Metastatic Infiltration of Nervous Tissue and Periosteal Nerve Sprouting in Multiple Myeloma-Induced Bone Pain in Mice and Human.

Multiple myeloma (MM) is a neoplasia of B plasma cells that often induces bone pain. However, the mechanisms underlying myeloma-induced bone pain (MIBP) are mostly unknown. Using a syngeneic MM mouse model, we show that periosteal nerve sprouting of calcitonin gene-related peptide (CGRP+) and growth associated protein 43 (GAP43+) fibers occurs concurrent to the onset of nociception and its blockade provides transient pain relief. MM patient samples also showed increased periosteal innervation. Mechanistically, we investigated MM induced gene expression changes in the dorsal root ganglia (DRG) innervating the MM-bearing bone of male mice and found alterations in pathways associated with cell cycle, immune response and neuronal signaling. The MM transcriptional signature was consistent with metastatic MM infiltration to the DRG, a never-before described feature of the disease that we further demonstrated histologically. In the DRG, MM cells caused loss of vascularization and neuronal injury, which may contribute to late-stage MIBP. Interestingly, the transcriptional signature of a MM patient was consistent with MM cell infiltration to the DRG. Overall, our results suggest that MM induces a plethora of peripheral nervous system alterations that may contribute to the failure of current analgesics and suggest neuroprotective drugs as appropriate strategies to treat early onset MIBP.SIGNIFICANCE STATEMENT Multiple myeloma (MM) is a painful bone marrow cancer that significantly impairs the quality of life of the patients. Analgesic therapies for myeloma-induced bone pain (MIBP) are limited and often ineffective, and the mechanisms of MIBP remain unknown. In this manuscript, we describe cancer-induced periosteal nerve sprouting in a mouse model of MIBP, where we also encounter metastasis to the dorsal root ganglia (DRG), a never-before described feature of the disease. Concomitant to myeloma infiltration, the lumbar DRGs presented blood vessel damage and transcriptional alterations, which may mediate MIBP. Explorative studies on human tissue support our preclinical findings. Understanding the mechanisms of MIBP is crucial to develop targeted analgesic with better efficacy and fewer side effects for this patient population.

Brain perfusion estimation by Tikhonov model-free deconvolution in a long axial field of view PET/CT scanner exploring five different PET tracers.

PURPOSE: New total-body PET scanners with a long axial field of view (LAFOV) allow for higher temporal resolution due to higher sensitivity, which facilitates perfusion estimation by model-free deconvolution. Fundamental tracer kinetic theory predicts that perfusion can be estimated for all tracers despite their different fates given sufficiently high temporal resolution of 1 s or better, bypassing the need for compartment modelling. The aim of this study was to investigate whether brain perfusion could be estimated using model-free Tikhonov generalized deconvolution for five different PET tracers, [15O]H2O, [11C]PIB, [18F]FE-PE2I, [18F]FDG and [18F]FET. To our knowledge, this is the first example of a general model-free approach to estimate cerebral blood flow (CBF) from PET data. METHODS: Twenty-five patients underwent dynamic LAFOV PET scanning (Siemens, Quadra). PET images were reconstructed with an isotropic voxel resolution of 1.65 mm3. Time framing was 40 × 1 s during bolus passage followed by increasing framing up to 60 min. AIF was obtained from the descending aorta. Both voxel- and region-based calculations of perfusion in the thalamus were performed using the Tikhonov method. The residue impulse response function was used to estimate the extraction fraction of tracer leakage across the blood-brain barrier. RESULTS: CBF ranged from 37 to 69 mL blood min-1 100 mL of tissue-1 in the thalamus. Voxelwise calculation of CBF resulted in CBF maps in the physiologically normal range. The extraction fractions of [15O]H2O, [18F]FE-PE2I, [11C]PIB, [18F]FDG and [18F]FET in the thalamus were 0.95, 0.78, 0.62, 0.19 and 0.03, respectively. CONCLUSION: The high temporal resolution and sensitivity associated with LAFOV PET scanners allow for noninvasive perfusion estimation of multiple tracers. The method provides an estimation of the residue impulse response function, from which the fate of the tracer can be studied, including the extraction fraction, influx constant, volume of distribution and transit time distribution, providing detailed physiological insight into normal and pathologic tissue.

Assessment of cerebral drug occupancy in humans using a single PET-scan: A [11C]UCB-J PET study.

PURPOSE: Here, we evaluate a PET displacement model with a Single-step and Numerical solution in healthy individuals using the synaptic vesicle glycoprotein (SV2A) PET-tracer [11C]UCB-J and the anti-seizure medication levetiracetam (LEV). We aimed to (1) validate the displacement model by comparing the brain LEV-SV2A occupancy from a single PET scan with the occupancy derived from two PET scans and the Lassen plot and (2) determine the plasma LEV concentration-SV2A occupancy curve in healthy individuals. METHODS: Eleven healthy individuals (five females, mean age 35.5 [range: 25-47] years) underwent two 120-min [11C]UCB-J PET scans where an LEV dose (5-30 mg/kg) was administered intravenously halfway through the first PET scan to partially displace radioligand binding to SV2A. Five individuals were scanned twice on the same day; the remaining six were scanned once on two separate days, receiving two identical LEV doses. Arterial blood samples were acquired to determine the arterial input function and plasma LEV concentrations. Using the displacement model, the SV2A-LEV target engagement was calculated and compared with the Lassen plot method. The resulting data were fitted with a single-site binding model. RESULTS: SV2A occupancies and VND estimates derived from the displacement model were not significantly different from the Lassen plot (p = 0.55 and 0.13, respectively). The coefficient of variation was 14.6% vs. 17.3% for the Numerical and the Single-step solution in Bland-Altman comparisons with the Lassen plot. The average half maximal inhibitory concentration (IC50), as estimated from the area under the curve of the plasma LEV concentration, was 12.5 µg/mL (95% CI: 5-25) for the Single-Step solution, 11.8 µg/mL (95% CI: 4-25) for the Numerical solution, and 6.3 µg/mL (95% CI: 0.08-21) for the Lassen plot. Constraining Emax to 100% did not significantly improve model fits. CONCLUSION: Plasma LEV concentration vs. SV2A occupancy can be determined in humans using a single PET scan displacement model. The average concentration of the three computed IC50 values ranges between 6.3 and 12.5 µg/mL. The next step is to use the displacement model to evaluate LEV occupancy and corresponding plasma concentrations in relation to treatment efficacy. CLINICAL TRIAL REGISTRATION: NCT05450822. Retrospectively registered 5 July 2022 https://clinicaltrials.gov/ct2/results? term=NCT05450822&Search=Search.

Bracing in severe skeletally immature adolescent idiopathic scoliosis: does a holding strategy change the surgical plan?

PURPOSE: The purpose of the study was to assess the changes in flexibility during night-time bracing in skeletally immature adolescent idiopathic scoliosis (AIS) with curves in the surgical range. MATERIALS AND METHODS: We included a consecutive cohort of 89 AIS patients with curves ≥ 45° and an estimated growth potential. All patients were eventually treated with fusion surgery, and all patients had side-bending radiographs prior to both bracing and surgery. Curves were classified as structural or non-structural curves according to Lenke at both timepoints. RESULTS: The main curve progressed by a mean of 12 ± 10° and the secondary curve by 8 ± 8°. Flexibility of the main curve decreased from 50 ± 19% to 44 ± 19% (p = 0.001) and the underlying curve from 85 ± 21% to 77 ± 22% (p = 0.005). In 69 patients (79%), the Lenke category did not progress during bracing. In 14 patients (15%), the progression in Lenke type occurred in the thoracic region (i.e., Lenke type 1 to type 2), while six patients (7%) progressed in the lumbar region (i.e., type 1 to type 3). In the 69 patients that did not progress, we found that the last touched vertebra moved distally by one or two levels in 26 patients. CONCLUSIONS: This is the first study to describe that curve flexibility decreases during bracing in severe AIS. However, this had only a modest impact on the surgical strategy. Bracing as a holding strategy can be applied, but the risk of losing flexibility in the lumbar spine should be outweighed against the risks of premature fusion surgery.

The effect of Providence night-time bracing on the sagittal profile in adolescent idiopathic scoliosis.

PURPOSE: Adolescent idiopathic scoliosis (AIS) is characterized by coronal scoliosis and often a sagittal hypokyphosis. The effect of bracing on the sagittal profile is not well understood. The aim of this study is to assess the effect of night-time bracing on the sagittal profile in patients with AIS. METHODS: We retrospectively included AIS patients with a main curve of 25-45° treated with a night-time brace in our institution between 2005 and 2018. Patients with estimated growth potential based on either Risser stage, hand X-rays, or menarchal status were included. Coronal and sagittal radiographic parameters were recorded at both brace- initiation and -termination. Patients were followed until surgery or one year after brace termination. Results were compared to a published cohort of full-time braced patients. RESULTS: One hundred forty-six patients were included. Maximum thoracic kyphosis (TK) increased 2.5° (± 9.7) (p = 0.003), corresponding to a 3.5-fold relative risk increase post bracing in TK compared to a full-time brace cohort. Twenty-seven percent (n = 36) of the patients were hypokyphotic (T4/T12 < 20°) at brace initiation compared with 19% (n = 26) at brace termination (p = 0.134). All other sagittal parameters remained the same at follow-up. We found no association between progression in the coronal plane and change in sagittal parameters. CONCLUSION: This is the first study to indicate that night-time bracing of AIS does not induce hypokyphosis. We found a small increase in TK, with a substantially lower risk of developing flat back deformity compared to full-time bracing. The coronal curve progression was not coupled to a change in TK.

The influence of night-time bracing on curve progression is not affected by curve magnitude in adolescent idiopathic scoliosis: a study of 299 patients.

BACKGROUND AND PURPOSE: The efficacy of bracing larger curves in adolescent idiopathic scoliosis (AIS) patients is uncertain. We aimed to assess the influence of night-time bracing in AIS patients with main curves exceeding 40° Cobb angle at brace initiation. METHODS: We reviewed AIS patients treated with nighttime braces between 2005 and 2018. Patients with curves ≥ 25° and estimated growth potential were included. Patients were monitored with radiographs from brace initiation until brace weaning at skeletal maturity. Patients were grouped based on curve magnitude at initial evaluation: a control group (25-39°) and a large-curves group (≥ 40°). Progression was defined as > 5° increase. RESULTS: We included 299 patients (control group, n = 125; large-curves group, n = 174). In the control group, 65 (52%) patients progressed compared with 101 (58%) in the large-curves group (P = 0.3). The lower-end vertebra (LEV) shifted distally post-bracing in 41 (23%) patients in the largecurves group. Patients with progressive large curves were younger (age 13.2 [SD 1.5] vs. 13.9 [SD 1.1], P = 0.009) and more premenarchal (n = 36 [42%] vs. n = 6 [9%], P < 0.001) compared with non-progressive large curves. CONCLUSION: Progression risk in patients with curves exceeding 40° treated with night-time bracing is similar to smaller curves. The LEV moved distally in almost one-fourth of the larger curves, possibly affecting fusion levels in cases of surgery.

Impaired Bone Fracture Healing in Type 2 Diabetes Is Caused by Defective Functions of Skeletal Progenitor Cells.

The mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high-fat diet and insulinopenia induced by treatment with streptozotocin, we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by -28.4 ± 7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06 ± 38.71%, and increased density of SCA1+ (+74.99 ± 29.19%) but not Runx2+ osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82 ± 33.05%), senescence gene signature (≈106.66 ± 34.03%), and LAMIN B1- senescent cell density (+225.18 ± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9 ± 6.2% which was inversely correlated with glucose levels (R2 = 0.48, P < .004) and callus adipose tissue area (R2 = .3711, P < .01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast-related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycemia per se and not just insulin levels is detrimental for bone healing.

Microfibrillar-associated protein 4 as a potential marker of acute relapse in inflammatory demyelinating diseases of the central nervous system: Pathological and clinical aspects.

BACKGROUND: Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein not previously described in the human central nervous system (CNS). OBJECTIVES: We determined MFAP4 CNS expression and measured cerebrospinal fluid (CSF) and serum levels. METHODS: Tissue was sampled at autopsy from patients with acute multiple sclerosis (MS) (n = 3), progressive MS (n = 3), neuromyelitis optica spectrum disorder (NMOSD) (n = 2), and controls (n = 9), including 6 healthy controls (HC). MFAP4 levels were measured in 152 patients: 49 MS, 62 NMOSD, 22 myelin oligodendrocyte glycoprotein-associated disease (MOGAD), and 19 isolated optic neuritis (ION). RESULTS: MFAP4 localized to meninges and vascular/perivascular spaces, intense in the optic nerve. At sites of active inflammation, MFAP4 reactivity was reduced in NMOSD and acute MS and less in progressive MS. CSF MFAP4 levels were reduced during relapse and at the onset of diseases (mean U/mL: MS 14.3, MOGAD 9.7, and ION 14.6 relative to HC 17.9. (p = 0.013, p = 0.000, and p = 0.019, respectively). Patients with acute ON (n = 68) had reduced CSF MFAP4 (mean U/mL: 14.5, p = 0.006). CSF MFAP4 levels correlated negatively with relapse severity (rho = -0.41, p = 0.017). CONCLUSION: MFAP4 immunoreactivity was reduced at sites of active inflammation. CSF levels of MFAP4 were reduced following relapse and may reflect disease activity.

Liraglutide treatment is associated with progression of coronary artery fibrous plaque: a prospective 1-year follow-up study in asymptomatic patients with type 2 diabetes.

OBJECTIVE: The objective of this study was to assess the association between clinically indicated liraglutide treatment and coronary artery plaque progression during 1-year follow-up in asymptomatic diabetes. METHODS: Patients were divided into a group receiving liraglutide (Lira+) and a group not receiving liraglutide (Lira-). Coronary computed tomography angiography (CCTA) was performed to assess total atheroma volume (TAV) and subtypes of plaque volumes (dense calcium, fibrous, fibrous-fatty, and necrotic core plaque) and the plaque progression during one year follow-up. RESULTS: Fifty-five patients (27%) receiving liraglutide and 149 (73%) how did not were included. Changes in TAV during 1-year of follow-up were similar in the two groups (38 ± 180 (Lira+) vs. -1 ± 160 mm3 (Lira-), P = 0.13). A greater increase in fibrous plaque volume was seen in the Lira + vs. the Lira- group (34 ± 129 vs. -2 ± 101 mm3, P = 0.04). Changes over 1-year in the other plaque subtypes were similar in the two groups. Treatment duration of liraglutide was not associated with changes in TAV. CONCLUSION: In patients with T2D without known prior coronary artery disease, liraglutide treatment was associated with a significant increase in coronary artery fibrous plaque volume during 1-year follow-up.

Does Risser stage accurately predict the risk of curve progression in patients with adolescent idiopathic scoliosis treated with night-time bracing?

PURPOSE: Risser stage is widely used as a marker for skeletal maturity (SM) and thereby an indirect measure for the risk of progression of adolescent idiopathic scoliosis (AIS). The Scoliosis Research Society recommends bracing for Risser stages 0-2 as Risser stage 3 or above is considered low risk. Very few studies have assessed the risk of progression during bracing in Risser stages 3-4. The objective of the current study is to determine if Risser stages 3-4 provide a meaningful cutoff in terms of progression risk in patients with AIS treated with night-time bracing. METHODS: AIS patients treated with night-time brace from 2005 to 2018 with a Cobb angle between 25 and 40 degrees and Risser stages 0-4 were retrospectively included. Curve progression (> 5 degrees increase) was monitored until surgery or SM. Skeletal maturity was defined as either 2 years postmenarchal, no height development or closed ulnar epiphyseal plates on radiographs. RESULTS: One hundred and thirty-five patients were included (Risser stages 0-2: n = 86 and 3-4: n = 49). Overall, radiographic curve progression occurred in 52% while progression beyond 45 degrees was seen in 35%. The progression rate in the Risser 0-2 group was 60% and 37% in the Risser 3-4 group (p = 0.012). In multivariate logistic regression analysis, adjusted for Risser stages and age, only premenarchal status showed a statistically significant association with progression (OR: 2.68, 95%CI 1.08-6.67). CONCLUSION: Risser stage does not provide a clinically meaningful differentiation of progression risk in AIS patients treated with a night-time brace. Risk assessment should include other more reliable measures of skeletal growth potential.

First cases of SARS-CoV-2 BA.2.86 in Denmark, 2023.

We describe 10 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant BA.2.86 detected in Denmark, including molecular characteristics and results from wastewater surveillance that indicate that the variant is circulating in the country at a low level. This new variant with many spike gene mutations was classified as a variant under monitoring by the World Health Organization on 17 August 2023. Further global monitoring of COVID-19, BA.2.86 and other SARS-CoV-2 variants is highly warranted.

The Collateral Effect of Enhanced Recovery After Surgery Protocols on Spine Patients With Neuromuscular Scoliosis.

INTRODUCTION: Enhanced recovery after surgery (ERAS) protocols are often specific to a specific type of surgery without assessing the overall effect on the ward. Previous studies have demonstrated reduced length of stay (LOS) with ERAS protocols in patients with adolescent idiopathic scoliosis (AIS), although the patients are often healthy and with few or no comorbidities. In 2018, we used ERAS principles for patients undergoing AIS surgery with a subsequent 40% reduced LOS. The current study aims to assess the potential collateral effect of LOS in patients surgically treated for neuromuscular scoliosis admitted to the same ward and treated by the same staff but without a standardized ERAS protocol. METHODS: All patients undergoing neuromuscular surgery 2 years before and after ERAS introduction (AIS patients) with a gross motor function classification score of 4 to 5 were included. LOS, intensive care stay, and postoperative complications were recorded. After discharge, all complications leading to readmission and mortality were noted with a minimum of 2 years of follow-up using a nationwide registry. RESULTS: Forty-six patients were included; 20 pre-ERAS and 26 post-ERAS. Cross groups, there were no differences in diagnosis, preoperative curve size, pulmonary or cardiac comorbidities, weight, sex, or age. Postoperative care in the intensive care unit was unchanged between the two groups (1.2 vs 1.1; P = 0.298). When comparing LOS, we found a 41% reduction in the post-ERAS group (11 vs 6.5; P < 0.001) whereas the 90-day readmission rates were without any significant difference (45% vs 34% P = 0.22) We found no difference in the 2-year mortality in either group. CONCLUSION: The employment of ERAS principles in a relatively uncomplicated patient group had a positive, collateral effect on more complex patients treated in the same ward. We believe that training involving the caregiving staff is equally important as pharmacological protocols.

Emerging Digital Practices Supporting Student-Centered Learning Environments in Higher Education: A Review of Literature and Lessons Learned from the Covid-19 Pandemic.

The aim of this paper is two-fold: firstly, to provide an overview of emerging digital practices that support collaborative learning, competency development, and digital literacy for student-centered learning environments in higher education during the rapid digital transition caused by pandemic-related lockdowns across the world, and secondly, to analyze and discuss how systematic reviews of generalized themes and trends can be combined with contextualized experiences and the lessons learned from the Covid-19 crisis to inform the digital transformation of higher education, with a particular focus on bridging the gap between campus-based teaching and online learning and on the identification of the digital competencies that teachers and students must acquire during the continuing shift into a 'new normal' for post-pandemic educational practices. This study was motivated by questions and findings emerging from an early reactive case study conducted by three of this paper's co-authors (Lyngdorf et al., 2021a). By reviewing the full texts of 18 articles, this study provides a systematic literature review which maps the general landscape of the online, hybrid, and blended digital practices applied in existing student-centered learning environments in higher education since the onset of the pandemic. Furthermore, this mapping is used to revisit data and findings from the earlier reactive study of emerging digital practices in a specific problem- and project-based learning (PBL) environment. This study's findings highlight critical factors and barriers related to emerging practices which support students' interactions with teachers, content, and each other, as well as the emerging competencies that these practices will require. The paper concludes with a discussion of the main findings and their implications for further research and practice.

Serum Neutralization of SARS-CoV-2 Omicron BA.1 and BA.2 after BNT162b2 Booster Vaccination.

The SARS-CoV-2 Omicron variant BA.2 sublineage is rapidly replacing earlier Omicron lineages, suggesting BA.2 has increased vaccine evasion properties. We measured neutralization titers of authentic BA.1 and BA.2 isolates in serum samples from persons who received the BNT162b2 booster vaccine. All samples neutralized BA.1 and BA.2 at equal median values.

Omicron BA.5 Neutralization among Vaccine-Boosted Persons with Prior Omicron BA.1/BA.2 Infections.

Worldwide, millions of persons have received multiple COVID-19 vaccinations and subsequently recovered from SARS-CoV-2 Omicron breakthrough infections. In 2 small, matched cohorts (n = 12, n = 24) in Denmark, we found Omicron BA.1/BA.2 breakthrough infection after 3-dose BNT162b2 vaccination provided improved Omicron BA.5 neutralization over 3-dose vaccination alone.

Intracellular uropathogenic Escherichia coli are undetectable in urinary bladders after oral mecillinam treatment: An experimental study in a pig model of cystitis.

OBJECTIVES: Experiments in murine models of urinary tract infection (UTI) show that uropathogenic Escherichia coli (UPEC) form bacterial reservoirs in the bladder tissue that can survive beta lactam antibiotics and give rise to reinfection. The observed reinfection cascade suggests intracellular bacterial persistence as a possible explanation for recurrent UTI in humans. To test this hypothesis in an animal model closer to humans, we here investigated whether UPEC infecting the bladders of experimentally inoculated pigs are able to survive standard oral mecillinam treatment. Moreover, we analyzed the infected pig bladders by microscopy for the presence of intracellular UPEC colonies. METHODS: Seven pigs were experimentally inoculated with the UPEC cystitis strain, UTI89, to induce cystitis. After 5 days of infections, a 3-day oral treatment with the extracellularly active ß-lactam, mecillinam, was initiated. The infection was monitored with regular urine and blood samples. When terminated, whole bladders were removed and homogenized to quantify viable intracellular bacteria. In addition, two pigs were inoculated with UTI89pMAN01 constitutively expressing green fluorescent protein and the bladders subsequently analyzed by microscopy for bacterial location and morphology. RESULTS: Experimental inoculation resulted in cystitis in all animals. After 3-day treatment with mecillinam, no viable UPEC were detectable in urine or bladder homogenates. Microscopy analysis of pig bladders at 12 h post infection, revealed no detectable intracellular bacterial colonies and no filamentous UPEC phenotypes were identified. CONCLUSIONS: Pigs experimentally infected with UPEC completely clear their infection upon mecillinam treatment, which contrasts earlier findings from similar experiments in mice. Moreover, the hallmarks of induced UTI in mice, i.e. intracellular bacterial communities and bacterial filamentation, could not be identically reproduced in a pig model of acute UTI. This result suggests that significant differences might exist between UTI in mice and larger mammals, and therefore perhaps also between mice and humans. Additional studies are needed to reveal details on the Escherichia coli acute UTI pathogenesis cascade in larger mammals to assess to which extent observations in mice can be transferred to humans.

Myeloma-bone marrow adipocyte axis in tumour survival and treatment response.

Multiple myeloma is an incurable cancer of the bone marrow that is dependent on its microenvironment, including bone marrow adipocytes (BMAds). Here, we discuss our findings that the reciprocal interaction of myeloma cells and BMAds, leads to myeloma cell survival and induces metabolic dysfunction and senescence-associated secretory phenotype in BMAds.

Catabolic activity of osteoblast lineage cells contributes to osteoclastic bone resorption in vitro.

Osteoblast lineage cells in human bone were recently shown to colonize eroded bone surfaces and to closely interact with osteoclasts. They proved to be identical to reversal cells and are believed to differentiate into bone-forming osteoblasts thereby coupling resorption and formation. However, they also exert catabolic activity that contributes to osteoclastic bone resorption, but this has not received much attention. Herein, we used co-cultures of primary human osteoblast lineage cells and human osteoclasts derived from peripheral blood monocytes to investigate whether a catabolic activity of osteoblast lineage cells could impact on osteoclastic bone resorption. Through a combination of immunofluorescence, in situ hybridization and time-lapse experiments, we show that MMP-13-expressing osteoblast lineage cells are attracted to and closely interact with bone-resorbing osteoclasts. This close interaction results in a strong and significant increase in the bone resorptive activity of osteoclasts - especially those making trenches. Importantly, we show that osteoclastic bone resorption becomes sensitive to inhibition of matrix metalloproteinases in the presence, but not in the absence, of osteoblast lineage cells. We propose that this may be due to the direct action of osteoblast-lineage-derived MMP-13 on bone resorption.

Escherichia coli type-1 fimbriae are critical to overcome initial bottlenecks of infection upon low-dose inoculation in a porcine model of cystitis.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models' natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.

Transcriptional Dynamics of Hepatic Sinusoid-Associated Cells After Liver Injury.

BACKGROUND AND AIMS: Hepatic sinusoidal cells are known actors in the fibrogenic response to injury. Activated hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, and Kupffer cells are responsible for sinusoidal capillarization and perisinusoidal matrix deposition, impairing vascular exchange and heightening the risk of advanced fibrosis. While the overall pathogenesis is well understood, functional relations between cellular transitions during fibrogenesis are only beginning to be resolved. At single-cell resolution, we here explored the heterogeneity of individual cell types and dissected their transitions and crosstalk during fibrogenesis. APPROACH AND RESULTS: We applied single-cell transcriptomics to map the heterogeneity of sinusoid-associated cells in healthy and injured livers and reconstructed the single-lineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted gene correlation network analysis of the HSC trajectory led to the identification of core genes whose expression proved highly predictive of advanced fibrosis in patients with nonalcoholic steatohepatitis (NASH). Among the core members of the injury-repressed gene module, we identified plasmalemma vesicle-associated protein (PLVAP) as a protein amply expressed by mouse and human HSCs. PLVAP expression was suppressed in activated HSCs upon injury and may hence define hitherto unknown roles for HSCs in the regulation of microcirculatory exchange and its breakdown in chronic liver disease. CONCLUSIONS: Our study offers a single-cell resolved account of drug-induced injury of the mammalian liver and identifies key genes that may serve important roles in sinusoidal integrity and as markers of advanced fibrosis in human NASH.