Mechanisms by which sialylated milk oligosaccharides impact bone biology in a gnotobiotic mouse model of infant undernutrition.

Undernutrition in children is a pressing global health problem, manifested in part by impaired linear growth (stunting). Current nutritional interventions have been largely ineffective in overcoming stunting, emphasizing the need to obtain better understanding of its underlying causes. Treating Bangladeshi children with severe acute malnutrition with therapeutic foods reduced plasma levels of a biomarker of osteoclastic activity without affecting biomarkers of osteoblastic activity or improving their severe stunting. To characterize interactions among the gut microbiota, human milk oligosaccharides (HMOs), and osteoclast and osteoblast biology, young germ-free mice were colonized with cultured bacterial strains from a 6-mo-old stunted infant and fed a diet mimicking that consumed by the donor population. Adding purified bovine sialylated milk oligosaccharides (S-BMO) with structures similar to those in human milk to this diet increased femoral trabecular bone volume and cortical thickness, reduced osteoclasts and their bone marrow progenitors, and altered regulators of osteoclastogenesis and mediators of Th2 responses. Comparisons of germ-free and colonized mice revealed S-BMO-dependent and microbiota-dependent increases in cecal levels of succinate, increased numbers of small intestinal tuft cells, and evidence for activation of a succinate-induced tuft cell signaling pathway linked to Th2 immune responses. A prominent fucosylated HMO, 2'-fucosyllactose, failed to elicit these changes in bone biology, highlighting the structural specificity of the S-BMO effects. These results underscore the need to further characterize the balance between, and determinants of, osteoclastic and osteoblastic activity in stunted infants/children, and suggest that certain milk oligosaccharides may have therapeutic utility in this setting.

The Impact of Actotoxumab Treatment of Gnotobiotic Piglets Infected With Different Clostridium difficile Isogenic Mutants.

Nosocomial infections with Clostridium difficile are on the rise in the Unites States, attributed to emergence of antibiotic-resistant and hypervirulent strains associated with greater likelihood of recurrent infections. In addition to antibiotics, treatment with Merck anti-toxin B (TcdB) antibody bezlotoxumab is reported to reduce recurrent infections. However, treatment with anti-toxin A (TcdA) antibody actotoxumab was associated with dramatically increased disease severity and mortality rates in humans and gnotobiotic piglets. Using isogenic mutants of C. difficile strain NAPI/BI/027 deficient in TcdA (A-B+) or TcdB (A+B-), and the wild type, we investigated how and why treatment of infected animals with anti-TcdA dramatically increased disease severity. Contrary to the hypothesis, among piglets treated with anti-TcdA, those with A+B- infection were disease free, in contrast to the disease enhancement seen in those with wild-type or A-B+ infection. It seems that the lack of TcdA, through either deletion or neutralization with anti-TcdA, reduces a competitive pressure, allowing TcdB to freely exert its profound effect, leading to increased mucosal injury and disease severity.

Maternal western diet causes inflammatory milk and TLR2/4-dependent neonatal toxicity.

For all newborn mammals, mother's milk is the perfect nourishment, crucial for their postnatal development. Here we report that, unexpectedly, maternal western diet consumption in mice causes the production of toxic milk that contains excessive long chain and saturated fatty acids, which triggers ceramide accumulation and inflammation in the nursing neonates, manifested as alopecia. This neonatal toxicity requires Toll-like-receptors (TLR), but not gut microbiota, because TLR2/4 deletion or TLR4 inhibition confers resistance, whereas germ-free mice remain sensitive. These findings unravel maternal western diet-induced inflammatory milk secretion as a novel aspect of the metabolic syndrome at the maternal offspring interface.

Antibiotics impair murine hematopoiesis by depleting the intestinal microbiota.

Bone marrow suppression is an adverse effect associated with many antibiotics, especially when administered for prolonged treatment courses. Recent advances in our understanding of steady-state hematopoiesis have allowed us to explore the effects of antibiotics on hematopoietic progenitors in detail using a murine model. Antibiotic-treated mice exhibited anemia, thrombocytosis, and leukopenia, with pronounced pan-lymphopenia as demonstrated by flow cytometric analysis of peripheral blood. Bone marrow progenitor analysis revealed depletion of hematopoietic stem cells and multipotent progenitors across all subtypes. Granulocytes and B cells were also diminished in the bone marrow, whereas the number of CD8+ T cells increased. Reductions in progenitor activity were not observed when cells were directly incubated with antibiotics, suggesting that these effects are indirect. Hematopoietic changes were associated with a significant contraction of the fecal microbiome and were partially rescued by fecal microbiota transfer. Further, mice raised in germ-free conditions had hematopoietic abnormalities similar to those seen in antibiotic-treated mice, and antibiotic therapy of germ-free mice caused no additional abnormalities. The effects of antibiotics were phenocopied in Stat1-deficient mice, with no additional suppression by antibiotics in these mice. We conclude that microbiome depletion as a result of broad-spectrum antibiotic treatment disrupts basal Stat1 signaling and alters T-cell homeostasis, leading to impaired progenitor maintenance and granulocyte maturation. Methods to preserve the microbiome may reduce the incidence of antibiotic-associated bone marrow suppression.

Acute Alpha-Naphthylisothiocyanate-induced Liver Toxicity in Germfree and Conventional Male Rats.

Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study.

Microbiota Protects Mice Against Acute Alcohol-Induced Liver Injury.

BACKGROUND: Our aim is to investigate the physiological relevance of the intestinal microbiota in alcohol-induced liver injury. Chronic alcohol abuse is associated with intestinal bacterial overgrowth, increased intestinal permeability, and translocation of microbial products from the intestine to the portal circulation and liver. Translocated microbial products contribute to experimental alcoholic liver disease. METHODS: We subjected germ-free and conventional C57BL/6 mice to a model of acute alcohol exposure that mimics binge drinking. RESULTS: Germ-free mice showed significantly greater liver injury and inflammation after oral gavage of ethanol (EtOH) compared with conventional mice. In parallel, germ-free mice exhibited increased hepatic steatosis and up-regulated expression of genes involved in fatty acid and triglyceride synthesis compared with conventional mice after acute EtOH administration. The absence of microbiota was also associated with increased hepatic expression of EtOH-metabolizing enzymes, which led to faster EtOH elimination from the blood and lower plasma EtOH concentrations. Intestinal levels of EtOH-metabolizing genes showed regional expression differences and were overall higher in germ-free mice relative to conventional mice. CONCLUSIONS: Our findings indicate that absence of the intestinal microbiota increases hepatic EtOH metabolism and the susceptibility to binge-like alcohol drinking.

Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation.

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.

Microbiome Resilience despite a Profound Loss of Minority Microbiota following Clindamycin Challenge in Humanized Gnotobiotic Mice.

Antibiotics are known to induce gut dysbiosis and increase the risk of antibiotic resistance. While antibiotic exposure is a known risk factor leading to compromised colonization resistance against enteric pathogens such as Clostridioides difficile, the extent and consequences of antibiotic perturbation on the human gut microbiome remain poorly understood. Human studies on impacts of antibiotics are complicated by the tremendous variability of gut microbiome among individuals, even between identical twins. Furthermore, antibiotic challenge experiments cannot be replicated in human subjects for a given gut microbiome. Here, we transplanted feces from three unrelated human donors into groups of identical germfree (GF) Swiss-Webster mice, and examined the temporal responses of the transplanted microbiome to oral clindamycin challenge in gnotobiotic isolators over 7 weeks. Analysis of 177 longitudinal fecal samples revealed that 59% to 81% of human microbiota established a stable configuration rapidly and stably in GF mice. Microbiome responses to clindamycin challenge was highly reproducible and microbiome-dependent. A short course of clindamycin was sufficient to induce a profound loss (∼one third) of the microbiota by disproportionally eliminating minority members of the transplanted microbiota. However, it was inadequate to disrupt the global microbial community structure or function, which rebounded rapidly to resemble its pre-treatment state after clindamycin discontinuation. Furthermore, the response of individual microbes was community-dependent. Taken together, these results suggest that the overall gut microbiome structure is resilient to antibiotic perturbation, the functional consequences of which warrant further investigation. IMPORTANCE Antibiotics cause imbalance of gut microbiota, which in turn increase our susceptibility to gastrointestinal infections. However, how antibiotics disrupt gut bacterial communities is not well understood, and exposing healthy volunteers to unnecessary antibiotics for research purposes carries clinical and ethical concerns. In this study, we used genetically identical mice transplanted with the same human gut microbiota to control for both genetic and environmental variables. We found that a short course of oral clindamycin was sufficient to eliminate one third of the gut bacteria by disproportionally eliminating minority members of the transplanted microbiota, but it was inadequate to disrupt the overall microbial community structure and function, which rebounded rapidly to its pre-treatment state. These results suggest that gut microbiome is highly resilient to antibiotic challenge and degradation of the human gut ecosystem may require repeated or prolonged antibiotic exposure.

Effects of gut microflora on pharmacokinetics of hesperidin: a study on non-antibiotic and pseudo-germ-free rats.

Hesperidin is a biologically active flavanone glycoside occurring abundantly in citrus fruits. In the present study, effects of intestinal microflora on pharmacokinetics of hesperidin were investigated using a pseudo-germ-free rat model treated with antibiotics. After administration of hesperidin to rats, hesperetin, hesperetin glucuronides, and metabolites postulated to be eriodictyol, hemoeriodictyol, and their glucuronides were detected in urine while hesperetin glucuronide was predominantly found in plasma. The plasma concentration-time profile of hesperetin was compared between non-antibiotic-exposed and pseudo-germ-free rats administered this compound. The maximal concentration (C(max)) values of hesperetin in non-antibiotic-exposed and pseudo-germ-free rats were 0.58 and 0.20 µg/ml, respectively, and area under the curve (AUC) values were 6.3 and 2.8 µg-h/ml, respectively. Thus, systemic exposure as evidenced by AUC and C(max) was significantly higher in normal compared to pseudo-germ-free rats. Fecal ß-glucosidase activities of non-antibiotic-exposed and pseudo-germ-free rats were 0.21 and 0.11 nmol/min/mg, while fecal α-rhamnosidase activities were 0.37 and 0.12 nmol/min/mg, respectively. The rate of hesperidin transformation to hesperetin was 6.9 and 2.9 nmol/min/g in fecal samples in non-antibiotic-exposed and pseudo-germ-free rats, respectively. Taken together, these results showed that pharmacokinetic differences between non-antibiotic-exposed and pseudo-germ-free rats may be attributed to differing hesperidin uptake, as well as alterations in metabolic activities of intestinal flora.

Host Microbiota Regulates Central Nervous System Serotonin Receptor 2C Editing in Rodents.

Microbial colonization of the gastrointestinal tract plays a crucial role in the development of enteric and central nervous system functionality. The serotonergic system has been heavily implicated in microbiota-gut-brain axis signaling, particularly in proof-of-principle studies in germ-free (GF) animals. One aspect of the serotonergic system that has been left unexplored in relation to the microbiota is the unique ability of the serotonin receptor 2C (5-HT2C) to undergo post-transcriptional editing, which has been implicated in decreased receptor functionality. We investigated whether GF mice, with absent microbiota from birth, have altered 5-HT2C receptor expression and editing in the brain, and if colonization of the microbiota is able to restore editing patterns. Next, we investigated whether microbiota depletion later in life using a chronic antibiotic treatment could affect 5-HT2C receptor editing patterns in rats. We found that GF mice have an increased prevalence of the edited 5-HT2C receptor isoforms in the amygdala, hypothalamus, prefrontal cortex, and striatum, which was partially normalized upon colonization post-weaning. However, no alterations were observed in the hypothalamus after microbiota depletion using an antibiotic treatment in adult rats. This suggests that alterations in the microbiome during development, but not later in life, could influence 5-HT2C receptor editing patterns. Overall, these results demonstrate that the microbiota affects 5-HT2C receptor editing in the brain and may inform novel therapeutic strategies in conditions in which 5-HT2C receptor editing is altered, such as depression.

Microbiota alter metabolism and mediate neurodevelopmental toxicity of 17ß-estradiol.

Estrogenic chemicals are widespread environmental contaminants associated with diverse health and ecological effects. During early vertebrate development, estrogen receptor signaling is critical for many different physiologic responses, including nervous system function. Recently, host-associated microbiota have been shown to influence neurodevelopment. Here, we hypothesized that microbiota may biotransform exogenous 17-ßestradiol (E2) and modify E2 effects on swimming behavior. Colonized zebrafish were continuously exposed to non-teratogenic E2 concentrations from 1 to 10 days post-fertilization (dpf). Changes in microbial composition and predicted metagenomic function were evaluated. Locomotor activity was assessed in colonized and axenic (microbe-free) zebrafish exposed to E2 using a standard light/dark behavioral assay. Zebrafish tissue was collected for chemistry analyses. While E2 exposure did not alter microbial composition or putative function, colonized E2-exposed larvae showed reduced locomotor activity in the light, in contrast to axenic E2-exposed larvae, which exhibited normal behavior. Measured E2 concentrations were significantly higher in axenic relative to colonized zebrafish. Integrated peak area for putative sulfonated and glucuronidated E2 metabolites showed a similar trend. These data demonstrate that E2 locomotor effects in the light phase are dependent on the presence of microbiota and suggest that microbiota influence chemical E2 toxicokinetics. More broadly, this work supports the concept that microbial colonization status may influence chemical toxicity.

The toxicity of copper contaminated soil using a gnotobiotic Soil Multi-species Test System (SMS).

A gnotobiotic multi-species study was designed to consist of a food-web of soil-dwelling animals. The food-web was exposed to five concentrations copper (Cu) spiked soil for three exposure durations i.e. 28, 56 and 84 days. Based on multivariate analysis the food-web was significantly affected by Cu exposure at and above 300 mg Cu kg(-1) soil (lowest tested concentration). The number of animals present in the 2500 mg Cu kg(-1) (highest tested concentration) was at all sampling occasions below the starting point level. Based on analysis of the individual species the lowest 10% effect concentration (EC10) observed was 50 mg Cu kg(-1) soil, for Enchytraeus crypticus. Using the EC10 for the individual species the HC5 (Hazard Concentration at the 5% level) was estimated to be between 25 and 36 mg Cu kg(-1) soil, depending on the exposure duration. A similar experiment but using a reduced design was performed employing soil contaminated with Cu in the field more than 80 years ago. The trend in the field-contaminated soil was similar to that observed for the spiked soil.

Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis.

The intestinal barrier encompasses structural, permeability and immune aspects of the gut mucosa that, when disrupted, may contribute to chronic inflammation. Although gnotobiotic studies have demonstrated the effects of microbiota on mucosal and systemic immunity, as well as intestinal barrier architecture and innate immune characteristics, its impact on barrier function remains unclear. We compared germ-free and conventional mice, as well as mice colonized with human fecal microbiota that were followed for 21 days post-colonization. Colonic barrier structure was investigated by immunohistochemistry, molecular and electron microscopy techniques. Permeability was assessed in colon tissue by Ussing chambers, and by serum LPS and MDP detection using TLR4- and NOD2-NFκB reporter assays. Microbiota profile was determined by Illumina 16S rRNA gene sequencing. Low dose dextran sodium sulfate was administered to assess microbiota-induced barrier changes on resistance to colonic injury. Permeability to paracellular probes and mucus layer structure resembled that of conventional mice by day 7 post-colonization, coinciding with reduced claudin-1 expression and transient IL-18 production by intestinal epithelial cells. These post-colonization adaptations were associated with decreased systemic bacterial antigen exposure and reduced susceptibility to intestinal injury. In conclusion, commensal colonization promotes physiological barrier structural and functional adaptations that contribute to intestinal homeostasis.

Physiological mechanisms of sustained fumagillin-induced weight loss.

Current obesity interventions suffer from lack of durable effects and undesirable complications. Fumagillin, an inhibitor of methionine aminopeptidase-2, causes weight loss by reducing food intake, but with effects on weight that are superior to pair-feeding. Here, we show that feeding of rats on a high-fat diet supplemented with fumagillin (HF/FG) suppresses the aggressive feeding observed in pair-fed controls (HF/PF) and alters expression of circadian genes relative to the HF/PF group. Multiple indices of reduced energy expenditure are observed in HF/FG but not HF/PF rats. HF/FG rats also exhibit changes in gut hormones linked to food intake, increased energy harvest by gut microbiota, and caloric spilling in the urine. Studies in gnotobiotic mice reveal that effects of fumagillin on energy expenditure but not feeding behavior may be mediated by the gut microbiota. In sum, fumagillin engages weight loss-inducing behavioral and physiologic circuits distinct from those activated by simple caloric restriction.

Influence of Lactobacillus reuteri L26 Biocenol™ on immune response against porcine circovirus type 2 infection in germ-free mice.

Probiotic bacteria are frequently used for prevention of bacterial infections of the gastrointestinal tract, but there are only limited studies on their efficacy against viral gut infections in animals. The aim of this study was to investigate the effect of probiotic Lactobacillus reuteri L26 BiocenolTM on the innate and adaptive immune responses in germ-free Balb/c mice, experimentally infected by porcine circovirus type 2 (PCV2), which confers immunosuppressive effect. A total of 30 six-week-old female mice were divided into 3 groups and animals in experimental group LPCV (n=10) were inoculated with L. reuteri L26, animals in the control group (C; n=10) and experimental group PCV (n=10) received sterile De Man-Rogosa-Sharpe broth for 7 days. Subsequently, mice from both experimental groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). Virus load in faeces, ileum and mesenteric lymph nodes (MLN); as well as gene expression of selected cytokines, immunoglobulin A (IgA) and polymeric Ig receptor (PIgR) in the ileum, and percentage of CD8+, CD19+ and CD49b+CD8- cells in the MLN were evaluated. Our results showed that L. reuteri significantly decreased the amount of PCV2 in faeces and in the ileum, and up-regulated the gene expression of chemokines, interferon (IFN)-γ, IgA and PIgR in the ileum. Increased IFN-γ mRNA level was accompanied by higher proportion of natural killer cells and up-regulated IgA and PIgR gene expressions were in accordance with significantly higher percentage of CD19+ lymphocytes in the MLN. These findings indicate that probiotic L. reuteri has an antiviral effect on PCV2 in the intestine which is mediated by stimulation of local gut immune response.

Susceptibility of germ-free rats to the hepatotoxic effects of dimethylnitrosamine or dimethylamine plus sodium nitrite administered orally.

The influence of intestinal microflora on the hepatotoxic effects of dimethylnitrosamine (DMN) or dimethylamine (DMA) plus NaNO2 was studied by comparing the degree of liver necrosis and the levels of serum alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) in germ-free and conventional male Wistar rats (320 to 340 g). In one experiment, both germ-free and conventional rats were intubated with DMN in respective doses of 8, 9, and 10 mg/kg of body weight, while in another experiment, both groups were intubated with DMA (1500 mg/kg) plus NaNO2 (100 mg/kg). In both experiments, 48 hr after intubation, there was a marked difference in the degree of liver necrosis and the levels of serum GPT and GOT between the groups. In particular, a dose of 8 mg of DMN or 1500 mg of DMA plus 100 mg of NaNO2 produced severe liver necrosis in the majority of germ-free rats, while the same dose did not produce any detectable liver necrosis in the majority of conventional rats. At a dose of 8 mg, serum GPT and GOT levels were raised to 22 and 15 times normal values, respectively, in germ-free rats, but only to about twice the normal values for both levels in conventional rats. At the combination dose of DMA plus NaNO2, the levels of serum GPT and GOT were raised to 40 and 30 times normal values, respectively, in germ-free rats, while both levels remained almost normal in conventional rats. Thus, the results indicated that the liver of the germ-free state was far more susceptible to the acute toxic effects of DMN as well as DMA plus NaNO2 administration at a certain dose range than was the liver of the conventional state, suggesting the influence of the absence of microflora.

Modulation of a Circulating Uremic Solute via Rational Genetic Manipulation of the Gut Microbiota.

Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease.

Response of germfree rat colonic mucous cells to peroral endotoxin application.

During microbial colonization, mucin-releasing goblet cells of germ-free (GF) rats proliferate and upregulate their mucin synthesis, thus improving the intestinal mucus barrier. The present study determined the significance of bacterial membrane constituents for this development. A single dose of lipopolysaccharide (LPS) (35 micrograms/100 g body weight) and lipid A (3.5 micrograms/100 g body weight, respectively), was perorally administered to GF AS/Ztm rats. One, 3 and 5 days later, sections of the proximal and distal colon served for characterization of mucin-secreting goblet cells, released mucins were isolated in parallel. Maximal goblet cell diameters were evidenced at day 3. LPS generated a maximal goblet cell hyperplasia one day after challenge, lipid A stimulated the goblet cell proliferation continuously up to day 5. Three days after challenge with one of the stimuli, either, intracellular mucins had shifted significantly to neutral constituents. In addition, mucins, adherent to the colon mucosa and submerged to the luminal content, respectively, then were augmented. At day 5, adherent mucins were similar to the controls, while luminal, soluble constituents had further increased. Histometrical and biochemical methods evidenced a transient, inflammatory response of mucin-secreting cells, followed by an upregulated release of immature mucins.

Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions.

Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes. Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing. Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo. This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL. The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense action of S-oligos. We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.