Cell-free Fluid Research Articles

High fat intake by ponies reduces both apparent digestibility of dietary cellulose and cellulose fermentation by faeces and isolated caecal and colonic contents

Inclusion of extra fat in the diet of horses, at the expense of an iso-energetic amount of non-structural carbohydrates, has been shown to decrease the apparent digestibility of the various dietary fiber fractions, but the mechanism was unknown. It was hypothesized that inclusion of extra fat in the diet depresses the caecal and/or colon microbial degradation of fiber, leading to a decrease in fiber digestion. The hypothesis was tested using six ponies fed either low fat or a high fat ration. In the ponies, the high fat ration lowered apparent NDFom digestibility by 0.089. The 12 ponies were killed and intestinal contents isolated to be used as inoculum with in vitro gas production technique. In general, groups mean maximum gas production from either cellulose or xylan by caecal, colon and faecal bacteria was lower when the ponies had been fed the high fat diet. Cumulative gas production by caecal fluid with xylan as substrate after 20 h of incubation was depressed when the donor animals had been fed the high fat diet. With cellulose as substrate, gas production by caecal contents was lowered by on average 20% after fat feeding of the ponies. There was a diet effect of cell-free caecal fluid on gas production from cellulose by a standard inoculum: fat feeding had an inhibitory impact. It is concluded that fat feeding in ponies inhibits microbial activity in the caecum, which in turn leads to a decrease in fiber digestibility.

Suppression of Alveolar Macrophage Apoptosis Prolongs Survival of Rats and Mice withPneumocystisPneumonia

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40-60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.

Macrophage-Activation Inhibitor Factor from L5178Y Murine Lymphoma and Formation of Hepatic Abscesses in BALB/c Mice

Normal mice are naturally resistant to hepatic abscesses produced by Listeria monocytogenes. A macrophage-activation inhibitor factor (MAIF) isolated from the cell-free ascitic fluid of L5178Y lymphoma-bearing mice inhibited the lipopolysaccharide-induced production of nitric oxide (NO) by the macrophages. Because macrophages are also involved in the immune response towards L. monocytogenes, the present study had the objective of investigating whether MAIF was also capable of allowing L. monocytogenes to form hepatic abscesses. BALC/c mice were inoculated intrahepatically with 5 x 10(5) bacteria. Experimental groups were treated daily with subcutaneous or intraperitoneal doses of 0, 1, 5 or 10 microg of MAIF/g of body weight. One dose was applied before inoculating bacteria and the remaining three doses 24, 48, and 72 h after inoculating bacteria. The development of hepatic abscess was analyzed 24 h after the last administration of MAIF. All treated mice (but not controls) developed hepatic abscess showing no differences regarding MAIF administration route. These results suggest a possible MAIF in vivo inhibition of NO macrophage production that allows L. monocytogenes hepatic abscess development in mice.

Amniotic fluid cytokine profile in association with fetal hyperechogenic bowel

Objectives Fetal hyperechogenic bowel (FEB) is associated with infection, chromosomal abnormalities and poor fetal outcome. FEB may result from an intrauterine fetal bowel cytokine-mediated inflammatory response. Since alterations in the levels of the cytokines interleukin (IL)-6, IL-8, IL-10, tumour necrosis factor (TNF)-α and interferon (IFN)-γ are associated with pregnancy complications and necrotizing enterocolitis, this study aimed: (i) to determine their involvement in the pathophysiology of FEB and (ii) to identify their role as amniotic fluid markers of this condition. Study design In this prospective case-control study, amniotic fluid was collected by transabdominal amniocentesis from pregnant women with fetuses presenting ( n = 10) – or not ( n = 30) – with FEB during routine 18–20 week ultrasound scans. Cell-free amniotic fluid samples were analysed for cytokine concentrations by fluid-phase multiplex immunoassay. Data were compared by Mann–Whitney U-tests and Pearson correlations. Results Amniotic fluid IL-8 levels were significantly higher in the FEB group. There was a positive correlation between IL-6 and each of IL-8 and INF-γ, as well as between IL-8 and IL-10, and TNF-α and INF-γ. Conclusions FEB likely ensues from a fetal inflammatory process involving IL-8 and, possibly, IL-6 and IL-10. This indicates the potential of immunomodulatory therapy in the management of FEB.

Lung cytotoxicity of combined exposure to refractory ceramic fibres and cigarette smoke.

Changes in some lung cytotoxic parameters after exposure to refractory ceramic fibres (RCF) or to cigarette smoke (S) and after combined exposure to RCF+S were studied in male Wistar rats in order to evaluate their potential adverse health effects. Four groups of rats were treated as follows : 1) intratracheally instilled by saline solution (0.4 ml); 2) intratracheally instilled by 4 mg of RCF; 3) exposed only to S (85 mg of total particulate matter/m(3) air ) for two hours daily; 4) exposed to RCF+S. After 6 months the animals were exsanguinated and the bronchoalveolar lavage (BAL) was perfomed. Viability and phagocytic activity of alveolar macrophages (AM), activity of lactate dehydrogenase (LDH) in cell-free BAL fluid (cf-BALF), acid phosphatase (ACP) and cathepsin D (CATD) in cfBALF, in BALF cells and in the lung tissue were estimated. Viability of AM was depressed by every type of exposure with RCF+S effect being at least additive. Phagocytic activity of AM increased in the presence of RCF. No significant changes in LDH activity were found. Activities of lysosomal enzymes measured in the lung tissue homogenates were not significantly changed, but those in the cfBALF increased especially after exposure to S with most expressive increase in BALF cells after exposure to S and RCF+S. In the case of CATD the effect of RCF+S was more than additive. The results point out to the persistence of the RCF exposure cytotoxic effects and their amplification by cigarette smoke.

Chromosomal Instability in Amniocytes From Fetuses of Mothers Who Smoke

Tobacco contains many mutagenic compounds, and tobacco-specific metabolites have recently been found in fetal blood and cell-free amniotic fluid. This study examined possible genotoxic effects of maternal smoking on amniotic fluid cells. Chromosomal instability in the form of chromosomal gaps and breaks was sought along with structural chromosomal abnormalities. Amniocytes were obtained by amniocentesis in a prospective series of 25 women who had smoked 10 or more cigarettes a day for at least 10 years and in 25 nonsmokers. Two independent observers assessed chromosomal stability in routine chromosome spreads in a blinded manner, and relevant break points were identified by G-banding. Chromosomal instability was found in 10.5% of smokers and 8.0% of control subjects, and chromosomal lesions in 15.7% of smokers and 10.1% of control subjects. Rates of structural chromosomal abnormalities were 12.1% and 3.5%, respectively. Deletions and translocations were the most common structural abnormalities in both groups. No significant age effect was noted. Analysis of all 689 break points showed that band 1 lq23, a band commonly implicated in hematopoietic malignancies, was the most affected chromosomal region. Increased chromosomal instability has been identified in amniocytes from women who smoke at least half a pack of cigarettes a day for 10 years or longer. Band 11q23, which is implicated in leukemogenesis, is apparently very sensitive to tobacco compounds. This band contains the genes ATM (cell prolymphocytic leukemia); PLZF (leukemia acute, promyelocytic; PLZF/RARA type); and MLL (leukemia, myeloid/lymphoid, or mixed lineage). Possibly, transplacental exposure to tobacco smoke is associated with an increased risk of hematopoietic malignancies in children.

Presence of Filterable and Nonfilterable Cell-Free mRNA in Amniotic Fluid

Much current research focuses on the properties and clinical applications of circulating nucleic acids (1). The recent discovery of cell-free RNA in the plasma and serum of cancer patients has generated considerable interest (2)(3)(4)(5)(6). Circulating RNA is surprisingly stable, and Ng et al. (7) recently showed that a considerable proportion of plasma mRNA species is particle associated and thus possibly protected from nuclease degradation (8). Fetal-derived mRNA has also been found in the plasma and serum of pregnant women (9)(10)(11) and in amniotic fluid (12), and has many potential clinical applications (13)(14). Amniotic fluid is routinely collected during amniocentesis for fetal chromosome analysis or fetal lung maturity studies. However, little is known regarding the biology of circulating fetal mRNA or fetal mRNA in amniotic fluid. In this report, we explore whether cell-free fetal nucleic acids in amniotic fluid have properties similar to circulating DNA and mRNA. Expanding on the work of Ng et al. (7), we hypothesized that cell-free fetal mRNA in amniotic fluid might be present in a particle-associated form and could thus be filterable, whereas the non-particle–associated form of DNA would be present in such high concentrations that there would be no significant reduction in its quantity by filtration. Additionally, we wished to compare the quantities of nucleic acids in amniotic fluid containing cells with the quantities in filtered and unfiltered cell-free supernatant. We hypothesized that whole amniotic fluid containing amniocytes would contain a significantly larger amount of DNA and RNA than cell-free amniotic fluid. This study was performed with Institutional Review Board approval from Tufts-New England Medical Center. Seven amniotic fluid samples with a minimum volume of 3 mL each were obtained from women undergoing scheduled amniocenteses. One sample originated from a woman …

Isolation of an N-alkylated Benzylamine Derivative from Pseudomonas putida BTP1 as Elicitor of Induced Systemic Resistance in Bean

Root treatment of Phaseolus vulgaris with the nonpathogenic Pseudomonas putida BTP1 led to significant reduction of the disease caused by the pathogen Botrytis cinerea on leaves. The molecular determinant of P. putida BTP1 mainly responsible for the induced systemic resistance (ISR) was isolated from cell-free culture fluid after growth of the strain in the iron-poor casamino acid medium. Mass spectrometry analyses performed on both the bacterial product and synthetic analogues revealed a polyalkylated benzylamine structure, with the quaternary ammonium substituted by methyl, ethyl, and C13 aliphatic groups responsible for the relative hydrophobicity of the molecule. The specific involvement of the N-alkylated benzylamine derivative (NABD) in ISR elicitation was first evidenced by testing the purified compound that mimicked the protective effect afforded by crude supernatant samples. The evidence was supported by the loss of elicitor activity of mutants impaired in NABD biosynthesis. Our experiments also showed that other iron-regulated metabolites secreted by the strain are not involved in ISR stimulation. Thus, these results indicate a wider variety of Pseudomonas determinants for ISR than reported to date.

Folate Cross-Feeding Supports Symbiotic Homoacetogenic Spirochetes

Treponema primitia, an H2-consuming CO2-reducing homoacetogenic spirochete in termite hindguts, requires an exogenous source of folate for growth. Tetrahydrofolate (THF) acts as a C1 carrier in CO2-reductive acetogenesis, a microbially mediated process important to the carbon and energy requirements of termites. To examine the hypothesis that other termite gut microbes probably supply some form of folate to T. primitia in situ, we used a bioassay to screen for and isolate folate-secreting bacteria from hindguts of Zootermopsis angusticollis, which is the host of T. primitia. Based on morphology, physiology, and 16S rRNA gene sequences, the major folate secretors were identified as strains of Lactococcus lactis and Serratia grimesii. During growth, these isolates secreted 5-formyl-THF at levels up to 146 ng/ml, and their cell-free culture fluids satisfied the folate requirement of T. primitia strains in vitro. Analysis of Z. angusticollis hindgut fluid revealed that 5-formyl-THF was the only detectable folate compound and occurred at an in situ concentration (1.3 mug/ml) which was more than sufficient to support the growth of T. primitia. These results imply that cross-feeding of 5-formyl-THF by other community members is important for growth of symbiotic hindgut spirochetes and thus termite nutrition and survival.

Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 1—Screening and characterization of the antibacterial compounds

A total of 87 lactic acid bacteria (LAB) (36 Lactobacillus sakei, 22 Enterococcus faecium, 16 Lactococcus garvieae, 11 Vagococcus carniphilus and 2 Enterococcus sp.) isolated from a small-scale facility producing traditional dry sausages were screened for antagonistic activity against other LAB and some spoilage and pathogenic microorganisms, also isolated from the same processing facility, except Listeria innocua (in lieu of Listeria monocytogenes) and Escherichia coli. The final goal was to investigate LAB antibacterial activity within the facility microbial ecosystem and to select interesting strains for the role of bio-preservatives. Twenty-one Ec. faecium, 6 Vc. carniphilus, 4 Lc. garvieae, 3 Lb. sakei and 2 Enterococcus sp. were shown to inhibit the growth of some indicator microorganisms in an agar well diffusion assay. Except 2 Lb. sakei and an Enterococcus sp., all these isolates exhibited antibacterial activity against L. innocua, but only 3 Ec. faecium, 5 Vc. carniphilus and 3 Lc. garvieae displayed also antagonistic activity against Staphylococcus aureus. The 5 Vc. carniphilus isolates were also found to be inhibitory for the Gram-negative bacterium Hafnia alvei. Isolates displaying antibacterial activity against L. innocua and/or Sc. aureus were investigated for the nature of antibacterial compounds synthesized against these indicator microorganisms. Bacteriocin-like production could be detected only on agar plated in overlay assays, and was unsuccessfully researched in cell-free culture supernatant fluids under conditions that eliminate acid and hydrogen peroxide inhibition. Results also showed that a Lb. sakei isolate displayed an additional inhibitory effect by H 2O 2 against L. innocua. These isolates will be investigated for their ability to repress the growth of undesirable bacteria in biofilms, i.e., the real mode of bacterial attachment. This is the first report on bacteriocin-like from Vc. carniphilus and on bacteriocin-like from Lc. garvieae active against both L. innocua and Sc. aureus species.

Defence function of pigmentocysts in the karyorelictid ciliate Loxodes striatus

The karyorelictid ciliate Loxodes striatus has pigment granules which are similar in size, structure and distribution to the pigmentocysts in the heterotrich ciliates, Blepharisma japonicum and Stentor coeruleus, which are known to be extrusomes for chemical defence against predators. We examined whether the pigment granules of L. striatus are also defensive organelles. We showed that: (1) pigment granules of L. striatus are extrusive organelles; (2) bleached cells of L. striatus produced by inducing a massive discharge of pigment granules are more vulnerable than normally pigmented cells to the raptorial ciliate Dileptus margaritifer and the turbellarian Stenostomum sphagnetorum, while they are indistinguishable from intact cells in external morphology and the capacity to grow; (3) the cell-free fluid (CFF) which contains the pigment discharged from pigment granules of L. striatus induced in D. margaritifer behavioural and pathological reactions which are essentially the same as those observed in the interaction with L. striatus, and this effect of the CFF disappeared when the pigment was bleached by light. We conclude that pigment granules of L. striatus are extrusomes for chemical defence against predators, and that the defence is based on the toxic pigment contained in these organelles.

Global Gene Expression Analysis of the Living Human Fetus Using Cell-Free Messenger RNA in Amniotic Fluid

No molecular biological tests are available to monitor the ongoing development of human fetuses in vivo. To determine whether cell-free fetal messenger RNA (mRNA) in amniotic fluid can be detected using oligonucleotide microarrays to study large-scale gene expression in living human fetuses, with analysis of sex, gestational age, and fetal pathology as variables. Four samples of cell-free amniotic fluid were analyzed from pregnant women between 20 and 32 weeks' gestation and undergoing amnioreduction for polyhydramnios associated with twin-twin transfusion syndrome or hydrops fetalis (cases). The control consisted of 6 pooled amniotic fluid samples from women at 17 weeks' gestation and undergoing genetic amniocentesis. After extraction from the normally discarded fraction of amniotic fluid, RNA was amplified twice, labeled, and analyzed using gene expression microarrays. Relative mRNA expression in cell-free samples of amniotic fluid from fetuses with polyhydramnios at different gestational ages vs cell-free amniotic fluid from a pooled control. Thirty-six percent of 22,283 probe sets represented on the arrays were present in the cell-free amniotic fluid, and a median of 20% of all probe sets differed between cases and the pooled control. Only male samples expressed 1 Y chromosome transcript. The expression of some developmental transcripts, such as surfactant proteins, mucins, and keratins, changed with gestational age by up to 64-fold. A water transporter gene transcript was increased up to 18-fold in both twin-twin transfusion samples. Placental gene transcripts were not present in any samples. This pilot study demonstrates that cell-free fetal mRNA can be extracted from amniotic fluid and successfully hybridized to gene expression microarrays. Preliminary analysis suggests that gene expression changes can be detected in fetuses of different sexes, gestational age, and disease status. Cell-free mRNA in amniotic fluid appears to originate from the fetus and not the placenta.

Black Tea Protects Thymocytes in Tumor-Bearing Animals by Differential Regulation of Intracellular ROS in Tumor Cells and Thymocytes

The accumulated in vitro evidence indicates that many tumors induce T-cell apoptosis as a mechanism of inhibiting antitumor activity. This downregulation of cell-mediated immune functions occurring at the late stages of the disease may be causally related to the thymic involution, because the thymus is the major site of T-cell maturation, extensive proliferation, and differentiation. Our results showed that in Erhlich's ascites carcinoma cell (EAC)-bearing mice, the number of EAC was inversely proportional to the thymocyte count in the host's thymus, which is the primary immune organ. Further studies indicated the presence of tumor-induced thymocyte apoptosis in EAC bearers. Black tea prolonged the survival of the tumor bearer by successfully restricting tumor progression as well as protecting the thymus from tumor insult. In fact, black tea inhibited thymic apoptosis while inducing programmed cell death of EAC. Interestingly, the tea regulated the oxidant status differentially in EAC and thymocytes--i.e., it reduced the EAC-induced reactive oxygen species (ROS) generation in the thymus while activating the same in the EAC. A similar effect of black tea was obtained when thymocytes were cultured in the presence of cell-free ascitic fluid, thereby indicating that black tea could directly reduce oxidative stress, an activity independent of its tumoricidal property. As a result, the maturation block in thymocyte subpopulations in tumor bearers was ameliorated significantly in black tea-treated animals. Our results demonstrate that black tea protects thymocytes in the tumor bearer by regulating the intracellular ROS in tumor cells and thymocytes differentially, thereby strengthening its candidacy in future anticancer therapeutic regimens.

Quantification of free RNA in serum and bronchial lavage: a new diagnostic tool in lung cancer detection?

Circulating cell-free nucleic acids have been detected in serum. In cancer patients levels of free DNA seem to be higher than in non-tumor controls and the detectable nucleic acids are partly of tumor origin. We asked whether free RNA can be detected as well in cell-free bronchial lavage fluid (BLF) supernatant, and whether quantification of free RNA allows to discriminate between tumor and non-tumor patients. 73 patients with lung cancer (NSCLC n=62, SCLC n=11) and 56 patients with non-malignant lung diseases were included. The RNA was isolated from 1 mL serum and lavage, respectively with the Quiamp MinElute Virus Vacuum Kit (Qiagen, Germany). A real-time quantitative RT-PCR assay was used for transcript quantification of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Intact RNA was detectable in cell-free supernatants of BLF from 126/129 patients and was investigated in all 64 serum samples. RNA levels were higher in the cell-free supernatant of BLF than in serum. RNA concentration in the BLF from tumor patients was higher than in patients with a benign lung disease (p=0.009). In conclusion, quantification of intact RNA isolated from BLF supernatant and from serum might become a valuable tool for differentiation between tumor and non-tumor patients.

Novel Type of Heme-Dependent Oxygenase Catalyzes Oxidative Cleavage of Rubber (Poly- cis -1,4-Isoprene)

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2--and--H2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40 degrees C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.

An Inexpensive Sedimentation Chamber for the Preparation of Cytologic Specimens of Cerebrospinal Fluid

An inexpensive sedimentation chamber to obtain cytologic specimens of cerebrospinal fluid (CSF) is described. The device, which has a total cost of about $5.00 can be built in few minutes. The device permits the cytologic study of specimens of CSF in clinics, where because of economic constraints, a cytocentrifuge is not available. The device permits the study of the CSF cells on either air-dried or wet smears even under field conditions, and the results obtained are consistent. Also, the device permits to retrieve the cell-free fluid for its use in chemical or immunologic procedures.

Pseudomonas aeruginosaprotease IV degrades surfactant proteins and inhibits surfactant host defense and biophysical functions

Pulmonary surfactant has two distinct functions within the lung: reduction of surface tension at the air-liquid interface and participation in innate host defense. Both functions are dependent on surfactant-associated proteins. Pseudomonas aeruginosa is primarily responsible for respiratory dysfunction and death in cystic fibrosis patients and is also a leading pathogen in nosocomial pneumonia. P. aeruginosa secretes a number of proteases that contribute to its virulence. We hypothesized that P. aeruginosa protease IV degrades surfactant proteins and results in a reduction in pulmonary surfactant host defense and biophysical functions. Protease IV was isolated from cultured supernatant of P. aeruginosa by gel chromatography. Incubation of cell-free bronchoalveolar lavage fluid with protease IV resulted in degradation of surfactant proteins (SP)-A, -D, and -B. SPs were degraded in a time- and dose-dependent fashion by protease IV, and degradation was inhibited by the trypsin-like serine protease inhibitor Nalpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK). Degradation by protease IV inhibited SP-A- and SP-D-mediated bacterial aggregation and uptake by macrophages. Surfactant treated with protease IV was unable to reduce surface tension as effectively as untreated surfactant, and this effect was inhibited by TLCK. We speculate that protease IV may be an important contributing factor to the development and propagation of acute lung injury associated with P. aeruginosa via loss of surfactant function within the lung.

Earthworm leukocytes kill HeLa, HEp-2, PC-12 and PA317 cells in vitro

Earthworm coelomic fluid contains biologically active molecules and leukocytes that participate in phagocytosis, encapsulation. Presumably they synthesize and secrete several effector modulators of innate immune responses such as antibacterial molecules, cytotoxic proteins and cytokines. Several lytic molecules have been detected in coelomic fluid previously but it is not yet clear which are actually released from the coelomocytes. Our aim was to analyze the cytotoxic effects of coelomocytes on mammalian target cells and to provide evidence that the lytic factors originate from coelomocytes. Cell-free coelomic fluid, supernatants of short-term cultured coelomocytes, and lysates from coelomocytes—derived by mechanical and detergent extraction—were used in cytotoxicity assays performed on different mammalian standard tumor cell lines and mouse fibroblasts. We used native and denaturized (using proteinase K, and trypsin digestions, or heat-inactivation) coelomocyte lysates (CCL). The viability controls of targeted cells were made by measuring photometrically and analyzing by inverted microscopy. According to our results the coelomic fluid, the supernatant of cultured coelomocytes, and the CCL significantly decreased ratios of living cells compared to controls in a dose-dependent manner. Our experiments performed with CCLs suggest that coelomocytes are responsible for the productions of cytotoxic components presumably proteins.

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge.

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Earthworm leukocytes kill HeLa, HEp-2, PC-12 and PA317 cells in vitro

Earthworm coelomic fluid contains biologically active molecules and leukocytes that participate in phagocytosis, encapsulation. Presumably they synthesize and secrete several effector modulators of innate immune responses such as antibacterial molecules, cytotoxic proteins and cytokines. Several lytic molecules have been detected in coelomic fluid previously but it is not yet clear which are actually released from the coelomocytes. Our aim was to analyze the cytotoxic effects of coelomocytes on mammalian target cells and to provide evidence that the lytic factors originate from coelomocytes. Cell-free coelomic fluid, supernatants of short-term cultured coelomocytes, and lysates from coelomocytes—derived by mechanical and detergent extraction—were used in cytotoxicity assays performed on different mammalian standard tumor cell lines and mouse fibroblasts. We used native and denaturized (using proteinase K, and trypsin digestions, or heat-inactivation) coelomocyte lysates (CCL). The viability controls of targeted cells were made by measuring photometrically and analyzing by inverted microscopy. According to our results the coelomic fluid, the supernatant of cultured coelomocytes, and the CCL significantly decreased ratios of living cells compared to controls in a dose-dependent manner. Our experiments performed with CCLs suggest that coelomocytes are responsible for the productions of cytotoxic components presumably proteins.