Evaluation of a transbronchial cryoprobe for the ablation of pulmonary nodules: an in vitro pilot study.

BACKGROUND: Ablation of malignant pulmonary nodules is a novel therapeutic option for patients who cannot undergo surgery. Current transthoracic approaches cause pneumothorax and/or bleeding in a significant number of cases. OBJECTIVE: Our purpose with this study was to evaluate cryoablation under in vitro conditions with a commercially available cryosurgery system. METHODS: We used ballistic gelatin to model the thermal conduction of lung tissue. The cryoprobe was inserted in the ballistic gelatin with two thermal sensors, they were placed 0.5 cm and 1.0. cm from the probe, respectively, temperature was measured on both sides. We used single-, double- and triple-freeze protocols to see if we could freeze it to -20 °C. RESULTS: We achieved - 18.6 ± 3.26 °C on the closer sensor (sensor 1) and - 3.7 ± 4.61 °C on the sensor further away (sensor 2) after 15 min using the single-freeze protocol. Using the dual-freeze protocol, we achieved - 23.2 ± 2,23 °C on sensor 1 and - 16.5 ± 2.82 °C on sensor 2. With the triple-freeze protocol we obtained - 23.5 ± 2.38 °C on sensor 1 and - 19.05 ± 3.22 °C on sensor 2. CONCLUSION: With dual-freeze, values above - 20 °C were achieved using nearer sensor data, but a plateau phase occurred as with continuous freezing. Using triple freeze, we reached - 20 °C at a distance of 0.5 cm from the probe, but not at 1 cm; therefore, we did not expand the diameter of the predicted necrosis zone.

CRISPR/Cas9-Based Mutagenesis of Histone H3.1 in Spinal Dynorphinergic Neurons Attenuates Thermal Sensitivity in Mice.

Burn injury is a trauma resulting in tissue degradation and severe pain, which is processed first by neuronal circuits in the spinal dorsal horn. We have recently shown that in mice, excitatory dynorphinergic (Pdyn) neurons play a pivotal role in the response to burn-injury-associated tissue damage via histone H3.1 phosphorylation-dependent signaling. As Pdyn neurons were mostly associated with mechanical allodynia, their involvement in thermonociception had to be further elucidated. Using a custom-made AAV9_mutH3.1 virus combined with the CRISPR/cas9 system, here we provide evidence that blocking histone H3.1 phosphorylation at position serine 10 (S10) in spinal Pdyn neurons significantly increases the thermal nociceptive threshold in mice. In contrast, neither mechanosensation nor acute chemonociception was affected by the transgenic manipulation of histone H3.1. These results suggest that blocking rapid epigenetic tagging of S10H3 in spinal Pdyn neurons alters acute thermosensation and thus explains the involvement of Pdyn cells in the immediate response to burn-injury-associated tissue damage.

In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

Passage through the Ocular Barriers and Beneficial Effects in Retinal Ischemia of Topical Application of PACAP1-38 in Rodents.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has two active forms, PACAP1-27 and PACAP1-38. Among the well-established actions are PACAP's neurotrophic and neuroprotective effects, which have also been proven in models of different retinopathies. The route of delivery is usually intravitreal in studies proving PACAP's retinoprotective effects. Recently, we have shown that PACAP1-27 delivered as eye drops in benzalkonium-chloride was able to cross the ocular barriers and exert retinoprotection in ischemia. Since PACAP1-38 is the dominant form of the naturally occurring PACAP, our aim was to investigate whether the longer form is also able to cross the barriers and exert protective effects in permanent bilateral common carotid artery occlusion (BCCAO), a model of retinal hypoperfusion. Our results show that radioactive PACAP1-38 eye drops could effectively pass through the ocular barriers to reach the retina. Routine histological analysis and immunohistochemical evaluation of the Müller glial cells revealed that PACAP1-38 exerted retinoprotective effects. PACAP1-38 attenuated the damage caused by hypoperfusion, apparent in almost all retinal layers, and it decreased the glial cell overactivation. Overall, our results confirm that PACAP1-38 given in the form of eye drops is a novel protective therapeutic approach to treat retinal diseases.

LRIG2 mutations cause urofacial syndrome.

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract.

Complement Fixation Test: An Update of an Old Method for Diagnosis of Bullous Pemphigoid.

The complement fixation test (CFT) is a method traditionally used for diagnosis of gestational pemphigoid. Its performance in diagnosis of bullous pemphigoid (BP) has not been investigated in a large patient cohort. The aim of this single-centre, retrospective, serological case-control study of 300 patients with BP and 136 control patients was to analyse its operating characteristics. CFT was found to have a sensitivity of 71.7% and a specificity of 100%. Furthermore, CFT diagnosed 20 of 46 patients with BP (43.5%) who were negative for both BP180 and BP230 enzyme-linked immunoassays (ELISAs), 31 of 66 patients (47.0%) who were negative for indirect immunofluorescence of the oesophagus, 5 of 14 patients (35.7%) who were serologically negative for all investigated serological assays, and 7 of 18 patients (38.9%) in whom direct immunofluorescence was negative. Combination of CFT with all other serological assays resulted in a sensitivity of 95.3%. In conclusion, CFT is suitable for the diagnosis of BP, and can help to diagnose serologically challenging cases.

Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure.

Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Exome sequencing identifies a REEP1 mutation involved in distal hereditary motor neuropathy type V.

The distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of neurodegenerative disorders affecting the lower motoneuron. In a family with both autosomal-dominant dHMN and dHMN type V (dHMN/dHMN-V) present in three generations, we excluded mutations in all genes known to be associated with a dHMN phenotype through Sanger sequencing and defined three potential loci through linkage analysis. Whole-exome sequencing of two affected individuals revealed a single candidate variant within the linking regions, i.e., a splice-site alteration in REEP1 (c.304-2A>G). A minigene assay confirmed complete loss of splice-acceptor functionality and skipping of the in-frame exon 5. The resulting mRNA is predicted to be expressed at normal levels and to encode an internally shortened protein (p.102_139del). Loss-of-function REEP1 mutations have previously been identified in dominant hereditary spastic paraplegia (HSP), a disease associated with upper-motoneuron pathology. Consistent with our clinical-genetic data, we show that REEP1 is strongly expressed in the lower motoneurons as well. Upon exogeneous overexpression in cell lines we observe a subcellular localization defect for p.102_139del that differs from that observed for the known HSP-associated missense mutation c.59C>A (p.Ala20Glu). Moreover, we show that p.102_139del, but not p.Ala20Glu, recruits atlastin-1, i.e., one of the REEP1 binding partners, to the altered sites of localization. These data corroborate the loss-of-function nature of REEP1 mutations in HSP and suggest that a different mechanism applies in REEP1-associated dHMN.

Childhood lichen planus pemphigoides: report of two cases treated successfully with systemic glucocorticoids and dapsone.

We describe two boys, ages 8 and 7 years, who developed lichen planus pemphigoides. In one of the boys, a henna tattoo probably triggered the disease. Therapy with systemic glucocorticoids and dapsone was successful.

Repeated diagnostic ultrasound exposure modifies the structural properties of CA1 dendrites and alters the hippocampal transcriptome.

The development of neurons is regulated by several spatiotemporally changing factors, which are crucial to give the ability of neurons to form functional networks. While external physical stimuli may impact the early developmental stages of neurons, the medium and long-term consequences of these influences have yet to be thoroughly examined. Using an animal model, this study focuses on the morphological and transcriptome changes of the hippocampus that may occur as a consequence of fetal ultrasound examination. We selectively labeled CA1 neurons of the hippocampus with in-utero electroporation to analyze their morphological features. Furthermore, certain samples also went through RNA sequencing after repetitive ultrasound exposure. US exposure significantly changed several morphological properties of the basal dendritic tree. A notable increase was also observed in the density of spines on the basal dendrites, accompanied by various alterations in individual spine morphology. Transcriptome analysis revealed several up or downregulated genes, which may explain the molecular background of these alterations. Our results suggest that US-derived changes in the dendritic trees of CA1 pyramidal cells might be connected to modification of the transcriptome of the hippocampus and may lead to an increased dendritic input.

Methionine sources and genotype affect embryonic intestinal development, antioxidants, tight junctions, and growth-related gene expression in chickens.

Methionine (Met) is an essential and first limiting amino acid in the poultry diet that plays a significant role in chicken embryonic development and growth. The present study examined the effect of in ovo injection of DL-Met and L-Met sources and genotypes on chicken embryonic-intestinal development and health. Fertilized eggs of the two genotypes, TETRA-SL layer hybrid (TSL) - commercial layer hybrid and Hungarian Partridge colored hen breed (HPC) - a native genotype, were randomly distributed into four treatments for each genotype. The treatment groups include the following: 1) control non-injected eggs (NoIn); 2) saline-injected (SaIn); 3) DL-Met injected (DLM); and 4) L-Met injected (LM). The in ovo injection was carried out on 17.5 d of embryonic development; after hatching, eight chicks per group were sacrificed, and the jejunum was extracted for analysis. The results showed that both DLM and LM groups had enhanced intestinal development as evidenced by increased villus width, villus height, and villus area (P < 0.05) compared to the control. The DLM group had significantly reduced crypt depth, glutathione content (GSH), glutathione S-transferase 3 alpha (GST3), occludin (OCLN) gene expression and increased villus height to crypt depth ratio in the TSL genotype than the LM group (P < 0.05). The HPC genotype has overexpressed insulin-like growth factor 1 (IGF1) gene, tricellulin (MD2), occludin (OCLN), superoxide dismutase 1 (SOD1), and GST3 genes than the TSL genotype (P < 0.05). In conclusion, these findings showed that in ovo injection of Met enhanced intestinal development, and function, with genotypes responding differently under normal conditions. Genotypes also influenced the expression of intestinal antioxidants, tight junction, and growth-related genes.

Do not trust the pedigree: reduced and sex-dependent penetrance at a novel mutation hotspot in ATL1 blurs autosomal dominant inheritance of spastic paraplegia.

The hereditary spastic paraplegias (HSPs), a group of neurodegenerative movement disorders, are among the genetically most heterogeneous clinical conditions. Still, the more than 50 forms known so far apparently explain less than 80% of cases. The present study identified two large HSP families, which seemed to show an autosomal recessive and an X-linked inheritance pattern. A set of genetic analyses including exome sequencing revealed plausible mutations only when assuming incomplete/sex-dependent penetrance of adjacent alterations in the autosomal dominant HSP gene ATL1 (c.1243C>T and c.1244G>A, respectively). By screening of additional HSP patients for the presence of these alterations, we identified three more cases and obtained additional evidence for reduced penetrance. Bisulfate sequencing and haplotype analysis indicated that c.1243C and c.1244G constitute a mutational hotspot. Our findings suggest that misinterpretation of inheritance patterns and, consequently, misselection of candidate genes to be screened in gene-focused approaches contribute to the apparently missing heritability in HSP and, potentially, in other genetically heterogeneous disorders.

Comparative study of direct and indirect immunofluorescence and of bullous pemphigoid 180 and 230 enzyme-linked immunosorbent assays for diagnosis of bullous pemphigoid.

BACKGROUND: Direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA) are used for the laboratory diagnosis of bullous pemphigoid (BP). OBJECTIVE: The diagnostic value of DIF and IIF on rabbit and monkey esophagus or human salt-split skin and commercial ELISAs was assessed. METHODS: This was a single-center retrospective study where 313 patients with BP were compared with 488 control subjects. RESULTS: DIF was the most sensitive test (90.8%) whereas sensitivities for IIF on rabbit esophagus, IIF on monkey esophagus, IIF on salt-split skin, BP180 ELISA, and BP230 ELISA were 76.0%, 73.2%, 73.3%, 72.0%, and 59.0%, respectively. The sensitivity of the serologic tests was 88.8% altogether. The specificities for DIF, IIF on rabbit esophagus, IIF on monkey esophagus, IIF on salt-split skin, BP180 ELISA, and BP230 ELISA were 98%, 96.5%, 97.1%, 100%, 94.1%, and 99.2%, respectively. LIMITATIONS: The retrospective nature of study was a limitation. Correlation of diagnostic data with clinical manifestations or disease course was not possible. CONCLUSIONS: In suspected BP, both serologic tests and DIF have to be performed because of a sensitivity issue. Although the ELISAs had a relatively low sensitivity, the serologic tests altogether almost reached the level of sensitivity of DIF. The specificities of all assays were excellent.

Progressive nodular histiocytosis.

Progressive nodular histiocytosis (PNH) represents a very rare type of non-Langerhans cell histiocytosis (NLCH). It can be distinguished from other types by the occurrence of yellow to red-brown papules and nodules measuring a few centimeters in size. Histologically the nodules represent spindle-cell xanthogranulomas. We report on two new cases of PNH with novel clinical symptoms. In the first case, the patient had microcytic anemia due to massive iron deposition in the nodules and a progressively worsening limp due to involvement of the soles. The complex therapeutic challenge was effectively managed by successive surgical ablation of all visible lesions. The second patient had epibulbar infiltrates. The clinical variability of PNH leads to considerable overlap with other forms of NLCH.

The link between the sphingolipid rheostat and obstructive sleep apnea.

Chronic inflammation induced by hypoxia during sleep is an important mechanism of microvascular damage in OSA patients. In this study, we investigated the role of the sphingosine rheostat, which has diverse inflammatory effects. Thirty-seven healthy subjects and 31 patients with OSA were recruited. We collected data on demographics and comorbidities. Plasma sphingosine-1-phosphate and ceramide antibody concentrations were measured by ELISA. The results were compared between the OSA and control groups, and the correlations between these measurements and markers of disease severity and comorbidities were explored. Ceramide antibody levels were significantly elevated in OSA patients (892.17 ng/ml) vs. controls (209.55 ng/ml). S1P levels were also significantly higher in patients with OSA (1760.0 pg/ml) than in controls (290.35 pg/ml, p < 0.001). The ceramide antibody concentration showed correlations with BMI (ρ = 0.25, p = 0.04), CRP (ρ = 0.36, p = 0.005), AHI (ρ = 0.43, p < 0.001), ODI (ρ = 0.43, p < 0.001), TST90% (ρ = 0.35, p = 0.004) and the lowest oxygen saturation (ρ =  0.37, p = 0.001) in the whole study population but not when patients with OSA were analyzed separately. The elevated ceramide antibody and sphingosine-1-phosphate concentrations in patients suffering from OSA suggests their involvement in the pathomechanism of OSA and its comorbidities.

MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives.

Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for identifying copy number mutations in diagnostic and research settings. The occurrence of false-positive deletion findings and the underlying causes are well recognized, whereas false-positive duplication/amplification findings have not been appreciated so far. We here present three pertinent cases which were only identified on extended, nonstandard secondary analyses. We also offer and experimentally validate a potential explanation. Our findings imply that MLPA data indicating gain of genomic sequence require validation on an independent sample or by an independent method.

Poly(amino acid) based fibrous membranes with tuneable in vivo biodegradation.

In this work two types of biodegradable polysuccinimide-based, electrospun fibrous membranes are presented. One contains disulfide bonds exhibiting a shorter (3 days) in vivo biodegradation time, while the other one has alkyl crosslinks and a longer biodegradation time (more than 7 days). According to the mechanical measurements, the tensile strength of the membranes is comparable to those of soft the connective tissues and visceral tissues. Furthermore, the suture retention test suggests, that the membranes would withstand surgical handling and in vivo fixation. The in vivo biocompatibility study demonstrates how membranes undergo in vivo hydrolysis and by the 3rd day they become poly(aspartic acid) fibrous membranes, which can be then enzymatically degraded. After one week, the disulfide crosslinked membranes almost completely degrade, while the alkyl-chain crosslinked ones mildly lose their integrity as the surrounding tissue invades them. Histopathology revealed mild acute inflammation, which diminished to a minimal level after seven days.

Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15.Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle's balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type.