Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Diversity in the Ammonia-Oxidizing Nitrifier Population of a Soil

Multiple genera of ammonia-oxidizing chemoautotrophic nitrifiers in a soil were detected, isolated, and studied by means of modified most-probable-number (MPN) techniques. The soil examined was a Waukegon silt loam treated with ammonium nitrate or sewage effluent. The genera Nitrosomonas and Nitrosospira were found to occur more commonly than the genus Nitrosolobus. Three different MPN media gave approximately the same overall ammonia oxidizer counts within statistical error after prolonged incubation but differed markedly in ratios of Nitrosomonas to Nitrosospira. Selectivity and counting efficiency of MPN media were studied by observing the growth response of representative pure cultures isolated from the soil. Selectivity was evident in each medium with respect to all strains tested, and the media differed greatly in incubation times required to obtain maximum counts.     

Ocular Tropism of Respiratory Viruses

SUMMARY

Respiratory viruses (including adenovirus, influenza virus, respiratory syncytial virus, coronavirus, and rhinovirus) cause a broad spectrum of disease in humans, ranging from mild influenza-like symptoms to acute respiratory failure. While species D adenoviruses and subtype H7 influenza viruses are known to possess an ocular tropism, documented human ocular disease has been reported following infection with all principal respiratory viruses. In this review, we describe the anatomical proximity and cellular receptor distribution between ocular and respiratory tissues. All major respiratory viruses and their association with human ocular disease are discussed. Research utilizing in vitro and in vivo models to study the ability of respiratory viruses to use the eye as a portal of entry as well as a primary site of virus replication is highlighted. Identification of shared receptor-binding preferences, host responses, and laboratory modeling protocols among these viruses provides a needed bridge between clinical and laboratory studies of virus tropism.     

Effects of Buprenorphine Treatment on Influenza Pathogenesis in the Ferret (Mustela putorius furo)

Ferrets are the gold-standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical signs similar to those seen in infected people. Because the presence and progression of clinical signs can be useful in infectious disease research, uncertainty in how analgesics alter research outcomes or compromise characteristics of disease progression have outweighed the concern regarding animal discomfort from these symptoms. Nonetheless, the principles of animal research require consideration of refinements for this important model for IAV research. Opioids offer a possible refinement option that would not directly affect the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that use ferrets, 12 ferrets were inoculated intranasally with the A(H3N2) IAV A/Panama/2007/1999 and divided into 3 treatment groups (n = 4 each), of which 2 groups received buprenorphine treatments on different schedules and the third received a saline control. The duration and location of viral replication, lymphohematopoietic changes, and clinical signs were comparable across all groups at all time points. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through day 5 after inoculation, and peak viral titers from the upper respiratory tract did not differ between ferrets receiving buprenorphine treatments on either schedule. Compared with the saline group, ferrets receiving buprenorphine exhibited transient weight loss and pyrexia, but all groups ultimately achieved similar peaks in both of these measurements. Collectively, these findings support the continued evaluation of buprenorphine as a refinement for IAV-challenged ferrets.

Despite decades of international research and the availability of public health countermeasures, including vaccines and antivirals, influenza viruses remain a persistent threat to human and animal health.^26,35 Influenza A viruses (IAV) exhibit a diverse range of virulence, exist in several host reservoirs, and can show rapid rates of antigenic change.²⁶ As a result, IAV are associated with both seasonal epidemics and occasional pandemics in humans,³⁵ and animal infections with IAV have become key for understanding multifactorial traits that include pathogenicity, transmissibility, and vaccine efficacy. Due to their relatively small size, adaptability to the research setting, and similarities to human lung anatomy and physiology, ferrets provide an excellent model for respiratory diseases in humans and are a valuable small-animal model for such studies.^8,30 Data generated from ferrets are included in numerous risk-assessment rubrics evaluating the pandemic potential of novel and emerging influenza viruses, including those established by the Centers for Disease Control and Prevention and the World Health Organization.^14,51The study of influenza virus in ferrets dates back to the early 1930s, when this species was first found to be susceptible to influenza virus.⁴⁴ Ferrets are naturally susceptible to both human and zoonotic IAV.⁴⁷ After infection, ferrets present with clinical signs like those of humans; these signs are often not recapitulated in other species, such as mice and guinea pigs.^28,39,46 The severity and spectrum of clinical signs associated with influenza virus–inoculated ferrets can vary, depending on the virus strain, route and dose of inoculation, and various host parameters.⁵ Whereas influenza viruses with low virulence in ferrets may cause only acute pyrexia and mild to moderate weight loss, isolates with high virulence can cause severe, systemic illness with gastrointestinal and neurologic symptoms.⁴The 3Rs, replace, reduce, refine, encourage investigation of how research involving animals can be conducted in more humane ways.^2,13,37,41 Analgesia for symptoms of influenza in ferrets represents an opportunity for refinement, but this intervention could confound research assessing disease progression. NSAID and corticosteroids are often prescribed to treat the clinical signs associated with influenza in humans.⁴³ These interventions could alter the inflammatory cascade and subsequent pathophysiology of the disease, thus reducing the validity of studies designed to characterize and compare influenza viruses.^6,43 NSAID reportedly inhibit nuclear factor κB, a regulator of inflammatory processes that is involved in viral RNA synthesis.^25,27 In addition, NSAID have been found to increase survival rates in influenza virus-infected mice.⁵³ Therefore, the use of NSAID may be problematic in studies investigating the pathogenesis of influenza viruses.Buprenorphine, an opioid, is an established analgesic in ferrets that can be administered either intravascularly, intramuscularly, or subcutaneously at 0.01 to 0.05 mg/kg with an analgesic duration of 6 to 12 h.^{11,16,24,38,52} Historically buprenorphine has been described as a partial µ receptor agonist and κ and γ receptor antagonist,^22,29,40,48 but the drug recently was described to behave as a full µ agonist.³⁶ The ceiling effect of analgesia and the immunosuppressive effects reported with other opioids have not been documented to occur with buprenorphine.^15,36,42 However, the use of buprenorphine does have the possibility of adverse effects, including sedation, weight loss, constipation, and respiratory depression.^{10,15,16,22,23,34,42} Nonetheless, buprenorphine is a commonly prescribed analgesic for numerous small mammalian species used in research settings.^20,22,40Given that influenza is an ongoing threat to human and animal health and because no replacement is available for data gained with the ferret model, pain mitigation options for research conducted in this species must be addressed. To date, concerns about altering the course of the disease have precluded the evaluation of refinements options in IAV-infected ferrets. The goal of the current study was to assess the effects of buprenorphine treatments on the pathogenesis of a seasonal IAV in ferrets; this assessment was achieved by comparing virus-inoculated ferrets that were either sham-treated or that received buprenorphine according to 2 different dosing schedules. We hypothesized that buprenorphine treatments would not affect experimental readouts, including morbidity, viral shedding, lymphopenia, and seroconversion in convalescent serum; these parameters are commonly measured during IAV research. Study results indicate that buprenorphine did not uniformly or significantly modulate disease progression, peak viral titers in the upper respiratory tract, or clinical responses used to characterize viral pathogenicity in ferrets.     

Simultaneous storage of medical images in the spatial and frequency domain: A comparative study

Background  

Digital watermarking is a technique of hiding specific identification data for copyright authentication. This technique is adapted here for interleaving patient information with medical images, to reduce storage and transmission overheads.     

Randomly distributed crossovers may generate block-like patterns of linkage disequilibrium: an act of genetic drift

There is considerable interest in identifying and characterizing block-like patterns of linkage disequilibrium (LD; haplotype blocks) in the human genome as these may facilitate the identification of complex disease genes via genome-wide association studies. Although recombination hot-spots have been suggested as the primary mechanism to explain the block-like pattern of LD, other forces, such as genetic drift, may also be important. To this end, we have studied the effect of various recombination models on patterns of LD by using extensive simulations. As expected, haplotype blocks were observed under a model allowing recombination hot-spots. However, we also observed similar block-like patterns in the models where recombination crossovers are randomly and uniformly distributed, and we demonstrate that these blocks are generated by genetic drift. We caution that genetic drift may be an alternative mechanism (in addition to recombination hot-spots) that can lead to block-like patterns of LD. Our findings highlight the necessity of characterizing haplotype blocks in world-wide populations.     

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2.

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.     

Epethelial Presence of Trueperella pyogenes Predicts Site-Level Presence of Cranial Abscess Disease in White-Tailed Deer (Odocoileus virginianus)

Cranial/intracranial abscess disease is an emerging source of significant mortality for male white-tailed deer (Odocoileus virginianus). Most cases of cranial/intracranial abscess disease are associated with infection by the opportunistic pathogen Trueperella pyogenes although the relationship between the prevalence of the bacteria and occurrence of disease is speculative. We examined 5,612 hunter-harvested deer from 29 sites across all physiographic provinces in Georgia for evidence of cranial abscess disease and sampled the forehead, lingual, and nasal surfaces from 692 deer. We used polymerase chain reaction (PCR) to determine presence of T. pyogenes from these samples. We found T. pyogenes prevalence at a site was a predictor for the occurrence of cranial abscess disease. Prevalence of T. pyogenes did not differ between samples from the nose or tongue although prevalence along the forehead was greater for males than females (p = 0.04), particularly at sites with high occurrence of this disease. Socio-sexual behaviors, bacterial prevalence, or physiological characteristics may predispose male deer to intracranial/cranial abscess disease. Determination of factors that affect T. pyogenes prevalence among sites may help explain the occurrence of this disease among populations.     

Pathogenesis of avian influenza (H7) virus infection in mice and ferrets: enhanced virulence of Eurasian H7N7 viruses isolated from humans

Before 2003, only occasional case reports of human H7 influenza virus infections occurred as a result of direct animal-to-human transmission or laboratory accidents; most of these infections resulted in conjunctivitis. An increase in isolation of avian influenza A H7 viruses from poultry outbreaks and humans has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. To better understand the pathogenesis of H7 viruses, we have investigated their ability to cause disease in mouse and ferret models. Mice were infected intranasally with H7 viruses of high and low pathogenicity isolated from The Netherlands in 2003 (Netherlands/03), the northeastern United States in 2002-2003, and Canada in 2004 and were monitored for morbidity, mortality, viral replication, and proinflammatory cytokine production in respiratory organs. All H7 viruses replicated efficiently in the respiratory tracts of mice, but only Netherlands/03 isolates replicated in systemic organs, including the brain. Only A/NL/219/03 (NL/219), an H7N7 virus isolated from a single fatal human case, was highly lethal for mice and caused severe disease in ferrets. Supporting the apparent ocular tropism observed in humans following infection with viruses of the H7 subtype, both Eurasian and North American lineage H7 viruses were detected in the mouse eye following ocular inoculation, whereas an H7N2 virus isolated from the human respiratory tract was not. Therefore, in general, the relative virulence and cell tropism of the H7 viruses in these animal models correlated with the observed virulence in humans.