Active specific immunotherapy with Newcastle-diseasevirus-modified autologous tumor cells following resection of liver metastases in colorectal cancer

Summary A group of 23 colorectal cancer patients were treated by a new type of active specific immunotherapy (ASI) following complete surgical resection of liver metastases (R0 resection). For ASI treatment we used a vaccine consisting of 1 × 10⁷ autologous, irradiated (200 Gy) metastases-derived tumor cells incubated with 32 hemagglutination units (HU) of Newcastle disease virus (NDV). The adjuvant vaccine therapy was started 2 weeks after surgery and was repeated five times at 14-days intervals followed by one boost 3 months later. The delayed-type hypersensitivity (DTH) skin reactions to the vaccine were measured as well as the DTH reactions to a challenge test of 1 × 10⁷ non-virus-modified autologous tumor cells from liver metastases or 1 × 10⁷ autologous normal liver cells. In addition 32 HU NDV alone and a standard antigen test (Merieux test) were applied pre- and post-vaccination. The vaccination was well tolerated. In 13 of 23 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. Nine patients (40%) experienced an increased DTH reactivity against autologous tumor cells following vaccination, while 17% or fewer showed an increased reactivity to Merieux test antigens, NDV, or normal liver cells. The increased antitumor response was not correlated to responsiveness to NDV alone, autologous liver cells, enzymes and culture medium used for vaccine preparation or standard antigens (Merieux test). After a follow-up of at least 18 months 61% of the vaccinated patients developed tumor recurrence in comparison to 87% of a matched control groups from the same institution that had been only surgically treated. The results of this phase II trial are encouraging and should stimulate further prospective randomized studies.     

A small deletion and an adjacent base exchange in a potential stem-loop region of the neurofibromatosis 1 gene

Summary A single-strand conformational polymorphism found in the DNA of a patient with neurofibromatosis 1 (NF1) was shown to be caused by a deletion of a CCACC or CACCT sequence and an adjacent transversion, located about 500 base pairs downstream from the region that codes for a functional domain of the NF1 gene product. This mutation could also be detected in the patient and in his affected daughter by heteroduplex analysis. The deletion removes the proximal half of a small potential stem-loop and interrupts the reading frame in exon 1. A severely truncated protein with a grossly altered carboxy terminus lacking one third of its sequence is expected to be formed from the mutant allele.     

Cell culture studies on neurofibromatosis (von Recklinghausen)

Summary The growth of strains of fibroblasts derived from patients with neurofibromatosis (NF) was compared with that of strains from appropriate controls in culture medium containing 1% or 15% fetal calf serum. The means of the ratios of final to initial cell numbers do not differ significantly between NF strains and control strains. Weakly significant differences are, however, obtained after conversion of the results to mean numbers of cell population doublings, the NF strains showing the higher numbers. The ratios of final to initial amounts of protein also differ significantly under both sets of growth conditions. High growth parameters occur significantly more frequently among our smaple of 11 NF strains than among our sample of 13 control strains. The possibility of the expression of the NF genotype(s) on the level of the cultured fibroblast-like cells and the possible causes of the large ranges of inter-and intra-individual variations of the results are discussed.     

A new human colon carcinoma line

Supported by the “Tumorzentrum Heidelberg-Mannheim”.     

The second case of a t(17;22) in a family with neurofibromatosis type 1: sequence analysis of the breakpoint regions

A reciprocal t(17;22)(q11.2;q11.2) was found in a female patient with neurofibromatosis type 1 (NF1) and in her affected daughter. Sequence analysis of cloned junction fragments traversing the breakpoints allowed the identification of the structures involved in the rearrangement. Aberrant bands in Southern hybridizations of restriction enzyme-digested DNA of the patient pointed to the disruption of the NF1 gene in intron 31. Semispecific polymerase chain reaction analysis of the genomic DNA of the patient with the specific primer anchored at NF1 exon 31 was used to obtain the breakpoint-spanning fragment of the derivative chromosome 17. The intron 31 sequence turned out to be interrupted within a large irregular (AT) repeat. The chromosome 22-derived sequence of the der(17) junction fragment allowed us to identify cosmids of the corresponding region from a chromosome 22-specific cosmid library. With the support of the breakpoint-spanning cosmids, the chromosome 22 region upstream of the fragment carried by the der(17) was characterized. Primers deduced from the sequence of this upstream region were used in combination with a primer in NF1 intron 31 distal to the breakpoint on chromosome 17 to amplify the der(22) junction fragment. The structure of the junction sequences suggested that the translocation had arisen by unequal homologous recombination between (AT)-rich repeats on chromosome 22 and on chromosome 17 in intron 31 of the NF1 gene. However, our data support the assumption of additional rearrangements prior to, or in the course of, the recombination event, leading to a loss of the sequences between the involved (AT) repeats on chromosome 22. In the direct vicinity of these (AT) repeats, two members of a previously undescribed low-copy repetitive sequence have been found, copies of which are also present on human chromosome 13. Received: 27 August 1996 / Revised: 7 October 1996     

Single-cell PCR performed with neurofibroma Schwann cells reveals the presence of both alleles of the neurofibromatosis type 1 (NF1) gene

It is commonly held that Schwann cells (SC) are the progenitor cells of benign neurofibromas. To test for loss of heterozygosity (LOH) at the neurofibromatosis 1 (NF1) gene locus, three intragenic polymorphic markers were analyzed after polymerase chain reaction amplification, starting from 98 single SC isolated from primary cultures of neurofibromas of five informative NF1 patients. The patterns obtained did not provide evidence for LOH at the NF1 gene. LOH by nondisjunction, large deletions, or somatic recombination in SC seems not to be the mechanism of generation of neurofibromas.     

Evidence against a (2)n synchronous increase of spermatogonia to produce spermatocytes in drosophila hydei

The numbers of primary spermatocytes within cysts as well as numbers of postmeiotic spermatids in bundles in Drosophila hydei were determined. Within the contents of a single testis the cysts of primary spermatocytes are found to contain 5–11 germ cells. Furthermore, the number of spermatocytes per cyst is age-dependent, in that pupae have a mean of 8.1 cells whereas fertile adult males have a mean of 7.1 cells. Counts of spermatids in section of testes add further support to the view that the primary spermatocytes, from which the spermatids originated, were not formed in a strict geometric progression.     

Kontinuierliche Messung der Assimilation und Atmung mariner Algen mittels der elektrochemischen Sauerstoffbestimmung

Zusammenfassung 1. Eine Apparatur zur kontinuierlichen Messung von Assimilation und Atmung mariner Organismen nach dem Prinzip der elektrochemischen Sauerstoffbestimmung wird beschrieben.2. Die O₂-Messung erfolgt in einem geschlossenen System mittels einer stabilisierten, membranüberzogenen Pt-Elektrode (Clark-Prinzip) mit folgenden Eigenschaften: Die Einstellzeit auf 90% des Endwertes beträgt 14 bis 16 sec; die Drift liegt über Tage bei 0,8%, über Wochen bei 2%. Im Bereich von 0 bis 10 mg O₂/l ist die Beziehung zwischen Depolarisationsstrom und O₂-Konzentration linear. Die Eichkurve schneidet den Nullpunkt.3. Mit der beschriebenen Anordnung können Sauerstoffänderungen von 0,0035 mg O₂ erfaßt werden. Es werden Beispiele für die Anwendung von Assimilations- und Atmungsmessungen an der BraunalgeFucus vesiculosus L. angeführt.

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Continuous measurement of assimilation and respiration of marine algae, employing electro-chemical oxygen determination

An apparatus is described for the continuous measurement of assimilation and respiration of marine organisms, employing the principle of electro-chemical oxygen determination. The measurement of O₂ takes place in a closed system with a stabilized membrane-covered Pt-electrode (Clark-principle), having the following properties: The electrode gives 90% of the full response in 14 to 16 seconds; drift over a period of a few days amounts to about 0.8%, over a few weeks, to about 2%. In the range from 0 to 10 mg O₂/l, the relationship between the depolarization current and O₂-concentration is linear. The calibration curve goes through the origin. With the described assembly, variations in oxygen changes down to 0.0035 mg can be detected. An example of the usefulness of the new method is shown on the basis of assimilation and respiration measurements on the algaFucus vesiculosus L.

     

Cell culture studies on neurofibromatosis (von Recklinghausen)

Summary Cell cultures grown from peripheral neurofibromas of three patients suffering from sporadic peripheral neurofibromatosis (NF) were analysed cytogenetically at early in vitro passages. The NF-cultures exhibited a 6.7-fold higher frequency of aneuploid mitoses, including pseudodiploids, than the control cultures derived from the skin of three healthy donors. The predominant numerical anomaly was monosomy 22. Several, as yet unidentified marker chromosomes occurred in the NF-cultures, which also showed a much higher level of unstable chromosomal anomalies. The role of monosomy 22 in tumorigenesis of meningiomas and neurofibromas is discussed.