PN5

Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In the present study, we investigated PN5, an AMP derived from Pinus densiflora needles, as a potential anti-infective agent against multidrug-resistant (MDR) Salmonella Typhimurium. PN5 exhibited potent antibacterial activity without cytotoxicity, retained stability across diverse pH and temperature conditions, inhibited biofilm formation, and disrupted bacterial membranes. It eliminated intracellular S. Typhimurium in infected RAW 264.7 macrophages, reducing bacterial survival and promoting clearance. Mechanistically, PN5 modulated host immunity by regulating NF-κB and MAPK signaling. In a murine S. Typhimurium infection model, PN5 significantly improved survival by enhancing bacterial clearance and suppressing pro-inflammatory cytokines. These findings establish PN5 as a dual-function antimicrobial agent combining direct bactericidal activity with immune modulation, providing therapeutic potential against MDR bacterial infections.

Infantile spasms are the typical seizures of West syndrome, an infantile epileptic encephalopathy with poor outcomes. There is an increasing need to identify more effective and better tolerated treatments for infantile spasms. We have optimized the rat model of infantile spasms due to structural etiology, the multiple-hit rat model, for therapy discovery. Here, we test three compounds administered after spasms induction in the multiple hit model for efficacy and tolerability. Specifically, postnatal day 3 (PN3) male Sprague-Dawley rats were induced by right intracerebral injections of doxorubicin and lipopolysaccharide. On PN5 p-chlorophenylalanine was given intraperitoneally (i.p.). Daily monitoring of weights and developmental milestones was done and rats were intermittently video monitored. A blinded, randomized, vehicle-controlled study design was followed. The caspase 1 inhibitor VX-765 (50-200 mg/kg i.p.) and the GABAB receptor inhibitor CGP35348 (12.5-100 mg/kg i.p.) each was administered in different cohorts as single intraperitoneal injections on PN4, using a dose- and time-response design with intermittent monitoring till PN5. 17β-estradiol (40 ng/g/day subcutaneously) was given daily between PN3-10 and intermittent monitoring was done till PN12. None of the treatments demonstrated acute or delayed effects on spasms, yet all were well tolerated. We discuss the implications for therapy discovery and challenges of replication trials.

Antibiotic-resistant bacteria have become a public health problem. Thus, antimicrobial peptides (AMPs) have been evaluated as substitutes for antibiotics. Herein, we investigated PN5 derived from Pinus densiflora (pine needle). PN5 exhibited antimicrobial activity without causing cytotoxic effects. Based on these results, we examined the mode of action of PN5 against Gram-negative and -positive bacteria. PN5 exhibited membrane permeabilization ability, had antimicrobial stability in the presence of elastase, a proteolytic enzyme, and did not induce resistance in bacteria. Bacterial lipopolysaccharide (LPS) induces an inflammatory response in RAW 264.7 macrophages. PN5 suppressed proinflammatory cytokines mediated by NF-κB and mitogen-activated protein kinase signaling. In C57BL/6J mice treated with LPS and d-galactosamine, PN5 exhibited anti-inflammatory activity in inflamed mouse livers. Our results indicate that PN5 has antimicrobial and anti-inflammatory activities and thus may be useful as an antimicrobial agent to treat septic shock caused by multidrug-resistant (MDR) Escherichia coli without causing further resistance. IMPORTANCE Antibiotic-resistant bacteria are a global health concern. There is no effective treatment for antibiotic-resistant bacteria, and new alternatives are being suggested. The present study found antibacterial and anti-inflammatory activities of PN5 derived from Pinus densiflora (pine needle), and further investigated the therapeutic effect in a mouse septic model. As a mechanism of antibacterial activity, PN5 exhibited the membrane permeabilization ability of the toroidal model, and treated strains did not develop drug resistance during serial passages. PN5 showed immunomodulatory properties of neutralizing LPS in a mouse septic model. These results indicate that PN5 could be a new and promising therapeutic agent for bacterial infectious disease caused by antibiotic-resistant strains.

OBJECTIVE: To test whether anti-inflammatory and antioxidant drugs that inhibit the nuclear factor kappa light chain enhancer of activated B cells (NF-kB), celastrol and edaravone, suppress spasms and improve developmental outcomes in the multiple-hit rat model of refractory infantile spasms (IS) due to structural lesions. METHODS: Postnatal day 3 (PN3) Sprague-Dawley rats were treated according to the multiple-hit IS model protocol. Using a randomized, blinded, vehicle-controlled, dose- and time-response study design, we tested the effects of single celastrol [1, 2, or 4 mg/kg intraperitoneally (i.p.), 10-14 rats/group] or edaravone (1, 10 or 30 mg/kg i.p., 14-17 rats/group) injections vs their vehicles on behavioral and electroclinical spasms and developmental milestones. Video-EEG monitoring was done on PN6-7 (n = 11-12 rats/group). Pulse celastrol treatment effects (PN4: 4 mg/kg, PN5-6: 2 mg/kg/day i.p.) were determined on spasms, developmental milestones and Barnes maze. Celastrol and edaravone pharmacokinetics in plasma and neocortex were assessed. Linear mixed model analysis of raw or normalized log-transformed spasm frequencies, considering repeated observations was used. RESULTS: Single (2-4 mg/kg i.p) or pulse celastrol, but not edaravone, reduced behavioral and electroclinical spasms frequencies within 5hrs. Pulse celastrol did not affect spasm-freedom, survival, developmental milestones or Barnes maze performance. Celastrol had erratic i.p. absorption with maximum concentrations observed between 2-4 h, when effects on spasms were seen. Edaravone had low blood-to-brain permeability. CONCLUSIONS: Celastrol's efficacy on spasms is partially explained by its better brain penetration than edaravone's. NFkB inhibitors may be useful in treating drug-resistant IS but delivery methods with improved bioavailability and brain permeability are needed.

Infantile spasms are seizures manifesting in infantile epileptic encephalopathies that are associated with poor epilepsy and cognitive outcomes. The current therapies are not always effective or are associated with serious side effects. Early cessation of spasms has been proposed to improve long-term outcomes. To identify new therapies for infantile spasms with rapid suppression of spasms, we are using the multiple-hit rat model of infantile spasms, which is a model of refractory infantile spasms. Here, we are testing the efficacy and tolerability of a single dose of the galanin receptor 1 preferring analog, NAX 5055, in the multiple-hit model of spasms. To induce the model, postnatal day 3 (PN3) male Sprague-Dawley rats underwent right intracerebral infusions of doxorubicin and lipopolysaccharide; p-chlorophenylalanine was then injected intraperitoneally (i.p.) at PN5. After the onset of spasms at PN4, 11-14 rats/group were injected i.p. with either NAX 5055 (0.5, 1, 2, or 4mg/kg) or vehicle. Video monitoring for spasms included a 1h pre-injection period, followed by 5h of recording post-injection, and two 2h sessions on PN5. The study was conducted in a randomized, blinded manner. Neurodevelopmental reflexes were assessed daily as well as at 2h after injection. Respiratory function, heart rate, pulse distension, oximetry and blood glucose were measured 4h after injection. The relative expression of GalR1 and GalR2 mRNA over β-actin in the cerebral cortex and hippocampus was determined with real time reverse transcription polymerase chain reaction. There was no acute effect of NAX 5055 on spasm frequency after the single dose of NAX 5055 (n=11-13 rats/group, following exclusions). Neurodevelopmental reflexes, vital signs, blood glucose measured 4h post-injection, and survival were not affected. A reduction in pulse and breath distention of unclear clinical significance was observed with the 7mg/kg NAX 5055 dose. GalR1 mRNA was present in the cerebral cortex and hippocampus of PN4 and adult rats. The hippocampal - but not the cortical - GalR1 mRNA expression was significantly lower in PN4 pups than in adults. GalR1 mRNA was also at least 20 times less abundant in the PN4 cortex than GalR2 mRNA. In conclusion, a single dose of NAX 5055 has no acute efficacy on spasms or toxicity in the multiple hit rat model of medically refractory infantile spasms. Our findings cannot exclude the possibility that repetitive NAX 5055 administration may show efficacy on spasms. The higher expression of GalR2 in the PN4 cortex suggests that GalR2-preferring analogs may be of interest to test for efficacy on spasms.

Nitroglycerin (NTG) in situ reduces the pressure of the upper anal sphincter (UAS). We have tested the effects of NTG on the UAS of patients with terminal constipation. We studied two groups of constipated patients. Group 1 consisted of 11 patients (nine females and two males) with hypertonicity of the UAS (> 70 mm Hg); age was 49.5 +/- 15.6 years. Group 2 consisted of 10 patients (nine females and one male) without hypertonicity; age was 40.1 +/- 14.1 years. Group 3 consisted of eight asymptomatic controls (four females and four males); age was 51.7 +/- 6.9 years. After a 10-minute resting pressure recording of the UAS with a water-filled balloon, the probe was pulled through the outside and the UAS was assessed after spreading 5 mg of placebo and then 5 mg of NTG on the balloon. Resting pressure (RP), delay of the pressure decrease (DP), pressure after five minutes either during the NTG (PN5) or placebo (PP5) period, and mean duration of the pressure decrease (MD) were measured. None of the subjects experienced a decrease of PP5 vs. RP. All patients in Group 1 (106.2 vs. 38.4 mm Hg), Group 2 (57.9 vs. 31.4 mm Hg), and controls (62.2 vs. 33.7 mm Hg) experienced a significant decrease of pressure of the UAS (P < 0.005). Delay of the pressure decrease was less than two minutes, with wide interindividual variability of duration of the pressure decrease. Mild side effects--anal pain and transient headache--were reported in five patients. In situ NTG significantly reduced UAS Pressure in all groups. NTG has to be evaluated in anal pathology, especially in patients with hypertonic sphincter terminal constipation or acute hypertonicity of the sphincter due to a fissure.