Papain Administration Research Articles

ILC2 require cell-intrinsic ST2 signals to promote type 2 immune responses.

The initiation of type 2 immune responses at mucosal barriers is regulated by rapidly secreted cytokines called alarmins. The alarmins IL-33, IL-25 and TSLP are mainly secreted by stromal and epithelial cells in tissues and were linked to chronic inflammatory diseases, such as allergic lung inflammation, or to resistance against worm infections. Receptors for alarmins are expressed by a variety of immune cells, including group 2 innate lymphoid cells (ILC2s), an early source of the type 2 cytokines, such as IL-5 and IL-13, which have been linked to atopic diseases and anti-worm immunity as well. However, the precise contribution of the IL-33 receptor signals for ILC2 activation still needs to be completed due to limitations in targeting genes in ILC2. Using the newly established Nmur1 iCre-eGFP mouse model, we obtained specific conditional genetic ablation of the IL-33 receptor subunit ST2 in ILC2s. ST2-deficient ILC2s were unresponsive to IL-33 but not to stimulation with the alarmin IL-25. As a result of defective ST2 signals, ILC2s produced limited amounts of IL-5 and IL-13 and failed to support eosinophil homeostasis. Further, ST2-deficient ILC2s were unable to expand and promote the recruitment of eosinophils during allergic lung inflammation provoked by papain administration. During infection with Nippostrongylus brasiliensis, ILC2-intrinsic ST2 signals were required to mount an effective type 2 immune response against the parasite leading to higher susceptibility against worm infection in conditional knockout mice. Therefore, this study argues for a non-redundant role of cell-intrinsic ST2 signals triggering proper activation of ILC2 for initiation of type 2 immunity.

Effect of papain enzyme administration in pH alteration, VEGF phosphorylation, and its impact on collagen degradation using a rat model of abnormal scarring


 
 
 
 Purpose: To analyze the effect of enzyme papain administration, after its injection into abnormal scars of Rattus norvegicus rats, on pH alteration (ΔpH) of scar tissue, vascular endothelial growth factor (VEGF) expression levels, and determine their impact on collagen degradation.
 Methods: This study used Rattus norvegicus as an experimental animal. The pH measurement of the scar tissue was carried out before papain injection at 12th weeks while abnormal scar was established after papain injection at 15th weeks. Expression of vascular endothelial growth factor (VEGF) was examined by Western blot technique, while hydroxyproline levels were measured using QuickZyme Total Collagen Assay.
 Results: Changes in ΔpH, VEGF expression, and hydroxyproline levels in the treatment group were significant (p < 0.05) compared to control. Path analysis showed a direct relationship between the parameters (p < 0.01), except for the correlation between VEGF and hydroxyproline value (p = 0.23).
 Conclusion: Papain enzyme administration increased ΔpH value of abnormal scar tissue. A high dose of papain also causes an increase of collagen degradation process, as a response to angiogenesis deflation.
 
 
 

Migration of Lung Resident Group 2 Innate Lymphoid Cells Link Allergic Lung Inflammation and Liver Immunity.

Group 2 innate lymphoid cells (ILC2s) are tissue resident in the lung and activated by inhaled allergens via epithelial-derived alarmins including IL-33. Activated ILC2s proliferate, produce IL-5 and IL-13, and induce eosinophilic inflammation. Here, we report that intranasal IL-33 or the protease allergen papain administration resulted in increased numbers of ILC2s not only in the lung but also in peripheral blood and liver. Analyses of IL-33 treated parabiosis mice showed that the increase in lung ILC2s was due to proliferation of lung resident ILC2s, whereas the increase in liver ILC2s was due to the migration of activated lung ILC2s. Lung-derived ILC2s induced eosinophilic hepatitis and expression of fibrosis-related genes. Intranasal IL-33 pre-treatment also attenuated concanavalin A-induced acute hepatitis and cirrhosis. These results suggest that activated lung resident ILC2s emigrate from the lung, circulate, settle in the liver and promote type 2 inflammation and attenuate type 1 inflammation.

Subcutaneous Allergic Sensitization to Protease Allergen Is Dependent on Mast Cells but Not IL-33: Distinct Mechanisms between Subcutaneous and Intranasal Routes.

Protease activity of papain, a plant-derived occupational allergen homologous to mite major allergens, is essential to IgE/IgG1 production and lung eosinophilia induced by intranasal papain administration in mice, and IL-33 contributes to these responses. In this work, we investigate skin and Ab responses induced by s.c. papain administration into ear lobes and responses induced by subsequent airway challenge with papain. Subcutaneous papain injection induced swelling associated with increased epidermal thickness, dermal inflammation, serum IgE/IgG1 responses, and Th2 cytokine production in draining lymph node cells restimulated in vitro. These responses were markedly less upon s.c. administration of protease inhibitor-treated papain. Results obtained by using mast cell-deficient mice and reconstitution of tissue mast cells suggested the contribution of mast cells to papain-specific IgE/IgG1 responses and eosinophil infiltration. The responses were equivalent between wild-type and IL-33(-/-) mice. After the subsequent airway challenge, the s.c. presensitized wild-type mice showed more severe lung eosinophilia than those without the presensitization. The presensitized IL-33(-/-) mice showed modest lung eosinophilia, which was absent without the presensitization, but its severity and IgE boost by the airway challenge were markedly less than the presensitized wild-type mice, in which protease activity of inhaled papain contributed to the responses. The results suggest that mechanisms for the protease-dependent sensitization differ between skin and airway and that cooperation of mast cell-dependent, IL-33-independent initial sensitization via skin and protease-induced, IL-33-mediated mechanism in re-exposure via airway to protease allergens maximizes the magnitude of the transition from skin inflammation to asthma in natural history of progression of allergic diseases.

ICOS promotes group 2 innate lymphoid cell activation in lungs

Group 2 innate lymphoid cells (ILC2s) are newly identified, potent producers of type 2 cytokines, such as IL-5 and IL-13, and contribute to the development of allergic lung inflammation induced by cysteine proteases. Although it has been shown that inducible costimulator (ICOS), a costimulatory molecule, is expressed on ILC2s, the role of ICOS in ILC2 responses is largely unknown. In the present study, we investigated whether the interaction of ICOS with its ligand B7-related protein-1 (B7RP-1) can promote ILC2 activation. Cytokine production in ILC2s purified from mouse lungs was significantly increased by coculture with B7RP-1-transfected cells, and increased cytokine production was inhibited by monoclonal antibody-mediated blocking of the ICOS/B7RP-1 interaction. ILC2 expansion and eosinophil influx induced by papain, a cysteine protease antigen, in mouse lungs were significantly abrogated by blocking the ICOS/B7RP-1 interaction. Dendritic cells (DCs) in the lungs expressed B7RP-1 and the number of DCs markedly increased with papain administration. B7RP-1 expression on lung DCs was reduced after papain administration. This downregulation of B7RP-1 expression may be an indication of ICOS/B7RP-1 binding. These results indicate that ILC2s might interact with B7RP-1-expressing DCs in allergic inflammatory lung, and ICOS signaling can positively regulate the protease allergen-induced ILC2 activation followed by eosinophil infiltration into the lungs.

The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development

Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.

Listeria infection inhibits IgE production in regional lymph nodes by suppressing chemotaxis of basophils to lymph nodes

Infection with Listeria induces a dominant shift to the Th1 immune response and inhibits the Th2 response. Papain is frequently utilized in animal models of allergies. Papain administration induces chemotaxis of basophils to regional lymph nodes (LNs) and production of interleukin (IL)-4 by basophils, resulting in a Th2-dominant status and increased IgE production in LNs. In this model, production of immunoglobulin (Ig) E by LN cells is primarily controlled by IL-4 produced by basophils. Based on this model, it was postulated that Listeria monocytogenes (Lm) infection suppresses IgE production by LN cells. Therefore, the effects of Lm infection on a papain-induced mouse model of allergies were investigated. Following s.c. injection of papain, basophils transiently migrated to draining LNs because of the effects of chemokine (C-C) motif ligand (CCL) 24 and secreted IL-4, inducing a Th2 response. Lm infection blocked recruitment of basophils into the popliteal LNs by inhibiting CCL24 production. Papain-induced class switch recombination (CSR) to IgE is inhibited by Lm infection, whereas CSR to IgG1 is not affected by the same treatment. Therefore, the CSR of IgG1 to IgE is basophil-dependent, whereas the CSR of IgM to IgG1 is basophil-independent. Hence, Lm infection suppresses CSR to IgE without affecting CSR to IgG1.

Bronchoscopic lung volume reduction using tissue engineering principles

Am J Respir Crit Care Med 2003;167:771–8. Ingenito EP, Berger RL, Henderson AC, et al. Comments: In this study, the investigators use a novel procedure to successfully achieve bronchoscopic lung volume reduction (BLVR) in a sheep model of emphysema. The basic goal was to initiate a remodeling process in the target areas of the lung so that the damaged and hyperinflated lung is remodeled and transformed into a contracted scar. The authors used a biologic approach to achieve this goal. The principle was to use an enzymatic primer solution to remove epithelial cells from the target area and to provide a modified hydrogel scaffold containing glycosaminoglycans and a synthetic polyamine to promote fibroblast attachment and collagen synthesis. First, the investigators developed an animal model for advanced homogeneous emphysema. This was achieved by making sheep inhale papain under general anesthesia. The BLVR procedure was apparently performed 2 weeks after papain administration. First, 10 mL of Bistech Primer Reagent (0.25% trypsin in buffer–phosphate saline) was introduced into the selected segmental bronchus through the working channel. After leaving it in place for 2 minutes, suction was applied to remove as much residual solution as possible. Then, 10 mL of Bistech Washout Solution containing 10% heat-inactivated fetal bovine serum was introduced in the same segment to neutralize the residual primer. Suction was again applied after 2 minutes of dwell-time. Lastly, 10 mL of fibrin hydrogel suspension and 1 mL of thrombin crosslinker were delivered simultaneously at this site using a double-lumen catheter system. The procedure was repeated at 6 different treatment sites. The individual treatment needed 5 to 7 minutes and the entire BLVR procedure required 30 to 45 minutes. The investigators performed detailed respiratory physiology parameters and chest computed tomography scans immediately before and 3 and 9 weeks after the BLVR procedure. After 9 weeks, the animals were killed and autopsy was performed. The study involved 6 sheep, all of which displayed hyperinflation, a decrease in elastic recoil pressure, a decrease in diffusion capacity, and an increase in airway resistance after papain administration. The BLVR was well tolerated and none of the animals developed acute complications such as hypoxemia, hemoptysis, bronchospasm, or respiratory distress. Three weeks post-BLVR procedure, the respiratory physiological parameters showed a reduction in TLC from 3.63 ± 0.42 to 3.01 ± 0.32 L (P = 0.02), FRC from 2.02 ± 0.27 to 1.66 ± 0.37 L (P = 0.05), and RV from 1.43 ± 0.48 to 0.63 ± 0.17 L (P = 0.002). Elastic recoil pressure improved after BLVR. The diffusion capacity showed a trend toward improvement post-BLVR. The benefits in respiratory physiology parameters were maintained at 9 weeks. Serial computed tomography scans showed collapse, loss of volume, and development of localized linear scars in the treatment areas. Autopsy findings confirmed well-demarcated scar tissue with extensive collagen deposition, fibroblasts, and mononuclear cells. There was no suggestion of pneumonia, granuloma, tissue necrosis, or abscess formation. The results of this study were very impressive. Desired goals were achieved in experimental animals with a relatively short procedure without needing surgery or introduction of complicated mechanical prosthesis. Using a similar experimental model of emphysema, the same group of investigators has attempted BLVR through mechanical sealing of bronchi with fibrin (Am J Respir Crit Care Med 2001;164:295–301). Although 55% of experimental animals developed desired lung collapse and focal scarring, sterile abscesses developed in 15% of animals in that study. The new approach based on a bioengineering principle appears to be safer and more effective than their prior method. Several limitations of this study should be noted. First, the BLVR procedure was performed within a short time after inducing experimental emphysema. Without a controlled group, the possibility that residual inflammatory changes from recent papain administration confounded the results of BLVR cannot be ruled out. Whether equally impressive results can be achieved in longstanding and established emphysema remains to be seen. Second, an animal experiment cannot establish that improvement in respiratory mechanics will translate into benefits in clinically relevant end points such as mortality, exercise capacity, and quality of life. Lastly, the long-term safety of inducing scar tissue in the lung needs to be established. The main concern is the risk of future development of scar carcinoma. Is this technique ready for use in human subjects? The procedure appears to be simple and free of any major short-term complications in animal subjects. However, the safety of the procedure needs to be established in human subjects in phase I studies. There always remains a possibility that human response could differ from that of sheep. Nevertheless, given the disease burden worldwide and limited benefits of current medical and surgical therapy, it seems appropriate to start exploring novel approaches in human subjects such as that used in the current study.

Growth changes in the craniofacial complex of the rat after prolonged papain administration

The purpose of the study was to evaluate by radiographic and biometric means the effects of prolonged papain administration on growth of the craniofacial complex, to gain further insight into the importance of cranial cartilage for skull enlargement during rapid skeletal growth (25 to 70 days of age). Fifty 21-day-old male Lewis strain rats were divided randomly into three groups. Group 1 ( n = 20) received 45 daily injections intraperitoneally of a 2% solution of crude papain in normal saline from 25 to 70 days of age. Group 2 ( n = 20) were given 30 daily injections at the same dose level from 40 to 70 days of age, and group 3 ( n = 10) served as untreated controls. Animals were weighed daily until 70 days of age and then every other day until killed at day 120. Submental-vertex and lateral skull radiographs were obtained at 25, 40, 53, 70, 90, and 120 days of age with standardized skull positioning and radiographic settings. Cephalometric measurements of 22 linear dimensions were made in each of 360 cephalograms, and the data analyzed statistically with Student's t test. Growth velocity curves were evaluated for evidence of catch-up growth. Body-weight gain by papain-treated groups was less than for controls (p < 0.001). Skull and nasal lengths, middle cranial base length, and sphenoidal length were significantly shorter (p < 0.001). Neurocranial length was also shortened, whereas neurocranial height and supraoccipital height increased. Reduction of skull dimensions was generally more pronounced in the earlier injected group. The effectiveness of catch-up growth was not constant and was more successful in the later injected group. Catch-up growth was effective for posterior nasal, neurocranial and supraoccipital heights, cranial width, and posterior cranial base length. The incomplete nature of catch-up growth may have been due to the particular timing and severity of the insult, the frequency of administration of the enzyme and the peak velocity of dimensional growth relative to the time of papain administration. (A M J O RTHOD D ENTOFAC O RTHOP 1994;105:270-8.)

Morphometric changes in growth of the rat pelvis after papain administration.

The proteolytic enzyme, papain, was given systematically to evaluate the short- and long-term effects of inhibition of endochondral bone formation on pelvic growth, with emphasis on the innominate bone. Ninety-eight Lewis-strain male rats, used concurrently for craniofacial growth studies, were divided into two groups. Thirty rats from Group I (n = 48) received 2% crude papain i.p. daily from 25-40 days-of-age and were euthanized at 40, 54, and 70 days-of-age. Thirty-five rats from Group II (n = 50) were given papain at the same dose from 25-70 days-of-age and were euthanized at 26, 40, 54, 70, and 120 days. The remaining animals in both groups were the controls. Standardized dorsoventral pelvic radiographs were taken of all 98 animals. Ten linear dimensions were measured on each and the data evaluated statistically. A reduction both in size and rate of growth of the bony pelvis was found. All the anteroposterior and most of the transverse pelvic dimensions were significantly shorter, to a greater extent in the prolonged papain group. Bi-ischial width was increased, perhaps to compensate for pelvic shortening and to accommodate the pelvic contents. The findings may contribute to our better understanding of abnormal endochondral bone growth in the pelvis.

Effects of prolonged papain administration on craniofacial cartilaginous growth of young rats—A histological study

Effects of prolonged papain administration on craniofacial cartilaginous growth of young rats—A histological study

Pulmonary vascular structural and functional changes in papain-induced emphysema in dogs.

We evaluated the pulmonary circulatory effects of emphysema produced by papain in 6 dogs that received 4 weekly treatments of papain administered by aerosolization. A control group of 6 dogs received saline treatments. Hemodynamic measurements were performed during normoxic and hypoxic (10% oxygen) ventilation approximately 3 and 6 months after treatment. Baseline hemodynamic parameters were comparable in both groups and were unchanged at 6 months in the control group. In the papain-treated group at 6 months, there were significant reductions in PaO2 (95 +/- 4 to 79 +/- 3 mm Hg, p less than 0.005) and increases in mean pulmonary artery pressure (12 +/- 1 to 18 +/- 3 mm Hg, p less than 0.05) and pulmonary vascular resistance (2.0 +/- 0.3 to 6.2 +/- 2.1 units, p less than 0.01). Morphometric evaluation demonstrated increased medial thickness in muscular pulmonary arteries less than 50 mu in size. Morphologic evaluation confirmed the presence of panlobular emphysema with papain administration. We conclude that chronic emphysema produced in dogs by aerosol administration of papain results in elevated pulmonary artery pressure, which is characterized pathologically by medial hypertrophy of small pulmonary arteries. This model appears suitable for further study of the pathogenesis of pulmonary vascular disease associated with chronic lung disease.

Changes in pulmonary connective tissue after a single intratracheal instillation of papain in the rat.

The effect of a single intratracheal instillation of 2 mg of papain on metabolic changes of collagen and elastin in lungs was studied in rats labeled with [14C]proline in vivo. The total amount, concentration, specific and total radioactivity of both proteins were examined during the 28 days after papain administration. In the early period (1 to 3 days) a loss of elastin content (about 50% of control; p less than 0.01) was found, whereas collagen content increased (p less than 0.05). Simultaneously, the specific as well as the total radioactivity of both proteins was enhanced (p less than 0.01) which indicated an accelerated biosynthesis and metabolic turnover. This fact was reflected in the increased amounts of both collagen and elastin in the lungs 14 and 28 days after instillation of the enzyme. The changes in connective tissue proteins accompanied a large increase in lung weight (200% that of the control rats; p less than 0.01) immediately after the exposure. Toward the end of the 28-day period, the increase was smaller. The enzymatic damage probably induced the formation of nonfunctional elastin and fibrous scars as a consequence of the healing process.

Biochemical studies on rat liver Golgi apparatus. IV. Effects of various treatments in vivo on the Golgi apparatus.

The effects of various treatments in vivo on the intracellular contents of the Golgi apparatus and microsomes (endoplasmic reticulum) in rat liver were studied. Partial hepatectomy increased the content of Golgi apparatus. Laparotmy also increased the content of Golgi apparatus, but to a lesser extent than partial hepatectomy. In contrast, the content of microsomes remained unchanged by these treatments. On the other hand, the plasma seromucoid content was markedly increased by laparotomy, but unchanged by partial hepatectomy. Papain administration also caused an increase in the content of Golgi apparatus. The contents of both organelles were increased by the injection of phenobarbital. These results indicate that the control mechanisms of proliferation of Golgi apparatus are different from those of endoplasmic reticulum. These findings are discussed in relation to the functions of the Golgi apparatus, and it is suggested that the major function of the organelle at a given time is determined by the major metabolic demands at that time.

Papain-induced lung injury: Alterations in connective tissue metabolism without emphysema

The acute effects of the deposition of papain in the lungs of 4-month-old rabbits have been investigated with an amount not sufficient to cause emphysematous changes. During the first 2 days of in vivo deposition when hemorrhaging and inflammation occur, the total amounts of collagen and elastin in the lung, into which the papain had been instilled, did not change but the concentrations of collagen and elastin decreased 20 and 25% respectively. The rates of synthesis of collagen and other proteins doubled. By 4 weeks, collagen and elastin concentrations returned to, or were slightly above, control levels. The histological appearance and mean linear intercepts of lung sections and the compliance curves of excised lungs of the treated animals were similar to controls by 2 weeks after papain administration. The rabbit lung thus appears able to repair mild injury due to proteolytic enzymes.

The changes in craniofacial growth following papain administration in young mice.

The effect on craniofacial growth after repeated papain administrations was studied in young mice. The length of the upper face and lower face were both seriously affected whereas the length of the neurocranium, the anterior height of the upper and lower face were moderately affected.The posterior height of the upper face and the height of the neurocranium were not influenced by papain injections.

Immunosuppressive activity of the 3a-1 fraction of papain.

The maximal nonlethal dose of the 3a-1 fraction of papain, determined by use of 9-week-old white albino rabbits, was 10 mg per injection, administered intravenously. The immunosuppressive activity of the 3a-1 fraction of papain was studied by its inhibition of sheep red blood cell hemolysin, bacterial agglutinin, and horse serum precipitin. Immune suppression was observed when papain injections preceded antigen injections by 12 to 18 hr. The enzyme preparation was analyzed by use of cellulose acetate and starch-gel electrophoresis and by the micro-Kjeldahl method. Starch-gel electrophoresis of serum samples revealed qualitative alterations in the alpha(-1) region 7 hr after papain administration. Changes were also observed in the urinary aminopolysaccharide content.

The effect of papain on epiphysial cartilage in rachitic rats: histologic, autoradiographic and microradiographic studies.

Epiphysial cartilage from rachitic and that from nonrachitic rats showed largely similar reactions to single administration of papain, as was demonstrated both histologically and by autoradiographic examination following injection of radiosulphate. Rachitic rats which had received multiple injections of papain exhibited severe, perhaps irreversible lesions of the epiphysial cartilage zones. Healing of rickets, was not observed in rats which had received vitamin D and a rachitogenic diet in conjunction with papain. Intensive capillary invasion of the epiphysial cartilage was noted, however, in rats which had been transferred to a normal diet plus vitamin D in conjunction with papain administration. Rachitic rats treated solely with papain showed no mineralization in the cartilage zone. In those rats which received vitamin D and a continued rachitogenic diet, on the other hand, mineralization was regularly observed in that part of the cartilage zone which comprised both proliferative cells and neighboring hypertrophic cells; the remainder of the hypertrophic cartilage was not mineralized. The animals which had been transferred to a normal diet supplemented by vitamin D showed, in some areas, a mineralization pattern similar to that, described above. In other areas, mineralization was observed throughout the hypertrophic cartilage and even quite deep into the proliferative zone. Mineralization of rachitic cartilage may occur despite a greatly reduced chondroitin sulphate content and the presence of severely damaged hypertrophic cells. The findings suggest that mineralization of the epiphysial cartilage is a determinant factor in its resorption.

Skeletal Changes in Suckling Rats Induced by Prolonged Papain Administration

A crude papain suspension when injected intraperitoneally into suckling rats for twenty-three to sixty-nine days produced malformation of the sternum, rib cage, vertebral column, and caudad ribs. Permanent cartilaginous disorganization and epiphyseal-plate closure resulted in shortened long bones and vertebral bodies. Rigidity of the vertebral column was produced by degeneration of the nucleus pulposus, characterized by precipitation of eosinophilic necrotic debris and final replacement by hyaline fibrocartilage.