Lacticaseibacillus rhamnosus GG DSM 33156 effects on pathogen defence in the upper respiratory tract: a randomised, double-blind, placebo-controlled paediatric trial.

Acute upper respiratory tract infections (URTIs) are caused by numerous viruses and bacteria. URTIs can be a cause of morbidity and are among the most common reasons for visiting healthcare practitioners and prescribing antibiotics to children in addition to causing absenteeism from school and work. Oral intake of Lacticaseibacillus rhamnosus GG DSM 33156 has shown beneficial health effects in several clinical trials, primarily relating to immune function and gastrointestinal health in children and adults. It has also been suggested that oral intake of L. rhamnosus GG DSM 33156 can reduce the incidence rate and alleviate symptoms of URTIs in children. We here report the results of a randomised, double-blind, placebo-controlled trial of 619 children aged 2-6 years conducted at a single centre in Scotland. The children, who were in day care or primary school, were followed over a 16-week intervention period with 309 randomised in the active group and 310 in the placebo group. The parents or guardians reported a daily healthcare status and any presumed episodes of URTI, which were subsequently confirmed by a general practitioner. The investigational product was well tolerated in the trial. Although a general trend towards a beneficial effect was observed, this trial did not demonstrate that L. rhamnosus GG DSM 33156 significantly reduced the incidence of URTIs, diagnosed by a general practitioner according to prespecified criteria (primary endpoint). Moreover, none of the secondary efficacy endpoints were met. Applying a Ward's hierarchical clustering, two separate clusters, focussing on four quality of life-related endpoints, were identified. Cluster 1 was associated with more severe URTI characteristics than cluster 2. Cluster 2 was significantly enriched with children who consumed the product, indicating that the symptoms children experience during an URTI are alleviated by the intake of L. rhamnosus GG DSM 33156. The study is registered at ClinicalTrials.gov ID: NCT03636191.

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Functional significance of PMM2 mutations in mildly affected patients with congenital disorders of glycosylation Ia.

PURPOSE: Congenital disorders of glycosylation (CDG) result from mutations in N-glycan biosynthesis. Mutations in phosphomannomutase (PMM2) cause CDG-Ia. Here, we report four clinically mild patients and their mutations in PMM2. METHODS: Analysis of the PMM2 cDNA and gene revealed the mutations affecting the glycosylation efficiency. RESULTS: The patients have 30% to 50% normal PMM activity in fibroblasts due to different mutations in PMM2, and we studied the effect of each mutation on the PMM activity in a Saccharomyces cerevisiae expression system. CONCLUSIONS: Each patient carried a severe mutation that decreased the PMM activity to less than 10% as well as a relatively mild mutation. A new mutation, deletion of base 24, changed the reading frame. The C9Y, C241S, and L32R mutations showed 27% to 45% activity when expressed in the eukaryotic expression system, and the more severe D148N was shown to be thermolabile.

Balancing N-linked glycosylation to avoid disease.

Complete loss of N-glycosylation is lethal in both yeast and mammals. Substantial deficiencies in some rate-limiting biosynthetic steps cause human congenital disorders of glycosylation (CDG). Patients have a range of clinical problems including variable degrees of mental retardation, liver dysfunction, and intestinal disorders. Over 60 mutations in phosphomannomutase (encoded by PMM2) diminish activity and cause CDG-Ia. The severe mutation R141H in PMM2 is lethal when homozygous, but heterozygous in about 1/70 Northern Europeans. Another disorder, CDG-Ic, is caused by mutations in ALG6, an alpha 1,3glucosyl transferase used for lipid-linked precursor synthesis, yet some function-compromising mutations occur at a high frequency in this gene also. Maintenance of seemingly deleterious mutations implies a selective advantage or positive heterosis. One possible explanation for this is that production of infective viruses such as hepatitis virus B and C, or others that rely heavily on host N-glycosylation, is substantially inhibited when only a tiny fraction of their coat proteins is misglycosylated. In contrast, this reduced glycosylation does not affect the host. Prevalent functional mutations in rate-limiting glycosylation steps could provide some resistance to viral infections, but the cost of this insurance is CDG. A balanced glycosylation level attempts to accommodate these competing agendas. By assessing the occurrence of a series of N-glycosylation-compromising alleles in multi-genic diseases, it may be possible to determine whether impaired glycosylation is a risk factor or a major determinant underlying their pathology.

Real-time optical coherence tomography of the anterior segment at 1310 nm.

BACKGROUND: Recent advances in high-speed scanning technology have enabled a new generation of optical coherence tomographic (OCT) systems to perform imaging at video rate. Here, a handheld OCT probe capable of imaging the anterior segment of the eye at high frame rates is demonstrated for the first time. OBJECTIVE: To demonstrate real-time OCT imaging of anterior segment structures. DESIGN: Survey of anterior segment structures in normal human subjects. SETTING: Laboratory. MAIN OUTCOME MEASURES: Achieving real-time imaging of the anterior segment, satisfactory image quality, and convenience of a handheld probe. RESULTS: Optical coherence tomographic imaging of the anterior segment of the eyes of human subjects was performed using 1310-nm wavelength light with an image rate of 8 frames per second. Imaging trials demonstrated clear resolution of corneal epithelium and stroma, sclerocorneal junction, sclera, iris pigment epithelium and stroma, and anterior lens capsule. The anterior chamber angle was clearly visualized. Limited imaging of the ciliary body was performed. Real-time imaging of pupillary constriction in response to light stimulus was also performed. CONCLUSION: High-speed OCT at 1310-nm wavelength is a potentially useful technique for noninvasive assessment of anterior segment structures. CLINICAL RELEVANCE: Our results suggest that real-time OCT has potential applications in glaucoma evaluation and refractive surgery.

High-resolution endoscopic imaging of the GI tract: a comparative study of optical coherence tomography versus high-frequency catheter probe EUS.

BACKGROUND: Both optical coherence tomography (OCT) and catheter probe EUS (CPEUS) are candidates for high-resolution imaging of the GI wall, but their potential roles in this clinical context have not been investigated. METHODS: OCT and CPEUS were used to image normal-appearing portions of the GI tract at the same sites. CPEUS was performed with a 20-MHz or a new 30-MHz catheter probe. RESULTS: Forty-four histologically confirmed normal sites in 27 patients were evaluated. With OCT, mucosa and muscularis mucosa were clearly seen at all sites. Except for stomach, OCT demonstrated the submucosa in all sites. OCT penetration ranged from 0.7 to 0.9 mm. Microscopic structures such as esophageal glands, intestinal villi, colonic crypts, and blood vessels were easily identified. CPEUS penetration ranged from 10 mm to 20 mm, and 5 to 7 distinct layers were discernible. However, both mucosa and submucosa were seen as thin layers without microscopic detail. CONCLUSION: OCT resolution is superior to high-frequency CPEUS, but depth of penetration is limited to mucosa and submucosa. OCT images the major structural components of the mucosa and submucosa whereas CPEUS does not. Potentially, OCT and high-frequency CPEUS may be complementary for clinical imaging.

Photothermal coagulation of blood vessels: a comparison of high-speed optical coherence tomography and numerical modelling.

Optical-thermal models that can accurately predict temperature rise and damage in blood vessels and surrounding tissue may be used to improve the treatment of vascular disorders. Verification of these models has been hampered by the lack of time- and depth-resolved experimental data. In this preliminary study, an optical coherence tomography system operating at 4-30 frames per second was used to visualize laser irradiation of cutaneous (hamster dorsal skin flap) blood vessels. An argon laser was utilized with the following parameters: pulse duration 0.1-2.0 s, spot size 0.1-1.0 mm, power 100-400 mW. Video microscopy images were obtained before and after irradiations, and optical-thermal modelling was performed on two irradiation cases. Time-resolved optical coherence tomography and still images were compared with predictions of temperature rise and damage using Monte Carlo and finite difference techniques. In general, predicted damage agreed with the actual blood vessel and surrounding tissue coagulation seen in images. However, limitations of current optical-thermal models were identified, such as the inability to model the dynamic changes in blood vessel diameter that were seen in the optical coherence tomography images.

Functional analysis of novel mutations in a congenital disorder of glycosylation Ia patient with mixed Asian ancestry.

Congenital disorders of glycosylation (CDG) are caused by autosomal recessive mutations in genes affecting N-glycan biosynthesis. Mutations in the PMM2 gene, which encodes the enzyme phosphomannomutase (mannose 6-phosphate <--> mannose 1-phosphate), give rise to the most common form: CDG-Ia. These patients typically present with dysmorphic features and neurological abnormalities, cerebellar hypoplasia, ataxia, hypotonia, and coagulopathy, in addition to feeding problems. However, the clinical symptoms vary greatly. The great majority of known CDG-Ia patients are of European descent where the most common mutant alleles originated. This ethnic bias can also be explained by lack of global awareness of the disorder. Here we report an Asian patient with prominent systemic features that we diagnosed with CDG-Ia resulting from two new mutations in the PMM2 gene (310C --> G resulting in L104V and an intronic mutation IVS1-1G --> A). The latter mutation seems to result in lower mRNA levels, and the L104V has been functionally analyzed in a yeast expression system together with known mutations. The Filipino and Cambodian origins of the parents show that CDG-Ia mutations occur in these ethnic groups as well as in Caucasians.

Genetic and metabolic analysis of the first adult with congenital disorder of glycosylation type Ib: long-term outcome and effects of mannose supplementation.

We report the diagnosis and follow-up of two sibs reported in 1980 with recurrent venous thromboses and protein-losing enteropathy; one sib with biopsy-proven hepatic fibrosis died at age 5. The combination of symptoms was suggestive of the recently characterized congenital disorder of glycosylation type Ib (CDG-Ib), which is caused by a deficiency of the enzyme phosphomannose isomerase (PMI). An abnormal serum transferrin isoelectric focusing (IEF) pattern and a reduced PMI activity confirmed the diagnosis of CDG-Ib. Furthermore, mutational analysis of the MPI gene revealed two missense mutations, 419 T --> C (I140T) and 636 G --> A (R219Q), a single base substitution in intron 5, 670 + 9G --> A, as well as a polymorphism 1131A --> C (V377V) in both sibs. The surviving 33-year-old sib has had no further symptoms following childhood. Short-term low-dose oral mannose supplementation improved her transferrin IEF pattern and normalized her antithrombin III activity, further substantiating the beneficial effect of mannose in CDG-Ib. When her mannose blood level was measured, she showed a lower steady-state level but a faster mannose clearance rate. These results suggest that the clinical manifestations of PMI deficiency, although serious in childhood, can improve with age, even without mannose therapy, and allow for a normal adult life. However, the long-term prognosis may vary from patient to patient.

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.