In-vitro model to mimic T cell subset change in human PDAC organoid co-culture.

PURPOSE: Immunotherapies have largely failed as treatment options for pancreatic ductal adenocarcinoma (PDAC). In this field, clinical translational studies into personalized treatment are of fundamental importance. In our study, we model tumor-cell immune-cell interactions in a co-culture of primary human PDAC organoids and matched peripheral blood mononuclear cells (PBMCs). METHODS: Using flow cytometry, we evaluated changes in T cell subtypes upon co-culture of patient-derived PDAC organoids and matched PBMCs. RESULTS: After co-culturing PDAC organoids with PBMCs, we observed changes in CD4+, CD8+ and Treg cell populations. We observed favorable clinical outcome in patients whose PBMCs reacted to the co-culture with organoids. CONCLUSION: This experimental model allows to investigate interactions between patient derived PDAC organoids and their PBMCs. This co-culture system could serve as a preclinical platform to guide personalized therapeutic strategies in the future.

Tailed forisomes of Canavalia gladiata: a new model to study Ca2+-driven protein contractility.

BACKGROUND AND AIMS: Forisomes are Ca(2+)-dependent contractile protein bodies that form reversible plugs in sieve tubes of faboid legumes. Previous work employed Vicia faba forisomes, a not entirely unproblematic experimental system. The aim of this study was to seek to establish a superior model to study these intriguing actuators. METHODS: Existing isolation procedures were modified to study the exceptionally large, tailed forisomes of Canavalia gladiata by differential interference contrast microscopy in vitro. To analyse contraction/expansion kinetics quantitatively, a geometric model was devised which enabled the computation of time-courses of derived parameters such as forisome volume from simple parameters readily determined on micrographs. KEY RESULTS: Advantages of C. gladiata over previously utilized species include the enormous size of its forisomes (up to 55 microm long), the presence of tails which facilitate micromanipulation of individual forisomes, and the possibility of collecting material repeatedly from these fast-growing vines without sacrificing the plants. The main bodies of isolated Canavalia forisomes were box-shaped with square cross-sections and basically retained this shape in all stages of contraction. Ca(2+)-induced a 6-fold volume increase within about 10-15 s; the reverse reaction following Ca(2+)-depletion proceeded in a fraction of that time. CONCLUSIONS: The sword bean C. gladiata provides a superior experimental system which will prove indispensable in physiological, biophysical, ultrastructural and molecular studies on the unique ATP-independent contractility of forisomes.

Biomimetic actuators: where technology and cell biology merge.

The structural and functional analysis of biological macromolecules has reached a level of resolution that allows mechanistic interpretations of molecular action, giving rise to the view of enzymes as molecular machines. This machine analogy is not merely metaphorical, as bio-analogous molecular machines actually are being used as motors in the fields of nanotechnology and robotics. As the borderline between molecular cell biology and technology blurs, developments in the engineering and material sciences become increasingly instructive sources of models and concepts for biologists. In this review, we provide a--necessarily selective--summary of recent progress in the usage of biological and biomimetic materials as actuators in artificial environments, focussing on motors built from DNA, classical cellular motor systems (tubulin/kinesin, actin/myosin), the rotary motor F1F0-ATPase and protein-based 'smart' materials.

Reversible calcium-regulated stopcocks in legume sieve tubes.

Sieve tubes of legumes (Fabaceae) contain characteristic P-protein crystalloids with controversial function. We studied their behavior by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media containing either Ca(2)+ or chelators. Sr(2)+ and Ba(2)+, but not Mg(2)+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with which to control sieve tube conductivity.

Novel approach in plastid transformation.

Engineering the nuclear genome of plants is perceived to be associated with problems regarding biosafety and the stability of expression of the transgene. Alternative transformation strategies using the genomic outfit of the plastid promise to be more successful in this respect. Over the past few years progress has been made in screening procedures, and plastid transformation technology has allowed function to be assigned to open reading frames, massive expression of insecticidal agents and proteins involved in herbicide resistance, and the accumulation of biopolymers. Recently, the design of a novel femtoinjection technique that allows injection into chloroplasts has provided the opportunity to further manipulate and understand chloroplastic gene expression.

High-throughput scanning of the rat genome using interspersed repetitive sequence-PCR markers.

We report the establishment of a hybridization-based marker system for the rat genome based on the PCR amplification of interspersed repetitive sequences (IRS). Overall, 351 IRS markers were mapped within the rat genome. The IRS marker panel consists of 210 nonpolymorphic and 141 polymorphic markers that were screened for presence/absence polymorphism patterns in 38 different rat strains and substrains that are commonly used in biomedical research. The IRS marker panel was demonstrated to be useful for rapid genome screening in experimental rat crosses and high-throughput characterization of large-insert genomic library clones. Information on corresponding YAC clones is made available for this IRS marker set distributed over the whole rat genome. The two existing rat radiation hybrid maps were integrated by placing the IRS markers in both maps. The genetic and physical mapping data presented provide substantial information for ongoing positional cloning projects in the rat.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.

Use of animal models to search for candidate genes associated with essential hypertension.

The use of inbred genetically hypertensive animal models enables the dissection of the underlying complex genetic traits into its individual components, and thus the elucidation and characterization of causative genes and gene variants. In addition, genetically hypertensive animal models will also be useful for the investigation of genetic characteristics that influence the effectiveness of antihypertensive therapy with specific pharmacologic agents. This report will discuss three different strategies that have recently been used for the identification of candidate gene loci or candidate genes for hypertension. The possibility to transfer of genetic data derived in animal models to human hypertension will also be considered.

[C-reactive protein and relative lymphocytopenia: early markers of acute myocardial infarction?]. / C-reaktives Protein und relative Lymphozytopenie: Frühmarker des akuten Myokardinfarkts?

OBJECTIVE: Recent data suggest that relative lymphocytopenia and elevated C-reactive protein (CRP) are early markers of myocardial infarction. We tested these two parameters to predict myocardial infarction before elevation of creatine kinase. METHODS: Over a two-year period, 260 patients presented at the emergency room of Männedorf Hospital with suspicion of unstable angina or myocardial infarction. 197 patients were excluded because of intercurrent conditions associated with an acute-phase response or changes in leukocyte counts, as well as patients with established myocardial infarction (creatine kinase elevation at entry). The remaining 63 patients were reviewed for relative lymphocytopenia (< 20.3%) and C-reactive protein levels > 5 mg/l at admission. RESULTS: Elevated levels of C-reactive protein were found in 8 of 20 patients (40%) with unstable angina and in 29 of 43 patients (67%) with myocardial infarction. A value for C-reactive protein > 5 mg/l on admission had a sensitivity of 67% and a predictive value of 78% for subsequent myocardial infarction. Relative lymphocytopenia was found in 2 patients (10%) with unstable angina and in 19 patients (44%) with myocardial infarction. The positive predictive value of both markers diagnosing myocardial infarction was 93% compared to 78% of elevated CRP or 90% of relative lymphocytopenia. In contrast, the sensitivity of both markers combined was 33%. CONCLUSIONS: At present, elevation of C-reactive protein and relative lymphocytopenia allow early diagnosis of myocardial infarction. However, the markers' sensitivity is relatively low.

Angiotensin-converting enzyme and angiotensinogen gene polymorphisms and heart rate variability in twins.

Decreased heart rate variability (HRV) is associated with congestive heart failure, post-myocardial infarction, ventricular arrhythmias, sudden cardiac death, and advancing age. A deletion/insertion polymorphism in the angiotensin-converting enzyme (ACE) gene and a substitution (M235T) in the angiotensinogen gene have been associated with risk for heart disease. The aim of this study was to determine the heritability of HRV and related parameters in monozygotic and dizygotic twins and to assess the influence of ACE and angiotensinogen polymorphisms. We studied 95 MZ pairs and 46 DZ pairs. We measured HRV and related parameters, ACE and angiotensinogen levels, plasma norepinephrine, ACE, and angiotensinogen genotypes. We found that HRV and related parameters were significantly influenced by genetic variability, although nonshared genetic effects were also important. Angiotensinogen and plasma norepinephrine were generally correlated with decreased HRV, whereas ACE was correlated with perturbances of normal rhythmic HRV. Nevertheless, the DD ACE genotype was associated with increased HRV (p <0.05), whereas angiotensinogen polymorphisms had no effect. We conclude that HRV and related parameters are in part heritable. Interestingly, the DD ACE genotype is associated with increased HRV.

Integration of foreign DNA and its consequences in mammalian systems.

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.

Angiotensin-converting enzyme and angiotensinogen gene polymorphisms, plasma levels, cardiac dimensions. A twin study.

We tested the hypotheses that angiotensin-converting enzyme insertion/deletion (I/D) and angiotensinogen 235 methionine/threonine (M/T) substitution gene polymorphisms influence angiotensin-converting enzyme and angiotensiongen serum concentrations and cardiac dimensions in 91 monozygotic and 41 dizygotic twin pairs. Cardiac dimensions were determined echocardiographically. Angiotensin-converting enzyme levels were 24 +/- 11, 43 +/- 18, and 58 +/- 24 U/L for the II, ID, and DD genotypes, respectively (P < .01). Posterior wall thickness was 8.1 +/- 1.3, 8.6 +/- 1.7, and 8.9 +/- 1.9 mm for these genotypes (P < .05). Angiotensin-converting enzyme levels were correlated with posterior wall thickness (r = .15, P < .05). The intrapair differences in angiotensin converting enzyme levels for monozygotic, concordant dizygotic, and discordant dizygotic twins were 1.36 +/- 1.6, 1.86 +/- 1.6, and 17.25 +/- 4.3 U/L, respectively. The angiotensinogen M/T genotypes exerted no influence on cardiac dimensions or on angiotensinogen concentrations. The additive genetic effect on angiotensin-converting enzyme levels (0.49), on posterior wall thickness (0.26), and on septum thickness (0.37) was significant (P < .01), although shared and nonshared environmental effects were also identified. Our data confirm the impressive effect that the angiotensin-converting enzyme D allele exerts on angiotensin-converting enzyme plasma levels. Furthermore, our data also suggest that the angiotensin-converting enzyme gene locus is primarily responsible for angiotensin-converting enzyme plasma levels. Our twin study also indicates that the angiotensin-converting enzyme gene locus is genetically linked to posterior wall thickness. The correlation between angiotensin-converting enzyme levels and posterior wall thickness suggests that this effect is exerted by angiotensin-converting enzyme. We were unable to demonstrate genetic linkage between the angiotensinogen gene locus and cardiac dimensions in this study.

The structure of adenovirus type 12 DNA integration sites in the hamster cell genome.

Foreign DNA can integrate into the genomes of mammalian cells, and this process plays major roles in viral oncogenesis and in the generation of transgenic organisms and will be important in evolving regimens for human somatic gene therapy. In the present study, the insertion sites of adenovirus type 12 (Ad12) DNA genomes have been analyzed in detail in the Ad12-transformed hamster cell line T637, its revertants, which have lost most of the >20 Ad12 genome equivalents integrated chromosomally in cell line T637, and in the Ad12-induced tumor T191. Some of these junction sites have been molecularly cloned, and the nucleotide sequences at the sites of transition between viral and cellular DNAs have been determined. The sites of linkage between the hamster cellular and the foreign (viral) DNA are characterized by the frequent occurrence of patch homologies between the recombination partners. The cellular junction sites investigated here are not transcriptionally active. One of the cellular DNA sequences abutting the right Ad12 DNA terminus in cell line T637 (os2) is represented only once in the hamster genome and has a strikingly low abundance of 5'-CG-3' dinucleotide sequences. One 5'-GCGC-3' sequence close to the Ad12 DNA integration site is heavily methylated in normal cells, Ad12-transformed cells, and Ad12-induced tumor cells. The second such sequence is more remote from the junction site, is partly methylated in BHK21 hamster cells, and shows differences in methylation in different Ad12-transformed cell lines. This site is unmethylated in liver DNA. The cellular DNA sequence at the site of Ad12 linkage in the tumor T191 exhibits homologies to highly repetitive sequences of the Alu family and to an origin of hamster DNA replication containing an Alu element. A number of junction sites between Ad12 DNA and hamster or mouse DNA in Ad12-transformed cell lines or Ad12-induced tumor cell lines, investigated here and previously, are characterized by stem-loop structures encompassing the junction sites.

[Role of hepatotropic viruses in liver pathology in Southwestern Cameroon]. / Die Rolle hepatotroper Viren in der Leberpathologie Südwestkameruns.

Between 1990 and 1992 (2 years), 102 patients with clinical liver pathology underwent standardized clinical, pathological, sonographic and serologic investigations (HAV, HBV, HCV, HDV autoantibodies and tumor markers). During the same period seroepidemiological studies with the same parameters as above were performed on the following control groups: healthy pregnant women (n = 383), blood donors (n = 85), HIV-positive individuals (n = 93), and hospitalized patients in all age groups with minor ailments unrelated to liver pathology (n = 108). The results are discussed in detail. Virtually all adults had HAV infection. HBV and HCV infection appears to play a major role in chronic liver pathology in southern Cameroon. The two infections frequently occur together (over 40% of liver cases) and correlate significantly with liver cirrhosis. The marked prevalence of HBV and HCV markers in healthy pregnant women is of epidemiological concern due to the potential for vertical transmission of the infection (immunization). Endemic infections such as falciparum malaria are probably responsible for unspecific stimulation of the immune system, which is reflected in a generally marked prevalence of autoimmune markers in liver patients and controls, since histologically there was no evidence of autoimmune liver disease.

Selective loss of unmethylated segments of integrated Ad12 genomes in revertants of the adenovirus type 12-transformed cell line T637.

We have studied the stability of integrated adenovirus type 12 (Ad12) DNA sequences and the relation of foreign DNA persistence to the state of methylation of this DNA. In the Ad12-transformed hamster cell line T637, multiple copies of Ad12 DNA are chromosomally integrated. Some of these integrated viral genomes are rearranged in that internal parts of the viral DNA have become juxtaposed to its left terminus. Fluorescent in situ hybridization analyses demonstrate that the Ad12 DNA in cell line T637 and in some of its revertants is located at one site on one of the hamster chromosomes. Major portions of the integrated viral genomes in cell line T637 have become extensively de novo methylated in specific patterns. Most of the rearranged Ad12 DNA sequences in the T637 genome are un- or hypomethylated. In the morphological revertants of the Ad12-transformed hamster cell line T637, the majority of the integrated Ad12 genomes has been lost. Surprisingly, we have found that the un- or hypomethylated rearranged viral sequences have been selectively lost, in contrast to some of the methylated sequences that are stably retained.

Liver pathology in rural south-west Cameroon.

In a prospective study, 102 hospital patients with liver disease were evaluated in West Cameroon, Africa. Blood donors, pregnant women and patients without liver disease served as controls. A total of 757 individuals were tested for markers of hepatitis A, B, C and D and for immunological markers (autoantibodies, procollagen III, alpha-foetoprotein, CA50 antigen, alpha-1-antitrypsin and antibodies to human immunodeficiency virus types 1 and 2). One-third of the liver disease patients had focal lesions on ultrasound examination. Histologically, 20 cases of cirrhosis, 14 cases of chronic hepatitis, 15 hepatocellular carcinomas and 17 cases of acute hepatitis were detected. All hepatic patients and virtually all controls had had a previous hepatitis A virus infection. Over 85% of adult patients and controls had at least one marker of hepatitis B virus infection. Over 30% of patients with liver disease had markers of possible hepatitis B virus replication. Antihepatitis C virus antibody was present in 18% of hepatic patients and in 6% of controls. Hepatitis C virus infection seems to play an important role in the development of chronic liver pathology; 40% of cirrhotic patients had a combined hepatitis B and C virus infection. Serum autoantibodies were frequently found and were not correlated with the presence of autoimmune liver disease.

[Therapy in hepatic ascites]. / Therapie bei hepatischem Aszites.

This is a review on the pathogenesis, precipitating causes, analyses, complications, prognosis and therapeutic possibilities of hepatic ascites for practical purposes. Among the possible analyses, granulocyte count, difference between serum albumin and ascitic albumin, cytologic evaluation and bacterial culture in blood-culture media are the most promising. Spontaneous bacterial peritonitis often shows no local clinical signs and carries a bad prognosis, unless looked for and treated. The hallmark of treatment of hepatic ascites consists of reduced sodium intake, diuretics and total paracentesis. Peritoneovenous shunt, TIPPS and hepatic transplantation are options in refractory cases.

The initiation of de novo methylation of foreign DNA integrated into a mammalian genome is not exclusively targeted by nucleotide sequence.

The de novo methylation of foreign DNA integrated into the mammalian genome is a fundamental process whose mechanism has not yet been elucidated. We have studied de novo methylation in adenovirus type 12 (Ad12) genomes inserted into the genomes of Ad12-induced hamster tumor cells. De novo methylation of Ad12 DNA, which is not methylated in the virion, is initiated in two paracentrally located regions and spreads from there across the integrated Ad12 genomes. (i) After extensive cultivation of cloned Ad12-induced hamster tumor cell lines, the same segments in integrated Ad12 DNA in different cell lines become methylated or remain unmethylated, depending on their positions in the viral genome. (ii) When Ad12 DNA or Ad12 DNA fragments are transfected into hamster cells and permanent cell lines are established by selection for the cotransfected neomycin phosphotransferase gene, patterns of de novo methylation in terminally or internally located segments of Ad12 DNA are different from those in Ad12-induced tumor cell lines. (iii) A detailed study on the topology of the integrated viral genomes in the Ad12-transformed hamster cell lines T637 and A2497-3 and in the Ad12-induced hamster tumors T191, T1111(1), and T181 has been performed. Some of the integrated viral genomes are inserted into the cellular genome in an orientation colinear with the virion genome; others have been rearranged. An originally internally located Ad12 DNA segment has become transposed to the left-terminal sequences of the viral genome in several cell lines and tumors. In the complete Ad12 genomes, the internally located PstI-D fragment becomes extensively methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences. When this DNA segment has been juxtaposed to the left-terminal, hypomethylated fragment of Ad12 DNA in rearranged genomes, the PstI-D fragment remains unmethylated. We therefore reason that the initiation of de novo methylation in integrated Ad12 DNA cannot be directed exclusively by the nucleotide sequence. Other parameters, such as site of integration, conformation of integrates, mode of cell selection, or chromatin structure related to transcriptional activity, may play decisive roles.