Development of Squamous Cell Carcinoma After Pulmonary Aspergillosis in the Native Lung of a Lung Transplant Recipient: A Case Report.

Lung transplant recipients have a significant incidence of posttransplant lung nodules. Such nodules can occur from various etiologies, both in the lung allograft or in the native lung. They most commonly originate from infections, such as Pseudomonas or Aspergillus species, or from posttransplant lymphoproliferative disorder. Lung cancer is challenging to diagnose in a native lung, especially with an underlying fibrotic disease. We present a case of a 75-year-old woman who presented with classic clinical features of pulmonary aspergillosis in the native right lung with idiopathic pulmonary fibrosis 5 years after left-sided single-lung transplant. She required a right lower lobectomy and antifungal treatment with isavuconazonium sulfate and inhaled amphotericin. A persistent right upper lobe lung nodule was noted during surveillance imaging and was initially presumed to be recurrent Aspergillus infection; however, growth of the nodule and change in its characteristics prompted additional examination. A navigational bronchoscopic biopsy was positive for squamous cell carcinoma. Her options for stage IIIA squamous cell carcinoma were limited to chemotherapy with paclitaxel and carboplatin plus radiation. Although initial surveillance scans showed adequate tumor response, metastatic squamous cell carcinoma was found in the liver 6 months later. She was eventually transitioned to palliative care. This case highlights the importance of a high index of suspicion for examination of nodules in the native lung of lung transplant recipients, even in cases of a known diagnosis, owing to the high morbidity and mortality associated with primary lung cancer in this population.

Predicting carotid artery disease and plaque instability from cell-derived microparticles.

OBJECTIVES: Cell-derived microparticles (MPs) are small plasma membrane-derived vesicles shed from circulating blood cells and may act as novel biomarkers of vascular disease. We investigated the potential of circulating MPs to predict (a) carotid plaque instability and (b) the presence of advanced carotid disease. METHODS: This pilot study recruited carotid disease patients (aged 69.3 ± 1.2 years [mean ± SD], 69% male, 90% symptomatic) undergoing endarterectomy (n = 42) and age- and sex-matched controls (n = 73). Plaques were classified as stable (n = 25) or unstable (n = 16) post surgery using immunohistochemistry. Blood samples were analysed for MP subsets and molecular biomarkers. Odds ratios (OR) are expressed per standard deviation biomarker increase. RESULTS: Endothelial MP (EMP) subsets, but not any vascular, inflammatory, or proteolytic molecular biomarker, were higher (p < .05) in the unstable than the stable plaque patients. The area under the receiver operator characteristic curve for CD31(+)41(-) EMP in discriminating an unstable plaque was 0.73 (0.56-0.90, p < .05). CD31(+)41(-) EMP predicted plaque instability (OR = 2.19, 1.08-4.46, p < .05) and remained significant in a multivariable model that included transient ischaemic attack symptom status. Annexin V(+) MP, platelet MP (PMP) subsets, and C-reactive protein were higher (p < .05) in cases than controls. Annexin V(+) MP (OR = 3.15, 1.49-6.68), soluble vascular cell adhesion molecule-1 (OR = 1.64, 1.03-2.59), and previous smoking history (OR = 3.82, 1.38-10.60) independently (p < .05) predicted the presence of carotid disease in a multivariable model. CONCLUSIONS: EMP may have utility in predicting plaque instability in carotid patients and annexin V(+) MPs may predict the presence of advanced carotid disease in aging populations, independent of established biomarkers.

The APOL1 genotype of African American kidney transplant recipients does not impact 5-year allograft survival.

Apolipoprotein L-1 (APOL1) gene variants are associated with end-stage renal disease in African Americans (AAs). Here we investigate the impact of recipient APOL1 gene distributions on kidney allograft outcomes. We conducted a retrospective analysis of 119 AA kidney transplant recipients, and found that 58 (48.7%) carried two APOL1 kidney disease risk variants. Contrary to the association seen in native kidney disease, there is no difference in allograft survival at 5-year posttransplant for recipients with high-risk APOL1 genotypes. Thus, we were able to conclude that APOL1 genotypes do not increase risk of allograft loss after kidney transplantations, and carrying 2 APOL1 risk alleles should not be an impediment to transplantation.

A novel SNP in COMT is associated with alcohol dependence but not opiate or nicotine dependence: a case control study.

BACKGROUND: It is well established that COMT is a strong candidate gene for substance use disorder and schizophrenia. Recently we identified two SNPs in COMT (rs4680 and rs165774) that are associated with schizophrenia in an Australian cohort. Individuals with schizophrenia were more than twice as likely to carry the GG genotype compared to the AA genotype for both the rs165774 and rs4680 SNPs. Association of both rs4680 and rs165774 with substance dependence, a common comorbidity of schizophrenia has not been investigated. METHODS: To determine whether COMT is important in substance dependence, rs165774 and rs4680 were genotyped and haplotyped in patients with nicotine, alcohol and opiate dependence. RESULTS: The rs165774 SNP was associated with alcohol dependence. However, it was not associated with nicotine or opiate dependence. Individuals with alcohol dependence were more than twice as likely to carry the GG or AG genotypes compared to the AA genotype, indicating a dominant mode of inheritance. The rs4680 SNP showed a weak association with alcohol dependence at the allele level that did not reach significance at the genotype level but it was not associated with nicotine or opiate dependence. Analysis of rs165774/rs4680 haplotypes also revealed association with alcohol dependence with the G/G haplotype being almost 1.5 times more common in alcohol-dependent cases. CONCLUSIONS: Our study provides further support for the importance of the COMT in alcohol dependence in addition to schizophrenia. It is possible that the rs165774 SNP, in combination with rs4680, results in a common molecular variant of COMT that contributes to schizophrenia and alcohol dependence susceptibility. This is potentially important for future studies of comorbidity. As our participant numbers are limited our observations should be viewed with caution until they are independently replicated.

Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction.

The localization of HIV-1 DNA in renal tissues is critically important for understanding pathogenesis of HIV-associated nephropathy (HIVAN), but the clarification has been technically challenging. We applied in situ polymerase chain reaction (IS-PCR) to human renal tissues to demonstrate viral entry into the renal epithelial cells in vivo. To test the specificity of this method and to determine the cell types infected, we used IS-PCR followed by in situ hybridization (ISH) and IS-PCR followed by immunohistochemistry and histochemical counterstains. Brief 2 hour fixation in 4% paraformaldehyde had 92.9% sensitivity and 100% specificity for detection of viral DNA in renal biopsies of HIVAN patients, compared to 70.8% sensitivity and 66.7% specificity in renal biopsies fixed overnight in 10% formalin. Under optimized conditions, the only signals detectable in HIV-1 seronegative cases were false positives attributable to renal tubular apoptosis. In HIVAN cases, positive signal was observed in podocytes, parietal cells, renal tubular cells, and interstitial leukocytes. Immunohistochemical co-labeling for pan-T cell and macrophage markers revealed that the interstitial leukocytes with positivity for HIV-1 DNA included both T cells and macrophages. Application of ISH after IS-PCR showed the same distribution of signal as observed using IS-PCR alone, confirming the specificity of the technique. IS-PCR is a powerful technique to detect viral DNA in human tissue sections, but requires proper use of negative controls to set optimal fixation, protein digestion, and amplification conditions.

Implementation of open and closed kinetic chain quadriceps strengthening exercises after anterior cruciate ligament reconstruction.

When working with athletes that have recently undergone anterior cruciate ligament (ACL) reconstructive surgery, a common goal of athletic trainers, strength and conditioning coaches, physicians, and physical therapists is to restore quadriceps strength while protecting the reconstructed ACL and patellofemoral joint from unnecessary stresses. Quadriceps strengthening exercises are often referred to as occurring in the open kinetic chain (OKC) or closed kinetic chain (CKC). Currently, there is little agreement in the literature as to whether only CKC exercises or a combination of OKC and CKC exercise should be performed after ACL reconstruction to strengthen the quadriceps. We believe that a combination of OKC and CKC exercises can be used to effectively and safely strengthen the quadriceps after ACL reconstruction. The purposes of this review are to examine the scientific literature currently available for the effects of OKC and CKC exercise on ACL strain and patellofemoral joint stress, and to present a sound rationale for using a combination of OKC and CKC exercises for quadriceps strengthening after ACL reconstruction. On the basis of our review, both OKC and CKC exercises can be modified and implemented for quadriceps strengthening after ACL reconstruction without causing excessive ACL strain or patellofemoral joint stress.

Medicine in long duration space exploration: the role of virtual reality and broad bandwidth telecommunications networks.

Safety of astronauts during long-term space exploration is a priority for NASA. This paper describes efforts to produce Earth-based models for providing expert medical advice when unforeseen medical emergencies occur on spacecraft. These models are Virtual Collaborative Clinics that reach into remote sites using telecommunications and emerging stereo-imaging and sensor technologies.

Complex vestibular macular anatomical relationships need a synthetic approach.

Mammalian vestibular maculae are anatomically organized for complex parallel processing of linear acceleration information. Anatomical findings in rat maculae are provided in order to underscore this complexity, which is little understood functionally. This report emphasizes that a synthetic approach is critical to understanding how maculae function and the kind of information they conduct to the brain.

Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection.

The anatomic heterogeneity of the nephron poses obstacles to microdissection of individual renal compartments for analysis of gene expression. We have systematically analyzed the effects of fixation time and nuclear staining on the ability to recover nucleic acid from individual renal compartments by laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. From 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubules were captured sequentially onto polymer films. DNA for beta-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditions for DNA amplification were brief (1- or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert's hematoxylin, Harris's hematoxylin, Mayer's hematoxylin, or methyl green, did not adversely affect the ability to extract and amplify DNA. For RNA extraction, glomeruli and tubules were microdissected from renal cryostat sections stored for up to 6 months. By RT-PCR, mRNA expression of the glomerulus-specific gene, Wilms' tumor-1, was identified in as few as 5 microdissected glomeruli and of the tubule-specific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate that the LCM method can successfully microdissect pure glomerular and tubular tissue compartments and that the optimal fixation and staining conditions are those employed routinely for renal biopsies, namely overnight formalin fixation and hematoxylin counterstain for DNA extraction, and cryostat sectioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool for the analysis of gene expression in specific renal compartments from archival human renal biopsies.

Changes in ribbon synapses and rough endoplasmic reticulum of rat utricular macular hair cells in weightlessness.

This study combined ultrastructural and statistical methods to learn the effects of weightlessness on rat utricular maculae. A principle aim was to determine whether weightlessness chiefly affects ribbon synapses of type II cells, since the cells communicate predominantly with branches of primary vestibular afferent endings. Maculae were microdissected from flight and ground control rat inner ears collected on day 13 of a 14-day spaceflight (F13), landing day (R0) and day 14 postflight (R14) and were prepared for ultrastructural study. Ribbon synapses were counted in hair cells examined in a Zeiss 902 transmission electron microscope. Significance of synaptic mean differences was determined for all hair cells contained within 100 section series, and for a subset of complete hair cells, using SuperANOVA software. The synaptic mean for all type II hair cells of F13 flight rats increased by 100%, and that for complete cells by 200%. Type I cells were less affected, with synaptic mean differences statistically insignificant in complete cells. Synapse deletion began within 8 h upon return to Earth. Additionally, hair cell laminated rough endoplasmic reticulum of flight rats was reversibly disorganized on R0. Results support the thesis that synapses in type II hair cells are uniquely affected by altered gravity. Type II hair cells may be chiefly sensors of gravitational and type I cells of translational linear accelerations.

Simulation studies of vestibular macular afferent-discharge patterns using a new, quasi-3-D finite volume method.

A quasi-three-dimensional finite-volume numerical simulator was developed to study passive voltage spread in vestibular macular afferents. The method, borrowed from computational fluid dynamics, discretizes events transpiring in small volumes over time. The afferent simulated had three calyces with processes. The number of processes and synapses, and direction and timing of synapse activation, were varied. Simultaneous synapse activation resulted in shortest latency, while directional activation (proximal to distal and distal to proximal) yielded most regular discharges. Color-coded visualizations showed that the simulator discretized events and demonstrated that discharge produced a distal spread of voltage from the spike initiator into the ending. The simulations indicate that directional input, morphology, and timing of synapse activation can affect discharge properties, as must also distal spread of voltage from the spike initiator. The finite volume method has generality and can be applied to more complex neurons to explore discrete synaptic effects in four dimensions.

New approaches to virtual environment surgery.

This research focused on two main problems: 1) low cost, high fidelity stereoscopic imaging of complex tissues and organs; and 2) virtual cutting of tissue. A further objective was to develop these images and virtual tissue cutting methods for use in a telemedicine project that would connect remote sites using the Next Generation Internet. For goal one we used a CT scan of a human heart, a desktop PC with an OpenGL graphics accelerator card, and LCD stereoscopic glasses. Use of multiresolution meshes ranging from approximately 1,000,000 to 20,000 polygons speeded interactive rendering rates enormously while retaining general topography of the dataset. For goal two, we used a CT scan of an infant skull with premature closure of the right coronal suture, a Silicon Graphics Onyx workstation, a Fakespace Immersive WorkBench and CrystalEyes LCD glasses. The high fidelity mesh of the skull was reduced from one million to 50,000 polygons. The cut path was automatically calculated as the shortest distance along the mesh between a small number of hand selected vertices. The region outlined by the cut path was then separated from the skull and translated/rotated to assume a new position. The results indicate that widespread high fidelity imaging in virtual environment is possible using ordinary PC capabilities if appropriate mesh reduction methods are employed. The software cutting tool is applicable to heart and other organs for surgery planning, for training surgeons in a virtual environment, and for telemedicine purposes.

Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.

There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Effect of gravity on vestibular neural development.

The timing, molecular basis, and morphophysiological and behavioral consequences of the interaction between external environment and the internal genetic pool that shapes the nervous system over a lifetime remain important questions in basic neuroscientific research. Space station offers the opportunity to study this interaction over several life cycles in a variety of organisms. This short review considers past work in altered gravity, particularly on the vestibular system, as the basis for proposing future research on space station, and discusses the equipment necessary to achieve goals. It is stressed that, in keeping with the international investment being made in this research endeavor, both the questions asked and the technologies to be developed should be bold. Advantage must be taken of this unique research environment to expand the frontiers of neuroscience.

Sites of recombinant adeno-associated virus integration.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.

Synaptic changes in rat maculae in space and medical imaging: the link.

Two different space life sciences missions (SLS-1 and SLS-2) have demonstrated that the synapses of the hair cells of rat vestibular maculae increase significantly in microgravity. The results also indicate that macular synapses are sensitive to stress. These findings argue that vestibular maculae exhibit neuroplasticity to macroenvironmental and microenvironmental changes. This capability should be clinically relevant to rehabilitative training and/or pharmacological treatments for vestibular disease. The results of this ultrastructural research also demonstrated that type I and type II hair cells are integrated into the same neuronal circuitry. The findings were the basis for development of three-dimensional reconstruction software to learn details of macular wiring. This software, produced for scientific research, has now been adapted to reconstruct the face and skull directly from computerized tomography scans. In collaboration with craniofacial reconstructive surgeons at Stanford University Medical Center, an effort is under way to produce a virtual environment workbench for complex craniofacial surgery. When completed, the workbench will help surgeons train for and simulate surgery. The methods are patient specific. This research illustrates the value of basic research in leading to unanticipated medical applications.

A national center for biocomputation: in search of a patient-specific interactive virtual surgery workbench.

This report describes the three-dimensional imaging and virtual environment technologies developed in NASA's Biocomputation Center for scientific purposes that have now led to applications in the field of medicine. A major goal is to develop a virtual environment surgery workbench for planning complex craniofacial and breast reconstructive surgery, and for training surgeons.

Morphological evidence for local microcircuits in rat vestibular maculae.

Previous studies suggested that intramacular, unmyelinated segments of vestibular afferent nerve fibers and their large afferent endings (calyces) on type I hair cells branch. Many of the branches (processes) contain vesicles and are presynaptic to type II hair cells, other processes, intramacular nerve fibers, and calyces. This study used serial section transmission electron microscopy and three-dimensional reconstruction methods to document the origins and distributions of presynaptic processes of afferents in the medial part of the adult rat utricular macula. The ultrastructural research focused on presynaptic processes whose origin and termination could be observed in a single micrograph. Results showed that calyces had 1) vesiculated, spine-like processes that invaginated type I cells and 2) other, elongate processes that ended on type II cells pre- as well as postsynaptically. Intramacular, unmyelinated segments of afferent nerve fibers gave origin to branches that were presynaptic to type II cells, calyces, calyceal processes, and other nerve fibers in the macula. Synapses with type II cells occurred opposite subsynaptic cisternae (C synapses); all other synapses were asymmetric. Vesicles were pleomorphic but were differentially distributed according to process origin. Small, clear-centered vesicles, approximately 40-60 nm in diameter, predominated in processes originating from afferent nerve fibers and basal parts of calyces. Larger vesicles approximately 70-120 nm in diameter having approximately 40-80 nm electron-opaque cores were dominant in processes originating from the necks of calyces. Results are interpreted to indicate the existence of a complex system of intrinsic feedforward (postsynaptic)-feedback (presynaptic) connections in a network of direct and local microcircuits. The morphological findings support the concept that maculae dynamically preprocess linear acceleratory information before its transmission to the central nervous system.