Presence Of Cell-free Supernatants Research Articles

Transcriptomic and metabolomic insights into the antimicrobial effect of Leuconostoc mesenteroides or lactic acid on pathogenic Gallibacterium anatis

Gallibacterium anatis (G. anatis) is an opportunistic poultry pathogen that poses a threat to human health via the food chain and can also lead to great economic losses in poultry industries. Our previous studies have demonstrated that the lactic acid-producing bacteria Leuconostoc mesenteroides QZ1178 can effectively inhibit the growth of G. anatis by acid production, but the mechanism remains unclear. The aim of the current research was to further investigate the molecular mechanism underlying this acid-induced antimicrobial effect. The TEM results showed that the cell membrane of G. anatis (GAC026) was damaged and that cells were lysed in the presence of cell-free supernatants from Leuconostoc mesenteroides (CFS) or lactic acid. Lactic acid showed a greater antimicrobial effect than CFS. In this study, the changes in the transcriptome and metabolic profile of G. anatis under acid stress at different stages were studied. Using culture medium supplemented with CFS (pH 3.6) or lactic acid (pH 3.6) at a 1:1 ratio, 677 differentially transcribed genes and 374 metabolites were detected in G. anatis. The interaction network of all identified differentially expressed genes and metabolites was constructed to outline the regulatory genes and dominant pathways in response to acid stress. The results of real-time reverse transcription quantitative PCR (RT‒qPCR) further confirmed the results of the transcriptomic analyses. Typically, succinate, citrate, L-malic acid, and oxaloacetate were reduced by acid stress in G. anatis, which suggested that lactic acid greatly disturbed energy metabolism. Overall, this work provides a comprehensive understanding of the stress response and cell death of G. anatis caused by lactic acid.Graphical

티베트 요거트에서 분리한 유산균의 병원성 세균 항균 효과 연구

Yogurt is produced by bacterial fermentation of milk and contains lactic acid bacteria (LAB), which produce various metabolites such as organic acid, hydrogen peroxide, and bacteriocin. This study aimed to investigate cell-free supernatants (CFS) of LAB isolated from Tibetan yogurt. CFS (TY1, TY2, TY3, TY4, TY5, TY6, and TY7) from selected strains of LAB were co-incubated with four different foodborne pathogenic bacteria, namely E. coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus. Inhibition of foodborne pathogenic bacterial growth was not affected in the presence of CFS (pH 6.5). In contrast, CFS without neutralization completely inhibited the growth of the bacteria. Furthermore, when the concentration of CFS (without neutralization) was changed to 1:4 and 1:8, a difference in inhibition was observed between Gram-positive and Gram-negative bacteria. CFS more effectively inhibited the growth of Gram-negative E. coli O157:H7 and S. Typhimurium than Gram-positive L. monocytogenes and S. aureus. These results suggest that organic acids in LAB may inhibit the growth of foodborne pathogenic bacteria, particularly Gram-negative bacteria.

국내 사찰 발효식품에서 분리한 유산균의 항균 특성 연구

Fermented foods of Korean temple are vegetable-based foods and their seasonings are limited to five pungent herbs. The aim of this study was to investigate cell-free supernatants (CFS) of lactic acid bacteria isolated from the fermented foods of Korean temples. Three selected CFS significantly inhibited the biofilm formation of Escherichia coli O157:H7 at 24 h, while one of the selected CFS markedly inhibited the biofilm formation at 72 h. However, only one CFS significantly attenuated the biofilm formation of Staphylococcus aureus at 24 h, but these CFS failed to inhibit the biofilm formation. Moreover, selected CFS effectively inhibited the growth of E. coli O157:H7, whereas the growth of S. aureus was not affected in the presence of CFS. These results suggest that CFS of lactic acid bacteria isolated from fermented foods of Korean temples shows antimicrobial activity agasint Gram-negative E. coli O157:H7, but not at Gramd-positive S. aureus.

Cell-FreeLactobacillussp Supernatants Modulate Staphylococcus aureus Growth, Adhesion and Invasion to Human Osteoblast (HOB) Cells.

The increase of antibiotic resistance has become a problem. Probiotic bacteria play an important role in preventive/supportive medicine. Therefore, we examined the inhibitory effects of four different Lactobacillus species' (L. acidophilus-La, L. plantarum-Lp, L. fermentum-Lf and L. rhamnosus-Lr) cell-free supernatants (CFSs) on growth, adhesion, invasion, and biofilm formation of Staphylococcus aureus and effects of S. aureus, CFSs, and S. aureus-CFSs co-existence on human osteoblast (HOB) cell viability. Growth alterations were measured spectrophotometrically. Adhesive/invasive bacterial counts were detected by colony counting. Biofilm was evaluated using microtiter plate assay. The MTT assay was used for detection of HOB cell viability. The growth of MSSA significantly (P < 0.01) decreased in the presence of two CFSs (Lf and Lr) (P < 0.01); the growth of MRSA significantly (P < 0.05) reduced in the presence of La CFSs. All tested CFSs were found to reduce adhesion and invasion of MSSA (P < 0.0001). The adhesion of MRSA was enhanced (P < 0.0001) in the presence of all CFSs except La and the invasion of MRSA was decreased (P < 0.01) in the presence of Lr and Lf CFSs. All tested CFSs were shown to inhibit biofilm formation significantly (P < 0.0001). The reduction of S. aureus infected HOB cell viability and exposed to all CFSs except Lr that was found to be significant (P < 0.0001). The viability of HOB cell during co-incubation with MSSA and CFSs was shown to be decreased significantly. However co-existence of MRSA and CFSs did not alter HOB cell viability. These results suggested that lactobacilli as probiotics have low protective effects on MRSA-infected host cells.

Ability of Three Lactic Acid Bacteria to Grow in Sessile Mode and to Inhibit Biofilm Formation of Pathogenic Bacteria.

In this study, we explored the effect of three lactic acid bacteria (LAB), i.e. Enterococcus sp CM9, Enterococcus sp CM18 and Enterococcus faecium H3, and their supernatants, on seven biofilm-forming pathogenic strains isolated from human urinary tract or nose infections. By quantitative biofilm production assay, a strong adherence ability of Enterococcus sp CM9 and Enterococcus sp CM18 was revealed while E. faecium H3 resulted to be moderately adherent. Inhibition tests demonstrated an antimicrobial activity of LAB against pathogens.The presence of cell free supernatant (CFS) of CM9 and CM18 strains significantly decreased the adhesion of S. aureus 10,850, S. epidermidis 4,296 and E. coli FSL24. The CFS of H3 strain was effective against S. epidermidis 4,296 and P. aeruginosa PA1FSL biofilms only. Biofilm formation of K. pneumoniae Kp20FSL, A. baumannii AB8FSL and ESBL+ E. coli FS101570 have not been affected by any CSF while P. aeruginosa PA1FSL biofilm increase in presence of CM9 and CM18 CFS.Confocal Laser Scanning Microscopy revealed that K. pneumoniae Kp20FSL biofilm was inhibited by Enterococcus sp CM9, when grown together.Our results suggest that the LAB strains and/or their bacteriocins can be considered as potential tools to control biofilm formation of some bacterial pathogens.

Characterization of the Potential Probiotic Vibrio sp. V33 Antagonizing Vibrio Splendidus Based on Iron Competition.

Background: Vibrio Splendidus Vs is an important aquaculture pathogen that can infect a broad host of marine organisms. In our previous study, an antagonistic bacterium Vibrio sp. V33 that possessed inhibitory effects on the growth and virulence of a pathogenic isolate V. splendidus Vs was identified.Objectives: Here, we further explored the antagonistic substances and antagonistic effects from the viewpoint of iron competition.Materials and Methods: The main antagonistic substances in the supernatants from Vibrio sp. V33 were identified using the bioassay-guided method. The response of V. splendidus Vs under the challenge of cell-free supernatant from Vibrio sp. V33 was determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and real-time reverse-transcription PCR.Results: The main antagonistic substances produced by Vibrio sp. V33 have low molecular weights, are water soluble, and are heat-stable substances. Meanwhile, the iron uptake rate of Vibrio sp. V33 was higher than that of V. splendidus Vs. In the presence of cell-free supernatant from Vibrio sp. V33, expressions of two functional genes, viuB and asbJ related to ferric uptake processes in V. splendidus Vs, were up-regulated, whereas furVs coding the ferric uptake repressor was suppressed below 0.5-fold. One gene coding phosphopyruvate hydratase does not change at mRNA level, but was up-regulated at protein level. Conclusions: Our results suggested that antagonistic effect of Vibrio sp. V33 on the pathogenic isolate V. splendidus Vs was partially due to the stronger ability of Vibrio sp. V33 to seize iron. This cell-free supernatant from Vibrio sp. V33 created an iron-limited milieu for V. splendidus Vs, which led to the changed expression profiles of genes that were related to iron uptake in V. splendidus Vs.

Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50μM, respectively. Violacein showed a MIC of 15μM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

Interspecies transfer of vancomycin, erythromycin and tetracycline resistance among Enterococcus species recovered from agrarian sources

BackgroundEnterococci are now well recognised for their ability to transfer antibiotic resistance and for their association with nosocomial infections, but less is known regarding their relevance in the wider environment. Enterococcus faecalis and Enterococcus faecium were isolated from a range of agrarian associated sources (low-flow water, septic tank, poultry litter, high flow water, slurry/soil) and were assessed for latent ability to transfer antimicrobial resistance.ResultsThe isolates were tested for phenotypic clumping in the presence of cell-free supernatant from other isolates. Some isolates were identified which demonstrated clumping, indicating that they possessed peptide sex pheromone conjugal machinery. All isolates were also tested for antibiotic resistance phenotypes using both disc diffusion and minimum inhibitory concentration (MIC) assays. These tests revealed that the enterococci demonstrated both phenotypic clumping and antibiotic resistance phenotypes. Based on these selection criteria, the isolates were identified as having the potential for horizontal gene transfer and were used to investigate the transfer of multiple antibiotic resistance phenotypes. Conjugal transfer of antibiotic resistance phenotypes was determined using a solid agar mating method followed by a standard antibiotic selection test resulting in different transfer patterns. An interspecies conjugal transfer of vancomycin resistance from E. faecalis to E. faecium was identified while the remaining reactions were within the same species. Transfer efficiencies ranging from 2 × 10−1 to 2.3 × 10−5 were determined based on the reactions of three donor isolates (MF06036, MF0410 and MF06035) and two recipient isolates (MW01105Rif and ST01109Rif), with the transfer of vancomycin, erythromycin and tetracycline resistance genes.ConclusionsThe conjugation reactions and selection conditions used in this study resulted in a variety of co-transferred resistance phenotypes suggesting the presence of different mobile elements in the set of natural isolates. This study highlights the potential for extensive horizontal gene transfer in a previously neglected reservoir for enterococci.

Inhibition of biofilm development and spoilage potential of Shewanella baltica by quorum sensing signal in cell-free supernatant from Pseudomonas fluorescens

The objective of this study was to in vitro evaluate the effect of a cell-free supernatant (CFS) containing quorum sensing (QS) signal of Pseudomonas fluorescens on the growth, biofilm development and spoilage potential of Shewanella baltica, and preliminarily assess the interactive influences of various chemically synthesized autoinducers on spoilage phenotypes of S. baltica. PF01 strain isolated from spoiled Pseudosciaen crocea was identified P. fluorescens. The addition of 25% and 50% CFS to S. baltica culture had no effect on the growth rate during the lag and exponential phase, however, caused cell decline during the stationary phase. The presence of CFS from P. fluorescens significantly inhibited biofilm development, and greatly decreased the production of trimethylamine (TMA) and biogenic amino in S. baltica. Various signal molecules of QS in the CFS of P. fluorescens culture were detected, including seven N-acyl-l-homoserine lactones (AHLs), autoinducer-2 (AI-2) and two diketopiperazines (DKPs). Exogenous supplement of synthesized seven AHLs containing in the CFS decreased biofilm formation and TMA production in S. baltica, while exposure to exogenous cyclo-(l-Pro-l-Leu) was showed to promote spoilage potential, which revealed that S. baltica also sense the two QS molecules. Furthermore, the stimulating effect of cyclo-(l-Pro-l-Leu) was affected when AHL was simultaneously added, suggesting that the inhibitory activity of spoilage phenotypes in S. baltica might be attributed to a competitive effect of these QS compounds in the CFS of P. fluorescens. The present studies provide a good basis for future research on the role of QS in the regulation of spoilage microbial flora.

Biological control of Alternaria alternata causing leaf spot disease of Aloe vera using two strains of rhizobacteria

Fungal pathogens causing the leaf spot disease of Aloe vera have been isolated from 40 randomly selected infected leaves, collected from different locations of Birbhum and Burdwan districts of West Bengal. All of the isolates showed similar morphological characteristics and the strain VBAV007, isolated from a severely infected Aloe leaf was identified as Alternaria alternata by D1/D2 region of 28S rRNA gene sequence homology. In addition to the destruction of leaf mesophyll tissues the pathogen also decreases the antimicrobial potential of A. vera gel. The commercially available fungicide mancozeb was effective at low concentration (100μg/ml) to control the pathogen whereas it can tolerate 1000μg/ml or more concentrations of bavistin. Two plant growth promoting rhizobacterial strains, viz. Burkholderia cenocepacia VBC7 and Pseudomonas poae VBK1 were able to produce prominent zones of inhibition against the pathogen in dual culture overlay plates. 89.3±1.22% and 81.5±2.67% inhibitions of conidial germination of the pathogen were noticed in the presence of cell free supernatant of VBK1 and VBC7 respectively. Radial growth assay also suggested prominent growth inhibition of VBAV007 by biocontrol strains. They induce mycelial breakage of pathogen as evidenced by scanning electron micrographs. Greenhouse challenge experiments also suggested excellent capabilities of biocontrol agents to reduce disease severity in good measure even after exposure to high concentration (3.1×104conidia/ml) of pathogenic spores. During in vivo field experiments 54.25±3.55% disease severity was observed for untreated plants, whereas only 11.69±1.25% and 15.22±2.64% disease severities were noticed in plants treated with VBK1 and VBC7 respectively. Since biocontrol organisms have the potential to decrease the disease severity, they also help to maintain the good health as well as antimicrobial potential of A. vera plants.

Impact of Cell-free Supernatant of Lactic Acid Bacteria on Putrescine and Other Polyamine Formation by Foodborne Pathogens in Ornithine Decarboxylase Broth.

Conversion of ornithine to putrescine by Salmonella Paratyphi A, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli was investigated in ornithine decarboxylase broth (ODB) using cell-free supernatants (CFSs) obtained from Leuconostoc mesenterodies subsp. cremoris, Pediococcus acidilactici, Lactococcus lactis subsp. lactis, Streptococcus thermophilus. Two groups of cell-free supernatants (25 or 50%) and control (only ODB) were prepared to investigate putrescine (PUT) and other polyamine formation by foodborne pathogens (FBPs). Significant differences (p < 0.05) were observed among the species for each amine. All of the CFSs reduced the formation of PUT by ≥65%. The production of cadaverine (CAD) was scarcely affected by the presence of CFSs, with the exception of the samples inoculated with L. monocytogenes. The variation in polyamine was found with respect to the control samples. Spermidine (SPD) was produced in lower amount in many samples, especially in Gram-negative FBPs, whereas spermine (SPN) increased drastically in the major part of the samples concerning the control. Histamine (HIS) was characterized by a marked concentration decrease in all of the samples, and tyramine (TYR) was accumulated in very low concentrations in the controls. Therefore, the ability of bacteria to produce certain biogenic amines such as HIS, TYR, PUT, and CAD has been studied to assess their risk and prevent their formation in food products. The results obtained from this study concluded that the lactic acid bacteria (LAB) strains with non-decarboxylase activity are capable of avoiding or limiting biogenic amine formation by FBP.

Understanding the crosstalk between ?d T cells and osteoclasts in patients with breast cancer

Event Abstract Back to Event Understanding the crosstalk between γδ T cells and osteoclasts in patients with breast cancer Shubhada Chiplunkar1*, Swati P. Phalke1, Vani Parmar2 and Rajendra A. Badwe2 1 ACTREC-TMC, India 2 Tata Memorial Hospital, India Bone metastasis is a common cause of morbidity in breast cancer patients. Aminobisphosphonates are widely used in treatment of bone metastasis and skeletal disorders. Aminobisphosphonates increase endogenous pool of isopentenyl pyrophosphate (IPP) of mevalonate pathway in tumor cells, which acts as potent antigen for γδ T cells. γδ T cells are unique subsets of T-lymphocytes that are activated by aminobisphosphonates and phosphoantigens (bromohydrin pyrophosphate, IPP). The present study aims at understanding the crosstalk between γδ T cells and osteoclasts in patients with breast cancer metastasis. Multicolor flow cytometry showed higher expression of RANKL on γδ T cells of breast cancer patients. Stimulation with aminobisphosphonate (zoledronate) increased RANKL expression on γδ T cells. Osteoclasts generated from CD14+ monocytes of breast cancer patients and healthy individuals in the presence of zoledronate activated γδ T cells showed reduced osteoclastogenesis as analysed by 23c6 mAb (CD51/61) and DAPI staining. Osteoclasts generated in the presence of cell free supernatants of activated γδ T cells showed reduced resorption area (pits) on Osteoclast Activity Assay Substrate. Cytokine analysis of activated γδ T cells showed presence of IFN-γ, antiosteoclastogenic cytokine. Zoledronate activated γδ T cells have high propensity to migrate to bone and inhibit osteoclastogenesis. Aminobisphosphonate activated γδ T cell based immunotherapy would be a promising approach to treat bone metastasis in breast cancer. Keywords: Aminobisphosphonates, γδ T cells, Osteoclasts, bone metastasis, breast cancer Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Chiplunkar S, Phalke SP, Parmar V and Badwe RA (2013). Understanding the crosstalk between γδ T cells and osteoclasts in patients with breast cancer. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01017 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Shubhada Chiplunkar, ACTREC-TMC, Navi Mumbai, Maharashtra, 410210, India, schiplunkar@actrec.gov.in Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shubhada Chiplunkar Swati P Phalke Vani Parmar Rajendra A Badwe Google Shubhada Chiplunkar Swati P Phalke Vani Parmar Rajendra A Badwe Google Scholar Shubhada Chiplunkar Swati P Phalke Vani Parmar Rajendra A Badwe PubMed Shubhada Chiplunkar Swati P Phalke Vani Parmar Rajendra A Badwe Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of L. plantarum cell-free extract and co-trimoxazole against Salmonella Typhimurium: a possible adjunct therapy

BackgroundFrequent and indiscriminate use of antibiotics has led to the development of multi-drug resistant bacterial strains. It necessitates the exploitation of alternative therapeutic strategies. In order to reduce the dose of antibiotic required and to decrease the associated side effects, the present study was aimed at evaluating the synergism, if any, between a conventional antibiotic, co-trimoxazole (CTZ)) and cell free supernatant (CFS) of a probiotic (L. plantarum) against S. Typhimurium NCTC 74. This antimicrobial combination was selected on the basis of antibiotic susceptibility pattern of Salmonella and L. plantarum.MethodsThe synergy was evaluated in terms of size of zone of inhibition, fractional inhibitory concentration index, time-kill assay (in-vitro) as well as macrophage functions (ex-vivo).ResultsThe concentration producing the same or higher antibacterial effect (size of zone of inhibition) was reduced to half when both the agents were used in combination with respect to the concentrations required when used separately. CTZ and CFS exhibited synergetic activity against Salmonella by checkerboard microtitre test and the time-kill test. Ex-vivo studies demonstrated a significantly higher intracellular killing of bacteria by macrophages treated with CFS (80 AU/ml) + (CTZ) (2 μg/ml) as compared to when treated with both separately at higher concentrations. Significant reduction in the extent of lipid peroxidation and nitrite levels generated by macrophages in presence of CFS and CTZ, in conjunction, further substantiated the synergistic efficacy of the combination.ConclusionsThe antimicrobial efficacy of this combination indicates that it may serve as the basis in developing alternative strategies to combat Salmonella infections.

Hydrolysis and association of leucine enkephalin to lymphomic and erythroleukaemic cell lines — III. Soluble enzymes

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of 3H-leucine enkephalin in the presence of cell-free supernatants. Results obtained indicate that in the presence of these supernatants leu-enkephalin rapidly disappears and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. Chromatograph analyses of cell supernatants indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In all three lines soluble enzymes are represented by a sizeable number (from 13 to 25) of distinct activities. The number of enzymes identified and their considerable total activity suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.