Cutaneous vascular calcifications in patients with chronic kidney disease and calcific uremic arteriolopathy: a cross-sectional study.

INTRODUCTION: Calcific uremic arteriolopathy is a life-threatening cutaneous condition in patients with chronic kidney disease. Often, clinical diagnosis is accompanied by histopathologic evaluations demonstrating vascular calcium deposits. We aimed to investigate the presence of cutaneous calcifications in non-lesional tissue in patients with chronic kidney disease, and the relation to systemic vascular calcification. METHODS: We investigated the presence of cutaneous vascular calcifications in non-lesional skin biopsies from patients with current or previous calcific uremic arteriolopathy and patients with different stages of chronic kidney disease without calcific uremic arteriolopathy, and explored their association with vascular calcification in other vascular beds. Systemic vascular calcification was examined by mammography and lumbar X-ray. RESULTS: Thirty-nine adults were enrolled (current or previous calcific uremic arteriolopathy, n = 9; end-stage chronic kidney disease, n = 12; chronic kidney disease stage 3b-4, n = 12; healthy controls, n = 6). All calcific uremic arteriolopathy patients had end-stage kidney disease. Cutaneous vascular calcifications were not present in any of the non-lesional skin punch biopsies. Breast arterial calcification was demonstrated in patients with calcific uremic arteriolopathy (75%) and chronic kidney disease (end-stage 67% and stage 3b-4 25%, respectively), but in none of the controls. All chronic kidney disease patients had systemic calcification on lumbar X-ray (median score 21, 22, and 15 in patients with calcific uremic arteriolopathy, end-stage kidney disease and chronic kidney disease stage 3b-4). The serum calcification propensity was significantly different between groups. DISCUSSION: Despite a high burden of systemic vascular calcification, cutaneous calcium deposits in non-lesional tissue could not be demonstrated histopathologically in patients with chronic kidney disease (with or without current or previous calcific uremic arteriolopathy). Further studies to determine whether these findings are representative or attributed to other factors are warranted.

Rationale and design of a prospective, clinical study of kidney biopsies in people with type 2 diabetes and severely increased albuminuria (the PRIMETIME 2 study).

INTRODUCTION: Diabetic kidney disease is a severe complication of diabetes. The diagnosis is based on clinical characteristics such as persistently elevated albuminuria, hypertension and decline in kidney function, although this definition is not specific to kidney disease caused by diabetes. The only way to establish an accurate diagnosis-diabetic nephropathy-is by performing a kidney biopsy. The histological presentation of diabetic nephropathy can be associated with a heterogeneous range of histological features with many pathophysiological factors involved demonstrating the complexity of the condition. Current treatment strategies aim to slow disease progression and are not specific to the underlying pathological processes.This study will investigate the prevalence of diabetic nephropathy in individuals with type 2 diabetes (T2D) and severely elevated albuminuria. The deep molecular characterisation of the kidney biopsy and biological specimens may pave the way for improved diagnostic accuracy and a better understanding of the pathological processes involved and may also reveal new targets for individualised treatment. METHODS AND ANALYSIS: In the PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy 2 study, research kidney biopsies will be performed in 300 participants with T2D, urine albumin/creatinine ratio ≥700 mg/g and estimated glomerular filtration ratio >30 mL/min/1.73 m2. Cutting-edge molecular technologies will be applied to the kidney, blood, urine, faeces and saliva samples for comprehensive multi-omics profiling. The associated disease course and clinical outcomes will be assessed by annual follow-up for 20 years. ETHICS AND DISSEMINATION: The Danish Regional Committee on Health Research Ethics and the Knowledge Center on Data Protection (in the Capital Region of Denmark) have granted approval for the study. The results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04916132.

Feasibility of Intratumoral Anti-PD1 as Treatment of Human Basal Cell Carcinoma: An Explorative Study with Adjuvant Ablative Fractional Laser.

The use of immune checkpoint inhibitors (ICI) is expanding with the approval for advanced/metastatic keratinocyte carcinoma; however, most tumors are non-aggressive. Local administration could broaden ICI, but adequate immune response might require an immune-attractive adjuvant such as ablative fractional laser (AFL). Accordingly, this study aimed to explore intratumoral injection of anti-PD1 with and without AFL in basal cell carcinoma (BCC), exploring anti-PD1 concentration, immune cell infiltration, tumor response, and safety. This open-label, proof-of-concept trial investigated intratumoral anti-PD1 + AFL combination therapy versus anti-PD1 or AFL monotherapy in 28 BCC patients. The primary endpoints were immune cell infiltration evaluated immunohistochemically and clinical tumor response after 3 months. The secondary outcomes were tumoral drug concentration and safety. The most robust response was obtained following intervention with combined anti-PD1+AFL, leading to a ~2.5-fold increase in CD3+ cells (p = 0.027), and tumor reduction ≥25% in 73%, including two tumors with complete remission. Upon anti-PD1 monotherapy, a slight decrease in CD3+ cells was observed while a non-significant increase following AFL was seen. Tumor reduction ≥25% was seen in 45% and 50%, respectively, after anti-PD1 and AFL monotherapy. The CD8/CD3 ratio remained unchanged after anti-PD1+AFL and anti-PD1 monotherapy, while AFL led to a decreased ratio. A non-significant decline in the Foxp3/CD3 ratio was observed for all groups. Side-effects were mild with no systemic drug concentration detected. Intratumoral anti-PD1 injection is feasible, and a single exposure to locally injected anti-PD1 with adjuvant AFL increased immune cell infiltration and reduction in BCC with limited side-effects.

Clinically-suspected cast nephropathy: A retrospective, national, real-world study.

Presentation with severe acute kidney injury due to cast nephropathy (CN) is a medical emergency in multiple myeloma (MM), with high risk of dialysis-dependent renal failure and death. Accrual of patients with CN into interventional studies is difficult, while phase III trials exclude patients with severe renal insufficiency. Real-world data are warranted. We assessed 2252 patients from the population-based Danish Multiple Myeloma Registry (DMMR) who were diagnosed between 2013 and 2017. We identified 204 patients with clinically-suspected CN, defined as serum creatinine concentration >177 µmol/L and serum free light chain (sFLC) concentration >1000 mg/L at the time of diagnosis. The median age was 72 years. Thirty-one percent of patients presented with dialysis-dependent renal failure. Kidney biopsies were performed in 19% of patients and showed CN in 74% of cases. Despite prompt initiation of bortezomib-based therapy in 94% of patients, 33% of patients died in the first year after diagnosis. Compared with the rest of the patients in the DMMR with symptomatic MM, patients with clinically-suspected CN had worse overall survival (OS) irrespective of transplant eligibility. Achievement of renal recovery was associated with deep reductions of involved sFLC. Achievement of very good partial response or better in the first line of therapy and/or deep reduction of involved sFLC at 3 months after initiation of therapy were associated with superior OS. In conclusion, MM patients presenting with clinically-suspected CN have an alarmingly high one-year mortality when treated with current standards of care. Early and deep hematologic response is crucial for survival.

Immune regulation by fibroblasts in tissue injury depends on uPARAP-mediated uptake of collectins.

Collectins such as mannose-binding lectin (MBL) and surfactant protein D (SP-D) become temporarily deposited in extravascular compartments after tissue injury and perform immune-stimulatory or inflammation-limiting functions. However, their turnover mechanisms, necessary to prevent excessive tissue damage, are virtually unknown. In this study, we show that fibroblasts in injured tissues undertake the clearance of collectins by using the endocytic collagen receptor uPARAP. In cellular assays, several types of collectins were endocytosed in a highly specific uPARAP-dependent process, not shared by the closely related receptor MR/CD206. When introduced into dermis or bleomycin-injured lungs of mice, collectins MBL and SP-D were endocytosed and routed for lysosomal degradation by uPARAP-positive fibroblasts. Fibroblast-specific expression of uPARAP governed endogenous SP-D levels and overall survival after lung injury. In lung tissue from idiopathic pulmonary fibrosis patients, a strong up-regulation of uPARAP was observed in fibroblasts adjacent to regions with SP-D secretion. This study demonstrates a novel immune-regulatory function of fibroblasts and identifies uPARAP as an endocytic receptor in immunity.

Renal amyloid A amyloidosis as a complication of hidradenitis suppurativa.

Rheumatic disease is the dominant cause of amyloid A (AA) amyloidosis, but other chronic inflammatory diseases may have similar consequences. Hidradenitis suppurativa (HS) is a relatively common, but little known skin disease characterized by chronic inflammation. Here we present a case of chronic HS leading to biopsy-verified severe renal AA amyloidosis and dialysis dependency.

The number of FoxP3+ cells in transbronchial lung allograft biopsies does not predict bronchiolitis obliterans syndrome within the first five years after transplantation.

BACKGROUND: An important limitation to the success of lung transplantation is the development of bronchiolitis obliterans syndrome (BOS). It has been hypothesized that regulatory T lymphocytes (Tregs) are related to the risk of BOS. We aim to evaluate whether the number of forkhead box P3 (FoxP3+) cells/mm(2) in lung allograft biopsies is a predictor of long-term outcome. MATERIALS AND METHODS: A total of 58 consecutive lung transplant patients were included in the study. For 233 routine surveillance biopsy samples, the numbers of FoxP3+ cells/mm(2) were assessed by immunohistochemical staining with antibodies against FoxP3. BOS scores were calculated for the first five yr after transplantation. RESULTS: We determined that acute rejection was related to the time elapsed from transplantation to BOS with hazard ratios of 3.18 (p = 0.02) and 3.73 (p = 0.04) when comparing the levels of acute rejection grade 1 and grade 2/3, respectively, to no rejection. According to a Cox regression analysis, the number of FoxP3+ cells/mm(2) was not predictive of time to BOS. DISCUSSION AND CONCLUSIONS: Our data indicate that the number of FoxP3+ cells in the lung allograft did not correlate with BOS-free survival time. Previous studies have been contradictory and included different time points. Our findings emphasize the importance of including a time factor.

Time elapsed after transplantation influences the relationship between the number of regulatory T cells in lung allograft biopsies and subsequent acute rejection episodes.

BACKGROUND: Regulatory T lymphocytes (Tregs) play an important role in acute rejection after lung transplantation. However, the importance of the time elapsed after transplantation on the Treg response requires further investigation. We aim to evaluate the change over time in the frequency of Tregs in lung allograft biopsies and to assess how Tregs relate to simultaneous and subsequent acute cellular rejection. MATERIALS AND METHODS: A total of 258 biopsy samples obtained 0.5, 1, 3, 12 and 24 months after transplantation from 58 consecutive lung transplant patients were included. The biopsies were scored for acute rejection according to the ISHLT criteria (A0-A4) and immunohistochemically stained with antibodies against FoxP3. RESULTS: There was a tendency for a decrease in the number of Tregs/mm2 with time. However, the previous levels of Tregs/mm2 did not have any significant effect on future levels of Tregs/mm2. For biopsies taken 0.5 and 1 month after transplantation, a significant correlation between Tregs/mm2 and the degree of acute rejection was found, and logistic regression analysis using updated values for Tregs/mm2 showed a significant relationship between Tregs/mm2 at 2 weeks and an A-score≥2 after 1 and 3 months. At later time points, this correlation disappeared. DISCUSSION AND CONCLUSION: Our data indicate that the time elapsed after transplantation is an important parameter influencing the Treg response after lung transplantation. This observation is in accordance with studies indicating a narrow therapeutic window for induction of tolerance by specifically targeting T-cells. The results also indirectly indicate that Tregs early after transplantation could have an impact on the long-term outcome.

The number of regulatory T cells in transbronchial lung allograft biopsies is related to FoxP3 mRNA levels in bronchoalveolar lavage fluid and to the degree of acute cellular rejection.

BACKGROUND: The transcription factor Forkhead Box P3 (FoxP3) is a marker of regulatory T cells (Tregs) - a subset of T cells known to suppress a wide range of immune responses. These cells are considered to be pivotal for the induction of tolerance to donor antigens in human allografts. We aimed to correlate the number of lymphocytes expressing FoxP3 in transbronchial biopsies from lung allografts with the FoxP3 expression in bronchoalveolar lavage fluid (BALF). In addition, we aimed to correlate the number of FoxP3+ cells in transbronchial biopsies with the degree of acute cellular rejection in lung allografts. MATERIALS AND METHODS: The expression of FoxP3 was evaluated using immunohistochemical staining in 40 lung allograft biopsies obtained from 23 patients. The number of Tregs was related to the FoxP3 mRNA levels as determined using qRT-PCR in corresponding BALF samples from the same patients. Furthermore, the number of Tregs was related to the degree of acute allograft rejection (according to ISHLT criteria, A0-A4). RESULTS: Regression analysis showed a significant concordance between the number of Tregs in lung tissue and the level of FoxP3 mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels in BALF (n=40, p=0.0001). In addition, we found a significant increase in the number of Tregs during acute allograft rejections of grades A2 and higher (median: 32.6Tregs/mm(2)) when compared to those of grades A1 and A0 (median: 4.9Tregs/mm(2)) (p=0.0002). DISCUSSION AND CONCLUSION: The association between the distribution of Tregs in transbronchial biopsies and the level of FoxP3 mRNA in BALF indicates that assessment of FoxP3 mRNA in BALF is a reliable non-invasive method for evaluating the number of Tregs in lung tissue. Furthermore, the association between the number of Tregs in lung tissue and the degree of acute cellular rejection shows that Tregs are recruited to the site of inflammation and may be involved in the regulation of acute rejection. Thus, Tregs may play a role in the cellular processes that affect lung allograft outcome.

Acute Myocardial Infarction and Pulmonary Diseases Result in Two Different Degradation Profiles of Elastin as Quantified by Two Novel ELISAs.

BACKGROUND: Elastin is a signature protein of the arteries and lungs, thus it was hypothesized that elastin is subject to enzymatic degradation during cardiovascular and pulmonary diseases. The aim was to investigate if different fragments of the same protein entail different information associated to two different diseases and if these fragments have the potential of being diagnostic biomarkers. METHODS: Monoclonal antibodies were raised against an identified fragment (the ELM-2 neoepitope) generated at the amino acid position '552 in elastin by matrix metalloproteinase (MMP) -9/-12. A newly identified ELM neoepitope was generated by the same proteases but at amino acid position '441. The distribution of ELM-2 and ELM, in human arterial plaques and fibrotic lung tissues were investigated by immunohistochemistry. A competitive ELISA for ELM-2 was developed. The clinical relevance of the ELM and ELM-2 ELISAs was evaluated in patients with acute myocardial infarction (AMI), no AMI, high coronary calcium, or low coronary calcium. The serological release of ELM-2 in patients with chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) was compared to controls. RESULTS: ELM and ELM-2 neoepitopes were both localized in diseased carotid arteries and fibrotic lungs. In the cardiovascular cohort, ELM-2 levels were 66% higher in serum from AMI patients compared to patients with no AMI (p<0.01). Levels of ELM were not significantly increased in these patients and no correlation was observed between ELM-2 and ELM. ELM-2 was not elevated in the COPD and IPF patients and was not correlated to ELM. ELM was shown to be correlated with smoking habits (p<0.01). CONCLUSIONS: The ELM-2 neoepitope was related to AMI whereas the ELM neoepitope was related to pulmonary diseases. These results indicate that elastin neoepitopes generated by the same proteases but at different amino acid sites provide different tissue-related information depending on the disease in question.

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues.

Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we found that ST6GalNAc-I is weakly expressed in normal gastric mucosa, but over-expressed in intestinal metaplasia, co-localized with sialyl-Tn. In gastric carcinomas ST6GalNAc-I was also associated with sialyl-Tn, but with heterogeneous staining and partial co-localization. Our results showed ST6GalNAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues.

Histological characteristics of human papilloma-virus-positive and -negative invasive and in situ squamous cell tumours of the penis.

A high prevalence of cervical cancer associated high-risk types of human papillomavirus (hrHPV) has been demonstrated in premalignant and invasive squamous cell lesions of the penis, but large studies correlating histological characteristics with HPV status are few in number. Tumour tissues from 145 patients with invasive (n = 116) or in situ (n = 29) penile squamous cell carcinoma were subjected to systematic histological evaluation and were PCR-tested for 14 hrHPV types and 23 low-risk HPV types. Around half (52%) of invasive and nine-tenths (90%) of in situ lesions were positive for an hrHPV type, of which HPV 16 was by far the predominant type (91% of hrHPV-positive lesions). In relation to histological characteristics, hrHPV positivity was statistically significantly more common in high-grade tumours, lesions dominated by small tumour cells, lesions with a high number of multinucleated cells and mitoses, and lesions with a small amount of parakeratosis. In conclusion, about half of invasive penile squamous carcinomas in this study were hrHPV-positive, most notably to HPV 16, and probably arose through in situ lesions whereas the other half of invasive penile lesions appeared to be unrelated to hrHPV. A number of histological characteristics differed significantly between hrHPV-positive and -negative invasive penile carcinomas.

Procollagen 1 - a marker of fibroblastic and fibrohistiocytic skin tumors.

BACKGROUND: When dealing with tumors of presumed fibroblastic or fibrohistiocytic origin, immunohistochemistry is traditionally done to exclude a tumor of non-fibroblastic or non-fibrohistiocytic nature, because a reliable marker of fibroblastic or fibrohistiocytic cell origin is still to be introduced. This study investigates whether procollagen 1 is a useful marker of skin tumors composed of cells derived from fibroblasts or fibrohistiocytes. MATERIALS AND METHODS: Twenty-nine different types of skin tumors, including tumors composed of spindle cells as well as some additional epithelial and melanocytic tumors, were stained immunohistochemically with antibodies against procollagen 1. The total number of cases tested was 154. RESULTS: Tumors composed of cells of fibroblastic or fibrohistiocytic origin in general showed a high expression of procollagen 1, whereas tumors composed of non-fibroblastic cells in general showed no expression. However, there were a few exceptions, e.g., leiomyosarcoma and desmoplastic melanoma. CONCLUSIONS: The study suggests that immunohistochemical staining for procollagen 1 is a valuable marker for skin tumors composed of spindle cells derived from fibroblasts or fibrohistiocytes. However, in cases of spindle cell tumors with a morphology suggesting malignancy, it is recommended to use a panel of antibodies, which besides procollagen 1 includes markers such as S100, CD68, CD34, h-caldesmon, and pancytokeratin.