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.

Dynamics of the interfacial film in bicontinuous microemulsions based on a partly ionic surfactant mixture: A neutron spin-echo study.

In a microemulsion system based on a mixture of nonionic and ionic surfactants the addition of alcohol instead of changing the temperature was used to tune the curvature of the surfactant interface. The influence of the addition of the short-chain alcohol 2-propanol in the system water-perchloroethylene- Marlowet IHF-2-propanol is studied using neutron spin-echo spectroscopy. In contrast to alcohols with long alkyl chains 2-propanol is no strong co-surfactant, but changes the properties of the solvents. The present contribution focuses on the bicontinuous phase in this system and a quantitative analysis of the obtained neutron spin-echo data is proposed within the theoretical framework given by Zilman and Granek for amphiphilic membranes. It turns out that, in addition to the local movements of the surfactant film, also a collective diffusional mode of the bicontinuous structure has to be taken into account. The presented approach allows to calculate the bending elastic constant κ of the film. The approach is subsequently applied to follow changes of κ as induced by changes of the alcohol concentration.

Structure of biodiesel based bicontinuous microemulsions for environmentally compatible decontamination: A small angle neutron scattering and freeze fracture electron microscopy study.

Most toxic industrial chemicals and chemical warfare agents are hydrophobic and can only be solubilized in organic solvents. However, most reagents employed for the degradation of these toxic compounds can only be dissolved in water. Hence, microemulsions are auspicious media for the decontamination of a variety of chemical warfare agents and pesticides. They allow for the solubilization of both the lipophilic toxics and the hydrophilic reagent. Alkyl oligoglucosides and plant derived solvents like rapeseed methyl ester enable the formulation of environmentally compatible bicontinuous microemulsions. In the present article the phase behavior of such a microemulsion is studied and the bicontinuous phase is identified. Small angle neutron scattering (SANS) and freeze fracture electron microscopy (FFEM) measurements are used to characterize the structure of the bicontinuous phase and allow for an estimation of the total internal interface. Moreover, also the influence of the co-surfactant (1-pentanol) on the structural parameters of the bicontinuous phase is studied with SANS.

In vivo characterization of progestins with reduced non-genomic activity in vitro.

Postmenopausal women that still have an uterus and suffer from hot flushes are treated with combinations of estrogens and progestins. Whereas estrogens are indispensable for treating postmenopausal symptoms, progestins are added to counteract the proliferative activity of estrogens on uterine epithelial cells. However, in the mammary gland, progestins, given together with estrogens, stimulate the proliferation of mammary epithelial cells. Therefore, progestins with reduced proliferative activity in the mammary gland would be of advantage for hormone therapy of postmenopausal women. In order to identify progestins with better tissue-selectivity, we exploited the activation of different signal transduction pathways by the classical progesterone receptor. We demonstrated that progestins with reduced non-genomic versus genomic activity in vitro show a better dissociation of uterine versus mammary gland effects in vivo than medroxyprogesterone acetate (MPA), a synthetic progestin that is widely used in hormone therapy.

CTF5--a new transcriptional activator of the NFI/CTF family.

NFI/CTF is a family of polypeptides involved in stimulating the initiation of adenovirus DNA replication and the activation of transcription driven by RNA polymerase II. Several naturally occurring NFI/CTF variants display distinctive transactivation activities in vivo. To define more precisely the role of the NFI/CTF family in regulating gene expression, we cloned the splice variant CTF5, analyzed transcriptional activation patterns in a yeast transcription assay, and compared it with other CTF proteins. CTF5, which lacks exons 9 and 10 including a CTD-like motif essential for transcriptional activation by full-length CTF1, enhances transcription to a greater extent than CTF1. In addition, CTF5 is even more active than CTF7, which lacks exons 7-9. These findings indicate that CTF proteins formed by differential splicing display a much broader range of transcriptional activities as observed previously.

Interaction of the COOH-terminal transactivation domain of p65 NF-kappa B with TATA-binding protein, transcription factor IIB, and coactivators.

We show that the transactivating COOH terminus of the p65 subunit of human transcription factor NF-kappa B directly binds the general transcription factors TFIIB and TATA-binding protein (TBP) in vitro. Interaction of p65 with TFIIB required the most COOH-terminal sequence repeat within TFIIB. A functional interaction of TFIIB with p65 was evident from assays in yeast cells. Cotransfection experiments in COS cells revealed that only overexpression of TBP was able to further stimulate p65-dependent transactivation of a reporter gene. The coexpression of neither TBP nor TFIIB was able to relieve squelching, indicating the involvement of additional factors in transactivation by p65. A cell-free assay using highly purified factors revealed a specific transcriptional stimulation through the COOH-terminal activation domain of NF-kappa B by at least one cofactor, PC1, isolated from HeLa cells. These data show that the potent acidic transactivation domains in the COOH terminus of p65 are able to functionally recruit various components of the basic transcription machinery as well as coactivators.

[Hepatocellular nodular hyperplasias, adenomas and carcinomas]. / Hepatozelluläre knotige Hyperplasien, Adenome und Karzinome.

Nodular hyperplasias ("hyperplasiomas") are new formations whose development as a required and regulated response can be traced either to compensatory reactions to the loss of cells (regeneration in a narrow sense) and to decreased cellular performance, or to primary growth impulses. Included in this group are: the "macroregenerative nodules" after extensive cell losses; solitary nodules of uncertain etiology; and the minute foci of "micronodular transformation" whose origin can be traced to a particular disturbance of the hepatic blood supply. The so-called "adenomatous hyperplasias" of the cirrhotic liver that have a tendency towards carcinomatous change are not included in this group and are perhaps better considered as "hyperplasiogenic adenomas". The so-called "focal nodular hyperplasia" too, it must be stressed, should be separated from the simple hyperplasias, for it is more closely related to the adenomas, but represents a new formation of limited growth potential. Morphologically it is conspicuously subdivided by multiple connective tissue bands and scars, but it is above all characterized by metaplastically derived neoductuli, and hence it is appropriately designated as a "combined nodule". Among the true uninodular adenomas there are several variants differing in their morphology,--the so-called "atypical" or "intermediate" forms, that can give rise to carcinomas. The hepatocellular carcinoma, that may arise in a variety of ways, presents multiple cytological and histological variants, but only the so-called "fibrolamellar carcinoma" presents also a clinical peculiarity. "Hepatoblastomas" differ from the common hepatocellular carcinomas by their origin in early childhood from immature early precursor cells and, in the later phases of life, from redifferentiated cells that can even give rise to mesenchymal elements. There is no evidence of the existence of particular pluripotential stem cells.

Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation.

The p65 subunit of the NF-kappa B transcription factor contains in its C-terminal 120 amino acids at least two transcription activation domains. One domain (TA1) is contained within only the 30 C-terminal amino acids. Structural studies employing CD and NMR spectroscopy revealed that the TA1 domain is unstructured. NMR analysis of a protein corresponding to the C-terminal 123 amino acids also showed a random coil conformation. However, a portion of TA1 was found to adopt an alpha-helical conformation in the presence of hydrophobic solvents. Transcriptional analysis of point mutants revealed the functional importance of two evolutionary conserved sequence repeats, which are located in the conditionally alpha-helical region of TA1. These repeats acted synergistically in transcription activation. The inhibitory effect of some mutants indicated secondary structure constraints on TA1 in intact cells. Inverting the sequence of two acidic activation domains significantly reduced their transactivating potential, suggesting that amino acid composition is not solely essential for activity; a defined primary structure is necessary as well. Acidic sequence motifs related in primary structure and squelching activity to those of TA1 are present in the activation domains of VP16, c-Rel, and several other transcription factors. We propose a model suggesting that primarily unstructured acidic activation domains can adopt a secondary structure upon contacting their target molecules by an "induced fit" mechanism.

Transcriptional activation of NFI/CTF1 depends on a sequence motif strongly related to the carboxyterminal domain of RNA polymerase II.

Initiation of RNA polymerase II-directed transcription is mediated by DNA sequence specific activator proteins interacting with components of the basal transcription machinery. NFI/CTF is a family of such binding proteins which have been shown to stimulate transcription via proline-rich activation domains. In order to identify residues crucial for its activator function, a pool of CTF1 mutants was cloned and fused to the bacterial repressor LexA. Transcriptional activation of these constructs was monitored in a Saccharomyces cerevisiae reporter assay. Our studies reveal the existence of a core domain in CTF1 between residues 463 and 508 essential for transcriptional activation functions. It contains the sequence motif SPTSPSYSP, which is strongly related to the heptapeptide repeat YSPTSPS present in the carboxyterminal domain (CTD) of RNA polymerase II. Removal of the entire CTD related motif, as well as substitution of key amino acids therein, abolish CTF1 mediated transcriptional activation.

Hepatic neoformations.

The aim of the paper is an accurate histologic description and illustration of those liver lesions that are usually summarized under the heading of "hepatic tumors and related subjects". For in some cases it may be unclear or at least controversial, whether the individual lesion is indeed an autonomous neoplasia or a malformation, regeneration or hyperplasia, the indifferent master term of neoformation is introduced, based on the fact that all of them are characterized by a cellular multiplication. According to common definitory practice the survey distinguishes between mesenchymal (angiomatous and non angiomatous) and epithelial neoformations. Among the latter hepatocellular and cholangiocellular types are distinguished, the criterium for differentiation being a phenomenological one, which is by no means identical with a histogenetical statement. The definition of subgroups mostly adheres to current nomenclatory usage; only occasionally--in the group of endothelial tumors--a novel term is employed, in view of brevity and coordination with the overall system of neoformations.

Transcriptional activation by CTF proteins is mediated by a bipartite low-proline domain.

Members of the CCAAT-binding transcription factor (CTF) family of proteins stimulate the initiation of adenovirus DNA replication and act as transcriptional activators. To investigate the mechanisms underlying CTF-mediated transactivation patterns, we expressed several natural CTF variants in Saccharomyces cerevisiae and determined their transactivating activities in enzymatic assays. CTF7, which lacks the entire proline-rich region previously thought to mediate transcriptional activation by CTF proteins, enhances transcription to a greater degree than full-length CTF1, which contains the putative activation domain. CTF2, which contains a partially deleted proline-rich activation region, does not stimulate transcription at all. These findings indicate that the proline-rich region of CTF proteins is not essential for transcriptional activation in yeast. Our studies also suggest a bipartite two-domain structure of CTF-type transcriptional activation domains.

[Tertiary gummatous syphilis of the liver--an unexpected disease today. Report of two cases]. / Die tertiäre gummatöse Lues der Leber--eine heute unerwartete Erkrankung. Bericht über zwei Fälle.

Today the luetic hepatitis and gummata of liver are very rare manifestations of a tertiary syphilis because of the antibiotic therapy. Therefore a luetic involvement of liver is a most unexpected and at first mostly misinterpreted finding. In the both represented cases foci in the liver were present, which were suspicious for a metastasizing malignoma and set off the search for an unknown primary tumor. In liver biopsies no malignant process was seen, but necroses with a granulomatous reaction at the border were found which were classifiable definitively only after serological investigations as gummata of liver. Especially by the presence of an additionally reactive hepatitis in the remaining liver parenchyma, possibly with some sarcoid-like granulomas, a tertiary gummatous syphilis should be taken into consideration. The regression of gummata in sonography and computertomography as well as the return to normal of the laboratory findings are important parameters in the follow-up to confirm the diagnosis under antibiotic therapy. The described cases stress the importance that even today the tertiary lues should be taken into consideration by liver foci of unknown origin and should be excluded serologically.

Epithelial and mixed hepatoblastoma in the adult. Histological observations and general considerations.

Two instances of hepatoblastoma in adults are reported, with one case representing a purely epithelial, the other a mixed epithelio-mesenchymal variant. The purely epithelial tumour, consisting entirely of so-called fetal elements, was present in the liver of a 35-year-old woman without any other hepatic changes, whereas the mixed tumour developed in the coarsely nodular liver of a 73-year-old man with a currently inactive cirrhosis. Besides the epithelial component, this case held predominantly indifferent spindle-shaped and histiocytic mesenchymal cells which frequently gave rise to osteoid and to bony trabeculae, and on occasion also to vascular structures, biliary ducts and even to groups of hepatocytes. The cytological and histological picture of both cases is comparable even in its details to that seen in the hepatoblastomas of early childhood. This concordance should be insisted upon as a diagnostic pre-condition if a mixed tumour in the adult, consisting of several components, is to be accepted as a hepatoblastoma. This similarity also enables us to recognize the purely epithelial variant of the hepatoblastoma in the older patient as belonging to this tumour category, and to separate it from other hepatic carcinomas. It is suggested that in these tumours the pluripotent cells, or the cells that have again become pluripotent, are derived from differentiated hepatocytes; no evidence in favour of the existence of a particular cellular subpopulation or of "stem cells" has been found.

Coumarin mercapturic acid isolated from rat urine indicates metabolic formation of coumarin 3,4-epoxide.

A coumarin mercapturic acid, N-acetyl-S-(3-coumarinyl)cysteine, has been identified in the urine of coumarin-treated rats. [14C]Coumarin was applied by gavage as a single dose to male Wistar rats (10-150 mg/kg body weight). Twenty-four-hour urine was collected, and the deproteinized concentrate was analyzed for radiolabeled metabolites by HPLC. The new mercapturic acid metabolite is supposed to result from oxidative biotransformation of coumarin to its 3,4-epoxide and subsequent coupling with glutathione.

Effect of ethanol on the induction of lung tumours by ethyl carbamate in mice.

Groups of 25 female NMRI-mice received daily doses of 0, 18, 36, 90, or 180 mg ethyl carbamate/kg body wt either in water or in 20% ethanol by gavage for 8 weeks. Another 8 weeks later, the animals were sacrificed and lung adenomas were counted. Ethyl carbamate was found to increase the number of lung adenomas per mouse dose-dependently in all dose groups. No significant differences, however, were observed between groups receiving ethyl carbamate in water or in 20% ethanol. Thus, ethanol had no effect on ethyl carbamate induced tumourigenesis.

Some histological remarks on the fibrolamellar carcinoma of the liver.

Investigations on five fibrolamellar carcinomas of the liver suggest that this tumor originates from purely epithelial proliferations, while the ensuing fibrous growth leading to lamellar formations is but a secondary event. Nevertheless, progressing fibrosis has a considerable influence on cell shape as the surrounded cell complexes are quasi immured, and their supply and transport procedures impaired. Its influence further evokes a compensatory increase of mitochondria so that, in advanced cases, these cells may be mistaken for genuine oncocytes, although the appraisal of an oncocytic tumor is not confirmed. At this point only, increased amount of fibrinogen-containing (endoplasmic) vacuoles and PAS positive globuli are interpreted as phenomena of cellular retention, and so is the accumulation of unexcretable copper. Ultimately, this fibrous incarceration will cause cell death, destruction and depletion resulting in abundant scarring especially in the center of the focus, without, however, signalling any close relationship with focal nodular hyperplasia. Excess fiber formation exerts a proliferation-inhibiting effect resulting in slower growth and consequently, in the more favorable prognosis of this tumor of distinctive and well-characterized morphology.

Continuous induction of unscheduled DNA synthesis by gamma irradiation.

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo-in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process--with centrifugation in alkaline sucrose a half-life of some minutes is found--an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation.

Poly(ADP-ribose)-synthesis and excision repair in light sensitive skin disorders.

Several data suggest a relationship of poly(ADP-ribose) (PAR) synthesis to DNA repair and the influence of some trace elements on the semiconservative and unscheduled DNA synthesis (UDS). Previously we found certain alterations in the UV-light induced UDS and in the contents of trace elements in the lymphocytes of patients with light sensitive skin disorders. In the recent study in polymorphic light eruption, cutaneous porphyrias and xeroderma pigmentosum the PAR synthesis and zinc, copper and manganese contents in the chromatin of the lymphocytes (measured by neutron activation analysis) were investigated. UV induced PAR synthesis was generally lower in the cells of polymorphic light eruption and especially in xeroderma pigmentosum with a reduced repair capacity whereas in cutaneous porphyrias no difference was observed. Some correlations occurred between the contents of trace elements studied and UDS as well in each group tested. It seems that PAR investigations throw new light upon our understanding of the pathomechanism of photodermatoses.