Community and genomic analysis of the human small intestine microbiota

Our intestinal tract is densely populated by different microbes, collectively called microbiota, of which the majority are bacteria. Research focusing on the intestinal microbiota often use fecal samples as a representative of the bacteria that inhabit the end of the large intestine. These studies revealed that the intestinal bacteria contribute to our health, which has stimulated the interest in understanding their dynamics and activities. However, bacterial communities in fecal samples are different compared to microbial communities at other locations in the intestinal tract, such as the small intestine. Despite that the small intestine is the first region where our food and intestinal microbiota meet, we know little about the bacteria in the small intestine and how they influence our overall well-being. This is mainly attributable to difficulties in obtaining samples with the small intestine being located between the stomach and the large intestine. Therefore, the work in this thesis aimed at providing a better understanding of the composition and dynamics of the human small intestinal microbiota and to provide insight in the metabolic potential as well as immunomodulatory properties of some of its typical commensal inhabitants. Small intestinal samples used in the work described in this thesis were collected from ileostomy subjects, individuals that had their large intestine surgically removed and the end of the small intestine connected to an abdominal stoma, providing access to luminal content of the small intestine. Considering the importance of molecular techniques in contemporary ecological surveys of microbial communities, first of all, two technologies, barcoded pyrosequencing and phylogenetic microarray analysis were compared in terms of their capacity to determine the bacterial composition in fecal and small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, the use of different primer pairs in the amplification step was assessed in terms of its impact on the outcome of microbial profiling. The analyses revealed that the different primer pairs and the two profiling technologies provide overall similar results for samples of fecal and terminal ileum origin. In contrast, the microbial profiles obtained for small intestinal samples by barcoded pyrosequencing and phylogenetic microarray analysesdiffered considerably. This is most likely attributable to the constraints that are intrinsic to the use of the microarray to enable the detection of predefined microbiota members only, which is due to the probe design that is largely based on large intestinal microbiota communities. However, the pyrosequencing technology also allows for identification of bacteria that were not in advance known to inhabit our intestinal tract. The pyrosequencing technology was used as the method of choice to study the total and active small intestinal communities in ileostoma effluent samples from four different subjects through sequencing the 16S ribosomal RNA gene (rDNA) and ribosomal RNA (rRNA) contentcombined with metatranscriptome analysis by Illumina sequencing of cDNA derived from enriched mRNAof the same sample set to investigate the activities of the small intestinal bacteria. The composition of the small intestinal bacterial communities as assessed from rDNA, rRNA, and mRNA patterns appeared to be similar, indicating that the dominant bacteria in the small intestine are also highly active in this ecosystem. Streptococcusspp. were among the bacterial species that were detected in each ileostoma effluent sample, albeit that their abundance varied greatly between samples from the same subject as well as samples from different subjects. Veillonellaspp. frequently co-occurred with Streptococcus spp., indicating that the Streptococcusand Veillonellapopulations play a prominent role in the human small intestine ecosystem and their co-occurrence suggests a metabolic relation between these genera. Therefore, cultivation and molecular typing methodologies were employed to zoom-in on these groups, which revealed that the richness of the small intestinal streptococci strongly exceeded the diversity that could be estimated on basis of 16S rRNA analyses, and could be extended to the genomic lineage level (anticipated to resemble strain-level). From ileostoma samples 3 different Streptococcusspecies were recovered belonging to the S. mitisgroup, S. bovisgroup, and S. salivariusgroup, which could be further divided in 7 genomic lineages. Notably, the Streptococcuslineages that were isolated displayed distinct carbohydrate utilization capacities, which may imply that their growth and relative community composition may respond quite strongly to differences in the dietary intake of simple carbohydrates over time. This notion is in good agreement with the observation that the Streptococcuslineage populations fluctuated in time with only one Streptococcuslineage being cultivated from both ileostoma samples collected in a one-year time frame. Conversely, the cultivated Veillonellaisolates from samples during that same time-interval consistently encompassed a single lineage. Furthermore, this Veillonellalineage could be isolated from both the oral cavity as well as the ileostoma effluent. Analogously, three Streptococcuslineages that belong to a single phylotype also appeared to be present in bacterial communities from the oral cavity as well as the small intestine. These observations suggest the representatives of the Veillonellaand Streptococcusgenera that are encountered in the oral and small intestinal microbial ecosystems are closely related and indicate that the oral microbiota may serve as an inoculum for the upper GI tract. The metabolic capacity of 6 small intestinal Streptococcus lineages, that were obtained from a single ileostoma effluent sample, was further investigated through the determination of genomic sequences of these lineages. The small-intestinal Streptococcusgenomes were found to encode different carbohydrate transporters and the necessary enzymes to metabolize different sugars, which was in excellent agreement with what carbohydrates could be used by representative strains of the Streptococcuslineages. To further our understanding how the different streptococci as representatives of the dominant small intestinal bacterial populations may influence our immune system, human dendritic cells were stimulated with strains of the different Streptococcuslineages to study their immunomodulatory properties. The Streptococcuslineages differed significantly in their capacity to modulate cytokine responses of blood-monocyte derived immature dendritic cells. As Streptococcusand Veillonellafrequently co-occur in the small intestinal ecosystem, pair-wise combinations of strains of these two species were also tested for their combined immunomodulatory properties. This resulted in considerably different cytokine responses as those that could be predicted from the stimulations with either Streptococcusor Veillonella, indicating that it is not trivial to predict gut mucosal associated immune responses and thatthe composition of the intestinal microbiota as a whole may have a distinct influence on an individual’s immune status. In conclusion, the work described this thesis provides an expansion to the accumulating knowledge on the human intestine microbiota. Whereas most studies focus on the microbiota present in the distal regions of the intestinal tract, this study targeted the microbiota of the poorly proximal regions of the intestine and also addressed its capacity to interact with the local mucosal tissue. The data presented here can be exploited to guide the design of future studies that aim to elucidate the interplay between diet, microbiota and the mucosal tissues in the human small intestinal tract.</p

Effect of Gender on Lower Extremity Kinematics During Rapid Direction Changes: An Integrated Analysis of Three Sports Movements

Anterior cruciate ligament (ACL) injury is a common sports injury, particularly in females. Gender differences in knee kinematics have been observed for specific movements, but there is limited information on how these findings relate to other joints and other movements. Here we present an integrated analysis of hip, knee and ankle kinematics across three movements linked to non-contact ACL injury. It was hypothesised that there are gender differences in lower extremity kinematics, which are consistent across sports movements. Ten female and ten male NCAA basketball players had three-dimensional hip, knee and ankle kinematics quantified during the stance phase of sidestep, side-jump and shuttle-run tasks. For each joint angle, initial value at contact, peak value and between-trial variability was obtained and submitted to a two-way mixed design ANOVA (gender and movement), with movement condition treated as a repeated measure. Females had higher peak knee valgus and lower peak hip and knee flexion, with the same gender differences also existing at the beginning of stance (

Assessing Vaginal Surgical Skills Using Video Motion Analysis

OBJECTIVE: To demonstrate the feasibility of using video motion analysis to quantitate a key step of vaginal hysterectomy and define measurable differences between novice and experienced surgical trainees during vaginal hysterectomy. METHODS: Analyses focused on clamping, transecting, and suturing the left uterosacral ligament. Using a cutoff of 25, trainees were grouped as experienced (n=14) and novice (n=9) based on the total number of vaginal hysterectomies performed by each trainee. Contrasting-groups analysis was used to determine cutoff values that separated novices from experts. RESULTS: Novice trainees took longer (112 seconds compared with 84 seconds) and had greater cumulative translational motion (92 cm compared with 49 cm, P=.05) while performing the task. Experienced trainees placed the Heaney clamp closer to a right angle to the vertical axis (experienced 96° compared with novice 109°, P=.03) while passing the needle through the uterosacral ligament. Trainees move from novice to experts when the steps occur in 112 or fewer seconds, cumulative translational motion is at or less than 75 cm, and the angle between the clamp to bladder retractor is at or below 105°. CONCLUSION: Video motion analysis is a feasible technique to quantify and compare surgical skills objectively during vaginal surgery. There are measurable differences between novice and more experienced surgical trainees performing vaginal hysterectomy that can be quantified using motion analysis

The Influence of Foot Positioning on Ankle Sprains

The goal of this study was to examine the influence of changes in foot positioning at touch-down on ankle sprain occurrence. Muscle model driven computer simulations of 10 subjects performing the landing phase of a side-shuffle movement were performed. The relative subtalar joint and talocural joint angles at touchdown were varied, and each subject-specific simulation was exposed to a set of perturbed floor conditions. The touchdown subtalar joint angle was not found to have a considerable influence on sprain occurrence, while increased touchdown plantar flexion caused increased ankle sprain occurrences. Increased touchdown plantar flexion may be the mechanism which causes ankles with a history of ankle sprains to have an increased susceptibility to subsequent sprains. This finding may also reveal a mechanism by which taping of a sprained ankle or the application of an ankle brace leads to decreased ankle sprain susceptibility

Effects of Foot Orthoses on Skeletal Motion During Running

Objective. To quantify the effects of medial foot orthoses on skeletal movements of the calcaneus and tibia during the stance phase in running. Design. Kinematic effects of medial foot orthoses (anterior, posterior, no support) were tested using skeletal (and shoe) markers at the calcaneus and tibia. Background. Previous studies using shoe and skin markers concluded that medially placed orthoses control/reduce foot eversion and tibial rotation. However, it is currently unknown if such orthoses also affect skeletal motion at the lower extremities. Methods. Intracortical Hofman pins with reflective marker triads were inserted under standard local anesthetic into the calcaneus and tibia of five healthy male subjects. The three-dimensional tibiocalcaneal rotations were determined using a joint coordinate system approach. Eversion (skeletal and shoe) and tibial rotation were calculated to study the foot orthoses effects. Results. Orthotic effects on eversion and tibial rotations were found to be small and unsystematic over all subjects. Differences between the subjects were significantly larger (pp\u3c0.05). Conclusions. This in vivo study showed that medially placed foot orthoses did not change tibiocalcaneal movement patterns substantially during the stance phase of running. Relevance Orthoses may have only small kinematic effects on the calcaneus and tibia (measured with bone pins) as well as on the shoes (measured with shoe markers) during running of normal subjects. Present results showed that orthotic effects were subject specific and unsystematic across conditions. It is speculated that orthotic effects during the stance phase of running may be mechanical as well as proprioceptive

Immunomodulatory Properties of Streptococcus and Veillonella Isolates from the Human Small Intestine Microbiota

The human small intestine is a key site for interactions between the intestinal microbiota and the mucosal immune system. Here we investigated the immunomodulatory properties of representative species of commonly dominant small-intestinal microbial communities, including six streptococcal strains (four Streptococcus salivarius, one S. equinus, one S. parasanguinis) one Veillonella parvula strain, one Enterococcus gallinarum strain, and Lactobacillus plantarum WCFS1 as a bench mark strain on human monocyte-derived dendritic cells. The different streptococci induced varying levels of the cytokines IL-8, TNF-a, and IL-12p70, while the V. parvula strain showed a strong capacity to induce IL-6. E. gallinarum strain was a potent inducer of cytokines and TLR2/6 signalling. As Streptococcus and Veillonella can potentially interact metabolically and frequently co-occur in ecosystems, immunomodulation by pair-wise combinations of strains were also tested for their combined immunomodulatory properties. Strain combinations induced cytokine responses in dendritic cells that differed from what might be expected on the basis of the results obtained with the individual strains. A combination of (some) streptococci with Veillonella appeared to negate IL-12p70 production, while augmenting IL-8, IL-6, IL-10, and TNF-a responses. This suggests that immunomodulation data obtained in vitro with individual strains are unlikely to adequately represent immune responses to mixtures of gut microbiota communities in vivo. Nevertheless, analysing the immune responses of strains representing the dominant species in the intestine may help to identify immunomodulatory mechanisms that influence immune homeostasis

Gait kinematic analysis in patients with a mild form of central cord syndrome

<p>Abstract</p> <p>Background</p> <p>Central cord syndrome (CCS) is considered the most common incomplete spinal cord injury (SCI). Independent ambulation was achieved in 87-97% in young patients with CCS but no gait analysis studies have been reported before in such pathology. The aim of this study was to analyze the gait characteristics of subjects with CCS and to compare the findings with a healthy age, sex and anthropomorphically matched control group (CG), walking both at a self-selected speed and at the same speed.</p> <p>Methods</p> <p>Twelve CCS patients and a CG of twenty subjects were analyzed. Kinematic data were obtained using a three-dimensional motion analysis system with two scanner units. The CG were asked to walk at two different speeds, at a self-selected speed and at a slower one, similar to the mean gait speed previously registered in the CCS patient group. Temporal, spatial variables and kinematic variables (maximum and minimum lower limb joint angles throughout the gait cycle in each plane, along with the gait cycle instants of occurrence and the joint range of motion - ROM) were compared between the two groups walking at similar speeds.</p> <p>Results</p> <p>The kinematic parameters were compared when both groups walked at a similar speed, given that there was a significant difference in the self-selected speeds (p < 0.05). Hip abduction and knee flexion at initial contact, as well as minimal knee flexion at stance, were larger in the CCS group (p < 0.05). However, the range of knee and ankle motion in the sagittal plane was greater in the CG group (p < 0.05). The maximal ankle plantar-flexion values in stance phase and at toe off were larger in the CG (p < 0.05).</p> <p>Conclusions</p> <p>The gait pattern of CCS patients showed a decrease of knee and ankle sagittal ROM during level walking and an increase in hip abduction to increase base of support. The findings of this study help to improve the understanding how CCS affects gait changes in the lower limbs.</p

Effectiveness of computer-navigated minimally invasive total hip surgery compared to conventional total hip arthroplasty: design of a randomized controlled trial

BACKGROUND: Moderate to severe osteoarthrosis is the most common indication for Total Hip Arthroplasty (THA). Minimally Invasive Total Hip Surgery (MIS) and computer-navigated surgery were introduced several years ago. However, the literature lacks well-designed studies that provide evidence of superiority of computer-navigated MIS over a conventional THA technique. Hence, the purpose of this study is to compare (cost)effectiveness of computer-navigated MIS with a conventional technique for THA. It is our hypothesis that computer-navigated MIS will lead to a quicker recovery during the early postoperative period (3 months), and to an outcome at least as good 6 months postoperatively. We also hypothesize that computer-navigated MIS leads to fewer perioperative complications and better prosthesis positioning. Furthermore, cost advantages of computer-navigated MIS over conventional THA technique are expected. METHODS/DESIGN: A cluster randomized controlled trial will be executed. Patients between the ages of 18 and 75 admitted for primary cementless unilateral THA will be included. Patients will be stratified using the Charnley classification. They will be randomly allocated to have computer-navigated MIS or conventional THA technique. Measurements take place preoperatively, perioperatively, and 6 weeks and 3 and 6 months postoperatively. Degree of limping (gait analysis), self-reported functional status and health-related quality of life (questionnaires) will be assessed preoperatively as well as postoperatively. Perioperative complications will be registered. Radiographic evaluation of prosthesis positioning will take place 6 weeks postoperatively. An evaluation of costs within and outside the healthcare sector will focus on differences in costs between computer-navigated MIS and conventional THA technique. DISCUSSION: Based on studies performed so far, few objective data quantifying the risks and benefits of computer-navigated MIS are available. Therefore, this study has been designed to compare (cost) effectiveness of computer-navigated MIS with a conventional technique for THA. The results of this trial will be presented as soon as they become available