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.

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).

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.

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.

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.

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.

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.