MRSA Infection in the Thigh Muscle Leads to Systemic Disease, Strong Inflammation, and Loss of Human Monocytes in Humanized Mice

MRSA (Methicillin-resistant Staphylococcus aureus) is the second-leading cause of deaths by antibiotic-resistant bacteria globally, with more than 100,000 attributable deaths annually. Despite the high urgency to develop a vaccine to control this pathogen, all clinical trials with pre-clinically effective candidates failed so far. The recent development of “humanized” mice might help to edge the pre-clinical evaluation closer to the clinical situation and thus close this gap. We infected humanized NSG mice (huNSG: (NOD)-scid IL2Rγnull mice engrafted with human CD34+ hematopoietic stem cells) locally with S. aureus USA300 LAC* lux into the thigh muscle in order to investigate the human immune response to acute and chronic infection. These mice proved not only to be more susceptible to MRSA infection than wild-type or “murinized” mice, but displayed furthermore inferior survival and signs of systemic infection in an otherwise localized infection model. The rate of humanization correlated directly with the severity of disease and survival of the mice. Human and murine cytokine levels in blood and at the primary site of infection were strongly elevated in huNSG mice compared to all control groups. And importantly, differences in human and murine immune cell lineages surfaced during the infection, with human monocyte and B cell numbers in blood and bone marrow being significantly reduced at the later time point of infection. Murine monocytes in contrast behaved conversely by increasing cell numbers. This study demonstrates significant differences in the in vivo behavior of human and murine cells towards S. aureus infection, which might help to sharpen the translational potential of pre-clinical models for future therapeutic approaches

Next-generation humanized NSG-SGM3 mice are highly susceptible to Staphylococcus aureus infection

Humanized hemato-lymphoid system mice, or humanized mice, emerged in recent years as a promising model to study the course of infection of human-adapted or human-specific pathogens. Though Staphylococcus aureus infects and colonizes a variety of species, it has nonetheless become one of the most successful human pathogens of our time with a wide armory of human-adapted virulence factors. Humanized mice showed increased vulnerability to S. aureus compared to wild type mice in a variety of clinically relevant disease models. Most of these studies employed humanized NSG (NOD-scid IL2Rgnull) mice which are widely used in the scientific community, but show poor human myeloid cell reconstitution. Since this immune cell compartment plays a decisive role in the defense of the human immune system against S. aureus, we asked whether next-generation humanized mice, like NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) with improved myeloid reconstitution, would prove to be more resistant to infection. To our surprise, we found the contrary when we infected humanized NSG-SGM3 (huSGM3) mice with S. aureus: although they had stronger human immune cell engraftment than humanized NSG mice, particularly in the myeloid compartment, they displayed even more pronounced vulnerability to S. aureus infection. HuSGM3 mice had overall higher numbers of human T cells, B cells, neutrophils and monocytes in the blood and the spleen. This was accompanied by elevated levels of pro-inflammatory human cytokines in the blood of huSGM3 mice. We further identified that the impaired survival of huSGM3 mice was not linked to higher bacterial burden nor to differences in the murine immune cell repertoire. Conversely, we could demonstrate a correlation of the rate of humanization and the severity of infection. Collectively, this study suggests a detrimental effect of the human immune system in humanized mice upon encounter with S. aureus which might help to guide future therapy approaches and analysis of virulence mechanisms

Introducing a novel model for simulating large loop excision of the transformation zone (LLETZ) using 3D printing technique

Purpose: Electrosurgery is the gold-standard procedure for the treatment of cervical dysplasia. The quality of the outcome depends on the accuracy of performance, which underlines the role of adequate training of surgeons, especially, as this procedure is often performed by novice surgeons. According to our knowledge, medical simulation has up until now lacked a model, which focuses on realistically simulating the treatment of cervical dysplasia with the concerning anatomy. Methods and result: In our work, we present a model created using 3D printing for holistically simulating diagnostic, as well as surgical interventions of the cervix, as realistically as possible. Conclusion: This novel simulator is compared to an existing model and both are evaluated. By doing so, we aim to provide novice gynecologists with standardized and high-quality simulation models for practicing to improve their proficiency. © 2021, The Author(s)

Influence of (R)- and (S)-methadoneconcentrations in the steady-state to the occurrence of withdrawal symptoms and craving in a methadone detoxifikation program

Einfluss der (R)- und (S)-Methadonkonzentrationen im Steady State auf das Auftreten von Entzugssymptomen und Craving bei Opiatabhängigen während der stationären Detoxifikationsbehandlung: Die Methadon-Substitutionsbehandlung ist die weltweit am häufigsten angewandte Therapieform bei Heroinabhängigen. Obwohl nur das (R)-Enantiomer therapeutisch wirksam ist, wird Methadon vorwiegend als Racemat eingesetzt. Bei der Detoxifikationsbehandlung mit Methadon („warmer Entzug“) wird die Dosisanpassung zur Erreichung optimaler Methadonplasmaspiegel durch eine Reihe von Faktoren erschwert: Die komplexe Metabolisierung über das Cytochrom P450-System, die genetischen Unterschiede der einzelnen Patienten, die individuellen Begleitmedikationen und das unterschiedliche Ausmaß des vorherigen Drogenkonsums. In der vorliegenden Studie wur­den die (R)- und (S)-Methadonplasmaspiegel gemessen, und vier Ratinginstrumente erfassten parallel dazu Entzugssymptomatik, das Craving, die Befindlichkeit und die Konzentrations­fähigkeit während der stationären Entgiftungsbehandlung von Opiatabhängigen. Bei der Auswertung der Ergebnisse ließ sich kein signifikanter Zusammenhang zwischen den Methadon-Plasmakonzentrationen und der Entzugssymptomatik, dem Craving, der Befindlichkeit oder der Konzentrationsleistung nachweisen. Bestätigt wurden die große interindividuelle Variabilität der Plasmakonzentrationen von Methadon und die Stereoselektivität des Methadon-Metabolismus. Es konnte gezeigt werden, dass die enantioselektive Bestimmung der Methadon­plasmaspiegel notwendig ist, um aussagekräftige Ergebnisse erzielen zu können, da das (R)-Methadon das aktive Enantiomer darstellt und das Verhältnis (R)/(S)-Methadon individuell von etwa 25/75 bis 75/25 schwanken kann. Häufig auftretende Entzugssymptome wie z.B. „Innere Unruhe“, „Schwitzen“, „Motorische Unruhe“ oder „Schlafstörungen“ konnten in dieser Studie bestätigt werden. Die verwendete Ent­zugscheckliste eignet sich gut, um besonders belastete Patienten „herauszufiltern“, die dann adäquat behandelt werden können. Die Ratinginstrumente für das Craving und die Befindlichkeit eignen sich ebenfalls gut, um überdurchschnittlich gefährdete Patienten zu erkennen oder um in Studien Patientengruppen zu vergleichen. Im Weiteren war im zeitlichen Verlauf ein signifikanter Unterschied zwischen dem Craving am Tag und in der Nacht zu beobachten. Das nächtliche Craving dürfte aufgrund der dann seltener stattfindenden „Drogengespräche“, mangelnder Patientencompliance und des Schlafes reduziert aufgetreten sein. Die Befindlichkeit der Patien­ten ergab in drei Skalen („Gute-Schlechte Stimmung“, „Ruhe-Unruhe“ und „Frustrationstoleranz“) eine breite Streuung. Die „Wachheit-Müdigkeit“ Skala zeigte im Verlauf einen zunehmend „müden, schläfrigen und schlappen“ Zustand an, was sich mit der untersuchten „Schlafqualität“ deckte. Dieser, meist lang andauernde, Befindlichkeitszustand („Hypersensibilisierung gegenüber Reizen von außen“) sollte während der Entgiftungsbehandlung mit besonderem Augenmerk er­fasst werden und mit empathischem Handeln vom Stationsteam aufgefangen werden. Die Auswertung der Plasmaspiegel unter Berücksichtigung der Begleitmedikation ergab einen si­gnifikanten Unterschied im Verlauf der Entgiftung zwischen einer Patientengruppe mit und einer Gruppe ohne Doxepingabe. Eine Hemmung der Isoenzyme CYP1A2, CYP2C9 und CYP2C19 durch Doxepin dürfte für die erhöhte Konzentration von (S)-Methadon eine Rolle spielen. Diesen Enzymen ist folglich beim Metabolismus von (R)-Methadon eine untergeordnete Rolle zuzu­schreiben. Aufgrund der vorliegenden Daten bleibt so bei gleichzeitiger Gabe von Doxepin der Abbau von (R)-Methadon im Gegensatz zu (S)-Methadon unbeeinflusst. Letzte Gewissheit könnten in vivo Studien an genotypisierten Probanden unter strenger Kontrolle der Begleitmedi­kation erbringen. Durch ein therapeutisches Drug Monitoring, wie es in der vorliegenden Studie durchgeführt wurde, könnte die Methadondosierung den Einflüssen der Begleitmedikation und der individu­ellen Stoffwechsellage der Patienten angeglichen werden. Dadurch ließe sich die Behandlung optimal auf die einzelnen Patienten einstellen, um bei einigen mit kürzeren, bei anderen mit längeren Entgiftungen eine höhere Haltequote und damit mehr erfolgreich abgeschlossene Be­handlungen erzielen zu können.Influence of (R)- and (S)-methadoneconcentrations in the steady-state to the occurrence of withdrawal symptoms and craving in a methadone detoxifikation program: The methadone maintenance treatment is the most frequently applied therapy with heroin addicts in the world. Although the (R)-enantiomer is responsible for the therapeutic effect, methadon is predominantly used as racemat. In methadone detoxifikation programs the accommodation of the optimal methadone plasma level is very difficult because of the following factors: the complex metabolism over the cytochrome P450-system, the genetic differences of the individual patients, the individual parallel medications and the different extent of the previous drug consumption. This study measured (R)- and (S)-methadon plasma levels, and parallel the withdrawal symptoms, the craving, the existential orientation and the concentration ability were seized by four rating instruments during a stationary methadone detoxifikation program. Results: During the evaluation we saw no significant correlation between the methadon plasmalevels and the withdrawal symptoms, the craving or the ability of concentration. The large inter-individual variability of the methadone plasma concentrations and the stereoselektive pharmacokinetics of methadon metabolism were confirmed. It could be shown that the enantioselektive determination of the methadonplasmalevels is necessary, in order to be able to obtain meaningful results, because the (R)-methadon represents the active enantiomer and the relationship between (R)/(S)-methadon has a individual variability from approximately 25/75 to 75/25. Frequently arising withdrawals symptoms like „internal unrest“, „sweating“, „motor unrest“ or „sleep disturbances“ could be confirmed in this study. The used withdrawal checklist is suitable well to filter loaded patients, who can be adequately treated then. The rating instruments for the craving and the existential orientation is suitable well, in order to recognize above-average endangered patients or compare groups of patients in studies. In addition a significant difference between the craving during the day and the craving at night could be observed. The reduced nocturnal craving might have arisen because of the more rarely taken place „drug discussions“, the compliance of the patients and the sleep. The existential orientation resulted a broad dispersion in three scales („property-bad tendency“, „quiescent-unrest“ and „frustration tolerance“). The „awake-tiredness“ scale indicated increasingly „a tired, sleepy and slack“ condition in the process, which is in agreement with the examined „sleep quality“. This usually long persisting state of affair („hypersensibilisation in relation to external attractions“) should be seized during the detoxifikation program with special attention and the medical attendant should act with empathy. The evaluation of the methadone plasma levels with consideration of the parallel medication resulted a significant difference in the process of the detoxifikation between a group of patients along and a group without doxepin. An inhibition of the isoenzymes CYP1A2, CYP2C9 and CYP2C19 by doxepin might play a role for the increased concentration of (S)-methadone. Therefore these enzymes has a subordinated role in the metabolism of (R)-methadone. Due to the available data the dismantling of the (R)-methadon remains uninfluenced with simultaneous gift of doxepin, contrary to (S)-methadone. In vivo studies at genotyping patients under strict control of the parallel medication could probably furnish last certainty. By therapeutic drug monitoring, how it was accomplished in the available study, the dose of methadone could be adapted to the influences of the parallel medication and the individual metabolism of the patients. Hence the treatment could be adjusted optimal, individually for patients with shorter, as well as patients with longer detoxification to obtain a higher retaining ratio and more successful treatments could be achieved

Construction of a balanced lethal systems for Salmonella typhi Ty21a

Ziel der Arbeit ist unter anderem die Entwicklung von Vakzinen gegen maligne Neoplasien auf der Basis von attenuierten Bakterien. Sie dienen hierbei als Träger von tumorspezifischen Antigenen. Diese können mit Hilfe des E. coli Hämolysin-a-Sekretionssystems von Salmonella-Bakterienstämmen sezerniert werden und eine spezifische Immunreaktion induzieren. Sowohl die Gene, die für das Sekretionssystem kodieren als auch die Gensequenzen des tumorspezifische Antigens sind bei dem Projekt auf dem detailliert beschriebenen Antigendelivery Plasmid pMO kodiert. Die bis dato angewandte Methode der pMO-Plasmidstabilisierung mit Hilfe von Antibiotikaresistenzgenen beinhaltet jedoch zahlreiche, beschriebene Probleme und wird seitens der Behörden in Impfstämmen zunehmend restriktiv gehandhabt. Im Rahmen der Entwicklung eines bakteriellen Tumorimpfstoffes war es daher das Ziel dieser Arbeit ein System zur Stabilisierung des Antigendelivery Plasmids pMO zu etablieren, das auf den Einsatz von Antibiotikaresistenzgenen verzichtet.Development of a system to stabilize plasmids in bacteria, by using a balanced lethal system, Development of tumor vaccines by using Salmonella bacteri

The JMU-SalVac-System: A Novel, Versatile Approach to Oral Live Vaccine Development

Salmonella enterica Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of any bacterial live vector vaccine correlates with its ability to express and present sufficient antigen, the genes for antigen expression are traditionally located on plasmids with antibiotic resistance genes for stabilization. However, for use in humans, antibiotic selection of plasmids is not applicable, leading to segregational loss of the antigen-producing plasmid. Therefore, we developed an oral Ty21a-based vaccine platform technology, the JMU-SalVac-system (Julius-Maximilians-Universität Würzburg) in which the antigen delivery plasmids (pSalVac-plasmid-series) are stabilized by a ΔtyrS/tyrS+-based balanced-lethal system (BLS). The system is made up of the chromosomal knockout of the essential tyrosyl-tRNA-synthetase gene (tyrS) and the in trans complementation of tyrS on the pSalVac-plasmid. Further novel functional features of the pSalVac-plasmids are the presence of two different expression cassettes for the expression of protein antigens. In this study, we present the construction of vaccine strains with BLS plasmids for antigen expression. The expression of cytosolic and secreted mRFP and cholera toxin subunit B (CTB) proteins as model antigens is used to demonstrate the versatility of the approach. As proof of concept, we show the induction of previously described in vivo inducible promoters cloned into pSalVac-plasmids during infection of primary macrophages and demonstrate the expression of model vaccine antigens in these relevant human target cells. Therefore, antigen delivery strains developed with the JMU-SalVac technology are promising, safe and stable vaccine strains to be used against mucosal infections in humans

Targeting breast cancer stem cells with HER2-specific antibodies and natural killer cells

Breast cancer is the most common cancer among women worldwide. Every year, nearly 1.4 million new cases of breast cancer are diagnosed, and about 450.000 women die of the disease. Approximately 15-25% of breast cancer cases exhibit increased quantities of the trans-membrane receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2) on the tumor cell surface. Previous studies showed that blockade of this HER2 proto-oncogene with the antibody trastuzumab substantially improved the overall survival of patients with this aggressive type of breast cancer. Recruitment of natural killer (NK) cells and subsequent induction of antibody-dependent cell-mediated cytotoxicity (ADCC) contributed to this beneficial effect. We hypothesized that antibody binding to HER2-positive breast cancer cells and thus ADCC might be further improved by synergistically applying two different HER2-specific antibodies, trastuzumab and pertuzumab. We found that tumor cell killing via ADCC was increased when the combination of trastuzumab, pertuzumab, and NK cells was applied to HER2-positive breast cancer cells, as compared to the extent of ADCC induced by a single antibody. Furthermore, a subset of $CD44^{high}CD24^{low}HER2^{low}$ cells, which possessed characteristics of cancer stem cells, could be targeted more efficiently by the combination of two HER2-specific antibodies compared to the efficiency of one antibody. These in vitro results demonstrated the immunotherapeutic benefit achieved by the combined application of trastuzumab and pertuzumab. These findings are consistent with the positive results of the clinical studies, CLEOPATRA and NEOSPHERE, conducted with patients that had HER2-positive breast cancer. Compared to a single antibody treatment, the combined application of trastuzumab and pertuzumab showed a stronger ADCC effect and improved the targeting of breast cancer stem cells

Anti-CD39 and anti-CD73 antibodies A1 and 7G2 improve targeted therapy in ovarian cancer by blocking adenosine-dependent immune evasion

The ectonucleotidases CD39 and CD73 degrade ATP to adenosine which inhibits immune responses via the $A_{2A}$ adenosine receptor (ADORA2A) on T and NK cells. The current study investigates the potential therapeutic use of the specific anti CD39- and anti CD73-antibodies A1 (CD39) and 7G2 (CD73) as these two ectonucleotidases are overexpressed in ovarian cancer (OvCA). As expected, NK cell cytotoxicity against the human ovarian cancer cell lines OAW-42 or SK-OV-3 was significantly increased in the presence of A1 or 7G2 antibody. While this might partly be due to antibody-dependent cell-mediated cytotoxicity, a luciferase-dependent assay for quantifying biologically active adenosine further showed that A1 and 7G2 can inhibit CD39 and CD73-dependent adenosine-generation. In turn, the reduction in adenosine levels achieved by addition of A1 and 7G2 to OAW-42 or SK-OV-3 cells was found to de-inhibit the proliferation of $CD4^+$ T cells in coculture with OvCA cells. Likewise, blocking of CD39 and CD73 on OvCA cells via A1 and 7G2 led to an increased cytotoxicity of alloreactive primed T cells. Thus, antibodies like A1 and 7G2 could improve targeted therapy in ovarian cancer not only by specifically labeling overexpressed antigens but also by blocking adenosine-dependent immune evasion in this immunogenic malignancy

Evaluating a novel 3D printed model for simulating Large Loop Excision of the Transformation Zone (LLETZ)

Background Electrosurgical excisions are common procedures for treating cervical dysplasia and are often seen as minor surgeries. Yet, thorough training of this intervention is required, as there are considerable consequences of inadequate resections, e.g. preterm birth, the risk of recurrence, injuries and many more. Unfortunately, there is a lack of sufficiently validated possibilities of simulating electrosurgeries, which focus on high fidelity and patient safety. Methods A novel 3D printed simulator for examination and electrosurgical treatment of dysplastic areas of the cervix was compared with a conventional simulator. Sixty medical students experienced a seminar about cervical dysplasia. Group A underwent the seminar with the conventional and Group B with the novel simulator. After a theoretical introduction, the students were randomly assigned by picking a ticket from a box and went on to perform the hands-on training with their respective simulator. Each student first obtained colposcopic examination training. Then he or she performed five electrosurgical excisions (each). This was assessed with a validated score, to visualize their learning curve. Furthermore, adequate and inadequate resections and contacts between electrosurgical loop and vagina or speculum were counted. Both groups also assessed the seminar and their simulator with 18 questions (Likert-scales, 1–10, 1 = strongly agree / very good, 10 = strongly disagree / very bad). Group B additionally assessed the novel simulator with four questions (similar Likert-scales, 1–10). Results Nine of 18 questions showed statistically significant differences favoring Group B (p < 0.05). Group B also achieved more adequate R0-resections and less contacts between electrosurgical loop and vagina or speculum. The learning curves of the performed resections favored the novel simulator of Group B without statistically significant differences. The four questions focusing on certain aspects of the novel simulator indicate high appreciation of the students with a mean score of 1.6 points. Conclusion The presented novel simulator shows several advantages compared to the existing model. Thus, novice gynecologists can be supported with a higher quality of simulation to improve their training and thereby patient safety

Evaluating the value of a 3D printed model for hands-on training of gynecological pelvic examination

Background Simulation in the field of gynecological pelvic examination with educational purposes holds great potential. In the current manuscript we evaluate a 3D printed model of the female pelvis, which improves practical teaching of the gynecological pelvic examination for medical staff. Methods We evaluated the benefit of a 3D printed model of the female pelvis (Pelvisio®) as part of a seminar (“skills training”) for teaching gynecological examination to medical students. Each student was randomly assigned to Group A or B by picking a ticket from a box. Group A underwent the skills training without the 3D printed model. Group B experienced the same seminar with integration of the model. Both groups evaluated the seminar by answering five questions on Likert scales (1–10, 1 = “very little” or “very poor”, 10 equals “very much” or “very good”). Additionally, both groups answered three multiple-choice questions concerning pelvic anatomy (Question 6 to 8). Finally, Group B evaluated the 3D printed model with ten questions (Question 9 to 18, Likert scales, 1–10). Results Two of five questions concerning the students’ satisfaction with the seminar and their gained knowledge showed statistically significant better ratings in Group B (6.7 vs. 8.2 points and 8.1 vs. 8.9 points (p < 0.001 and p < 0.009). The other three questions showed no statistically significant differences between the traditional teaching setting vs. the 3D printed model (p < 0.411, p < 0.344 and p < 0.215, respectively). The overall mean score of Question 1 to 5 showed 8.4 points for Group B and 7.8 points for Group A (p < 0.001). All three multiple-choice questions, asking about female pelvic anatomy, were answered more often correctly by Group B (p < 0.001, p < 0.008 and p < 0.001, respectively). The mean score from the answers to Questions 9 to 18, only answered by Group B, showed a mean of 8.6 points, indicating, that the students approved of the model. Conclusion The presented 3D printed model Pelvisio® improves the education of female pelvic anatomy and examination for medical students. Hence, training this pivotal examination can be supported by a custom designed anatomical model tailored for interactive and explorative learning