Theaflavin-3,3′-Digallate and Lactic Acid Combinations Reduce Herpes Simplex Virus Infectivity

The present study examined the efficacy of using multiple mechanisms as part of a topical microbicide to inactivate herpes simplex virus (HSV) by combining theaflavin-3,3′-digallate (TF-3) and lactic acid (LA) over the pH range of 4.0 to 5.7 to mimic conditions in the female reproductive tract. Six clinical isolates of HSV-2 and two clinical isolates of HSV-1 were almost completely inactivated when TF-3 (100 μM) was present with LA over the pH range of 4.5 to 5.7, whereas four additional HSV-1 clinical isolates required TF-3 concentrations of 250 to 500 μM for comparable virus titer reduction. LA (1%) alone at pH 4.0 reduced the titers of laboratory and clinical isolates of HSV-1 and HSV-2 by ≥5 log₁₀, but most LA-dependent antiviral activity was lost at a pH of ≥4.5. When HSV-1 and HSV-2 were incubated at pH 4.0 without LA virus titers were not reduced. At pH 4.0, HSV-1 and HSV-2 titers were reduced 5 log₁₀ in 20 min by LA alone. TF-3 reduced HSV-2 titers by 5 log₁₀ in 20 to 30 min at pH 4.5, whereas HSV-1 required 60 min for comparable inactivation. Mixtures of TF-3 and LA stored at 37°C for 1 month at pH 4.0 to 5.7 maintained antiviral activity. Semen, but not cervical vaginal fluid, decreased LA-dependent antiviral activity at pH 4.0, but adding TF-3 to the mixture reduced HSV titers by 4 to 5 log₁₀. These results indicate that a combination microbicide containing TF-3 and LA could reduce HSV transmission.     

Novel Composite Efficacy Measure To Demonstrate the Rationale and Efficacy of Combination Antiviral–Anti-Inflammatory Treatment for Recurrent Herpes Simplex Labialis

Historically, the primary target for research and treatment of recurrent herpes simplex labialis (HSL) has been limited to inhibiting herpes simplex virus (HSV) replication. Antiviral monotherapy, however, has proven only marginally effective in curtailing the duration and severity of recurrent lesions. Recently, the role of inflammation in the progression and resolution of recurrences has been identified as an additional target. This was evaluated in a randomized study comparing combination topical 5% acyclovir-1% hydrocortisone cream (AHC) with 5% acyclovir alone (AC; in the AHC vehicle) and the vehicle. The efficacy of each topical therapy was evaluated for cumulative lesion size—a novel composite efficacy endpoint incorporating episode duration, lesion area, and proportion of nonulcerative lesions. In that study, cumulative lesion area was significantly decreased with AHC compared with AC (25% decrease; P < 0.05) and the vehicle (50% decrease; P < 0.0001). As research continues in this arena, cumulative lesion area should be included as a measure of efficacy in clinical trials of recurrent HSL therapies.     

Structure-Microbicidal Activity Relationship of Synthetic Fragments Derived from the Antibacterial α-Helix of Human Lactoferrin

There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial α-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial α-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.Steadily increasing antimicrobial drug resistance has become a worldwide problem. For instance, resistance to multiple antibiotics can be found among pathogens such as staphylococci, pneumococci, Pseudomonas species, and extended-spectrum β-lactamase (ESBL)-producing strains of Enterobacteriaceae. Also, resistance to antimycotics, such as the azoles, has climbed significantly. Thus, there is a need for new treatment therapies.Human lactoferrin (LF), a single-chain iron-binding glycoprotein, is a major protein in milk and mucosal secretions. It is also found in the secondary granulae of neutrophils. It is a multifunctional molecule participating in the innate host defense, expressing both antimicrobial and antiinflammatory activities in vivo (16-18, 34).Iron depletion and destabilization of the bacterial cell wall are two mechanisms suggested to be involved in the antimicrobial activity of lactoferrin. In Gram-negative bacteria, destabilization could partly be due to its binding capacity to lipid A and porins (3, 4, 10, 25, 37). Binding to lipopolysaccharide (LPS) has been suggested to release LPS from the bacterial membrane. A third mechanism of its antimicrobial activity is the release of microbicidal fragments upon enzymatic hydrolysis of LF (4).The pepsin-derived bactericidal LF fragment, lactoferricin (Lfcin), consists of residues 1-47 or 49 from the N-terminal end, comprising two antimicrobial domains (residues 1-11 and 18-40) (4, 19, 26). Another recently discovered antimicrobial region of the LF molecule comprises residues 153-183 (32). A smaller peptide sequence of LFcin (residues 21-31), corresponding to a part of the first α-helix of the LF molecule, has been studied in more detail (5). This 11-amino-acid-long peptide exhibited bactericidal activity against Escherichia coli and caused disruption of the outer membrane of E. coli O111 (6, 27). The amphipathic α-helix region also comprises a part of the LPS and glycosaminoglycan binding region of LF, as determined by site-directed mutagenesis at positions 28-34 in the LF molecule (9, 23). The amino acid residues 35-40 constitute a β-sheet region in the native LF molecule (2, 15). Although LFcin may not have a well-developed secondary structure in solution, it may adopt a helical structure when bound to membranes, analogous to other antimicrobial peptides (15).In the present study, the aim was to investigate the structure-microbicidal activity relationship of peptide fragments based on the antibacterial α-helix (amino acid residues 14-31) of LF, using E. coli, Staphylococcus aureus, and Candida albicans as test organisms. The experimental strategy included downsizing the helical region, followed by alanine scanning and amino acid substitutions.     

Suppression of Herpes Simplex Virus Type 1 (HSV-1)–induced Pneumonia in Mice by Inhibition of Inducible Nitric Oxide Synthase (iNOS,NOS2)

Intranasal Herpes simplex virus type 1 (HSV-1) infection of mice caused pneumonia. Manifestations of the disease included: histological pneumonitis, pulmonary influx of lymphocytes, decreased pulmonary compliance, and decreased survival. Immunohistochemical staining demonstrated iNOS induction and the nitrotyrosine antigen in the lungs of infected, but not uninfected mice, suggesting that nitric oxide contributes to the development of pneumonia. To elucidate the role of nitric oxide in the pathogenesis of HSV-1 pneumonia, infected mice were treated either with the inhibitor of nitric oxide synthase activity, N^G-monomethyl-l-arginine (l-NMMA), or, as a control, with PBS or d-NMMA. l-NMMA treatment decreased the histological evidence of pneumonia and reduced the bronchoalveolar lavage lymphocyte number to one-quarter of the total measured in control-treated mice. l-NMMA treatment significantly improved survival and pulmonary compliance of HSV-1–infected mice. Strikingly, the l-NMMA–mediated suppression of pneumonia occurred despite the presence of a 17-fold higher pulmonary viral titer. Taken together, these data demonstrated a previously unrecognized role of nitric oxide in HSV-1–induced pneumonia. Of note, suppression of pneumonia occurred despite higher pulmonary virus content; therefore, our data suggest that HSV-1 pneumonia is due to aspects of the inflammatory response rather than to direct viral cytopathic effects.Herpes simplex virus type 1 (HSV-1)¹ pneumonia is of considerable clinical significance, particularly in newborns and immunocompromised patients (1–6), but the pathogenesis is poorly understood. A broader mechanistic understanding of the disease would provide a basis for prophylactic and therapeutic strategies in HSV-1 pneumonia patients (7).Nitric oxide (NO) produced by the inducible isoform of NO-synthase (iNOS, NOS2) plays a multifunctional role in the cytotoxicity of activated macrophages, in the pathophysiology of septic shock, and in the suppression of cardiac function (8, 9). Studies of iNOS-deficient mutant mice demonstrated that NO contributes to endotoxic shock and to the immune response to bacteria and parasites (10, 11). Experiments with N^G-monomethyl-l-arginine (l-NMMA) or aminoguanidine, inhibitors of nitric oxide synthase (NOS), showed the involvement of NO in influenza virus pneumonia (12), graft versus host disease (13), experimental autoimmune encephalomyelitis (14), and alloreactivity (15).NO production can be beneficial as an antiviral effector mechanism against HSV-1 and other viruses (16–19). On the other hand, NO may also be detrimental by contributing to pathology during viral infections (20–22); for example, NO induced by gp41 of HIV-1 contributes to the dementia seen in AIDS patients (23).NO is crucial for the pathogenesis of influenza virus pneumonia in mice (12). Inhibition of iNOS by l-NMMA treatment improved survival of virus-infected mice without exerting an effect on the pulmonary viral titer. Thus, NO is important for the development of influenza virus pneumonia but not as an antiviral effector mechanism (12). In contrast, HSV-1 is susceptible to NO (18, 19). Furthermore, NO is produced during HSV-1 infection (24). Therefore, we wanted to determine whether iNOS inhibition in HSV-1–induced pneumonia would lead to a higher pulmonary viral burden and exacerbation of pneumonitis, or alternatively, to a reduction in proinflammatory effects of NO with suppression of pneumonitis. Therefore, inhibition of NO production could affect the development of viral pneumonitis in opposite ways: it could worsen the disease by diminishing the antiviral effect of NO, or it could improve the pneumonitis by reducing a proinflammatory mechanism.Our data suggest that NO is an important mediator of HSV-1 pneumonia. Inhibition of NO production by l-NMMA treatment led to a significantly improved survival rate and to a strong suppression of HSV-1–induced pneumonitis despite the presence of a higher pulmonary virus content. These results suggest that iNOS inhibition is beneficial; the proinflammatory effects of NO are more important for the pathology of HSV-1 pneumonia than the antiviral effects of NO.     

E Pluribus Unum: 50 Years of Research,Millions of Viruses,and One Goal—Tailored Acceleration of AAV Evolution

Fifty years ago, a Science paper by Atchison et al. reported a newly discovered virus that would soon become known as adeno-associated virus (AAV) and that would subsequently emerge as one of the most versatile and most auspicious vectors for human gene therapy. A large part of its attraction stems from the ease with which the viral capsid can be engineered for particle retargeting to cell types of choice, evasion from neutralizing antibodies or other desirable properties. Particularly powerful and in the focus of the current review are high-throughput methods aimed at expanding the repertoire of AAV vectors by means of directed molecular evolution, such as random mutagenesis, DNA family shuffling, in silico reconstruction of ancestral capsids, or peptide display. Here, unlike the wealth of prior reviews on this topic, we especially emphasize and critically discuss the practical aspects of the different procedures that affect the ultimate outcome, including diversification protocols, combinatorial library complexity, and selection strategies. Our overall aim is to provide general guidance that should help users at any level, from novice to expert, to safely navigate through the rugged space of directed AAV evolution while avoiding the pitfalls that are associated with these challenging but promising technologies.     

Lentivirus Mediated Delivery of Neurosin Promotes Clearance of Wild-type α-Synuclein and Reduces the Pathology in an α-Synuclein Model of LBD

Neurosin is a predominant serine protease in the central nervous system (CNS) and has been shown to play a role in the clearance of α-synuclein (α-syn) which is centrally involved in the pathogenesis of Parkinson''s disease (PD) and dementia with Lewy bodies (DLB). Although it has been previously shown that neurosin and α-syn colocalize and that neurosin degrades α-syn aggregates in vitro, it is not clear if neurosin is dysregulated in the brains of patients with PD/DLB and to what extent delivery of neurosin into the CNS might ameliorate the deficits associated with α-syn accumulation in vivo. We analyzed the levels of neurosin in the brains of patients with PD/DLB and in α-syn transgenic (tg) models. With increased accumulation of α-syn, we observed decreased neurosin expression. Lentiviral vector (LV) driven expression of neurosin in neuronal cell cultures reduced the accumulation of wild type but not A53T α-syn and prevented α-syn associated toxicity. Neuropathological analysis following delivery of LV-Neurosin to α-syn tg mice resulted in reduced accumulation of α-syn and reversal of neurodegenerative alterations in wild type but not A53T α-syn tg mice. Therefore, viral vector driven expression of neurosin may warrant further investigation as a potential therapeutic tool for DLB.     

Toward a theory of “hypnotic” behavior: Replication and extension of experiments by barber and co-workers (1962–65) and Hilgard and Tart (1966)

Responses to test suggestions (e.g., hallucination and amnesia) were assessed under the following treatments: motivational instructions alone; hypnotic procedure with motivational instructions; and imagination-control. Comparisons were made across independent groups, each tested under one treatment, and also within the same Ss tested twice under various combinations of the treatments. Although Ss were suggestible under the imagination-control treatment, both the motivational instructions alone and the hypnotic procedure given together with the motivational instructions raised suggestibility above the control level. The hypnotic-motivational treatment tended to produce an increment in suggestibility which went slightly beyond that attributable to the motivational instructions. The latter increment is interpreted as due to the slightly greater effectiveness of the hypnotic procedure in defining the situation as one in which unusual manifestations, such as hallucination and amnesia, are within Ss' capabilities and definitely expected by E.     

Synthetic cathinones in Southern Germany – characteristics of users,substance-patterns,co-ingestions,and complications

Objective: To define the characteristics of synthetic cathinone users admitted to hospital including clinical and laboratory parameters and the complications of use.

Design: Retrospective single-center study of patients treated for acute cathinone intoxication and complications of cathinone use between January 2010 and January 2016.

Setting: A specialized clinical toxicology unit at an academic tertiary care center in Southern Germany serving a population of about 4 million.

Patients and methods: 81 consecutive patients with laboratory-confirmed use of cathinones who presented for acute intoxication or complications of cathinone use were retrospectively analyzed.

Results and conclusions: The patients were predominantly male (64%, 52/81) with a median age of 34 years. 60 were admitted for signs of acute intoxication while 21 suffered from complications of cathinone use. 70% of acutely intoxicated patients had an increased creatinine phosphokinase. Only a minority of patients presented with a sympathomimetic toxidrome. Three patients had infectious complications, 10 prolonged psychosis, 6 rhabdomyolyses and/or kidney failure, and two patients died. Based on presentations, cathinone use has increased with the first cases seen in 2010. Opiates/opioids are the main co-ingested drugs of abuse. The pattern of cathinone use shifted from methylone in 2010/2011 to 3,4-methylenedioxypyrovalerone (MDPV) and 3-methylmethcathinone (3-MMC) in 2014/2015. We conclude that in our setting “typical“ cathinone users are males in their thirties. They are seldom drug naïve and regularly co-ingest illicit drugs. Preventive measures have to be tailored to these difficult to reach patients. Present efforts to educate young clubbers in their late teens may fail to reach the pertinent demographic.     

Quantitative Assessment of Age-Associated Alterations in Brain Vasculature in Wild-Type Mice and in Bigenic Mice that Model Alzheimer’s Disease

Molecular Imaging and Biology - Vascular dysfunction is a major hallmark of Alzheimer’s disease (AD). However, studies that investigated vascular dysfunction in mice modeling AD using...     

Natalizumab and the role of α4-integrin antagonism in the treatment of multiple sclerosis

Natalizumab is a powerful new therapy with a novel mechanism of action for the treatment of multiple sclerosis. In a randomized, double-blind, Phase III study (the AFFIRM [Natalizumab Safety and Efficacy in Relapsing-Remitting Multiple Sclerosis] study), natalizumab monotherapy 300 mg intravenous every 4 weeks reduced the risk of sustained disability progression by 42% and annualized relapse rate by 68% over 2 years (both p < 0.001 versus placebo). Natalizumab was approved in the US in November 2004 for the treatment of relapsing multiple sclerosis, but was voluntarily withdrawn in February 2005 due to three cases of progressive multifocal leukoencephalopathy. Following a safety evaluation and regulatory review, the US FDA approved natalizumab as monotherapy for the treatment of relapsing multiple sclerosis in June 2006 generally for patients who have had an inadequate response to, or are unable to tolerate, alternative treatments.     

Reversal of Vanadium-induced Toxicity by Combination Therapy of Tiferron and α-tocopherol in Rat during Pregnancy and their Fetuses

Objective. The aim of this study was to analyze the effect of tiferron (sodium 4, 5-dihydroxybenzene-1, 3-disulfonate) per se and combination with α-tocopherol against vanadium induced developmental toxicity. Vanadium, as vanadyl sulphate pentahydrate, was evaluated for embryotoxic/fetotoxic effect in female albino rats (Sprague Dawley). Methods. The compound was administered by gavage to pregnant animals at a dose of 15 mg/kg/day, p.o. on day 6-15 of pregnancy (organogenesis). Tiferron was given on day 16-18 as chelating agent. Cesarean sections were performed on day 19 of gestation. Results. Maternal toxicity was observed, the level of sugar in the blood decreased, while we observed an increase in serum protein, serum alkaline phosphatase and serum transaminase activity. Level of lipid peroxidation showed enhances value in fetal and maternal liver. Vanadium induced inhibition in glycogen contents. Protein contents were decreased in vital organs where as increased in uterus and placenta. There was increased activity of acid phosphatase with the concomitant decline in alkaline phosphatase, adenosine triphosphatase and succnic dehydrogenase after vanadium intoxication. Toxicant caused severe alteration in histopathological observation of maternal and fetal liver, kidney, uterus and placenta proving its toxic consequences at cellular level. Tiferron along with α-tocopherol dramatically reversed alterations of all variables towards control rather than individual treatment. Conclusion. The combination therapy of tiferron and α-tocopherol played a beneficial role in reducing vanadium induced developmental toxicity.     

Substrate inhibition of lysosomal hydrolases: α-Galactosidase A and β-glucocerebrosidase

ObjectiveWe have investigated the kinetics of α-galactosidase A and β-glucocerebrosidase deficient in Fabry and Gaucher diseases, respectively.Design and methodsWe have performed spectrofluorymetric measurements of the activity of enzymes using a derivative of 4-methylumbelliferone as a substrate and a human T-cell line as a source of enzymes.ResultsWe have observed the substrate inhibition effect, which is related to temperature.ConclusionsThe diagnostic procedures for Fabry and Gaucher diseases used now in laboratory practice neglect temperature-dependent substrate inhibition, which may significantly reduce the sensitivity of enzyme activity determinations.     

Fetal death associated with the use of 3,4-MDPHP and α-PHP

Introduction: The second largest group of new drugs monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) is synthetic cathinones. Substances that are controlled by the law are immediately replaced by new uncontrolled derivatives that cause constant and dynamic changes on the drug market. Some of the most recent synthetic cathinones that have appeared on the “legal highs” market are 3,4-methylenedioxy-α-pyrrolidinohexanophenone (3,4-MDPHP) and α-pyrrolidinohexanophenone (α-PHP).

Case history: A 21-year-old woman in the 36th week of pregnancy presented with psychomotor agitation. Fetal demise was demonstrated and a caesarean delivery performed.

Methods: The analyses were carried out by liquid chromatography with mass spectrometry (LC–MS/MS). The analytes were isolated from the biological material by liquid–liquid extraction with n-butyl chloride.

Results: 3,4-MDPHP and α-PHP were detected and quantified in both the fetus’ and the mothers blood, as well as in the mothers urine samples. The determined concentrations of 3,4-MDPHP and α-PHP were, 76?ng/mL and 12?ng/mL in the fetal blood sample, 16?ng/mL and traces in the mothers blood, and 697?mg/mL and 136?ng/mL in the mothers urine, respectively.

Discussion: The presented case demonstrates that 3,4-MDPHP and α-PHP transfers from maternal blood to fetal blood. Blood concentrations of these compounds were higher in the fetus than in the mother. Based on the known effects of these substances and the patient’s presentation and clinical course, it would seem that these substances contributed to the fetal death.

Conclusions: The detected substances transfer from maternal to fetal circulation, and synthetic cathinone blood concentration can be higher in the fetus than in the mother. This along with the fact immature metabolic ability makes a fetus more vulnerable to cathinones intoxication than adults.     

The α-tocopherol status and expression of α-tocopherol-related proteins in methionine-choline deficient rats treated with vitamin E

Non-alcoholic fatty liver disease is the most common liver disorder in developed countries, and its incidence is increasing in all population groups. As an antioxidant, vitamin E is effective in the treatment of non-alcoholic fatty liver disease, although the mechanism is still unclear. Methionine-choline deficient Wistar rats (n = 5) used as an experimental model of non-alcoholic fatty liver disease were fed a vitamin E-enriched diet (500 mg/kg) for 4 weeks. The effects were assessed by measuring lipid peroxidation, α-tocopherol levels, and the expression of α-tocopherol-related proteins in the liver. In vitamin E-treated methionine-choline deficient rats, lipid peroxidation was reduced, but liver histopathological changes were not improved. Hepatic α-tocopherol levels in these rats were significantly elevated compared to normal rats treated with vitamin E. Expression of liver α-tocopherol transfer protein in vitamin E-treated methionine-choline deficient rats was significantly repressed compared to methionine-choline deficient rats. The expression of liver cytochrome P450 4F2 and ATP-binding cassette transporter protein 1, involved in metabolism and transport of α-tocopherol, respectively, was significantly repressed in vitamin E-treated methionine-choline deficient rats. In methionine-choline deficient rats, vitamin E treatment altered the hepatic α-tocopherol-related protein expression, which may affect α-tocopherol status in the liver, leading to reduced lipid peroxidation.     

Molecular characterization and expression analysis of porcine integrins αvβ3, αvβ6 and αvβ8 that are potentially involved in FMDV infection

In the present study, we report the sequences and characterization of the porcine integrin cDNAs encoding αv, β3, β6 and β8 subunits and compare them to those of other species. The coding sequences for the porcine αv, β3, β6 and β8 subunits were found to be 3141, 2289, 2367 and 2304 nucleotides in length, encoding 1046, 762, 788 and 767-amino-acid-residue protein, respectively. The porcine integrin αv, β3, β6 and β8 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic trees showed that the porcine αv, β3, β6 and β8 were clustered into the Artiodactyla group, together with those of camels, sheep, and cattle, that are susceptible to FMDV infection. Real-time RT-PCR was used to investigate expression of the integrins αvβ3, αvβ6 and αvβ8 in different tissues of pigs in order to determine the role of these receptors in tissue tropism. Expression analysis showed that αvβ6 and αvβ8 mRNA expression were detected at high levels in tissues known to support replication of FMDV. Tissue distribution pattern of αvβ3 mRNA seems to be unrelated to the known tissue tropism of FMDV. This study provided the first data of porcine integrins for the further studies of the FMDV pathogenesis in pigs.