Neocortex and Allocortex Respond Differentially to Cellular Stress In Vitro and Aging In Vivo

In Parkinson’s and Alzheimer’s diseases, the allocortex accumulates aggregated proteins such as synuclein and tau well before neocortex. We present a new high-throughput model of this topographic difference by microdissecting neocortex and allocortex from the postnatal rat and treating them in parallel fashion with toxins. Allocortical cultures were more vulnerable to low concentrations of the proteasome inhibitors MG132 and PSI but not the oxidative poison H₂O₂. The proteasome appeared to be more impaired in allocortex because MG132 raised ubiquitin-conjugated proteins and lowered proteasome activity in allocortex more than neocortex. Allocortex cultures were more vulnerable to MG132 despite greater MG132-induced rises in heat shock protein 70, heme oxygenase 1, and catalase. Proteasome subunits PA700 and PA28 were also higher in allocortex cultures, suggesting compensatory adaptations to greater proteasome impairment. Glutathione and ceruloplasmin were not robustly MG132-responsive and were basally higher in neocortical cultures. Notably, neocortex cultures became as vulnerable to MG132 as allocortex when glutathione synthesis or autophagic defenses were inhibited. Conversely, the glutathione precursor N-acetyl cysteine rendered allocortex resilient to MG132. Glutathione and ceruloplasmin levels were then examined in vivo as a function of age because aging is a natural model of proteasome inhibition and oxidative stress. Allocortical glutathione levels rose linearly with age but were similar to neocortex in whole tissue lysates. In contrast, ceruloplasmin levels were strikingly higher in neocortex at all ages and rose linearly until middle age. PA28 levels rose with age and were higher in allocortex in vivo, also paralleling in vitro data. These neo- and allocortical differences have implications for the many studies that treat the telencephalic mantle as a single unit. Our observations suggest that the topographic progression of protein aggregations through the cerebrum may reflect differential responses to low level protein-misfolding stress but also reveal impressive compensatory adaptations in allocortex.     

Culture-Independent Identification of Microorganisms That Respond to Specified Stimuli

A new approach that permits culture-independent identification of microorganisms that respond to specified stimuli was developed. This approach was illustrated by examination of microorganisms that grew in response to various nutrient supplements added to soil. A thymidine nucleotide analog, bromodeoxyuridine (BrdU), and supplements were added to soil and incubated for 3 days. DNA was extracted from the soil, and the newly synthesized DNA was isolated by immunocapture of the BrdU-labeled DNA. The unique perspective this approach offers was demonstrated by comparing the microbial community structures obtained from total soil DNA and the BrdU-labeled fraction in an rRNA gene (rDNA) analysis. The traditional total DNA analysis revealed no notable differences between the treatments, whereas the BrdU-labeled DNA showed significantly different banding patterns between the nutrient supplement treatments and compared with total DNA banding patterns. PCR primers were developed to specifically amplify the intergenic region of an rDNA sequence unique to the BrdU analysis of a phosphate supplement treatment. Amplification of DNA from all treatments using these primers showed that it was unique to the phosphate treatment and that it was present in both the total DNA and BrdU-labeled DNA fractions. This result demonstrates the promise of this new strategy, because it was able to permit identification of a sequence from a phosphate-responsive organism that was not discernable in the traditional total DNA community structure analysis.     

Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload

Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO₄; 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.     

In Vitro Susceptibility of Selected Bacteria to Cefaclor

Cefaclor is an orally absorbed cephalosporin antibiotic chemically and pharmacologically similar to cephalexin. It appears to be more active than cephalexin against susceptible strains. The in vitro sensitivity of 230 clinical bacterial isolates to cefaclor was studied. Most isolates of S. aureus, K. pneumoniae, E. coli, and indole negative Proteus species were inhibited at clinically attainable serum and urine concentrations. Like cephalexin, cefaclor was less active against isolates of Enterobacter species, indole positive Proteus species and enterococci although many of these isolates were inhibited at concentrations achievable in urine.     

In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins

The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.     

Susceptibility of Phytopathogenic Bacteria to Wheat Purothionins In Vitro

Purothionins are basic polypeptides with antimicrobial properties that are present in the endosperm of wheat and other cereal species. Susceptibility to wheat purothionins among phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas, Agrobacterium, Erwinia, and Corynebacterium has been investigated. Sensitive strains have been found in all of these genera except Agrobacterium (the only strain of A. tumefaciens available proved to be resistant). Minimal inhibitory concentrations (MIC) with partially purified crude purothionins ranged from 1 μg/ml for C. sepedonicum (C.5) to 540 μg/ml for E. amylovora (E.3). Minimal bactericidal concentrations (MBC) were not higher than twice the MIC value, except for C. poinsettiae (C.4) (MBC/MIC = 8). Purothionins α and β, obtained by carboxymethyl-cellulose column chromatography, were tested against P. solanacearum (P.2) and X. phaseoli (X.2); α purothionin was more active than β against X.2, and β more active than α against P.2. This suggests a relationship between polypeptide sequence and specificity of action.     

An In Vitro Model of Hematopoietic Injury In Chronic Hypoplastic Anemia

Mice treated with high-dose busulfan develop a ‘latent’ form of bone marrow failure characterized by near-normal peripheral blood counts and marrow cellularity, but marked reductions in marrow pluripotent stem cells (CFUs) and myeloid progenitor cells (CFUc). Spleen cell suspensions from control and ‘latent’ mice were placed in liquid culture in the presence of colony-stimulating activity. Cells were harvested at intervals up to 14 days and sub-cultured in agar to assay for CFUc. Baseline splenic CFUc did not differ significantly between control and ‘latent’ mice. Splenic CFUc from control mice increased 50-fold and reached a peak at day 10 in liquid culture. In contrast, splenic CFUc from ‘latent’ mice increased only 7-fold and reached a peak at day 3. Our results indicate that although splenic CFUc are present in normal numbers in ‘latent’ mice, their proliferative capacity is markedly reduced, either as the result of defective CFUc self-renewal or defective feed-in from CFUs or both.     

An In Vitro Model of Skeletal Muscle Volume Regulation

Introduction

Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response.

Methods

The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay.

Results

Medium osmolality was increased by addition of up to 200mM sucrose. Addition of 200mM sucrose resulted in mean cell shrinkage of 44±1% after 30mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%).

Conclusion

We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death.     

In Vitro Utilization of Amylopectin and High-Amylose Maize (Amylomaize) Starch Granules by Human Colonic Bacteria

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M_r) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.     

MALDI-TOF Identification of the Human Gut Microbiome in People with and without Diarrhea in Senegal

Background

In Africa, there are several problems with the specific identification of bacteria. Recently, MALDI-TOF mass spectrometry has become a powerful tool for the routine microbial identification in many clinical laboratories.

Methodology/Principal Findings

This study was conducted using feces from 347 individuals (162 with diarrhea and 185 without diarrhea) sampled in health centers in Dakar, Senegal. Feces were transported from Dakar to Marseille, France, where they were cultured using different culture conditions. The isolated colonies were identified using MALDI-TOF. If a colony was unidentified, 16S rRNA sequencing was performed. Overall, 2,753 isolates were tested, allowing for the identification of 189 bacteria from 5 phyla, including 2 previously unknown species, 11 species not previously reported in the human gut, 10 species not previously reported in humans, and 3 fungi. 2,718 bacterial isolates (98.8%) out of 2,750 yielded an accurate identification using mass spectrometry, as did the 3 Candida albicans isolates. Thirty-two bacterial isolates not identified by MALDI-TOF (1.2%) were identified by sequencing, allowing for the identification of 2 new species. The number of bacterial species per fecal sample was significantly higher among patients without diarrhea (8.6±3) than in those with diarrhea (7.3±3.4; P = 0.0003). A modification of the gut microbiota was observed between the two groups. In individuals with diarrhea, major commensal bacterial species such as E. coli were significantly decreased (85% versus 64%), as were several Enterococcus spp. (E. faecium and E. casseliflavus) and anaerobes, such as Bacteroides spp. (B. uniformis and B. vulgatus) and Clostridium spp. (C. bifermentans, C. orbiscindens, C. perfringens, and C. symbosium). Conversely, several Bacillus spp. (B. licheniformis, B. mojavensis, and B. pumilus) were significantly more frequent among patients with diarrhea.

Conclusions/Significance

MALDI-TOF is a potentially powerful tool for routine bacterial identification in Africa, allowing for a quick identification of bacterial species.     

Interactions of Anaerobic Bacteria with Dental Stem Cells: An In Vitro Study

Background

In patients with periodontitis, it is highly likely that local (progenitor) cells encounter pathogenic bacteria. The purpose of this in vitro study was to elucidate how human dental follicle stem cells (hDFSC) react towards a direct challenge with anaerobic periodontal pathogens under their natural oxygen-free atmosphere. HDFSC were compared to human bone marrow mesenchymal stem cells (hBMSC) and differentiated primary human gingival fibroblasts (hGiF), as well as permanent gingival carcinoma cells (Ca9-22).

Methodology/Principal Findings

The different cell types were investigated in a co-culture system with Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). The viability of the cells and pathogens under anaerobic conditions, as well as interactions in terms of adherence and internalization, were examined. Additionally, the release of pro-inflammatory interleukin-8 (IL-8) and anti-inflammatory interleukin-10 (IL-10) was quantified via enzyme-linked immunosorbent assay. The bacteria adhered less efficiently to hDFSC compared to Ca9-22 (P. gingivalis: 0.18% adherence to hDFSC; 3.1% adherence to Ca9-22). Similar results were observed for host cell internalization (F. nucleatum: 0.002% internalization into hDFSC; 0.09% internalization into Ca9-22). Statistically significantly less IL-8 was secreted from hDFSC after stimulation with F. nucleatum and P. gingivalis in comparison with hGiF (F. nucleatum: 2080.0 pg/ml – hGiF; 19.7 pg/ml – hDFSC). The IL-10 response of the differentiated cells was found to be low in relation to their pro-inflammatory IL-8 response.

Conclusions/Significance

The results indicate that dental stem cells are less prone to interactions with pathogenic bacteria than differentiated cells in an anaerobic environment. Moreover, during bacterial challenge, the stem cell immune response seems to be more towards an anti-inflammatory reaction. For a potential future therapeutic use of hDFSC, these findings support the idea of a save application.     

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.     

In Vivo Emission of Dinitrogen by Earthworms via Denitrifying Bacteria in the Gut

Earthworms emit the greenhouse gas nitrous oxide (N₂O), and ingested denitrifiers in the gut appear to be the main source of this N₂O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N₂), the end product of complete denitrification. When [¹⁵N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N₂ (and also labeled N₂O) under in vivo conditions; emission of N₂ by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N₂ per h per g (fresh weight), respectively. Isolated gut contents also produced [¹⁵N]nitrate-derived N₂ and N₂O under anoxic conditions. N₂ is formed by N₂O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [¹⁵N]nitrate-derived N₂ by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N₂O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N₂ (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N₂ per h per g (fresh weight), respectively; these emission rates approximated that of N₂O. These collective observations indicate that (i) earthworms emit N₂ concomitant with the emission of N₂O via the in situ activity of denitrifying bacteria in the gut and (ii) N₂O is quantitatively an important denitrification-derived end product under in situ conditions.     

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.     

An In Vitro Inhibition Test That Predicts Toxicity of Bacterial Pathogen Combinations in the Colorado Potato Beetle

Many insect bacterial pathogens are not toxic enough for field control. Combinations of bacteria may increase toxicity. Bacteria toxic to Colorado potato beetle , Photorhabdus luminescens, Chromobacterium violaceum and Serratia marcescens , were tested in pair-wise combinations in an in vitro double streak test to determine bacterial compatibility. Only C. violaceum and S. marcescens grew to confluency. Their combined toxicity in vivo was additive. Other bacterial combinations had clear zones between bacterial streaks indicating inhibition. In the insect, the combined toxicity was less than the most toxic bacteria of the pair. For these strains, this in vitro test predicted compatibility in the insect.     

Focus: Microbiome: The Influence of the Gut Microbiota on Host Physiology: In Pursuit of Mechanisms

The results generated from the NIH funded Human Microbiome Project (HMP) are necessarily tied to the overall mission of the agency, which is to foster scientific discoveries as a basis for protecting and improving health. The investment in the HMP phase 1 accomplished many of its goals including the preliminary characterization of the human microbiome and the identification of links between microbiome diversity and disease states. Going forward, the next step in these studies must involve the identification of the functional molecular elements that mediate the positive influence of a eubiotic microbiome on health and disease. This review will focus on recent advances describing mechanistic events in the intestine elicited by the microbiome. These include symbiotic bacteria-induced activation of redox-dependent cell signaling, the bacterial production of short chain fatty acids and ensuing cellular responses, and the secretion of bacteriocins by bacteria that have anti-microbial activities against potential pathogens.     

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.     

An In Vitro Model for the Study of the Macrophage Response Upon Trichophyton rubrum Challenge

Diversity in the macrophage models currently employed in immunology studies may lead to opposed results and interpretations. In this study, we aimed to analyze the suitability of J774 macrophage-like cells as a model for the interaction between the dermatophyte Trichophyton rubrum and macrophages. J774 cells were competent in fungal phagocytosis, but succumbed to hyphal growth. Nevertheless, they could also secrete IL-1β in response to the dermatophyte. On the opposite direction, inflammatory, thioglycollate-induced peritoneal macrophages did not succumb to fungal growth and showed no significant IL-1β production. The proteomic profiling of these cells uncovered vimentin and plastin-2 as proteins whose abundance was altered by the fungal interaction. Our study indicates that this cell line could be an interesting tool in the investigation of T. rubrum infection biology.