MS

thesisThe term specific capsular reaction has been used in this thesis instead of capsular swelling reaction or "quelling" because no data were available to indicate that an increase in capsular size occurred when specific antiserum and encapsulated staphylococci were combined. When specific antiserum and organisms were mixed, the antigen-antibody reaction occurred only at the surface of the capsule. However, the cell wall, cytoplasmic matrix, and nuclear material were not affected. The present study showed that erythrocytes sensitized with capsular material prepared from the wound strain of S. aureus were agglutinated by homologous staphylococcal antiserum. Gel double-diffusion tests showed three distinct precipitation lines when the partially purified capsular Material was diluted 1:1,000 in buffered saline pH 7.2 and only one precipitation line when the partially purified capsular material was diluted 1:10,000. However. since the partially purified capsular material used for the sensitization of rabbit erythrocytes was diluted 1:10,000 in buffered saline and since it showed only one precipitation line at this concentration in gel double-diffusion tests. it was justifiable to ascribe the sensitization of rabbit erythrocytes as probably due to the capsular antigen. The specificity of the hemagglutination reaction was clearly shown by inhibition of hemagglutination by microgram quantities of the partially purified capsular material prepared from the wound strain of S. aureus. Capsular material from the encapsulated wound strain and a nonencapsulated variant of it was detected in culture supernatant fluids by inhibition of indirect hemagglutination. The encapsulated wound strain retained capsular material on its surface as shown by the specific capsular reaction while the nonencapsulated variant did not. The increased virulence of the encapsulated wound strain for embrjonated hens' eggs as compared to the nonencapsulated variant derived from it was believed to be due to a better retention of capsular material. In the present work the specific capsular reaction was used exclusively to demonstrate capsules on the Smith diffuse variant and to demonstrate their absence on the compact variant of the Smith strain of S. aureus. Morse (1962a), Koenig (1962) and Modd and DeCourcy (1965) were unable to elicit a specific capsular reaction using the Smith strain. but workers in this laboratory succeeded in this endeavor by employing both rooster and rabbit antistaphylococeal serum. Morse used the Smith diffuse strain of S, aureus in all of his experiments (personal communication). India ink preparations of this strain showed that the organism was surrounded by an envelope structure (Morse, 1962a). Mudd and DeCourcy (1965) reported that the encapsulated Smith strain of S.aureus grew as a compact colony in normal rabbit serum. He reported that this staphylococcus was also surrounded by an envelope structure as demonstrated by the India ink method. Although this thesis does not dispute the validity of these observations, his data tend to obscure the basic question, namely, whether or not the virulent form of the Smith strain is encapsulated and grows as a diffuse colony in serum or plasma soft agar. Growth of the Smith strain of S. aureus in normal rabbit serum soft agar as a compact colony has usually been interpreted (Koenig, 1962; Koenig et al., 1962a, 1962b; Koenig and Melly, 1965) as an indication of nonencapsulation and characteristic of the avirulent form of the Smith strain of S. aureus, but Mudd and DeCourcy (1965) reported that their culture of the encapsulated Smith strain of S. aureus grew as a compact colony in normal rabbit serum soft agar. Electron photomicrographs of the Smith diffuse variant of S. aureus showed the presence of a capsule (Koenig and Melly, 1965). However, Koenig (1962) was unable to find any difference in the diffuse or compact Smith strains of S. aureus when these strains were examined in India ink preparations. To use wet India ink preparations as the only criterion for the demonstration of capsules of S. aureus could lead to confusion. It should be re-emphasized that the most definitiive method for the demonstration of capsules of S. aureus is the specific capsular reaction. In his research Mudd (1965) maintained that the encapsulated Smith strain of S. aureus should be considered as the prototype of encapsulated S, aureus strains and that the wound strain isolated in this laboratory should be considered as a representative of a phenomenon which he called the extracellular peripheral precipitation reaction (EPPR). The latter term, EPPR, seems to be only a circumlocution used to describe the specific capsular reaction,, Mudd and DeCourcy (1965) would exclude the wound strain of S. aureus as being truly encapsulated because they would restrict true encapsulation to virulent naturally occurring strains. The wound strain in its encapsulated state was obtained by the method employed by Bigger„ Boland and 0'Mera (1927). Its virulence in embryonated hens' eggs is indisputable (Wiley. 1961)„ Naturally occurring encapsulated S. aureus of the wound mucoid type-have been isolated from rabbits, roosters, mice, and humans. Each isolate exhibited a positive specific capsular reaction upon initial isolation (Wiley, 1961, 1963). Wiley (1961) devised and employed a virulence test in embryonated hens' eggs which is well suited to the testing of the virulence of S. aureus isolates. In this test, the wound strain behaved as a virulent strain with an LD50 of less than 500 organisms. The finding of anticapsular antibodies that react with the wound strain in normal rabbits, roosters, mice, and humans (Wiley, 1961, 1963) further supports the validity of this position. Apparently, the wound strain is a major capsular type of S. aureus, but not the only one. Evidence presented in this thesis indicates, for the first time, that the Smith diffuse variant is also encapsulated, as shown by the specific capsular reaction0 The wound mucoid strain and the RLM strain of Price and Kneeland represent members of a major capsular type against which antibodies are widely distributed (Wiley, 1961, 1963). No detectable differences were found between the wound mucoid strain and the Smith diffuse variant, regarding the amount of hemolysin present in cultures. Differences in the amount of coagulase formed by each strain were not great enough to explain the difference in virulence observed. Apparently, the best explanation is that the mice were resistant to the wound strain because they carried it„ and they had elaborated antibodies against it. Our tests did not reveal that mice carried the Smith capsular type of S, aureus, nor could we detect anticapsular antibodies against the latter

PhD

dissertationThe Smith diffuse variant and the wound mucoid strain of Staphylococcus aureus were shown to exhibit serologically distinct capsules. The Welwood and K-6 strains of S. aureus were tested to determine their capsular types. Both Welwood and K-6 were found to be representative of the Smith capsular type. An additional 13 isolated of S. aureus from mice were tested. Gel double-diffusion tests and immunoelectrophoresis of staphylococcal antigens disclosed the probable existence of at least two additional capsular types. Passive hemagglutination tests carried out with cells sensitized with 1 mg of antigen per ml showed a multiplicity of cross-reacting antigens. However, cells sensitized either with 0.1 or 0.05 mg of antigen per ml and reacted with antisera absorbed with 10 or 1 ug/ml of homologous antigen showed the presence of specific antigen of extracts from each strain of S. aureus. Corroborative evidence for multiplicity of capsular types was obtained by the specific capsular reaction. At least four capsular types of S. aureus were found. The prototypic strains for these antigens are the RLM or wound strain, the Smith diffuse strain, and mouse strains designated 36T and 43R. It was proposed to designated these types 1, 2, 3, and 4, respectively. Three serologically active components in partially purified capsular material (PPCM) from the wound strain S. aureus were separated by column chromatography on Sepharose 6B. Chemical analyses of the components following acid hydrolysis showed no significant qualitative differences in their amino acid contents. The 73-80 ml pool showed 37% glucosamine and 36% reducing sugars. A significant difference was noticed in the glucosamine and reducing sugar values of the 73-80 ml pool as compared with the other pools. Immunoelectrophoretic analysis showed the 73-80 ml pool contained a single serologically active component which migrated anodally. Gel diffusion investigations confirmed the presence of the same serologically active component in PPCM by showing the present of a reaction of identity between the PPCM and 73-80 ml pool when they were reacted with specific antiserum.. Antiserum absorption studies revealed that the 58-72 ml, 73-/0 ml, and 81-100 ml pools all contained capsular antigen. The most active pool in the anticapsular antibody absorption test was the 73-80 ml pool. This fraction was twice as active as the 58-72 ml pool and eight times more active than the 81-100 ml pool in the absorption of anticapsular antibodies

CAPSULE PRODUCTION AND VIRULENCE AMONG STRAINS OF STAPHYLOCOCCUS AUREUS

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74661/1/j.1749-6632.1974.tb41493.x.pd

Topical clobetasol for the treatment of toxic epidermal necrolysis: study protocol for a randomized controlled trial.

BackgroundToxic epidermal necrolysis (TEN) is a rare systemic allergic drug eruption with high patient mortality. Currently, no established treatments have been shown to be effective for TEN beyond supportive care. Prior studies of systemic corticosteroids have yielded conflicting data, with some showing a possible benefit and others reporting in increased mortality. However, topical steroids have shown promise for treatment of ocular sequelae of TEN, such as scarring and vision loss. We have designed a randomized controlled trial to evaluate topical clobetasol for treatment of the epidermal manifestations of TEN. In addition, we propose genetic studies to characterize the TEN transcriptome and alterations in cutaneous gene expression that might occur following topical steroid treatment.Methods/designThis split-body randomized, double-blind, placebo-controlled Phase IIa proof-of-concept trial will evaluate the safety and efficacy of once-daily topical clobetasol applied to the skin of patients with TEN. This multicenter trial will recruit a total of 15 patients between the ages of 12 and 85 from the University of California Davis Medical Center and Shriners Hospital for Children inpatient burn units. Designated treatment areas on opposite sides of the body will be treated with blinded clobetasol 0.05% ointment or control petrolatum ointment daily for 14 days. On day 3 of therapy, a biopsy will be taken from the treated area for genetic studies. The primary study aims will be to establish the safety of topical clobetasol treatment and determine the time to cessation of skin detachment for the control and clobetasol-treated areas. Secondary endpoints will evaluate efficacy using parameters such as time to 90% re-epithelialization and percentage of affected skin at 0, 3, 6, 9, 12 and 15 days. Genomic DNA and RNA will be obtained from biopsy samples, to characterize the TEN transcriptome and identify changes in gene expression after topical steroid treatment.DiscussionTopical steroids have shown promise for treating ocular complications of TEN, but to date have not been evaluated for cutaneous manifestations of the disease. This trial will investigate clinical and molecular outcomes of topical clobetasol application and hopefully provide insight into the disease pathophysiology.Trial registrationClinicalTrials.gov NCT02319616. https://clinicaltrials.gov/ct2/show/NCT02351037

Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation.

Given that unnatural sugar expression is metabolically achieved, the kinetics and disposition of incorporation can lend insight into the temporal and localization preferences of sialylation across the cell surface. However, common detection schemes lack the ability to detail the molecular diversity and distribution of target moieties. Here we employed a mass spectrometric approach to trace the placement of azido sialic acids on membrane glycoconjugates, which revealed substantial variations in incorporation efficiencies between N-/O-glycans, glycosites, and glycosphingolipids. To further explore the propensity for sialylation, we subsequently mapped the native glycome of model epithelial cell surfaces and illustrate that while glycosylation sites span broadly across the extracellular region, a higher number of heterogeneous glycoforms occur on sialylated sites closest to the transmembrane domain. Beyond imaging techniques, this integrative approach provides unprecedented details about the frequency and structure-specific distribution of cell surface sialylation, a critical feature that regulates cellular interactions and homeostatic pathways

Dietary supplementation with Bifidobacterium longum subsp. infantis (B. infantis) in healthy breastfed infants: study protocol for a randomised controlled trial.

BackgroundThe development of probiotics as therapies to cure or prevent disease lags far behind that of other investigational medications. Rigorously designed phase I clinical trials are nearly non-existent in the field of probiotic research, which is a contributing factor to this disparity. As a consequence, how to appropriately dose probiotics to study their efficacy is unknown. Herein we propose a novel phase I ascending dose trial of Bifidobacterium longum subsp. infantis (B. infantis) to identify the dose required to produce predominant gut colonisation in healthy breastfed infants at 6 weeks of age.Methods/designThis is a parallel-group, placebo-controlled, randomised, double-blind ascending dose phase I clinical trial of dietary supplementation with B. infantis in healthy breastfed infants. The objective is to determine the pharmacologically effective dose (ED) of B. infantis required to produce predominant (>50 %) gut colonisation in breastfed infants at 6 weeks of age. Successively enrolled infant groups will be randomised to receive two doses of either B. infantis or placebo on days 7 and 14 of life. Stool samples will be used to characterise the gut microbiota at increasing doses of B. infantis.DiscussionProbiotic supplementation has shown promising results for the treatment of a variety of ailments, but evidence-based dosing regimes are currently lacking. The ultimate goal of this trial is to establish a recommended starting dose of B. infantis for further efficacy-testing phase II trials designed to evaluate B. infantis for the prevention of atopic dermatitis and food allergies in at-risk children.Trial registrationClinicaltrials.gov # NCT02286999 , date of trial registration 23 October 2014

Infection-generated electric field in gut epithelium drives bidirectional migration of macrophages.

Many bacterial pathogens hijack macrophages to egress from the port of entry to the lymphatic drainage and/or bloodstream, causing dissemination of life-threatening infections. However, the underlying mechanisms are not well understood. Here, we report that Salmonella infection generates directional electric fields (EFs) in the follicle-associated epithelium of mouse cecum. In vitro application of an EF, mimicking the infection-generated electric field (IGEF), induces directional migration of primary mouse macrophages to the anode, which is reversed to the cathode upon Salmonella infection. This infection-dependent directional switch is independent of the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. The switch is accompanied by a reduction of sialic acids on glycosylated surface components during phagocytosis of bacteria, which is absent in macrophages challenged by microspheres. Moreover, enzymatic cleavage of terminally exposed sialic acids reduces macrophage surface negativity and severely impairs directional migration of macrophages in response to an EF. Based on these findings, we propose that macrophages are attracted to the site of infection by a combination of chemotaxis and galvanotaxis; after phagocytosis of bacteria, surface electrical properties of the macrophage change, and galvanotaxis directs the cells away from the site of infection

2D Visualization of the Psoriasis Transcriptome Fails to Support the Existence of Dual-Secreting IL-17A/IL-22 Th17 T Cells.

The present paradigm of psoriasis pathogenesis revolves around the IL-23/IL-17A axis. Dual-secreting Th17 T cells presumably are the predominant sources of the psoriasis phenotype-driving cytokines, IL-17A and IL-22. We thus conducted a meta-analysis of independently acquired RNA-seq psoriasis datasets to explore the relationship between the expression of IL17A and IL22. This analysis failed to support the existence of dual secreting IL-17A/IL-22 Th17 cells as a major source of these cytokines. However, variable relationships amongst the expression of psoriasis susceptibility genes and of IL17A, IL22, and IL23A were identified. Additionally, to shed light on gene expression relationships in psoriasis, we applied a machine learning nonlinear dimensionality reduction strategy (t-SNE) to display the entire psoriasis transcriptome as a 2-dimensonal image. This analysis revealed a variety of gene clusters, relevant to psoriasis pathophysiology but failed to support a relationship between IL17A and IL22. These results support existing theories on alternative sources of IL-17A and IL-22 in psoriasis such as a Th22 cells and non-T cell populations

Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice.

Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF(+) macrophages in response to αCD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with αCD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon αCD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT