Correlative 3D Structured Illumination Microscopy and Single-Molecule Localization Microscopy for Imaging Cancer Invasion.

Super-resolution microscopy methods enable resolution of biological molecules in their cellular or tissue context at the nanoscale. Different methods have their strengths and weaknesses. Here we present a method that enables correlative confocal, structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM) imaging of structures involved in formation of invadopodia on the same sample. This enables up to four colors to be visualized in three dimensions at a resolution of between 120 and 10 nm for SIM and SMLM, respectively.

Implementing Integrated Mental Health and Chaplain Care in a National Quality Improvement Initiative.

This column describes the development, implementation, and outcomes of a quality improvement learning collaborative that aimed to better integrate chaplaincy with mental health care services at 14 participating health care facilities evenly distributed across the U.S. Department of Veterans Affairs and Department of Defense. Teams of health care chaplains and mental health professionals from participating sites sought to improve cross-disciplinary service integration in six key domains: screening, referrals, assessment, communication and documentation, cross-disciplinary training, and role clarification. Chaplains and mental health providers across all facilities at participating sites were significantly more likely post-collaboration to report having a clear understanding of how to collaborate and to report using a routine process for screening patients who could benefit from seeing a professional from the other discipline. Foundational efforts to enhance cross-disciplinary awareness and screening practices between chaplains and mental health professionals appear particularly promising.

Cutting edge: T cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity.

Autoimmune diseases can be reduced or even prevented if proinflammatory immune responses are appropriately down-regulated. Receptors (such as CTLA-4), cytokines (such as TGF-beta), and specialized cells (such as CD4+CD25+ T regulatory cells) work together to keep immune responses in check. T cell Ig mucin (Tim) family proteins are key regulators of inflammation, providing an inhibitory signal that dampens proinflammatory responses and thereby reducing autoimmune and allergic responses. We show in this study that reducing Tim-3 signaling during the innate immune response to viral infection in BALB/c mice reduces CD80 costimulatory molecule expression on mast cells and macrophages and reduces innate CTLA-4 levels in CD4+ T cells, resulting in decreased T regulatory cell populations and increased inflammatory heart disease. These results indicate that regulation of inflammation in the heart begins during innate immunity and that Tim-3 signaling on cells of the innate immune system critically influences regulation of the adaptive immune response.

IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart.

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rbeta1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-alpha and IFN-gamma levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-alpha and IFN-gamma during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-gamma deficiency resulted in significantly increased viral replication and significantly reduced TNF-alpha and IFN-gamma levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-gamma production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-gamma deficiency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoimmune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease.

Interferon-gamma protects against chronic viral myocarditis by reducing mast cell degranulation, fibrosis, and the profibrotic cytokines transforming growth factor-beta 1, interleukin-1 beta, and interleukin-4 in the heart.

Inflammatory fibrosis is a characteristic feature of myocarditis, dilated cardiomyopathy (DCM), and congestive heart failure. Th1-type immune responses, mediated by interleukin (IL)-12-induced interferon (IFN)-gamma, are believed to exacerbate autoimmune diseases including myocarditis. In this study, we examined the effect of IL-12R beta 1 and IFN-gamma deficiency on the development of chronic CB3-induced myocarditis using knockout mice. We found increased chronic CB3-induced myocarditis (14.1 to 43.1%, P < 0.001); pericarditis (1.5 to 7.6%, P < 0.001); fibrosis (9.7 to 27.4%, P < 0.05); and the profibrotic cytokines transforming growth factor-beta(1), IL-1 beta, and IL-4 in the hearts of IFN-gamma-deficient mice. All mice infected with CB3 developed DCM, but IFN-gamma-deficient mice developed a fibrous, adhesive pericarditis associated with increased numbers of degranulating mast cells (MCs) in the pericardium (26.6 to 45.9%, P < 0.01), increased histamine levels (716 to 1930 ng/g of heart, P < 0.01), and reduced survival (100 to 43%). In contrast, IL-12R beta 1 deficiency did not significantly alter the development of chronic myocarditis. Thus, IFN-gamma protects against the development of severe chronic myocarditis, pericarditis, and DCM after CB3 infection by reducing MC degranulation, fibrosis, and the profibrotic cytokines transforming growth factor-beta(1), IL-1 beta, and IL-4 in the heart.

Emergency repair of type A aortic dissection in type IV Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome type IV is a distinctive syndrome in which thin and fragile skin, premature ageing, bruising and scarring are combined with lethal or life-threatening arterial weakness. Aortic rupture either at the aortic root and arch, or sometimes lower down the artery, are particularly characteristic. Even quite minor injury can produce dangerous vascular tearing and damage. Technical difficulties encountered in arterial repair or venous ligature are particularly worrying. The authors report the treatment of a ruptured type A aortic dissection associated with Ehlers-Danlos syndrome where the extreme fragility of the tissues and tendency to bleed posed a difficult task for the surgeon.

The C-terminal domain of the Bordetella pertussis autotransporter BrkA forms a pore in lipid bilayer membranes.

BrkA is a 103-kDa outer membrane protein of Bordetella pertussis that mediates resistance to antibody-dependent killing by complement. It is proteolytically processed into a 73-kDa N-terminal domain and a 30-kDa C-terminal domain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BrkA is also a member of the autotransporter family of proteins. Translocation of the N-terminal domain of the protein across the outer membrane is hypothesized to occur through a pore formed by the C-terminal domain. To test this hypothesis, we performed black lipid bilayer experiments with purified recombinant protein. The BrkA C-terminal protein showed an average single-channel conductance of 3.0 nS in 1 M KCl. This result strongly suggests that the C-terminal autotransporter domain of BrkA is indeed capable of forming a pore.

Studies on the innervation of human renal allografts.

Kidneys are innervated by a plexus of nerves around the renal artery, which is disrupted by transplantation. This is a report of a comparison of the nerves in human renal allografts and normal kidneys. There were many sympathetic ganglia around normal renal arteries but none around transplanted vessels, although equal numbers of ganglia were present in hilar tissues of normal and transplanted kidneys. An immunohistological study with an antibody to synaptophysin showed that the number of synapses in transplanted ganglia was severely reduced. Immunohistological studies on allograft kidneys using antibodies to various neurofilament proteins and the Schwann cell marker S100 showed a marked reduction in neurofilament proteins shortly after transplantation with subsequent partial recovery, and a lesser reduction in S100. Renal allografts have structurally abnormal innervation but are not completely denervated.

A time-saving method for performing a clinical remount procedure with complete dentures.

Occlusal disharmony is one cause of uncomfortable dentures. Remounting complete dentures to eliminate occlusal discrepancies at the time of delivery has several advantages. This time-saving method for clinically remounting complete dentures is easy and quick and helps preserve denture supporting tissues.

Communicating patients' esthetic needs to the dental laboratory.

A visual aid in the form of a lined transparent plastic screen is attached to an earbow type of facebow and placed on the face. The screen can be adjusted to several horizontal tilts from the horizontal aspect in a clockwise or counterclockwise direction. The screen helps the dentist visualize horizontal and vertical facial characteristics that facilitate alignment of anterior restorations. This information can be sent to the laboratory, which can interpret the data with a smaller version of the adjustable screen.

Scanning and transmission electron microscopy of Candida albicans chlamydospores.

A simple, convenient method of growing large quantities of Candida albicans chlamydospores on a cellulose dialysis membrane has been developed. Long, narrow, cylindrical suspensor cells bearing spherical to ovoid chlamydospores were observed. Ultrastructural observations showed the chlamydospore to have a bilayered cell wall made up of an outer electron-transport primary layer and an inner electron-dense secondary layer, a large portion of the total cell volume occupied by a single large vacuole and several smaller vacuoles, and cytoplasmic organelles typical of those observed in the yeast-like cell. There are structural similarities between the region of chlamydospore-suspensor cell connection and septa observed in budding yeast-like cells.

A bilaterally balanced occlusal scheme for patients with arch width and curvature discrepancies.

A technique is described that will allow the patient maximum tooth contact in bilateral balanced occlusion without compromising esthetics or mechanics for a patient with an underdeveloped retrognathic and normal or oversized maxillae (Figs. 4 and 5). A disadvantage is maintaining the upper posterior teeth in the mold during the boil-out procedure, but this problem may be minimized with careful execution of the laboratory procedures.

Use of color microfiche cards in teaching preclinical prosthodontics.

Color microfiche afford a low-cost method for providing students with a complete series of color illustrations depicting procedures to be performed in their preclinical laboratory exercises.

Use of the remount jig as an aid in relining upper dentures.

A remount jig record is used to reposition the teeth of an upper denture reline in their original position. A face-bow record is used to position the upper edentulous cast in the correct relation to cranial planes. The impression may be developed as is done in any other impression procedure, and the repositioning can be accomplished in the laboratory with reasonable assurance that the upper denture is positioned occlusally as it was when the denture was constructed originally. This procedure relieves the dentist of the monumental task of making a reline impression and adjusting the position of the denture in one three-minute operation.

Transverse septum formation in budding cells of the yeastlike fungus Candida albicans.

Septum formation is initiated in Candida albicans by an electron transparent primary septum, which is then thickened on both sides to form secondary septa. Primary and secondary septa are incorporated into the bud scar, and secondary septum material only is incorporated into the birth scar.