Active specific immunotherapy in hepatic metastasis.

The immune system takes note of the presence of a malignant tumor but in most cases an effective defense reaction is hardly likely to come about. In patients with a solid tumor a tumor-directed immune response will usually be manifested as sensitization of T-lymphocytes to different tumor associated antigens (TAA). There is hope, however, that deeper insights into the mechanisms which ensure the selective clonal expansion and differentiation of antigen-sensitized lymphocytes and an appropriate stimulation of tumor specific effector cells will make it possible to inhibit or at least to impede the outgrowth of metastases following the surgical resection of the primary tumor. Active specific immunization (ASI) is one way to activate tumor specific T-or B-lymphocytes. In this paper, we give a short survey on the state of the art of ASI, outline newer approaches to improve its effectiveness and summarize results of clinical studies with ASI in patients with malignant melanoma or colorectal carcinoma. Among the epidemiologically important tumors, malignant melanoma is that one which seems to be most immunogenic and therefore has been studied intensively. The reason we refer to colorectal carcinoma is that we ourselves have some preliminary experiences with ASI in patients with colon carcinoma.

Expression of Thomsen-Friedenreich-related antigens in primary and metastatic colorectal carcinomas. A reevaluation.

BACKGROUND: Expression of the pancarcinoma Thomsen-Friedenreich (TF) carbohydrate antigen or, more correctly, hapten, in colorectal carcinomas is not generally agreed on. Furthermore, its suggested role in liver metastasis so far has not been substantiated by direct immunohistochemical evidence. METHODS: Cryostat sections from 52 primary tumors (20 with adjacent transitional mucosa), 22 liver metastases of colorectal carcinomas, and 17 samples of normal mucosae were examined immunohistologically with a panel of at least two monoclonal antibodies (mAbs) each to TF, to its precursor, Tn, and to sialosyl-Tn, among them two newly developed anti-TF mAbs. RESULTS: Of the primary colorectal carcinomas, 60% expressed TF. Staining was more intense with TF-alpha/beta-reactive than with exclusively TF-alpha- or TF-beta-reactive mAbs. Normal and transitional mucosae were negative. Liver metastases were positive for TF in a significantly higher percentage of cases (91%) than primary carcinomas. Patients with TF-positive primary tumors had a significantly higher risk to develop liver metastases compared with patients with TF-negative tumors (57% vs. 14%, respectively). Tn and sialosyl-Tn were expressed concomitantly in most primary (85%) and metastatic (95%) colorectal carcinomas. These antigens also were detected in transitional mucosae (Tn in 25%, sialosyl-Tn in 55% of cases). Normal mucosae were negative. CONCLUSIONS: These results prove unequivocally the presence of exposed TF epitopes in a majority of colorectal carcinomas in which both anomers of TF are expressed. These data further suggest that TF favors liver metastasis and that its expression in primary colorectal carcinomas is a significant risk factor for the development of liver metastasis.

Cadmium-binding proteins from a tunicate, Pyura stolonifera.

The determination of cadmium levels in tissues of the tunicate Pyura stolonifera collected from uncontaminated sites revealed highest concentrations in the liver. After keeping P. stolonifera under laboratory conditions in Cd-containing water for 15 days, cadmium accumulated most markedly in liver tissue. Liver tissue of Cd-exposed specimens was used for the isolation of Cd-binding proteins. Five Cd-binding proteins, which differ in their chromatographic properties, could be purified. At least four of these Cd-binding proteins are heat stable and cysteine-rich. The N-terminal sequence (Met-Asp-Pro-Cys-Asn-Cys-Ala-Glu...) of at least two of these peptides resembles fish (plaice) metallothionein. Unlike vertebrate metallothioneins, P. stolonifera Cd-binding proteins are not N-terminally blocked by acetylation of methionine.

[Significance of serum beta-hCG as a tumor marker for stomach carcinoma]. / Zur Bedeutung von beta-hCG im Serum als Tumormarker für das Magenkarzinom.

INTRODUCTION: Recent investigations indicate that in 50% of patients with gastric cancer, beta-hCG-positive cells can be found in the tumour by immunohistochemical investigations. The objective of this study was to investigate how often beta-hCG-immunoreactive gastric carcinomas were accompanied by an elevation in serum beta-hCG, that could have been used as a course control variable. METHODS: In 54 patients with gastric carcinoma a monoclonal antibody directed against beta-hCG was used for immunohistochemical marking in the APAAP system. The evaluation was graded positive or negative. In parallel, serum beta-hCG was determined preoperatively using an enzyme immunoassay (MEIA). Tumour stage, grading and tumour localization were determinants in the evaluation. RESULTS: We found that 41% (22 of 54) of the carcinomas induced a positive immunohistochemical response to beta-hCG, regardless of their location in the stomach. In relation to tumour stage, a positive beta-hCG immunoreactivity was apparent in 27% (6/22) of tumours without lymph node or distant metastases (T1-4N0M0), in 54% (7/13) of tumours with lymph node and without distant metastases (T1-4N > or = 1M0) and in 47% (9/35) of tumours with distant metastases. Poorly differentiated tumours (G3-4) were positive in 42% (15/36) and well-differentiated tumors (G1-2) in 39% (7/18) of cases. In only 1 patient was the beta-hCG level in serum elevated, however. CONCLUSIONS: beta-hCG-Positive gastric carcinomas are found more frequently in advanced tumour stages and poorly differentiated carcinomas. These carcinomas, however, seem not to excrete beta-hCG in sufficient amounts to produce measurable serum values. Therefore, beta-hCG cannot be used a prognostic factor or for course control. The relevance of beta-hCG expression of tumour cells to the patients' prognosis remains obscure.

Isolation and primary structures of neuropeptides of the AKH/RPCH family from various termite species.

We have isolated neuropeptides of the AKH/RPCH family from extracts of whole heads of four termite species (Mastotermes darwiniensis, Microhodotermes viator, Hodotermes mossambicus, and Trinervitermes trinervoides) using the effect of mobilizing lipids in Locusta migratoria for bioassay. Isolation was essentially achieved by two steps of reversed-phase chromatography (on phenyl-support followed by C-18). The peptides were identified by Edman degradation after deblocking with oxoprolyl peptidase. Each termite species contained only one AKH/RPCH family member. The primary structure in M. darwiniensis and T. trinervoides is pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, a peptide previously found mainly in cockroaches and code named Pea-CAH-I. The peptide from M. viator has the primary sequence pGlu-Ile-Asn-Phe-Thr-Pro-Asn-Trp-NH2; it is a novel member of the family and is code-named Miv-CC (Microhodotermes viator corpus cardiacum peptide). Phylogenetic relations between the known cockroach and mantid AKH/RPCH octapeptides and the termite peptides from this study could be revealed employing the parsimony method. Based on a computer analysis, using PAUP 3.1.1., we concluded that termites are plesiomorphic with regard to cockroaches, and mantids are the sister taxon to the termite/cockroach group.

[Mitochondrial morphogenesis in insects during meiosis and spermatogenesis: a comparative study between modern high resolution microscopy and historical data]. / Mitochondrienmorphogenese bei Insekten während Meiose und Spermiogenese: Eine vergleichende Studie zwischen moderner Hochauflösungsmikroskopie und historischen Daten.

Spermatogenesis in cysts isolated from testes of Drosophila and morphogenesis of male germ cell mitochondria were observed in vitro and compared with their first description in the literature. Since Drosophila started to be used for laboratory research only at the beginning of the 20th century, publications dealing with other insects have been consulted for earlier periods. Formerly, results were usually obtained from squashed preparations. This technique causes many artifacts and only occasionally can intact cysts be observed. It was only after tissue culture technique and microscopes improved that in vitro observation of differentiating cysts became possible. However, thanks to acute observational skills and careful reasoning, former researchers already had a correct picture of the early development of male germ cell mitochondria. Modern high resolution microscopy confirmed their results.

Active specific immunotherapy with Newcastle-disease-virus-modified autologous tumor cells following resection of liver metastases in colorectal cancer. First evaluation of clinical response of a phase II-trial.

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 (RO resection). For ASI treatment we used a vaccine consisting of 1 x 10(7) 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 x 10(7) non-virus-modified autologous tumor cells from liver metastases or 1 x 10(7) 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.

In vitro and clinical characterisation of a Newcastle disease virus-modified autologous tumour cell vaccine for treatment of colorectal cancer patients.

A virus-modified autologous tumour cell vaccine prepared from human colorectal cancer cells is described. After dissociation an average of 5 x 10(7) cells/g tissue were obtained from primary tumours and 9 x 10(7)/g tissue from metastases with an average viability of 72% and 51%, respectively. Following irradiation (200 Gy), inactivation of the proliferative activity of the cells was demonstrated by their degeneration in tissue culture and the absence of incorporation of 3H-labelled thymidine. One third of the cells were still metabolically active, as shown by the incorporation of 3H-uridine and a mixture of 3H-aminoacids. The dissociated cells expressed MHC class I and II antigens in a qualitatively similar way to tissue sections. Epithelium-specific antigens (detected by MAb HEA125) were expressed on an average of more than 75% cells of the suspension, while leucocyte-specific antigens (detected by MAb CD53) were expressed on an average of less than 25% cells. The vaccine was prepared by admixing the nonlytic strain Ulster of Newcastle disease virus (NDV) with the tumour cell suspension. The NDV adsorption at tumour cells was shown by electron microscopy. Clinically, the treatment with the vaccine was associated with an increased sensibilisation against autologous tumour cells, measured by DTH skin reactivity. First results in 23 patients with colorectal liver metastases who underwent "curative" liver resection followed by vaccination show a clear correlation between the induced increase of DTH skin reaction against autologous tumour cells and the recurrence-free interval. No correlation was found for DTH reaction caused by standard antigens (Mérieux test), NDV alone or autologous normal liver tissue. The results demonstrate the possibility of preparing immunogenic virus-modified autologous tumour cell vaccine from colorectal cancer tissue, which could be used for cancer therapy.

Postoperative active specific immunization in colorectal cancer patients with virus-modified autologous tumor-cell vaccine. First clinical results with tumor-cell vaccines modified with live but avirulent Newcastle disease virus.

Sixteen patients with colorectal carcinoma Dukes' Stage B2, C, or D were treated with an autologous virus-modified tumor-cell vaccine after potential curative tumor resection (R0-Resection). An inoculum of 1 X 10(7) cells incubated with 32 hemagglutination units of nonirradiated Newcastle disease virus (NDV) was given intracutaneously up to four times at 10-day intervals. The delayed-type hypersensitivity (DTH) skin reaction was measured. The vaccination was well tolerated. In 11 of 16 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. A challenge test using autologous tumor cells without NDV after the vaccination cycle revealed a specific antitumor sensibilization in 12 patients. The DTH response was not due to bacterial contamination or sensibility to the virus. Histologic examination of the vaccination site showed a dense infiltration of predominantly helper T-lymphocytes. We conclude that in most of the patients treated active, specific immunization led to a specific antitumor sensitivity.

Postoperative active specific immunization in curatively resected colorectal cancer patients with a virus-modified autologous tumor cell vaccine.

Active specific immunotherapy was performed in a phase I study in 20 colorectal cancer patients after surgical resection of the tumor. An autologous tumor cell vaccine surface modified by Newcastle disease virus (NDV) was used, which showed the following characteristics. After mechanical and enzymatic dissociation of the tumor tissue an average of 5 x 10(7) cells/g tissue was obtained. According to trypan blue dye exclusion assay the average viability was 72%. Following irradiation (200 Gy) the inactivation of proliferative activity of the cells could be demonstrated by the absence of incorporation of 3H-labelled thymidine. The cells were, however, still metabolically active as shown by the incorporation of [3H]-uridine and a mixture of 3H-labelled amino acids. Epithelium-specific antigens (detected by mAb HEA125) were expressed on more than 75% cells of the cell suspension indicating a high amount of (epithelium-derived) tumor cells. In order to increase the immunogenicity of the tumor cells the suspended cells were infected by the nonlytic, apathogenic Ulster strain of NDV. The successful modification of tumor cells with NDV could be shown by electron microscopy. Three weeks postoperatively cells were thawed, virus-modified, and inoculated intradermally in the upper thigh. Several cell and virus concentrations were tested in each patient. As control, tumor cells without NDV, NDV alone and normal colon mucosa were used. The number of tumor cells ranged from 2 x 10(6) up to 2 x 10(7) cells and NDV concentrations from 4 to 64 hemagglutination units (HU) were tested. Sixteen patients responded with a delayed-type hypersensitivity (DTH) skin reaction to the vaccine. The best DTH reaction, measured 24 h following vaccination, was obtained using a vaccine consisting of 1 x 10(7) tumor cells and 32 HU NDV (median induration of 8 mm). Response to NDV alone was seen in 2 patients only (median induration of 3 mm); 12 patients responded to tumor cells (1 x 10(7) alone (median induration of 4 mm). Of 10 patients tested with normal colorectal mucosa, 4 responded with a median induration of 3.5 mm. DTH responses to the vaccine of 1 x 10(7) tumor cells and 32 HU NDV increased throughout the repeated vaccinations to a median induration of 9.5 mm at the end of the therapy. No severe side-effects in the course of the immunotherapy, except for mild fever in 4/20 patients, were observed. The results of our phase I study show that this type of autologous colorectal tumor cell vaccine is ready for a large clinical trial to prove its efficacy.

A group of non-Y encoded Drosophila hydei primary spermatocyte nuclear glycoproteins exhibits epitopes depending on formation of Y chromosomal giant lampbrush loops.

In wild-type Drosophila hydei (genotype X/Y) four different primary spermatocyte nuclear glycoproteins, classified as non-Y encoded because of their occurrence in X/O genotypes, were demonstrated to possess a few epitopes that depended on formation of the Y chromosomal giant lampbrush loops threads (th; Mr 55,000 proteins) or pseudonucleolus (ps; Mr 38,000, 58,000 and 98,000 proteins). The epitopes reacted with lectins and/or antibodies in vitro lectin-/immunoreplica of primary spermatocyte total nuclear protein), and were lacking in mutants not possessing the respective loops. Those dependent on ps reacted with human sera. Epitopes restricted to proteins from th-forming spermatocytes reacted with lectin Con A (specific for D-Man and/or D-Glc) and antibodies directed against mouse immunoglobulins (AIA). In situ experiments (immunofluorescence microscopy of primary spermatocyte nuclei) revealed antibody cross-reactions with the respective loops. The reagents stained the distal (fused) sections and proximal (compact) parts of ps (human sera) or the proximal (compact) parts of th (AIA). Reaction with the latter loops was significantly repressed after absorption of AIA with the L-Fuc carbohydrate unit, classifying the AIA as fucosyl specific, and the epitopes along th as L-Fuc carbohydrate units.

The hormonal coordination of cuticulin deposition and morphogenesis in Drosophila imaginal discs in vivo and in vitro.

Cuticulin is the first layer of the insect cuticle to be deposited and is laid down as a continuous inelastic sheet over the apical surface of cuticle-secreting cells. During metamorphosis in Drosophila melanogaster, imaginal discs deposit the cuticulin layer of the pupal cuticle between 3 and 7 hr after puparium formation. This is a period of rapid morphogenesis involving cell shape changes and cell rearrangements. We have examined cuticulin deposition in vivo and in vitro with a view to understanding the coordination of cuticulin deposition with morphogenesis. We find that the optimum hormonal regimen (of the steroid hormone, 20-hydroxyecdysone) for the completion of both morphogenesis and cuticulin deposition in vitro parallels the changes in hormone titer observed in vivo. We also find that cuticulin is deposited last over cell boundaries, thereby allowing cell rearrangements to occur as cuticulin is laid down. We have identified in vitro conditions under which cuticulin deposition is completed precociously, inhibiting further morphogenesis. Cytochalasin B and colchicine do not inhibit cuticulin deposition and we therefore conclude that an intact cytoskeleton is not necessary for secretion of this extracellular structure. Finally, we present a preliminary protocol for the partial purification of cuticulin synthesized in vitro by mass isolated discs.

The effects of cytochalasin B and colchicine on the morphogenesis of mitochondria in Drosophila hydei during meiosis and early spermiogenesis. An in vitro study.

Morphogenesis of mitochondria in male germ cells in cultivated cytocysts begins in early prophase I at which time mitochondria thicken and become ordered along the spindle apparatus during meiosis. At the end of the second meiotic division they aggregate to form the Nebenkern. In the presence of colchicine or cytochalasin B mitochondria are able to begin differentiation, although the correct course of meiosis is not guaranteed. In medium supplemented with colchicine they undergo normal thickening but do not aggregate, in a pattern known from untreated cultures. This may indicate that microtubules are involved in the aggregation process of mitochondria as colchicine is known to inhibit microtubule formation. Moreover, in cell cultures treated with cytochalasin B mitochondrial aggregation does occur; it is concluded that microfilaments, which are sensitive to cytochalasin B, do not play a detectable role in the aggregation of mitochondria.

In vitro spermatogenesis in Drosophila. I. Development of isolated spermatocyte cysts from wild-type D. hydei.

In vitro spermatogenesis of isolated single spermatocyte cysts of Drosophila hydei was studied by microscopic observations and time-lapse cinematography. Cysts of spermatocytes isolated during diplotene develop as far as the coiling stage of spermatid differentiation. The existence of an interphase between meiosis I and meiosis II is, for the first time, documented. Meiosis, Nebenkern formation, and elongation of spermatids occur just as in D. melanogaster; however, an individualization cone, as described for D. melanogaster, can not be detected.

Scanning electron microscopic observations on cells grown in vitro. V. Human diploid cells can attach also with their upper surface.

When small glass particles are seeded on top of human diploid cells in a monolayer the cells attach firmly to the glass with their upper surface. In combination with this attachment fingerlike protrusions and small leading lamellae are formed de novo from the normally completely protrusion-free upper surface. These results are in contradition to findings from Vasiliev and Gelfand [10]. Our results indicate that attachment and adhesiveness must not unequivocally be in relation to the presence and formation of cellular protrusions.

Scanning electron microscopic observations on cells grown in vitro. IV. HeLa cells do not have an upperside-underside polarity.

Small glass particles are added to HeLa monolayer cell cultures. Landing on top of a flattened cell fingerlike protrusions are surrounding the particles. Later on leading lamellae are formed on the upperside of the cell and the glass becomes more and more surrounded until it appears to be "incorporated" completely. The glass particles become attached so firmly that by detaching them the cell surface is destroyed. This speaks against the assumption that cells in monolayers are polarized.

Ultrastructure of aggregates of tumor and normal cells and of mixed aggregates.

Contact areas have been studied in pure aggregates of either normal and malignant cells and mixed aggregates contaning both cell types. The structures of both cell types are compared. Tumor cells form looser connections than normal cells. The contanct areas of tumor cells resemble those of embryonic and mitotic cells. In a mixed aggregate sorted out, the two cell types are arranged in two regions: the "HeLa" - and the "decidua region." The contact areas and the cells in these regions do not differ from those of the pure aggregates. If tumor and normal cells are in direct connection with each other, they show remarkable changes: the tumor cells lose their spherical appearance as well as parts of their organelles and become ameboid. The normal cells from a dense layer of filamentous material beneath the membrane in the neighborhood of the tumor cell.

Studies on coexistence and intercellular communication. Scanning electron microscopy for early stages of co-culturing of normal mesothelial cells and mouse ascites tumor cells in vitro.

Mouse ascites tumor cells (MAT-cells) were co-cultured with mesothelial cells of the mouse. During early stages of coexistence (30-60 min) the mesothelial cells show fingerlike protrusions formed mostly at their flanks pointing towards MAT-cells in the neighbourhood. Obviously there is a directional response of the mesothelial cells to the existence of MAT-cells. The mesothelial cells are the most active partner at the beginning of coexistence. Later on MAT-cells also develop fingerlike protrusions which grow towards the mesothelial cells. A network of fingerlike protrusions is formed in the region between MAT-cells and mesothelial cells when they come near to each other. The advantages of the system serving as a model for investigations on intercellular communication are discussed.