Antibiotic Treatment of Clostridium difficile Carrier Mice Triggers a Supershedder State,Spore-Mediated Transmission,and Severe Disease in Immunocompromised Hosts

Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88^−/−) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures.Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that can reside asymptomatically in the intestinal tract of humans (6, 11, 42). The use of antibiotics that spare C. difficile but suppress the intestinal microbiota allows C. difficile to proliferate (23), potentially leading to intestinal damage, inflammation, and clinical disease (9). In most cases stopping antibiotic therapy is often sufficient to prevent or reverse disease symptoms in immunocompetent individuals (5, 32). However, in immunocompromised hospital patients, particularly the elderly, intestinal disease can quickly develop after antibiotic treatment, with clinical outcomes ranging from mild diarrhea to pseudomembraneous colitis to multiple-organ dysfunction syndrome (17, 31).Unlike most pathogens, C. difficile produces a metabolically dormant spore form that is excreted by infected patients (43, 63). The infective spores persist in the environment and are highly resistant to commonly used disinfectants (24). Indeed, environmental spore contamination in a hospital results in a reservoir for transmission (21, 46) that can lead to a proportional increase in the percentage of the patient population colonized by C. difficile (33). As a result, C. difficile is endemic in many hospitals, and outbreaks are difficult to contain, highlighting the compelling need to understand the spore-mediated infection cycle and the factors that lead to C. difficile transmission (24, 59).Most studies of C. difficile pathogenesis in animals have focused on the acute stage of infection (13, 38, 45, 57), so many aspects of the C. difficile infection cycle have not been investigated in detail, including intestinal carriage, interactions with the microbiota, the role of spores in transmission, and host susceptibility to severe disease. Recent reports that various virulent C. difficile ribotypes can colonize several mammalian hosts (25, 30, 48, 51, 52) led us to reinvestigate the mouse as a model for the C. difficile infection cycle. We found that C. difficile strain M68, a representative ribotype that frequently causes human disease (19), was very proficient at persisting in inbred mice. Here we describe the impact of antibiotics on the composition of the intestinal microbiota of murine C. difficile M68 carriers and reveal that antibiotics can inadvertently trigger high-level spore excretion and remarkably efficient host-to-host transmission of C. difficile. Further, we demonstrate that transmission of C. difficile to immunocompetent mice leads to self-limiting intestinal disease, whereas transmission to Myd88^−/− mice, which are defective in a key signaling pathway in the innate immune response, leads to severe intestinal disease and multiple-organ dysfunction syndrome, potentially mimicking the situation in humans.     

Modulation of Vacuolar pH Is Required for Replication of Edwardsiella ictaluri in Channel Catfish Macrophages

Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H⁺-ATPases by use of the specific inhibitor bafilomycin A₁ abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.     

Clinical Assessment of Scapular Positioning in Musicians: An Intertester Reliability Study

Context:

The reliability of the measurement of the distance between the posterior border of the acromion and the wall and the reliability of the modified lateral scapular slide test have not been studied. Overall, the reliability of the clinical tools used to assess scapular positioning has not been studied in musicians.

Objective:

To examine the intertester reliability of scapular observation and 2 clinical tests for the assessment of scapular positioning in musicians.

Design:

Intertester reliability study.

Setting:

University research laboratory.

Patients or Other Participants:

Thirty healthy student musicians at a single university.

Main Outcome Measure(s):

Two assessors performed a standardized observation protocol, the measurement of the distance between the posterior border of the acromion and the wall, and the modified lateral scapular slide test. Each assessor was blinded to the other''s findings.

Results:

The intertester reliability coefficients (κ) for the observation in relaxed position, during unloaded movement, and during loaded movement were 0.41, 0.63, and 0.36, respectively. The κ values for the observation of tilting and winging at rest were 0.48 and 0.42, respectively; during unloaded movement, the κ values were 0.52 and 0.78, respectively; and with a 1-kg load, the κ values were 0.24 and 0.50, respectively. The intraclass correlation coefficient (ICC) of the measurement of the acromial distance was 0.72 in relaxed position and 0.75 with the participant actively retracting both shoulders. The ICCs for the modified lateral scapular slide test varied between 0.63 and 0.58.

Conclusions:

Our results demonstrated that the modified lateral scapular slide test was not a reliable tool to assess scapular positioning in these participants. Our data indicated that scapular observation in the relaxed position and during unloaded abduction in the frontal plane was a reliable assessment tool. The reliability of the measurement of the distance between the posterior border of the acromion and the wall in healthy musicians was moderate.     

Ameliorating effect of microdoses of a potentized homeopathic drug,Arsenicum Album,on arsenic-induced toxicity in mice

Background  

Arsenic in groundwater and its accumulation in plants and animals have assumed a menacing proportion in a large part of West Bengal, India and adjoining areas of Bangladesh. Because of the tremendous magnitude of the problem, there seems to be no way to tackle the problem overnight. Efforts to provide arsenic free water to the millions of people living in these dreaded zones are being made, but are awfully inadequate. In our quest for finding out an easy, safe and affordable means to combat this problem, a homeopathic drug, Arsenicum Album-30, appears to yield promising results in mice. The relative efficacies of two micro doses of this drug, namely, Arsenicum Album-30 and Arsenicum Album-200, in combating arsenic toxicity have been determined in the present study on the basis of some accepted biochemical protocols.     

抗血管生成因子Angiostatin与Endostatin作用下肿瘤血管生成的二维数值模拟

目的数值模拟抗血管生成因子Angiostatin和Endostatin对肿瘤血管生成的影响。方法建立肿瘤内外血管生成的二维离散数学模型。模型耦合两种抗血管生成因子Angiostatin和Endostatin的抑制效应,数值模拟在促血管生成因子诱导下肿瘤微血管网生成,讨论血管生成抑制因子的影响。结果抗血管生成因子Angiostatin对肿瘤内外血管网络生成的速度和成熟度有抑制作用。抗血管生成因子Angiostatin和Endostatin耦合作用时,在肿瘤血管生成的早期有明显的抑制效应;在肿瘤血管生成的中后期,它们可以降低肿瘤血管化程度。结论本文模型能够较好的模拟抗血管生成因子Angiostatin和Endostatin对内皮细胞迁移和增殖的抑制作用。     

Validation of the Hamilton Depression Rating Scale and Montgommery and Asberg Rating Scales in terms of AGECAT depression cases

OBJECTIVE. To validate the Hamilton Depression (17) and Montgommery and Asberg Depression Scales as research instruments in older depressed community residents. DESIGN. External validation against GMS/AGECAT case level in the recruitment of older community residents for an antidepressant trial. ANALYSES. Receiver operator curves were generated for each rating scale, using GMS/AGECAT case level in external criterion. The sensitivity, specificity, positive and negative predictive values of both rating instruments were examined in the whole sample and age and gender subgroups. MADRS and HAM-D cut-off scores differentiating GMS/AGECAT cases from subcases were identified. RESULTS. HAM-D cut-off score of 16 and MADRS score of 21 were identified as differentiating case from sub-case. Diagnostic accuracy of both instruments was good, reflecting good sensitivity and specificity across both genders and sub-age groups. CONCLUSIONS. Both scales performed well in this population. These scores provide researchers with externally validated and clinically relevant cut-off scores in designing trials in the management of older depressed community residents.     

Benzo[a]pyrene at an environmentally relevant dose is slowly absorbed by, and extensively metabolized in, tracheal epithelium

While tobacco smoke has been conclusively identified as a lung carcinogen, there is much debate over which smoke constituent(s) are primarily responsible for its carcinogenicity. Previous studies in our laboratory suggested that highly lipophilic carcinogens are slowly absorbed in the thicker epithelium of the conducting airways, potentially allowing for substantial local metabolism. The bioactivation of polycyclic aromatic hydrocarbons in airway epithelium may, hence, be important in tobacco smoke-induced carcinogenesis. In the present study, the hypothesis of slow absorption and substantial local metabolic activation of highly lipophilic carcinogen in airway epithelium was tested in dogs. A single dose of tritiated benzo[a]pyrene (BaP) dissolved in a saline/phospholipid suspension was instilled in the trachea, just anterior to the carina. At intervals of a few minutes up to 30 min over a 3-h period, blood samples were drawn from the azygous vein, which drains the area around the point of instillation, and from the systemic circulation. Tissue samples were taken at the end of the experiment. The concentration of BaP with depth into the tracheal mucosa was determined with autoradiography. BaP was slowly absorbed into the trachea with a half-time of approximately 73 min, which is consistent with diffusion-limited passage through the epithelium and lead to local doses in the tracheal epithelium that were more than a 1000-fold those of other tissues. The long retention of BaP in the epithelium provided the local metabolizing enzymes with high substrate levels over a long period, resulting in extensive metabolism. At 3 h after the exposure, 23% of the BaP-equivalent activity remained in the tracheal mucosa. Of this fraction, 13% was parent compound, 28% was organic extractable, 31% was water-soluble, and 28-7% of the instilled dose was bound to tracheal tissues. These results explain the tendency of highly lipophilic carcinogens, such as BaP, to induce tumors at the site of entry and, furthermore, indicate that the highly lipophilic components of tobacco smoke and polluted air may be the most important contributors to lung tumors of the conducting airways.      

Need for follow up in coeliac disease

The use of follow up studies was evaluated in 128 patients with coeliac disease during their first visit to a department for adults. The original diagnosis had been made in childhood in all patients. Fifty eight (45%) of the subjects were following a gluten free diet, 23 (18%) were following a gluten free diet but with occasional gluten consumption, and 47 (37%) had adopted an unrestricted, gluten containing diet for a mean of 11.2 years. There was no correlation in individual subjects between the presence of symptoms, biochemical and immunological abnormalities, severity of histological findings, and the amount of dietary gluten, despite the greater frequency of symptoms in the group following an unrestricted diet than in the other two groups. Short stature and epilepsy with cerebral calcifications only occurred in patients following an unrestricted diet. As only diagnosis based on two or three biopsy samples and regular follow up correlated positively with dietary compliance, it is suggested that a histologically confirmed diagnosis of coeliac disease and regular lifelong follow up are essential in the management of these patients.     

Elderly Patients with Suppressed Serum TSH but Normal Free Thyroid Hormone Levels Usually Have Mild Thyroid Overactivity and are at Increased Risk of Developing Overt Hyperthyroidism

The clinical and biochemical characteristics of 15 elderly patientswith low levels of thyrotrophin (TSH) (<0.1 mU/L) but normalfree tri-iodothyronine, (T3) and free thyroxine (T4) (groupS) were compared with 10 euthyroid subjects (group E) and 10hyperthyroid patients (group T). Free T3 and free T4 were significantlyhigher (p<0.05) in group S(6.3±0.5 and 18.6±1.0pmol/l, respectively) than in group E(4.6±0.3, 12.6+0.6).In common with elderly hyperthyroid patients (group T)patientsin group S had few signs or symptoms of thyrotoxocosis, butthe Wayne score (clinical index of hyperthyroidism) was higherin group S than in euthyroid subjects (p<0.05). Thyroid microsomal,thyrogolobulin or thyrotrophin receptor antibodies were commonin group T (n=9)but not in groups S(n=2) or E(n=1). This suggestsa low prevalence of Graves' disease in group S compared to groupT. Combined thyrotrophin releasing hormone (TRH; 200 µgi.v.) and gonadotrophin releasing hormone GnRH; 100 µgi.v.) tests were performed; no cases of low TSH due to hypopituitarismwere identified in group S. During a mean of 7.9 (4–12)months of observation TSH reverted to the normal range (>0.2mU/L)in 7 of 15 patients in group S; thyroid hormone concentrationsrose above the normal range in four, however, only two patientsrequired treatment for hyperthyroidism. It is unlikely thatthe suppressed TSH of patients in group S was due to mild thyroidhormone excess; although this is often a transitory phenomenon,these patients are at increased risk of developing overt hyperthyroidism.