Nonrandom Distribution of Pseudomonas aeruginosa and Staphylococcus aureus in Chronic Wounds

The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.Chronic wounds, such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers, are an increasing problem worldwide. One to 2% of the population in developed countries develops chronic wounds, a condition associated with severe patient suffering, the loss of employment, a reduced quality of life, and high costs to the health care system (13). Detailed knowledge about chronic wounds is required in order to develop better wound treatment and management strategies.A normal wound healing process involves four main phases: (i) coagulation, (ii) inflammation, (iii) cell proliferation and repair of the matrix, and (iv) epithelialization and remodeling of the scar tissue (23). However, chronic wounds are believed to be captured in the inflammatory phase, where persistent influx and elevated activity of polymorphonuclear neutrophils (PMNs) occur (1). Although PMNs play a critical role in the host defense and wound healing, they release cytolytic enzymes, free oxygen radicals, inflammatory mediators, and matrix metalloproteases, which cause local tissue damage in the host (22, 23, 26).It is known that the microflora of chronic wounds comprises multiple species. In a bacterial profiling study, Gjødsbol et al. found that chronic venous leg ulcers harbored Staphylococcus aureus (in 93.5% of the ulcers), Enterococcus faecalis (71.7%), Pseudomonas aeruginosa (52.2%), coagulase-negative staphylococci (45.7%), Proteus species (41.3%), and anaerobic bacteria (39.1%) (12). S. aureus and P. aeruginosa are opportunistic pathogenic bacteria and are widely known to cause chronic biofilm-based infections in their hosts. S. aureus is most commonly isolated from chronic wounds (8, 12, 15, 17) and, in certain situations, can express a number of potential virulence factors and surface proteins which promote its adherence to the damaged tissue and decrease neutrophil functions and immune responses of the host (10, 11). P. aeruginosa often causes biofilm-based chronic infections and expresses virulence factors, in particular, rhamnolipid, that can eliminate the activity of PMNs (4, 16). A number of studies have demonstrated that P. aeruginosa is frequently present in chronic wounds (12, 17) and have provided evidence that the bacteria are located in aggregates enclosed in extracellular polymeric matrix material as found in biofilms (17). Furthermore, chronic wounds that harbored P. aeruginosa were larger than those that did not, and the healing process also seemed to be more severely hindered for those wounds (12, 14, 20).Biofilms are bacterial aggregates enclosed in a self-produced extracellular polymeric matrix (6, 21, 25). In clinical environments biofilms can form on dead or living tissues, mucosal surfaces, or the surfaces of medical devices in the host. The bacteria in biofilms often display characteristics different from those of their planktonic counterparts, such as increased resistance to the activities of the host immune system and tolerance to antimicrobial treatments (7). Such characteristics are important, since biofilms are involved in many chronic bacterial infections. Recent studies have shown the presence of bacterial biofilms in chronic wounds (9, 15, 17). Although the role of biofilms in chronic wounds is not yet fully understood, it is believed that their existence may be one of the reasons for impaired wound healing (4, 16).We previously demonstrated that there is a lack of correlation between the bacteria detected by standard culturing and those detected directly by peptide nucleic acid (PNA)-based fluorescence in situ hybridization (FISH) in chronic wound samples (17). While S. aureus was detected more frequently by swab sample cultivation than by PNA-FISH, the opposite was true for P. aeruginosa. This lack of correlation between detection by swab sample cultivation and PNA-FISH may be due to the ability of the different bacterial species to colonize different regions of chronic wounds. Swab sample cultivation identifies the microorganisms present in the surface region of the wound but may not detect microorganisms located inside the wound bed. Accordingly, in the present report, we present evidence that S. aureus primarily colonizes the region of chronic wounds which is close to the surface, whereas P. aeruginosa primarily colonizes the deeper regions of chronic wounds. The ability of P. aeruginosa to colonize the deeper regions of chronic wounds may be due to the ability of this organism to produce virulence factors which destroy PMNs (4, 16), and it may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.     

A two‐step one‐pot radiosynthesis of the potent dopamine D2/D3 agonist PET radioligand [11C]MNPA

(R)‐(?)‐2‐[¹¹C]Methoxy‐N‐n‐propylnorapomorphine ([¹¹C]MNPA ([¹¹C]2)) is an agonist radioligand of interest for imaging D₂/D₃ receptors in vivo. Here we sought to develop an improved radiosynthesis of this radioligand. Reference 2 was synthesized in nine steps with an overall yield of about 5%, starting from codeine. Trimethylsilyldiazomethane proved to be a practical improvement in comparison to diazomethane in the penultimate methylation step. A protected precursor for radiolabeling ((R)‐(?)‐2‐hydroxy‐10,11‐acetonide‐N‐n‐propylnoraporphine, 4) was prepared from (R)‐(?)‐2‐hydroxy‐N‐n‐propylnorapomorphine (1) in 30% yield. [¹¹C]2 was prepared from 4 via a two‐step one‐pot radiosynthesis. The first step, methylation of 4 with [¹¹C]methyl triflate, occurred in quantitative radiochemical yield. The second step, deprotection of the catechol moiety with HCl and heat, yielded 60–90% of [¹¹C]2 giving an overall incorporation yield from [¹¹C]methyl triflate of 60–90%. In a typical run more than 1 GBq of [¹¹C]2, was produced from carbon‐11 generated from a 10‐min proton irradiation (16 MeV; 35 µA) of nitrogen–hydrogen target gas. The radiochemical purity of [¹¹C]2 was > 99% and specific radioactivity at the time of injection was 901±342 GBq/µmol (n=10). The total synthesis time was 35–38 min from the end of radionuclide production. The identity of [¹¹C]2 was confirmed by comparing its LC‐MS/MS spectrum with those of reference 2 and (R)‐(?)‐10‐methoxy‐2,11‐dihydroxy‐N‐n‐propylnoraporphine. Copyright © 2009 John Wiley & Sons, Ltd.     

Therapy with pembrolizumab in treatment-naïve patients with nonmetastatic,mismatch repair deficient colorectal cancer

Therapy with immune checkpoint inhibitors (ICI) is effective in patients with metastatic mismatch-repair deficient (dMMR) colorectal cancer (CRC); however, data on treatment with neoadjuvant ICI in patients with locally advanced CRC are limited. From March 2019 to June 2020, five Danish oncological centers treated 10 patients with a treatment-naïve dMMR CRC with preoperative pembrolizumab, 9 with a nonmetastatic, unresectable colon cancer and 1 with a locally advanced rectum cancer. All 10 patients were evaluated regularly at a multidisciplinary team (MDT) meeting, and they all had a radical resection after a median of 8 cycles (range 2-13) of pembrolizumab. A microscopic evaluation of the resected tumors revealed no remaining tumor cells in five patients, while five still had tumor cells present. The patients were given no additional therapy. No recurrences were reported after a median follow-up of 26 months (range 23-38.5 months). Biopsies from Danish patients with CRC are routinely screened for dMMR proteins. In 2017, data from the Danish Colorectal Cancer Group showed that 19% (565/3000) of the patients with colon cancer and 1.5% (19/1279) of those with rectum cancer had an dMMR tumor. Among the patients with MMR determination, 26% (99/384) patients had a T4 dMMR colon cancer; thus, the 10 patients treated with neoadjuvant pembrolizumab comprised about 9% of the patients with a T4 dMMR colon cancer (9/99) and 5% of patients with dMMR rectal cancer (1/19). Therapy with pembrolizumab was feasible and effective. Larger prospective trials are needed to confirm our findings.     

An evaluation of a high‐pressure 11CO carbonylation apparatus

[¹¹C]Carbon monoxide (¹¹CO) is a versatile building block for the synthesis of Positron Emission Tomography (PET) radioligands. However, the difficulty of trapping ¹¹CO in a small solvent volume has limited its utility. We here report an evaluation of a simple, fully automated high‐pressure synthesizer prototype for the use in ¹¹C‐carbonylation reactions. [¹¹C]Carbon monoxide was easily prepared by online reduction of [¹¹C]carbon dioxide using either Mo(s) or Zn(s) as the reducing agent. The conversion yield of ¹¹CO was >99% when zinc was used as the reducing agent, and the corresponding value for Mo was approximately 71%. When the Zn or Mo column was constantly kept under inert atmosphere, no significant decrease in reducing properties was observed for more than 100 ¹¹CO productions. However, in our hands, Mo reductant was much easier to service. A total of nine functional groups were successfully radiolabeled using the ¹¹CO synthesizer prototype. All measured radiochemical yields exceeded 37%, and the ¹¹CO trapping efficiency was generally above 90%, except for the Suzuki coupling where the trapping efficiency was 80%. This high‐pressure synthesizer using [¹¹C]carbon monoxide as the labeling precursor is easy to operate allowing for ¹¹C‐carbonylation reactions to be performed in a high yield and in a routinely fashion.     

Cockroach allergen in house dust

The cockroach allergen (Bla g 1) content was determined in the floor dust of 46 homes with recent cockroach extermination in Amsterdam, The Netherlands. IgE antibodies to Blattella germanica , house-dust mite, cat dander, dog dander, and a mixture of molds were determined in venous blood samples of 46 children (4-12 years) and one of their biologic parents (24-54 years). Specific IgE to cockroach was also determined in a sample of the general population studied in a previous case-control study, one group ( n =20) with and three groups ( n =76) without history of cockroach infestation of the home. Cockroach allergen was detected in floor dust from 44% of the homes, with levels up to 3899 ng Bla g 1/g. Seven of the 46 adults and only one of the 46 children studied had positive RAST to cockroach. Geometric mean cockroach allergen concentrations in living room and master bedroom of sensitized adults were similar to those of nonsensitized adults. In the groups of children without a history of cockroach infestation of the home, positive RAST against cockroach was observed in 16% of the children with respiratory symptoms, in 4% of the children without respiratory symptoms, and in 48% of the children with two or more positive RAST to other allergens. Of the 18 children with positive RAST against cockroach, only one had a history of cockroach infestation of the home and 16 (89%) had also positive RAST against house-dust mite.     

Erythrocyte folate before and during treatment with lithium

We determined erythrocyte folate in normal subjects (blood donors) as well as in manic--depressive patients before the start of prophylactic lithium treatment and at various times during the treatment. Before treatment the patients' erythrocyte folate was 25 per cent lower and after 6 months of treatment it was 10 per cent lower than the control value. Thereafter patient values and control values did not differ. Lithium treatment accordingly normalised on abnormal folate balance, but it is also possible that lithium in itself may have elevated erythrocyte folate. This is supported by animal data. Rats were given ordinary food and food containing lithium. After eight weeks erythrocyte folate was 27 per cent higher in the lithium-treated rats than in the controls. Our study does not support the notion that folate supplements are required during lithium therapy.     

Myocardial Ischemia Induced by 5-Fluorouracil: A Prospective Electrocardiographic and Cardiac Biomarker Study

BackgroundCardiotoxicity induced by 5‐fluorouracil (5‐FU) is well known but poorly understood. In this study, we undertook ECG recording (Holter) and analyses of the biomarkers troponin and copeptin in patients receiving 5‐FU to increase our understanding of the cardiotoxicity.Subjects, Materials, and MethodsPatients with colorectal or anal cancer that received first‐time treatment with 5‐FU‐based chemotherapy were prospectively included. Holter recording, clinical evaluation, 12‐lead electrocardiogram, and assessment of plasma concentrations of troponin I and copeptin were performed before (control) and during 5‐FU treatment (intervention).ResultsA total of 108 patients were included, 82 with colorectal and 26 with anal cancer. The proportion of patients with myocardial ischemia on Holter recording was significantly higher during the first 5‐FU infusion (14.1%) than before (3.7%; p = .001). The ischemic burden per day (p = .001), the number of ST depression episodes per day (p = .003), and the total duration of ischemic episodes per day (p = .003) were higher during the first 5‐FU infusion than before, as was plasma copeptin (p < .001), whereas plasma troponin I was similar (p > 0.999). Six patients (5.6%) developed acute coronary syndromes and two (1.8%) developed symptomatic arrhythmias during 5‐FU treatment.Conclusion5‐FU infusion is associated with an increase in the number of patients with myocardial ischemia on Holter recording. According to biomarker analyses, 5‐FU is associated with an increase in copeptin, but rarely with increases in cardiac troponin I. However, 5%–6% of the patients developed acute coronary syndromes during treatment with 5‐FU.Implications for PracticeSymptomatic 5‐fluorouracil (5‐FU) cardiotoxicity occurs in 0.6%–19% of patients treated with this drug, but a small electrocardiographic (Holter) study has revealed silent myocardial ischemia in asymptomatic patients, suggesting a more prevalent subclinical cardiac influence. This study demonstrated a significant increase in the number of patients with myocardial ischemia on Holter recording during 5‐FU treatment and an increase in ischemic burden. Cardiac biomarker analyses suggested that 5‐FU infusion results in endogenous stress (increased copeptin) but rarely induces myocyte injury (no change in troponin). These findings suggest a more prevalent cardiac influence from 5‐FU and that Holter recording is an important tool in the evaluation of patients with suspected cardiotoxicity from 5‐FU.