Molecular monitoring of the development of intestinal microbiota in Japanese infants.

The faecal microbiota of 166 healthy Japanese newborns was analysed periodically from day 1 after birth until the age of 3 years by using the reverse transcription-quantitative PCR. Faecal pH and the organic acid concentration were also examined. Colonisation by both facultative anaerobes and strict anaerobes was confirmed in 95% of the meconium tested. Bifidobacterium-predominant microbiota was established subsequently in most of the infants by 3 months after birth. Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium catenulatum group and Bifidobacterium bifidum were the species mainly detected. Intergroup correlation analysis revealed that the bifidobacterial population levels, but not other strict anaerobe groups, were found to be negatively correlated with those of the Enterobacteriaceae from 7 days until 3 months after birth. Faecal pH was maintained at about 6 until 6 months after birth and reached 6.6 at 3 years after birth. The initial concentration of faecal organic acids (19 µM/g of faeces) just after birth increased until 3 years after birth to the level of 111 µM/g of faeces. Early start of feeding formula milk promoted colonisation by obligate anaerobes such as the Clostridium coccoides group, the Clostridium leptum subgroup, Prevotella, and Atopobium cluster during the 3 months after birth. Population levels of the bifidobacteria until 1 month after birth and those of the Bacteroides fragilis group until 6 months after birth were lower in infants delivered by Caesarean section than in those delivered normally. The results suggested that both earlier start of feeding of formula milk and the mode of infant delivery were found to be important in the development of intestinal microbiota in early infancy.

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei.

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.

Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice.

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.

Initiation of protein synthesis by a labeled derivative of the Lactobacillus casei DN-114 001 strain during transit from the stomach to the cecum in mice harboring human microbiota.

Although studies on the survival of bacteria in the digestive tract have been reported in the literature, little data are available on the physiological adaptation of probiotics to the digestive environment. In previous work, a transcriptional fusion system (i.e., luciferase genes under the control of a deregulated promoter) was used to demonstrate that a derivative of the Lactobacillus casei DN-114 001 strain, ingested in a fermented milk and thus exhibiting initially a very weak metabolic activity, synthesized proteins de novo after its transit in the digestive tract of mice harboring human microbiota (known as human-microbiota-associated mice). With the same genetic system and animal model, we here investigate for the first time the ability of L. casei to reinitiate synthesis in the different digestive tract compartments. In this study, most ingested L. casei cells transited from the stomach to the duodenum-jejunum within 1 h postingestion. No luciferase activity was observed in these digestive tract compartments after the first hour. At later times, the bulk of bacteria had transited to the ileum and the cecum. Luciferase synthesis was detected between 1.5 and 2.0 h postingestion at the ileal level and from 1.5 h to at least 6.0 h postingestion in the cecum, where the activity remained at a maximum level. These results demonstrate that ingested L. casei (derivative of the DN-114 001 strain) administered via a fermented milk has already reinitiated protein synthesis when it reaches the ileal and cecal compartments.

Lactobacillus casei is able to survive and initiate protein synthesis during its transit in the digestive tract of human flora-associated mice.

Live Lactobacillus casei is present in fermented dairy products and has beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine if L. casei DN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora-associated (HFA) mouse model. L. casei DN-114 001 was genetically modified by the introduction of erm and lux genes, encoding erythromycin resistance and luciferase, respectively. For this modified strain (DN-240 041), light emission related to luciferase expression could easily be detected in the contents of the digestive tract. When inoculated into the digestive tract of HFA mice, L. casei (DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture of L. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable 1 day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5-day-old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. These results suggest that in HFA mice L. casei DN-240 041, and thus probably L. casei DN-114 001, is able to initiate new protein synthesis during its transit with the diet. The beneficial properties of L. casei-fermented milk for human health might be related to this protein synthesis in the digestive tract.

[Radiotherapy of lung cancer: the inspiration breath hold with spirometric monitoring]. / Radiothérapie des cancers du poumon: le blocage en inspiration sous contrôle spirométrique.

A CT acquisition during a free breathing examination generates images of poor quality. It creates an uncertainty on the reconstructed gross tumour volume and dose distribution. The aim of this study is to test the feasibility of a breath hold method applied in all preparation and treatment days. Five patients received a thoracic radiotherapy with the benefit of this procedure. The breathing of the patient was measured with a spirometer. The patient was coached to reproduce a constant level of breath-hold in a deep inspiration. Video glasses helped the patients to fix the breath-hold at the reference level. The patients followed the coaching during preparation and treatment, without any difficulty. The better quality of the CT reconstructed images resulted in an easier contouring. No movements of the gross tumour volume lead to a better coverage. The deep breath hold decreased the volume of irradiated lung. This method improves the reproducibility of the thoracic irradiation. The decrease of irradiated lung volume offers prospects in dose escalation and intensity modulation radiotherapy.

[Thoracic radiotherapy and control of respiration: current perspectives]. / Radiothérapie thoracique et contrôle de la respiration: perspectives actuelles.

Three-dimensional conformal radiotherapy (3D CRT) is adversely affected by setup error and organ motion. In thoracic 3D CRT, breathing accounts for most of intra-fraction movements, thus impairing treatment quality. Breath control clearly exhibits dosimetric improvement compared to free breathing, leading to various techniques for gated treatments. We review benefits of different breath control methods--i.e. breath-holding or beam gating, with spirometric, isometric or X-ray respiration sensor--and argument the choice of expiration versus inspiration, with consideration to dosimetric concerns. All steps of 3D-CRT can be improved with breath control. Contouring of organs at risk (OAR) and target are easier and more accurate on breath controlled CT-scans. Inter- and intra-fraction target immobilisation allows smaller margins with better coverage. Lung outcome predictors (NTCP, Mean Dose, LV20, LV30) are improved with breath-control. In addition, inspiration breath control facilitates beam arrangement since it widens the distance between OAR and target, and leaves less lung normal tissue within the high dose region. Last, lung density, as of CT-scan, is more accurate, improving dosimetry. Our institution's choice is to use spirometry driven, patient controlled high-inspiration breath-hold; this technique gives excellent immobilization results, with high reproducibility, yet it is easy to implement and costs little extra treatment time. Breath control, whatever technique is employed, proves superior to free breathing treatment when using 3D-CRT. Breath control should then be used whenever possible, and is probably mandatory for IMRT.

[Are there physical, technical and morphological limits to conformational radiotherapy?]. / Y a-t-il des limites physiques, techniques et morphologiques à la radiothérapie conformationnelle?

Conformal radiotherapy results from several technical and data processing advances. The treatment planning and the daily treatments have benefited from a significant improvement of the quality control. However, all the steps of the process contain various limitations of varying effects. It appear important to identify, describe and exploit those limits to better drive the procedures and optimise the quality. Identifying the limits of the conformal radiotherapy should be an important help to implement intensity modulation radiotherapy.

[Dosimetric evaluation of conformal radiotherapy: conformity factor]. / Evaluation dosimétrique d'une radiothérapie conformationnelle: le facteur de conformation.

The aim of three-dimensional conformal therapy (3DCRT) is to treat the Planning Target Volume (PTV) to the prescribed dose while reducing doses to normal tissues and critical structures, in order to increase local control and reduce toxicity. The evaluation tools used for optimizing treatment techniques are three-dimensional visualization of dose distributions, dose-volume histograms, tumor control probabilities (TCP) and normal tissue complication probabilities (NTCP). These tools, however, do not fully quantify the conformity of dose distributions to the PTV. Specific tools were introduced to measure this conformity for a given dose level. We have extended those definitions to different dose levels, using a conformity index (CI). CI is based on the relative volumes of PTV and outside the PTV receiving more than a given dose. This parameter has been evaluated by a clinical study including 82 patients treated for lung cancer and 82 patients treated for prostate cancer. The CI was low for lung dosimetric studies (0.35 at the prescribed dose 66 Gy) due to build-up around the GTV and to spinal cord sparing. For prostate dosimetric studies, the CI was higher (0.57 at the prescribed dose 70 Gy). The CI has been used to compare treatment plans for lung 3DCRT (2 vs 3 beams) and prostate 3DCRT (4 vs 7 beams). The variation of CI with dose can be used to optimize dose prescription.

[Conformal radiotherapy of prostatic cancer: a general review]. / Radiothérapie conformationnelle des cancers de la prostate: revue générale.

Recent progress in radiotherapeutic management of localized prostate cancer is reviewed. Clinical aspects--including dose-effect beyond 70 Gy, relative role of conformal radiation therapy techniques and of early hormonal treatment--are discussed as well as technical components--including patient immobilization, organ motion, prostate contouring, beam arrangement, 3-D treatment planning and portal imaging. The local control and biological relapse-free survival rates appear to be improved by high dose conformal radiotherapy from 20 to 30% for patients with intermediate and high risk of relapse. A benefit of overall survival is expected but not yet demonstrated. Late reactions, especially the rectal toxicity, remain moderate despite the dose escalation. However, conformal radiotherapy demands a high precision at all steps of the procedure.

[Definition of prostatic contours using tomodensitometric slices: study of differences among radiotherapists and between examinations]. / Définition des contours prostatiques sur coupes tomodensitométriques: étude des discordances entre radiothérapeutes et entre examens.

Accuracy of conformal treatment planning for prostatic radiotherapy is based on the contours of target volumes (prostate +/- seminal vesicles) and normal tissues (rectum and bladder), drawn on CT (computed tomography) images by radiation oncologists. The interpretation of a given CT image can be different from one radiation oncologist to another, and may change in time with the state of filling of the bladder and of the rectum during the treatment. In order to quantify these variations, 12 patients treated with conformal radiotherapy for prostate carcinoma (pelvis 40 Gy/20 sessions + prostate 30 Gy/15 sessions) had two series of CT at one month intervals. Contouring of prostate, rectum and bladder were performed independently on each CT by two radiation oncologists. The first CT scan (planning CT) and the first series of contours (planning contours) were used for treatment planning. The contours of the second scan were compared to the planning contours after image fusion based on manual superimposition of bony anatomy of the two sets of CT images. Coherence ratio were defined to measure discrepancies in prostate volumes between radiation oncologists (RCE) and between scans (RCT). The mean RCE was 38 +/- 7% (1 standard deviation). Those discrepancies were primarily located at the prostate apex and at the interface between bladder and prostate and between rectum and prostate. The mean RCT was 42 +/- 8% (1 sigma). Those discrepancies were due to the prostate motion related to the state of filling of the rectum and bladder. For bladder and rectal walls, less important differences were observed between the two radiation oncologists for the same CT (4.5% for rectal volume receiving 65 Gy or more, 3% for bladder volume receiving 65 Gy or more). However, important differences in bladder and rectal volumes receiving 65 Gy or more (16% and 7% respectively) were noted for the same patient from a CT to another due to the variation in bladder or rectal filling. New techniques for planning CT acquisition are needed to decrease the discrepancies due to contouring. The treatment must, as far as possible, be delivered with an empty bladder and rectum in order to ensure a good reproduction of the initially planned treatment.

A model for the lateral penumbra in water of a 200-MeV proton beam devoted to clinical applications.

An experimental approach for modeling the lateral penumbra of a proton beam has been investigated. Measurements were made with a silicon diode in a water tank. Several geometrical configurations (phantom position, collimator-to-surface distance, collimator diameter, bolus thickness, air gap, etc.) and beam characteristics (range, modulation, etc.) have been studied. The results show that the lateral penumbra is almost independent of the beam modulation and the diameter of the collimator. The use of scaled variables for depth and penumbra allows us to represent the increase in penumbra with depth for any configuration with a second order polynomial function, provided that the penumbra at the entrance of the medium and at the depth of the range are known.

[Conformational radiotherapy in cancers of the prostate: contribution of pelvic immobilization and new fiducial markers]. / Radiothérapie conformationnelle des cancers de la prostate: apport de la contention pelvienne et de nouveaux repères de positionnement.

PURPOSE: To reduce errors in the positioning of patients treated with external conformal radiotherapy for prostate cancer, we evaluated both the use of an immobilization device and new fiducial markers. MATERIALS AND METHODS: The immobilization device consisted of an individual mold made of polyurethan foam. Two sets of skin markers located on the anterior tibial surfaces were used to identify the pelvic isocenter. The patient's position was evaluated by orthogonal port films which were then compared with the original simulation film. RESULTS: Results are presented with respect to orthogonal axes. Comparison with classic procedures without immobilization showed that use of the mold and new fiducial markers led to a decrease in set-up errors which were less than 5 mm. CONCLUSION: With the use of an immobilization device and optimized techniques for patients' positioning, conformal radiotherapy of prostate cancer is more accurate.

Reversible changes in cerebral activity associated with acidosis in preterm neonates.

Computerized online EEG monitoring in ventilated preterm infants less than 32 weeks' gestation enabled evaluation of the effect of acidosis on cerebral function. All episodes of acidosis were found to be associated with changes in the levels of cerebral activity. In 21 of the 32 episodes, EEG activity returned to pre-acidosis levels after therapeutic intervention. The duration of EEG abnormality was related to the severity of acidosis. However, the time to recovery of the EEG after therapeutic procedures was not related to duration of the EEG change.

The effect of pethidine on the neonatal EEG.

Thirty-two preterm infants were monitored with an on-line cotside EEG system for periods of up to nine days. Changes in the normal pattern of discontinuity of the EEG were seen in association with pethidine administration. The duration of the EEG suppression after pethidine administration was greatest after the first dose and progressively less with subsequent doses. It was not related to the gestational maturity or postnatal age at which the dose was given. This system of EEG analysis allowed recognition of this previously undescribed effect of pethidine on the neonatal EEG, and should have application to monitoring the effects of brain function of other drugs in routine neonatal practice.

A new system for cotside display and analysis of the preterm neonatal electroencephalogram.

Continuous EEG monitoring is considered to be a sensitive indicator of changes in cerebral function. This paper describes a system for cotside display and analysis of EEG discontinuity and amplitude in one-minute epochs, with provision for simultaneous recording of the EEG. It has been successfully used to monitor continuously the EEGs of more than 30 babies in a neonatal intensive care unit for periods of up to nine days. This system rapidly provides indications of changes in cerebral function, which can allow early intervention and possible prevention of morbidity.

Continuous EEG monitoring of neonatal seizures: diagnostic and prognostic considerations.

We recruited 275 full term and preterm infants into a prospective evaluation of continuous four channel electroencephalographic (EEG) monitoring in the diagnosis and prognosis of neonatal seizures. EEG seizure activity was found in 55 infants; clinical signs were completely simultaneous in only 12 of these, they were present but limited in another 20, and were completely absent in the remaining 23. EEG seizure activity, with or without clinical signs, were equally associated with serious cerebral lesions and with adverse clinical outcome. The four channel EEG recording provided sufficient data on abnormality to be prognostically specific in 79% of the 43 infants who either died or had serious neurological impairment.

Clinical and EEG response to anticonvulsants in neonatal seizures.

During a two year period prospective continuous electroencephalographic (EEG) monitoring of 275 infants identified seizure activity in 55 cases, 31 of whom were treated with anticonvulsant drugs on clinical grounds. EEG and clinical response was complete in only two and equivocal in another six. Clinical response with persistent EEG seizures occurred in 13 and neither clinical nor EEG response in 10. There was no significant improvement in the generally poor neurological outcome compared with that in 24 infants whose seizures were not treated because of limited or absent clinical manifestations. Background EEG abnormality (as an index of associated cerebral dysfunction) was a guide to potential lack of response to anticonvulsant drugs; it was also predictive of subsequent clinical outcome irrespective of treatment. This study shows that commonly used anticonvulsant drugs (phenobarbitone, paraldehyde, phenytoin, and diazepam) have little effect on seizure control or neurological outcome in neonatal seizures associated with haemorrhagic, hypoxic, or ischaemic cerebral lesions. In view of the variable clinical appearance of EEG seizure activity, continuous EEG monitoring should be an essential feature of further study of neonatal anticonvulsant treatment.

Predictive value of early continuous electroencephalogram monitoring in ventilated preterm infants with intraventricular hemorrhage.

The contribution of early continuous four-channel EEG monitoring to the evaluation of intraventricular hemorrhage in acutely ill preterm infants mechanically ventilated for acute respiratory distress was assessed in a prospective study of 54 infants of less than 34 weeks' gestation. Early abnormal EEG results correlated significantly with later outcome. They often preceded ultrasound evidence of hemorrhage and provided prognostically significant functional correlation with the grade of hemorrhage. Continuous EEG monitoring allows collection of significant data with minimal interference and could contribute to clinical management of high-risk preterm infants.