Apmis

Elsborg, Søren H.; Pedersen, Gitte A.; Madsen, Mia G.; Keller, Anna K.; Nørregaard, Rikke; Nejsum, Lene N.

doi: 10.1111/apm.13329pmid: 37211896

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin‐embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re‐staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal‐like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin‐embedded tissue was used for multiplex IF staining with increased Z‐resolution. In summary, this method offers time‐saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

Ernstsen, Christina V.; Riishede, Andreas; Iversen, Anne Kristine S.; Bay, Lene; Bjarnsholt, Thomas; Nejsum, Lene N.

doi: 10.1111/apm.13332pmid: 37267058

Chronic wounds are defined as wounds that fail to proceed through the normal phases of wound healing; a complex process involving different dynamic events including migration of keratinocytes in the epidermis. Chronic wounds are estimated to affect 1–2% of the human population worldwide and are a major socioeconomic burden. The prevalence of chronic wounds is expected to increase with the rising number of elderly and patients with diabetes and obesity, who are at high risk of developing chronic wounds. Since E‐cadherin and the water channel aquaporin‐3 are important for both skin function and cell migration, and aquaporin‐3 is furthermore involved in wound healing of the skin demonstrated by impaired wound healing in aquaporin‐3‐null mice, we hypothesized that E‐cadherin and aquaporin‐3 expression may be dysregulated in chronic wounds. Therefore, we investigated the expression of E‐cadherin and aquaporin‐3 in biopsies from the edges of chronic wounds from human patients. This was accomplished by immunohistochemical stainings of E‐cadherin and aquaporin‐3 on serial sections followed by qualitative evaluation of staining patterns, which revealed low expression of both E‐cadherin and aquaporin‐3 at the wound edge. Future studies are needed to reveal if this downregulation is associated with the pathophysiology of chronic wounds.

Pichler Sekulic, Simona; Sekulic, Miroslav

doi: 10.1111/apm.13340pmid: 37337415

Native cardiac valves in the setting of chronic injury may become thickened and disrupted by dystrophic calcification, which impede valve structure/function, and there may be evidence of chondromatous (i.e., cartilaginous, CM) metaplasia admixed with dystrophic calcification. In order to characterize the presence of CM in native cardiac valves – with particular focus upon aortic valves – a retrospective review of the histologic features of 46 native aortic valves (identified from 1094 sequentially reviewed native valves of all types) containing CM were focused upon, as well as correlation with other histopathologic features, and clinical and echocardiographic findings. The prevalence rate of CM was low, and greatest among aortic valves, less in mitral valves, and never identified in tricuspid or pulmonic valves. CM in aortic valves was less commonly identified in patients with a history of autoimmune disease. The rate of CM increased with degree of valve thickening and/or calcification. The proportion of aortic valves with CM increased with an increasing degree of stenosis and decreasing degree of regurgitation, and aortic valves with CM were more commonly associated with a smaller aortic valve area, and greater peak and mean gradients. The rate of osseous metaplasia, arterial vessels, capillary bed formation, and chronic inflammation were greater in aortic valves with CM compared to valves without. CM within aortic valves is an infrequent albeit identifiable histopathologic alteration associated with chronic valve disease alongside changes in valve thickening and calcification.

Katzenstein, Terese L.; Faurholt‐Jepsen, Daniel; Qvist, Tavs; Jensen, Peter Østrup; Pressler, Tacjana; Johansen, Helle Krogh; Kolpen, Mette

doi: 10.1111/apm.13331pmid: 37294911

Ceftolozane‐tazobactam is a new β‐lactam/β‐lactamase inhibitor combination approved by the U.S. Food and Drug Administration in 2019 for the treatment of hospital‐acquired and ventilator‐associated pneumonia. The combination is a particularly potent inhibitor of penicillin‐binding proteins with higher affinity than other β‐lactam agents. Persons with cystic fibrosis (pwCF) often harbour resistant Gram‐negative bacteria in the airways and need antibiotics to prevent declining lung function. To test whether the introduction of ceftolozane‐tazobactam in the period 2015–2020 led to a bacterial population level increase in cephalosporin resistance in a Danish CF population. In vitro, activity of ceftolozane‐tazobactam was evaluated by susceptibility testing of clinical Pseudomonas aeruginosa isolated from pwCF from January 1, 2015, to June 1, 2020. Six thousand three hundred thirty two isolates collected from 210 adult pwCF were included. Thirty pwCF were treated with ceftolozane‐tazobactam at least once. Ceftolozane‐tazobactam exposure did not increase cephalosporin resistance on an individual or population level. However, resistance to ceftolozane‐tazobactam was recorded despite no prior exposure in four pwCF. Compared to ceftazidime, ceftolozane‐tazobactam had a better in vitro activity on P. aeruginosa. The percentage of non‐mucoid P. aeruginosa isolates susceptible to ceftolozane‐tazobactam were higher or equal to 5 other β‐lactams. Ceftolozane‐tazobactam expands the armamentaria against P. aeruginosa with acceptable levels for a selection of drug resistance.

Sølund, Christina; Pedersen, Martin S.; Fahnøe, Ulrik; Filskov, Jonathan; Jenssen, Håvard; Weis, Nina; Schønning, Kristian; Bukh, Jens

doi: 10.1111/apm.13335pmid: 37355962

The introduction of direct‐acting antiviral (DAA) treatment of hepatitis C virus (HCV) infected patients has greatly increased treatment success rates. However, viral response kinetics to DAA treatment may depend on pre‐existing resistance‐associated substitutions (RASs) in HCV. The aim of this study was to describe how pre‐existing RASs affect DAA treatment‐induced reduction in HCV RNA titers in HCV genotypes 1‐ and 3‐infected individuals. Patients with HCV genotype 1 infection (N = 31) treated with either sofosbuvir/ledipasvir/ribavirin or paritaprevir/ombitasvir/ritonavir/dasabuvir/ribavirin and HCV genotype 3‐infected patients (N = 16) treated with either sofosbuvir/daclatasvir/ribavirin or sofosbuvir/ribavirin were analyzed. HCV RNA levels were determined at baseline and frequently during treatment, and RAS profiles were obtained by deep sequencing at baseline. In total, 33/47 (70.2%) of the patients had baseline RASs. However, treatment‐specific RASs were detected at baseline only in 12.9% and 18.8% of HCV genotypes 1‐ and 3‐infected patients, respectively. In genotype 1‐infected individuals, reduction in HCV RNA titer during the first week of treatment was not affected by evidence of either treatment‐specific RASs or cirrhosis or treatment regimen. In genotype 3‐infected individuals receiving sofosbuvir/daclatasvir/ribavirin, the presence of daclatasvir‐specific NS5A RASs at baseline correlated with a reduced decline of HCV RNA in the first treatment week. For both genotypes 1‐ and 3‐infected individuals, cirrhosis but not treatment‐specific RAS were associated with the time of clearance of HCV RNA. It is, however, important to note that this study involves DAA regimens that were used only during the original introduction of interferon‐free DAA‐based treatments.

Kuzma, Jozef; Zavala‐Meneses, Sofía Guadalupe; Skultety, Ludovit; Chmelar, Dittmar; Ficík, Jozef; Palcová, Lenka

doi: 10.1111/apm.13338pmid: 37355959

A total of, 78 Clostridium septicum (CLSE) isolates were screened for genes encoding: α‐toxin, flagellin, and resistance to vancomycin (VANg). The isolates were also tested for their ability to form biofilm and their antibiotic susceptibility. All isolates were positive for α‐toxin and flagellin genes. However, only 19 isolates (24.3%) showed prevalence for VANg. We observed the strongest capacity to form a biofilm (100%) in isolates from patients with oncologic or septic and febrile diagnoses. This percentage was also very high in patients with colitis and gastrointestinal hemorrhage (72.7%). No less than 43 isolates showed antibiotic resistance, and 21 were multidrug‐resistant (MDR). Interestingly, our studies showed a correlation between antibiotic resistance and biofilm formation. A statistically significant difference was observed between biofilm‐forming MDR isolates and those with low/no biofilm‐forming ability. However, the most impressive observation was the correlation with mortality rate. While the overall mortality rate for CLSE infections was 16.7% (13/78), the mortality rate for patients infected with MDR isolates forming biofilm moderately or strongly reached 38.1% (8/21). This number increased even further when only infections with the biofilm‐forming VANg‐positive isolates were considered (61.5%; 8/13). Therefore, the ability of a VANg‐positive CLSE isolate to form a biofilm has been suggested as a biomarker of poor prognosis.

Jangir, Puneet; Kalra, Sapna; Tanwar, Sunita; Bari, Vinay Kumar

doi: 10.1111/apm.13336pmid: 37337929

Multidrug resistance Candida auris is a dangerous fungal pathogen that is emerging at an alarming rate and posing serious threats to public health. C. auris is associated with nosocomial infections that cause invasive candidiasis in immunocompromised patients. Several antifungal drugs with distinct mechanisms of action are clinically approved for the treatment of fungal infections. The high rates of intrinsic and acquired drug resistance, particularly to azoles, reported in characterized clinical isolates of C. auris make treatment extremely problematic. In systemic infections, azoles are the first‐line treatment for most Candida species; however, the increasing use of drugs results in the frequent emergence of drug resistance. More than 90% of the clinical isolates of C. auris is shown to be highly resistant to azole drugs especially fluconazole, with some strains (types) resistant to all three classes of commonly used antifungals. This presents a huge challenge for researchers in terms of completely understanding the molecular mechanism of azole resistance to develop more efficient drugs. Due to the scarcity of C. auris therapeutic alternatives, the development of successful drug combinations provides an alternative for clinical therapy. Taking advantage of various action mechanisms, such drugs in combination with azole are likely to have synergistic effects, improving treatment efficacy and overcoming C. auris azole drug resistance. In this review, we outline the current state of understanding about the mechanisms of azole resistance mainly fluconazole, and the current advancement in therapeutic approaches such as drug combinations toward C. auris infections.