Expression of the Calcium-Sensing Receptor on Normal and Abnormal Parathyroid and Thyroid Tissue.

INTRODUCTION: Current imaging techniques have several limitations in detecting parathyroid glands. We have investigated the calcium-sensing receptor (CaSR) as a potential target for specifically labeling parathyroid glands for radiologic detection. For accurate imaging it is vital that a large differential expression exists between the target tissue and adjacent structures. We sought to investigate the relative abundance of the CaSR in normal and abnormal parathyroid tissue, as well as normal and abnormal thyroid. METHODS: Existing clinical specimens were selected that represented a wide variety of pathologically and clinically confirmed malignant and benign thyroid and parathyroid specimens. Sections were stained for the CaSR using immunohistochemistry and scored for intensity and abundance of expression. (H score = intensity scored from 0 to 3 multiplied by the % of cells at each intensity. Range 0-300). RESULTS: All parathyroid specimens expressed the CaSR to a high degree. Normal parathyroid had the highest H score (271, s.d. 25.4). Abnormal parathyroid specimens were slightly lower but still much higher than normal thyroid (H score 38.3, s.d. 23.3). Medullary thyroid cancer also expressed the CaSR significantly higher than normal thyroid (H score 182, s.d. 69.1, P < 0.001) but below parathyroid levels. Hürthle cell carcinoma expressed the CaSR to a lesser degree but higher than normal thyroid (H score 101, s.d. 46.4, P = 0.0037). CONCLUSIONS: The CaSR is differentially expressed on parathyroid tissue making it a feasible target for parathyroid imaging. False positives might be anticipated with medullary and Hürthle cell cancers.

Fat-Free Spindle Cell Lipoma of the Scalp: Lipoma Without a Lipogenic Component.

ABSTRACT: Spindle cell lipoma (SCL) is a rare, benign subcutaneous neoplasm that typically occurs on the upper trunk or neck of middle-aged men. The diagnosis of SCL is often straightforward due to its characteristic clinical presentation and classic histologic features of admixed mature adipocytes and CD-34 positive bland spindle cells. However, the diagnosis can be elusive when low-fat and free-fat patterns occur. Due to the lack of lipogenic content and morphologic overlap, this rare tumor is often mistaken for other benign and malignant soft tissue tumors. The authors present the case of a middle-aged man with a fat-free SCL of the temporal scalp. To our knowledge, this is the first reported case in the literature ofa fat-free SCL involving the temporal scalp. With careful attention to the clinical context, histologic features, immunohistochemical profile, and cytogenetic abnormalities, the proper diagnosis of SCL without a lipogenic component can be achieved.

Relationship between the Viable but Nonculturable State and Antibiotic Persister Cells.

Bacteria have evolved numerous means of survival in adverse environments with dormancy, as represented by "persistence" and the "viable but nonculturable" (VBNC) state, now recognized to be common modes for such survival. VBNC cells have been defined as cells which, induced by some stress, become nonculturable on media that would normally support their growth but which can be demonstrated by various methods to be alive and capable of returning to a metabolically active and culturable state. Persister cells have been described as a population of cells which, while not being antibiotic resistant, are antibiotic tolerant. This drug-tolerant phenotype is thought to be a result of stress-induced and stochastic physiological changes as opposed to mutational events leading to true resistance. In this review, we describe these two dormancy strategies, characterize the molecular underpinnings of each state, and highlight the similarities and differences between them. We believe these survival modes represent a continuum between actively growing and dead cells, with VBNC cells being in a deeper state of dormancy than persister cells.

A 61-year-old man with erythematous forearm papules three months after liver transplantation.

A 61-year-old Caucasian man presented with papules on his left forearm and hand three months after liver transplantation: images from physical exam, pathology, and microbiology are presented. Skin biopsy confirmed the presence of fungal elements within the hair shaft, which is consistent with Majocchi granuloma, also known as nodular granulomatous perifolliculitis. A combination of fungal culture, microscopic morphology, and gene sequencing was used to identify the causative organism. The patient recovered with appropriate systemic antifungal therapy.

Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin.

The human pathogen Vibrio vulnificus is the leading cause of seafood-related deaths in the United States. Strains are genotyped on the basis of alleles that correlate with isolation source, with clinical (C)-genotype strains being more often implicated in disease and environmental (E)-genotype strains being more frequently isolated from oysters and estuarine waters. Previously, we have shown that the ecologically distinct C- and E-genotype strains of V. vulnificus display different degrees of chitin attachment, with C-genotype strains exhibiting reduced attachment relative to their E-genotype strain counterparts. We identified type IV pili to be part of the molecular basis for this observed genotypic variance, as E-genotype strains exhibit higher levels of expression of these genes than C-genotype strains. Here, we used a C-genotype quorum-sensing (QS) mutant to demonstrate that quorum sensing is a negative regulator of type IV pilus expression, which results in decreased chitin attachment. Furthermore, calcium depletion reduced E-genotype strain attachment to chitin, which suggests that calcium is necessary for proper functioning of the type IV pili in E-genotype strains. We also found that starvation or dormancy can alter the efficiency of chitin attachment, which has significant implications for the environmental persistence of V. vulnificus. With the increasing incidence of wound infections caused by V. vulnificus, we investigated a subset of E-genotype strains isolated from human wound infections and discovered that they attached to chitin in a manner more similar to that of C-genotype strains. This study enhances our understanding of the molecular and physical factors that mediate chitin attachment in V. vulnificus, providing insight into the mechanisms that facilitate the persistence of this pathogen in its native environment.

Bridging the gap between viable but non-culturable and antibiotic persistent bacteria.

Microbial dormancy is a widespread phenomenon employed by bacteria to evade environmental threats including antibiotics. This intrinsic mechanism of antibiotic tolerance has drawn special attention to the role of dormancy in human disease, particularly in regards to recurrent infections. Two dormancy states, the viable but non-culturable state and bacterial persistence, both produce antibiotic-tolerant populations capable of withstanding prolonged lethal treatment. Currently described as two distinct forms of dormancy, they are rarely discussed in the same context. We argue here that these two dormant states are closely related phenomena which are part of a shared 'dormancy continuum'. This discussion is intended to stimulate discourse about these seemingly different but very similar dormant states.

Serum Survival of Vibrio vulnificus: Role of Genotype, Capsule, Complement, Clinical Origin, and in Situ Incubation.

Virulence of the human pathogen, V. vulnificus, is associated with encapsulation, serum complement resistance, and genotype. The C-genotype of this bacterium is correlated (>90%) with virulence and with isolation source (clinical settings). E-genotype strains are highly correlated with environmental isolation (93%) but appear less virulent. In this study, we characterized the importance of genotype, encapsulation, serum complement, and in situ exposure to estuarine water on the survival of the two genotypes in human serum. Results confirmed the superior ability of C-genotype strains to survive exposure to human serum, as well as the significance of complement, and revealed that lack of capsule allowed serum killing of both C- and E-genotypes. Cells incubated in situ responded similarly to cells incubated in vitro with the exception of E-environmental strains. Interestingly, our studies found that those cells of the E-genotype, typically considered non-pathogenic, which were isolated from wound infections demonstrated serum survival similar to that of virulent, C-genotype, strains.

Interspecific quorum sensing mediates the resuscitation of viable but nonculturable vibrios.

Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.

Implications of chitin attachment for the environmental persistence and clinical nature of the human pathogen Vibrio vulnificus.

Vibrio vulnificus naturally inhabits a variety of aquatic organisms, including oysters, and is the leading cause of seafood-related death in the United States. Strains of this bacterium are genetically classified into environmental (E) and clinical (C) genotypes, which correlate with source of isolation. E-genotype strains integrate into marine aggregates more efficiently than do C-genotype strains, leading to a greater uptake of strains of this genotype by oysters feeding on these aggregates. The causes of this increased integration of E-type strains into marine "snow" have not been demonstrated. Here, we further investigate the physiological and genetic causalities for this genotypic heterogeneity by examining the ability of strains of each genotype to attach to chitin, a major constituent of marine snow. We found that E-genotype strains attach to chitin with significantly greater efficiency than do C-genotype strains when incubated at 20°C. Type IV pili were implicated in chitin adherence, and even in the absence of chitin, the expression level of type IV pilin genes (pilA, pilD, and mshA) was found to be inherently higher by E genotypes than by C genotypes. In contrast, the level of expression of N-acetylglucosamine binding protein A (gbpA) was significantly higher in C-genotype strains. Interestingly, incubation at a clinically relevant temperature (37°C) resulted in a significant increase in C-genotype attachment to chitin, which subsequently provided a protective effect against exposure to acid or bile, thus offering a clue into their increased incidence in human infections. This study suggests that C- and E-genotype strains have intrinsically divergent physiological programs, which may help explain the observed differences in the ecology and pathogenic potential between these two genotypes.

Survival of Vibrio vulnificus genotypes in male and female serum, and production of siderophores in human serum and seawater.

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. Several studies have demonstrated that V. vulnificus infections are enhanced when host iron availability is increased, such as occurs with chronic liver disease. Because of the gender difference seen in these infections, we examined whether there was a difference in the survival in both male and female serum by both the C (clinical) and E (environmental) genotypes of V. vulnificus. We further determined the significance of the catecholate and hydroxamate siderophores produced by this pathogen during both human infections and while in its natural estuarine environment. We found that only C-genotype strains were capable of growth in human serum, regardless of inoculum size, with growth in male and female serum being equal. We found the catecholate outer membrane receptor gene (vuuA) to be expressed significantly more than that for the hydroxamate siderophore (fhuA) when the cells were exposed to human serum, regardless of the genotype. When cells were exposed to natural seawater, fhuA showed increased expression over time, while vuuA showed decreased expression. Our data suggest that the catecholate siderophore is important during human infections, whereas the hydroxamate siderophore may be more important in the estuarine environment this pathogen inhabits.

Integration of Vibrio vulnificus into marine aggregates and its subsequent uptake by Crassostrea virginica oysters.

Marine aggregates are naturally forming conglomerations of larvacean houses, phytoplankton, microbes, and inorganics adhered together by exocellular polymers. In this study, we show in vitro that the bacterial pathogen Vibrio vulnificus can be concentrated into laboratory-generated aggregates from surrounding water. We further show that environmental (E-genotype) strains exhibit significantly more integration into these aggregates than clinical (C-genotype) strains. Experiments where marine aggregates with attached V. vulnificus cells were fed to oysters (Crassostrea virginica) resulted in greater uptake of both C and E types than nonaggregated controls. When C- and E-genotype strains were cocultured in competitive experiments, the aggregated E-genotype strains exhibited significantly greater uptake by oyster than the C-genotype strains.