FLT3-directed UniCAR T-cell therapy of acute myeloid leukaemia.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape, which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform 'UniCAR' is currently under early clinical investigation. Recently, the first proof of concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off switch of UniCAR T cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T cells for the therapy of AML.

The thermal conductivity of cortical and cancellous bone.

Surgical interventions close to vulnerable structures, such as nerves, require precise handling of surgical instruments and tools. These tools not only pose the risk of mechanical damage to soft tissues, but they also generate heat, which can lead to thermal necrosis of bone or soft tissues. Researchers and engineers are trying to improve those tools through experimentation and simulations. To simulate temperature distributions in anatomical structures, reliable material constants are needed. Therefore, this study aimed at investigating the thermal conductivity of cortical and cancellous bone. Accordingly, a custom-made steady-state experimental setup was designed and validated. 6 bovine and 3 human cortical bone samples, as well as 32 bovine cancellous bone samples, with variable bone volume fraction were tested. The cancellous bone samples were scanned by micro-computed tomography (µCT) and micro-finite element (µFE) voxel models were created to calculate iteratively the thermal conductivity of the bone marrow. The experimental results provided 0.64 ± 0.04 W/mK for bovine cortical bone and 0.68 ± 0.01 W/mK for human cortical bone. A linear dependency of thermal conductivity on bone volume fraction was found for cancellous bone [R-square (R2) = 0.8096, standard error of the estimates (SEE) = 0.0355 W/mK]. The thermal conductivity of the bone marrow was estimated to be 0.42 ± 0.05 W/mK. These results will help to improve thermal finite element simulations of the human skeleton and aid the development of new surgical tools or procedures.

Dynamic MR defecography of the posterior compartment: Comparison with conventional X-ray defecography.

PURPOSE: The goal of this study was to compare conventional X-ray defecography and dynamic magnetic resonance (MR) defecography in the diagnosis of pelvic floor prolapse of the posterior compartment. MATERIAL AND METHODS: Fifty women with a mean age of 65.5 years (range: 53-72 years) who underwent X-ray defecography and MR defecography for clinical suspicion of posterior compartment dysfunction, were included in this retrospective study. X-ray defecography and dynamic MR defecography were reviewed separately for the presence of pelvic organ prolapse. The results of the combination of X-ray defecography and MR defecography were used as the standard of reference. Differences in sensitivities between X-ray defecography and MR defecography were compared using the McNemar test. RESULTS: With the gold standard, we evidenced a total of 22 cases of peritoneocele (17 elytroceles, 3 hedroceles and 2 elytroceles+hedroceles), including 15 cases of enterocele, 28 patients with rectocele including 16 that retained contrast, 37 cases of rectal prolapse, and 11 cases of anismus. The sensitivities of X-ray defecography were 90.9% for the diagnosis of peritoneocele, 71.4% for rectocele, 81.1% for rectal prolapse and 63.6% for anismus. The sensitivities of MR defecography for the same diagnoses were 86.4%, 78.6%, 62.2% and 63.6%, respectively. For all these pathologies, no significant differences between X-ray defecography and MR defecography were found. CONCLUSION: Dynamic MR defecography is equivalent to X-ray defecography for the diagnosis of abnormalities of the posterior compartment of the pelvic floor.

Instrument flight to the inner ear.

Surgical robot systems can work beyond the limits of human perception, dexterity and scale making them inherently suitable for use in microsurgical procedures. However, despite extensive research, image-guided robotics applications for microsurgery have seen limited introduction into clinical care to date. Among others, challenges are geometric scale and haptic resolution at which the surgeon cannot sufficiently control a device outside the range of human faculties. Mechanisms are required to ascertain redundant control on process variables that ensure safety of the device, much like instrument-flight in avionics. Cochlear implantation surgery is a microsurgical procedure, in which specific tasks are at sub-millimetric scale and exceed reliable visuo-tactile feedback. Cochlear implantation is subject to intra- and inter-operative variations, leading to potentially inconsistent clinical and audiological outcomes for patients. The concept of robotic cochlear implantation aims to increase consistency of surgical outcomes such as preservation of residual hearing and reduce invasiveness of the procedure. We report successful image-guided, robotic CI in human. The robotic treatment model encompasses: computer-assisted surgery planning, precision stereotactic image-guidance, in-situ assessment of tissue properties and multipolar neuromonitoring (NM), all based on in vitro, in vivo and pilot data. The model is expandable to integrate additional robotic functionalities such as cochlear access and electrode insertion. Our results demonstrate the feasibility and possibilities of using robotic technology for microsurgery on the lateral skull base. It has the potential for benefit in other microsurgical domains for which there is no task-oriented, robotic technology available at present.

Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts.

The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide 'proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo.

Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts.

Bispecific antibodies (bsAbs) engaging T cells are emerging as a promising immunotherapeutic tool for the treatment of hematologic malignancies. Because their low molecular mass, bsAbs have short half-lives. To achieve clinical responses, they have to be infused into patients continously, for a long period of time. As a valid alternative we examined the use of mesenchymal stromal cells (MSCs) as autonomous cellular machines for the constant production of a recently described, fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. The immortalized human MSC line SCP-1 was genetically modified into expressing bsAb at sufficient amounts to redirect T cells efficiently against CD33 presenting target cells, both in vitro and in an immunodeficient mouse model. Moreover, T cells of patients suffering from acute myeloid leukemia (AML) in blast crisis eliminated autologous leukemic cells in the presence of the bsAb secreting MSCs over time. The immune response against AML cells could be enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through CD137L expression on MSCs. This study demonstrates that MSCs have the potential to be used as cellular production machines for bsAb-based tumor immunotherapy in the future.

Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia.

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

The rubber hand illusion increases heat pain threshold.

BACKGROUND: Accumulating evidence shows that manipulations of cortical body representation, for example, by simply viewing one's own body, can relieve pain in healthy subjects. Despite the widespread use of the rubber hand illusion (RHI) as an effective experimental tool for the manipulation of bodily awareness, previous studies examining the analgesic effect of the RHI have produced conflicting results. METHOD: We used noxious heat stimuli to induce finger pain in 29 healthy subjects, and we recorded the participants' pain thresholds and subjective pain ratings during the RHI and during the control conditions. Two control conditions were included in our experiment - a standard one with reduced illusion strength (asynchronous stroking control) and an additional one in which the participants viewed their own hand. RESULTS: Raw data showed that both the RHI and the vision of the own hand resulted in slightly higher pain thresholds than the asynchronous stroking control (illusion: 47.79 °C; own-hand: 47.99 °C; asynchronous: 47.52 °C). After logarithmic transformation to achieve normality, paired t-tests revealed that both increases in pain threshold were significant (illusion/asynchronous: p = 0.036; own-hand/asynchronous: p = 0.007). In contrast, there was no significant difference in pain threshold between the illusion and the own-hand conditions (p = 0.656). Pain rating scores were not log-normal, and Wilcoxon singed-rank tests found no significant differences in pain ratings between the study conditions. CONCLUSION: The RHI increases heat pain threshold and the analgesic effect of the RHI is comparable with that of seeing one's own hand. The latter finding may have clinical implications.

Costimulation improves the killing capability of T cells redirected to tumor cells expressing low levels of CD33: description of a novel modular targeting system.

Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.

[Cerebral listeria abscess in a patient with gastric cancer]. / Zerebraler Listerienabszess bei einer Patientin mit Magenkarzinom.

UNLABELLED: HISTORY AND PRESENTATION ON ADMISSION: A 70-year-old woman with a history of gastric cancer and palliative chemotherapy was admitted with disturbed consciousness. INVESTIGATIONS: The cranial CT showed a tumor in the left parietocccipital region so that a cerebral metastasis was suspected. However further investigations including cranial MRT, liquor and blood culture collection showed that the immunocompromised patient suffered from a Listeria monocytogenes sepsis with subsequent meningitis and a cerebral abscess. TREATMENT AND COURSE: During an antibiotic treatment with ampicillin and gentamycin a rapid neurological improvement and normalization of the liquor findings was achieved. CONCLUSIONS: Listeria monocytogenes is a rare but relevant cause of systemic inflammations in immunocompromised patients. The differentiation between cerebral abscess and metastasis in cancer patients can be complex but is important concerning differential therapeutic approaches.

Social cognition and Theory of Mind in patients with relapsing-remitting multiple sclerosis.

BACKGROUND: Theory of Mind (ToM) is an ability to understand and interpret another person's beliefs, emotions, and intentions. ToM requires both cognitive and emotional perspective taking and is deficient in several neuropsychiatric disorders all connected with impaired social functioning. Cognitive and mood dysfunctions have been recognized as common symptoms in multiple sclerosis (MS). METHODS: We investigated social cognition in 40 ambulatory patients with MS compared to 35 healthy controls by using verbal and non-verbal ToM tests (Faux Pas, Baron-Cohen's Adult Eyes and Faces test) and Baron-Cohen's Empathy questionnaire. The effect of disability and disease duration on social cognition was also analyzed by multiple logistic regression analysis after adjusting for confounding factors of age, gender, intelligence, depression, and anxiety. RESULTS: Even when adjusted, patients with MS made significantly more mistakes in non-verbal test (adult Eyes Test), and more disabled patients performed worse in both verbal and non-verbal ToM tests (Eyes Test and Faux Pas) compared to controls. Patients with a shorter disease course described themselves as more empathetic. DISCUSSION: In the absence of marked cognitive decline and disability, patients with ambulatory MS had a deficit interpreting social situations and performing in interpersonal contexts.

Regional gray matter reduction and theory of mind deficit in the early phase of schizophrenia: a voxel-based morphometric study.

OBJECTIVE: We tested the association between theory of mind (ToM) performance and structural changes in the brains of patients in the early course of schizophrenia. METHOD: Voxel-based morphometry (VBM) data of 18 patients with schizophrenia were compared with those of 21 controls. ToM skills were assessed by computerized faux pas (FP) tasks. RESULTS: Patients with schizophrenia performed significantly worse in FP tasks than healthy subjects. VBM revealed significantly reduced gray matter density in certain frontal, temporal and subcortical regions in patients with schizophrenia. Poor FP performance of schizophrenics correlated with gray matter reduction in the left orbitofrontal cortex and right temporal pole. CONCLUSION: Our data indicate an association between poor ToM performance and regional gray matter reduction in the left orbitofrontal cortex and right temporal pole shortly after the onset of schizophrenia.

Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos.

The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.

Genetics of refractoriness to Plasmodium falciparum in the mosquito Anopheles stephensi.

We previously selected a line of the malaria vector mosquito Anopheles stephensi refractory (resistant) to the human malaria parasite Plasmodium falciparum, using in vitro infections with P. falciparum gametocytes. This report presents data on the genetic background of refractoriness. The results of F1-crosses and backcrosses show that refractoriness to P. falciparum in our A. stephensi line is autosomal and semi-dominant to susceptibility. The expression of refractoriness is apparently affected by a cytoplasmic factor. Interpretation of data from the crosses by quantitative trait locus analysis shows that one gene or two unlinked interacting autosomal genes, or groups of closely linked genes, are involved.

Two-dimensional gel analysis of midgut proteins of Anopheles stephensi lines with different susceptibility to Plasmodium falciparum infection.

Little is known about the composition of the mosquito midgut which plays a central role in the development and subsequent transmission of malaria parasites. As a first step towards the characterization of mosquito midgut molecules involved in the transmission of malaria parasites, we analysed two-dimensional gel electrophoresis patterns of the midgut proteins of sugar-fed and blood-fed Anopheles stephensi lines of different susceptibility to P. falciparum infection. Two lines fully susceptible and one line (Pb3-9A) of reduced susceptibility were used. In the refractory line ookinetes do develop but are only inefficiently transformed into oocysts (Feldmann & Ponnudurai, 1989). The protein profiles of midguts from all sugar-fed mosquito lines were similar. However, after blood feeding, the midgut of the fully susceptible lines contained proteins not found in the midgut of line Pb3-9A. Twenty-nine such proteins were detected and are candidates for involvement in the interaction between the mosquito midgut and P. falciparum.

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.