Initial Animal Studies Research Articles

Synthetic mimics of antimicrobial peptides

Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.

Biomarkers in the Assessment of Therapies for Familial Amyloidotic Polyneuropathy

The identification of specific biomarkers provides opportunities to develop early diagnostic parameters, monitor disease progression, and test drug efficiency in clinical trials. We previously demonstrated that in familial amyloidotic polyneuropathy (FAP) related to the abnormal extracellular tissue deposition of mutant transthyretin (TTR), inflammatory and apoptotic pathways are triggered in the presymptomatic stages of the disease, when nonfibrillar TTR deposits are present. In the present work, to better define biomarkers for future assessment of prophylactic and therapeutic drugs in the treatment of FAP, we extended the search for oxidative stress and apoptotic biomarkers to clinical samples and animal models presenting nonfibrillar and fibrillar TTR. We found that lipid peroxidation measured by hydroxynonenal, oxidative DNA damage measured by 8-hydroxy-2'-deoxyguanosine, and cellular redox homeostasis measured by glutaredoxin 1 were consistently increased in biopsy specimens from FAP patients and in tissues from transgenic mouse models presenting nonfibrillar TTR deposition. Death-receptor Fas, caspase-8, and Bax were also found to be increased, indicative of the involvement of death receptors in the observed apoptosis process. Removal of TTR deposition by an immunization protocol resulted in significant decreases of the selected markers we describe, corroborating the relationship between TTR deposition, oxidative stress, and apoptosis. Taken together, our results provide a robust biomarker profile for initial experimental animal studies and clinical trials to assess the application of the selected markers in therapies aimed at removal and/or inhibition of TTR polymerization.

The response of squamous cell carcinoma of the conjunctiva to treatment with photodynamic therapy

Abstract Purpose: Photodynamic therapy (PDT) was originally developed for tumor treatment. Based on initial promising animal studies, we investigated the clinical use of PDT for treating conjunctival neoplasia. Methods: Patients with newly diagnosed or recurrent conjunctival neoplasia / squamous cell carcinoma of the conjunctiva (SCC) were treated as part of an interventional case series. Patients received one to three treatments of 6 mg/m2 body surface area verteporfin (Visudyne, Novartis Ophthalmics, Switzerland) intravenously. The light dose was calculated as 50 J/cm2. All tumors were irradiated 1 minute after a bolus injection. Patients were followed weekly and then in monthly intervals up to 1 year. Retreatment was performed as early as one months after the initially treatment based on clinically and angiographic exam. Results: Angiographic occlusion of the tumor vasculature was usually observed one week after treatment. Signs of tumor regression were seen in all patients one month after treatment. Highly vascularized (papillomatous) lesions showed the most dramatic response to treatment. PDT was well tolerated with only minimal side‐effects including mild ocular irritaion, subconjunctival hemorrhage, chemosis and one case of infusion related back‐pain. No conjunctival scaring, thinning or scleral thinning were observed following treatments. Conclusions: Photodynamic therapy could potentially offer a less invasive approach to treating non‐melanotic conjunctival lesions in a subset of patients. However, larger studies are warranted to assess optimal treatment parameters and long‐term tumor control.

Conjugated linoleic acid: a functional nutrient in the different pathophysiological components of the metabolic syndrome?

Much attention has focused on the therapeutic potential of conjugated linoleic acid with the most abundant isomers being cis-9,trans-11 conjugated linoleic acid and trans-10,cis-12 conjugated linoleic acid. Initial animal studies associated conjugated linoleic acid with beneficial health properties, such as reducing the risk of cancer, diabetes, atherosclerosis, inflammation and obesity. This review has appraised the evidence in relation to the effect of conjugated linoleic acid on components of the metabolic syndrome (clinically or experimentally), in particular, obesity, insulin resistance, atherosclerosis and inflammation. More recent human conjugated linoleic acid supplementation studies have often shown conflicting and less convincing health benefits. The marked variation between studies may reflect the isomer-specific effect of the individual conjugated linoleic acid isomers, which can often have opposing effects. Detrimental effects have been observed in some studies, in particular after supplementation with the trans-10,cis-12 conjugated linoleic acid isomer. Further studies and long-term clinical trials will be required to determine the efficacy and safety of conjugated linoleic acid isomers before conjugated linoleic acid could be considered as a functional nutrient in humans.

Biological evaluation of tubulysin A: a potential anticancer and antiangiogenic natural product

Tubulysin A (tubA) is a natural product isolated from a strain of myxobacteria that has been shown to depolymerize microtubules and induce mitotic arrest. The potential of tubA as an anticancer and antiangiogenic agent is explored in the present study. tubA shows potent antiproliferative activity in a panel of human cancer cell lines irrespective of their multidrug resistance properties. It induces apoptosis in cancer cells but not in normal cells and shows significant potential antiangiogenic properties in several in vitro assays. It is efficacious in initial animal studies using a hollow fibre assay with 12 different human tumour cell lines. This study suggests that both in vitro and preclinical profiles of tubA may translate into clinically useful anticancer properties.

Preliminary findings from tests of a microwave applicator designed to treat Barrett's oesophagus

Barrett's oesophagus is considered to increase the risk of cancer 30-fold. Helical microwave antennas have been developed for ablative treatment of Barrett's. A microwave balloon applicator was tested in an initial animal study using adult white pigs. For treatment, a balloon filled with tissue-equivalent material encapsulated the antenna. A range of different treatment temperatures and durations was used to investigate a range of thermal ablations of the oesophageal epithelium. Eight animals were investigated, five non-survival and three with a 1-week survival period. The balloon was fitted with an array of temperature sensors, which gave an indication of the treatment in situ and allowed modifications to be performed in real time. Temperature data were recorded from all four quadrants of the balloon throughout and test sites were collected and analysed histologically. All experiments were successfully completed without perforation, serious adverse effects or death. Sites of discrete ulceration were induced in the survival tests, whereas the non-survival tests yielded little reproducible tissue modification. Results suggested that an activation temperature of ∼55 °C needed to be reached during the treatment for tissue damage to be induced. Once damage had been triggered the severity was related to the mean temperature attained during the treatment period. A mean temperature of 52 °C or more resulted in substantial damage, whilst a mean temperature of ∼50 °C resulted in the desired surface damage with sparing of subjacent tissues.

Angiotensin-Converting Enzyme Inhibition in Diabetic Nephropathy

Since the landmark multicenter trial in type I diabetics by Lewis et al. in 1993 ([1][1]), the use of angiotensin-converting enzyme (ACE) inhibitors to retard progression of diabetic nephropathy has become a widely accepted therapeutic strategy. Although initial animal studies determined that ACE

Investigation of Toxicity through Human Cases and Animal Experiments-Study of Bromopropanes-

In 1995, workers in a Korean electronic factory exhibited oligospermia or amenorrhea. We investigated the toxicity of 2-bromopropane used as an alternative to chlorofluorocarbons in the factory in animal experiments, and clarified that exposure to 2-bromopropane depletes spermatogenic cells in male rats and oocytes in female rats. Subsequently, we investigated the neurotoxicity of 2-bromopropane on the basis of the reported neuropathy in the Korean workers exposed to 2-bromopropane. For comparison, we employed 1-bromopropane, which is now used as a new alternative to chlorofluorocarbons. The results showed that 1-bromopropane is more neurotoxic than 2-bromopropane, causing harm to reproductive organs by inhibiting spermiation in the testis and impairing follicular development in the ovary. Shortly after the initial investigation of 1-bromopropane in animals, human cases were reported in the United States. Neurologic abnormalities in Chinese workers exposed to 1-bromopropane were also reported, such as the elongation of distal latency and lowered sense of vibration in the lower limbs. Thus, these serial studies revealed that 1-bromopropane is neurotoxic, but its dose-response relationship in humans remains unknown. In the investigation of 2-bromopropane toxicity, initial animal studies were designed on the basis of human studies, while in research on 1-bromopropane, animal studies preceded human studies and contributed to the prediction of toxicity in humans. However, the use of animal model is limited in its ability to predict the toxicity of chemicals introduced depending on species differences. Further studies should focus more on the differences and commonality between animals and humans in response to toxic agents.

"Topless" cardiopulmonary resuscitation. Fashion or science?

A decade after the onset of a discussion whether ventilation could be omitted from bystander basic life support (BLS) algorithms, the state of the evidence is reevaluated. Initial animal studies and a prospective randomized patient trial had suggested that omission of ventilation during the first minutes of lay cardiopulmonary resuscitation (CPR) did not impair patient outcomes. More recent studies demonstrate, however, that this may hold true only in very specific scenarios, and that the chest compression-only technique was never superior to standard BLS. Instead of calling basics of BLS training and practice into question, more and better training of lay persons and professionals appears mandatory, and targeted use of dispatcher-guided telephone CPR should be evaluated and, if it improves outcome, it should be encouraged. Future studies should focus much less on the omission but on the optimization of ventilation under the specific conditions of CPR.

Bone-marrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences.

Initial animal studies of single angiogenic agents, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), generated enthusiasm for the concept that these agents might enhance collateral development and thereby provide alternative therapies for patients with vascular disease not amenable to traditional revascularization. The enthusiasm, apparently justified by the subsequent results of small nonrandomized phase-I clinical trials, was then tempered by the subsequent disappointing results of randomized clinical trials. In light of these disappointing results, investigators have pursued alternative strategies in an attempt to improve tissue perfusion. One such strategy is the utilization of bone marrow-derived cell therapy. This review discusses mechanistic pathways mediating the effects of such cell therapy, summarizes the animal and early clinical experience, and speculates on the potential of genetic manipulation of bone marrow-derived cells in an attempt to further enhance their potency.

High-dose steroids for acute spinal cord injury in emergency medical services

Acute spinal cord injury (ASCI) is a potentially disabling condition that has been extremely resistant to treatment. Emergency medical services (EMS) providers are often among the first members of the health care team to treat victims of ASCI. Although initial human and animal studies indicated that highdose corticosteroid therapy might be an effective therapy for ASCI, subsequent human and animal studies have not been able to reproduce the beneficial effects described in the earlier research. Highdose corticosteroid therapy for the treatment of ASCI was introduced to prehospital care in the 1980s. It was postulated that early administration of corticosteroids could limit the severity of neurologic deficit associated with ASCI by stabilizing neuronal membranes and reducing inflammation and swelling. The recommended corticosteroid for acute spinal cord injury was methylprednisolone (MP), and the doses were large. Prior to this recommendation, many EMS services carried standard doses, typically 125 mg, of MP for use in severe allergic

AAV-mediated gene transfer to mouse lungs.

The ability of adeno-associated viral (AAV) vectors to promote persistent gene expression in nondividing cells in multiple somatic tissues of animals (1-4) makes them excellent tools for gene transfer. One tissue of interest for gene transfer is the lung epithelium, which is afflicted in cystic fibrosis (CF). However, although initial animal studies done with vectors based on AAV type 2 have demonstrated transduction in multiple cells types in the lung, the rates were modest in alveolar cells and much lower rates in airway epitheila and required high particle numbers (5-7). In contrast, an AAV6 encapsidated vector showed preferential transduction of epithelial cells in large and small airways (8) at rates that exceeded the 5% efficiency rate predicted to have a therapeutic value for CF gene therapy (9). In fact, recent studies using vectors based on other AAV types showed that types 1-6 have different tissue tropisms (10-15), and that types 5 and 6 are more efficient than type 2 in lung epithelium (8,14). In mouse lung, an AAV2 vector gave modest transduction rates.

Osteolysis: basic science.

Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has been gained on the basic science of periprosthetic bone loss. Initially termed cement disease, it now generally is accepted that, in most instances, osteolysis is a manifestation of an adverse cellular response to phagocytosable particulate wear and corrosion debris, possibly facilitated by local hydrodynamic effects. Tissue explant, animal, and cell culture studies have allowed us to compile an appreciation of the complexity of cellular interactions and chemical mediators involved in osteolysis. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular responses and effects on bone include prostaglandin E2, tumor necrosis factor-alpha, interleukin-1, and interleukin 6. However, an increasing number of other proinflammatory and antiinflammatory cytokines, prostenoids, and enzymes have been shown to play important roles in this process. The ultimate goal of basic research is to develop novel strategies for evaluation and treatment of patients with osteolysis. Although initial animal studies are promising for possible pharmacologic treatment and prevention of osteolysis, well-controlled human trials are required before agents such as bisphosphonates can be recommended for general clinical use.

Robotically assisted laparoscopic cholecystectomy: a pilot study.

Since the advent of laparoscopic surgery in 1987 and the introduction of robotics into medicine in 1991, medical technology has advanced to robotic applications in performing surgery. In our study, we investigated the feasibility of performing simple laparoscopic maneuvers and laparoscopic cholecystectomy using a robotic surgical system. The study used a ZEUS robotic system (Computer Motion Inc, Goleta, Calif), consisting of 3 interactive robotic arms fixed at the operating table and remotely controlled by the surgeon. After initial training, using a bench model and 3 isolated porcine livers to perform cholecystectomy, 7 female pigs underwent robotically assisted laparoscopic cholecystectomy. The surgeon, seated at the console, manipulated the 3-mm laparoscopic instruments and performed the surgery. Robotically assisted laparoscopic cholecystectomy was accomplished in all 7 pigs, with a mean operative time of 46 minutes (range, 30-62 minutes). There were no complications. The mean time to setup of the robotic system decreased from 30 minutes to 14 minutes. All the robotic maneuvers were performed without any particular difficulties, and the movements were stable, accurate, and reliable, with good control. Our preliminary experimental study showed that robotically reproduced laparoscopic maneuvers, such as tying, suturing, dissection, clipping, and cautery, seemed to be as accurate and as fast as maneuvers made without robotics. We conclude that our initial experimental and animal study confirmed the feasibility of robotically assisted laparoscopic cholecystectomy. Further reports are needed to show that robotics can be used for clinical applications in surgery.

Preclinical profiles of progestins used in formulations of oral contraceptives and hormone replacement therapy

Progestins used in oral contraceptive formulations available in the United States include norgestimate, desogestrel, norethindrone, norethindrone acetate, and levonorgestrel. Progestins used in the United States in continuous and intermittent formulations of hormone replacement therapy are norgestimate, medroxyprogesterone acetate, and norethindrone acetate. The chemical structure of a progestin determines its relative binding affinity for the progesterone and androgen receptors, as well as the sex hormone binding globulin in human serum, and determines its clinical profile. Overall, the properties of levonorgestrel or norethindrone acetate in this regard differ from norgestimate and are more conducive to androgenic stimulation. Estrogen replacement offers cardioprotective effects in postmenopausal women. Progestins are added to hormone replacement therapy to counteract the well-known increased risk of endometrial hyperplasia associated with the use of unopposed estrogen. Animal models show that for some parameters, including improvement of lipid profiles, progestins can diminish the cardioprotective effect of estrogen. Initial animal studies of norgestimate combined with estrogen do not show an attenuation of estrogenic effects.

Nitric oxide synthase inhibition as therapy for sepsis: a decade of promise.

Inhibition of nitric oxide synthase (NOS) has held promise as a novel and important therapeutic target for sepsis for over a decade. However, the question as to whether an inhibitor of NOS will prove to be efficacious in human septic shock remains unanswered. Review of the pertinent English-language medical literature. Observations of the induction of NOS (iNOS, NOS2) by proinflammatory cytokines led to the hypothesis that nitric oxide (NO) relaxes vascular smooth muscle, thereby producing vasodilation and hypotension in sepsis. Other effects of NO in vascular tone may be manifested by refractoriness to vasopressors or myocardial dysfunction. However, such negative effects of NO are balanced by the role of NO in maintaining microvascular perfusion and host defenses against invading pathogens. Initial animal studies of septic shock modulated by the administration of competitive inhibitors of NOS demonstrated that NOS inhibition might be beneficial therapy for clinical septic shock. However, subsequent animal studies have produced variable results, and a phase III clinical trial of a nonselective NOS inhibitor has failed to show benefit for therapy of septic shock. A better understanding of the effects of NOS and its inhibitors is needed as is an understanding of the underlying pathophysiology of sepsis. Moreover, a nontoxic, short-acting, titratable, specific inhibitor of NOS2 has yet to be identified and tested. Until then, efforts should be designed to describe more completely the role of NO in the pathophysiology of sepsis.

Clinical trials of a new class of therapeutic agents: antisense oligonucleotides

Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.

Potential of Gd-DTPA-mannan liposome particles as a pulmonary perfusion MRI contrast agent: an initial animal study.

Suga K, Mikawa M, Ogasawara N, et al. Potential of Gd-DTPA-mannan liposome particles as a pulmonary perfusion MRI contrast agent: An initial animal study. Invest Radiol 2001;36:136-145. A paramagnetic, particle-type MR contrast agent, (Gd-diethylenetriamine pentaacetic acid [DTPA]-mannan-cholesterol)-coated liposomes, was designed to localize in the lung by the mechanism of capillary blockade, and the potential of this agent for pulmonary perfusion MRI was experimentally investigated. Before and up to 60 minutes after slow injection of this contrast agent, MR images were sequentially acquired at 10-second intervals along the same transaxial plane of the lung by using a gradient-echo pulse sequence with a short echo time of 1.2 ms on a 1.5-T MR scanner. After the minimal dose for obtaining a sufficient lung enhancement effect was determined in five rabbits, the time course of the enhancement effect was evaluated in six dogs by arterial blood gas analysis. The efficacy of MRI for detecting perfusion defects was evaluated in seven other dogs with pulmonary embolism. Normal lungs were dose-dependently enhanced by this agent, and with a 2.0 mL/kg dose, dependent lungs were enhanced by more than 201%, with an average half-life of the enhancement effect of 35.7 +/- 5.3 minutes. With less than this dose (1.0-1.5 mL/kg), all of the embolized lung portions were clearly identified as perfusion defects. The prolonged enhancement effect allowed the acquisition of subsequent multisectional lung images, thus facilitating the assessment of anatomic location and extent of the perfusion defects. The reduction of PaO2 in room air after injection was within 5 mm Hg in both normal and embolized animals. These initial, experimental results show that paramagnetically labeled liposome particles may be a successful MR contrast agent for pulmonary perfusion imaging.

The Basic Science of Periprosthetic Osteolysis*†

Despite improvements in the techniques, materials, and fixation of total joint replacements, wear and its sequelae continue to be the main factors limiting the longevity and clinical success of arthroplasty. Since Charnley first recognized aseptic loosening in the early 1960s, a tremendous amount of information has been gained on the basic science of osteolysis. Tissue explant, animal, and cell culture studies have allowed development of an appreciation of the complexity of cellular interactions and chemical mediators involved in these processes. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular interactions and effects on bone primarily include PGE2, TNF-alpha, IL-1, and IL-6. Recent and ongoing work in the field of signaling pathways will continue to advance our understanding of the mechanisms of periprosthetic bone loss. Although initial animal studies are promising for the development of possible pharmacologic agents for the treatment and prevention of osteolysis, well controlled human trials are required.

Laparoscopic Lymph Node Dissection: Pelvic and Retroperitoneal

The application of laparoscopic procedures to the field of urology continues to expand at a rapid rate. The initial animal studies performed in the late 1980s were brought into the clinical arena by 1990. The first widely accepted procedure was laparoscopic pelvic lymph node dissection for the staging of prostate cancer. Since that time, numerous laparoscopic procedures have been developed and accepted. Herein we discuss laparoscopic pelvic lymph node dissection for the staging evaluation of cancer of the prostate with possible applications to the bladder, urethra, and penis. The technique of laparoscopic retroperitoneal lymph node dissection has been used for cancer of the testes and will also be described. The indications and a brief review of the postoperative results will also be discussed for each malignancy.