Reduction in activity and abundance of mitochondrial electron transport chain supercomplexes in pulmonary hypertension-induced right ventricular dysfunction.

Pulmonary hypertension (PH) results in RV hypertrophy, fibrosis and dysfunction resulting in RV failure which is associated with impaired RV metabolism and mitochondrial respiration. Mitochondrial supercomplexes (mSC) are assemblies of multiple electron transport chain (ETC) complexes that consist of physically associated complex I, III and IV that may enhance respiration and lower ROS generation. The goal of this study was to determine if mSCs are reduced in RV dysfunction associated with PH. We induced PH in Sprague-Dawley rats by Sugen/Hypoxia (3 weeks) followed by normoxia (4 weeks). Control and PH rats were subjected to echocardiography, blue and clear native-PAGE to assess mSC abundance and activity, and cardiomyocyte isolation to assess mitochondrial reactive oxygen species (ROS). mSC formation was also assessed in explanted human hearts with and without RV dysfunction. RV activity of CI and CIV and abundance of CI, CIII and CIV in mitochondrial mSCs was severely reduced in PH rats compared to control. There were no differences in total CI or CIV activity or abundance in smaller ETC assemblies. There were no changes in both RV and LV of expression of representative ETC complex subunits. PAT, TAPSE and RV Wall thickness significantly correlated with CIV and CI activity in mSC, but not total CI and CIV activity in the RV. Consistent with reduced mSC activity, isolated PH RV myocytes had increased mitochondrial ROS generation compared to control. Reduced mSC activity was also demonstrated in explanted human RV tissue from patients undergoing cardiac transplant with RV dysfunction. The right atrial pressure/pulmonary capillary wedge pressure ratio (RAP/PCWP, an indicator of RV dysfunction) negatively correlated with RV mSC activity level. In conclusion, reduced assembly and activity of mitochondrial mSC is correlated with RV dysfunction in PH rats and humans with RV dysfunction.

Macro and micro-sleep dysfunctions as translational biomarkers for Parkinson's disease.

Sleep disturbances are highly prevalent among patients with Parkinson's disease (PD) and often appear from the early-phase disease or prodromal stages. In this chapter, we will discuss the current evidence addressing the links between sleep dysfunctions in PD, focusing most closely on those data from animal and mathematical/computational models, as well as in human-based studies that explore the electrophysiological and molecular mechanisms by which PD and sleep may be intertwined, whether as predictors or consequences of the disease. It is possible to clearly state that leucine-rich repeat kinase 2 gene (LRRK2) is significantly related to alterations in sleep architecture, particularly affecting rapid eye movement (REM) sleep and non-REM sleep, thus impacting sleep quality. Also, decreases in gamma power, observed after dopaminergic lesions, correlates negatively with the degree of injury, which brings other levels of understanding the impacts of the disease. Besides, abnormal synchronized oscillations among basal ganglia nuclei can be detrimental for information processing considering both motor and sleep-related processes. Altogether, despite clear advances in the field, it is still difficult to definitely establish a comprehensive understanding of causality among all the sleep dysfunctions with the disease itself. Although, certainly, the search for biomarkers is helping in shortening this road towards a better and faster diagnosis, as well as looking for more efficient treatments.

Inhibitory interneurons show early dysfunction in a SOD1 mouse model of amyotrophic lateral sclerosis.

Few studies in amyotrophic lateral sclerosis (ALS) measure effects of the disease on inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology, and establish a long-term imbalance of inhibition/excitation. We directly assessed excitability and morphology of glycinergic (GlyT2 expressing) ventral lumbar interneurons from SOD1G93AGlyT2eGFP (SOD1) and wild-type GlyT2eGFP (WT) mice on postnatal days 6-10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), increased voltage and current threshold for firing action potentials, along with a marginal decrease in afterhyperpolarization duration. Primary neurites of ventral SOD1 inhibitory interneurons were larger in volume and surface area than WT. GlyT2 interneurons were then divided into three subgroups based on location: (1) interneurons within 100 µm of the ventral white matter, where Renshaw cells (RCs) are located, (2) interneurons interspersed with MNs in lamina IX, and (3) interneurons in the intermediate ventral area including laminae VII and VIII. Ventral interneurons in the RC area were the most profoundly affected, exhibiting more depolarized PICs and larger primary neurites. Interneurons in lamina IX had depolarized PIC onset. In lamina VII-VIII, interneurons were least affected. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory/inhibitory balance of MNs, modify motor output and provide early biomarkers of ALS. Therapeutics like riluzole that universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here. KEY POINTS: Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology, but their excitability has never been directly measured. We studied the excitability and morphology of glycinergic interneurons in early postnatal transgenic mice (SOD1G93A GlyT2eGFP). Interneurons were less excitable and had marginally smaller somas but larger primary neurites in SOD1 mice. GlyT2 interneurons were analysed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the most ventrally located interneurons. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory/inhibitory imbalance in ALS.

Enhanced nociceptive behavior and expansion of associated primary afferents in a rabbit model of cerebral palsy.

Spastic cerebral palsy (CP) is a movement disorder marked by hypertonia and hyperreflexia; the most prevalent comorbidity is pain. Since spinal nociceptive afferents contribute to both the sensation of painful stimuli as well as reflex circuits involved in movement, we investigated the relationship between prenatal hypoxia-ischemia (HI) injury which can cause CP, and possible changes in spinal nociceptive circuitry. To do this, we examined nociceptive afferents and mechanical and thermal sensitivity of New Zealand White rabbit kits after prenatal HI or a sham surgical procedure. As described previously, a range of motor deficits similar to spastic CP was observed in kits born naturally after HI (40 min at ~70%-80% gestation). We found that HI caused an expansion of peptidergic afferents (marked by expression of calcitonin gene-related peptide) in both the superficial and deep dorsal horn at postnatal day (P)5. Non-peptidergic nociceptive afferent arborization (labeled by isolectin B4) was unaltered in HI kits, but overlap of the two populations (peptidergic and non-peptidergic nociceptors) was increased by HI. Density of glial fibrillary acidic protein was unchanged within spinal cord white matter regions important in nociceptive transmission at P5. We found that mechanical and thermal nociception was enhanced in HI kits even in the absence of motor deficits. These findings suggest that prenatal HI injury impacts spinal sensory pathways in addition to the more well-established disruptions to descending motor circuits. In conclusion, changes to spinal nociceptive circuitry could disrupt spinal reflexes and contribute to pain experienced by individuals with CP.

Anxious Profile Influences Behavioral and Immunohistological Findings in the Pilocarpine Model of Epilepsy.

Anxiety and epilepsy have a complex bidirectional relationship, where a depressive/anxious condition is a factor that can trigger seizures which in turn can aggravate the depressive/anxious condition. In addition, brain structures such as the hippocampus and amygdala might have a critical relevance in both epilepsy and anxiety. The aim of the present work was to investigate the influence of different anxious profiles to epileptogenesis. Initially, animals were screened through the elevated plus-maze anxiety test, and then seizure development was evaluated using the pilocarpine model of epilepsy. There were no differences in the susceptibility to status epilepticus, mortality rate or frequency of spontaneous recurrent seizures between animals characterized as anxious as compared to the non-anxious animals. Next, we evaluated immunohistological patterns related to seizures and anxiety in various related brain areas. Despite a decrease in the density of neuropeptide Y and parvalbumin expression in epileptic animals, those presenting greater neuropeptide Y immunoreactivity in various brain regions, also showed higher spontaneous recurrent seizures frequency. Differences on the anxious profile showed to interfere with some of these findings in some regions. In addition, animals that were injected with pilocarpine, but did not develop status epilepticus, had behavioral and neuroanatomical alterations as compared to control animals, indicating its importance as an additional tool for investigating the heterogeneity of the epileptogenic response after an initial insult. This study allowed to better understand the association between anxiety and temporal lobe epilepsy and might allow for therapeutic targets to be developed to minimize the negative impacts associated with it.

Altered Motoneuron Properties Contribute to Motor Deficits in a Rabbit Hypoxia-Ischemia Model of Cerebral Palsy.

Cerebral palsy (CP) is caused by a variety of factors attributed to early brain damage, resulting in permanently impaired motor control, marked by weakness and muscle stiffness. To find out if altered physiology of spinal motoneurons (MNs) could contribute to movement deficits, we performed whole-cell patch-clamp in neonatal rabbit spinal cord slices after developmental injury at 79% gestation. After preterm hypoxia-ischemia (HI), rabbits are born with motor deficits consistent with a spastic phenotype including hypertonia and hyperreflexia. There is a range in severity, thus kits are classified as severely affected, mildly affected, or unaffected based on modified Ashworth scores and other behavioral tests. At postnatal day (P)0-5, we recorded electrophysiological parameters of 40 MNs in transverse spinal cord slices using whole-cell patch-clamp. We found significant differences between groups (severe, mild, unaffected and sham control MNs). Severe HI MNs showed more sustained firing patterns, depolarized resting membrane potential, and fired action potentials at a higher frequency. These properties could contribute to muscle stiffness, a hallmark of spastic CP. Interestingly altered persistent inward currents (PICs) and morphology in severe HI MNs would dampen excitability (depolarized PIC onset and increased dendritic length). In summary, changes we observed in spinal MN physiology likely contribute to the severity of the phenotype, and therapeutic strategies for CP could target the excitability of spinal MNs.

Animal models of developmental motor disorders: parallels to human motor dysfunction in cerebral palsy.

Cerebral palsy (CP) is the most common motor disability in children. Much of the previous research on CP has focused on reducing the severity of brain injuries, whereas very few researchers have investigated the cause and amelioration of motor symptoms. This research focus has had an impact on the choice of animal models. Many of the commonly used animal models do not display a prominent CP-like motor phenotype. In general, rodent models show anatomically severe injuries in the central nervous system (CNS) in response to insults associated with CP, including hypoxia, ischemia, and neuroinflammation. Unfortunately, most rodent models do not display a prominent motor phenotype that includes the hallmarks of spasticity (muscle stiffness and hyperreflexia) and weakness. To study motor dysfunction related to developmental injuries, a larger animal model is needed, such as rabbit, pig, or nonhuman primate. In this work, we describe and compare various animal models of CP and their potential for translation to the human condition.

Is Mossy Fiber Sprouting a Potential Therapeutic Target for Epilepsy?

Mesial temporal lobe epilepsy (MTLE) caused by hippocampal sclerosis is one of the most frequent focal epilepsies in adults. It is characterized by focal seizures that begin in the hippocampus, sometimes spread to the insulo-perisylvian regions and may progress to secondary generalized seizures. Morphological alterations in hippocampal sclerosis are well defined. Among them, hippocampal sclerosis is characterized by prominent cell loss in the hilus and CA1, and abnormal mossy fiber sprouting (granular cell axons) into the dentate gyrus inner molecular layer. In this review, we highlight the role of mossy fiber sprouting in seizure generation and hippocampal excitability and discuss the response of alternative treatment strategies in terms of MFS and spontaneous recurrent seizures in models of TLE (temporal lobe epilepsy).

ER Stress Induced by Tunicamycin Triggers α-Synuclein Oligomerization, Dopaminergic Neurons Death and Locomotor Impairment: a New Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons of the substantia nigra pars compacta (SNpc), leading to the major clinical abnormalities that characterize this disease. Although PD's etiology is unknown, α-synuclein aggregation plays a pivotal role in PD pathogenesis, which could be associated to some pathological processes such as oxidative stress, endoplasmic reticulum (ER) stress, impaired protein degradation, and mitochondrial dysfunction. Increasing experimental evidence indicates that ER stress is involved in PD, however most of the described results employed cultured cell lines and genetically modified animal models. In this study, we developed a new ER stress rat model employing the well-known ER stressor tunicamycin (Tm). To evaluate if ER stress was able to induce PD features, we performed an intranigral injection of Tm (0.1 µg/cerebral hemisphere) and animals (male Wistar rats) were analyzed 7 days post injection. The classical 6-OHDA neurotoxin model (1 µg/cerebral hemisphere) was used as an established positive control for PD. We show that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with DMSO-injected controls. Finally, both Tm and 6-OHDA treated animals presented increased levels of ER stress markers. Taken together, these findings show for the first time that the ER stressor Tm recapitulates some of the phenotypic characteristics observed in rodent models of PD, reinforcing the concept that ER stress could be an important contributor to the pathophysiology of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.

Maximal electroshock-induced seizures are able to induce Homer1a mRNA expression but not pentylenetetrazole-induced seizures.

OBJECTIVE: Homer1a is a protein that regulates metabotropic glutamate receptors involved in neural plasticity processes. Recently, we demonstrated that Homer1a mRNA is enhanced after pilocarpine-induced status epilepticus. Here, we investigated whether a single acute seizure triggered by means of pentylenetetrazole (PTZ) injection or maximal electroshock (MES) stimulation (2 different seizure models) would alter Homer1a expression in the hippocampus. METHODS: Male Wistar rats subjected to the PTZ or MES model were analyzed 2h, 8h, 24h, and 7days after seizure induction. Homer1a, mGluR1, and mGluR5 mRNA expression levels in hippocampal extracts were analyzed by quantitative PCR. RESULTS: Quantitative PCR revealed Homer1a overexpression at 2h after MES-induced tonic-clonic seizures compared to control, but the overexpression did not remain elevated after 8h. Pentylenetetrazole-induced seizures, in contrast, were not able to change Homer1a mRNA expression. No differences were observed at these time points after seizures for mGluR1 and mGluR5 mRNA expression in any of the models. SIGNIFICANCE: Our data indicate that the levels of Homer1a mRNA were transiently increased only after MES-induced tonic-clonic seizures (and not after PTZ-induced seizures). We suggest that Homer1a expression may be dependent on seizure intensity or on specific brain circuit activation. We suggest that Homer1a may contribute to counteract hyperexcitability processes.

Effects of anterior thalamic nucleus deep brain stimulation in chronic epileptic rats.

Deep brain stimulation (DBS) has been investigated for the treatment of epilepsy. In rodents, an increase in the latency for the development of seizures and status epilepticus (SE) has been reported in different animal models but the consequences of delivering stimulation to chronic epileptic animals have not been extensively addressed. We study the effects of anterior thalamic nucleus (AN) stimulation at different current intensities in rats rendered epileptic following pilocarpine (Pilo) administration. Four months after Pilo-induced SE, chronic epileptic rats were bilaterally implanted with AN electrodes or had sham-surgery. Stimulation was delivered for 6 h/day, 5 days/week at 130 Hz, 90 µsec. and either 100 µA or 500 µA. The frequency of spontaneous recurrent seizures in animals receiving stimulation was compared to that recorded in the preoperative period and in rats given sham treatment. To investigate the effects of DBS on hippocampal excitability, brain slices from animals receiving AN DBS or sham surgery were studied with electrophysiology. We found that rats treated with AN DBS at 100 µA had a 52% non-significant reduction in the frequency of seizures as compared to sham-treated controls and 61% less seizures than at baseline. Animals given DBS at 500 µA had 5.1 times more seizures than controls and a 2.8 fold increase in seizure rate as compared to preoperative values. In non-stimulated controls, the average frequency of seizures before and after surgery remained unaltered. In vitro recordings have shown that slices from animals previously given DBS at 100 µA had a longer latency for the development of epileptiform activity, shorter and smaller DC shifts, and a smaller spike amplitude compared to non-stimulated controls. In contrast, a higher spike amplitude was recorded in slices from animals given AN DBS at 500 µA.

Reduced hippocampal dentate cell proliferation and impaired spatial memory performance in aged-epileptic rats.

Increased adult neurogenesis is observed after training in hippocampal-dependent tasks and also after acutely induced status epilepticus (SE) although the specific roles of these cells are still a matter of debate. In this study, we investigated hippocampal cell proliferation and differentiation and the spatial learning performance in young or aged chronically epileptic rats. Status was induced by pilocarpine in 3 or 20-month old rats. Either 2 or 20 months later, rats were treated with bromodeoxyuridine (BrdU) and subsequently underwent to 8-day schedule of water maze (WM) tests. As expected, learning curves were faster in young than in aged animals (P < 0.001). Chronically epileptic animals exhibited impaired learning curves compared to age-matched controls. Interestingly, the duration of epilepsy (2 or 20 months) did not correlate with the memory impairment of aged-epileptic animals. The number of BrdU-positive cells was greater in young-epileptic subjects than in age-matched controls. In contrast, cell proliferation was not increased in aged-epileptic animals, irrespective of the time of SE induction. Finally, dentate cell proliferation was not related to performance in the WM. Based on the present results we conclude that even though aging and epilepsy lead to impairments in spatial learning, their effects are not additive.

Increased seizure susceptibility in mice 30 days after fluid percussion injury.

Traumatic brain injury (TBI) has been reported to increase seizure susceptibility and also contribute to the development of epilepsy. However, the mechanistic basis of the development of increased seizure susceptibility and epilepsy is not clear. Though there is substantial work done using rats, data are lacking regarding the use of mice in the fluid percussion injury (FPI) model. It is unclear if mice, like rats, will experience increased seizure susceptibility following FPI. The availability of a mouse model of increased seizure susceptibility after FPI would provide a basis for the use of genetically modified mice to study mechanism(s) of the development of post-traumatic epilepsy. Therefore, this study was designed to test the hypothesis that, mice subjected to a FPI develop increased seizure susceptibility to a subconvulsive dose of the chemoconvulsant, pentylenetetrazole (PTZ). Three groups of mice were used: FPI, sham, and naïve controls. On day 30 after FPI, mice from the three groups were injected with PTZ. The results showed that FPI mice exhibited an increased severity, frequency, and duration of seizures in response to PTZ injection compared with the sham and naïve control groups. Histopathological assessment was used to characterize the injury at 1, 3, 7, and 30 days after FPI. The results show that mice subjected to the FPI had a pronounced lesion and glial response that was centered at the FPI focus and peaked at 3 days. By 30 days, only minimal evidence of a lesion is observed, although there is evidence of a chronic glial response. These data are the first to demonstrate an early increase in seizure susceptibility following FPI in mice. Therefore, future studies can incorporate transgenic mice into this model to further elucidate mechanisms of TBI-induced increases in seizure susceptibility.

Effects of ethanol on hippocampal neurogenesis depend on the conditioned appetitive response.

Neurogenesis in the subgranular layer of the dentate gyrus (DG) has been suggested to underlie some forms of associative learning. The present study was undertaken to determine whether there was also a role of neurogenesis in the ethanol (EtOH)-induced conditioned place preference (CPP). Outbreed Swiss mice were conditioned with EtOH (2.0 g/kg) in one compartment of a non-biased place preference chamber and saline in the other compartment. This procedure produced three groups of mice: some developed a conditioned preference (EtOH_Cpp), others developed a conditioned avoidance (EtOH_Cpa) and still others demonstrated indifference to the context previously paired with ethanol (EtOH_Ind). BrdU (40 mg/kg, i.p.) was administered 4 hours after each session comprising the conditioning phase. When measured 24 hours following the CPP test, there was no effect of EtOH on doublecortin (DCX) expression or Fluoro Jade B staining. However, there were decreases in the number of BrdU+ and Ki-67+ cells in the EtOH_Cpa and EtOH_Ind groups, but not in the EtOH_Cpp group. Most of BrdU+ cells were co-labeled with DCX. Similarly, in another experiment, in that the perfusion was done 28 days after CPP test, most BrdU+ cells were co-localized with NeuN. These results suggest that conditioned appetitive response is able to maintain normal levels of neurogenesis in DG and might counteract ethanol-produced decreased cell proliferation/survival rate.

m1 Acetylcholine Receptor Expression is Decreased in Hippocampal CA1 region of Aged Epileptic Animals.

In the present study, we investigated the possible additive effects of epilepsy and aging on the expression of m1 muscarinic acetylcholine receptors (AChR) in the rat hippocampus. Young (3 months) and Aged (20 months) male, Wistar rats were treated with pilocarpine to induce status epilepticus (SE). Immunohistochemical procedure for m1 AChR detection was performed 2 months after pilocarpine-induced SE. In the CA1 pyramidal region m1 AChR staining was significantly decreased in aged epileptic animals when compared to young epileptic and aged control rats, indicating that the aging effect is worsened by the epileptic condition. However, the Nissl-stained cell analysis indicated that the number of pyramidal CA1 neurons was similarly reduced in both epileptic groups, young and aged animals. Therefore, our data suggest that the progressive reduction of m1 AChR expression in CA1 pyramidal cells of aged epileptic rats might bear relevance to the associated progressive cognitive impairment.

Bilateral anterior thalamic nucleus lesions are not protective against seizures in chronic pilocarpine epileptic rats.

AIMS: To investigate whether anterior thalamic nucleus (AN) lesions are protective against spontaneous recurrent seizures in the chronic phase of the pilocarpine model of epilepsy. METHODS: Two groups of rats were treated with bilateral AN radiofrequency thalamotomies or sham surgery 2 weeks after pilocarpine-induced status epilepticus. After the lesions, animals were videotaped from the 2nd to the 8th week after status epilepticus (total 180 h). RESULTS: During the 6 weeks of observation, no differences in the frequency of spontaneous seizures were found between animals that had bilateral AN lesions (n = 26; 3.1 +/- 0.6 seizures per animal) and controls (n = 25; 3.0 +/- 0.6 seizures per animal; p = 0.8). CONCLUSIONS: We conclude that AN thalamotomies were not effective in reducing the frequency of seizures during the chronic phase of the pilocarpine model of epilepsy.

Assessment of seizure susceptibility in pilocarpine epileptic and nonepileptic Wistar rats and of seizure reinduction with pentylenetetrazole and electroshock models.

PURPOSE: Pentylenetetrazole (PTZ) and maximal electroshock (MES) models are often used to induce seizures in nonepileptic control animals or naive animals. Despite being widely used to screen antiepileptic drugs (AEDs), both models have so far failed to detect potentially useful AEDs for treating drug-resistant epilepsies. Here we investigated whether the acute induction of MES and PTZ seizures in epileptic rats might yield a distinct screening profile for AEDs. METHODS: Status epilepticus (SE) was induced in adult male Wistar rats by intraperitoneal pilocarpine injection (Pilo, 320 mg/kg, i.p.). One month later, controls or naive animals (Cont) that did not develop SE postpilocarpine (N-Epi) and pilocarpine-epileptic rats (Epi) received one of the following: phenobarbital (PB, 40 mg/kg), phenytoin (PHT, 50 mg/kg), or valproic acid (VPA, 400 mg/kg). Thirty min later the animals were challenged with either subcutaneous MES or PTZ (50 mg/kg, s.c.). RESULTS: VPA, PB, and PHT were able to prevent MES in all groups tested (Cont, N-Epi, and Epi groups), whereas for the PTZ model, only the Cont group (naive animals) had seizure control with the same AEDs. In addition, Epi and N-Epi groups when challenged with PTZ exhibited a higher incidence of severe seizures (scores IV-IX) and SE (p < 0.05, Fisher's exact test). CONCLUSIONS: Our findings suggest that the induction of acute seizures with PTZ, but not with MES, in animals pretreated with pilocarpine (regardless of SE induction) might constitute an effective and valuable method to screen AEDs and to study mechanisms involved in pharmacoresistant temporal lobe epilepsy (TLE).

Effective topical treatment of subcutaneous murine B16F10-Nex2 melanoma by the antimicrobial peptide gomesin.

Gomesin is a potent antimicrobial peptide (AMP) isolated from hemocytes of the spider Acanthoscurria gomesiana. The present study aimed at determining whether gomesin exerted antitumor activity in vitro and in vivo. Topical treatment of subcutaneous murine melanoma with gomesin incorporated in a cream base significantly delayed tumor growth. A direct cytotoxicity of gomesin in murine melanoma B16F10-Nex2 cells and several human tumor cell lineages was observed in vitro, with IC(50) values below 5 microM. The beta-hairpin structure of gomesin with disulfide bridges seemed essential for optimal activity. d-Gomesin was equally active. A membrane-permeabilizing activity was suggested, as gomesin bound to the cell membrane and cytoplasmic lactate dehydrogenase was detected extracellularly. At doses causing partial growth of tumor cells, gomesin allowed internalization of macromolecules (immunoglobulins), which increased the cytotoxic effect. The in vivo antitumor effect of gomesin might also involve a cytotoxic effect on endothelial cells because cultured human endothelial cells were killed in vitro at a similar concentration range. This effect represents a novel and potential use for gomesin as a topical agent against unsuccessfully treated intradermal and epithelial skin cancers. To our knowledge, this is the first report on the successful topical use of AMPs in cancer treatment.