Improved Visualization of Oral Microbial Consortia.

Bacteria on the tongue dorsum (TD) form consortia tens to hundreds of microns in diameter organized around a core of epithelial cells. Whole-mount preparations have been instrumental in revealing their organization and specific microbial associations. However, their thickness and intricate 3-dimensional complexity present challenges for a comprehensive spatial analysis. To overcome these challenges, we employed a complementary approach: embedding in hydrophilic plastic followed by sectioning and postsectioning labeling. Samples were labeled by hybridization with multiplexed fluorescent oligonucleotide probes and visualized by spectral imaging and linear unmixing. Application of this strategy to TD biofilms improved the visualization of bacteria that were difficult to resolve in whole-mount imaging. Actinomyces, previously detected as patches, became resolved at the single-cell level. The filamentous taxa Leptotrichia and Lachnospiraceae, located at the core of the consortium, were regularly visualized whereas previously they were rarely detected when using whole mounts. Streptococcus salivarius, heterogeneously detected in whole mounts, were regularly and homogenously observed. Two-dimensional images provide valuable information about the organization of bacterial biofilms. However, they offer only a single plane of view for objects that can extend to hundreds of microns in thickness, and information obtained from such images may not always reflect the complexity of a 3-dimensional object. We combined serial physical sectioning with optical sectioning to facilitate the 3-dimensional reconstruction of consortia, spanning over 100 µm in thickness. Our work showcases the use of hydrophilic plastic embedding and sectioning for examining the structure of TD biofilms through spectral imaging fluorescence in situ hybridization. The result was improved visualization of important members of the human oral microbiome. This technique serves as a complementary method to the previously employed whole-mount analysis, offering its own set of advantages and limitations. Addressing the spatial complexity of bacterial consortia demands a multifaceted approach for a comprehensive and effective analysis.

Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

BACKGROUND: Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. METHODS: Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. RESULTS: Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. CONCLUSIONS: LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Course and outcome of acute limbic encephalitis with negative voltage-gated potassium channel antibodies.

BACKGROUND: Limbic encephalitis is a potentially treatable immunological condition. The presence of voltage-gated potassium channel antibodies (VGKC-Ab) in the cerebrospinal fluid (CSF) and serum of patients with the condition is a marker of the disease associated with a non-paraneoplastic form and good response to treatment. Recent work has highlighted absent serum VGKC-Ab and distinct immunology in patients with the paraneoplastic form of limbic encephalitis. METHODS: The cases of four patients with the typical clinical presentation, neuropsychological features and brain imaging of acute limbic encephalitis, in the absence of any evidence for associated cancer during a follow-up of at least 18 months are described here. RESULTS: All patients had negative testing for VGKC-Ab measured during their acute presentation. All patients made some recovery, although they were left with marked cognitive deficits and persistent seizures. CONCLUSION: These cases demonstrate that the absence of VGKC-Ab in limbic encephalitis does not necessarily imply a paraneoplastic form. Further work is required to establish the immunological basis for the disorder in these patients, and the optimal treatment regimen.

Down-regulation of prostasin serine protease: a potential invasion suppressor in prostate cancer.

BACKGROUND: Prostasin is a serine protease predominantly expressed in normal prostate epithelial cells. The biological function of prostasin has not been determined. METHODS: Western blot and RT-PCR analyses were used to examine the expression of prostasin in prostate cancer cell lines. Immunohistochemistry was used to evaluate prostasin protein expression in human prostate cancer. An in vitro Matrigel invasion assay was used to test the invasiveness of prostate cancer cell lines forced to express recombinant prostasin. RESULTS: Both prostasin protein and mRNA were found to be expressed in normal human prostate epithelial cells and a non-invasive human prostate cancer cell line, the LNCaP, but neither was found in invasive human prostate cancer cell lines DU-145 and PC-3. Prostasin mRNA expression was absent in invasive prostate cancer cell lines of a transgenic mouse model. Immunohistochemistry analysis showed that prostasin protein expression is down-regulated in high-grade prostate cancer. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68 and 42%, respectively. CONCLUSIONS: Our data indicate that prostasin may be implicated in normal prostate biology and is able to suppress prostate cancer invasion in vitro.

Hemodialysis patient beliefs by stage of fluid adherence.

Fluid limitations are difficult for hemodialysis patients to follow, and factors related to fluid adherence are not well known. The primary purpose of this study, which was guided by the health belief model and the transtheoretical model for behavior change, was to determine whether differences exist in perceived benefits, barriers, susceptibility, seriousness, or self-efficacy among individuals in different stages of fluid adherence. In a cross-sectional descriptive design, 148 persons (mean age=53.9 years; 52% male) were interviewed using a structured format. Hemodialysis patients in the action/maintenance stage perceived significantly more benefits to fluid adherence than persons in the precontemplation stage. Persons who were in the action/maintenance stage perceived they were significantly more susceptible to pulmonary edema than persons in the contemplation stage. Stage-appropriate interventions may be needed to target specific beliefs about fluid adherence.

Stressors, coping and depression in haemodialysis patients.

BACKGROUND: Depression is common in persons receiving outpatient haemodialysis, but little work has been done to explore the variables associated with depression. AIMS: The primary purposes of this study were to (i) examine relationships among stressors, coping and depression and (ii) test the mediating role of coping. DESIGN/METHODS: Data were collected at two points in time, three months apart in 1995/1996. The final convenience sample at Time 2 was 86 participants from two United States midwestern, inner-city dialysis units. Structured interviews were conducted using the Centre for Epidemiologic Studies Depression Scale, the haemodialysis stressor scale (HSS) and the coping strategy indicator. RESULTS: At Time 1 more psychosocial stressors were associated with greater use of problem-solving, social-support and avoidance coping. Both avoidance coping and more psychosocial stressors at Time 1 were related to depression at Time 2. Finally, avoidance coping was found to explain much of the relationship between psychosocial stressors and depression. CONCLUSIONS: Research is now needed that explicates the causal relationships among stress, coping and depression in haemodialysis patients.

Integrating theory and research into practice.

An experiential learning assignment was imPlemented in an undergraduate nursing course. The purpose of the assignment was to integrate theory and research into a nursing practice activity. The authors describe the development, implementation, and evaluation of the assignment. Evaluation data indicated that the assignment was an appropriate way to prepare beginning nursing students for building knowledge about nursing as a scholarly discipline.

Genomic structure and chromosome location of the mouse RelA p65 gene (Rela).

The RelA (p65) subunit of transcription factor NF-kappaB plays a critical role in development, and rela(-/-) knockout mice die in utero from massive liver apoptosis. Only partial sequences of the mouse Rela gene are available. We have determined the genomic structure of mouse Rela and promoter, and have mapped the gene to chromosome 19B1-3.

Self-care strategies to reduce fluid intake and control thirst in hemodialysis patients.

The purpose of this pilot study was to describe how often hemodialysis (HD) patients used particular self-care strategies to reduce fluid intake or relieve thirst, how effective each strategy was perceived to be, and whether they planned to use that strategy in the future. A cross-sectional descriptive design was used. The sample of 20 participants from one Midwest dialysis unit verbally responded to the self-care items that were presented. Most of these patients reported they regularly avoided the sun, took medications with mealtime fluid, limited salt on their foods, avoided salty foods, stayed busy, stayed away from fast food restaurants, drank ice-cold drinks, and drank only when thirsty to reduce fluid intake and relieve thirst. They also planned to use these strategies in the future. Interestingly, the strategies perceived as most effective--measuring daily allotted fluids in a pitcher to drink from all day, spacing liquids over the entire day, and measuring the amount of fluid they drank--were not the same as those they used most. Replication of these findings with larger samples is strongly recommended.

Quality of life in black hemodialysis patients.

The objectives of this study were to describe quality of life in a sample of black in-center hemodialysis patients, to identify relationships between quality of life (QOL) and selected demographic and illness variables, and to identify changes in quality of life over time. Data were collected at two points in time, 3 months apart. The data were obtained from 79 patients in two inner-city dialysis units and included persons new to dialysis. Structured interviews were conducted using the Quality of Life Index. On the average, these patients were satisfied with their QOL, although there was a large range of scores. Psychological/spiritual QOL (M = 4.27) was higher than health and functioning quality of life (M = 3.77) at both time periods. Younger age (M = 4.07) and more education (M = 4.00) were associated with poorer psychological/spiritual QOL at Time 1, whereas lower hematocrits (M = 3.55) and being new to dialysis (M = 3.41) were associated with poorer health and functioning QOL. The QOL of these black hemodialysis patients was fairly high and similar to the QOL previously reported for whites, suggesting that interventions to improve QOL in hemodialysis patients do not need to be tailored by race. In addition, findings suggest that nursing support may be the most needed during the early stages of dialysis.

Fragile X syndrome.

Fragile X syndrome is the most common inherited condition causing mental retardation in males. Females with the full mutation expansion can have milder signs of the disorder. Families with members who have been diagnosed with fragile X syndrome face concerns about the health of their newborn infant, decisions regarding family planning, and questions about the possibility that other family members could have this disorder. Neonatal nurses participate in assessment, health care management, counseling, and referral of the families regarding this syndrome.

Factors associated with treatment-related stressors in hemodialysis patients.

The objectives of this study were to describe the treatment-related stressors of incenter hemodialysis patients, to identify relationships between stressors and selected demographic and illness variables, and to identify changes in stressors over time. Data were collected at two points in time, 3 months apart. The data were obtained from 86 patients in 2 inner-city midwest dialysis units. Structured interviews were conducted using one open-ended question and the Hemodialysis Stressor Scale. The greatest stressors were fluid limitations, the length of dialysis, and vacation limitations. There was a consistent trend for almost all stressors to become more intense over time, with some specific stressors increasing significantly. Patients new to dialysis and those with more education had relatively more stressors. An important serendipitous finding was that some treatment-related stressors were not troublesome because subjects chose not to follow certain treatment recommendations. In-depth evaluation in relation to individual stressors is required before adequate individual intervention strategies can be developed. Nurses need to be educated about the factors that are stressful to patients, so they can support them appropriately.

Massive pulmonary embolus after surgery for renal cell carcinoma extending into the inferior vena cava: a case report.

Fatal massive pulmonary embolization following removal of an intracaval renal cell carcinoma was encountered. This case teaches that vena cava interruption should be considered as part of the perioperative management of patients with renal cell carcinoma tumor thrombus extending into the inferior vena cava.

Deletion status and intellectual impairment in Duchenne muscular dystrophy.

The authors collected Verbal, Performance and Full-scale IQs for 74 patients in whom complete analysis of the dystrophin gene for deletions and duplications had been performed. There was a significant difference in the mean Full-scale IQ between patients with deletions at the 5' and 3' ends of the gene, with no patients with 5' deletions having mental retardation. No relationship was established between mental retardation and the presence or absence of deletions or length of deletions, and similar deletions were observed in the presence and absence of mental retardation. Although distal deletions were more commonly associated with mental retardation, there was no clear evidence for a particular region of the dystrophin gene being specifically responsible for IQ. The intellectual deficit seen in DMD may be a consequence of cerebral hypoxia, ue to malfunction of smooth muscle dystrophin.

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 1. Trends across the clinical groups.

This multidisciplinary study was undertaken to record the variation in gene and protein expression in a large cohort of patients with well defined clinical phenotypes. The patients, whose ages ranged from 4 years to 66 years, spanned a wide range of disease severity. They represented the first 100 patients who had been examined in Newcastle, had undergone a muscle biopsy, and provided a blood sample for DNA analysis. The study had three aims: to observe any trends in the analyses across the clinical groups, to correlate gene and protein expression in individual patients, and to use the data collected to assess the relative usefulness of different techniques in the diagnosis and prognosis of patients with Duchenne and Becker dystrophy (DMD/BMD). In part 1, we describe the clinical assessment of the patients and the trends that were observed across the cohort. The patients were divided into seven groups. Group 1 had severe DMD (n = 21), group 2 had milder DMD (n = 20), group 3 were intermediate D/BMD patients (n = 9), group 4 had severe BMD (n = 5), and group 5 were more typical BMD patients (n = 31). Some patients were too young to be classified (n = 7) and a group of all the female patients were also classified separately (n = 7). The number of DMD and BMD patients was about equal, in accord with disease prevalence in the north of England, but an unusually high proportion were sporadic cases. Dystrophin labelling (performed with up to three antibodies) on both blots and sections increased gradually across the clinical groups. All histopathological indices, except the proportion of fat in biopsy sections, showed clear trends across the groups.

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 2. Correlations within individual patients.

This report is the second part of a trilogy from a multidisciplinary study which was undertaken to record the relationships between clinical severity and dystrophin gene and protein expression. The aim in part 2 was to correlate the effect of gene deletions on protein expression in individual patients with well defined clinical phenotypes. Among the DMD patients, most of the deletions/duplications disrupted the open reading frame, but three patients had in frame deletions. Some of the intermediate D/BMD patients had mutations which were frameshifting while others were in frame. All of the deletions/duplications in the BMD patients maintained the open reading frame and 25/26 deletions in typical BMD group 5 started with exon 45. The deletion of single exon 44 was the most common mutation in patients from groups 1 to 3. Dystrophin was detected in sections and blots from 58% of the DMD patients with a size that was compatible with synthesis from mRNA in which the reading frame had been restored. Certain deletions were particularly associated with the occurrence of limited dystrophin synthesis in DMD patients. For example, 9/11 DMD patients missing single exons had some detectable dystrophin labelling compared with 10/24 who had deletions affecting more than one exon. All patients missing single exon 44 or 45 had some dystrophin. Deletions starting or finishing with exons 3 or 51 (8/9) cases were usually associated with dystrophin synthesis whereas those starting or finishing with exons 46 or 52 (11/11) were not. Formal IQ assessments (verbal, performance, and full scores) were available for 47 patients. Mean IQ score among the DMD patients was 83 and no clear relationship was found between gene mutations and IQ. The mutations in patients with a particularly severe deficit of verbal IQ were spread throughout the gene.

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 3. Differential diagnosis and prognosis.

This report is the third part of a trilogy from a multidisciplinary study which was undertaken to investigate gene and protein expression in a large cohort of patients with well defined and diverse clinical phenotypes. The aim of part 3 was to review which of the analytical techniques that we had used would be the most useful for differential diagnosis, and which would provide the most accurate indication of disease severity. Careful clinical appraisal is very important and every DMD patient was correctly diagnosed on this basis. In contrast, half of the sporadic BMD patients and all of the sporadic female patients had received different tentative diagnoses based on clinical assessments alone. Sequential observations of quantitative parameters (such as the time taken to run a fixed distance) were found to be useful clinical indicators for prognosis. Intellectual problems might modify the impression of physical ability in patients presenting at a young age. Histopathological assessment was accurate for DMD but differentiation between BMD and other disorders was more difficult, as was the identification of manifesting carriers. Our data on a small number of women with symptoms of muscle disease indicate that abnormal patterns of dystrophin labelling on sections may be an effective way of differentiating between female patients with a form of limb girdle dystrophy and those carrying a defective Xp21 gene. Dystrophin gene analysis detects deletions/duplications in 50 to 90% of male patients and is the most effective non-invasive technique for diagnosis. Quantitative Western blotting, however, would differentiate between all Xp21 and non-Xp21 male patients. In this study we found a clear relationship between increased dystrophin abundance (determined by densitometric analysis of blots) and clinical condition, with a correlation between dystrophin abundance and the age at loss of independent mobility among boys with DMD and intermediate D/BMD. This indicates that blotting is the most sensitive and accurate technique for diagnosis and prognosis.

Coma arousal procedure: a therapeutic intervention in the treatment of head injury.

This study reports on the efficacy of a 'coma arousal procedure'. This procedure involved a programme of vigorous sensory stimulation administered to comatose patients by relatives using Comakits. An experimental group of 12 severely head-injured patients received the coma arousal procedure while a matched control group did not. Total duration of coma and weekly Glasgow Coma Scale Scores were recorded for the two groups. Results indicate that the total duration of coma was significantly shorter and that coma lightened more rapidly for the experimental group.

Visuospatial working memory in Parkinson's disease.

Visuospatial impairment is frequently reported in Parkinson's disease but the psychological mechanisms which subserve the impaired abilities and the way in which breakdown of the mechanisms leads to performance deficits have not been precisely delineated. This paper reports experimental investigations designed to test the hypothesis that the locus of the impairment is the visuospatial subsystem of working memory. Subjects were a group of sixteen patients with Parkinson's disease of mild to moderate severity and a matched control group. They performed complex visuospatial and verbal memory tasks. The Parkinsonian group were significantly slower than the control group when performing the visuospatial task. They were not significantly slower and made no more errors than the control group on the verbal task. The findings are compatible with the hypothesis that the visuospatial subsystem of working memory is impaired in Parkinson's disease. It is demonstrated that the impairment is not the result of a reduction in the capacity of this subsystem but is due to difficulty in utilising information stored in the subsystem to perform complex visuospatial tasks.

Simulation of ventilatory-induced stone movement and its effect on stone fracture during extracorporeal shock wave lithotripsy.

Because ventilation influences renal movement, we investigated the effect of stone motion on the efficiency of extracorporeal shock-wave lithotripsy (ESWL). Comparisons of the rates of fragmentation of an experimental model of renal calculi were made between simulated high-frequency jet ventilation at 100 breaths/min. with four-mm. stone movement as measured from the fluoroscope screen, conventional mechanical ventilation at 10 breaths/min. with 32-mm. stone movement as measured from the fluoroscope screen, and a static control. Fragmentation did not differ significantly between high-frequency jet ventilation and no ventilation (static control), but was significantly greater with high-frequency jet ventilation than with conventional ventilation.