Teaching Medical Students Rapid Ultrasound for shock and hypotension (RUSH): learning outcomes and clinical performance in a proof-of-concept study.

BACKGROUND: Point-of-care ultrasound (POCUS) is a critical diagnostic tool in various medical settings, yet its instruction in medical education is inconsistent. The Rapid Ultrasound for Shock and Hypotension (RUSH) protocol is a comprehensive diagnostic tool, but its complexity poses challenges for teaching and learning. This study evaluates the effectiveness of a single-day training in RUSH for medical students by assessing their performance in clinical scenarios. METHODS: In this prospective single-center observational proof-of-concept study, 16 medical students from Saarland University Medical Center underwent a single-day training in RUSH, followed by evaluations in clinical settings and on a high-fidelity simulator. Performance was assessed using a standardized scoring tool and time to complete the RUSH exam. Knowledge gain was measured with pre- and post-training written exams, and diagnostic performance was evaluated with an objective structured clinical examination (OSCE). RESULTS: Students demonstrated high performance in RUSH exam views across patients (median performance: 85-87%) and improved scanning times, although not statistically significant. They performed better on simulators than on live patients. Written exam scores significantly improved post-training, suggesting a gain in theoretical knowledge. However, more than a third of students could not complete the RUSH exam within five minutes on live patients. CONCLUSIONS: Single-day RUSH training improved medical students' theoretical knowledge and simulator performance but translating these skills to clinical settings proved challenging. The findings suggest that while short-term training can be beneficial, it may not suffice for clinical proficiency. This study underscores the need for structured and possibly longitudinal training programs to ensure skill retention and clinical applicability.

Prehospital Electrocardiogram Transmission and Prehospital Scene Time: A Retrospective Cohort Study.

Background: Telemedical transmission of prehospital electrocardiograms (ECGs) to a target clinic may improve clinical workflows and speed of intervention. However, whether ECG transmission delays prehospital workflows remains controversial. Therefore, we aimed to clarify whether ECG transmission prolongs prehospital scene time in patients diagnosed with acute coronary syndrome (ACS). Methods: We retrospectively included all patients diagnosed with ACS by prehospital emergency physicians from July 2016 to June 2019 at a single academic center. The primary endpoint was the effect of ECG transmission on prehospital scene time. The secondary endpoints were the effects of ECG diagnosis on prehospital scene time and quality of care. Multivariable regression was used to account for patients' age, physician specialty, completion of specialty training, and whether emergencies occurred throughout the day or night shifts as potential confounders. Results: A total of 1,106 cases were included, of which 154 ECG transmissions were performed. ECG transmission prolonged prehospital scene time by an average of 3 min: adjusted regression coefficient [95% confidence interval (95% CI)]: 3.24 (1.7-4.8), p < 0.001. Prehospital treatment time was not influenced by prehospital ECG-based diagnosis (ST-elevation myocardial infarction [STEMI] vs. non-ST-elevation ACS [NSTE-ACS]): adjusted regression coefficient (95% CI): 0.7 (-1.3 to 2.7), p = 0.490. Emergency physicians adhered to local standard operating procedures in 739 of 1,007 (73%) patients diagnosed with NSTE-ACS and 93 of 99 (94%) patients diagnosed with STEMI. A STEMI diagnosis compared with NSTE-ACS was associated with five times higher odds of adhering to standard operating procedures (odds ratio [95% CI]: 5.6 [2.7-14.6], p < 0.001). Conclusion: The observed delay of ∼3 min in the prehospital scene time by ECG transmission is clinically irrelevant. For patients prehospitally diagnosed with NSTE-ACS who do not meet STEMI criteria, adherence to standard operating procedures seems unjustifiably low and should be improved.

Racial and ethnic disparities in diagnostic efficacy of comprehensive genetic testing for sensorineural hearing loss.

Understanding racial and ethnic disparities in diagnostic rates of genetic testing is critical for health equity. We sought to understand the extent and cause of racial and ethnic disparities in diagnostic efficacy of comprehensive genetic testing (CGT) for sensorineural hearing loss (SNHL). We performed a retrospective cohort study at two tertiary children's hospitals on a diverse cohort of 240 consecutive pediatric patients (76% publicly insured, 82% non-White) with SNHL of unknown etiology who underwent CGT. Definite and possible genetic diagnoses were assigned for each patient, representing the likelihood of a genetic cause of hearing loss. Associations between diagnostic rates were examined. 3.8 ± 2.1 variants were detected per patient; this frequency did not vary between White/Asian and Hispanic/Black cohorts. Overall, 82% of variants were variants of uncertain significance (VUS). Compared with White and Asian subjects, variants identified among Hispanic and Black children were less likely to be classified as pathogenic/likely pathogenic (15% vs. 24%, p < 0.001), and Hispanic and Black children were less likely to have a definite genetic diagnosis (10% vs. 37%, p < 0.001). The adjusted odds ratio for definite genetic diagnosis in Black and Hispanic children compared with White and Asian children was 0.19. Expanding genetic diagnostic criteria to include predicted deleterious VUSs reduced these disparities between White/Asian and Hispanic/Black children, with comparable molecular diagnostic rates (41% vs. 38%, p = 0.72). However, in silico predictions are insufficiently valid for clinical use. Increased inclusion of underrepresented groups in genetic hearing-loss studies to clinically validate these variants is necessary to reduce racial and ethnic disparities in diagnostic efficacy of comprehensive genetic testing.

Pandemics and pediatric otolaryngology.

The objective is to describe how the COVID pandemic changed the epidemiology and management of pediatric otolaryngologic diseases, which may influence clinical decision-making in the future. Many changes were made to the structure of healthcare delivery to minimize transmission of coronavirus. As a result, there was a widespread adoption of telehealth. Additionally, guidelines were published with new protocols for evaluation and management of common pediatric otolaryngologic conditions, which in many circumstances, delayed or replaced surgical intervention. Now, as we evaluate the impact of these clinical changes, we have gained new understanding about the pathophysiology of certain pediatric conditions, namely otitis media, for which upper respiratory infection exposure may play a larger role than previously thought. As we have altered practice patterns for common pediatric otolaryngologic conditions, we recognize that ongoing research may help us determine if surgical interventions have been overutilized in the past and help guide clinical practice guidelines moving forward.

Preparing for the SARS-CoV-2 pandemic: creation and implementation of new recommendations.

During the SARS-CoV-2 pandemic in 2020, departments of anesthesiology worldwide have encountered new and unique challenges. In this short communication, we present and assess our recommendations for orotracheal intubation, a frequent high-risk procedure. We will point out that interdisciplinary cooperation with "non-patient care" departments like the Institute for Medical Microbiology and Hygiene tremendously helped us in creating this and other new, clear standards for anesthesiological procedures. Moreover, to reliably implement our newly created measures, we distributed incisive posters and organized comprehensive training sessions. Eventually, we summarize and analyze the occurring problems of our suggestions for intubation during their realization.

Transfusion Camp: a prospective evaluation of a transfusion education program for multispecialty postgraduate trainees.

BACKGROUND: The optimal method of providing transfusion medicine (TM) education has not been determined. Transfusion Camp was established in 2012 at the University of Toronto as a centrally delivered TM education program for postgraduate trainees. The impact of Transfusion Camp on knowledge, attitudes, and self-reported behavior was evaluated. METHODS: Didactic lectures (delivered locally, by webinar, or recorded) and locally facilitated team-based learning seminars were delivered over 5 days during the academic year to 8 sites: 7 in Canada and 1 in the United Kingdom. Knowledge assessment using a validated 20-question multiple-choice exam was conducted before and after Transfusion Camp. Attitudes and self-reported behavior were collected through a survey. RESULTS: Over 2 academic years (July 2016 to June 2018), 390 trainees from 16 different specialties (predominantly anesthesia, 41%; hematology, 14%; and critical care, 7%) attended at least 1 day of Transfusion Camp. The mean pretest score was 10.3 of 20 (±2.9; n = 286) compared with posttest score of 13.0 (±2.8; n = 194; p < 0.0001). Lower pretest score and greater attendance (4-5 days compared with 1-3 days) were associated with larger improvement in posttest score; delivery format, specialty, and postgraduate year were not. Trainees reported an improvement in self-rated abilities to manage TM scenarios; 95% rated TM knowledge as very or extremely important in providing patient care; and 81% indicated that they had applied learning from Transfusion Camp into clinical practice. CONCLUSIONS: Transfusion Camp increased TM knowledge, fostered a positive attitude toward TM, and enabled a self-reported positive impact on transfusion practice in postgraduate trainees. It is a novel and scalable approach to delivering TM education.

Process mining is an underutilized clinical research tool in transfusion medicine.

BACKGROUND: To understand inventory performance, transfusion services commonly use key performance indicators (KPIs) as summary descriptors of inventory efficiency that are graphed, trended, and used to benchmark institutions. STUDY DESIGN AND METHODS: Here, we summarize current limitations in KPI-based evaluation of blood bank inventory efficiency and propose process mining as an ideal methodology for application to inventory management research to improve inventory flows and performance. RESULTS: The transit of a blood product from inventory receipt to final disposition is complex and relates to many internal and external influences, and KPIs may be inadequate to fully understand the complexity of the blood supply chain and how units interact with its processes. Process mining lends itself well to analysis of blood bank inventories, and modern laboratory information systems can track nearly all of the complex processes that occur in the blood bank. CONCLUSION: Process mining is an analytical tool already used in other industries and can be applied to blood bank inventory management and research through laboratory information systems data using commercial applications. Although the current understanding of real blood bank inventories is value-centric through KPIs, it potentially can be understood from a process-centric lens using process mining.

[Comparison of the preclinical quality of analgesia of emergency physicians and paramedics based on trauma patients]. / Vergleich der präklinischen Analgesiequalität von Notarzt und Notfallsanitäter anhand traumatologischer Patienten.

BACKGROUND: Adequate prehospital pain management is a critical component of emergency medical services. With the introduction of the paramedic profession and the Paramedics Act in Germany, the basis for more extensive competencies of paramedics was established. In many emergency medical service areas it is thus possible for paramedics to perform analgesia and sedation with esketamine/midazolam according to pre-established instructions and/or standard operating procedures. This study assessed the quality of analgesia administered to trauma patients by paramedics compared to emergency medical service physicians. MATERIAL AND METHODS: The study included trauma patients who received prehospital administration of analgesia by either emergency medical service physicians or paramedics and were subsequently admitted to the central emergency department of the Saarland University Hospital. A standardized data collection form was used to collect information from the emergency service protocol and initial emergency department assessment. The evaluation employed descriptive statistical methods and a total of 207 completed records were analyzed. RESULTS: Both professional groups achieved significant pain reduction and fulfilled the criteria for effective pain management (pain reduction: emergency medical service physicians 5.5 ± 2.0/paramedic 4.4 ± 2.1, p < 0.001). Emergency medical service physicians, however, more frequently attained a higher reduction in numerical rating scale scores and administered oxygen. Notable differences were observed in the range of medications used and the dosages. CONCLUSION: This study could show that prehospital analgesia is comparable between emergency medical service physicians and paramedics in terms of effectiveness for trauma patients if the indications are correctly set, while observing pre-existing instructions. With their competencies paramedics are able to perform an effective and safe analgesic treatment within the framework of preformulated procedural instructions, which can be equal to that of an emergency medical service physician.

The E2F1/DNMT1 axis is associated with the development of AR negative castration resistant prostate cancer.

BACKGROUND: Research on castration resistant prostate cancer (CRPC) has focused primarily on functional alterations of the androgen receptor (AR). However, little is known about the loss of AR gene expression itself and the possible contribution of AR negative cells to CRPC. METHODS: Human and murine prostate cancer tissue microarrays (TMAs) were evaluated with antibodies specific for E2F1, DNA methyltransferase 1 or AR. The human prostate cancer TMA consisted of clinical samples ranging from normal tissue to samples of metastatic disease. The murine TMA was comprised of benign, localized or metastatic prostate cancer acquired from TRAMP mice treated with castration and/or 5'-Aza-2'-deoxycytidine (5Aza). RESULTS: Immunohistochemical analysis revealed increased nuclear DNMT1 staining in localized PCa (P < 0.0001) and metastatic PCa (P < 0.0001) compared to normal tissue. Examination of specific diagnoses revealed that Gleason seven tumors exhibited greater nuclear DNMT1 staining than Gleason six tumors (P < 0.05) and that metastatic tissue exhibited greater levels of nuclear DNMT1 than Gleason seven tumors (P < 0.01). Evaluation of the murine tissue cores revealed that 8.2% and 8.1% of benign tissue cores stained positive for E2F1 and DNMT1 respectively, while 97.0% were AR positive. Conversely, 81% and 100% of tumors were positive for E2F1 and DNMT1 respectively. This was in stark contrast to only 18% of tumors positive for AR. Treatment of mice with 5Aza reduced DNMT1 staining by 30%, while AR increased by 27%. CONCLUSIONS: These findings demonstrate that the E2F1/DNMT1 inhibitory axis of AR transcription is activated during the emergence of CRPC.

Harnessing oncolytic virus-mediated antitumor immunity in an infected cell vaccine.

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSV's extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.

Immunohistochemical and Molecular Genetic Analysis of Canine Digital Mast Cell Tumours.

Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs.

Comparative Study of Digital Squamous Cell Carcinoma in Giant, Standard, and Miniature Schnauzers.

In schnauzers, a breed predisposition to squamous cell carcinoma of the digit (dSCC) is well known. The aim of this study was to compare the clinical and macroscopic findings of dSCCs in giant (GSs), standard (SSs), and miniature schnauzers (MSs). METHODS: Pathology reports of 478 dSCCs from 417 schnauzers (227 GSs, 174 SSs, and 16 MSs) were retrospectively evaluated. RESULTS: The MSs were older than the SSs and GSs (p ≤ 0.01). The male GSs were predisposed to dSCC (p < 0.05). In the GSs, the nodular dSCCs were larger than in the MSs (p ≤ 0.05) and SSs (p ≤ 0.001). The digital SCCs were mostly diagnosed at the forelimbs, especially at digits 1, 2, and 5. At the hindlimbs, the affected toes differed between the GSs and SSs. Multiple dSCCs were more common in SSs than in GSs (p = 0.003). If dSCC was the cause of death, the survival time was shorter than in dogs dying from other diseases (p = 0.004). Metastases occurred in 20% of the cases and led to a significantly shorter survival time in both the GSs and SSs (p < 0.001). CONCLUSIONS: The results showed various differences in the dSCC depending on the size variant of the schnauzer.

The calcium channel modulator 2-APB hydrolyzes in physiological buffers and acts as an effective radical scavenger and inhibitor of the NADPH oxidase 2.

2-aminoethoxydiphenyl borate (2-APB) is commonly used as a tool to modulate calcium signaling in physiological studies. 2-APB has a complex pharmacology and acts as activator or inhibitor of a variety of Ca2+ channels and transporters. While unspecific, 2-APB is one of the most-used agents to modulate store-operated calcium entry (SOCE) mediated by the STIM-gated Orai channels. Due to its boron core structure, 2-APB tends to readily hydrolyze in aqueous environment, a property that results in a complex physicochemical behavior. Here, we quantified the degree of hydrolysis in physiological conditions and identified the hydrolysis products diphenylborinic acid and 2-aminoethanol by NMR. Notably, we detected a high sensitivity of 2-APB/diphenylborinic acid towards decomposition by hydrogen peroxide to compounds such as phenylboronic acid, phenol, and boric acid, which were, in contrast to 2-APB itself and diphenylborinic acid, insufficient to affect SOCE in physiological experiments. Consequently, the efficacy of 2-APB as a Ca2+ signal modulator strongly depends on the reactive oxygen species (ROS) production within the experimental system. The antioxidant behavior of 2-APB towards ROS and its resulting decomposition are inversely correlated to its potency to modulate Ca2+ signaling as shown by electron spin resonance spectroscopy (ESR) and Ca2+ imaging. Finally, we observed a strong inhibitory effect of 2-APB, i.e., its hydrolysis product diphenylborinic acid, on NADPH oxidase (NOX2) activity in human monocytes. These new 2-APB properties are highly relevant for Ca2+ and redox signaling studies and for pharmacological application of 2-APB and related boron compounds.

KITLG Copy Number Germline Variations in Schnauzer Breeds and Their Relevance in Digital Squamous Cell Carcinoma in Black Giant Schnauzers.

Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS (18 with and 4 without dSSC; 5 unknown), and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5-7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7-5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC.

Induction of CD4(+)CD25(+)Foxp3(-) regulatory T cells by Thy-1 stimulation of CD4(+) T cells.

Thy-1 (CD90) on mouse T cells has been reported to have both T-cell activating and regulatory roles. In this study, we show that monoclonal antibody (mAb)-mediated crosslinking of Thy-1 on CD4(+) mouse T-cells-induced regulatory T (T(reg)) cells that expressed CD25, CD39 and glucocorticoid-induced tumor necrosis factor receptor family-related gene, but not CD73, CD122 or Foxp3. The proliferation of CD4(+) T(responder) cells in response to anti-CD3/anti-CD28mAb-coated T-cell expander beads or syngeneic dendritic cells and soluble anti-CD3mAb was inhibited by Thy-1-induced T(reg) cells, in spite of elevated IL-2 levels in the co-cultures. Interestingly, stimulation with T-cell expander beads caused Thy-1-induced T(reg) cells to synthesize large amounts of interleukin-2 (IL-2). IL-10 was also elevated in co-cultures of activated T(responder) cells and Thy-1-induced T(reg) cells. However, mAb-mediated neutralization of IL-10 did not restore T(responder)-cell proliferation to control levels, which excluded IL-10 as a potential mediator of Thy-1-induced T(reg)-cell suppressor function. In addition, Thy-1-induced T(reg) cells did not inhibit IL-2-dependent proliferation of CTLL-2 cells, suggesting that IL-2 receptor signaling remained intact in the presence of Thy-1-induced T(reg) cells. We suggest that T(reg) cells induced by Thy-1 ligation in vivo may contribute to the maintenance of T-cell homeostasis.

Deprivation-based inequalities in under-18 conception rates and the proportion of under-18 conceptions leading to abortion in England, 1998-2010.

BACKGROUND: In 1999, the UK Government launched a strategy to reduce teenage pregnancy and geographical inequalities in teenage conception rates. This study investigates how associations between deprivation and under-18 conceptions, along with subsequent abortions, since changed as teenage pregnancy rates fell. METHODS: A data set was constructed from local authority Indices of Multiple Deprivation (IMD) scores and routine data on under-18 conception rates and the proportion of under-18 conceptions leading to abortion from 1998 to 2010. Correlation analysis (Pearson's r) was used to measure the association between each period of conception and abortion data and the relevant version of the IMD. Changes in these correlations over the period were tested for statistical significance. RESULTS: There remained a strong association between IMD and under-18 conception rates from 1998 (r = 0.782, P< 0.0001) to 2010 (r = 0.817, P< 0.0001) with no statistically significant change. A statistically significant decrease occurred in the inverse association between IMD and the proportion of under-18 conceptions leading to abortion from 1998 (r = -0.501, P< 0.0001) to 2010 (r = -0.332, P< 0.0001) CONCLUSIONS: While under-18 conceptions fell from 1998 to 2010, inequalities in rates between the most and least deprived local authorities remained undiminished. At the same time, abortion became an increasingly common outcome of under-18 conceptions.

Pathological Findings in Gastrointestinal Neoplasms and Polyps in 860 Cats and a Pilot Study on miRNA Analyses.

Background: Gastrointestinal masses in cats are of clinical relevance, but pathological studies with larger case numbers are lacking. Biomarkers such as miRNA have not yet been investigated in feline intestinal neoplasms. Methods: A retrospective analysis of pathology reports included 860 feline gastrointestinal masses. Immunohistochemistry was performed on 91 lymphomas, 10 sarcomas and 7 mast cell tumours (MCT). Analyses of miRNA-20b and miRNA-192 were performed on 11 lymphomas, 5 carcinomas and 5 control tissues by ddPCR. Results: The pathological diagnosis identified 679 lymphomas, 122 carcinomas, 28 sarcomas, 23 polyps, 7 MCT and 1 leiomyoma. Carcinomas and polyps were most commonly found in the large intestine, lymphomas were most commonly found in the stomach and small intestine and MCT only occurred in the small intestine. Besides the well-described small-cell, mitotic count <2 T-cell lymphomas and the large-cell B-cell lymphomas with a high mitotic count, several variants of lymphomas were identified. The values of miRNA-20b were found to be up-regulated in samples of all types of cancer, whereas miRNA-192 was only up-regulated in carcinomas and B-cell lymphomas. Conclusions: The histopathological and immunohistochemical (sub-)classification of feline intestinal masses confirmed the occurrence of different tumour types, with lymphoma being the most frequent neoplasm. Novel biomarkers such as miRNA-20b and miRNA-192 might have diagnostic potential in feline intestinal neoplasms and should be further investigated.

Molecular Genetic Investigation of Digital Melanoma in Dogs.

Canine digital melanoma, in contrast to canine oral melanoma, is still largely unexplored at the molecular genetic level. The aim of this study was to detect mutant genes in digital melanoma. Paraffin-embedded samples from 86 canine digital melanomas were examined for the BRAF V595E variant by digital droplet PCR (ddPCR), and for exon 11 mutations in c-kit. Furthermore, exons 2 and 3 of KRAS and NRAS were analysed by Sanger sequencing. Copy number variations (CNV) of KITLG in genomic DNA were analysed from nine dogs. The BRAF V595E variant was absent and in c-kit, a single nucleotide polymorphism was found in 16/70 tumours (23%). The number of copies of KITLG varied between 4 and 6. KRAS exon 2 codons 12 and 13 were mutated in 22/86 (25.6%) of the melanomas examined. Other mutually exclusive RAS mutations were found within the hotspot loci, i.e., KRAS exon 3 codon 61: 2/55 (3.6%); NRAS exon 2 codons 12 and 13: 2/83 (2.4%); and NRAS exon 3 codon 61: 9/86 (10.5%). However, no correlation could be established between histological malignancy criteria, survival times and the presence of RAS mutations. In summary, canine digital melanoma differs from molecular genetic data of canine oral melanoma and human melanoma, especially regarding the proportion of RAS mutations.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling.

Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4(+) T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca(2+) release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4(+)CD25(+) regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.