Systematic review and co-ordinate based meta-analysis to summarize the utilization of functional brain imaging in conjunction with human models of peripheral and central sensitization.

BACKGROUND AND OBJECTIVE: Functional magnetic resonance imaging, in conjunction with models of peripheral and/or central sensitization, has been used to assess analgesic efficacy in healthy humans. This review aims to summarize the use of these techniques to characterize brain mechanisms of hyperalgesia/allodynia and to evaluate the efficacy of analgesics. DATABASES AND DATA TREATMENT: Searches were performed (PubMed-Medline, Cochrane, Web of Science and Clinicaltrials.gov) to identify and review studies. A co-ordinate based meta-analysis (CBMA) was conducted to quantify neural activity that was reported across multiple independent studies in the hyperalgesic condition compared to control, using GingerALE software. RESULTS: Of 217 publications, 30 studies met the inclusion criteria. They studied nine different models of hyperalgesia/allodynia assessed in the primary (14) or secondary hyperalgesia zone (16). Twenty-three studies focused on neural correlates of hyperalgesic conditions and showed consistent changes in the somatosensory cortex, prefrontal cortices, insular cortex, anterior cingulate cortex, thalamus and brainstem. The CBMA on 12 studies that reported activation coordinates for a contrast comparing the hyperalgesic state to control produced six activation clusters (significant at false discovery rate of 0.05) with more peaks for secondary (17.7) than primary zones (7.3). Seven studies showed modulation of brain activity by analgesics in five of the clusters but also in four additional regions. CONCLUSIONS: This meta-analysis revealed substantial but incomplete overlap between brain areas related to neural mechanisms of hyperalgesia and those reflecting the efficacy of analgesic drugs. Studies testing in the secondary zone were more sensitive to evaluate analgesic efficacy on central sensitization at brainstem or thalamocortical levels. SIGNIFICANCE: Experimental pain models that provide a surrogate for features of pathological pain conditions in healthy humans and functional imaging techniques are both highly valuable research tools. This review shows that when used together, they provide a wealth of information about brain activity during pain states and analgesia. These tools are promising candidates to help bridge the gap between animal and human studies, to improve translatability and provide opportunities for identification of new targets for back-translation to animal studies.

Effects of sleep deprivation on cortical excitability: A threshold-tracking TMS study and review of the literature.

Objective: Insufficient sleep is linked to several health problems. Previous studies on the effects of sleep deprivation on cortical excitability using conventional transcranial magnetic stimulation (TMS) included a limited number of modalities, and few inter-stimulus intervals (ISIs) and showed conflicting results. This study aimed to investigate the effects of sleep deprivation on cortical excitability through threshold-tracking TMS, using a wide range of protocols at multiple ISIs. Methods: Fifteen healthy subjects (mean age ±â€¯SD: 36 ±â€¯3.34 years) were included. The following tests were performed before and after 24 h of sleep deprivation using semi-automated threshold-tacking TMS protocols: short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) at 11 ISIs between 1 and 30 ms, short interval intracortical facilitation (SICF) at 14 ISIs between 1 and 4.9 ms, long interval intracortical inhibition (LICI) at 6 ISIs between 50 and 300 ms, and short-latency afferent inhibition (SAI) at 12 ISIs between 16 and 30 ms. Results: No significant differences were observed between pre- and post-sleep deprivation measurements for SICI, ICF, SICF, or LICI at any ISIs (p < 0.05). As for SAI, we found a difference at 28 ms (p = 0.007) and 30 ms (p = 0.04) but not at other ISIs. Conclusions: Sleep deprivation does not affect cortical excitability except for SAI. Significance: This study confirms some of the previous studies while contradicting others.

The test-retest reliability of large and small fiber nerve excitability testing with threshold tracking.

Objective: Standard nerve excitability testing (NET) predominantly assesses Aα- and Aß-fiber function, but a method examining small afferents would be of great interest in pain studies. Here, we examined the properties of a novel perception threshold tracking (PTT) method that preferentially activates Aδ-fibers using weak currents delivered by a novel multipin electrode and compared its reliability with NET. Methods: Eighteen healthy subjects (mean age:34.06 ±â€¯2.0) were examined three times with motor and sensory NET and PTT in morning and afternoon sessions on the same day (intra-day reliability) and after a week (inter-day reliability). NET was performed on the median nerve, while PTT stimuli were delivered through a multipin electrode located on the forearm. During PTT, subjects indicated stimulus perception via a button press and the intensity of the current was automatically increased or decreased accordingly by Qtrac software. This allowed changes in the perception threshold to be tracked during strength-duration time constant (SDTC) and threshold electrotonus protocols. Results: The coefficient of variation (CoV) and interclass coefficient of variation (ICC) showed good-excellent reliability for most NET parameters. PTT showed poor reliability for both SDTC and threshold electrotonus parameters. There was a significant correlation between large (sensory NET) and small (PTT) fiber SDTC when all sessions were pooled (r = 0.29, p = 0.03). Conclusions: Threshold tracking technique can be applied directly to small fibers via a psychophysical readout, but with the current technique, the reliability is poor. Significance: Further studies are needed to examine whether Aß-fiber SDTC may be a surrogate biomarker for peripheral nociceptive signalling.

IMI2-PainCare-BioPain-RCT1: study protocol for a randomized, double-blind, placebo-controlled, crossover, multi-center trial in healthy subjects to investigate the effects of lacosamide, pregabalin, and tapentadol on biomarkers of pain processing observed by peripheral nerve excitability testing (NET).

BACKGROUND: Few new drugs have been developed for chronic pain. Drug development is challenged by uncertainty about whether the drug engages the human target sufficiently to have a meaningful pharmacodynamic effect. IMI2-PainCare-BioPain-RCT1 is one of four similarly designed studies that aim to link different functional biomarkers of drug effects on the nociceptive system that could serve to accelerate the future development of analgesics. This study focusses on biomarkers derived from nerve excitability testing (NET) using threshold tracking of the peripheral nervous system. METHODS: This is a multisite single-dose, subject and assessor-blind, randomized, placebo-controlled, 4-period, 4-way crossover, pharmacodynamic (PD), and pharmacokinetic (PK) study in healthy subjects. Biomarkers derived from NET of large sensory and motor fibers and small sensory fibers using perception threshold tracking will be obtained before and three times after administration of three medications known to act on the nociceptive system (lacosamide, pregabalin, tapentadol) and placebo, given as a single oral dose with at least 1 week apart. Motor and sensory NET will be assessed on the right wrist in a non-sensitized normal condition while perception threshold tracking will be performed bilaterally on both non-sensitized and sensitized forearm skin. Cutaneous high-frequency electrical stimulation is used to induce hyperalgesia. Blood samples will be taken for pharmacokinetic purposes and pain ratings as well as predictive psychological traits will be collected. A sequentially rejective multiple testing approach will be used with overall alpha error of the primary analysis split across the two primary outcomes: strength-duration time constant (SDTC; a measure of passive membrane properties and nodal persistent Na+ conductance) of large sensory fibers and SDTC of large motor fibers comparing lacosamide and placebo. The key secondary endpoint is the SDTC measured in small sensory fibers. Remaining treatment arm effects on key NET outcomes and PK modelling are other prespecified secondary or exploratory analyses. DISCUSSION: Measurements of NET using threshold tracking protocols are sensitive to membrane potential at the site of stimulation. Sets of useful indices of axonal excitability collectively may provide insights into the mechanisms responsible for membrane polarization, ion channel function, and activity of ionic pumps during the process of impulse conduction. IMI2-PainCare-BioPain-RCT1 hypothesizes that NET can serve as biomarkers of target engagement of analgesic drugs in this compartment of the nociceptive system for future Phase 1 clinical trials. Phase 2 and 3 clinical trials could also benefit from these tools for patient stratification. TRIAL REGISTRATION: This trial was registered 25/06/2019 in EudraCT ( 2019-000942-36 ).

Human surrogate models of central sensitization: A critical review and practical guide.

BACKGROUND: As in other fields of medicine, development of new medications for management of neuropathic pain has been difficult since preclinical rodent models do not necessarily translate to the clinics. Aside from ongoing pain with burning or shock-like qualities, neuropathic pain is often characterized by pain hypersensitivity (hyperalgesia and allodynia), most often towards mechanical stimuli, reflecting sensitization of neural transmission. DATA TREATMENT: We therefore performed a systematic literature review (PubMed-Medline, Cochrane, WoS, ClinicalTrials) and semi-quantitative meta-analysis of human pain models that aim to induce central sensitization, and generate hyperalgesia surrounding a real or simulated injury. RESULTS: From an initial set of 1569 reports, we identified and analysed 269 studies using more than a dozen human models of sensitization. Five of these models (intradermal or topical capsaicin, low- or high-frequency electrical stimulation, thermode-induced heat-injury) were found to reliably induce secondary hyperalgesia to pinprick and have been implemented in multiple laboratories. The ability of these models to induce dynamic mechanical allodynia was however substantially lower. The proportion of subjects who developed hypersensitivity was rarely provided, giving rise to significant reporting bias. In four of these models pharmacological profiles allowed to verify similarity to some clinical conditions, and therefore may inform basic research for new drug development. CONCLUSIONS: While there is no single "optimal" model of central sensitization, the range of validated and easy-to-use procedures in humans should be able to inform preclinical researchers on helpful potential biomarkers, thereby narrowing the translation gap between basic and clinical data. SIGNIFICANCE: Being able to mimic aspects of pathological pain directly in humans has a huge potential to understand pathophysiology and provide animal research with translatable biomarkers for drug development. One group of human surrogate models has proven to have excellent predictive validity: they respond to clinically active medications and do not respond to clinically inactive medications, including some that worked in animals but failed in the clinics. They should therefore inform basic research for new drug development.

Increased antioxidant response in medium-chain acyl-CoA dehydrogenase deficiency: does lipoic acid have a protective role?

BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is the most frequent fatty acid oxidation (FAO) defect in humans. MCAD-deficient fibroblasts are more resistant to oxidative stress-induced cell death than other FAO defects and healthy controls. METHODS: Herein we investigate the antioxidant response and mitochondrial function in fibroblasts from MCAD-deficient patients (c.985 A>G/c.985 A>G) and healthy controls. RESULTS: MCAD-deficient fibroblasts showed increased level of mitochondrial superoxide, while lipids were less oxidatively damaged, and higher amount of manganese superoxide dismutase were detected compared to healthy controls, showing forceful antioxidant system in MCADD. We showed increased maximal respiration and reserve capacity in MCAD-deficient fibroblasts compared to controls, indicating more capacity through the tricarboxylic acid (TCA) cycle and subsequently respiratory chain. This led us to study the pyruvate dehydrogenase complex (PDC), the key enzyme in the glycolysis releasing acetyl-CoA to the TCA cycle. MCAD-deficient fibroblasts displayed not only significantly increased PDC but also increased lipoylated PDC protein levels compared to healthy controls. CONCLUSIONS: Based on these findings, we raise the interesting hypothesis that increased PDC-bound lipoic acid, synthesized from accumulated octanoic acid in MCADD, may affect the cellular antioxidant pool in MCADD.

FLAD1-associated multiple acyl-CoA dehydrogenase deficiency identified by newborn screening.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as glutaric aciduria type II, is a mitochondrial fatty acid oxidation disorder caused by variants in ETFA, ETFB, and ETFDH. Recently, riboflavin transporter genes and the mitochondrial FAD transporter gene have also been associated with MADD-like phenotype. METHODS: We present a case of MADD identified by newborn biochemical screening in a full-term infant suggestive of both medium-chain acyl-CoA dehydrogenase deficiency and MADD. Urine organic acid GC/MS analysis was also concerning for both disorders. However, panel sequencing of ETFA, ETFB, ETFDH, and ACADM was unrevealing. Ultimately, a variant in the FAD synthase gene, FLAD1 was found explaining the clinical presentation. RESULTS: Exome sequencing identified compound heterozygous variants in FLAD1: NM_025207.4: c.[442C>T];[1588C>T], p.[Arg148*];[Arg530Cys]. The protein damaging effects were confirmed by Western blot. The patient remained asymptomatic and there was no clinical decompensation during the first year of life. Plasma acylcarnitine and urinary organic acid analyses normalized without any treatment. Riboflavin supplementation was started at 15 months. CONCLUSION: Newborn screening, designed to screen for specific treatable congenital metabolic diseases, may also lead to the diagnosis of additional, very rare metabolic disorders such as FLAD1 deficiency. The case further illustrates that even milder forms of FLAD1 deficiency are detectable in the asymptomatic state by newborn screening.

Deficiency of the mitochondrial sulfide regulator ETHE1 disturbs cell growth, glutathione level and causes proteome alterations outside mitochondria.

The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.

Short-chain acyl-CoA dehydrogenase deficiency: from gene to cell pathology and possible disease mechanisms.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an inherited disorder of mitochondrial fatty acid oxidation that is characterized by the presence of increased butyrylcarnitine and ethylmalonic acid (EMA) concentrations in plasma and urine. Individuals with symptomatic SCADD may show relatively severe phenotype, while the majority of those who are diagnosed through newborn screening by tandem mass spectrometry may remain asymptomatic. As such, the associated clinical symptoms are very diverse, ranging from severe metabolic or neuromuscular disabilities to asymptomatic. Molecular analysis of affected individuals has identified rare gene variants along with two common gene variants, c.511C > T and c.625G > A. In vitro studies have demonstrated that the common variants as well as the great majority of rare variants, which are missense variants, impair folding, that may lead to toxic accumulation of the encoded protein, and/or metabolites, and initiate excessive production of ROS and chronic oxidative stress. It has been suggested that this cell toxicity in combination with yet unknown factors can trigger disease development. This association and the full implications of SCADD are not commonly appreciated. Accordingly, there is a worldwide discussion of the relationship of clinical manifestation to SCADD, and whether SCAD gene variants are disease associated at all. Therefore, SCADD is not part of the newborn screening programs in most countries, and consequently many patients with SCAD gene variants do not get a diagnosis and the possibilities to be followed up during development.

Selected reaction monitoring mass spectrometry for relative quantification of proteins involved in cellular life and death processes.

Monitoring of proteins involved in cellular life and death processes is of high scientific interest since it permits the elucidation of functional changes in a variety of diseases. In this study, we have developed a nanoLC-MS/MS assay for the simultaneous detection and quantification of 24 selected proteins that are known to be important for cellular homeostasis. The Selected Reaction Monitoring (SRM) assay applies heavy-labeled peptide analogues for the relative quantification of proteins with central functions in cellular stress and metabolism, including many mitochondrial proteins. The assay includes proteins involved in the quality control of mitochondrial proteins, oxidative stress, respiratory chain, and fatty acid degradation, as well as the cytosolic glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase and ribosomal proteins. The assay can thus quantitate the balance between mitochondrial and cytosolic pathways, which is relevant in many disease states, and can be studied by comparing patient and control samples. The measured validation parameters showed satisfactory results for the proteins included in the analysis. The linear range of the monitored proteins was 0.01-20nM, with a median precision of less than 10%. The assay performed well in monitoring proteins in both cultured human skin fibroblast cells as well as in isolated peripheral blood mononuclear cells. We therefore believe that this assay is applicable for the study of cellular stress response in various types of cell defects and disease states.

Antimicrobial resistance in Salmonella spp. recovered from patients admitted to six different hospitals in Tehran, Iran from 2007 to 2008.

The objective of this study was to assess the genotypic diversity associated with antimicrobial susceptibility of Salmonella serovars isolated from patients arriving with diarrhoea to six hospitals of Tehran, Iran. During 2007-2008, a cross-sectional convenience study was performed. Stool samples were screened for the presence of Salmonella, serotyped, tested for antimicrobial susceptibility using disk diffusion and examined for the presence of relevant resistance genes and integrons by PCR. A total of 1,120 patients were screened for the presence of Salmonella. Out of 71 Salmonella isolates recovered, the following serovars were identified: 17 Typhi, 14 Paratyphi C, 13 Enteritidis, 11 Paratyphi B, 10 Paratyphi A and six Infantis. Most resistance was observed towards sulfamethoxazole (30%), tetracyclines (25%), nalidixic acid (22%), spectinomycin (17%), trimethoprim (15%), ampicillin (14%) and kanamycin (14%). The tetracycline resistance genes tet(A), tet(B), and tet(G) were found in 28%, 14% and 6% of the tetracycline resistant isolates, respectively. The genes aadA, aadB, strA, strB and aphA1-Iab were present in 83%, 55%, 34%, 1% and 17% of the aminoglycoside resistant isolates, respectively. Additionally, bla (PSE) and bla (TEM) ß-lactamase genes were detected in 63% and 18% of the ampicillin-resistant isolates. The 23 sulphonamide resistant isolates harboured sul1 and intI1 genes, typical to class 1 integrons. Nine of these isolates also yielded amplicons for intI2 (class 2 integrons). The presence of multi-drug resistant Salmonella may compromise the successful treatment of enteric infection diseases. The enforcement of strict prescription practices will help to minimise the emergence of resistance.

Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad.

Staphylococcus aureus is considered one of the most important food borne pathogens. A total of 111 isolates of S. aureus were cultured from raw milk samples during January 2009 to June 2009 from Tehran and Mashhad. The coagulase gene polymorphism and the prevalence of classical enterotoxin genes of S. aureus strains were determined by PCR-RFLP (restriction fragment length polymorphism) and Multiplex-PCR. Disk diffusion method was used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. Sixty-seven % of the isolates harboured one or more enterotoxin genes. The most prevalent gene was sec, found in 59 % of the isolates. Approximately 8% of the isolates were positive for sea, seb and sed genes. Only one isolate had see gene. The rate of coexistence of enterotoxin genes was 14%. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem, minocycline, oxacillin and vancomycin. They were resistant to ampicillin (64%), penicillin (56%), clindamycin (22%), tetracycline (22%), doxycycline (19%), teicoplanin (13%), rifampin (2%) and trimethoprim-sulfamethoxazole (2%). On the basis of coagulase gene analysis of 111 S. aureus isolates, the PCR products of 56 isolates were digested with Alu I that produced three distinct patterns. These data indicate the high prevalence of enterotoxigenic S. aureus in raw bovine milk in Tehran and Mashhad, and highlight the importance of proper quality control of dairy products for public health.

Discrimination of Entamoeba moshkovskii in patients with gastrointestinal disorders by single-round PCR.

Entamoeba moshkovskii and Entamoeba dispar are impossible to differentiate microscopically from the pathogenic species Entamoeba histolytica. There are limited data on the prevalence of these commensal parasites in Iran. We utilized a single-round PCR assay to determine the prevalence of E. moshkovskii, E. dispar, and E. histolytica in stool samples from Iranian patients infected with gastrointestinal disorders. After culturing of microscopy-positive isolates and extraction of DNA, PCR was carried out to differentiate the Entamoeba isolates. Out of 3,825 stool samples examined by microscopy, 58 specimens (1.52%) were infected with E. histolytica, E. dispar, or E. moshkovskii. By PCR, 2 E. histolytica (3.45%), 53 E. dispar (91.37%), 2 E. moshkovskii (3.45%), and one mixed E. dispar/E. moshkovskii infection (1.73%) were detected. In view of the reporting of E. moshkovskii in this study in Iran and the difficulty in discriminating this ameba from two similar Entamoeba spp. by microscopy, we recommend the single-round PCR assay as an alternative tool in routine diagnosis and in epidemiological studies of amebiasis.

A case-control study of Blastocystis hominis among Iranian population.

OBJECTIVES: A case-control study was conducted to investigate the epidemiology and clinical features of Blastocystis hominis among Iranian patients with and without GI symptoms. METHODS: Six hundred and seventy patients with GI and Six hundred and seventy patients without GI symptoms were enrolled as cases and controls respectively during 2006-07. Standard microscopic examinations following in vitro culture were used to examine the stool samples for presence of trophozoites and cysts of B. hominis. RESULTS: Infection with B. hominis occurred most commonly in those with GI symptoms (5.67 %) compared with those patients without GI symptoms (3.43 %). The most common symptom in case group was abdominal pain (86.84 %). B. hominis was mostly found with Giardia lamblia in case group and with Entamoeba coli in control group. In addition, there was no significant relation between the presence of GI symptoms and the incidence of B. hominis. CONCLUSION: B. hominis has long been described as a non-pathogenic protozoan parasite until recently, when claims have been made that it could result in pathogenic conditions. Thus, to confirm the complication is needed to additional study especially on molecular pathogenesis of this organism.