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Purpose The following is a comparative analysis on the treatment outcomes of corneal perforations using amniotic membrane transplantation (AMT) or penetrating keratoplasty (PK). Methods This monocentric retrospective study was performed at the Department of Ophthalmology, University Hospital Ulm, Germany. A total of 78 eyes of 78 patients were included. Thirty-nine eyes received an AMT, and 39 patients were treated with a PK. Primary outcome was recurrence of perforation. Secondary outcomes were patient mortality and visual acuity. Results No statistically significant difference was observed with regard to a recurrence of perforation between the two groups (26% in AMT vs 23% in PK, p > 0.99). The time of recurrences was within the first two years and did not differ statistically (p = 0.97). In addition, a proportional hazards model with cox regression regarding recurrent perforation showed no significant differences (p = 0.5). After AMT, 41% and after KP, 28% of the patients died during follow-up (p = 0.2), respectively. The Charlson Comorbidity Index (p p = 0.0002) were statistically significantly higher in those who were deceased. A mean follow-up of 485 ± 517 days was recorded. Conclusion Both surgical methods show good results and no statistically significant difference regarding recurrent perforation rate. About a third of the patients died during the follow-up period. The decision regarding the appropriate method should therefore be based on a combination of all factors.

Purpose Amniotic membrane (AM) is a popular treatment for ocular surface diseases due to its wound healing effects. First intraocular implantations showed good anatomical and functional results. Here, three cases of intravitreal AM transplantation and the morphological and functional outcome are described. The reaction to AM was examined by immunostaining and cellular reactions were investigated on retinal cells in-vitro. Methods: Three patients with epiretinal implanted AM during PPV for complicated retinal detachment are presented retrospectively. We investigated the AM-influence in-vitro on RPE cells (ARPE-19), Müller cells (Mio-M1), and retinoblasts (661W). An anti-histone DNA ELISA and live/dead assay for apoptosis, a BrdU ELISA for proliferation and a WST-1 assay for viability were performed. Following AM-removal, tissue-specific cellular responses were evaluated by light microscopy and immunohistochemical staining. Results: Despite severe vitreoretinal disease, good anatomical and functional outcomes were achieved in all three cases. Immunostaining of AM showed no immunological rejection. In-vitro, AM mediates no toxic or proliferating effect on RPE and Müller cells but a neuroprotective effect on differentiated retinoblasts. Conclusion: AM offers possible advantages treating severe vitreoretinal disease. The pathobiological mode of action was elucidated and we could not determine issues regarding rejection reactions and biocompatibility. Further studies are needed to assess clinical potential.

Forensic proof of venomous snakebite is challenging, particularly when the snake species involved is not endemic to the region in which the event occurred. This case report describes postmortem histopathological findings and venom detection following suicide by monocled cobra (Naja kaouthia) envenomation, at a location (Texas, USA) where that animal is not normally encountered. External, internal and histopathological examination was performed according to the standard of the local office of the medical examiner. Tissue homogenate and blood were assayed with a Naja-specific enzyme-linked immunosorbent assay (ELISA), using locally obtained coral snake (Micrurus) venom as negative control. External evidence of puncture marks, edema and erythema were associated with local evidence of microscopic necrosis, acute inflammation, vascular congestion and lymphadenitis. Cobra venom was present in cardiac serum and at higher concentrations in serum, soft tissue and lymph nodes near the puncture sites. In the absence of commercial kits for venom detection, confirmation of snake envenomation postmortem can be achieved by correlation of specific ELISA with local findings. Exclusion of related local snake venom is necessary to confirm the specificity of the assay.

Repeated intravitreal injections of methotrexate for proliferative vitreoretinopathy, a rare ocular condition that can cause vision loss, have shown beneficial effects in recent clinical studies. The purpose of this study was to develop a slow-release, long-term drug carrier composed of the polymer polylactide-co-glycolide and methotrexate that can be injected intravitreally.The required composition of the drug carrier was modeled using pharmacokinetic parameters based on current literature. Release kinetics were determined using an ocular pharmacokinetic model. Epiretinal PVR-membranes were harvested during pars plana vitrectomy and subsequently transferred to cell culture. The effect of the drug carrier on cell migration was investigated using time-lapse microscopy and a scratch-induced migration assay. The colorimetric WST-1-assay and a live-dead-assay were performed to determine viability, and the BrdU-assay was applied for proliferation.The release profile showed an initial and a final burst of methotrexate with an intervening steady state that lasted 9-11 weeks. It showed inhibitory effects on pathobiological processes in human PVR-cells in vitro. Cell velocity in the time-lapse assay, migration in the scratch assay (p = 0.001), and proliferation in the BrdU assay (p = 0.027) were reduced after addition of the drug carrier. These effects occurred without causing a reduction in viability in the WST-1 assay (p 0.99) and the live-dead assay.The methotrexate-loaded drug carrier can maintain a stable concentration for 9-11 weeks and influence the pathobiological process of PVR cells in vitro. Therefore, it represents a potential therapeutic orphan drug for PVR.

Although many HIV-infected patients have attained older age owing to the success of antiretroviral therapy (ART) in controlling viremia and increasing CD4 T cell counts, HIV continues to persist in several target cells. We have characterized 514 HIV-1 envelope V3 region sequences (94-96 amino acids [aa]) from 25 HIV-infected older patients' peripheral blood mononuclear cell DNA on long-term ART with controlled viremia (undetectable viral load) and improved CD4 T cell counts. Phylogenetic analysis revealed that the V3 region sequences of each patient formed distinct clusters that were well separated and discriminated from other patients' sequences. The coding potential of the V3 region, including several patient-specific amino acid motifs and functional domains, including the two cysteines sandwiching the V3 loop, the central GPGR motif with variation at one position in some sequences, the base GDIR motif, and the N-glycosylation sites were generally conserved. The patients' V3 region sequences contained amino acid motifs conferring affinity mostly for CCR5 coreceptor, suggesting R5 phenotype. There was a low degree of heterogeneity and lower estimates of genetic diversity in all 25 patients' V3 region sequences. Twelve of 25 patients' V3 region sequences were found to be under positive selection pressure. Analysis of the several cytotoxic T lymphocytes (CTL) epitopes showed variation, whereas some of known neutralizing antibodies (nAbs) epitopes showed conservation in patients' V3 region sequences. In conclusion, a low degree of genetic variability and maintenance of functional domains with R5 phenotypes, and variation in CTL and conservation of nAb epitopes were the hallmarks of V3 region sequences from our 25 virologically controlled HIV-infected older patients on long-term ART.

HIV-infected older individuals may have a diminished immune response because of exhaustion/immune aging of T-cells. Therefore, we have investigated HIV-specific CD4 and CD8 T-cell responses in 100 HIV-infected patients (HIV+) who have aged on long-term antiretroviral therapy (ART) and achieved controlled viremia (mostly undetectable viral load; 92 patients with

Age-related alterations in immunity have been linked to increased incidence of infections and decreased responses to vaccines in the aging population. Human peripheral blood monocytes are known to promote Ag presentation and antiviral activities; however, the impact of aging on monocyte functions remains an open question. We present an in-depth global analysis examining the impact of aging on classical (CD14+CD16−), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16+) monocytes. Monocytes sorted from nonfrail healthy adults (21–40 y) and old (≥65 y) individuals were analyzed after stimulation with TLR4, TLR7/8, and retinoic acid–inducible gene I agonists. Our data showed that under nonstimulated conditions, monocyte subsets did not reveal significant age-related alternations; however, agonist stimulated-monocytes from adults and old subjects did show differences at the transcriptional and functional levels. These alternations in many immune-related transcripts and biological processes resulted in reduced production of IFN-α, IFN-γ, IL-1β, CCL20, and CCL8, and higher expression of CX3CR1 in monocytes from old subjects. Our findings represent a comprehensive analysis of the influence of human aging on pattern recognition receptors signaling and monocyte functions, and have implications for strategies to enhance the immune response in the context of infection and immunization.

The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.

Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's “gold standard,” fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at −70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides , except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

Although the demographic revolution has produced hundreds of millions people aged 65 and older, a substantial segment of that population is not enjoying the benefits of extended healthspan. Many live with multiple chronic conditions and disabilities that erode the quality of life. The consequences are also costly for society. In the United States, the most costly 5% of Medicare beneficiaries account for approximately 50% of Medicare's expenditures. This perspective summarizes a recent workshop on biomedical approaches to best extend healthspan as way to reduce age-related dysfunction and disability. We further specify the action items necessary to unite health professionals, scientists, and the society to partner around the exciting and palpable opportunities to extend healthspan.

Background Close relationships play an integral role in human development, and robust evidence links marital separation and divorce to poor health outcomes. Social integration may play a key role in this association. In many ways, the study of marital separation and divorce provides an ideal model system for a more complete understanding of the association between life stress and physical health. Purpose The current study investigated associations among objectively measured social integration, psychological distress, and biomarkers of immune health in recently separated adults (N = 49). Methods We collected four measures of immune functioning-interleukin-6, C-reactive protein, and antibody titers to latent cytomegalovirus and Epstein-Barr virus-that were combined to yield a viral-Immune Risk Profile. To assess how variability in social integration is associated with immunological correlates following the end of a marriage, we incorporated observational ecological momentary assessment data using a novel methodology (the Electronically Activated Recorder). Results We found that objectively measured social behaviors are associated with concurrent viral-Immune Risk Profile scores over and above the effects of psychological distress and that psychological distress may be linked to biomarkers of immune health through social integration. Conclusions This research expands current knowledge of biomarkers of immune health after divorce and separation and includes a new methodology for objective measures of social engagement.

Frailty is an increasingly recognized syndrome resulting in age-related decline in function and reserve across multiple physiologic systems. It presents as a hyperinflammable state, characterized by high vulnerability for adverse health outcomes, such as disability, falls, hospitalization, institutionalization, and mortality. The prevalence of Frailty Syndrome (FS) is of potentially enormous significance, as it potentially affects 20-30% of adults older than 75. Cellular and molecular basis of frailty has not been elucidated. The objective of this review is to discuss recent advances in: (i) the potential cellular and molecular basis of Frailty Syndrome, including development of new models to study it; (ii) the human and animal measures of Frailty Syndrome; and (iii) the development of objective cross-species correlates to aid the basic understanding, diagnosis, treatment and rehabilitation of Frailty Syndrome in older adults.

The impact of intrinsic aging upon human peripheral blood T cell subsets remains incompletely quantified and understood. This impact must be distinguished from the influence of latent persistent microorganisms, particularly CMV, which has been associated with age-related changes in the T cell pool. In a cross-sectional cohort of 152 CMV-negative individuals, aged 21–101 y, we found that aging correlated strictly to an absolute loss of naive CD8, but not CD4, T cells but, contrary to many reports, did not lead to an increase in memory T cell numbers. The loss of naive CD8 T cells was not altered by CMV in 239 subjects (range 21–96 y), but the decline in CD4+ naive cells showed significance in CMV+ individuals. These individuals also exhibited an absolute increase in the effector/effector memory CD4+ and CD8+ cells with age. That increase was seen mainly, if not exclusively, in older subjects with elevated anti-CMV Ab titers, suggesting that efficacy of viral control over time may determine the magnitude of CMV impact upon T cell memory, and perhaps upon immune defense. These findings provide important new insights into the age-related changes in the peripheral blood pool of older adults, demonstrating that aging and CMV exert both distinct and joint influence upon blood T cell homeostasis in humans.

The mechanisms regulating memory CD8+ T cell function and homeostasis during aging are unclear. CD8+ effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8+ T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8+ T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8+ T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA−CD45RO+ CMV-specific CD8+ T cells induced CD45RA expression while Ag activated cells remained CD45RO+. This raises the possibility that non-specific cytokine–driven accumulation of CMV-specific CD8+CD45RA+ T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe–host interactions. We developed a rhesus macaque model for studying Neisseria –host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria , as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.

West Nile Virus arrived in North America just over 12 years ago, and since that time, much has been learned about its pathology and transmission of infection and the factors affecting prevalence. Key developments uncovered critical risk factors for severe neurologic disease and revealed successful host defense strategies. At present, only equine vaccines are available; human vaccines remain in various stages of clinical trials. This review discusses the advances in and limitations of the current research related to the emergence and continued dissemination of West Nile virus throughout the Americas.

Several studies have shown an increased accumulation of terminally differentiated T cells during HIV infection, suggestive of exhaustion/senescence, causing dysregulation of T cell homeostasis and function and rapid HIV disease progression. We have investigated whether long-term antiretroviral therapy (ART), which controls viremia and restores CD4 T cell counts, is correlated with reduction in terminally differentiated T cells, improved ratios of naïve to memory and function of T cells in 100 virologically controlled HIV-infected patients. We show that while the median frequencies of terminally differentiated CD4+ and CD8+ T cells (CD28-, CD27-, CD57+ and CD28-CD57+), were higher in the virologically controlled HIV-infected patients' cohort compared with uninfected individuals' cohort, the frequencies of these cells significantly decreased with increasing CD4 T cell counts in HIV-infected patients. Although, the naïve CD4+ and CD8+ T cells were lower in HIV patients' cohort than uninfected cohort, there was a significant increase in both naïve CD4+ and CD8+ T cells with increasing CD4 T cell counts in HIV-infected patients. The underlying mechanism behind this increased naïve CD4+ and CD8+ T cells in HIV-infected patients was due to an increase in recent thymic emigrants, CD4+CD31+, as compared to CD4+CD31-. The CD4+ T cells of HIV-infected patients produced cytokines, including IL-2, IL-10 and IFN-γ comparable to uninfected individuals. In conclusion, virologically controlled HIV-infected patients on long-term ART show a significant reduction in terminally differentiated T cells, suggestive of decreased exhaustion/senescence, and improvement in the ratios of naïve to memory and function of T cells.

A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.

In the majority of cases, infection with hepatitis C virus (HCV) becomes chronic and is often associated with impaired innate and adaptive immune responses. The mechanisms underlying viral persistence and lack of protective immunity are poorly understood. Considering that dendritic cells (DCs) play critical roles in initiating and modulating immune responses, we explored the effect of HCV proteins on DC gene and protein expression, phenotype, and function. Human DCs were generated following plastic adherence of monocytes and culture with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) from normal subjects. Autologous nonadherent peripheral blood mononuclear cells were infected with vaccinia constructs expressing various HCV proteins (core-E1, NS5A, NS5B) or an irrelevant protein β-galactosidase (β-gal) as the control, induced to undergo apoptosis, then co-cultured with DCs. Between 2% and 10% of the genes probed in a cDNA nylon array were differentially regulated within DCs that had engulfed HCV proteins. In particular, the presence of intracellular NS5A led to increased transcriptional and protein expression of IL-8 (CXCL-8), a chemokine with proinflammatory and anti-interferon properties, and impaired interferon induction of signal transducers and activators of transcription 1 (STAT1) serine and tyrosine and STAT2 tyrosine phosphorylation. Conclusion: These data provide novel mechanisms by which HCV subverts antiviral host immunity. (HEPATOLOGY 2007;45:1422–1432.)

Aging leads to dysregulation of multiple components of the immune system that results in increased susceptibility to infections and poor response to vaccines in the aging population. The dysfunctions of adaptive B and T cells are well documented, but the effect of aging on innate immunity remains incompletely understood. Using a heterogeneous population of peripheral blood mononuclear cells (PBMCs), we first undertook transcriptional profiling and found that PBMCs isolated from old individuals (≥ 65 years) exhibited a delayed and altered response to stimulation with TLR4, TLR7/8, and RIG-I agonists compared to cells obtained from adults (≤ 40 years). This delayed response to innate immune agonists resulted in the reduced production of pro-inflammatory and antiviral cytokines and chemokines including TNFα, IL-6, IL-1β, IFNα, IFNγ, CCL2, and CCL7. While the major monocyte and dendritic cell subsets did not change numerically with aging, activation of specific cell types was altered. PBMCs from old subjects also had a lower frequency of CD40+ monocytes, impaired up-regulation of PD-L1 on monocytes and T cells, and increased expression of PD-L2 and B7-H4 on B cells. The defective immune response to innate agonists adversely affected adaptive immunity as TLR-stimulated PBMCs (minus CD3 T cells) from old subjects elicited significantly lower levels of adult T-cell proliferation than those from adult subjects in an allogeneic mixed lymphocyte reaction (MLR). Collectively, these age-associated changes in cytokine, chemokine and interferon production, as well as co-stimulatory protein expression could contribute to the blunted memory B- and T-cell immune responses to vaccines and infections.

Hepatitis C virus (HCV)-related liver failure is the leading indication for liver transplantation worldwide. After transplantation, virological recurrence is the rule, but the spectrum of histological injury is wide, ranging from the development of allograft cirrhosis within a few years to minimal hepatitis despite long-term follow-up. The immunological correlates of this variable natural history are poorly understood. Here, we studied the kinetics of the cellular immune responses, viral replication, and allograft histology in 24 patients who had undergone liver transplantation for HCV-related liver failure. Using direct ex vivo methodologies (i.e., interferon-gamma ELISPOT and major histocompatibility complex class I–peptide tetrameric complexes), we found that patients who experienced viral eradication after antiviral therapy showed restoration of HCV-specific T-cell responses, whereas patients with progressive HCV recurrence that failed to respond to therapy showed declining frequencies of these viral-specific effector cells. The cytotoxic T lymphocytes that peripherally reconstituted after transplantation were clonotypically identical to those present within the recipient explant liver, defined at the level of the T-cell receptor beta chain (one epitope/one clone). Moreover, the subset of patients who spontaneously demonstrated minimal histologic recurrence had more vigorous CD4+ T-cell responses in the first 3 months, particularly targeting nonstructural proteins. We provide evidence that T-cell responses emerge after liver transplantation, and their presence correlates with improved histological and clinical outcomes. In conclusion, these results may help identify patients more likely to develop severe HCV recurrence and therefore benefit from current antiviral therapy, as well as provide a rationale for the future use of novel immunotherapeutic approaches. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;41:72–81.)

By necessity, human liver transplantation is performed across HLA barriers. As a result, intracellular infection of the allograft presents a unique immunologic challenge for the recipient’s immune system. In this study, we describe the presence of HLA-A2-restricted, hepatitis C virus (HCV)-specific CD8+ T cells in liver transplant recipients in whom the allograft is HLA-A2 positive and the recipient is HLA-A2 negative. These memory-effector T cells are recipient derived and recognize HCV peptide uniquely in the context of HLA-A2. Furthermore, these cells were absent before the transplant, suggesting that the allograft is capable of selectively expanding naive CD8+ T cells. The in vitro specificity to donor HLA allele-restricted CD8+ T cells suggests that these cells may function to control HCV spread in the allograft.

Humans are the only known natural host for the hepatitis C virus and the majority of cases result in chronic infection. Identification of acute infection is difficult because symptoms are often subtle or absent. Thus, much remains to be learned about the early mechanisms conferring resolution of infection. The study of spontaneously resolved individuals has revealed a multispecific and sustained antigen-specific immune response, including CD4+ and CD8+ T cells. Evidence is mounting that a cooperative immune response involving both innate and adaptive immunity may be required to successfully eradicate hepatitis C virus infection.

In order to assess the effect of in vitro models on the expression of key genes known to be implicated in the development or progression of cancer, we quantified by real-time quantitative PCR the expression of 28 key genes in three bladder cancer tissue specimens and in their derived cell lines, studied either as one-dimensional single cell suspensions, two-dimensional monolayers or three-dimensional spheroids. Global analysis of gene expression profiles showed that in vitro models had a dramatic impact upon gene expression. Remarkably, quantitative differences in gene expression of 2–63-fold were observed in 24 out of 28 genes among the cell models. In addition, we observed that the in vitro model which most closely mimicked in vivo mRNA phenotype varied with both the gene and the patient. These results provide evidence that mRNA expression databases based on cancer cell lines, which are studied to provide a rationale for selection of therapy on the basis of molecular characteristics of a patient's tumour, must be carefully interpreted. British Journal of Cancer (2002) 86, 1283–1289. DOI: 10.1038/sj/bjc/6600239 www.bjcancer.com © 2002 Cancer Research UK

The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum . Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR , which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT , a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5′-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum . We also show in this study that the angT gene, found downstream of angR , intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.

Objectives/Hypothesis: The present study determined whether a 125 mm2 electrode would produce lower and more reliable electrical taste thresholds than a 25 mm2 electrode when measurement occurred in a medial tongue region relatively sparse in taste bud numbers. We hypothesized this would be the case and that the obtained threshold values would be higher than those previously reported for anterior and lateral tongue regions. Study Design And Methods: Sixteen college-age subjects were tested twice, once using the 25 mm2 electrode and once using the 125 mm2 electrode on each of two sessions separated by 2 to 16 days. The order of presentation of the sessions was counterbalanced across subjects. Measurement was confined to the medial tongue, 0.7 cm lateral to the median furrow. Results: As hypothesized, the larger electrode resulted in lower and more reliable threshold values than the smaller electrode (respective median threshold values = 20.06 μA & 33.59 μA, P = .001; respective test-retest rs = 0.78 [P < .001] and 0.46 [P < .05]). Also as hypothesized, the threshold values were higher (i.e., sensitivity lower) than previously reported for anterior and lateral tongue regions. Conclusions: The magnitude and reliability of electrical taste thresholds depends on the tongue region examined and the size of the electrodes used. These results suggest that relatively large electrodes should be considered for electrogustometric threshold testing, particularly when lingual regions not highly populated with taste buds are evaluated.

Objective To enhance arboviral surveillance and laboratory capacity to establish a surveillance baseline for the emerging threat of Dengue fever in the Arizona-Mexico border region. Introduction West Nile Virus (WNV) and dengue virus (DENV) are both arboviruses which are transmitted to humans by an infected mosquito bite during blood-meal feeding. The clinical presentations of non-neuroinvasive WNV and dengue fever are similar, and symptoms may include acute onset of high fever, headache, myalgia, arthralgia, nausea, vomiting, and often a maculopapular rash. More serious manifestations of these viruses include fatal encephalitis and meningitis in WNV patients and fatal hemorrhagic disease in dengue patients. Over the last decade, WNV has spread rapidly across North America, reaching Arizona in 2004, and has become a significant cause of human illness since that time. Even though dengue has been described as primarily a disease of the tropics and sub-tropical areas, there is a small but significant risk for dengue outbreaks in the continental United States as evidenced by surveillance efforts in Texas that identified local dengue transmission in 2005. In recent years, outbreaks of dengue have occurred in Mexico border states, most notably Sonora in 2010. That same year, Arizona had the highest incidence of WNV cases in the U.S. including number of neuroinvasive disease cases, total cases, and number of deaths per state. The emergence of DENV and WNV as important public health problems maybe have been due to non-effective mosquito control, global demographic changes (urbanization and population growth), increased air travel, and inadequate surveillance. Methods Vector mapping: Mapping techniques will be utilized to visually depict Aedes aegypti populations captured from previous seasonal public health environmental vector trapping programs. Laboratory capacity: Multi-state laboratory training by CDC Dengue Branch was held in October 2012. Surveillance: The WNV cases that present to medical services for WNV testing and reported to public health officials are the most severe nueroinvasive cases. Much less is understood about the non-neuroinvasive cases with often present with non-descript symptoms. Results Vector mapping: Comparative densities of Ae. aegypti with academic partners of the Entomology and Public Health conducting a study capturing Ae. aegypti may help to enhance environmental programs. Laboratory Capacity: The laboratory training will cover conventional serological methods as well as recently FDA cleared molecular RT-PCR. Participants will include public health laboratory personnel working in molecular and serology diagnostics and other binational partners. Surveillance: A convenient seroprevalence study at sentinel-hospital site of symptomatic patients presenting in Arizona border hospital sites will be performed to better understand circulating levels of arboviral infections. Conclusions Appropriate and timely response to surveillance data is the key to identification human and animal disease associated with WNV, DENV, and other arboviruses. The mosquito vector Ae. aegypti is well established widespread and thriving in Arizona yet there is no autochthonous transmission of DENV identified to date. The results from this study will identify gaps and potential prevention and control measures for emerging infectious diseases including WNV and DENV in Arizona.

Vibrio anguillarum virulence is associated with the presence of a plasmid-mediated iron-uptake system expressed under iron-limiting conditions, which consists of the siderophore anguibactin and specific iron-transport proteins. This system is maximally expressed under iron-limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE-like enzymatic function that may play a role in anguibactin biosynthesis. In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron-transport genes fatA and fatB. We also show that transcription of angR is repressed by Fur under iron-rich conditions. In addition, we present evidence that anguibactin itself enhanced transcription of the iron-transport genes fatA and fatB, independently of AngR and the trans-acting factor (TAF) product(s). The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron-transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.

Vibrio anguillarum Fur mutants, 775met9 and 775met11, were characterized. V. anguillarum 775met9 had a change of D to G at position 104 located in the carboxy terminus resulting in impaired Fur activity.Computer analysis predicts perturbation of an alpha-helix in the carboxy terminus which may interfere with Fur protein conformation. Strain 775met11 had a change in the start codon resulting in no protein synthesis. The mutants are unstable, and reversion to the wild type occurs frequently.

This article describes the development of the New York City Gay and Lesbian Anti-Violence Project (AVP) since 1980. After a brief historical overview of the emergence of the victims' modern crime movement and a discussion of how the gay and lesbian victims' movement is related to it, AVP's treatment and service interventions are described. These include crisis intervention immediately after an attack and a wide variety of follow-up services, including short-term professional counseling, peer counseling, and group counseling. The AVP also provides advocacy services, including police advocacy, court monitoring, and advocacy with other human service organizations. In describing the AVP's activities, the article highlights unique problems faced by lesbian and gay survivors of assault.

Acute infection with hepatitis C virus (HCV) rarely is identified, and hence, the determinants of spontaneous resolution versus chronicity remain incompletely understood. In particular, because of the retrospective nature and unknown source of infection in most human studies, direct evidence for emergence of escape mutations in immunodominant major histocompatibility complex class I–restricted epitopes leading to immune evasion is extremely limited. In two patients infected accidentally with an identical HCV strain but who developed divergent outcomes, the total lack of HCV-specific CD4+ T cells in conjunction with vigorous CD8+ T cells that targeted a single epitope in one patient was associated with mutational escape and viral persistence. Statistical evidence for positive Darwinian selective pressure against an immunodominant epitope is presented. Wild-type cytotoxic T lymphocytes persisted even after the cognate antigen was no longer present.

The Department of Homeland Security (DHS) has vast amounts of data available, but its ultimate value cannot be realized without powerful technologies for knowledge discovery to enable better decision making by analysts. Past evidence has shown that terrorist activities leave detectable footprints, but these footprints generally have not been discovered until the opportunity for maximum benefit has passed. The challenge faced by the DHS is to discover the money transfers, border crossings, and other activities in advance of an attack and use that information to identify potential threats and vulnerabilities. The data to be analyzed by DHS comes from many sources ranging from news feeds, to raw sensors, to intelligence reports, and more. The amount of data is staggering; some estimates place the number of entities to be processed at 1015. The uses for the data are varied as well, including entity tracking over space and time, identifying complex and evolving relationships between entities, and identifying organization structure, to name a few. Because they are ideal for representing relationship and linkage information, semantic graphs have emerged as a key technology for fusing and organizing DHS data. A semantic graph organizes relational data by using nodes to represent entities and edgesmore » to connect related entities. Hidden relationships in the data are then uncovered by examining the structure and properties of the semantic graph.« less

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum , a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di( o -hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA - angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.