Morphological and developmental studies of the snake trypanosome Trypanosoma hydrae Ayala, Atkinson, Vakalis, 1983 in experimentally infected hosts and in culture

Trypanosoma hydrae from the broad-banded watersnake, Nerodia fasciata confluens, underwent development in the freshwater leech, Placobdella parasitica. Epimastigotes and transitional stages were present only in the crop and gastric caecum. Only one metacyclic form was observed. The potential of leeches as vectors is discussed. Two broad-banded watersnakes were infected by inoculation with culture forms of T. hydrae maintained on NNN medium. Parasitemias varied from 10(5)/ml to 10(6)/ml with dividing forms seen only rarely. A broad and a slender form of T. hydrae are described from the peripheral blood of a watersnake 7 months after an experimental infection.     

Trypanosoma acomys (Wenyon, 1909): Continuous Culturing with a Mouse Fibroblast Cell-Line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37° C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 × 10⁴, 1.5 × 10⁵ and 7.4 × 10⁵ parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 × 10⁶ parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

New Culture Medium for Maintenance of Tsetse Tissues and Growth of Trypanosomatids*

SYNOPSIS. A new culture medium (SM), based on the amino-acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre-emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ～ 10⁶ procyclics/ml yielded 4–5 × 10⁷ organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ～ 2 × 10⁷ after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ～ 4.5 × 10⁷ cells/ml. Promastigotes of Leishmania donovani, cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ～ 4 × 10⁷ cells/ml.     

Cultivation of Trypanosoma theileri at 37 C in Partially Defined Media*

SYNOPSIS. Trypanosoma theileri has become the 1st mammalian trypanosome to be serially cultivated in a blood- and cell-free medium at 37 C. This was accomplished by progressive simplification of an initial culture medium containing whole bovine blood. Fifty-one subcultures in 4- to 5-ml quantities of monophasic liquid media were made over a period of 1 year. The final medium contained hemin and a defined modification of McCoy 5a tissueculture medium, supplemented with a peptone solution. Hemin was essential and T. theileri grew best with ～1 mg/100 ml. Counts reached 8 × 10⁶ organisms/ml in 3–4 days. Seventy-ml volumes of medium supported growth of T. theileri to 3-4 × 10⁶ organisms/ml. Early in the study trypomastigotes were the predominant nondividing form, but they were absent when the medium was being rapidly modified. Trypomastigotes reappeared when the medium was stabilized at the end of the study. Elimination of serum antigens from the medium should greatly facilitate antigenic analysis of T. theileri.     

Assortative mating in mixed swarms of the mosquito Anopheles gambiae s.s. M and S molecular forms,in Burkina Faso,West Africa

The molecular form composition of Anopheles gambiae Giles s.s. (Diptera: Culicidae) mating swarms and the associated mating pairs (copulae) were investigated during two rainy seasons (July to October, 2005 and July to November, 2006) in the villages of Soumousso and Vallée du Kou (VK7). Although the habitats of these villages differ markedly, sympatric populations of M and S molecular forms of An. gambiae s.s. occur in both places periodically. The main aim was to assess the degree to which these molecular forms mate assortatively. In Soumousso, a wooded savannah habitat, the majority of swarm samples consisted of only S‐form males (21/28), although a few M‐form males were found in mixed M‐ and S‐form swarms. In VK7, a rice growing area, the majority of swarm samples consisted of only M‐form males (38/62), until October and November 2006, when there were nearly as many mixed‐form as single‐form swarms. Overall, ～60% of M‐ and S‐form swarms were temporally or spatially segregated; the two forms were effectively prevented from encountering each other. Of the remaining 40% of swarms, however, only about half were single‐form and the rest were mixed‐form. Of the 33 copulae collected from mixed‐form swarms, only four were mixed‐form pairs, significantly fewer than expected by random pairing between forms (χ² = 10.34, d.f. = 2, P < 0.01). Finally, all specimens of inseminated females were of the same form as the sperm contained within their spermatheca (n = 91), even for the four mixed‐form copulae. These findings indicate that assortative mating occurs within mixed‐form swarms, mediated most probably by close‐range mate recognition cues.     

Comparative study of effects of cadmium cations in free and chelated forms on activity of glutathione S-transferase, growth, and endocytosis in culture of the infusoriumTetrahymena pyriformis

Effects of cadmium cations in free (Cd²⁺) and chelated with EDTA (Cd²⁺-EDTA) forms were studied on growth, endocytosis, and activity of glutathione S-transferase (GT) in the free-living infusoriaTetrahymena pyriformis. It is shown that the cytotoxicity of Cd²⁺ in the free form at a concentration of 10 μM is much higher than of the Cd²⁺-EDTA complex at the equimolar concentration. Even at a low concentration (2 μM), Cd²⁺ produces an inhibition of the growth rate and endocytosis in theT. pyriformis culture, while the Cd²⁺-EDTA complex suppresses these functions insignificantly. Cd²⁺ in the free form at concentrations of 10 and 100 μM reduced activity of glutathione S-transferase by 39 and 61%. The chelated Cd²⁺-EDTA complex at these concentrations inhibited the GT activity by 5 and 55%, respectively.     

Control of green mould of citrus by using Trichoderma harzianum,humic acid or garlic

Penicillium digitatum, an aggressive fungus causes post-harvest decay of mandarin sweet orange and Washington navel. In vitro Trichoderma harzianum or humic acid (HA) or powdered cloves of garlic caused inhibition of fungal growth of isolates P₁ and P₂. Under storage conditions, the fruit citrus is protected by using T. harzianum with standard volume 2.0?ml (9.6?×?10⁶?conidia/ml) and application 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum spore suspension (1.0?×?10⁶?spores/ml) compared to control. Spraying the fruit citrus by standard volume of 2.0?ml of either HA or powder cloves of garlic 1% on each fruit 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum (1.0?×?10⁶ spores/ml) compared to control. The lowest percentage of disease incidence and disease severity were associated with powder of cloves garlic and followed by HA and T. harzianum during two growing seasons compared with the untreated and control.     

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation,Fluorescent Antibody Staining,and Polymerase Chain Reaction

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 10⁴ to 10⁶ cell/100 ml in all 14 samples, while colony counting method detected 10 to 10³ CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.     

Brown root rot of tomato, and its control, in Crete

Symptoms similar to those regarded as typical of brown root rot disease have been noticed in tomato and eggplant in Crete. Infected plants showed symptoms only during the winter. Three fungi Pyrenochaeta lycopersici, Colletotrichum coccodes and Fusarium oxysporum were consistently isolated from infected tomato roots and proved to be pathogenic to young tomato seedlings. Pycnidia of P. lycopersici were produced when small pieces of infected young tomato roots were plated out on tomato juice agar (Oxoid) and incubated at 23 °C under continuous white light. Good control of the disease was achieved when soil was fumigated with dazomet 98% (70g/m²), methyl isothiocyanate (MIC) 20%+ dichloropropane-dichloropropene mixture (DD) 80% (-Di-Trapex; 50 ml/m²), methyl bromide (75 g/m²), and vapam (200 ml/m²) + ethoprophos 10% (10 g/m²) or when solarisation was applied (soil covered by 50 μm transparent polyethylene sheet) during the whole period of August and September. Dazomet (30 g/m²) or ethoprophos 10% (10 g/m²) when combined with solarisation did not improve its effectiveness. MIC 20%+ DD 80% emulsified (Di-Trapex e.c, 50 ml/m²) was ineffective.     

Optimization of formation and regeneration of protoplasts from biocontrol agents ofTrichoderma species

The optimal conditions necessary for a large yield and a high frequency of regeneration of protoplasts isolated from the biocontrol agentsTrichoderma koningii andT. harzianum were investigated. Protoplast yields were 1.2×10⁸/ml fromT. koningii and 6×10⁷/ml fromT. harzianum when 20-h mycelial culture was treated with a lytic enzyme solution containing Novozym 234 (15 mg/ml), sucrose (0.6 M) and citrate phosphate buffer (0.02 M), pH 5.6 at 31°C. When the protoplasts were grown in the regeneration medium containing yeast extract (1.5%), 1 I of Mandel's salts, pH 5.6, and glucose (0.8 M), a high frequency of regeneration of the protoplast was obseved: 66% forT. koningii and 45% forT. harzianum. Two patterns of regeneration were observed. First, the hyphae arose directly from the regenerated protoplast mother cell. Second, a chain of bud cells developed from the protoplast and subsequently generating hyphae generally protruded from the terminal bud cells.     

Effects of DNA Topology on Transformation Efficiency of Bacillus subtilis ISW1214 by Electroporation

We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonuclease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6±0.6) × 10⁵ transformants per μg DNA higher than that ((2.0±0.3) × 10⁴ transformants per μg DNA) for the cr DNA, using the DNA concentration of 20 μg/ml at an electric field strength of 7kV/cm and a capacitance of 10 μF with a single decayed pulse. The transformation efficiency (TE) for the In DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

Agglutination of Trypanosoma cruzi by Concanavalin A*

Epimastigotes of Trypanosoma cruzi obtained in culture agglutinate readily with low concentrations of concanavalin A (Con A). Agglutination was linear with time up to 10 min providing that the initial cell density was greater than 1 × 10⁸ cells/ml. Under these conditions, the percentage agglutination was dependent on the Con A concentration. Agglutination was inhibited by α-methyl D-mannoside, α-D-mannose, and α-D-glucose. Pretreatment of cells with trypsin had no effect on the epimastigote agglutinations. Blood forms (trypomastigotes) of T. cruzi did not agglutinate even in the presence of 100 times more Con A. Results suggest differences in membrane structure between blood forms and cultured epimastigotes of T. cruzi. These membrane differences might be related to the different pathogenic properties of both cell forms of T. cruzi.     

Trypanosoma vivax: Adaptation of Two East African Stocks to Laboratory Rodents1

Two Trypanosoma vivax stocks from East Africa have been adapted to rats and mice. Adaptation was induced by rapid passage at two- to four-day intervals in sublethally irradiated rats. After 200 such passages, the two stocks gave rise to parasitemias of 10⁹–10¹⁰ trypanosomes/ml in peripheral blood, and the infection was fatal in 90% of the rats. By passaging the rat-adapted T. vivax into normal mice at two- to three-day intervals for over 200 passages, the two stocks also became pathogenic to mice. One of the stocks was also capable of maintenance in non-irradiated rats. The two stocks displayed a marked degree of pleomorphism in irradiated and non-irradiated rats and mice. In the early rising parasitemia, the organisms were predominantly short, with a well formed undulating membrane, a pointed posterior end, and a large terminal kinetoplast. As parasitemia approached its peak, the organisms transformed into long, slender forms with an inconspicuous undulating membrane, an elongated posterior end, and a sub-terminal kinetoplast. The short forms associated with the early, rising parasitemia were more infective for mice than the long forms encountered at peak parasitemia. Although the two rodent-adapted stocks retained their pathogenicity for goats, neither the original stocks nor their corresponding rodent-adapted stocks could be cyclically transmitted by tsetse flies. The availability of these stocks will greatly facilitate investigations on East African T. vivax which would otherwise be difficult to carry out in experimental rodents.     

Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium,on polyethyleneterephthalate microporous membranes,as an in vitro model

Summary Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells). Cells are cultivated in a serum-free medium, containing 1μg/ml insulin, 1 ng/ml epidermal growth factor, 10μg/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen. Cells rapidly adhere, grow, and form confluent monolayers; after 15 days, scanning electron microscopy reveals numerous uniform microvilli. Domes, which develop on nonporous substrata, are absent on high porosity membranes. Culture medium from upper and lower compartments of microplate inserts and cell lysates were immunoprecipitated after labeling with [³H]glucosamine and leucine; analysis was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. [³H]transferrin is found mainly in the lower compartment and in cells; [³H]apolipoprotein B is released in both compartments, and fibronectin almost entirely recovered in the lower compartment; [³H]transferrin receptors and ferritin are only present in cell lysates. Binding experiments also show that transferrin receptors are accessible from the lower compartment. These results suggest that CaCo2 cells, cultivated in synthetic medium on membranes of appropriate porosity, could provide an in vitro model of the intestinal barrier, with the upper compartment of the culture insert corresponding to the apical pole facing the intestinal lumen and the lower one to the basal pole in contact with blood. This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (grant 3.4551.88) and the Walloon Region.     

Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀ = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹ CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.     

Studies on the Development of Metacyclic Trypanosoma brucei sspp. Cultivated at 27°C with Insect Cell Lines1

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T. b. rhodesiense were grown at 27°C in 25-cm² flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 × 10⁵ metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Cloning and Expression of a Rat Acetylcholinesterase Subunit: Generation of Multiple Molecular Forms and Complementarity with a Torpedo Collagenic Subunit

Abstract: We obtained a cDNA clone encoding one type of catalytic subunit of acetylcholinesterase (AChE) from rat brain (T subunit). The coding sequence shows a high frequency of (G + C) at the third position of the codons (66%), as already noted for several AChEs, in contrast with mammalian butyrylcholinesterase. The predicted primary sequence of rat AChE presents only 11 amino acid differences, including one in the signal peptide, from that of the mouse T subunit. In particular, four alanines in the mouse sequence are replaced by serine or threonine. In northern blots, a rat AChE probe indicates the presence of major 3.2-and 2.4-kb mRNAs, expressed in the CNS as well as in some peripheral tissues, including muscle and spleen. In vivo, we found that the proportions of G₁, G₂, and G₄ forms are highly variable in different brain areas. We did not observe any glycolipid-anchored G₂ form, which would be derived from an H subunit. We expressed the cloned rat AChE in COS cells: The transfected cells produce principally an amphiphilic G₁^a form, together with amphiphilic G₂^a and G₄^a forms, and a nonamphiphilic G₄^na form. The amphiphilic G₁^a and G₂^a forms correspond to type II forms, which are predominant in muscle and brain of higher vertebrates. The cells also release G₄^na, G₂^a, and G₁^a in the culture medium. These experiments show that all the forms observed in the CNS in vivo may be obtained from the T subunit. By cotransfecting COS cells with the rat T subunit and the Torpedo collagenic subunit, we obtained chimeric collagentailed forms. This cross-species complementarity demonstrates that the interaction domains of the catalytic and structural subunits are highly conserved during evolution.     

Characterization of Na+, K+-ATPase in Cultured and Separated Neuronal and Glial Cells from Rat Cerebellum

Abstract: The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na ⁺, K⁺- ATPase; EC 3.6.1.3) were examined in cultures and peri- karya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synapto- somal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na ⁺, K⁺-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3–15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the a'strocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na ⁺, K⁺-ATPase (apparent A_j values of about 10–⁷M and 10–⁴-10–⁵M, respectively), the high- affinity form accounting for 60–75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na ⁺,K ⁺ - ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na⁺,K⁺-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na ⁺, K⁺-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into acc.ount the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na⁺, K⁺-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of ⁸⁶Rb into “live cells” was estimated as a measure of in situ ion pump activity. Besides the interaction with ouabain, the K⁺ dependence of the Na⁺, K⁺-ATPase activity was also investigated in crude particulate preparations from cultured cerebellar neurones and astrocytes. Differences were observed as nearly maximal enzyme activity was obtained in the as- trocyte preparations at 1 mM KCl, when only about one- third of the maximal activity was displayed by the cultured nerve cells.     

Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Objective Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T_reg). To study the role of T_reg in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10⁺ T_reg type 1 (Tr1) was established. Methods An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4⁺CD25⁻ T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) ± 1 nM rapamycin. Autologous iDC and CD4⁺CD25⁻ T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays ± neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-β and IFN-γ in ELISA. Results In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3⁺CD4⁺CD25⁻IL2Rβ⁺IL2Rγ⁺FoxP3⁺CTLA-4⁺IL-10⁺ cells with suppressor activity (mean suppression ± SD = 58 ± 12%). These suppressor cells produced IL-10 (mean ± SD = 535 ± 12 pg/ml) and TGF-β (mean ± SD = 512 ± 38 pg/ml), but no IL-4 or IFN-γ. Suppressor function of co-cultures correlated with the percent of expanding IL-10⁺ Tr1 cells (r ² = 0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent. Conclusion The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4⁺CD25^highFoxP3⁺ nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.