Efficacy Of Potential Therapeutic Agents Research Articles

Of mice and men: Laboratory murine models for recapitulating the immunosuppression of human sepsis.

Prolonged immunosuppression is increasingly recognized as the major cause of late phase and long-term mortality in sepsis. Numerous murine models with different paradigms, such as lipopolysaccharide injection, bacterial inoculation, and barrier disruption, have been used to explore the pathogenesis of immunosuppression in sepsis or to test the efficacy of potential therapeutic agents. Nonetheless, the reproducibility and translational value of such models are often questioned, owing to a highly heterogeneric, complex, and dynamic nature of immunopathology in human sepsis, which cannot be consistently and stably recapitulated in mice. Despite of the inherent discrepancies that exist between mice and humans, we can increase the feasibility of murine models by minimizing inconsistency and increasing their clinical relevance. In this mini review, we summarize the current knowledge of murine models that are most commonly used to investigate sepsis-induced immunopathology, highlighting their strengths and limitations in mimicking the dysregulated immune response encountered in human sepsis. We also propose potential directions for refining murine sepsis models, such as reducing experimental inconsistencies, increasing the clinical relevance, and enhancing immunological similarities between mice and humans; such modifications may optimize the value of murine models in meeting research and translational demands when applied in studies of sepsis-induced immunosuppression.

Profibrotic Signaling Pathways and Surface Markers Are Up-Regulated in Fibroblasts of Human Striae Distensae and in a Mouse Model System.

Striae distensae are common disfiguring cutaneous lesions but lack effective treatments because of an incomplete understanding of their pathophysiology. Dermal fibroblasts likely play an important role. The authors investigate the cellular-molecular features distinguishing fibroblasts from human striae distensae and normal skin. The authors also develop a mouse model of striae distensae. Human striae distensae and normal skin samples were compared for tensile strength and histologic structure. Fibroblasts from striae distensae and normal skin were isolated by fluorescence-activated cell sorting for gene expression analysis. Immunofluorescence staining and fluorescence-activated cell sorting were used to confirm gene expression data at the protein level. A mouse model of striae distensae formation was created by administering corticosteroids and mechanically loading the dorsal skin. Human striae distensae exhibited reduced tensile strength, more disordered collagen fibers, and epidermal atrophy compared to human normal skin. There were 296 up-regulated genes in striae distensae fibroblasts, including the profibrotic lineage and surface marker CD26. Up-regulated genes were involved in profibrotic and mechanoresponsive signaling pathways (TGFβ and FAK-PI3-AKT-signaling). In contrast, 571 genes were down-regulated, including CD74 and genes of the AMPK pathway. Increased CD26 and decreased CD74 expression was confirmed by fluorescence-activated cell sorting and immunofluorescence. Similar cutaneous histologic and gene expression changes were induced in hypercortisolemic mice by mechanically loading the dorsal skin. Fibroblasts from human striae distensae exhibit increased profibrotic and decreased antifibrotic signaling. CD26 and CD74 are promising surface markers that may be targeted therapeutically. The authors' mouse model of striae distensae can be used as a platform to test the efficacy of potential therapeutic agents. Striae distensae are common disfiguring cutaneous lesions whose etiology remains elusive, which has hindered development of effective treatment strategies. Dermal fibroblasts likely play an important role. The authors sought to elucidate the key cellular-molecular pathways distinguishing fibroblasts in striae distensae from those in normal skin.

IL-1β promotes disc degeneration and inflammation through direct injection of intervertebral disc in a rat lumbar disc herniation model

BACKGROUND CONTEXTLumbar intervertebral disc herniation (LDH) is a common disease that causes low back pain, radiating leg pain, and sensory impairment. Preclinical studies rely heavily upon standardized animal models of human diseases to predict clinical treatment efficacy and to identify and investigate potential adverse events in human subjects. The current method for making the LDH model involves harvesting the nucleus pulposus (NP) from autologous coccygeal discs and applying to the lumbar nerve roots just proximal to the corresponding dorsal root ganglion. However, this surgical method generates a model that exhibits very different characteristics of disc herniation than that observed in human. PURPOSETo produce a rat LDH model that better resembles disc herniation in humans and a standardized and uniform LDH model using Interleukin-1 beta (IL-1β). STUDY DESIGNExperimental rat LDH model. METHODSWe exposed the L5–6 disc dorsolaterally on the right side through hemi-laminectomy without nerve compression. Herniation was initiated by puncturing the exposed disc with a 30-gauge needle at a depth of 4 mm. Interleukin-1 beta (IL-1β) was injected simultaneously to heighten the pathological processes of disc degeneration, including inflammatory responses, matrix destruction, and herniation of the NP. We performed histological staining to assess morphological changes, immunohistochemistry to analyze inflammation- and pain-related expression within and around the puncture site of the L5–6 disc, and real-time polymerase chain reaction to examine expression of markers for degenerative processes. In addition, we performed locomotor tests on the rats. RESULTSWe found that the IL-1β groups showed that the border between the annulus fibrosis and nucleus pulposus was severely interrupted compared to that of the control (puncture only) group. And, the injection of IL-1β leads to accelerated disc degeneration and inflammation in a more consistent manner in LDH model. Functional deficit was consistently induced by puncturing and injection of IL-1β in the exposed disc. CONCLUSIONSThe method proposed here can be used as an index to control the severity of disc degeneration and inflammation through the injected IL-1β concentration concurrent with surgically induced herniation. CLINICAL SIGNIFICANCEOur proposed model may facilitate research in drug development to evaluate the efficacy of potential therapeutic agents for disc herniation and neuropathic pain and may also be used for nonclinical studies to more accurately assess the effectiveness of various treatment strategies according to the severity of disc degeneration.

Preliminary information on prevention of infections caused by SARS-COV-2 virus in endoscopic laboratories.

On 11 March 2020, the Director-General of the World Health Organization (WHO) announced COVID-19 (Coronavirus Disease 2019) as a global pandemic Currently, no vaccines are available and there is little evidence of the efficacy of potential therapeutic agents. Furthermore, there is presumably no pre-existing immunity in the population to the new coronavirus, and it is as-sumed that everyone in the population is susceptible. The aim of the procedures described in the article is to minimize the risk of human-to-human transmission of the SARS-CoV-2 (Severe acute respiratory syndrome - coronavirus 2) virus during procedures carried out in endoscopic laboratories. SARS-CoV-2 infection can be asymptomatic, cause severe pneumonia, or lead to death. Symptoms of COVID-19 range from none (asymptomatic) to severe pneumonia and it can be fatal. Case studies to-date indicate that this infection causes a mild illness (i.e. pneumonia or mild pneumonia) in approximately 80% of cases, and most cases recove; 14% have a more severe illness, 6% experience a critical illness. The vast majority of the most serious illnesses and deaths have occurred among the elderly and people with other chronic underlying diseases. Average progression times include: • in mild cases: from the onset of symptoms to recovery in almost 2 weeks; • in severe cases: from the onset of symptoms to recovery in 3-6 weeks, and from symptoms to death in 2-8 weeks. Special precautions should be taken and procedures followed when performing invasive medical procedures in endoscopic laboratories in patients with specific or clinically probable SARS-CoV-2 infection. This article contains up-to-date information as at 04/04/2020.

NURR1 deficiency is associated to ADHD-like phenotypes in mice

The transcription factor NURR1 regulates the dopamine (DA) signaling pathway and exerts a critical role in the development of midbrain dopaminergic neurons (mDA). NURR1 alterations have been linked to DA-associated brain disorders, such as Parkinson’s disease and schizophrenia. However, the association between NURR1 defects and the attention-deficit hyperactivity disorder (ADHD), a DA-associated brain disease characterized by hyperactivity, impulsivity and inattention, has never been demonstrated. To date, a comprehensive murine model of ADHD truly reflecting the whole complex human psychiatric disorder still does not exist. NURR1-knockout (NURR1-KO) mice have been reported to exhibit increased spontaneous locomotor activity, but their complete characterization is still lacking. In the present study a wide-ranging test battery was used to perform a comprehensive analysis of the behavioral phenotype of the male NURR1-KO mice. As a result, their hyperactive phenotype was confirmed, while their impulsive behavior was reported for the first time. On the other hand, no anxiety and alterations in motor coordination, sociability and memory were observed. Also, the number of mDA expressing tyrosine hydroxylase, a rate-limiting enzyme of catecholamines biosynthesis, and DA level in brain were not impaired in NURR1-KO mice. Finally, hyperactivity has been shown to be recovered by treatment with methylphenidate, the first line psychostimulant drug used for ADHD. Overall, our study suggests that the NURR1 deficient male mouse may be a satisfactory model to study some ADHD behavioral phenotypes and to test the clinical efficacy of potential therapeutic agents.

Corneal toxicity induced by vesicating agents and effective treatment options.

The vesicating agents sulfur mustard (SM) and lewisite (LEW) are potent chemical warfare agents that primarily cause damage to the ocular, skin, and respiratory systems. However, ocular tissue is the most sensitive organ, and vesicant exposure results in a biphasic injury response, including photophobia, corneal lesions, corneal edema, ulceration, and neovascularization, and may cause loss of vision. There are several reports on ocular injury from exposure to SM, which has been frequently used in warfare. However, there are very few reports on ocular injury by LEW, which indicate that injury symptoms appear instantly after exposure and faster than SM. In spite of extensive research efforts, effective therapies for vesicant-induced ocular injuries, mainly to the most affected corneal tissue, are not available. Hence, we have established primary human corneal epithelial cells and rabbit corneal organ culture models with the SM analog nitrogen mustard, which have helped to test the efficacy of potential therapeutic agents. These agents will then be further evaluated against in vivo SM- and LEW-induced corneal injury models, which will assist in the development of potential broad-spectrum therapies against vesicant-induced ocular injuries.

Ultrasensitive detection of PrP(Sc) in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrP(Sc)) in the central nervous system. The pathological features and biochemical properties of PrP(Sc) in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrP(Sc). This method involves amplifying PrP(Sc) by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrP(C) substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrP(Sc) in the CSF by serial PMCA, and the CSF levels of PrP(Sc) tended to increase with disease progression. In addition, PrP(Sc) was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrP(Sc) in non-human primate models of CJD.

On the importance of long-term functional assessment after stroke to improve translation from bench to bedside

Despite extensive research efforts in the field of cerebral ischemia, numerous disappointments came from the translational step. Even if experimental studies showed a large number of promising drugs, most of them failed to be efficient in clinical trials. Based on these reports, factors that play a significant role in causing outcome differences between animal experiments and clinical trials have been identified; and latest works in the field have tried to discard them in order to improve the scope of the results. Nevertheless, efforts must be maintained, especially for long-term functional evaluations. As observed in clinical practice, animals display a large degree of spontaneous recovery after stroke. The neurological impairment, assessed by basic items, typically disappears during the firsts week following stroke in rodents. On the contrary, more demanding sensorimotor and cognitive tasks underline other deficits, which are usually long-lasting. Unfortunately, studies addressing such behavioral impairments are less abundant. Because the characterization of long-term functional recovery is critical for evaluating the efficacy of potential therapeutic agents in experimental strokes, behavioral tests that proved sensitive enough to detect long-term deficits are reported here. And since the ultimate goal of any stroke therapy is the restoration of normal function, an objective appraisal of the behavioral deficits should be done.

Systems medicine and the integration of bioinformatic tools for the diagnosis of alzheimer's disease

Because of the changes in demographic structure, the prevalence of Alzheimer's disease is expected to rise dramatically over the next decades. The progression of this degenerative and terminal disease is gradual, with the subclinical stage of illness believed to span several decades. Despite this, no therapy to prevent or cure Alzheimer's disease is currently available. Early disease detection is still important for delaying the onset of the disease with pharmacological treatment and/or lifestyle changes, assessing the efficacy of potential therapeutic agents, or monitoring disease progression more closely using medical imaging. Sensitive cerebrospinal-fluid-derived marker candidates exist, but given the invasiveness of sample collection their use in routine diagnostics may be limited. The pathogenesis of Alzheimer's disease is complex and poorly understood. There is thus a strong case for integrating information across multiple physiological levels, from molecular profiling (metabolomics, lipidomics, proteomics and transcriptomics) and brain imaging to cognitive assessments. To facilitate the integration of heterogeneous data, such as molecular and image data, sophisticated statistical approaches are needed to segment the image data and study their dependencies on molecular changes in the same individuals. Molecular profiling, combined with biophysical modeling of molecular assemblies associated with the disease, offer an opportunity to link the molecular pathway changes with cell- and tissue-level physiology and structure. Given that data acquired at different levels can carry complementary information about early Alzheimer's disease pathology, it is expected that their integration will improve early detection as well as our understanding of the disease.

Contribution of Inflammatory Processes to Nerve Cell Toxicity by Bilirubin and Efficacy of Potential Therapeutic Agents

Hyperbilirubinemia is a common condition in neonatal life, where elevated levels of unconjugated bilirubin (UCB) may lead to adverse neurologic outcomes, namely in the presence of inflammatory features. In this review, we summarize recent concepts on UCB damage to brain cells and associated neuroinflammation research. Exposure of astrocytes and microglia to UCB initiates an inflammatory response with the release of proinflammatory cytokines, such as TNF-alpha, IL-1beta and IL-6, accumulation of extracellular glutamate and a time-dependent cell death. Moreover, undifferentiated cells revealed to be particularly susceptible to UCB-induced immunostimulation pointing to a mechanism that may preside to the vulnerability evidenced by premature newborns. Evaluation of intracellular mechanisms of astrocyte and microglia to UCB revealed that TNF-alpha and IL-1beta pathways as well as MAPK and NF-kappaB signaling cascades are key mediators of both cytokine production and cell toxicity observed upon UCB challenge. Understanding these mechanisms is essential for the development of new strategies targeting UCB-induced neurotoxicity. Thus, a therapeutic approach for the prevention or amelioration of neurological deficits resulting from moderate to severe hyperbilirubinemia, may consist on the use of immunomodulators, such as IL-10 that showed ability to suppress the release of cytokines from astrocytes exposed to UCB, glycoursodeoxycholic acid (GUDCA) that abrogated both UCB-stimulated cytokine secretion and UCB-induced loss of cell survival, and minocycline that evidenced a unique role in preventing neurodegeneration in in vitro and in vivo models. Novel pharmacological strategies may reduce the incidence of UCB encephalopathy and prevent minor cerebral lesions that may result in mental illness.

Improvements of the Stroke Model Guidelines Animal body weight and long-term functional concerns

Despite extensive research efforts in the field of cerebral ischemia, numerous disappointments came out. Indeed, even if experimental studies showed up a huge number of promising drugs, most of them unfortunately failed to be efficient in clinical trials. Based on these reports, factors that play a significant role in causing outcome variation were identified and recently reviewed in the Rodent Stroke Model Guidelines for Preclinical Studies. Herein, we provide improvements to this first edition as regards to the functional evaluation section, especially regarding long-term evaluation. Indeed, as in clinical practice (Rothrock et al 1995), animals display a certain degree of spontaneous recovery after stroke (Hunter et al 1998; Roof et al 2001; Zausinger et al 2000; Zhang et al 2000). Typically, the neurological impairment, assessed by basic items, usually disappears during the initial week following stroke in rodents (Bederson et al 1986). As a contrary, more demanding sensorimotor and cognitive tasks underline other deficits, usually long-lasting. Unfortunately, studies addressing such behavioral impairments are less abundant. Therefore, because the characterization of long-term functional recovery is critical for evaluating the efficacy of potential therapeutic agents in experimental stroke, behavioral tests that proved to be sensitive enough to detect long-term deficits are reported here. Overall, since the ultimate goal of any stroke therapy is the restoration of normal functions, an objective appraisal of the behavioral deficits should be done.

Improvements of the Stroke Model Guidelines – Animal body weight and long-term functional concerns

Despite extensive research efforts in the field of cerebral ischemia, numerous disappointments came out. Indeed, even if experimental studies showed up a huge number of promising drugs, most of them unfortunately failed to be efficient in clinical trials. Based on these reports, factors that play a significant role in causing outcome variation were identified and recently reviewed in the Rodent Stroke Model Guidelines for Preclinical Studies. Herein, we provide improvements to this first edition as regards to the functional evaluation section, especially regarding long-term evaluation. Indeed, as in clinical practice (Rothrock et al 1995), animals display a certain degree of spontaneous recovery after stroke (Hunter et al 1998; Roof et al 2001; Zausinger et al 2000; Zhang et al 2000). Typically, the neurological impairment, assessed by basic items, usually disappears during the initial week following stroke in rodents (Bederson et al 1986). As a contrary, more demanding sensorimotor and cognitive tasks underline other deficits, usually long-lasting. Unfortunately, studies addressing such behavioral impairments are less abundant. Therefore, because the characterization of long-term functional recovery is critical for evaluating the efficacy of potential therapeutic agents in experimental stroke, behavioral tests that proved to be sensitive enough to detect long-term deficits are reported here. Overall, since the ultimate goal of any stroke therapy is the restoration of normal functions, an objective appraisal of the behavioral deficits should be done.

M17 human neuroblastoma cell as a cell model for investigation of Botulinum Neurotoxin A activity and evaluation of BoNT/A specific antibody

Since BoNTs are potential biothreat agents, efforts are being made to develop therapeutic countermeasures for which sensitive in vitro models are required to evaluate the efficacy of potential therapeutic agents. In this report, we tested human neuroblastoma cell lines for their susceptibility to BoNT toxicity and demonstrated the application of these cells to measure the neutralising activity of a BoNT-specific antibody. We demonstrated that SK-N-BE(2)M17 cells are highly sensitive to BoNT/A and /C intoxication and can be used as a cell-based assay to investigate the molecular mechanism of BoNT/A intoxication and to quantitate the neutralising activity of antibodies. [Received 26 October 2007; Accepted 12 December 2007]

Quantitative histological analysis of amyloid deposition in Alzheimer’s double transgenic mouse brain

The development of transgenic mice has created new opportunities for the generation of animal models of human neurodegenerative diseases where previously there was no animal counterpart. The first successful transgenic mouse model of Alzheimer’s disease expressed increased levels of mutant human amyloid precursor protein, exhibiting neuritic-type amyloid deposits and behavioral deficits at six to nine months of age. More recently, it was shown that transgenic mice expressing both mutant human amyloid precursor protein and presenilin 1 exhibit neuritic-type amyloid deposits and behavioral deficits in as little as 12 weeks. This accelerated Alzheimer phenotype greatly reduces the time necessary to conduct preclinical drug trials, as well as animal housing costs. The purpose of this study was to quantify the deposition of amyloid in five regions of the cortex and two regions of the hippocampus of transgenic mice expressing amyloid precursor protein (K670N, M671L) and presenilin 1 (M146L) mutations at various ages, using quantitative methods of confocal laser scanning microscopy and image analysis. Amyloid burden, expressed as the percentage area occupied by thioflavin S-positive amyloid deposits, increased an average of 179-fold from 12 to 54 weeks of age (0.02±0.01% to 3.57±0.29%, mean±S.E.M., respectively) in five regions of the cortex and two of the hippocampus. This was a function of increases in both deposit number and size. This transgenic mouse provides an ideal animal model for evaluating the efficacy of potential therapeutic agents aimed at reducing amyloid deposition, such as inhibitors of amyloid fibril formation or secretase inhibitors.

Biological markers in the diagnosis and treatment of ALS

The care of patients with amyotrophic lateral sclerosis (ALS), which has classically focused on treatment of symptomatology, has now entered an encouraging new era of therapy targeted at the pathophysiology of the disease. However, an objective measure of disease progression and therapeutic response is sorely needed. Quantitative neuromuscular examinations, measurement of pulmonary function, disability scales, and even survival, are limited by variability due to a number of poorly controlled factors. Quantitative electromyography, positron emission tomography scanning, and magnetic cortical stimulation, provide potential objective indicators of disease progression, but require a large number of patients and a long observation period for adequate statistical power. We have examined the role of magnetic resonance spectroscopic imaging in detecting acute changes in motor cortical metabolism in response to riluzole therapy. N-acetylaspartate (NAA), the most prominent signal in proton spectra of normal brain, is a neuron-specific molecule. ALS patients were found to experience a significant increase in the NAA/creatine ratio within 3 weeks of initiation of riluzole therapy. As glutamate can trigger the generation of reactive oxygen species in neurons, we speculate that acute changes in NAA levels may reflect oxidative injury to mitochondria where NAA is synthesised. The advent of a useful test for upper motor neuron metabolic compromise may provide an objective, non-invasive, short duration measure with which to screen the efficacy of potential therapeutic agents for ALS.

Evaluation of the efficacy of potential therapeutic agents at protecting against nitric oxide synthase-mediated mitochondrial damage in activated astrocytes

Within the central nervous system, nitric oxide is an important physiological messenger [1]. However, when synthesized excessively in neurones, cell death may occur [2]. An impairment of mitochondrial cytochrome oxidase and subsequent cellular energy depletion seems to be a likely mechanism for this neurotoxicity [3]. Within neurones, nitric oxide is synthesized by the constitutive, Ca 2+-dependent form of nitric oxide synthase (nNOS) [4]. Astrocytes, however, possess both the constitutive [5]and the inducible Ca 2+-independent NOS (iNOS) [6, 7], which is expressed by endotoxin and/or cytokines [8]. In vitro, activation of nNOS rapidly produces neuronal cell death [2]. In contrast to neurones, following induction of iNOS, astrocytes synthesize large quantities of nitric oxide [3, 9], but cell death is not apparent despite marked damage to mitochondrial cytochrome oxidase [3]. The resistance of astrocytes to nitric oxide synthase-mediated cell damage may be due to their ability to increase their glycolytic rate when mitochondrial ATP synthesis is compromised [3]. On the basis of this phenomenon, we propose that activated astrocytes represent a suitable system for studying the efficacy of potential therapeutic agents at protecting from nitric oxide synthase-mediated mitochondrial damage [10].

Establishment of a rabbit short-term dry eye model.

The dry eye syndrome is a chronic disease which can become a serious threat to useful vision. However, there is only a limited understanding regarding effective treatment or prevention of this disease. Establishing an effective mode of treatment requires the use of a satisfactory animal dry eye model. Ideally, such a model should rapidly determine the effectiveness of agents that inhibit the damaging effects of this syndrome. This paper presents a short-term dry eye model using rabbits, which combines mechanical prevention of blinking and methylene blue staining. This model is not intended to be a precise representation of the dry eye syndrome, since this disorder has recently become recognized to involve a primary pathological process of the corneal and conjunctival epithelium. However, by using this model, clinical signs of dry eye can be observed after a few hours in the form of acute desiccation. Corneal damage can easily be evaluated both qualitatively by methylene blue staining scores, and quantitatively by chronic assay. Visually observed corneal epithelial thinning was confirmed by scanning electron microscopy (SEM) to be due to loss of epithelial integrity. Using a 3% chondroitin sulfate solution, an already proven effective agent for dry eye, this model effectively demonstrated an 80% inhibition in the development of methylene blue positive lesion after a period of only 2 hours. This short term dry eye model is valuable in primarily screening the efficacy of potential therapeutic agents in the prevention and treatment of dry eye.