Lysozyme Production Research Articles

Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors.

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or alpha- or beta-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.

Salivary Concentration of Secretory Leukocyte Protease Inhibitor, an Antimicrobial Protein, Is Decreased with Advanced Age

Background: Secretory leukocyte protease inhibitor (SLPI) exhibits antimicrobial activities that, in addition to other well-characterized proteins such as lysozyme and lactoferrin, is thought to play a critical role in mucosal defenses. Although elderly individuals are particularly susceptible to mucosal infections, salivary production of SLPI has not been assessed in an aged cohort. Objectives: Hypothesizing that oral SLPI concentrations are reduced with advanced age, this cross-sectional study compared SLPI concentrations to concentrations of lysozyme, lactoferrin and total protein in unstimulated salivary secretions of healthy, community-dwelling 79+-year-old and younger adults. Methods: Study participants were 45 non-hospitalized dentate adults aged 79–89 (23 elderly) or 21–51 years (22 non-elderly). Home-based interviews and clinical examinations determined dentate status and confirmed the absence of dentures, oral mucosal disease, anti-infective medication use, irradiation therapy for head and neck cancer and self-perceived xerostomia. Whole unstimulated saliva was collected from all subjects and analyzed for antimicrobial protein concentration by enzyme-linked immunosorbent assay and for total protein content by the bicinchoninic acid method. Bivariate and multivariate (generalized linear modeling) analyses evaluated the relationships between age, gender and salivary protein concentrations. Results: Mean salivary levels of SLPI and lysozyme were lower in elderly compared with non-elderly subjects (p < 0.001), unlike lactoferrin and total protein levels. Similar results were obtained when concentrations of the individual proteins were normalized to the total protein concentration, suggesting that glandular production of SLPI and lysozyme preferentially decreases with aging. Gender differences were detected only for SLPI concentrations; males had lower SLPI levels than females regardless of age (p < 0.01). Generalized linear models confirmed that age (p < 0.001) and gender (p < 0.05) were each associated with the SLPI concentration and together accounted for 50% of the variation in SLPI concentration in this population. Conclusion: These findings indicate that SLPI production is diminished among healthy community-dwelling older adults, particularly elderly males. Further investigation should determine the impact of decreased local SLPI production on the increased risk of oral mucosal disease with advanced age.

Enhanced lysozyme production in Atlantic salmon (Salmo salar L.) macrophages treated with yeast β-glucan and bacterial lipopolysaccharide

Atlantic salmon head kidney macrophages grown in the presence of particulate yeast β-glucan and bacterial lipopolysaccharide (LPS) showed increased production of lysozyme in the culture supernatants compared to non-treated controls. The increased lysozyme production started at day 3 and was five- to six-fold higher compared to controls at day 6 in culture. β-Glucan showed an approximate linear dose–response curve between 1 and 250μg ml−1whereas LPS showed a dose–response curve with a well-defined optimum concentration (10μg ml−1). The increase in lysozyme activity was accompanied by an accumulation of lysozyme gene transcript in the stimulated cells. Recombinant human tumor necrosis factor alpha, known for its ability to stimulate lysozyme in human macrophages and to elevate respiratory burst activity of rainbow trout macrophages, failed to stimulate lysozyme production of Atlantic salmon macrophages. Macrophages isolated from fish suffering from a non-lethal Ichthyobodo necator infection displayed a highly increased ability to produce lysozyme in response to both β-glucan and LPS. As in higher vertebrates, lysozyme production may reflect the differentiation stage of the Atlantic salmon macrophages as well as a direct activation of lysozyme gene transcription by biological response modifiers. The rather late increase in lysozyme production induced by β-glucan and LPS may thus be explained by stimulation of differentiation of the macrophages in culture eventually combined with direct activation of transcription of the lysozyme gene.

L-Glutamate production by lysozyme-sensitive Corynebacterium glutamicum ltsA mutant strains

BackgroundA non-pathogenic species of coryneform bacteria, Corynebacterium glutamicum, was originally isolated as an L-glutamate producing bacterium and is now used for fermentative production of various amino acids. A mutation in the C. glutamicum ltsA gene caused susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production.ResultsThe characteristics of eight lysozyme-sensitive mutants which had been isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis were examined. Complementation analysis with the cloned wild-type ltsA gene and DNA sequencing of the ItsA region revealed that four mutants had a mutation in the ltsA gene. Among them, two mutants showed temperature-sensitive growth and overproduced L-glutamate at higher temperatures, as well as the previously reported ltsA mutant. Other two showed temperature-resistant growth: one missense mutant produced L-glutamate to some extent but the other nonsense mutant did not. These two mutants remained temperature-resistant in spite of introduction of ltsA::kan mutation that causes temperature-sensitive growth in the wild-type background.ConclusionsThese results indicate that a defect caused by the ltsA mutations is responsible for temperature-sensitive growth and L-glutamate overproduction by C. glutamicum. The two temperature-resistant mutants seem to carry suppressor mutations that rendered cells temperature-resistance and abolished L-glutamate overproduction.

Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice.

To assess the role of lysozyme in pulmonary host defense in vivo, transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated. Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar (BAL) lavage fluid was increased 2- or 4-fold relative to that in WT mice. Lung structure and cellular composition of BAL were not altered by the expression of lysozyme. Lysozyme activity in BAL was significantly increased (6.6- and 17-fold) in 5-wk-old animals from each transgenic line. To determine whether killing of bacteria was enhanced by expression of rat lysozyme, 5-wk-old transgenic mice and WT littermates were infected with 10(6) CFU of group B streptococci or 10(7) CFU of a mucoid strain of Pseudomonas aeruginosa by intratracheal injection. Killing of group B streptococci was significantly enhanced (2- and 3-fold) in the mouse transgenic lines at 6 h postinfection and was accompanied by a decrease in systemic dissemination of pathogen. Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines (5- and 30-fold). Twenty-four hours after administration of Pseudomonas aeruginosa, all transgenic mice survived, whereas 20% of the WT mice died. Increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo, and was associated with decreased systemic dissemination of pathogen and increased survival following infection.

A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production.

The Corynebacterium glutamicum mutant KY9714, originally isolated as a lysozyme-sensitive mutant, does not grow at 37 degrees C. Complementation tests and DNA sequencing analysis revealed that a mutation in a single gene of 1,920 bp, ltsA (lysozyme and temperature sensitive), was responsible for its lysozyme sensitivity and temperature sensitivity. The ltsA gene encodes a protein homologous to the glutamine-dependent asparagine synthetases of various organisms, but it could not rescue the asparagine auxotrophy of an Escherichia coli asnA asnB double mutant. Replacement of the N-terminal Cys residue (which is conserved in glutamine-dependent amidotransferases and is essential for enzyme activity) by an Ala residue resulted in the loss of complementation in C. glutamicum. The mutant ltsA gene has an amber mutation, and the disruption of the ltsA gene caused lysozyme and temperature sensitivity similar to that in the KY9714 mutant. L-Glutamate production was induced by elevating growth temperature in the disruptant. These results indicate that the ltsA gene encodes a novel glutamine-dependent amidotransferase that is involved in the mechanisms of formation of rigid cell wall structure and in the L-glutamate production of C. glutamicum.

Effect of pH on hen egg white lysozyme production and evolution of a recombinant strain of Aspergillusniger

An Aspergillus niger strain (B1) transformed to produce mature hen egg white lysozyme (HEWL) from a glucoamylase fusion protein under control of the A. niger glucoamylase promoter was grown in glucose-limited chemostat culture at a dilution rate of 0.07 h −1 at various pH values. Maximum HEWL production (9.3 mg g −1; specific production rate=0.65 mg g −1 per h) was obtained at pH 4.5. However, in chemostat culture, HEWL production was not stable at any pH tested. After 240 h in steady state, specific production decreased to only 0.03±0.01 and 0.24±0.02 mg g −1 per h at pH 6.5 and 4.5, respectively. Some isolates removed from the chemostat cultures had lost copies of the HEWL gene and when grown in shake flask cultures all of the isolates produced less HEWL than the parental strain. Morphological mutants with similar phenotypes were isolated at all pHs, but their rate of increase in the population was pH dependent, with cultures at low pH (<4.5) being more morphologically stable than cultures at high (>4.5) pH. The selective advantage of these mutants was also generally dependent on pH. Both yellow pigment producing mutants and brown sporulation mutants had higher selective advantages over the parental strain at high than at low pH, regardless of the pH at which they were isolated. However, the selective advantage of densely sporulating mutants was independent of pH.

High-level production of heterologous protein by engineered yeasts grown in cottage cheese whey.

Cottage cheese whey is a cheese industry by-product still rich in proteins and lactose. Its recycling is seldom cost-effective. In this work we show that the lactose-utilizing yeast Kluyveromyces lactis, engineered for production of recombinant human lysozyme, can be grown in cottage cheese whey, resulting in high-level production of the heterologous protein (125 microg/ml).

2-Deoxy- d-Glucose–Induced Metabolic Stress Enhances Resistance to Listeria monocytogenes Infection in Mice

Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy- d-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF 1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG–induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.

Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in chicken phagocytes

Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease, and is in development for use in poultry mycoplasma air sacculitis. In order to provide an understanding of clinical efficacy, the in vitro interaction of tilmicosin with three types of chicken phagocytes (MQ-NCSU macrophages, monocyte-macrophages, and heterophils) was evaluated. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the cellular (Cc) to the extracellular (Ce) drug concentration (Cc:Ce). Tilmicosin was avidly accumulated by heterophils (Cc: Ce 138 at 4 h incubation vs 32 and 66, respectively, in MQ-NCSU and monocyte-macrophages) with 61 to 88% localized in the lysosomes. Uptake was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors. However, phagocytosis of Pasteurella multocida and lipopolysaccharide exposure increased tilmicosin uptake by the chicken phagocytes. Upon removal of extracellular tilmicosin, 50% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in antibiotic-free medium, 30% remained cell-associated. Opsonized P. multocida significantly enhanced the release of tilmicosin from all three types of chicken phagocytes. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme, avidin, and beta-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro observations of cellular pharmacology suggest a complex interaction between phagocytes and tilmicosin that contribute to clinical efficacy.

Intracellular accumulation, subcellular distribution and efflux of tilmicosin in swine phagocytes.

Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease. As the concentrations of tilmicosin are generally low in swine lung tissue, the interaction of tilmicosin with three types of swine phagocytes (monocyte-macrophages, alveolar macrophages, and neutrophils) was evaluated to provide an understanding of clinical efficacy. After incubation with radiolabelled tilmicosin, uptake was determined and expressed as the ratio of the intracellular (Ci) to the extracellular (Ce) drug concentration (Ci/Ce). Tilmicosin was avidly accumulated by the swine phagocytes (Ci/Ce 48-69 at 4 h incubation) with 51 to 85% localized in the lysosomes. Uptake was dependent on cell viability, temperature and pH, but was not influenced by the metabolic inhibitors, sodium cyanide or potassium fluoride. However, lipopolysaccharide (LPS) exposure increased tilmicosin uptake by the swine phagocytes. In neutrophils, upon removal of extracellular tilmicosin, 60% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in drug-free medium, 25% remained cell-associated. In contrast, after 4 h of incubation in drug-free medium, 60% and 45% of tilmicosin remained cell-associated, within alveolar macrophages and monocyte-derived macrophages, respectively. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme and beta-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro interactions of tilmicosin with swine phagocytes suggest an integral role in effecting clinical efficacy.

Ultrastructure of the binary parotid glands in the free-tailed bat, Tadarida thersites. I. Principal parotid gland.

Many species of bats have two sets of submandibular glands, principal and accessory. The accessory gland may resemble the principal one but more often shows wide morphological divergence. The free-tailed bat, Tadarida thersites, is very unusual in that it has two sets of parotid glands rather than binary submandibular glands. We studied the ultrastructure of the principal parotid gland to establish a baseline for comparison with the accessory parotid. Two specimens of adult free-tailed bats, one male and one female, were live-trapped in western Kenya. Parotid glands were fixed for electron microscopy using a protocol expressly designed for field fixation and then embedded by conventional means. Histologically, the principal parotid is a typical serous gland. The secretory granules of the endpiece cells have an unusual substructure in that they contain variable numbers of lucent halos and one or several spherules. Intercalated duct cells contain a significant number of dense, serous-like granules. Striated ducts have the usual basal configuration of mitochondria and folded plasma membranes, but the supranuclear cytoplasm contains many small, dense granules, so that these ducts resemble the granular convoluted tubules found in the submandibular glands of many families of rodents. The apices of the duct cells have a peculiar contour--the luminal surfaces obliquely invaginate into the apical cytoplasm, so that in thin section the luminal membranes appear to be underlaid by a layer of vacuoles. Although the principal parotid gland of the free-tailed bat shows some distinctive, species-specific ultrastructural features, it basically is similar to the parotid gland in two other molossid bats, Tadarida brasiliensis and Molossus molossus. The distinctive features in the principal parotid gland of T. thersites might relate to its feeding on hard-bodied insects and perhaps to the production of lysozyme.

Electrosynthetic immobilisation of proteins for bioanalysis

Use of electrosynthetic methodology allows the production of hen egg-white lysozyme (HEWL) either mononitrated at tyrosine 23 or bisnitrated at tyrosines 20 and 23, but never nitrated at tyrosine 53. This is a different sequence from that obtained by the chemical nitrating agent tetranitromethane, and when reduced by dithionite, the selectively modified enzyme can be anchored at pH 5 via the unique aromatic amino group to magnetic beads or other suitable matrices. HEWL so immobilised loses less than 10% of cell-wall lytic activity compared with the approximately 50% loss of activity when immobilised by conventional methodology at pH 9 via essentially random reaction at lysine residues and other functionalities which are nucleophilic at this pH. This result offers promise as a general method for selective protein immobilisation in biosensors and similar applications. © 1998 John Wiley & Sons, Ltd.

Up-regulation of lysozyme production in colonic adenomas and adenocarcinomas.

The presence of lysozyme protein in some gastric adenomas and adenocarcinomas has been well documented. There have been relatively few studies investigating the presence of lysozyme in tumours of the large intestine and they show contrasting results. We aim to investigate the cellular source and expression of lysozyme in colonic adenomas and adenocarcinomas. We randomly selected 29 and 27 colonic adenomas and adenocarcinomas, respectively. Using in-situ hybridization (ISH) and immunohistochemistry (IHC), we found an up-regulation of lysozyme in the dysplastic epithelium of all the adenomas studied, with more than 80% of cases expressing moderate to strong signals. Although the up-regulation of lysozyme was also observed in adenocarcinomas, only 30% of the cases showed moderate to strong signals, mostly with an uneven distribution. Down-regulation of lysozyme in the severely dysplastic and invasive foci were noted in some cases of adenoma with malignant transformation. Normal colonic glands were consistently negative for lysozyme at both the mRNA and the protein level, but inflamed and immature regenerative colonic epithelium at the crypt base showed positive signals in a similar pattern to those observed in the dysplastic epithelium of the adenomas. Our results confirm that colonic epithelium can produce lysozyme and its expression is up-regulated in the dysplastic epithelium in adenomas and in invasive cancer cells. It is interesting that regenerative colonic epithelium showed a similar pattern of lysozyme expression as in adenomas. The loss of lysozyme secreting phenotype in most of the invasive tumours suggests that lysozyme may not confer an advantage to tumour progression.

Production and Purification of an Active Bovine Lysozyme in Tobacco (Nicotiana tabacum): Utilization of Value-Added Crop Plants Traditionally Grown under Intensive Agriculture

The goals of this study were to express bovine lysozyme in tobacco and to purify the protein with a scaleable process to >90% homogeneity while retaining antimicrobial characteristics. Results showed that the enzyme was expressed at levels equivalent to 1−1.5% of total fraction 2 protein in each of five different transformant groups. The enzyme was subsequently purified to 93% homogeneity using an easily scaleable process while retaining high activity. It was concluded that tobacco was an excellent choice for expression of the recombinant protein and that the purification process developed in this study demonstrates methodology for isolation of high-value enzymes from tobacco and other crop plants. Keywords: Transgenic; lysozyme; recombinant protein; antimicrobial; Nicotiana tabacum

The Catalytic Domain of Protein Kinase C-δ in Reciprocal δ and ∊ Chimeras Mediates Phorbol Ester-induced Macrophage Differentiation of Mouse Promyelocytes

The overexpression of protein kinase C-delta (PKC-delta), but not PKC-epsilon, enables the mouse myeloid cell line 32D to differentiate into macrophages when treated with phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine the domain of PKC-delta that is responsible for this isotype-specific function, cDNAs that encode reciprocal chimeras of PKC-delta and -epsilon (PKC-delta epsilon and PKC-epsilon delta) were constructed by exchanging regulatory and kinase domains using polymerase chain reaction technology. Both chimeras were stably expressed in 32D cells using the pLTR expression vector and displayed protein kinase activity upon TPA treatment. TPA treatment of L epsilon delta, cells that overexpressed the PKC-epsilon delta chimera, induced a dramatically increased cell volume, surface adherence, surface expression of Mac-1 and Mac-3, lysozyme production, and phagocytosis. These are the characteristics of the macrophage phenotype found in TPA-treated 32D cells that overexpressed PKC-delta. In contrast, little effect was seen in L delta epsilon, 32D cells that overexpressed PKC-delta epsilon, with or without TPA treatment. A PKC inhibitor directed toward the catalytic domain of PKC, GF109203X, and a selective inhibitor of PKC-delta, Rottlerin, blocked the TPA-induced differentiation of PKC-epsilon delta-overexpressing 32D cells. These results demonstrate that the catalytic domain of PKC-delta contains the primary determinants for its activity in phorbol ester-induced macrophage differentiation.

Bacteriocidal qualities of ixodid tick (Acarina: Ixodidae) salivary cement plugs and their changes under the influence of a viral tick-borne pathogen.

The abundance of bacteriocidal compounds contained in the salivary cement plug of ixodid ticks was changed because of the reproduction of tick-borne encephalitis virus (TBEV) in their bodies. The size of TBEV-infected Ixodes persulcatus Shulze lytic zone surrounding the cement plug enlarged to that of naive ticks, whereas Micrococcus lysodeikticus (Cohn) lytic zones induced by the cement plugs of TBEV-infected Amblyomma hebraeum Koch nymphs or Rhipicephalus appendiculatus Neumann females were reduced, compared with those produced by noninfected specimens. It is possible that an increase of lysozyme production by the primary TBEV vectors (Ixodinae) infected salivary gland cells, compared with suppression of the bacteriocidal qualities of saliva of ticks that are not TBEV vectors in nature (Amblyomminae), is an indication of the specificity of a I. persulcatus-TBEV interface.

Influence of soluble suture factors on in vitro macrophage function

Suture materials may interact with immune competent cells and thereby affect localized immunity. Macrophages are central to the inflammatory response and coordinate wound healing. They are also involved in the clearance of foreign material, bacteria and malignant cells. We studied the influence of soluble factors associated with silk, steel, nylon, polyglactin, polydioxanone and chromic catgut sutures on macrophage adherence, phagocytosis and the production of lysozyme and tumour necrosis factor. Soluble factors from suture materials influenced macrophage behaviour in vitro causing cellular activation, functional impairment and alterations in secreted levels of the cytokine tumour necrosis factor and the bactericidal agent lysozyme. Of the six materials studied, polyglactin had the most extreme effect, causing significant inhibition of cell adherence and lysozyme production. Silk also exerted a considerable effect on macrophages, significantly inhibiting adherence. In contrast, steel and polydioxanone media caused minimal inhibition of macrophage function although, as with all materials, they did activate the cells. This study has demonstrated that sutures release immunotoxic factors which considerably influence macrophage behaviour in vitro. These effects may have important clinical implications.

Long-term lipid-based total parenteral nutrition activates mononuclear cells and modulates membrane lipid composition in pigs.

In previous studies we have found lung granulomas in pigs on long-term soybean-based total parenteral nutrition (TPN). In the present study we have investigated activities and membrane lipids of cells known to participate in a granulomatous process. Pigs were given TPN with soybean emulsion for 7 weeks, controls were given saline intravenously and consumed a similar caloric load in a standard oral diet. Spontaneous nitroblue tetrazolium (NBT) reduction increased 204% in peripheral blood mononuclear cells (MNC) from TPN animals compared to controls (p < 0.05), and 161% (p = 0.05) in alveolar macrophages. The spontaneous lymphocyte mitogen response (LMR) rate in MNC increased 299% (p < 0.05). Endotoxin-stimulated procoagulant activity in MNC tended to increase in TPN animals. Acid phosphatase and lysozyme production in alveolar macrophages were not significantly changed. The serum neopterin level at the end of the observation was 7.0 nmol l-1 in TPN animals compared to 3.9 nmol l-1 in controls (p < 0.01), while interleukin-1 and tumour necrosis factor were not detectable. These findings indicated in vivo activation of peripheral blood monocytes, lymphocytes and alveolar macrophages in pigs on TPN. The membrane lipid contents of linoleic acid (18:2, n-6) increased, while arachidonic acid (20:4, n-6) tended to decrease both in MNC and lung tissue. Thus, linoleic acid conversion did not lead to accumulation of arachidonate in the cell membranes. Direct effects on monocytes, lymphocytes and alveolar macrophages of TPN particles, and altered cell membrane function, due to linoleic acid enrichment, are tentative explanations for the raised cell responses. Such cell activation may have contributed to the formation of granulomas.

Phenotype, genotype and cytokine production in acute leukemia involving progenitors of dendritic Langerhans' cells

To date no hematopoietic progenitors of dendritic Langerhans' cells (DLC), which represent an highly efficient class of antigen presenting cells, have been identified or the cytokines they elaborate have been defined. Here we describe an acute leukemia patient whose blasts (90–96% in peripheral blood and bone marrow) had a phenotype consistent with putative progenitors of DLC. The patient was treated with ara-C and VP-16 but did not achieve remission. The blasts had lobulated nuclei, no cytoplasmic vacuolation or Auer rods and were weakly positive for acid phosphatase and non-specific esterase and negative for PAS, granzyme A, dipeptidyl aminopeptidase IV, ATPase/ADPase and lysozyme production. The blasts were positive for CD1a, CD4, CD16, CD35, HLADR, HLADQ, CD11b, CD11c, CD14, CD33, CD34, CD11a, CD71, CD19, CD25, IL-2Rβ and negative for CD2, CD7, CD8, CD10, CD22, CD56, CD57, surface or cytoplasmic CD3, TCRδ and TCRβ, HTLV-1p19 and P-glycoprotein. On liquid culture with or without 5 × 10 −9M 12- O-tetradecanoylphorbol-13-acetate (TPA) for 3 days, the blasts formed aggregates of proliferating and elongating cells on the wall of the flasks with a decline in CD34, numerous dendritic processes appeared on the cells and there was strong positivity for ATPase/ADPase, but no other changes in phenotype. No macrophages were observed, indicating derivation from separate DLCs. Cytogenetic analysis showed chromosomal abnormalities and electron microscopy showed Birbeck granules. Southern blotting of DNA showed rearangement of one allele for both JH and TCRβ but not HTLV-1 related sequences. Culture supernatants from blasts cultured with or without TPA showed the production of large amounts of IL-8, IL-6, TNF-α, MIP-1α, IL-10 and interferon γ and modest amounts of IL-1α, GM-CSF and stem cell factor. The presence not only of CD1a, HLADR, HLADQ and many other characteristics including Birbeck granules, but also differentiation along the lines of DLC with appearance of dendritic processes on the cells and expression of ATPase/ADPase activity, indicate that the leukemic blasts in our patient represented at leukemic counterpart of normal progenitors of DLC and the leukemia a new entity which could possibly be classified as AML-M8. Lastly, many pro-inflammatory cytokines produced by DLC could contribute to inflammation and IL-10 to immunosuppression.