The possibility of using xenogeneic phagocytes in wound treatment.

Metamorphosis in the insect larva is associated with disintegration, engulf and digestion of larval tissues. These processes are accompanied by a significant shift in physiological parameters like high activity of hydrolytic enzymes and decrease of pH. In the way, the metamorphosing larva resembles the processes occurring in the wound at the stage of inflammation. Based on this thesis, we put forward the idea of the possibility of using insect phagocytes in the wound treatment. The search for a suitable insect cell line and the study of its properties were the purpose of the work. The abilities of insect phagocytes to retain viability and functional activity under conditions physiological for humans were also investigated. We found that blue blowfly Calliphora vicina larvae had histolysocytes, a specialized population of professional phagocytes involved in the histolysis. In vitro, histolysocytes possess high phagocytic activity to fragments of vertebrate soft tissues and debris. These cells retain viability and functional activity for a long time under conditions that are physiological for vertebrate cells. Moreover histolysocytes can realize the humoral control over the bacteria through the synthesis of antimicrobial peptides. So histolysocytes have the potential to be used as xenogeneic phagocytes in the wound treatment. The data obtained allow proceeding to experiments on laboratory animals for studying the effect of such therapy on the wound healing process.

Neonatal Reserpine Administration Produces Widespread Neuronal Losses and ⍺-Synuclein Inclusions in a Rat Model.

Historically, reserpine was widely used as an antihypertensive drug. However, severe motor and non-motor symptoms such as dyskinesia and depression led to the discontinuation of reserpine as a first-line treatment for hypertension. Reserpine functions by inhibiting vesicular monoamine transporter 2 (VMAT2), reducing sequestration of monoamines into synaptic vesicles. The consequent reduction in monoamines, most notably dopamine, serotonin and norepinephrine, in the central nervous system, causes well-defined symptoms such as catalepsy, hypoactivity and sedation in animals, and these motor and non-motor symptoms are well defined for reserpine treatment. However, no gross neuropathological changes in response to reserpine treatment have been reported previously in any animal model. In contrast, reducing VMAT2 expression in genetically modified VMAT2 LO mice leads to the production of ⍺-synuclein-positive aggregates and progressive nigrostriatal neuronal loss. These VMAT2 LO mice have reduced VMAT2 functionality during critical brain developmental stages and this could be the key to producing a reserpine model with matching histopathologies. The aim of this study was therefore to investigate the effect of neonatal reserpine administration on brain histology. We report here that a single dose of 5 mg kg-1 reserpine administered subcutaneously to neonatal rats on postnatal day 3 leads to widespread neuronal loss in various brain regions including the substantia nigra pars compacta, ventral tegmental area, striatum, hippocampus, locus coeruleus, amygdala and cerebral cortex, and the presence of ⍺-synuclein-positive inclusions in the substantia nigra pars compacta and the dorsal striatum within 30 days of administration.

Inhibition of the futalosine pathway for menaquinone biosynthesis suppresses Chlamydia trachomatis infection.

Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow-spectrum antibiotic targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through liquid chromatography-tandem mass spectrometry. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with the futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development.

Doxycycline inhibits α-synuclein-associated pathologies in vitro and in vivo.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn). Doxycycline, a tetracyclic antibiotic shows neuroprotective effects, initially proposed to be due to its anti-inflammatory properties. More recently, an additional mechanism by which doxycycline may exert its neuroprotective effects has been proposed as it has been shown that it inhibits amyloid aggregation. Here, we studied the effects of doxycycline on aSyn aggregation in vivo, in vitro and in a cell free system using real-time quaking induced conversion (RT-QuiC). Using H4, SH-SY5Y and HEK293 cells, we found that doxycycline decreases the number and size of aSyn aggregates in cells. In addition, doxycycline inhibits the aggregation and seeding of recombinant aSyn, and attenuates the production of mitochondrial-derived reactive oxygen species. Finally, we found that doxycycline induces a cellular redistribution of aggregates in a C.elegans animal model of PD, an effect that is associated with a recovery of dopaminergic function. In summary, we provide strong evidence that doxycycline treatment may be an effective strategy against synucleinopathies.

Modulators of Neuroinflammation Have a Beneficial Effect in a Lafora Disease Mouse Model.

Lafora disease (LD; OMIM#274780) is a fatal rare neurodegenerative disorder characterized by generalized epileptic seizures and the presence of polyglucosan inclusions (PGs), called Lafora bodies (LBs), typically in the brain. LD is caused by mutations in two genes EPM2A or EPM2B, which encode respectively laforin, a glucan phosphatase, and malin, an E3-ubiquitin ligase. Much remains unknown about the molecular bases of LD and, unfortunately, appropriate treatment is still missing; therefore patients die within 10 years from the onset of the disease. Recently, we have identified neuroinflammation as one of the initial determinants in LD. In this work, we have investigated anti-inflammatory treatments as potential therapies in LD. With this aim, we have performed a preclinical study in an Epm2b-/- mouse model with propranolol, a ß-adrenergic antagonist, and epigallocatechin gallate (EGCG), an antioxidant from green tea extract, both of which displaying additional anti-inflammatory properties. In vivo motor and cognitive behavioral tests and ex vivo histopathological brain analyses were used as parameters to assess the therapeutic potential of propranolol and EGCG. After 2 months of treatment, we observed an improvement not only in attention defects but also in neuronal disorganization, astrogliosis, and microgliosis present in the hippocampus of Epm2b-/- mice. In general, propranolol intervention was more effective than EGCG in preventing the appearance of astrocyte and microglia reactivity. In summary, our results confirm the potential therapeutic effectiveness of the modulators of inflammation as novel treatments in Lafora disease.

Transient Howell-Jolly-body-like cytoplasmic inclusions in neutrophils after severe intoxication with alpha-lipoic acid.

After intravenous supplementation of an unintentionally high dose of the antioxidant alpha-lipoic acid (ALA), a 53-year-old female complained of myalgia, chills and nausea, and showed signs of haemorrhagic diathesis. The laboratory findings were excessive hyperferritinemia, leukoerythroblastosis, severe thrombocytopenia, elevated liver enzymes and impaired coagulation. The toxicological tests resulted in an ALA serum concentration of 10 280 µg/L. The peripheral blood film of the patient showed some neutrophil dysplasia with unusual small dark-blue stained round cytoplasmic inclusions resembling 'Howell-Jolly-body-like' (HJBL) cytoplasmic inclusions, aptly named due to the morphologic similarity to their erythrocytic counterparts. Such HJBL inclusions are occasionally associated with acquired immunodeficiency, or immunosuppressive or cytostatic treatment. An association with ALA intoxication has not been described before. There are only a few reports on unintentional, harmful and lethal intoxications with ALA. The underlying molecular background of its toxicity on liver function or haematopoiesis is not yet known in detail, but ALA seems to interact with enzyme functions, e.g. with mitochondrial enzyme-complexes, possibly due to its pro-oxidant potential at high doses.

Endoplasmic Reticulum Stress Signalling Induces Casein Kinase 1-Dependent Formation of Cytosolic TDP-43 Inclusions in Motor Neuron-Like Cells.

Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation.

C9-ALS/FTD-linked proline-arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation.

A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

Cell models of lipid-rich α-synuclein aggregation validate known modifiers of α-synuclein biology and identify stearoyl-CoA desaturase.

Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion.

Lafora disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a-/- mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a-/- mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy.

Polyphenolic flavonoid (Myricetin) upregulated proteasomal degradation mechanisms: Eliminates neurodegenerative proteins aggregation.

Major neurodegenerative disorders are characterized by the formation of misfolded proteins aggregates inside or outside the neuronal cells. Previous studies suggest that aberrant proteins aggregates play a critical role in protein homeostasis imbalance and failure of protein quality control (PQC) mechanism, leading to disease conditions. However, we still do not understand the precise mechanisms of PQC failure and cellular dysfunctions associated with neurodegenerative diseases caused by the accumulation of protein aggregates. Here, we show that Myricetin, a flavonoid, can eliminate various abnormal proteins from the cellular environment via modulating endogenous levels of Hsp70 chaperone and quality control (QC)-E3 ubiquitin ligase E6-AP. We have observed that Myricetin treatment suppresses the aggregation of different aberrant proteins. Myricetin also enhances the elimination of various toxic neurodegenerative diseases associated proteins from the cells, which could be reversed by the addition of putative proteasome inhibitor (MG132). Remarkably, Myricetin can also stabilize E6-AP and reduce the misfolded proteins inclusions, which further alleviates cytotoxicity. Taken together these findings suggested that new mechanistic and therapeutic insights based on small molecules mediated regulation of disturbed protein quality control mechanism, which may result in the maintenance of the state of proteostasis.

Monitoring and control strategies for inclusion body production in E. coli based on glycerol consumption.

The Gram-negative bacterium E. coli is the host of choice for the production of a multitude of recombinant proteins in industry. Generally, cultivation is easy, media are cheap and a high product titer can be obtained. However, harsh induction procedures using IPTG as inducer are often referred to cause stress reactions, leading to a phenomenon known as metabolic burden and expression of inclusion bodies. In this contribution, we present different strategies for determination of critical timepoints for product stability in an E. coli IB bioprocess. As non-controlled feeding during induction regularly led to undesired product loss, we applied physiological feeding control. We found that the feeding strategy has indeed high impact on IB productivity. However, high applied qs,C increased IB product titer, but subsequently stressed the cells and finally led to product degradation. Calculating the cumulated glycerol uptake of the cells during induction phase (dSn), we found an empirical value, which serves as a strong indicator for process performance and can be used as process analytical tool. We tested different approaches starting from offline control. Glycerol accumulation could be used as trigger to establish a model-based approach to predict titer and viable cell concentration for a model protein. This straight forward control and model-based approach is high beneficial for upstream development and for increasing stability.

Prenatal Betamethasone interferes with immune system development and alters target cells in autoimmune diabetes.

Non-genetic factors are crucial in the pathogenesis of type 1 diabetes (T1D), a disease caused by autoimmunity against insulin-producing ß-cells. Exposure to medications in the prenatal period may influence the immune system maturation, thus altering self-tolerance. Prenatal administration of betamethasone -a synthetic glucocorticoid given to women at risk of preterm delivery- may affect the development of T1D. It has been previously demonstrated that prenatal betamethasone administration protects offspring from T1D development in nonobese diabetic (NOD) mice. The direct effect of betamethasone on the immature and mature immune system of NOD mice and on target ß-cells is analysed in this paper. In vitro, betamethasone decreased lymphocyte viability and induced maturation-resistant dendritic cells, which in turn impaired γδ T cell proliferation and decreased IL-17 production. Prenatal betamethasone exposure caused thymus hypotrophy in newborn mice as well as alterations in immune cells subsets. Furthermore, betamethasone decreased ß-cell growth, reduced C-peptide secretion and altered the expression of genes related to autoimmunity, metabolism and islet mass in T1D target tissue. These results support the protection against T1D in the betamethasone-treated offspring and demonstrate that this drug alters the developing immune system and ß-cells. Understanding how betamethasone generates self-tolerance could have potential clinical relevance in T1D.

Proteomic Study of a Parkinson's Disease Model of Undifferentiated SH-SY5Y Cells Induced by a Proteasome Inhibitor.

Parkinson's disease (PD) is one of the most common nervous system degenerative diseases. However, the etiology of this disease remains elusive. Here, a proteasome inhibitor (PSI)-induced undifferentiated SH-SY5Y PD model was established to analyze protein alterations through proteomic study. METHODS: Cultured undifferentiated SH-SY5Y cells were divided into a control group and a group treated with 2.5 µM PSI (PSI-treated group). An methyl thiazolyl tetrazolium (MTT) assay was applied to detect cell viability. Acridine orange/ethidium bromide (AO/EB), α-synuclein immunofluorescence and hematoxylin and eosin (H&E) staining were applied to evaluate apoptosis and cytoplasmic inclusions, respectively. The protein spots that were significantly changed were separated, analyzed by 2D gel electrophoresis and DIGE De Cyder software, and subsequently identified by MALDI-TOF mass spectrometry and database searching. RESULTS: The results of the MTT assay showed that there was a time and dose dependent change in cell viability following incubation with PSI. After 24 h incubation, PSI resulted in early apoptosis, and cytoplasmic inclusions were found in the PSI-treated group through H&E staining and α-synuclein immunofluorescence. Thus, undifferentiated SH-SY5Y cells could be used as PD model following PSI-induced inhibition of proteasomal function. In total, 18 proteins were differentially expressed between the groups, 7 of which were up-regulated and 11 of which were down-regulated. Among them, 5 protein spots were identified as being involved in the ubiquitin proteasome pathway-induced PD process. CONCLUSIONS: Mitochondrial heat shock protein 75 (MTHSP75), phosphoglycerate dehydrogenase (PHGDH), laminin binding protein (LBP), tyrosine 3/tryptophan 5-monooxygenase activation protein (14-3-3ε) and YWHAZ protein (14-3-3ζ) are involved in mitochondrial dysfunction, serine synthesis, amyloid clearance, apoptosis process and neuroprotection. These findings may provide new clues to deepen our understanding of PD pathogenesis.

Citalopram Reduces Aggregation of ATXN3 in a YAC Transgenic Mouse Model of Machado-Joseph Disease.

Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is a fatal polyglutamine disease with no disease-modifying treatment. The selective serotonin reuptake inhibitor citalopram was shown in nematode and mouse models to be a compelling repurposing candidate for Machado-Joseph disease therapeutics. We sought to confirm the efficacy of citalopram to decrease ATXN3 aggregation in an unrelated mouse model of Machado-Joseph disease. Four-week-old YACMJD84.2 mice and non-transgenic littermates were given citalopram 8 mg/kg in drinking water or water for 10 weeks. At the end of treatment, brains were collected for biochemical and pathological analyses. Brains of citalopram-treated YACMJD84.2 mice showed an approximate 50% decrease in the percentage of cells containing ATXN3-positive inclusions in the substantia nigra and three examined brainstem nuclei compared to controls. No differences in ATXN3 inclusion load were observed in deep cerebellar nuclei of mice. Citalopram effect on ATXN3 aggregate burden was corroborated by immunoblotting analysis. While lysates from the brainstem and cervical spinal cord of citalopram-treated mice showed a decrease in all soluble forms of ATXN3 and a trend toward reduction of insoluble ATXN3, no differences in ATXN3 levels were found between cerebella of citalopram-treated and vehicle-treated mice. Citalopram treatment altered levels of select components of the cellular protein homeostatic machinery that may be expected to enhance the capacity to refold and/or degrade mutant ATXN3. The results here obtained in a second independent mouse model of Machado-Joseph disease further support citalopram as a potential drug to be repurposed for this fatal disorder.

Heat shock promotes inclusion body formation of mutant huntingtin (mHtt) and alleviates mHtt-induced transcription factor dysfunction.

PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in individuals with and models of Huntington's disease (HD). The role of IB versus diffusible mHtt in neurotoxicity remains unclear. Using a ponasterone (PA)-inducible cell model of HD, here we evaluated the effects of heat shock on the appearance and functional outcome of Htt103QExon1-EGFP expression. Quantitative image analysis indicated that 80-90% of this mHtt protein initially appears as "diffuse" signals in the cytosol, with IBs forming at high mHtt expression. A 2-h heat shock during the PA induction reduced the diffuse signal, but greatly increased mHtt IB formation in both cytosol and nucleus. Dose- and time-dependent mHtt expression suggested that nucleated polymerization drives IB formation. RNA-mediated knockdown of heat shock protein 70 (HSP70) and heat shock cognate 70 protein (HSC70) provided evidence for their involvement in promoting diffuse mHtt to form IBs. Reporter gene assays assessing the impacts of diffuse versus IB mHtt showed concordance of diffuse mHtt expression with the repression of heat shock factor 1, cAMP-responsive element-binding protein (CREB), and NF-κB activity. CREB repression was reversed by heat shock coinciding with mHtt IB formation. In an embryonic striatal neuron-derived HD model, the chemical chaperone sorbitol similarly promoted the structuring of diffuse mHtt into IBs and supported cell survival under stress. Our results provide evidence that mHtt IB formation is a chaperone-supported cellular coping mechanism that depletes diffusible mHtt conformers, alleviates transcription factor dysfunction, and promotes neuron survival.

Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.

Diagnosis and treatment of MYH9-RD in an Australasian cohort with thrombocytopenia.

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (döhle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if döhle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells.

Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist; 5CPPSS-50, an LXRα inhibitor; and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism.

Serological Response Predicts Normalization of Cerebrospinal Fluid Abnormalities at Six Months after Treatment in HIV-Negative Neurosyphilis Patients.

This study aimed to determine whether a serological response could predict the normalization of cerebrospinal fluid (CSF) abnormalities at 6 months after treatment in human immunodeficiency virus (HIV)-negative neurosyphilis patients. A total of 123 neurosyphilis patients were recruited at baseline, 58 of these patients undergoing treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment were included in the follow-up study. Before treatment, the CSF rapid plasma reagin (RPR) titer, CSF Treponema pallidum particle agglutination (TPPA) titer, CSF leukocyte count, and CSF protein concentration were correlated with both serum RPR and TPPA titers. At 6 months after treatment, 28 and nine patients achieved serological responses of RPR and TPPA tests, respectively. The sensitivities of the serological response of RPR and TPPA tests for identifying the normalization of CSF abnormalities were 60.0∼83.3% and 17.1~22.2%, respectively; and 75.0∼91.3% of patients showing serological response of RPR test also achieved CSF normalization, suggesting that the serological response could predict CSF normalization to some degree. Particularly, in patients with ≥8-fold decreases in the serum RPR titer, the CSF RPR, CSF leukocyte count, and CSF protein concentration had normalized, and follow-up lumbar puncture could be reduced considering the resolution of neurological symptoms.