of papain treated with iodacetamide is attributable to a portion of the enzyme in the disulfide form which has not undergone alkylation. It is this portion that is reactivated in cartilage in vivo and initiates the enzymatic breakdown of the protein-polysaccharide complex which forms a major component of the matrix. Evidence presented in support of these conclusions indicates that, contrary to an earlier hypothesis, papain acetamide is not reactivated in vivo. Following intravenous injection in amounts up to 4 mg./kg., active and inactive papain leaves the circulation at a rate proportional to the concentration, and it is likely that the initial rate of disappearance represents equilibration with the extracellular space. Following injection in the active or inactive form, a high proportion of papain in serum is bound to protein in the alpha globulin fraction. It is believed that in the case of fully active papain, the proportion which is not bound to alpha globulin becomes attached to other proteins of serum in extracellular fluid, such as albumin, by a process of enzyme substrate combination, and is thus prevented from diffusing into cartilage. In the case of inactive papain, a comparable excess remains free to enter cartilage, where it initiates depletion of matrix following reactivation within the tissue. These conclusions provide an explanation for the failure of fully active papain to cause depletion of cartilage matrix in vivo; the widespread changes seen after the injection of papain inactivated by iodoacetamide or by simple reversible oxidation are attributable to a small proportion of the injected material which enters cartilage in the disulfide form in a concentration of approximately 2 micrograms/gm. wet weight. The possibility that such a small amount of protease, when reactivated, can produce changes in cartilage matrix has been confirmed by studies on the effects of papain on isolated cartilage and chondromucoprotein in vitro. It has been shown that severe local injury results when active papain is injected into the skin of a rabbit in low concentrations. Since a much higher concentration of papain can be attained in the circulation without obvious adverse effects, it is evident that binding of the protease by alpha globulin and possibly other serum proteins may exemplify a mechanism whereby the tissues are protected from injury following entry into the circulation of other potentially harmful agents, such as proteolytic enzymes derived from cells or bacteria.
PMCID: PMC2137314  PMID: 13737507
2.  Lung Structure and Function with Age in Normal Rats and Rats With Papain Emphysema 
Journal of Clinical Investigation  1973;52(11):2921-2927.
Intrapulmonary deposition of the proteolytic enzyme papain produces a lesion resembling emphysema in experimental animals. The natural history of this lesion has not been well defined. The present study was performed to evaluate changes in lung structure and function with aging in normal rats and rats exposed to an aerosol of papain at 2 mo of age. Groups of control and papain-exposed animals were studied at 4, 8, and 18 mo of age. The parameters of lung function studied were specific airways' conductance (Gaw/TGV), diffusing capacity per unit of alveolar volume (DLco/VA), diffusing capacity (DLco), and functional residual capacity (FRC). Morphometric parameters were the postfixation lung volume (VL) and mean chord length (LM); internal surface area (ISA) and ISA extrapolated to both the mean VL of the corresponding papain group and a VL of 10 ml (ISA10) were calculated.
At 4 mo of age LM and FRC were significantly increased and ISA, DLco/VA, and DLco were significantly reduced in the papain group. At 8 mo of age LM was significantly increased and ISA was significantly decreased in the papain group: physiologic studies were not performed in this group. At 18 mo of age LM was significantly increased and DLco/VA, DLco, and ISA were significantly decreased. Neither progression nor healing of the lesion was observed despite similar lung growth in both groups.
This study demonstrates that a single proteolytic lung injury produces a fixed deficit of lung parenchyma. Progressive lung destruction may require repeated or continuous lung injury.
PMCID: PMC302560  PMID: 4748515
3.  Long-Term Exposure to Silica Dust and Risk of Total and Cause-Specific Mortality in Chinese Workers: A Cohort Study 
PLoS Medicine  2012;9(4):e1001206.
A retro-prospective cohort study by Weihong Chen and colleagues provides new estimates for the risk of total and cause-specific mortality due to long-term silica dust exposure among Chinese workers.
Human exposure to silica dust is very common in both working and living environments. However, the potential long-term health effects have not been well established across different exposure situations.
Methods and Findings
We studied 74,040 workers who worked at 29 metal mines and pottery factories in China for 1 y or more between January 1, 1960, and December 31, 1974, with follow-up until December 31, 2003 (median follow-up of 33 y). We estimated the cumulative silica dust exposure (CDE) for each worker by linking work history to a job–exposure matrix. We calculated standardized mortality ratios for underlying causes of death based on Chinese national mortality rates. Hazard ratios (HRs) for selected causes of death associated with CDE were estimated using the Cox proportional hazards model. The population attributable risks were estimated based on the prevalence of workers with silica dust exposure and HRs. The number of deaths attributable to silica dust exposure among Chinese workers was then calculated using the population attributable risk and the national mortality rate. We observed 19,516 deaths during 2,306,428 person-years of follow-up. Mortality from all causes was higher among workers exposed to silica dust than among non-exposed workers (993 versus 551 per 100,000 person-years). We observed significant positive exposure–response relationships between CDE (measured in milligrams/cubic meter–years, i.e., the sum of silica dust concentrations multiplied by the years of silica exposure) and mortality from all causes (HR 1.026, 95% confidence interval 1.023–1.029), respiratory diseases (1.069, 1.064–1.074), respiratory tuberculosis (1.065, 1.059–1.071), and cardiovascular disease (1.031, 1.025–1.036). Significantly elevated standardized mortality ratios were observed for all causes (1.06, 95% confidence interval 1.01–1.11), ischemic heart disease (1.65, 1.35–1.99), and pneumoconiosis (11.01, 7.67–14.95) among workers exposed to respirable silica concentrations equal to or lower than 0.1 mg/m3. After adjustment for potential confounders, including smoking, silica dust exposure accounted for 15.2% of all deaths in this study. We estimated that 4.2% of deaths (231,104 cases) among Chinese workers were attributable to silica dust exposure. The limitations of this study included a lack of data on dietary patterns and leisure time physical activity, possible underestimation of silica dust exposure for individuals who worked at the mines/factories before 1950, and a small number of deaths (4.3%) where the cause of death was based on oral reports from relatives.
Long-term silica dust exposure was associated with substantially increased mortality among Chinese workers. The increased risk was observed not only for deaths due to respiratory diseases and lung cancer, but also for deaths due to cardiovascular disease.
Please see later in the article for the Editors' Summary
Editors' Summary
Walk along most sandy beaches and you will be walking on millions of grains of crystalline silica, one of the commonest minerals on earth and a major ingredient in glass and in ceramic glazes. Silica is also used in the manufacture of building materials, in foundry castings, and for sandblasting, and respirable (breathable) crystalline silica particles are produced during quarrying and mining. Unfortunately, silica dust is not innocuous. Several serious diseases are associated with exposure to this dust, including silicosis (a chronic lung disease characterized by scarring and destruction of lung tissue), lung cancer, and pulmonary tuberculosis (a serious lung infection). Moreover, exposure to silica dust increases the risk of death (mortality). Worryingly, recent reports indicate that in the US and Europe, about 1.7 and 3.0 million people, respectively, are occupationally exposed to silica dust, figures that are dwarfed by the more than 23 million workers who are exposed in China. Occupational silica exposure, therefore, represents an important global public health concern.
Why Was This Study Done?
Although the lung-related adverse health effects of exposure to silica dust have been extensively studied, silica-related health effects may not be limited to these diseases. For example, could silica dust particles increase the risk of cardiovascular disease (diseases that affect the heart and circulation)? Other environmental particulates, such as the products of internal combustion engines, are associated with an increased risk of cardiovascular disease, but no one knows if the same is true for silica dust particles. Moreover, although it is clear that high levels of exposure to silica dust are dangerous, little is known about the adverse health effects of lower exposure levels. In this cohort study, the researchers examined the effect of long-term exposure to silica dust on the risk of all cause and cause-specific mortality in a large group (cohort) of Chinese workers.
What Did the Researchers Do and Find?
The researchers estimated the cumulative silica dust exposure for 74,040 workers at 29 metal mines and pottery factories from 1960 to 2003 from individual work histories and more than four million measurements of workplace dust concentrations, and collected health and mortality data for all the workers. Death from all causes was higher among workers exposed to silica dust than among non-exposed workers (993 versus 551 deaths per 100,000 person-years), and there was a positive exposure–response relationship between silica dust exposure and death from all causes, respiratory diseases, respiratory tuberculosis, and cardiovascular disease. For example, the hazard ratio for all cause death was 1.026 for every increase in cumulative silica dust exposure of 1 mg/m3-year; a hazard ratio is the incidence of an event in an exposed group divided by its incidence in an unexposed group. Notably, there was significantly increased mortality from all causes, ischemic heart disease, and silicosis among workers exposed to respirable silica concentrations at or below 0.1 mg/m3, the workplace exposure limit for silica dust set by the US Occupational Safety and Health Administration. For example, the standardized mortality ratio (SMR) for silicosis among people exposed to low levels of silica dust was 11.01; an SMR is the ratio of observed deaths in a cohort to expected deaths calculated from recorded deaths in the general population. Finally, the researchers used their data to estimate that, in 2008, 4.2% of deaths among industrial workers in China (231,104 deaths) were attributable to silica dust exposure.
What Do These Findings Mean?
These findings indicate that long-term silica dust exposure is associated with substantially increased mortality among Chinese workers. They confirm that there is an exposure–response relationship between silica dust exposure and a heightened risk of death from respiratory diseases and lung cancer. That is, the risk of death from these diseases increases as exposure to silica dust increases. In addition, they show a significant relationship between silica dust exposure and death from cardiovascular diseases. Importantly, these findings suggest that even levels of silica dust that are considered safe increase the risk of death. The accuracy of these findings may be affected by the accuracy of the silica dust exposure estimates and/or by confounding (other factors shared by the people exposed to silica such as diet may have affected their risk of death). Nevertheless, these findings highlight the need to tighten regulations on workplace dust control in China and elsewhere.
Additional Information
Please access these websites via the online version of this summary at
The American Lung Association provides information on silicosis
The US Centers for Disease Control and Prevention provides information on silica in the workplace, including links to relevant US National Institute for Occupational Health and Safety publications, and information on silicosis and other pneumoconioses
The US Occupational Safety and Health Administration also has detailed information on occupational exposure to crystalline silica
What does silicosis mean to you is a video provided by the US Mine Safety and Health Administration that includes personal experiences of silicosis; Dont let silica dust you is a video produced by the Association of Occupational and Environmental Clinics that identifies ways to reduce silica dust exposure in the workplace
The MedlinePlus encyclopedia has a page on silicosis (in English and Spanish)
The International Labour Organization provides information on health surveillance for those exposed to respirable crystalline silica
The World Health Organization has published a report about the health effects of crystalline silica and quartz
PMCID: PMC3328438  PMID: 22529751
4.  Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro 
Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein endothelial cells (HUVEC) in vitro.
Cell viability after prolonged treatment with papain was investigated by life cell staining and lactate dehydrogenase release assay. Angiogenic activation was assessed by ELISA against phosphorylated proteins AKT, MEK1/2, ERK1/2, SAPK/JNK and p38-MAPK. Growth inhibition was determined by means of an MTT-assay and cell migration by means of a scratch assay. Capability to form a capillary network was investigated using a tube formation assay.
Papain did not induce proteolysis or cell detachment of HUVEC in a concentration range between 0 and 25 μg/mL. Four hours treatment with 10 μg/mL papain resulted in a reduced susceptibility of endothelial cells to activation by VEGF as determined by phosphorylation levels of Akt, MEK1/2, SAPK/JNK. Papain exerted a distinct inhibitory effect on cell growth, cell migration and tube formation with inhibition of tube formation detectable at concentrations as low as 1 μg/mL. Bromelain and ficin displayed similar effects with regard to cell growth and tube formation.
Papain showed a strong anti-angiogenic effect in VEGF activated HUVEC. This effect may be due to interference with AKT, MEK1/2 and SAPK/JNK phosphorylation. Two other plant derived cysteine proteases displayed similar inhibition of HUVEC cell growth and tube formation. These findings indicate that plant proteolytic enzymes may have potential as preventive and therapeutic agents against angiogenesis related human diseases.
PMCID: PMC3849051  PMID: 24053149
Bromelain; Papain; Ficin; Angiogenesis; Endothelial cells; VEGF; Plant proteolytic enzymes
The intravenous injection of crystalline papain into young rabbits results in depletion of cartilage matrix throughout the body, with loss of rigidity and collapse of the ears, provided the enzyme is inactivated by oxidation or sulfhydryl blocking agents prior to administration. Cysteine-activated crystalline papain, when injected intravenously, produces little or no change in cartilage. The changes which occur in cartilage following an injection of inactivated crystalline papain are indistinguishable from those produced by crude papain. Activation of crude papain by cysteine prior to injection results in loss of its capacity to produce in vivo changes in cartilage. The progressive changes which take place in cartilage in vivo also occur in vitro in isolated rabbit ears removed shortly after an injection of crude papain or inactivated crystalline papain. In vitro ear collapse occurs rapidly at 37°C. and does not occur at 4°C. Collapse is enhanced by exposing the cartilage to cysteine and prevented by exposure to iodoacetamide or p-chloromercuribenzoate. The direct action of crystalline papain on plates of normal cartilage, in vitro, results in the same gross and histological changes which were observed in vivo. The direct action is accelerated by cysteine and inhibited by iodoacetamide or p-chloromercuribenzoate. The intravenous injection of iodoacetamide-treated bromelin produces the same in vivo changes in cartilage as papain. Untreated bromelin has no demonstrable effect on cartilage. It is suggested that the reason for the failure of activated papain to enter cartilage, after being injected intravenously, is that it probably reacts with a substrate or substrates in the blood. Oxidized or otherwise inactivated papain, in contrast, is readily taken up by cartilage and there converted to its active form.
PMCID: PMC2136872  PMID: 13575673
The catabolism of homologous and heterologous 7S gamma globulin fragments obtained by pepsin and papain digestion was studied in rabbits, guinea pigs, and mice. The elimination from the circulation of I* labeled gamma globulin fragments was followed and the urinary excretion of the total and protein-bound I* activity determined. Evidence is presented that the molecular structure responsible for the catabolism of 7S gamma globulin is located in papain fragment III. The elimination of papain fragment III was slow and closely related to the intact gamma globulin, whereas the pepsin fragment and papain fragments I and II were rapidly eliminated and catabolized in all species examined. Prolonged incubation with cysteine altered papain fragment III as shown by a rapid catabolism of a large portion of incubated fragment III within 24 hours after injection. Small amounts of intact RGG and RGG papain fragment III were excreted as protein-bound I* activity in the urine. On the other hand, large amounts of the pepsin fragment and papain fragments I and II of RGG were excreted as protein-bound I* activity in the urine. The possibility of a molecular structure present in papain fragment III, which may be responsible for tubular reabsorption in the kidney, is discussed. The rate of urinary excretion of fragments obtained from RGG was different from that of fragments obtained from gamma globulin of several other species. In general, small amounts of the pepsin fragment and papain fragment III obtained from gamma globulin other than RGG were excreted as protein-bound I* activity. The amounts of fragment I* excreted as protein-bound I* activity depended on the species in which it was injected, as well as the source of the gamma globulin. The rapid catabolism of the pepsin fragment and papain fragments I or II which bear antibody-combining sites suggest that their use for the prophylactic treatment of tetanus and diphtheria in man is limited.
PMCID: PMC2137959  PMID: 14271318
7.  Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent 
The Journal of General Physiology  1989;94(2):213-232.
We have determined the time course of Na channel inactivation in clonal pituitary (GH3) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole- cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near -40 mV), and a much smaller increase for large ones (approximately 1.5x at +40 mV). For technical reasons it was sometimes convenient to study outward INa recorded with no Na+ outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNa-V curve after papain, obtained by dividing the INa-V curve by the IIV curve, was left- shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papain. The rate of the inactivation step was found to vary only slightly from -100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from +20 to +60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by less than 10% from -100 to +60 mV. The data are considered in terms of existing models of the Na channel.
PMCID: PMC2228939  PMID: 2551998
8.  Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation 
Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.
PMCID: PMC4471117  PMID: 25705851
9.  Actinopyga lecanora Hydrolysates as Natural Antibacterial Agents 
Actinopyga lecanora, a type of sea cucumber commonly known as stone fish with relatively high protein content, was explored as raw material for bioactive peptides production. Six proteolytic enzymes, namely alcalase, papain, pepsin, trypsin, bromelain and flavourzyme were used to hydrolyze A. lecanora at different times and their respective degrees of hydrolysis (DH) were calculated. Subsequently, antibacterial activity of the A. lecanora hydrolysates, against some common pathogenic Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas sp.) were evaluated. Papain hydrolysis showed the highest DH value (89.44%), followed by alcalase hydrolysis (83.35%). Bromelain hydrolysate after one and seven hours of hydrolysis exhibited the highest antibacterial activities against Pseudomonas sp., P. aeruginosa and E. coli at 51.85%, 30.07% and 30.45%, respectively compared to the other hydrolysates. Protein hydrolysate generated by papain after 8 h hydrolysis showed maximum antibacterial activity against S. aureus at 20.19%. The potent hydrolysates were further fractionated using RP-HPLC and antibacterial activity of the collected fractions from each hydrolysate were evaluated, wherein among them only three fractions from the bromelain hydrolysates exhibited inhibitory activities against Pseudomonas sp., P. aeruginosa and E. coli at 24%, 25.5% and 27.1%, respectively and one fraction of papain hydrolysate showed antibacterial activity of 33.1% against S. aureus. The evaluation of the relationship between DH and antibacterial activities of papain and bromelain hydrolysates revealed a meaningful correlation of four and six order functions.
PMCID: PMC3546722  PMID: 23222684
Actinopyga lecanora; bioactive peptides; antibacterial activity; proteolytic enzyme
10.  Increase in non-specific bronchial hyperresponsiveness as an early marker of bronchial response to occupational agents during specific inhalation challenges. 
Thorax  1996;51(5):472-478.
BACKGROUND: Specific bronchial reactivity to occupational agents may decline after exposure in the workplace ceases leading to falsely negative specific inhalation challenges. A study was carried out to assess prospectively whether increases in nonspecific bronchial hyperresponsiveness could be useful in detecting the bronchial response to occupational agents during specific inhalation challenges. METHODS: Specific inhalation challenges were performed in 66 subjects with possible occupational asthma due to various agents. After a control day the subjects were challenged with the suspected agent for up to two hours on the first test day. Those subjects who did not show an asthmatic reaction were rechallenged on the next day for 2-3 hours. The provocative concentration of histamine causing a 20% fall (PC20) in the forced expiratory volume in one second (FEV1) was assessed at the end of the control day as well as six hours after each challenge that did not cause a > or = 20% fall in FEV1. The subjects who had a significant (> or = 3.1-fold) reduction in PC20 value at the end of the second challenge day were requested to perform additional specific inhalation challenges. RESULTS: The first test day elicited an asthmatic reaction in 25 subjects. Of the other 41 subjects five (12%, 95% confidence interval (CI) 4% to 26%) exhibited a > or = 3.1-fold fall in the PC20 value after the inhalation challenge and developed an asthmatic reaction during the second (n = 3) or third (n = 2) challenge exposure. The offending agents included persulphate (n = 1), wood dust (n = 2), isocyanate (n = 1), or amoxycillin (n = 1). These five subjects had left their workplace for a longer period (mean (SD) 21 (14) months) than those who reacted after the first specific inhalation challenge (8 (11) months). CONCLUSIONS: The increase in non-specific bronchial hyperresponsiveness after a specific inhalation challenge can be an early and sensitive marker of bronchial response to occupational agents, especially in subjects removed from workplace exposure for a long time. Non-specific bronchial hyperresponsiveness should be systematically assessed after specific inhalation challenges in the absence of changes in airway calibre.
PMCID: PMC473590  PMID: 8711673
11.  Immobilization of Papain on Chitin and Chitosan and Recycling of Soluble Enzyme for Deflocculation of Saccharomyces cerevisiae from Bioethanol Distilleries 
Enzyme Research  2015;2015:573721.
Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1–10% w·v−1), polyethyleneimine (0.5% v·v−1), and tripolyphosphate (1–10% w·v−1) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L−1. Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.
PMCID: PMC4299301  PMID: 25628895
The effects of papain protease and of vitamin A on explanted limb bone rudiments from 7- and 13-day chick embryos and fetal mice have been studied and compared. The incubation of cartilaginous rudiments from 7-day chick embryos in a solution containing papain and cysteine resulted in complete loss of the metachromasia of the cartilage matrix within 1 hour; explants treated in this fashion recovered normal metachromatic staining properties when grown in normal medium for 4 days. The incubation of 7-day chick cartilage rudiments in a solution containing papain without cysteine resulted in partial loss of metachromasia from cartilage within 1 hour; the addition of vitamin A to the solution did not enhance the effect of papain during this period. The addition of papain to the culture medium in which 7-day chick embryo cartilage rudiments were grown resulted in uniform loss of the metachromasia of the cartilage matrix; similar explants grown in the presence of excess vitamin A also showed loss of the metachromasia of cartilage, but certain regions of the cartilage were affected earlier and more severely than others. Changes in cartilage cells, including loss of glycogen, occurred when the rudiment was grown in medium containing excess vitamin A, but not when it was grown in the presence of papain. Bone rudiments from 13-day chick embryos showed changes in cartilage similar to those seen in 7-day chick embryo rudiments when grown in the presence of papain or of excess vitamin A; the existing bone was not affected under these conditions. When grown in the presence of papain or excess vitamin A, the cartilage of late fetal mouse bone underwent changes similar to those already described in chick embryo rudiments. In contrast to the chick embryo rudiments, those from the fetal mouse showed rapid resorption of bone when grown in the presence of excess vitamin A. Papain had no effect on bone from either source. The changes seen in cartilage of explants grown in the presence of vitamin A and papain together were greater than those seen with either agent alone. The changes seen in fetal mouse bone grown in the presence of vitamin A were not enhanced by the additional presence of papain. On the basis of these observations, it is suggested that the changes in cartilage seen in experimental hypervitaminosis A may be the result of activation of a proteolytic enzyme or enzymes with properties similar to papain.
PMCID: PMC2137281  PMID: 13698767
13.  Impaired Angiotensin Conversion and Bradykinin Clearance in Experimental Canine Pulmonary Emphysema 
Journal of Clinical Investigation  1981;67(1):201-209.
Chronic hypoxic lung diseases are associated with abnormal blood pressure regulation. Because the lung is the principal site of angiotensin conversion and because hypoxia decreases converting enzyme activity, we examined whether angiotensin converting enzyme activity was impaired in lung disease. 12 dogs received a 6 wk course of aerosolized and intratracheal papain that produced moderate panlobular emphysema. These dogs and 24 control dogs were anesthetized and sampling catheters were placed under fluoroscopic control. Angiotensin conversion was measured by a blood pressure response bioassay. Pulmonary converting enzyme activity was also assessed by infusing bradykinin (BK) and using radioimmunoassay to measure the instantaneous clearance of BK and the concentration of BK in the pulmonary artery which first produced spillover of BK into left atrial blood. Angiotensin conversion was reduced in the emphysematous dogs to 81.1% (13.2 SD) from 92% (6 SD) in the control dogs (P < 0.01). Instantaneous clearance of BK in the emphysematous dogs was only slightly reduced (93%), despite reduction in their Pao2 to 75 mm Hg, indicating that the greatest proportion of the perfused vascular bed was exposed to alveolar Po2 of >90 mm Hg. However, the barrier to BK passage provided by the lung, and measured by the spillover level, was reduced ¼ to ½ that observed in control animals. That the defect was promptly corrected by supplemental oxygen indicates that regional pulmonary vascular converting enzyme activity had been impaired by regional alveolar hypoxia, which permitted some peptide to pass through the lungs unmetabolized. Determination of peptide metabolism in the lungs may provide a useful measure of regional alveolar hypoxia and may lead to new ways of assessing lung injury.
PMCID: PMC371588  PMID: 6256412
14.  Occupational asthma and rhinitis due to Western red cedar (Thuja plicate), with special reference to bronchial reactivity 
Gandevia, B., and Milne, J. (1970).Brit. J. industr. Med.,27, 235-244. Occupational asthma and rhinitis due to Western red cedar (Thuja plicata), with special reference to bronchial reactivity. With the increasing use of Western, or Canadian, red cedar (Thuja plicata) in the timber industry, a distinctive respiratory syndrome of rhinitis and asthma has been observed with increasing frequency in clinical and industrial practice. Six cases of asthma and four of rhinitis are described in some detail; the onset of symptoms some hours after exposure, the nocturnal predominance of symptoms, especially of cough, and their persistence for days or weeks after cessation of exposure may conspire to make diagnosis difficult if the occupational hazard is not appreciated.
Both immediate and late skin reactions to extracts of Western red cedar were mild or absent, and serum precipitins were absent in the two cases in which they were sought. Positive bronchial reactions, reflected in serial estimations of ventilatory capacity, occurred in response to provocative inhalations of extracts of the cedar dust, commonly at four to six hours and at night, rarely within the first hour. In some instances, a single provocative exposure to the nebulized extract over 90 seconds was shown to produce exacerbations of asthma for two or three successive nights, with normal or reduced ventilatory capacity during the intervening days. Regularly recurring asthma after an isolated exposure has not previously been documented, and is perhaps of fundamental importance to the understanding of non-occupational asthma. Bronchial reactions were not observed to house dust extract, to which patients consistently showed dermal sensitivity. Symptoms subsided gradually when exposure was avoided, but there was considerable individual variation as to how much exposure could be tolerated without relapse; symptomatic therapy, with or without specific hyposensitization, did not adequately control the symptoms.
PMCID: PMC1009138  PMID: 5448121
The administration of large amounts of vitamin A to rabbits has been shown to result in depletion of cartilage matrix. The normal basophilic, metachromatic, and Alcian blue staining properties of the matrix are lost, especially in articular and epiphyseal cartilage. The cartilage cells remain intact, but are reduced in size. These changes sometimes appeared as early as 48 hours after the initiation of daily injection of 1 million units of vitamin A, and were usually well established by 5 days. Some rabbits failed to show changes in cartilage, even after 5 daily injections. Increased amounts of material presumed to be chondroitin sulfate were present in the sera of vitamin A-treated rabbits, usually by 72 hours after the first injection. This was demonstrated by a turbidimetric procedure using hexamminecobaltic chloride. In rabbits given sulfur-35 (Na2S35O4) 5 days before the initiation of vitamin A treatment, it was shown that sulfur-35 was lost from articular and epiphyseal cartilage. This was associated with an increase in the non-dialyzable sulfur-35 in both serum and in the cobalt-precipitable material. These rabbits also excreted more sulfur-35 than rabbits not given vitamin A. There was a reduction in sulfur-35 activity in chondromucoprotein extracted from the ear cartilage of vitamin A-treated rabbits. The changes are interpreted as indicating that the administration of large amounts of vitamin A to rabbits results in removal of chondroitin sulfate from cartilage matrix. The administration of small amounts of crude papain causes histologic changes in cartilage that are remarkably similar to those seen in vitamin A-treated rabbits. The possibility is suggested that the changes in cartilage produced by administration of vitamin A to rabbits may be the result of activation of a proteolytic enzyme or enzymes, with properties similar to those of papain.
PMCID: PMC2137286  PMID: 13776507
16.  Health and environmental consequences of the world trade center disaster. 
Environmental Health Perspectives  2004;112(6):731-739.
The attack on the World Trade Center (WTC) created an acute environmental disaster of enormous magnitude. This study characterizes the environmental exposures resulting from destruction of the WTC and assesses their effects on health. Methods include ambient air sampling; analyses of outdoor and indoor settled dust; high-altitude imaging and modeling of the atmospheric plume; inhalation studies of WTC dust in mice; and clinical examinations, community surveys, and prospective epidemiologic studies of exposed populations. WTC dust was found to consist predominantly (95%) of coarse particles and contained pulverized cement, glass fibers, asbestos, lead, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polychlorinated furans and dioxins. Airborne particulate levels were highest immediately after the attack and declined thereafter. Particulate levels decreased sharply with distance from the WTC. Dust pH was highly alkaline (pH 9.0-11.0). Mice exposed to WTC dust showed only moderate pulmonary inflammation but marked bronchial hyperreactivity. Evaluation of 10,116 firefighters showed exposure-related increases in cough and bronchial hyperreactivity. Evaluation of 183 cleanup workers showed new-onset cough (33%), wheeze (18%), and phlegm production (24%). Increased frequency of new-onset cough, wheeze, and shortness of breath were also observed in community residents. Follow-up of 182 pregnant women who were either inside or near the WTC on 11 September showed a 2-fold increase in small-for-gestational-age (SGA) infants. In summary, environmental exposures after the WTC disaster were associated with significant adverse effects on health. The high alkalinity of WTC dust produced bronchial hyperreactivity, persistent cough, and increased risk of asthma. Plausible causes of the observed increase in SGA infants include maternal exposures to PAH and particulates. Future risk of mesothelioma may be increased, particularly among workers and volunteers exposed occupationally to asbestos. Continuing follow-up of all exposed populations is required to document the long-term consequences of the disaster.
PMCID: PMC1241968  PMID: 15121517
17.  SQ-standardized house dust mite immunotherapy as an immunomodulatory treatment in patients with asthma 
Allergy  2011;66(2):178-185.
Specific immunotherapy is the only treatment with the potential to prevent progression of the allergic disease and the potential to cure patients. The immunomodulatory ability of SQ-standardized house dust mite (HDM) subcutaneous immunotherapy (SCIT) was investigated in patients with allergic asthma.
Fifty-four adults with HDM-allergic asthma were randomized 1 : 1 to receive SQ-standardized HDM SCIT (ALK) or placebo for 3 years. At baseline, and after 1, 2 and 3 years of treatment, the lowest possible inhaled corticosteroid dose required to maintain asthma control was determined, followed by determinations of nonspecific and HDM-allergen-specific bronchial hyperresponsiveness, late asthmatic reaction (LAR), immediate and late-phase skin reactions, and immunological response.
SQ-standardized HDM SCIT provided a statistically significantly higher HDM-allergen tolerance (P < 0.05 vs placebo) in terms of a 1.6-fold increase in PD20 (HDM-allergen inhalation challenge), a 60-fold increase in skin test histamine equivalent HDM-allergen concentrations, reduced immediate- and reduced or abolished late-phase skin reactions, as well as fewer patients with LAR. PD20 (methacholine inhalation challenge) increased initially and was similar between groups. House dust mite SCIT induced an initial increase in serum HDM-allergen-specific IgE (P = 0.028 vs placebo), which then declined to baseline value. House dust mite SCIT induced an increase in components blocking IgE binding to allergen [ΔIgE-blocking factor: 0.31; 95% CI of (0.26; 0.37)] after 1 year that remained constant after 2 and 3 years (P < 0.0001 vs placebo).
SQ-standardized HDM SCIT induced a consistent immunomodulatory effect in adults with HDM-allergic asthma; the humoral immune response was changed and the HDM-allergen tolerance in lung and skin increased.
PMCID: PMC3039748  PMID: 20883456
allergic asthma; bronchial hyperresponsiveness; house dust mite; immune response; immunotherapy
18.  Effect of fluticasone 250 μg/salmeterol 50 μg and montelukast on exhaled nitric oxide in asthmatic patients 
Monitoring noninvasive biomarkers of inflammation is an important adjunct in asthma therapy.
The goal of the present study was to identify airway and alveolar site(s) of inflammation using exhaled nitric oxide (NO) as a marker in asthmatic patients, and to evaluate the NO response to maintenance fluticasone 250 μg/salmeterol 50 μg (F/S) and add-on montelukast 10 mg (M).
Thirty (24 women) nonsmoking, mild to moderate asthmatic patients were studied, mean age (± SD) 43±9 years, treated with F/S for more than one year. All were clinically stable for longer than eight weeks and had not taken oral corticosteroids and/or leukotriene antagonists for eight weeks before the present study. Spirometry, Juniper asthma symptom score, fractional exhaled NO (FENO) 100 mL/s, bronchial NO and alveolar NO concentration (CANO) were measured in a single-blind, nonrandomized crossover study.
Visit 1: baseline F/S; visit 2: after four weeks of F/S plus M; visit 3: after four weeks of S plus M; and visit 4: after four weeks of S only. Values in asthmatic patients were also compared with 34 nonsmoking age-matched healthy controls with normal lung function.
After 180 μg aerosolized metered dose inhaler albuterol, the forced expiratory volume in 1 s at baseline was 2.6±0.8 L (86%±16% of the predicted value) and the forced expiratory volume in 1 s over the forced vital capacity was 77%±9% (mean ± SD), and was similar at visits 2 to 4. Juniper scores were mildly abnormal at visits 1 to 3, but significantly worse (P=0.03) at visit 4 versus visits 1 to 3. FENO values at visits 1 to 3 were similar but significantly increased (P=0.007) at visit 4. Bronchial NO was higher (P=0.03) at visit 4, versus visits 1 and 2, and was no different at visit 3. Compared with the healthy subjects, FENO and bronchial NO values were abnormal (greater than the normal mean plus 2 SD) in 33% of asthmatic patients at visits 1 to 3. CANO was similar for visits 1 to 4. CANO was abnormal (greater than the normal mean + 2 SD) in 20% of asthmatic patients.
In clinically stable asthmatic patients, despite controller treatment including moderate-dose inhaled corticosteroids and add-on M, 33% of mild to moderate asthmatic patients have ongoing nonsuppressed bronchial sites of increased NO production, compared with healthy control subjects. These controllers have no effect on CANO, which was abnormal in 20% of the asthmatic patients studied. The addition of add-on M to baseline moderate-dose inhaled corticosteroid did not further reduce total exhaled, bronchial and/or alveolar NO production.
PMCID: PMC2677951  PMID: 18551200
Asthma; Exhaled nitric oxide; Montelukast; Salmeterol/fluticasone
19.  Airway hyperresponsiveness and bronchial mucosal inflammation in T cell peptide‐induced asthmatic reactions in atopic subjects 
Thorax  2007;62(9):750-757.
Subjects with allergic asthma develop isolated late asthmatic reactions after inhalation of allergen‐derived T cell peptides. Animal experiments have shown that airway hyperresponsiveness (AHR) is CD4+ cell‐dependent. It is hypothesised that peptide inhalation produces increases in non‐specific AHR and a T cell‐dominant bronchial mucosal inflammatory response.
Bronchoscopy, with bronchial biopsies and bronchoalveolar lavage (BAL), was performed in 24 subjects with cat allergy 6 h after aerosol inhalation of short overlapping peptides derived from Fel d 1, the major cat allergen. Biopsy specimens and BAL fluid were studied using immunohistochemistry and ELISA.
Twelve of the 24 subjects developed an isolated late asthmatic reaction without a preceding early (mast cell/histamine‐dependent) reaction characteristic of whole allergen inhalation. These responders had significant between‐group differences (responders vs non‐responders) in the changes (peptide vs diluent) in AHR (p = 0.007) and bronchial mucosal CD3+ (p = 0.005), CD4+ (p = 0.006) and thymus‐ and activation‐regulated chemokine (TARC)+ (p = 0.003) but not CD8+ or CD25+ cells or eosinophils, basophils, mast cells and macrophages. The between‐group difference for neutrophils was p = 0.05 but with a non‐significant within‐group value (peptide vs diluent, responders, p = 0.11). In BAL fluid there was a significant between‐group difference in TARC (p = 0.02) but not in histamine, tryptase, basogranulin, C3a or C5a, leukotrienes C4/D4/E4, prostaglandins D2 or F2α.
Direct activation of allergen‐specific airway T cells by peptide inhalation in patients with atopic asthma leads to increased AHR with local increases in CD3+ and CD4+ cells and TARC but no significant changes in eosinophils or basophil/mast cell products. These findings support previous animal experiments which showed a CD4+ dependence for AHR.
PMCID: PMC2117301  PMID: 17389757
20.  Effect of low-level laser therapy on the expression of inflammatory mediators and on neutrophils and macrophages in acute joint inflammation 
Arthritis Research & Therapy  2013;15(5):R116.
Inflammation of the synovial membrane plays an important role in the pathophysiology of osteoarthritis (OA). The synovial tissue of patients with initial OA is characterized by infiltration of mononuclear cells and production of proinflammatory cytokines and other mediators of joint injury. The objective was to evaluate the effect of low-level laser therapy (LLLT) operating at 50 mW and 100 mW on joint inflammation in rats induced by papain, through histopathological analysis, differential counts of inflammatory cells (macrophages and neutrophils), as well as gene expression of interleukin 1-beta and 6 (IL-1β and IL-6), and protein expression of tumor necrosis factor alpha (TNFα).
Male Wistar rats (n = 60) were randomly divided into four groups of 15 animals, namely: a negative control group; an inflammation injury positive control group; a 50 mW LLLT group, subjected to injury and treated with 50 mW LLLT; and a 100 mW LLLT group, subjected to injury and treated with 100 mW LLLT. The animals were subject to joint inflammation (papain solution, 4%) and then treated with LLLT (808 nm, 4 J, 142.4 J/cm2, spot size 0.028 for both groups). On the day of euthanasia, articular lavage was collected and immediately centrifuged; the supernatant was saved for analysis of expression of TNFα protein by enzyme-linked immunosorbent assay and expression of IL-1β and IL-6 mRNA by real-time polymerase chain reaction. A histologic examination of joint tissue was also performed. For the statistical analysis, analysis of variance with Tukey's post-hoc test was used for comparisons between each group. All data are expressed as mean values and standard deviation, with P < 0.05.
Laser treatment with 50 mW was more efficient than 100 mW in reducing cellular inflammation, and decreased the expression of IL-1β and IL-6. However, the 100 mW treatment led to a higher reduction of TNFα compared with the 50 mW treatment.
LLLT with 50 mW was more efficient in modulating inflammatory mediators (IL-1β, IL-6) and inflammatory cells (macrophages and neutrophils), which correlated with the histology that showed a reduction in the inflammatory process.
PMCID: PMC3979014  PMID: 24028507
21.  Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain 
PLoS ONE  2013;8(12):e80490.
The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×104 cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.
PMCID: PMC3861243  PMID: 24349001
22.  The Intestinal Brush Border Membrane in Diabetes 
Journal of Clinical Investigation  1977;60(1):181-188.
Diabetes stimulates the functional activity of the intestinal brush border membrane with enhancement of both hydrolytic enzyme activity and membrane transport systems. To determine the mechanism of this effect, we studied the effects of streptozotocin diabetes on the metabolism of one membrane protein, sucrase-isomaltase, which increases its activity in diabetes. The protein was purified and an antiserum prepared. Sucrase-isomaltase from control and diabetic rats was immunologically identical as shown by Ouchterlony double-diffusion analysis of papain-solubilized mucosal proteins. The increase in sucrase enzyme activity in diabetic animals (31.0±1.4 U SEM 5 days after streptozotocin vs. 13.1±1.0 in controls) was the consequence of increased enzyme protein and not an alteration in catalytic efficiency as demonstrated by quantitative immunoprecipitin reactions.
To account for increased sucrase-isomaltase protein in diabetes we studied papain-solubilized mucosal proteins labeled by injection of [14C]carbonate and [14C]leucine and analyzed incorporation into sucrase-isomaltase protein (anti-serum precipitable) and total protein (trichloroacetic acid precipitable). We found that diabetes did not affect the decay of labeled total protein, but prolonged the decay of labeled sucrase-isomaltase. t½ of sucrase-isomaltase was 4.4 h in control animals after [14C]carbonate injection and 8.8 and 10.2 h, respectively, 2 and 5 days after induction of streptozotocin diabetes. We obtained similar results in experiments with [14C]leucine with diabetes increasing t½ from 6 to 13.6 h. Diabetes did not appear to increase the rate of addition of sucrase-isomaltase to the brush border membrane, since it did not affect the 10- and 60-min incorporations of isotope into sucrase-isomaltase protein relative to incorporation into total protein and did not alter rate constants for synthesis calculated from the t½ and the change in enzyme mass over time.
Thus, enhanced sucrase activity in the diabetic animal is the consequence of an increase in sucrase-isomaltase protein which develops because of a decrease in its rate of degradation.
PMCID: PMC372356  PMID: 141462
23.  Ventilatory effects of aerosol gentamicin. 
Thorax  1978;33(1):54-56.
Bronchial provocation tests with gentamicin solution, 40 mg/ml, and with the drug vehicle solution alone were carried out in 29 subjects aged 19 to 66 years. There were 18 subjects with bronchial asthma, four with chronic bronchitis, four with primary carcinoma of the lung, and three with no chest disease. Two millilitres of each of the two test solutions was given to each subject, in duplicate, via a nebuliser driven by a Bird Mark 8 respirator. Ventilatory function (FEV1 and VC) was measured before and after each inhalation, and changes were expressed as percentage variations from baseline. Seven subjects, all from the asthmatic group, developed at least one immediate FEV1 fall of 20% or more. The reactions ranged up to 71% and occurred to both test solutions. There was a trend towards greater reactions to the vehicle. In two subjects pretreatment with salbutamol and sodium cromoglycate did not modify these reactions. In three of the seven, inhalation of 2 ml normal saline produced FEV1 falls of 25% to 30%, but these falls were not as great as each subject's reactions to the test solutions. Skin prick tests using the gentamicin solution were negative in all subjects. These results show that substantial obstructive reactions may occur in some asthmatic subjects after inhalation of gentamicin. The reactions appear to be non-immunological in nature and may be due to an irritant effect of the drug vehicle.
PMCID: PMC470845  PMID: 644539
24.  Expiratory flow limitation and the response to breathing a helium-oxygen gas mixture in a canine model of pulmonary emphysema. 
Journal of Clinical Investigation  1984;73(5):1321-1334.
The pathophysiology of reduced maximum expiratory flow in a canine model of pulmonary emphysema was studied, and the results interpreted in terms of the wave-speed theory of flow limitation. According to this theory, maximum expiratory flow is related both to the cross-sectional area and compliance at an airway site where a critical gas velocity is first reached ("choke-point") and to gas density. Pulmonary emphysema was produced by the repeated instillations of the enzyme papain into the airways of six dogs. In five control dogs, a saline solution was instilled. During forced vital capacity deflation, in an open-chest preparation, maximum expiratory flow, choke-point locations, and the response to breathing an 80:20 helium/oxygen gas mixture were determined at multiple lung volumes. To locate choke-points, a pressure measuring device was positioned in the airway to measure lateral and end-on intrabronchial pressures, from which the relevant wave-speed parameters were obtained. In general, the reduced maximum expiratory flow in emphysema can be explained by diminished lung elastic recoil pressure and by altered bronchial pressure-area behavior, which results in a more peripheral location of choke-points that have smaller cross-sectional areas than controls. With respect to the density dependence of maximum expiratory flow, this response did not differ from control values in four dogs with emphysema in which frictional pressure losses upstream from choke-points did not differ on the two gas mixtures. In two dogs with emphysema, however, upstream frictional pressure losses were greater on helium/oxygen than on air, which resulted in a smaller cross-sectional area on helium/oxygen; hence density dependence decreased.
PMCID: PMC425154  PMID: 6715539
25.  Papain-gel Degrades Intact Nonmineralized Type I Collagen Fibrils 
Scanning  2009;31(6):253-258.
Papain-gel has been utilized as a chemomechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papaingel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain-gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM-based nanoindentation. AFM images showed that the papain-gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two-fold drop from the values of normal dentin (E= 20 +/− 1.9, H = 0.8 +/− 0.08 GPa) was found after four applications (E = 9.7 +/− 3.2, H = 0.24 +/− 0.1 GPa) ( P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain-gel.
PMCID: PMC2939036  PMID: 20205185
collagen; papain; dentin; AFM; mechanical properties

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