Marcelo Couto

Cationic defensins arise from charge-neutralized propeptides: a mechanism for avoiding leukocyte autocytotoxicity?

Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mammalian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic granules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil‐specific rabbit defensin cDNAs that code for preproprotein precursors to the mature defensins NP‐3a, NP‐4, and NP‐5. The preprodefensins NP‐3a, NP‐4, and NP‐5, like the previously characterized preprodefensins, lack consensus sequences for N‐linked glycosylation, suggesting that defensins are targeted to lysosome‐like granules by a mechanism not dependent on the mannose‐6‐phosphate receptor. Analysis of all seven known myeloid prodefensins revealed a structure wherein an anionic propiece neutralizes the cationicity of the mature peptide. Because defensins apparently require cationic epitopes for cell membrane permeabilization and cytotoxicity, charge neutralization of mature peptides by their anionic propieces may prevent autocytotoxicity during defensin synthesis and processing.

INDs for PET Molecular Imaging Probes—Approach by an Academic Institution

We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes (while the FDA uses the terminology PET drugs, we are using “PET imaging probes,” “PET probes,” or “probes” as the descriptive terms). The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [18 F]fluoro-arabinofuranosylcytosine (FAC) analog PET probes to phase 1 clinical trials. In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner.

Anatomy, Physiology, and Behavior

Publisher Summary The rabbit is a popular model in laboratory animal medicine due to its relatively large size and docile nature. Though rabbits are sometimes mistakenly thought of as rodents, they are in their own Order, Lagomorpha. Among the many breeds of rabbits, by far the most popular in research is the New Zealand White rabbit. Research in these rabbits has contributed to advances in cardiology, orthopedics, dentistry, immunology, and more. This chapter outlines the anatomy and physiology of rabbits. Rabbits have thin, delicate skin that is generously covered with both underfur and guard hairs. The rabbits skin, similar to the rats, has blood vessels immediately under the dermis. Unlike the rat, however, the fascia superficialis is well differentiated due to the elastic fibers and dense collagen content. Hair grows in waves starting from the ventrum and grows dorsally and caudally. Thick fur covers the feet in place of footpads. The fragile skin can tear easily and must be handled carefully during any manipulation. Rabbits are social, nocturnal animals that, despite over 2000 years of domestication, still have a highly developed prey instinct.

Comparison of the protective effect of a commercially available western diamondback rattlesnake toxoid vaccine for dogs against envenomation of mice with western diamondback rattlesnake (Crotalus atrox), northern Pacific rattlesnake (Crotalus oreganus oreganus), and southern Pacific rattlesnake (Crotalus oreganus helleri) venom.

OBJECTIVE To evaluate effectiveness of a commercially available toxoid manufactured from western diamondback (WD) rattlesnake (Crotalus atrox) venom against envenomation of mice with WD, northern Pacific (NP) rattlesnake (Crotalus oreganus oreganus), and southern Pacific (SP) rattlesnake (Crotalus oreganus helleri) venom. ANIMALS 90 specific pathogen-free female mice. PROCEDURES Mice were allocated into 3 cohorts (30 mice/cohort). Mice received SC injections of C atrox toxoid (CAT) vaccine (n = 15/group) or adjuvant (15/group) at day 0 and again at 4 weeks. At 8 weeks, mice were challenge-exposed with 1 of 3 venoms. Survival until 48 hours was evaluated by use of log-rank analysis of survival curves and the z test for proportions. RESULTS 6 of 15 WD-challenged CAT-vaccinated mice, 3 of 15 NP-challenged CAT-vaccinated mice, and 0 of 15 SP-challenged CAT-vaccinated mice survived until 48 hours. All adjuvant-only vaccinates survived ≤ 21 hours. Mean survival time of CAT vaccinates was longer than that of adjuvant-only vaccinates for all venoms (1,311 vs 368 minutes for WD, 842 vs 284 minutes for NP, and 697 vs 585 minutes for SP). Results of the z test indicated a significantly increased survival rate for vaccinates exposed to WD rattlesnake venom but not for vaccinates exposed to NP or SP rattlesnake venom. Log-rank analysis revealed a significant difference between survival curves of vaccinated versus unvaccinated mice exposed to NP but not WD or SP venom. CONCLUSIONS AND CLINICAL RELEVANCE CAT vaccination improved survival rate and survival time after challenge exposure with WD rattlesnake venom and may offer limited protection against NP rattlesnake venom but did not provide significant cross-protection against SP rattlesnake venom.

In vivo physiological research in the US: regulatory and ethical issues

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Open Access Animal Physiology 2015:7 1–11 Open Access Animal Physiology Dovepress

Response to Protocol Review Scenario: the economy of research.

1. Public Health Service. Policy on Humane Care and Use of Laboratory Animals (US Department of Health and Human Services, Washington, DC, 1986; amended 2002). 2. Animal Welfare Act and Animal Welfare Regulations. Part 2, Subpart C, Research Facilities. 3. Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals 8th edn. (National Academies Press, Washington, DC, 2011). 4. Russell, W.M.S. & Burch, R.L. The Principles of Humane Experimental Technique (Methuen, London, 1959).

Acute abdominal distension in a spinally transected rat.

More than 11,000 Americans sustain spinal cord injuries (SCIs) every year1. SCIs are a major source of morbidity worldwide, with the loss of ambulation being a major consequence of these injuries2. Studying how to restore basic life functions is one of the goals of spinal cord research. Research laboratories currently use spinally transected rats as a model to study strategies for restoring spinal cord function after injury. Rats are often used as the model of choice in the study of SCI because their neuromuscular system is similar to humans3. Also they are small; relatively easy to acquire, maintain, and care for; and attract less attention from animal rights groups than phylogenetically higher species, such as cats, previously used in similar studies. This makes the rat the primary model for the study of spinal cord injury and regeneration3. This case report describes a rat that was in a study involving spinal cord transection (SCT) to study spinal column regeneration. A 9-month-old adult female SpragueDawley rat from a colony of spinally transected rats presented 10 days after spinal cord transection surgery with a 2-day history of abdominal distention. The surgery involved transecting the spinal cord at the mid-thoracic level4. After surgery, the rats were housed on a paper-based bedding (CareFRESH, International Absorbents, Inc., Ferndale, WA) as we found it minimized trauma compared with wood-based bedding. We filled the cage three-quarters of the way to the top with bedding to allow for the expression of natural burrowing behavior and to allow the rats easy access to water and feed in the microisolator wire lids. For environmental enrichment we put Nylabones (Nylabone Products, Neptune City, NJ) in the cage for gnawing, and provided a few pieces of Froot Loops cereal (Kellogg Company, Battle Creek, MI) as daily treats. To facilitate urinary and fecal evacuation, the research staff manually expressed the bladder and rectum twice a day. They also flexed the rear legs daily through their full range of motion to maintain muscle and tendon tone and elasticity. Upon presentation the rat had a severely distended abdomen. History and physical examination revealed decreased appetite, moderate dehydration, lethargy, and ocular-nasal porphyrin discharge. On palpation and percussion, the abdomen felt distended with gas. An abdominal tap yielded gas and 0.5 ml of brownish fluid. Cytologic examination of this fluid using a Wright-Giemsa stain revealed large numbers of bacteria, and a moderate number of mononuclear white blood cells, macrophages, and neutrophils containing intracellular bacteria. A complete blood count (CBC) showed severe leukocytopenia and anemia. A urinalysis yielded a specific gravity of 1.030 and a pH of 6.5, both within our in-house diagnostic laboratory’s normal reference range5. While the blood and urine samples were being processed, we initiated supportive care consisting of intravenous fluids and intravenous antibiotics. On the second day the rat was more active, although it exhibited pica, as evidenced by consumption of the shredded paper bedding. The abdominal bloating had returned and we did a second abdominal tap. This time it produced ~40 ml of brownish fluid (Fig. 1). Due to the grave prognosis, we euthanized the rat and submitted it for necropsy. Based on the clinical findings presented, what are your differential diagnoses? What is the cause of the brown abdominal fluid? Is it related to the spinal cord surgery?