Development of a Novel Fluorescent-Based Lateral Flow Assay for the Detection of Neisseria gonorrhoeae at the Point of Care.

BACKGROUND: Neisseria gonorrhoeae (NG) has acquired significant resistance, primarily due to extensive and unwarranted antibiotic utilization over several decades. This resistance has largely been associated with the syndromic management of sexually transmitted infections, particularly in low- and middle-income countries where affordable point of care tests are unavailable. To address this diagnostic gap, FIND has developed a low-cost lateral flow assay for the detection of NG at the point of care. METHODS: The early performance of the lateral flow assay was evaluated using frozen clinical samples. Limit of detection, inclusivity, and exclusivity studies were performed using well-characterized NG strains, common commensal genital microorganisms, and other Neisseria bacteria. Subsequently, clinical performance was evaluated at 2 sexual health clinics in Birmingham, Alabama. RESULTS: The observed limit of detection with reference NG strains was 5 × 103 CFU/mL. Inclusivity was demonstrated for 31 NG strains. Exclusivity testing showed no cross-reactivity with 28 non-Neisseria and nongonococcal Neisseria species; cross-reactivity was observed with Neisseria meningitidis, Neisseria lactamica, and Neisseria polysaccharea. The lateral flow assay demonstrated clinical sensitivity and specificity of 78.6% and 100% in female vaginal swabs and 100% and 89.7% in male urine, respectively. CONCLUSIONS: FIND has developed a lateral flow assay that aligns with the majority of the World Health Organization Target Product Profile criteria for confirming or excluding NG infection at the point of care. The NG lateral flow assay has now achieved design freeze (final device optimization) and is ready for technology transfer to a manufacturing partner. This test has the potential to support the shift in patient management from a syndromic to an etiological approach.

Infectivity-Enhanced, Conditionally Replicative Adenovirus for COX-2-Expressing Castration-Resistant Prostate Cancer.

BACKGROUND: The development of conditionally replicative adenoviruses (CRAds) for castration-resistant prostate cancer (CRPC), particularly neuroendocrine prostate cancer (NEPC), has two major obstacles: choice of control element and poor infectivity. We applied fiber-modification-based infectivity enhancement and an androgen-independent promoter (cyclooxynegase-2, COX-2) to overcome these issues. METHODS: The properties of the COX-2 promoter and the effect of fiber modification were tested in two CRPC cell lines (Du-145 and PC3). Fiber-modified COX-2 CRAds were tested in vitro for cytocidal effect as well as in vivo for antitumor effect with subcutaneous CRPC xenografts. RESULTS: In both CRPC cell lines, the COX-2 promoter showed high activity, and Ad5/Ad3 fiber modification significantly enhanced adenoviral infectivity. COX-2 CRAds showed a potent cytocidal effect in CRPC cells with remarkable augmentation by fiber modification. In vivo, COX-2 CRAds showed an antitumor effect in Du-145 while only Ad5/Ad3 CRAd showed the strongest antitumor effect in PC3. CONCLUSION: COX-2 promoter-based, infectivity-enhanced CRAds showed a potent antitumor effect in CRPC/NEPC cells.

Polytrauma-induced hepatic stress response and the development of liver insulin resistance.

Insulin resistance and metabolic dysfunction are common following injury. Polytrauma is defined as combined injuries to more than one body part or organ system, and is common in modern warfare, as well as automobile and industrial accidents. Polytrauma can include any combination of burn injury, fracture, hemorrhage, trauma to the extremities, and blunt or penetrating trauma. Multiple minor injuries are often more deleterious than a more severe single injury. To investigate the mechanisms of development of insulin resistance following injury, we have developed a rat model of polytrauma which combined soft tissue trauma with burn injury and penetrating gastrointestinal (GI) trauma. Male Sprague-Dawley rats were subjected to a laparotomy plus either a 15-18% total body surface area scald burn or a single puncture of the cecum (CLP) with a G30 needle, or the combination of both burn and CLP injuries (polytrauma). We examined the effects of polytrauma which increased markers of hepatic endoplasmic reticulum (ER) stress, and increased hepatic Trib3 mRNA levels coincident with reduced insulin-inducible insulin signaling. Phosphorylation/activation of the insulin receptor (IR) and AKT were decreased at 24, but not 6h following polytrauma. These results demonstrate a complex, time-dependent development of hepatic ER-stress and a diminished response to insulin, which were among the pathological sequelae following polytrauma.

Skeletal muscle atrogene expression and insulin resistance in a rat model of polytrauma.

Polytrauma is a combination of injuries to more than one body part or organ system. Polytrauma is common in warfare, and in automobile and industrial accidents. The combination of injuries can include burn, fracture, hemorrhage, and trauma to the extremities or specific organ systems. Resistance to anabolic hormones, loss of muscle mass, and metabolic dysfunction can occur following injury. To investigate the effects of combined injuries, we have developed a highly reproducible rodent model of polytrauma. This model combines burn injury, soft tissue trauma, and penetrating injury to the gastrointestinal (GI) tract. Adult, male Sprague-Dawley rats were anesthetized with pentobarbital and subjected to a 15-20% total body surface area scald burn, or laparotomy and a single puncture of the cecum with a G30 needle, or the combination of both injuries (polytrauma). In the current studies, the inflammatory response to polytrauma was examined in skeletal muscle. Changes in skeletal muscle mRNA levels of the proinflammatory cytokines TNF-α, IL-1ß, and IL-6 were observed following single injuries and polytrauma. Increased expression of the E3 ubiquitin ligases Atrogin-1/FBX032 and TRIM63/MuRF-1 were measured following injury, as was skeletal muscle insulin resistance, as evidenced by decreased insulin-inducible insulin receptor (IR) and AKT/PKB (Protein Kinase B) phosphorylation. Changes in the abundance of IR and insulin receptor substrate-1 (IRS-1) were observed at the protein and mRNA levels. Additionally, increased TRIB3 mRNA levels were observed 24 h following polytrauma, the same time when insulin resistance was observed. This may suggest a role for TRIB3 in the development of acute insulin resistance following injury.

Oncolytic adenovirus expressing interferon alpha in a syngeneic Syrian hamster model for the treatment of pancreatic cancer.

BACKGROUND: The addition of interferon (IFN) alpha to adjuvant chemoradiotherapy regimens resulted in remarkable improvements in survival for pancreatic cancer patients. However, systemic toxicities and insufficient levels of IFN at the tumor sites have limited its widespread adoption in treatment schemes. We have previously developed an IFN-expressing conditionally replicative oncolytic adenovirus and demonstrated its therapeutic effects both in vitro and in vivo. Here, the same vectors were tested in a syngeneic and immunocompetent Syrian hamster model to better understand the roles of adenoviral replication and of the pleiotropic effects of IFN on pancreatic tumor growth suppression. METHODS: Oncolytic adenoviruses expressing human or hamster IFN were designed and generated. Viral vectors were tested in vitro to determine qualitative and quantitative cell viability, cyclooxygenase 2 (Cox2) promoter activity, and IFN production. For the in vivo studies, subcutaneous hamster pancreatic cancer tumors were treated with 1 intratumoral dose of virus. Similarly, 1 intraperitoneal dose of virus was used to prolong survival in a carcinomatosis model. RESULTS: All cell lines tested demonstrated Cox2 promoter activity. The oncolytic potential of a replication competent adenovirus expressing the IFN cytokine was clearly demonstrated. These viruses resulted in significant tumor growth suppression and survival increases compared with controls in a hamster model. CONCLUSION: The profound therapeutic potential of an IFN-expressing oncolytic adenovirus for the treatment of pancreatic cancer was demonstrated in a syngeneic Syrian hamster model. These results strongly suggest the potential application of our viruses as part of combination regimens with other therapeutics.

A rat model of concurrent combined injuries (polytrauma).

Polytrauma, a combination of injuries to more than one body part or organ system, is common in modern warfare and in automobile and industrial accidents. The combination of injuries can include burn injury, fracture, hemorrhage, trauma to the extremities, and trauma to specific organ systems. To investigate the effects of combined injuries, we have developed a new and highly reproducible model of polytrauma. This model combines burn injury with soft tissue and gastrointestinal (GI) tract trauma. Male Sprague Dawley rats were subjected to a 15-20% total body surface area scald burn, or a single puncture of the cecum with a G30 needle, or the combination of both injuries (polytrauma). Unlike many 'double hit' models, the injuries in our model were performed simultaneously. We asked whether multiple minor injuries, when combined, would result in a distinct phenotype, different from single minor injuries or a more severe single injury. There were differences between the single injuries and polytrauma in the maintenance of blood glucose, body temperature, body weight, hepatic mRNA and circulating levels of TNF-α, IL-1ß and IL-6, and hepatic ER-stress. It has been suggested that models utilizing combinatorial injuries may be needed to more accurately model the human condition. We believe our model is ideal for studying the complex sequelae of polytrauma, which differs from single injuries. Insights gained from this model may suggest better treatment options to improve patient outcomes.

Regulation of hepatic insulin receptor activity following injury.

Impaired insulin receptor (IR) activity has been found in various models of insulin resistance, including models of injury or critical illness and Type 2 diabetes. However, mechanisms that modulate IR function remain unclear. With an animal model of critical-illness diabetes, we found insulin-induced IR tyrosine phosphorylation in the liver was impaired as early as 15 min following trauma and hemorrhage. Possible mechanisms for this defect were examined, including IR protein levels and IR posttranslational modifications. The total amounts of hepatic IRα and IRß subunits and the membrane localization of the IR were not altered by trauma and hemorrhage, and, likewise, no change in IR tyrosine nitration was found in the liver. However, there was a decrease in the level of protein O-linked ß-N-acetlyglucosamine (O-GlcNac) modification on Ser/Thr in the liver following trauma and hemorrhage. Inhibition of JNK increased IR O-GlcNac modification, implicating an involvement of JNK. These findings suggest that a balance between O-GlcNac modification and JNK-induced phosphorylation may exist, with decreased Ser/Thr O-GlcNac modification following trauma and hemorrhage, allowing JNK to phosphorylate the IR on neighboring Ser/Thr residues, which subsequently inhibits IR activity. The present studies suggest potential mechanisms of hemorrhage-induced defects in IR activity and a potential role for acutely decreased O-GlcNac and increased serine phosphorylation of the IR.

Generation of a novel, cyclooxygenase-2-targeted, interferon-expressing, conditionally replicative adenovirus for pancreatic cancer therapy.

BACKGROUND: Oncolytic adenoviruses provide a promising alternative for cancer treatment. Recently, adjuvant interferon (IFN)-alfa has shown significant survival benefits for pancreatic cancer, yet was impeded by systemic toxicity. To circumvent these problems adenovirus with high-level targeted IFN-alfa expression can be generated. METHODS: Conditionally replicative adenoviruses (CRAds) with improved virulence and selectivity for pancreatic cancer were generated. The vectors were tested in vitro, in vivo, and in human pancreatic cancer and normal tissue specimens. RESULTS: Adenoviral death protein and fiber modifications significantly improved oncolysis. CRAds selectively replicated in vitro, in vivo and showed persistent spread in cancer xenografts. They showed high-level replication in human pancreatic cancer specimens, but not in normal tissues. Improved IFN-CRAd oncolytic efficiency was shown. CONCLUSIONS: Optimized cyclooxygenase-2 CRAds show highly favorable effects in vitro and in vivo. We report a pancreatic cancer-specific, highly virulent, IFN-expressing CRAd, and we believe that adenovirus-based IFN therapy offers a new treatment opportunity for pancreatic cancer patients.

Role of tissue macrophages in the development of critical illness diabetes.

Macrophage-derived factors, including TNF-α, are known as important inducers of insulin resistance. However, the role of macrophages in insulin resistance in the liver is unclear. Hyperglycemia and insulin resistance commonly occur following acute injuries or critical illness, referred to as "critical illness diabetes." In the present studies, the roles of macrophages in hepatic insulin resistance following surgical trauma and hemorrhage were investigated. Intravenous administration of gadolinium chloride or clodronate-liposome resulted in depletion of macrophages in both liver and spleen of rats. Macrophage depletion by either gadolinium chloride or clodronate-liposome did not prevent the development of trauma and hemorrhage-induced insulin resistance in the liver of rats, as indicated by impaired hepatic insulin signaling following a 90-minute hemorrhage period. Similarly, hepatic insulin resistance still developed in rats after removal of the spleen (splenectomy). In contrast, macrophage depletion significantly reversed the hepatic insulin resistance several hours later, following resuscitation. As a comparison, splenectomy resulted in improvement in hepatic insulin signaling following resuscitation, but to a lesser extent, suggesting that both liver and spleen resident macrophages have a role in the continuation of hepatic insulin resistance following resuscitation. These studies demonstrated that the initial development of insulin resistance in liver is macrophage-independent in a rodent model of critical illness diabetes, whereas both liver and spleen macrophages have a role in the later maintenance of the insulin-resistant state, following resuscitation.

Adenovirus infection results in alterations of insulin signaling and glucose homeostasis.

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4-13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents.

In vivo bioimaging tracks conditionally replicative adenoviral replication and provides an early indication of viral antitumor efficacy.

In vivo monitoring of conditionally replicative adenovirus (CRAd) replication and assessing its correlation to CRAd biological effects are necessary for the clinical development of gene therapy. Noninvasive bioimaging is one current approach which can monitor in vivo CRAd replication and functional effect. Here we describe a novel cyclooxygenase-2 (Cox2) promoter-controlled CRAd that was modified to contain firefly luciferase in its E3 region; this modification permitted serial bioluminescence imaging of viral replication in vitro and in vivo. In vitro luciferase expression correlated with viral replication and cytolytic effect. In vivo bioluminescence imaging showed dynamic representation of the viral replication level in athymic nude mice bearing subcutaneous tumor xenografts. Importantly, in vivo luciferase bioluminescence measured 6 days after viral administration significantly correlated with CRAd antitumor effect at day 36. Thus, our system could detect viral replication and predict in vivo therapeutic outcome based on early imaging. Further development of this approach may improve patient safety, enhance clinical trial conduct, and provide mechanistic insight into CRAd function in vivo.

Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling.

BACKGROUND: Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated. METHODS: We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r). RESULTS: The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. CONCLUSIONS: Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.

Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.

To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

Infectivity-enhanced cyclooxygenase-2-based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment.

The employment of conditionally replicative adenoviruses (CRAd) constitutes a promising alternative for cancer treatment; however, in the case of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor-specific promoter and relative resistance to adenovirus infection have hampered the construction of CRAds with clinically applicable specificity and efficacy. By combining transcriptional targeting with infectivity enhancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) promoter-controlled replicative viral agents for the treatment of EAC. We used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral (Ad) fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob. The Cox-2 promoter was highly active in EAC, whereas showing no significant activity in Cox-2-negative cell lines and primary cells isolated from normal mouse esophagus and stomach. Evaluation of infectivity-enhanced vectors revealed that the transduction and virus-cell binding ability of Ad5/Ad3-chimera were significantly more efficient than that of unmodified and Arg-Gly-Asp (RGD)-modified vectors. All of the Cox-2 CRAds demonstrated replication and subsequent oncolysis in EAC cells but not in Cox-2-negative cells in vitro, thus confirming the dependence of their replication on the Cox-2 promoter activity. Ad5/Ad3 CRAds exhibited significantly improved oncolysis and progeny production compared with unmodified and RGD-modified vectors without sacrificing tumor selectivity. Whereas unmodified and RGD-modified CRAds showed insignificant therapeutic effect in vivo, Ad5/Ad3 CRAds remarkably suppressed tumor growth of established xenografts in mice. Thus, our studies have demonstrated that Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds are selective and potent agents for the treatment of EAC.