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.

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.

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.

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.

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.

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.

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.

The microvasculature of the small-intestinal mucosa of the rat: quantification of hemodynamic effects of topically applied cimetidine, ranitidine, somatostatin, and vasopressin.

The influence of topically administered cimetidine, ranitidine, somatostatin, and vasopressin on microvessels of the jejunal villus of the rat was investigated by means of an in vivo videomicroscopy technique. For all substances a dose-response curve concerning the change of vessel diameters was obtained. All four drugs induced a significant vasoconstriction at the capillary level. Maximum vasoconstriction was achieved with vasopressin. These results strongly suggest the presence and possibly importance of H2 receptors in the intestinal vascular bed of the rat.

[Persistent leg pain]. / Hartnäckige Beinschmerzen.

A 72 year old patient suddenly experienced severe lumbar pain irradiating into the right leg. Later on, weakness of the muscles thigh appeared. A thorough radiological investigation which showed degenerative alterations of the vertebral column did not supply an explanation. After a pathological titer against Borrelia burgdorferi was found in serum and radiculitis was detected on EMG, the diagnosis of Lyme-Borreliosis of the nervous system could be confirmed by analysis of the cerebrospinal fluid. Under intravenous antibiotic treatment with Ceftriaxone (2 to 4 g daily for three weeks) the symptoms regressed completely, and the pathological findings in the CSF regressed. The significance of some findings in CSF in relation to Borreliosis of the CNS.

[Diagnostic value of a diagnostic strip for determining urinary amylase]. / Diagnostische Wertigkeit eines Teststreifens für die Urinamylasebestimmung.

The semiquantitative determination of amylase in urine with a stick-method has produced controversial results as to its diagnostical significance. In our study we compared the result of the Rapignost-test with the simultaneously measures activity of enzymes (total amylase, pancreatic isoamylase in serum and urine and lipase) in 323 samples of serum and urine taken from 32 patients affected with acute pancreatitis. On the 1st day of hospitalization Rapignost was positive in only 17 out of 32 cases. Both sensitivity and specificity of the test depended of the values referred to. Near range limits and in cases with slight or average increase of the enzymes, the correspondence of Rapignost results were unsatisfactory. We found a good correspondence in cases with normal low and excessively high values of the enzymes. On the whole, Rapignost showed the best correspondence with the pancreas isoamylase in urine. The result of Rapignost was also positive in 5 out of 15 cases with extrapancreatic increased urinary amylase, which confirms its lack of organ specificity. From case to case, the -, and + + test results of Rapignost did not lead to any conclusions as to the absolute values of the enzymes in serum and urine. Our results show that Rapignost does not offer enough safety either to confirm or to exclude an acute pancreatitis, and especially it should not be used as a screening test in emergency diagnostics. The determination of the enzyme, in particular in serum, can not be eluded.

[Semiquantitative lipase determination--a useful screening test for pancreatitis?]. / Die semiquantitative Bestimmung der Lipase--ein nützlicher Screeningtest für die Pankreatitis?

In 233 sera, taken from 112 patients and 20 healthy blood-donors, we have compared RapiTex-Lipase, a semi quantitative immunochemical latex test for the determination of the lipase, with the conventional quantitative method of determination in order to establish its diagnostical value as a screening test for acute pancreatitis. It appeared that, with a positive result of the test increased values of lipase were found in 98% of the cases, but that a negative result of the test did not exclude an increased lipase. The sensitivity of the test as to the quantitative determination of the lipase depended directly on the activity of the lipase and turned out to be acceptable at 88% only with values 4 X higher than the upper limit of the normal range. With values 5 X higher and more, it reached 100%. Even if the test, due to its very high level of discrimination, did not show a reaction in every case of unspecific extrapancreatic hyperlipasemia, its organ-specificity for the pancreas was reduced and came to 80 to 96% in patients with renal insufficiency, diseases of the liver, abuse of alcohol and in various non-pancreatic intra- or extra-abdominal affections. In 16/18 patients with acute pancreatitis the test used for screening turned out positive (sensitivity 89%), two patients with values between 2 and 4 X the upper limit of the norm had a negative test.(ABSTRACT TRUNCATED AT 250 WORDS)

[Upper abdominal pain]. / Oberbauchschmerzen.

We report a female patient in whom the false diagnosis of acute pancreatitis was made, based on intensive epigastric pain and a four fold increase of amylase. The normal values for lipase and decreased values for the urinary amylase were suggestive of a constellation of macroamylasemia. Further investigations revealed a fresh ulcer in the bulbus duodeni as cause of the epigastric pain. With a therapy of cimetidine and antacids painlessness could be achieved within few days.

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.