A century of phage therapy: from novel discoveries about the past to the challenges of the today.

The discovery of bacterial viruses able to kill bacteria, named bacteriophages (phages), more than a century ago, contributed to combating bacterial infections during the first three decades of the 20th century. Vladimir Sertic, a Croatian microbiologist, was one of the pioneers in bacteriophages investigation, who performed significant scientific research on phage strains characterization, isolation, and classification. The important contribution of Vladimir Sertic can be seen from his private archive (from the 1930s) which contains hundreds of valuable archival materials that are today kept at the Department of Pharmacology School of Medicine, Zagreb. The Discovery of this archive was recently described by Lackovic and Toljan. Here we describe a further survey of this archive. In addition to many documents, it contains phage cocktails in a small commercial paper box, with instructions for usage, as clear evidence of their therapeutic application in the pre-antibiotic era. This revelation, not shown in the literature up to now, to the best of our knowledge, motivated us to present it to the wider scientific and professional community with this short article, giving an additional brief overview of the current perspective of phage investigation and therapeutic application.

Botulinum Toxin Type A in Dental Medicine.

Botulinum toxins (BoNTs) are a product of the bacteria Clostridium botulinum. By entering nerve endings, they cleave and inactivate SNARE proteins, which are essential for neurotransmitter release. Prevention of acetylcholine release at the neuromuscular junction causes long-lasting and potentially fatal flaccid paralysis-a major feature of botulism. However, an intramuscular injection of minute amounts of BoNTs, primarily type A (BoNT-A), has useful long-lasting muscle relaxation effects on spastic motor disorders. This characteristic of BoNT-A is widely used in neurology and cosmetics. Over the last few decades, it has been demonstrated that the functions of BoNT-A are not limited to muscle-relaxing or autonomic cholinergic effects but that it can act as an analgesic agent as well. More recently, it was revealed that this antinociceptive effect starts after entering the sensory nerve endings, where these agents are axonally transported to the central nervous system, suggesting that at least part of their analgesic effect might be of central origin. Because of its antinociceptive effect, BoNT-A is currently approved for treatment of chronic migraine; nonetheless, case reports and preclinical and clinical experiments indicating its benefit in numerous potential painful conditions have increased. In the field of dentistry, the US Food and Drug Administration approved BoNT-A for the treatment of sialorrhea only. Legal status of the use of BoNT-A in other countries is less known. However, there are controlled clinical trials suggesting its efficacy in other conditions, such as bruxism, temporomandibular disorders, and trigeminal neuropathic pain. Thereby, using criteria of the American Academy of Neurology, we critically reviewed the uses of BoNTs in oral medicine and found it effective for trigeminal neuralgia (category A) and probably effective in temporomandibular disorders and bruxism.

Role of central versus peripheral opioid system in antinociceptive and anti-inflammatory effect of botulinum toxin type A in trigeminal region.

BACKGROUND: Although botulinum toxin type A (BT-A) is approved for chronic migraine treatment, its site and mechanism of action are still elusive. Recently our group discovered that suppression of CGRP release from dural nerve endings might account for antimigraine action of pericranially injected BT-A. We demonstrated that central antinociceptive effect of BT-A in sciatic region involves endogenous opioid system as well. Here we investigated possible interaction of BT-A with endogenous opioid system within the trigeminal region. METHODS: In orofacial formalin test we investigated the influence of centrally acting opioid antagonist naltrexone (2 mg/kg, s.c.) versus peripherally acting methylnaltrexone (2 mg/kg, s.c.) on BT-A's (5 U/kg, s.c. into whisker pad) or morphine's (6 mg/kg, s.c.) antinociceptive effect and the effect on dural neurogenic inflammation (DNI). DNI was assessed by Evans blue-plasma protein extravasation. RESULTS: Naltrexone abolished the effect of BT-A on pain and dural plasma protein extravasation, whereas peripherally acting methylnaltrexone did not change either BT-A's effect on pain or its effect on dural extravasation. Naltrexone abolished the antinociceptive and anti-inflammatory effects of morphine, as well. However, methylnaltrexone decreased the antinociceptive effect of morphine only partially in the second phase of the test and had no significant effect on morphine-mediated reduction in DNI. CONCLUSIONS: Morphine acts on pain in trigeminal region both peripherally and centrally, whereas the effect on dural plasma protein extravasation seems to be only centrally mediated. However, the interaction of BT-A with endogenous opioid system, with consequent inhibition of nociceptive transmission as well as the DNI, occurs primarily centrally. SIGNIFICANCE: Botulinum toxin type A (BT-A)'s axonal transport and potential transcytosis suggest that its antinociceptive effect might involve diverse neurotransmitters at different sites of trigeminal system. Here we discovered that the reduction in pain and accompanying DNI involves the interaction of BT-A with central endogenous opioid system (probably at the level of trigeminal nucleus caudalis).

Botulinum toxin type A in motor nervous system: unexplained observations and new challenges.

In the motor system, botulinum toxin type A (BoNT/A) actions were classically attributed to its well-known peripheral anticholinergic actions in neuromuscular junctions. However, the enzymatic activity of BoNT/A, assessed by the detection of cleaved synaptosomal-associated protein 25 (SNAP-25), was recently detected in motor and sensory regions of the brainstem and spinal cord after toxin peripheral injection in rodents. In sensory regions, the function of BoNT/A activity is associated with its antinociceptive effects, while in motor regions we only know that BoNT/A activity is present. Is it possible that BoNT/A presence in central motor nuclei is without any function? In this brief review, we analyze this question. Limited data available in the literature warrant further investigations of BoNT/A actions in motor nervous system.

Antinociceptive action of botulinum toxin type A in carrageenan-induced mirror pain.

"Mirror pain" or mirror-image pain (MP) is pain opposite to the side of injury. Mechanism and frequency in humans are not known. There is no consent on therapy. Here we report that unilaterally injected botulinum toxin type A (BT-A) has bilateral effect in experimental MP, thus deserves to be investigated as therapy for this condition. We examined the localization of BT-A's bilateral antinociceptive action in MP induced by 3 % carrageenan intramuscular injection in Wistar rats. BT-A was applied peripherally (5 U/kg), into ipsilateral or contralateral hind paw pad (i.pl.) and centrally (1 U/kg), at spinal (intrathecally, i.t.) or supraspinal (intracisternally, i.c.) level. Additionally, we examined the involvement of central opioid and GABAergic systems, as well as the contribution of peripheral capsaicin-sensitive neurons to BT-A's bilateral antinociceptive effect. Ipsilateral i.pl. and i.t. BT-A reduced the bilateral mechanical sensitivity to von Frey filaments, while contralateral i.pl. and i.c. treatments had no effect on either tested side. Bilateral antinociceptive effect of ipsilateral i.pl. BT-A was prevented by µ-opioid antagonist naloxonazine (1.5 µg/10 µl) and GABAA antagonist bicuculline (1 µg/10 µl) if applied at the spinal level, in contrast to supraspinal application of the same doses. Local treatment of sciatic nerve with 2 % capsaicin 5 days following BT-A i.pl. injection caused desensitization of sciatic capsaicin-sensitive fibers, but did not affect bilateral antinociceptive effect of BT-A and the presence of cleaved SNAP-25 at the spinal cord slices. Present experiments suggest segmental actions of peripheral BT-A at spinal level, which are probably not solely dependent on capsaicin-sensitive neurons.

Effects of botulinum toxin type A facial injection on monoamines and their metabolites in sensory, limbic and motor brain regions in rats.

Despite its toxicity, botulinum neurotoxin type A (BTX-A) is a valuable therapeutic agent for several motor, autonomic and pain disorders. Numerous studies have described its peripheral as well as central effects. Using reversed-phase High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED) and gradient elution, we quantified the concentrations of dopamine (DA), noradrenaline (NA), serotonin (5-HT) and their metabolites in 10 brain regions, ipsilateral and contralateral from the site of unilateral BTX-A administration (5 U/kg) into the rat whisker pad. In regions associated with nociception and pain processing we also examined possible BTX-A effects in combination with formalin-induced inflammatory orofacial pain. The dominant BTX-A effects on the monoamines and their metabolites were insignificant. The only significant increase caused by BTX-A alone was that of NA in striatum and serotonin in hypothalamus. While antinociceptive effects of BTX-A are most probably not related to central monoamine concentrations, the localized increased NA and 5-HT concentrations might play a role in reported BTX-A efficacy for the treatment of depression.

Association of antinociceptive action of botulinum toxin type A with GABA-A receptor.

The mechanism of botulinum toxin type A (BTX-A) antinociceptive action in the central nervous system is little known. The potential interaction between BTX-A and GABAergic system has not been investigated previously. In the present study, we demonstrate prevention of BTX-A antinociceptive effect on formalin-induced inflammatory pain and partial sciatic nerve transection-induced mechanical allodynia by GABA-A antagonist bicuculline, thus suggesting association of the GABA-A receptors and antinociceptive action of BTX-A.

Dural neurogenic inflammation induced by neuropathic pain is specific to cranial region.

Up to now, dural neurogenic inflammation (DNI) has been studied primarily as a part of migraine pain pathophysiology. A recent study from our laboratory demonstrated the occurrence of DNI in response to peripheral trigeminal nerve injury. In this report, we characterize the occurrence of DNI after different peripheral nerve injuries in and outside of the trigeminal region. We have used the infraorbital nerve constriction injury model (IoNC) as a model of trigeminal neuropathic pain. Greater occipital nerve constriction injury (GoNC), partial transection of the sciatic nerve (ScNT) and sciatic nerve constriction injury (SCI) were employed to characterize the occurrence of DNI in response to nerve injury outside of the trigeminal region. DNI was measured as colorimetric absorbance of Evans blue plasma protein complexes. In addition, cellular inflammatory response in dural tissue was histologically examined in IoNC and SCI models. In comparison to the strong DNI evoked by IoNC, a smaller but significant DNI has been observed following the GoNC. However, DNI has not been observed either in cranial or in lumbar dura following ScNT and SCI. Histological evidence has demonstrated a dural proinflammatory cell infiltration in the IoNC model, which is in contrast to the SCI model. Inflammatory cell types (lymphocytes, plasma cells, and monocytes) have indicated the presence of sterile cellular inflammatory response in the IoNC model. To our knowledge, this is the first observation that the DNI evoked by peripheral neuropathic pain is specific to the trigeminal area and the adjacent occipital area. DNI after peripheral nerve injury consists of both plasma protein extravasation and proinflammatory cell infiltration.

Involvement of µ-opioid receptors in antinociceptive action of botulinum toxin type A.

Botulinum toxin A (BTX-A) is approved for treatment of chronic migraine and has been investigated in various other painful conditions. Recent evidence demonstrated retrograde axonal transport and suggested the involvement of CNS in antinociceptive effect of BTX-A. However, the mechanism of BTX-A central antinociceptive action is unknown. In this study we investigated the potential role of opioid receptors in BTX-A's antinociceptive activity. In formalin-induced inflammatory pain we assessed the effect of opioid antagonists on antinociceptive activity of BTX-A. Naltrexone was injected subcutaneously (0.02-2 mg/kg) or intrathecally (0.07 µg/10 µl-350 µg/10 µl), while selective µ-antagonist naloxonazine was administered intraperitoneally (5 mg/kg) prior to nociceptive testing. The influence of naltrexone (2 mg/kg s.c.) on BTX-A antinociceptive activity was examined additionally in an experimental neuropathy induced by partial sciatic nerve transection. To investigate the effects of naltrexone and BTX-A on neuronal activation in spinal cord, c-Fos expression was immunohistochemically examined in a model of formalin-induced pain. Antinociceptive effects of BTX-A in formalin and sciatic nerve transection-induced pain were prevented by non-selective opioid antagonist naltrexone. Similarly, BTX-A-induced pain reduction was abolished by low dose of intrathecal naltrexone and by selective µ-antagonist naloxonazine. BTX-A-induced decrease in dorsal horn c-Fos expression was prevented by naltrexone. Prevention of BTX-A effects on pain and c-Fos expression by opioid antagonists suggest that the central antinociceptive action of BTX-A might be associated with the activity of endogenous opioid system (involving µ-opioid receptor). These results provide first insights into the mechanism of BTX-A's central antinociceptive activity.

Behavioral and immunohistochemical evidence for central antinociceptive activity of botulinum toxin A.

Botulinum toxin A (BTX-A) is approved for treatment of different cholinergic hyperactivity disorders, and, recently, migraine headache. Although suggested to act only locally, novel observations demonstrated bilateral reduction of pain after unilateral toxin injection, and proposed retrograde axonal transport, presumably in sensory neurons. However, up to now, axonal transport of BTX-A from periphery to CNS was identified only in motoneurons, but with unknown significance. We assessed the effects of low doses of BTX-A injected into the rat whisker pad (3.5 U/kg) or into the sensory trigeminal ganglion (1 U/kg) on formalin-induced facial pain. Axonal transport was prevented by colchicine injection into the trigeminal ganglion (5 mM, 2 µl). To find the possible site of action of axonally transported BTX-A, we employed immunohistochemical labeling of BTX-A-truncated synaptosomal-associated protein 25 (SNAP-25) in medullary dorsal horn of trigeminal nucleus caudalis after toxin injection into the whisker pad. Both peripheral and intraganglionic BTX-A reduce phase II of formalin-induced pain. Antinociceptive effect of BTX-A was prevented completely by colchicine. BTX-A-truncated SNAP-25 in medullary dorsal horn (spinal trigeminal nucleus) was evident 3 days following the peripheral treatment, even with low dose applied (3.5 U/kg). Presented data provide the first evidence that axonal transport of BTX-A, obligatory for its antinociceptive effects, occurs via sensory neurons and is directed to sensory nociceptive nuclei in the CNS.

Spontaneous mesh evacuation per rectum after incisional ventral hernia repair.

We present a case of a 58-year-old man who was admitted to our hospital because of abdominal pain. He underwent incisional ventral hernia repair with intraabdominal mesh (ePTFE). On the day of admission, physical examination included the discovery of a foreign body in the rectum. There were no signs of acute abdomen. We induced stool, and the mesh came out with it. His further course was uneventful. Gastrografin series showed persisting fistula between the small intestine and colon, but without extralumination into the peritoneal cavity. The patient was discharged in good health and without signs of incisional ventral hernia.

Brain antioxidant capacity in rat models of betacytotoxic-induced experimental sporadic Alzheimer's disease and diabetes mellitus.

It is believed that oxidative stress plays a central role in the pathogenesis of metabolic diseases like diabetes mellitus (DM) and its complications (like peripheral neuropathy) as well as in neurodegenerative disorders like sporadic Alzheimer's disease (sAD). Representative experimental models of these diseases are streptozotocin (STZ)-induced diabetic rats and STZ-intracerebroventricularly (STZ-icv) treated rats, in which antioxidant capacity against peroxyl (ORAC(-ROO)*) and hydroxyl (ORAC(-OH)*) free radical was measured in three different brain regions (hippocampus, cerebellum, and brain stem) by means of oxygen radical absorbance capacity (ORAC) assay. In the brain of both STZ-induced diabetic and STZ-icv treated rats decreased antioxidant capacity has been found demonstrating regionally specific distribution. In the diabetic rats these abnormalities were not associated with the development of peripheral diabetic neuropathy. Also, these abnormalities were not prevented by the icv pretreatment of glucose transport inhibitor 5-thio-D-glucose in the STZ-icv treated rats, suggesting different mechanism for STZ-induced central effects from those at the periphery. Similarities in the oxidative stress alterations in the brain of STZ-icv rats and humans with sAD could be useful in the search for new drugs in the treatment of sAD that have antioxidant activity.

Central administration of alloxan impairs glucose tolerance in rats.

By means of oral glucose tolerance test (OGTT), we investigated glucose tolerance in rats pre-treated with intracerebroventricular and subcutaneous non-diabetogenic dose of betacytotoxic drug alloxan 7 days before OGTT. Being normoglycemic and normoinsulinemic pre-OGTT, at 30 minutes post-OGTT, alloxan intracerebroventricularly-treated rats had a lower glucose and a higher insulin plasma levels in comparison with controls or alloxan subcutaneously treated animals. Centrally administered alloxan seems to have brain related effect on the regulation of peripheral glucose tolerance and insulin secretion.

Botulinum toxin type A in experimental neuropathic pain.

A peripheral application of botulinum toxin type A (7 U/kg) has significantly reduced thermal and mechanical hypersensitivity in rats with the partial sciatic nerve transection as a classical model of surgical neuropathy.

Intracerebroventricular administration of betacytotoxics alters expression of brain monoamine transporter genes.

Intracerebroventricular (icv) administration of betacytotoxics alters brain monoamine neurotransmission, without producing hyperglycemia. By means of in situ hybridization, we have investigated the expression of dopamine, noradrenaline and serotonin transporter (DAT, NAT, 5-HTT, respectively) mRNAs, in the brain of alloxan- and streptozotcin-icv treated rats. DAT1 mRNA expression is increased in 1-week alloxan-icv treated rats in arcuate nucleus (+51%) and ventral medial bundle /VMB/ (+32%), and decrease in VMB of 4-week alloxan- (-53%) and streptozotocin- (-9%) icv treated rats, respectively. NAT1 mRNA expression in locus coeruleus is decreased in 4-week alloxan-icv treated rats (-35%) and increased in A1 cell group of 1- (+19%) and 4- (+14%) week streptozotocin-icv treated rats. 5-HTT mRNA expression in dorsal raphe nucleus is increased in 1- (+13%) and 4- (+21%) week alloxan-icv treated rats. Observed changes may suggest altered response to antidepressants in streptozotocin-icv treated rats, used as an animal model of sporadic Alzheimer's disease.

Vagal influence on compensatory ovarian growth is important only briefly after hemicastration.

Hemicastration induces growth of the remnant ovary in the rat. As evidenced by the effects of total abdominal vagotomy, vagal innervation markedly influences this compensatory ovarian growth. In the present experiments, vagotomy inhibited compensatory ovarian growth when performed immediately after hemicastration, but not when delayed until 4.5 hr after hemicastration. Brief exposure of subdiaphragmal portion of the vagi nerves to 2% lidocaine shortly before hemicastration also inhibited compensatory growth. Fifteen minutes after hemicastration, markedly elevated tissue concentrations of cyclic adenosine monophosphate (cAMP) were recorded in the remnant ovaries. This accumulation of cAMP was inhibited by vagotomy that preceded hemicastration, as well as by lidocaine pretreatment of the vagi nerves, and partly by vagotomy that followed 10 min after hemicastration. At 5 hr after hemicastration, tissue cAMP concentrations in the remnant ovaries were not elevated and were not affected by vagotomy. The present results suggest that vagal influence on the compensatory ovarian growth is important only during a short period of time after hemicastration (apparently shorter than 4.5 hr), and that it, at least briefly after hemicastration, includes neural input to the ovary.

Usage of the Internet pharmacology resources among European pharmacologists: a preliminary investigation.

The Internet is becoming an ever more important source of information in pharmacology and medicine. Little is known, however, about which Internet pharmacology resources are actually used by the pharmacologists - and to what extent - and how they estimate the Internet information quality and evaluation. This pilot study used an anonymous questionnaire, distributed among 250 mostly European (220/250) pharmacologists from 30 European countries attending The 2nd European Congress of Pharmacology, held in Budapest, Hungary, in 1999. According to study results, 93% of all participants use the Internet pharmacology resources: 56%, 35% and 9% of them on a daily, weekly and monthly basis, respectively. Among 56 pharmacological/medical free online databases offered, the general scientific databases were found to greatly prevail (Pub Med 60%, Evaluated MEDLINE 37%, Internet Grateful Med 29%, etc.), while drug monographs or toxicological databases were less used [e.g. ECDIN, RxList, National Toxicology Programme (NTP) < 10%]. Some 80% of the participants estimated the quality of the pharmacological information on the Internet as good or very good, while 20% thought the quality should be improved. Also, 35% of participants felt the need for improvement of the Internet pharmacological information evaluation, which should be the goal of pharmacology professional.

Duplex ultrasonography in diagnosis of spigelian hernia with incarcerated jejunal loop.

Spigelian hernia is a very rare anterior abdominal wall hernia with uncharacteristic symptoms and challenging diagnosis. The case of a 76-year-old male patient with colicky pain and vomiting lasting for 24 hours before admission to the hospital is presented. Physical examination of the patient revealed abdominal tenderness and a round-shaped tumefaction of 3 cm in size, located in the left lower abdominal quadrant. X-ray examination of the abdomen, obtained in left lateral position of the patient, showed small bowel ileus with distended jejunal loops. The abdominal ultrasound examination, followed by duplex ultrasonography, revealed a spigelian hernia with ischemic changes of strangulated bowel segment indicating incarceration of the herniated jejunal loop. Preoperative findings were confirmed by intraoperative diagnosis of spigelian hernia and incarcerated jejunal loop with ischemic changes and deserosation, followed by resection of the bowel segment involved and plastic surgical reconstruction of anterior abdominal wall. This case report highlights the role of duplex ultrasonography in the evaluation of circulatory status of potentially incarcerated bowel segment within hernial sac.

[Addictive substances and medical students in Zagreb]. / Sredstva ovisnosti i zagrebacki studenti medicine.

We surveyed 3 generations (1996-1998) of the 3rd year medical students at the University of Zagreb for their contact with tobacco, alcohol and illicit substances of abuse using an anonymous questionnaire. Of 464 participants 25-30% were smokers, 60-70% confirmed that they had got drunk and 31% that they had used illicit substances at least once in the lifetime (primarily cannabis). The proportion of participants confirming contact with cannabis was significantly higher in 1998 than in 1996--34.3% vs. 22.3% (p < 0.05). Comparing the data with a similar survey in 1989, it seems that there has been no significant change in the use of socially accepted substances (tobacco, alcohol), but the proportion of students in contact with illicit substances has increased three times. This increasing trend is in agreement with trends among medical students in Western countries. The students' knowledge about the hazards related to the most harmful substances was poor.