Diagnostic utility of combining PRAME and HMB-45 stains in primary melanocytic tumors.

BACKGROUND: Pathologists face ongoing challenges distinguishing between benign and malignant melanocytic tumors. PRAME (PReferentially expressed Antigen in Melanoma) has a demonstrated value distinguishing between these types of lesions. However, the sensitivity of single immunohistochemistry is variable. HMB-45 is another valuable marker, but on its own, has a limited ability in setting of primary melanocytic tumors. This study sought to evaluate the diagnostic potential of a dual panel combining PRAME and HMB-45 in the assessment of primary melanocytic tumors. METHODS: 259 tumors, of which 141 were benign nevi, 31 dysplastic nevi (either low- or high grade dysplasia), and further 87 malignant melanomas, were retrieved from the department's archives and assessed by two experienced dermatopathologists. New sections were stained with PRAME and HMB-45, respectively. For PRAME, a nuclear, and for HMB-45, a cytoplasmic staining, was considered positive and scored as described in the literature on a scale from 0 to 4+. Only dermal component was assessed on HMB-45 stain. RESULTS: PRAME was diffusely expressed in only 1 benign nevus, with focal expression in further 28 compared to 22 diffusely and 103 focally HMB-45-positive benign nevi. 5 high-grade dysplastic nevi showed diffuse PRAME expression in epidermal component, with varying degree of positivity in adjacent dermal compartment, and further 8 dysplastic nevi showed only focal expression. HMB-45 was diffusely expressed in only 2, with focal expression in 23, and no apparent positivity in remaining 6 dysplastic nevi. In invasive melanoma group, PRAME stained >75 % cells in 64/87 tumors, however, 10/87 melanomas were completely negative. HMB-45 was captured diffusely in 49/87 melanomas, 32 showed patchy expression, and 6 tumors were blank negative. Diffuse 4+ PRAME positivity showed superior sensitivity and specificity of 73,6 % and 96,5 %, respectively, compared to HMB-45, 56,3 % and 86,0 %, respectively. No nevi showed double 4+ positivity, however, the sensitivity for double positivity was only 49,4 %. CONCLUSION: Our results confirm the superiority of PRAME over HMB-45 in the differential diagnosis of melanocytic tumors. However, combined staining can significantly increase specificity, rendering a benign diagnosis more unlikely in a double 4+ diffuse positivity setting.

Detection of DZIP1L mutations by whole-exome sequencing in consanguineous families with polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported. METHODS: Four children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing. RESULTS: We identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L. CONCLUSIONS: In line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases. A higher resolution version of the Graphical abstract is available as Supplementary information.

On the Road to Digital Pathology in Denmark-National Survey and Interviews.

Digital pathology (DP) is changing pathology departments dramatically worldwide, yet globally, few departments are presently digitalized for the full diagnostic workflow. Denmark is also on the road to full digitalization countrywide, and this study aim to cover experiences during the implementation process in a national context. Thus, quantitative questionnaires were distributed to all pathology departments in Denmark (n = 13) and distributed to all professions including medical clinical directors, medical doctors (MD) and biomedical laboratory scientists (BLS). For a qualitative perspective, we interviewed four employees representing four professions. Data were collected in 2019-2020. From the questionnaire and interviews, we found strategies differed at the Danish departments with regards to ambitions, technological equipment, workflows, and involvement of type of professions. DP education was requested by personnel. Informants were in general positive toward the digital future but mainly had concerns regarding the political pressure to integrate DP before technological advances are sufficient for maintaining rational budgets, workflows, and for sustaining diagnostic quality. This study is a glance on the Danish implementation process in its early stages from personnel's point of view. It shows the complexity when large new workflow processes are to be implemented countrywide and with a large diversity of stakeholders like managers, MD, BLS, IT-professionals, and authorities. To ensure best technological and economical solutions and to maintain-or even optimize-diagnostic quality with DP and workflow alignment, we suggest superior inter- and intradepartmental communication. When implementing DP countrywide, a national working group is warranted with the variety of stakeholders represented.

Differential localization patterns of claudin 10, 16, and 19 in human, mouse, and rat renal tubular epithelia.

Functional properties of the paracellular pathway depend critically on the set of claudins (CLDN) expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX) syndrome caused by genetic variations in the CLDN10 gene and familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by genetic variations in the CLDN16 or CLDN19 genes. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, CLDN16, and CLDN19 in both paraffin-embedded and frozen kidney sections from the adult human, mouse, and rat using immunohistochemistry and immunofluorescence, respectively. Here, CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junctions in human and rodent kidneys. Weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in the cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidneys. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of the TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junctions almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidneys.NEW & NOTEWORTHY This study used immunohistochemistry and immunofluorescence to investigate the distribution of claudin 10, 16, and 19 in the human, mouse, and rat kidney. The findings showed distinct junctional pathways in both human and rodent kidneys, supporting the existence of different permeation profiles in all species investigated.

Coding practice in national and regional kidney biopsy registries.

BACKGROUND: Kidney biopsy registries all over the world benefit research, teaching and health policy. Comparison, aggregation and exchange of data is however greatly dependent on how registration and coding of kidney biopsy diagnoses are performed. This paper gives an overview over kidney biopsy registries, explores how these registries code kidney disease and identifies needs for improvement of coding practice. METHODS: A literature search was undertaken to identify biopsy registries for medical kidney diseases. These data were supplemented with information from personal contacts and from registry websites. A questionnaire was sent to all identified registries, investigating age of registries, scope, method of coding, possible mapping to international terminologies as well as self-reported problems and suggestions for improvement. RESULTS: Sixteen regional or national kidney biopsy registries were identified, of which 11 were older than 10 years. Most registries were located either in Europe (10/16) or in Asia (4/16). Registries most often use a proprietary coding system (12/16). Only a few of these coding systems were mapped to SNOMED CT (1), older SNOMED versions (2) or ERA-EDTA PRD (3). Lack of maintenance and updates of the coding system was the most commonly reported problem. CONCLUSIONS: There were large gaps in the global coverage of kidney biopsy registries. Limited use of international coding systems among existing registries hampers interoperability and exchange of data. The study underlines that the use of a common and uniform coding system is necessary to fully realize the potential of kidney biopsy registries.

Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy for Amyloid Screening in Patients with Suspected Systemic Amyloidosis.

Screening for systemic amyloidosis is typically carried out with abdominal fat aspirates with varying reported sensitivities. Fat aspirates are preferred for use in primary screening instead of organ biopsies as they are less invasive and thereby minimize the potential risk of complications. At Odense Amyloidosis Center, we performed a prospective study on whether the combined use of fat aspirate and tru-cut skin biopsy could increase the diagnostic sensitivity. Both fat aspirates and skin biopsies were screened with Congo Red staining, and positive biopsies were subsequently subtyped using immunoelectron microscopy and mass spectrometry. Seventy-six patients were included. In total, 24 patients had systemic amyloidosis (11 AL, 12 wtATTR, 1 AA), and 6 patients had localized amyloidosis. Combined fat aspirate and skin biopsy were Congo Red-positive in 15 patients (overall sensitivity (OS) 62.5%). Fat aspirates were positive in 14 patients (OS 58.3%), and the skin biopsy was positive in 5 patients (OS 20.8%). In only one patient did the skin biopsy add extra diagnostic information. The sensitivity differed between AL and ATTR amyloidosis-81.8% and 41.7%, respectively. Using skin biopsy as the only screening method is not recommended.

Low-intensity shockwave therapy in the treatment of diabetic nephropathy: a prospective Phase 1 study.

BACKGROUND: Low-intensity shockwave therapy (LI-SWT) is suggested as a therapy for promoting tissue regeneration. In pigs, it was recently found that LI-SWT improved renal function after ischaemic injury. Our objectives were to study glomerular filtration rate (GFR) and albuminuria in diabetic nephropathy (DN) after treatment with LI-SWT. The present pilot study reports on the clinical safety of LI-SWT in DN. METHODS: A total of 14 patients with diabetes mellitus and Stage 3 chronic kidney disease were recruited for this prospective, one-arm Phase 1 study. The patients were treated with six sessions of LI-SWT during a 3-week period. At each session, 3000 shockwaves were applied to each kidney with 0.265 mJ/mm2, extended focal size and 4 Hz. Follow-up visits were performed at 1, 3 and 6 months. RESULTS: In general, the treatment was well tolerated. Transient macroscopic haematuria was observed in three patients immediately after LI-SWT. The majority of patients experienced lower back tenderness lasting up to 2 days after treatment. There was no need for analgesic treatment. LI-SWT showed no negative effect on GFR and albuminuria. At baseline, median (interquartile range) GFR was 33.5 mL/min/1.73 m2 (27.8-43.8) compared with 36.0 mL/min/1.73 m2 (27.5-52.0) at 6 months follow-up. In parallel, median albuminuria was 256 mg/24 h (79-619) at baseline and tended to decrease to 137 mg/24 h (41-404) 6 months after LI-SWT. There was no statistical difference between baseline and follow-up results. CONCLUSIONS: LI-SWT is a safe treatment for DN. Inclusion of more patients is needed to determine whether LI-SWT can improve renal functional outcomes.

Proteomic Analysis of Renal Biomarkers of Kidney Allograft Fibrosis-A Study in Renal Transplant Patients.

Renal transplantation is the preferred treatment of end stage renal disease, but allograft survival is limited by the development of interstitial fibrosis and tubular atrophy in response to various stimuli. Much effort has been put into identifying new protein markers of fibrosis to support the diagnosis. In the present work, we performed an in-depth quantitative proteomics analysis of allograft biopsies from 31 prevalent renal transplant patients and correlated the quantified proteins with the volume fraction of fibrosis as determined by a morphometric method. Linear regression analysis identified four proteins that were highly associated with the degree of interstitial fibrosis, namely Coagulation Factor XIII A chain (estimate 18.7, adjusted p < 0.03), Uridine Phosphorylase 1 (estimate 19.4, adjusted p < 0.001), Actin-related protein 2/3 subunit 2 (estimate 34.2, adjusted p < 0.05) and Cytochrome C Oxidase Assembly Factor 6 homolog (estimate -44.9, adjusted p < 0.002), even after multiple testing. Proteins that were negatively associated with fibrosis (p < 0.005) were primarily related to normal metabolic processes and respiration, whereas proteins that were positively associated with fibrosis (p < 0.005) were involved in catabolic processes, cytoskeleton organization and the immune response. The identified proteins may be candidates for further validation with regards to renal fibrosis. The results support the notion that cytoskeleton organization and immune responses are prevalent processes in renal allograft fibrosis.

The epithelial Na+ channel α- and γ-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney.

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.

Nephrotic syndrome is associated with increased plasma K+ concentration, intestinal K+ losses, and attenuated urinary K+ excretion: a study in rats and humans.

The present study tested the hypotheses that nephrotic syndrome (NS) leads to renal K+ loss because of augmented epithelial Na+ channel (ENaC) activity followed by downregulation of renal K+ secretory pathways by suppressed aldosterone. The hypotheses were addressed by determining K+ balance and kidney abundance of K+ and Na+ transporter proteins in puromycin aminonucleoside (PAN)-induced rat nephrosis. The effects of amiloride and angiotensin II type 1 receptor and mineralocorticoid receptor (MR) antagonists were tested. Glucocorticoid-dependent MR activation was tested by suppression of endogenous glucocorticoid with dexamethasone. Urine and plasma samples were obtained from pediatric patients with NS in acute and remission phases. PAN-induced nephrotic rats had ENaC-dependent Na+ retention and displayed lower renal K+ excretion but elevated intestinal K+ secretion that resulted in less cumulated K+ in NS. Aldosterone was suppressed at day 8. The NS-associated changes in intestinal, but not renal, K+ handling responded to suppression of corticosterone, whereas angiotensin II type 1 receptor and MR blockers and amiloride had no effect on urine K+ excretion during NS. In PAN-induced nephrosis, kidney protein abundance of the renal outer medullary K+ channel and γ-ENaC were unchanged, whereas the Na+-Cl- cotransporter was suppressed and Na+-K+-ATPase increased. Pediatric patients with acute NS displayed suppressed urine Na+-to-K+ ratios compared with remission and elevated plasma K+ concentration, whereas fractional K+ excretion did not differ. Acute NS is associated with less cumulated K+ in a rat model, whereas patients with acute NS have elevated plasma K+ and normal renal fractional K+ excretion. In NS rats, K+ balance is not coupled to ENaC activity but results from opposite changes in renal and fecal K+ excretion with a contribution from corticosteroid MR-driven colonic secretion.

Coordination of Fusion and Trafficking of Pre-osteoclasts at the Marrow-Bone Interface.

Fusion is the final osteoclast differentiation step leading to bone resorption. In healthy trabecular bone, osteoclast fusion is restricted to bone surfaces undergoing resorption, and necessarily requires site-specific recruitment of mononucleated pre-osteoclasts originating from the bone marrow. However, the spatiotemporal mechanism coordinating recruitment and fusion is poorly investigated. Herein we identify a collagen/vascular network as a likely structure supporting this mechanism. We therefore used multiplex immunohistochemistry and electron microscopy on human iliac crest bone samples, in combination with functional assays performed in vitro with osteoclasts generated from healthy blood donors. First, we found that putative pre-osteoclasts are in close vicinity of a network of collagen fibers associated with vessels and bone remodeling compartment canopies. Based on 3D-reconstructions of serial sections, we propose that this network may serve as roads leading pre-osteoclasts to resorption sites, as reported for cell migration in other tissues. Importantly, almost all these bone marrow pre-osteoclasts, but only some osteoclasts, express the collagen receptor OSCAR, which is reported to induce fusion competence. Furthermore, differentiating osteoclasts cultured on collagen compared to mineral show higher fusion rates, higher expression of fusogenic cytokines, and a CD47 plasma membrane distribution pattern reported to be typical of a pre-fusion state-thus collectively supporting collagen-induced fusion competence. Finally, these in vitro assays show that collagen induces high cell mobility. The present data lead to a model where collagen fibers/vasculature support the coordination between traffic and fusion of pre-osteoclasts, by serving as a physical road and inducing fusion competence as well as cell mobility.

The effect of two exercise modalities on skeletal muscle capillary ultrastructure in individuals with type 2 diabetes.

Type 2 diabetes is associated with microvascular dysfunction, but little is known about how capillary ultrastructure is affected by exercise training. To investigate the effect of two types of exercise training on skeletal muscle capillary ultrastructure and capillarization in individuals with type 2 diabetes, 21 individuals with type 2 diabetes were allocated (randomized controlled trial) to 11 weeks of aerobic exercise training consisting of either moderate-intensity endurance training (END; n = 10) or low-volume high-intensity interval training (HIIT; n = 11). Skeletal muscle biopsies (m vastus lateralis) were obtained before and after the training intervention. At baseline, there was no difference in capillarization, capillary structure, and exercise hyperemia between the two groups. After the training intervention, capillary-to-fiber ratio increased by 8% ± 3% in the END group (P < 0.05) and was unchanged in the HIIT group with no difference between groups. Endothelium thickness increased (P < 0.05), basement membrane thickness decreased (P < 0.05), and the capillary lumen tended (P = 0.07) to increase in the END group, whereas these structural indicators were unchanged after HIIT. In contrast, skeletal muscle endothelial nitric oxide synthase (eNOS) increased after HIIT (P < 0.05), but not END, whereas there was no change in vascular endothelial growth factor (VEGF), superoxide dismutase (SOD)-2, or NADPH oxidase after both training protocols. In contrast to END training, HIIT did not alter capillarization or capillary structure in individuals with type 2 diabetes. In conclusion, HIIT appears to be a less effective strategy to treat capillary rarefaction and reduce basement thickening in type 2 diabetes.

Seminal human papillomavirus originates from the body surface and is not a frequent aetiological factor in azoospermia.

Human papillomavirus (HPV) DNA has been detected in the testis tissue of 6.5% of 185 men with non-obstructive azoospermia (NOA). Others have suggested that seminal HPV originates from contamination from the genital skin and mucosa. One hundred unselected azoospermic men and 43 normal men undergoing vasectomy were recruited. Testicular biopsies for HPV examination were collected from all the men. Additionally, the normal men undergoing vasectomy delivered a semen sample and had a swab for HPV examination taken from the genital skin before vasectomy. A piece of each Vas deferens obtained during the vasectomy was examined for the presence of HPV. Two of the primarily azoospermic men were shown to have cryptozoospermia. It was not possible to detect HPV in the testis tissue of any of the included 98 azoospermic men or the 43 proven fertile men. In the proven fertile men, HPV DNA was detected in the semen of 15 men (35%), on the genital skin of 28 men (65%), and in the Vas deferens in three cases (7%). In 13 (87%) men with HPV-positive semen samples, HPV DNA was also detected in the skin swabs, and in 11 men (73%), identical HPV genotypes were found in the two locations.

H+-ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney.

The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.

Early reversal cells in adult human bone remodeling: osteoblastic nature, catabolic functions and interactions with osteoclasts.

The mechanism coupling bone resorption and formation is a burning question that remains incompletely answered through the current investigations on osteoclasts and osteoblasts. An attractive hypothesis is that the reversal cells are likely mediators of this coupling. Their nature is a big matter of debate. The present study performed on human cancellous bone is the first one combining in situ hybridization and immunohistochemistry to demonstrate their osteoblastic nature. It shows that the Runx2 and CD56 immunoreactive reversal cells appear to take up TRAcP released by neighboring osteoclasts. Earlier preclinical studies indicate that reversal cells degrade the organic matrix left behind by the osteoclasts and that this degradation is crucial for the initiation of the subsequent bone formation. To our knowledge, this study is the first addressing these catabolic activities in adult human bone through electron microscopy and analysis of molecular markers. Periosteoclastic reversal cells show direct contacts with the osteoclasts and with the demineralized resorption debris. These early reversal cells show (1) ¾-collagen fragments typically generated by extracellular collagenases of the MMP family, (2) MMP-13 (collagenase-3) and (3) the endocytic collagen receptor uPARAP/Endo180. The prevalence of these markers was lower in the later reversal cells, which are located near the osteoid surfaces and morphologically resemble mature bone-forming osteoblasts. In conclusion, this study demonstrates that reversal cells colonizing bone surfaces right after resorption are osteoblast-lineage cells, and extends to adult human bone remodeling their role in rendering eroded surfaces osteogenic.

The epithelial sodium channel γ-subunit is processed proteolytically in human kidney.

The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved-to-full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved-to-full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAbinhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC.

Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia.

Plasma membrane Ca(2+)-ATPases (PMCAs) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial transient receptor potential vanilloid 5 Ca(2+) channel. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestines, whereas pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In the kidney, Pmca4 showed broad localization to the distal nephron. In the mouse, expression was most abundant in segments coexpressing the epithelial ransient receptor potential vanilloid 5 Ca(2+) channel. Significant, albeit lower, expression was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In the human kidney, a similar pattern of distribution was observed, with the highest PMCA4 expression in Na(+)-Cl(-) cotransporter-positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing to a housekeeping function of the pump in Ca(2+)-transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca(2+)-transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca(2+) transport.

Protective effects of surfactant protein D treatment in 1,3-ß-glucan-modulated allergic inflammation.

Surfactant protein D (SP-D) is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd(-/-)) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. ß-Glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-ß-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-ß-glucan. Allergic airway disease was induced in Sftpd(-/-) and Sftpd(+/+) mice by OVA sensitization and subsequent challenge with OVA, 1,3-ß-glucan, or OVA/1,3-ß-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-ß-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd(-/-) mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in bronchoalveolar lavage were unaffected by 1,3-ß-glucan, but were reversed with rfhSP-D treatment. 1,3-ß-Glucan treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in bronchoalveolar lavage, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-ß-Glucan reduced OVA-induced mucous cell metaplasia, T helper 2 cytokines, and IFN-γ production. rfhSP-D treatment further reduced mucous metaplasia and T helper 2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-ß-glucan-mediated neutrophilic inflammation. Our data suggest that treatment with high-dose SP-D protects from mold-induced exacerbations of allergic asthma.

Microfibrillar-associated protein 4 modulates airway smooth muscle cell phenotype in experimental asthma.

BACKGROUND: Recently, several proteins of the extracellular matrix have been characterised as active contributors to allergic airway disease. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein abundant in the lung, whose biological functions remain poorly understood. In the current study we investigated the role of MFAP4 in experimental allergic asthma. METHODS: MFAP4-deficient mice were subjected to alum/ovalbumin and house dust mite induced models of allergic airway disease. In addition, human healthy and asthmatic primary bronchial smooth muscle cell cultures were used to evaluate MFAP4-dependent airway smooth muscle responses. RESULTS: MFAP4 deficiency attenuated classical hallmarks of asthma, such as eosinophilic inflammation, eotaxin production, airway remodelling and hyperresponsiveness. In wild-type mice, serum MFAP4 was increased after disease development and correlated with local eotaxin levels. MFAP4 was expressed in human bronchial smooth muscle cells and its expression was upregulated in asthmatic cells. Regarding the underlying mechanism, we showed that MFAP4 interacted with integrin αvß5 and promoted asthmatic bronchial smooth muscle cell proliferation and CCL11 release dependent on phosphatidyloinositol-3-kinase but not extracellular signal-regulated kinase pathway. CONCLUSIONS: MFAP4 promoted the development of asthmatic airway disease in vivo and pro-asthmatic functions of bronchial smooth muscle cells in vitro. Collectively, our results identify MFAP4 as a novel contributor to experimental asthma, acting through modulation of airway smooth muscle cells.