The MGDS examination: a systematic approach. 3. Part II of the examination: diagnosis, treatment planning, execution of treatment, maintenance and appraisal, writing-up log diaries

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.     

Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues

The plasminogen activation (PA) system may participate in cancer invasion and metastasis. A series of breast cancer tissue specimens was analysed using in situ hybridization and immunohistochemistry. Urokinase-type plasminogen activator (u-PA) mRNA was detected in cancer cells and fibroblasts adjacent to them and its expression was found to be more intense in invasive than in intraductal regions. In invasive but not in intraductal regions, especially those with abundant stroma, plasminogen activator inhibitor-1 (PAI-1) mRNA was observed in cancer cells, fibroblasts, macrophages, and endothelial cells, and PAI-2 mRNA was present in cancer cells, and fibroblasts, macrophages, and lymphocytes around them. These PAI-1- and PAI-2-positive cancer cells were localized at the periphery of the invasive front. Immunohistochemistry yielded basically similar results. A retrospective study of surgically resected breast cancers from 73 patients revealed significant clinical differences associated with u-PA and PAI-2 expression in cancer cells, associated with a poor and a good prognosis, respectively. These findings indicate that breast cancer cells and fibroblasts express u-PA initially and then its inhibitors, and that this process is related to invasion. Expression of u-PA and PAI-2 in cancer cells themselves may serve to up-regulate and limit PA-mediated invasion and metastasis, respectively.     

Cystine metabolism in human fibroblasts. Comparison of normal, cystinotic, and gamma-glutamylcysteine synethetase-deficient cells

Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of gamma-glutamylcysteine synthetase (gamma-glutamylcysteine synthetase-deficient) in culture medium containing L-[35S]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione, gamma-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [35S]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione. Cystinotic fibroblasts incorporated [35S]cystine into the large intracellular cystine pool not found in normal or gamma-glutamylcysteine synthetase-deficient cells and also appeared to synthesize glutathione more slowly than normal cells. However, the radioactivity recovered as cystine was reduced greatly and the rate of [35S]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [35S]cystine. This suggests that the apparent reduced rate of glutathione synthesis observed in untreated cystinotic cells was a secondary effect caused by dilution of the [35S]cystine by the large pool of nonradioactive cystine. Cystinotic cells depleted of cystine by treatment with mercaptoethylamine reaccumulate 30 to 50% of their initial cystine in 24 hours in the absence of extracellular cystine. Both normal and cystinotic cells lose more than 90% of their intracellular glutathione in 24 hours in cystine-free medium. Both cell types can reutilize cysteine from glutathione for protein synthesis.     

Ceramide, a mediator of interleukin 1, tumour necrosis factor alpha, as well as Fas receptor signalling, induces apoptosis of rheumatoid arthritis synovial cells

OBJECTIVES: To examine the effects of ceramide, which is a lipid second messenger of cell surface receptors, including tumour necrosis factor alpha (TNF alpha), interleukin 1 (IL1), and Fas receptors, on rheumatoid arthritis (RA) synovial cells. METHODS: Synovial cells from RA patients and normal skin fibroblasts were cultured with cell permeable ceramide (C2-ceramide). Apoptosis was assessed by microscopic observation of morphological changes, nuclear staining, and DNA electrophoresis. DNA synthesis was examined by thymidine incorporation. RESULTS: C2-ceramide induced reversible morphological changes of synovial cells such as cell rounding within four hours. Subsequently, irreversible nuclear changes characteristic to apoptosis were observed at 48 hours. DNA synthesis was not promoted. The addition of ceramide exerted similar effects on cultured dermal fibroblasts. CONCLUSION: Ceramide induced apoptosis in RA synovial cells. Ceramide could be a second messenger specific for apoptosis of RA synovial cells.     

Individual differences in behavior following amphetamine, GBR-12909, or apomorphine but not SKF-38393 or quinpirole

Cultures of dissociated Edinger Westphal nuclei, dissected from embryonic chick brainstems, were screened immunohistochemically for a variety of non-neuronal cell markers. In young cultures, small clusters of cells were stained by the oligodendrocyte-specific antibodies 04 and 01. In older cultures, larger groups of cells were 04 and 01 positive, sheets of myelin-like membrane were elaborated, and immunoreactivity for proteolipid protein appeared. This sequence resembles that observed in well-characterized rodent brain cultures and suggests that oligodendrocytes in chick Edinger Westphal cultures differentiate in a pattern similar to rodent oligodendrocytes in culture. Variable numbers of cells were immunoreactive for glial fibrillary acidic protein. Many vimentin positive cells were observed, some of which morphologically resembled flat astrocytes. Together with the widespread presence of vimentin, large patches of fibronectin-like immunoreactivity suggested the presence of fibroblasts and/or endothelial cells. An anti-thymocyte polyclonal antibody stained a subset of cobblestone-shaped cells, possibly endothelial cells, in both Edinger Westphal cultures and control cultures of skin fibroblasts. Staining for smooth muscle myosin was detected in several patches of cells, tentatively identifying them as pericytes or smooth muscle cells. In conclusion, Edinger Westphal cultures contain a diverse and varying population of non-neuronal cells loosely organized in large, overlapping islands of cell types and including oligodendrocytes, astrocytes, possibly fibroblasts, endothelial cells, pericytes and/or smooth muscle cells.     

Effects of Cu, Zn-superoxide dismutase delivered by genetically modified skin fibroblasts on cold-induced skin edema in rats

The effects of Cu, Zn-superoxide disumutase (SOD) delivered by genetically modified skin fibroblasts on cold-induced skin edema were studied in rats. Cold-induced skin edema was induced on the dorsal skin following transplantation of ILSOD cells, genetically modified skin fibroblasts which release secretable SOD protein into the extracellular space. The degree of skin edema induced by cold injury was estimated by measuring the amounts of Evans' blue (EB) leaking into the injured skin following intravenously administration. The amounts of EB leakage were significantly reduced by transplantation of ILSOD cells relative to that observed following transplantation of host cells as a control. The degrees and durability of these effects of ILSOD cells were dependent on the number of cells transplanted. Also, the increases of lipid peroxidation following cold injury were significantly reduced by transplantation of ILSOD cells but not of host cells. These findings suggested that transplantation of ILSOD cells was a suitable delivery system for obtaining efficient and continuous effects of SOD. This strategy using genetically modified skin fibroblasts may also be useful as a drug delivery system for other therapeutic proteins.     

The ultrastructural examination of gingival fibromatosis

Hereditary gingival fibromatosis (GF) is a special type of fibrous overgrowth classified as non-inflammatory gingival enlargement. Microscopically, the connective tissue consists of coarse collagen bundles and fibroblasts. The ultrastructural examination of fibrous gingival hyperplasia reveals that fibroblasts phagocyte the mast cell granules and mast cells stimulate collagen synthesis which results in hyperplasia. In the ultrastructural examination of phenytoin-induced hyperplasia, fibroblasts, phagocytosing mast cell granules were also found. Based on these findings, the purpose of this study is to establish whether there is a relationship between fibroblasts and mast cells in GF. The gingival tissues of 5 patients with GF were examined ultrastructurally. In the connective tissue, well-defined bundles of collagen fibres were found together with fibroblasts and capillaries. There were mast cells around these capillaries which had collapsed lumens. The proximity of the mast cells and fibroblasts may indicate that mast cells play some role on collagen synthesis of fibroblasts.     

Study of the repairing process of gastric ulcer using multivariate analysis of bFGF-positive cells, haemodynamics, PAS-positive mucus amount and glandular index in the gastric mucosa

To investigate pathophysiological diversities in the repairing process of gastric ulcer, distribution density of basic fibroblast growth factor (bFGF)-positive fibroblasts and myofibroblasts and vascular endothelial cells, mucosal haemoglobin content, PAS-positive mucus amount and glandular index were compared in the peripheral region of an open ulcer (the unhealed group; n = 17), the central region of a red scar (the red scar group; n = 32) and the central region of the white scar (white scar group; n = 32). Density of bFGF-positive fibroblasts and myofibroblasts and vascular endothelial cells was highest in the unhealed group, followed by the red scar group, while the white scar group showed a low value close to control. Mucosal haemoglobin content was high in the red scar and unhealed groups. PAS-positive mucus amount in the unhealed and red scar groups showed lower values compared with that in the white scar group. Glandular index in the unhealed group was the lowest, followed by the red scar group, while the white scar group neared control values. Statistically significant correlations were observed between the density of bFGF-positive "fibroblasts and myofibroblasts' and density of bFGF-positive vascular endothelial cells, between the density of bFGF-positive vascular endothelial cells and mucosal haemoglobin content and between the PAS-positive mucus amount and glandular index. Discriminant analysis demonstrated that the unhealed group could be distinguished from the red and white scar groups, based on glandular index, density of bFGF-positive "fibroblasts and myofibroblasts', mucosal haemoglobin content and PAS-positive mucus amount, while the red scar group could be discriminated from the white scar group based on the density of bFGF-positive "fibroblasts and myofibroblasts', density of bFGF-positive vascular endothelial cells, glandular index, haemoglobin content and PAS-positive mucus amount. The white scar group was difficult to discriminate from control. Our present results show that the recovery of glandular formation and mucus production continues throughout the repairing process, whereas the acceleration of angiogenesis and granulation formation is observed only in unhealed ulcers and at the red scar stage.     

Expression of TGF-beta 1, PDGF and IGF-1 mRNA in lung of bleomycin-A5-induced pulmonary fibrosis in rats

OBJECTIVE: To investigate the influence of alveolar macrophages (AMs), fibroblasts and interstitial cells on development of lung fibrosis, and the interactions among TGF-beta 1 PDGF and IGF-1 and these cytokines-effects on lung fibrosis. MATERIAL AND METHODS: Expressions of TGF-beta 1, PDGF and IGF-1 mRNA in the lung cells and lung tissues in different stages of Bleomycin-A5-induced pulmonary fibrosis in rats were studied through Northern hybridization. RESULTS: The expressions of TGF-beta 1 and PDGF mRNA reached their peaks in AMs of pulmonary fibrosis in rats on the 7th day after Bleomycin-A5 instillation. It was similar with that in the lung tissues. IGF-1 mRNA remained relatively stable in AMs during the course. PDGF and IGF-1 mRNA increased gradually in fibroblasts, and reached the highest expressions in the interstitial cells. There was almost no TGF-beta 1 mRNA expression in all groups of fibroblasts. CONCLUSIONS: AMs are the main sources of TGF-beta 1 and PDGF in the lung tissues with fibrosis induced by Bleomycin-A5 AMs are activated in the first weekend and secrete TGF-beta 1 and PDGF to promote fibroblasts proliferation and fibrosis. As fibrosis developed, fibroblasts have established PDGF and IGF-1 autocrine and these three cytokines paracrine nets combined with the interstitial cells to promote lung fibrosis.     

Cultured, autologous nucleus pulposus cells induce functional changes in spinal nerve roots

STUDY DESIGN: Nerve conduction velocity in pig nerve roots was assessed after application of various preparations of nucleus pulposus and control. OBJECTIVE: To study whether cultured nucleus pulposus cells could reduce nerve conduction velocity after epidural application. SUMMARY OF BACKGROUND DATA: It is known that nucleus pulposus applied epidurally may reduce the nerve conduction velocity of the adjacent nerve roots and that this reduction seems to be related to the cells of the nucleus pulposus. METHODS: Nucleus pulposus cells and fibroblasts were cultured for 3 weeks, and various preparations were applied to the cauda equina in 29 pigs. The cells were always from the same animals from which they had been harvested. After 1 week, nerve conduction velocity was determined by local electrical stimulation. RESULTS: Application of live fibroblasts and conditioned culture medium from the nucleus pulposus cell culture dishes did not induce significant reduction of conduction velocity, compared with application of dead fibroblasts, which served as control. However, application of live and dead nucleus pulposus cells induced significant reductions. CONCLUSIONS: Application of nucleus pulposus cells reproduced the previously seen reduction in nerve conduction velocity induced by nonmodified nucleus pulposus. Because membranes of the nucleus pulposus cells had similar effects, it can be assumed that the effects are related to membrane-bound substances or structures.     

Male circumcision, sexually transmitted disease, and risk of HIV

The potential of enhanced chromatid damage, observed after X-irradiation of G2 phase, has been used to detect individuals genetically predisposed to cancer, utilising fibroblast/lymphocytes from these patients as well as fibroblasts derived from human tumours. Fibroblasts and/or lymphocyte samples of two autosomal recessive syndromes (xeroderma pigmentosum (XP), Fanconi's anaemia (FA)) and one congenital or acquired disorder, aplastic anaemia (AA), were employed for the G2 radiosensitivity assay. In addition, we have estimated the frequencies of spontaneously occurring chromosomal aberrations as well as G2 radiosensitivity of eight samples of fibroblasts/fibroblast-like cells (two normal, two colorectal carcinoma, two Wilms' tumour, one retinoblastoma and one polyposis coli), and three samples of lymphocytes (two normal and one from a lymphoma patient). The results obtained indicate that there were no differences between fibroblast cells derived from patients or tumours, except FA patients, in the frequency of spontaneously occurring chromosomal aberrations when compared to normal cells. Following X-irradiation we did not observe any significantly increased G2 radiosensitivity in FA and XP cells. Lymphocytes from AA and lymphoma patients, and all tumour cell lines except retinoblastoma, responded with increased frequencies of aberrations following G2 X-irradiation in comparison to cells derived from normal individuals. In our hands, the G2 sensitivity assay could not always discriminate cells from cancer-prone individuals from those of controls.     

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.     

Line-shape analysis of speed-dependent collisional width inhomogeneities in CO broadened by Xe, N2, and He

The electrophoretic method of Sch?gger and von Jagow (Anal. Biochem. 199, 233-231 (1991) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10(6) fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.     

Clinical spectrum, therapeutic management, and follow-up of ventricular tachycardia in infants and young children

Prostaglandins mediate many biological processes. Arachidonic acid, the common precursor for all prostaglandins, is released from membrane phospholipids by both secretory and cytoplasmic forms of phospholipase A2. Free arachidonate is converted to prostaglandin H2, the common precursor to all prostanoids, by prostaglandin synthase. Both mitogen-induced prostaglandin synthesis in fibroblasts and endotoxin-induced prostaglandin synthesis in macrophages require expression of the inducible prostaglandin synthase-2; arachidonate released in these contexts is unavailable to prostaglandin synthase-1 constitutively present in fibroblasts or macrophages. In contrast to the results for fibroblasts and macrophages, prostaglandin synthesis by activated mast cells is mediated by prostaglandin synthase-1. Mast cell activation also provokes release of secretory phospholipase A2 (sPLA2). We now demonstrate that sPLA2 released from activated mast cells can mobilize arachidonate from distal Swiss 3T3 cells. This arachidonate is then used by prostaglandin synthase-1 present in 3T3 cells for prostaglandin synthesis. We thus distinguish two pathways for prostaglandin synthesis: (i) an intracellular pathway by which arachidonate released following ligand stimulation is made available only to prostaglandin synthase-2, and (ii) a transcellular pathway by which sPLA2 of proximal cells mobilizes, in distal cells, arachidonate available to prostaglandin synthase-1. Molecular and pharmacologic approaches to modulating prostaglandin-mediated events will differ for these two pathways.     

Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro

An organotypic, tridimensional cell culture, also called a raft system, was used to study the influence of fibroblasts on epithelial carcinogenesis in a cell line derived from laryngeal squamous cell carcinoma and harboring a mutated p53. Differences between the effects of normal fibroblasts and those of tumor-derived fibroblasts were compared by means of fibroblasts taken from the normal skin and from the tumor of a cancer patient and cultivated with epithelial carcinoma cells in an organotypic culture. To study cell contact-mediated changes, the fibroblasts were either simply embedded in collagen matrix or additionally brought into direct contact with epithelial cells. Control epithelial cells were cultivated without any fibroblasts in an organotypic model. A protein panel [p53, p21, PCNA, bcl-2, Ki67, total cytokeratin (CK), CK 8, CK 10, CK 17, CK 18, CK 19, vimentin] involved in cell cycling and epithelial differentiation was assessed immunocytochemically in all organotypic cultures with fibroblasts, in tumor cells cultivated as a monolayer, and in the original tumor sample. The most dysplastic phenotype was obtained when tumor-derived fibroblasts were used in direct contact with epithelial cells, whereas the most benign phenotype was seen when skin fibroblasts had no contact with them. The intensive staining seen for p53 can be explained by p53 mutations also reflecting the weak expression of p21 and abundant expression of PCNA. The intensive Ki67 staining seen in all sections paralleled that of PCNA and marked active cellular proliferation. The CK staining pattern seen in cultured epithelia toward embryonic CKs, CK 8 and CK 18, suggested a simple epithelial phenotype. CK 19 was found only in the epithelium where no direct contacts had occurred. Vimentin expression increased when the raft epithelium was shifting toward a more benign phenotype. The results stress the importance of the origin of fibroblasts as well as the role of direct cellular contacts in modifying the epithelial phenotype even when the epithelial cells are malignant.     

Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease

Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disease that is characterized by lysosomal accumulation of sphingolipids, phospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we examined the hypothesis that the ML-IV phenotype might arise from abnormal transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycling were nearly identical in ML-IV and normal fibroblasts. A fluorescent analog of lactosylceramide (LacCer) was used to study plasma membrane lipid internalization and subsequent transport. Lipid internalization at 19 degreesC was similar in both cell types; however, 40-60 min after raising the temperature to 37 degreesC, the fluorescent lipid accumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts. Biochemical studies demonstrated that at these time points, hydrolysis of the lipid analog was minimal ( approximately 7%) in both cell types. A fluorescence ratio imaging assay was developed to monitor accumulation of fluorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts. By 60 min, LacCer apparently decreased in the lysosomes of normal fibroblasts but not in ML-IV cells, suggesting that lipid efflux from the lysosomes was also impaired. These results demonstrate that there is a defect in ML-IV fibroblasts that affects membrane sorting and/or late steps of endocytosis.     

Relative contribution of angiotensin II, bradykinin, and prostaglandins to the renal effects of converting enzyme inhibition in rats after chronic myocardial infarction

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblasts seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs (approximately 300 bp) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones.     

Reduced efficacy of allogeneic versus syngeneic fibroblasts modified to secrete cytokines as a tumor vaccine adjuvant

We examined the relative efficacy of allogeneic versus syngeneic fibroblasts admixed with tumor cells as a vaccine to induce antitumor T-cell reactivity. Allogeneic (3T3) or syngeneic (BLK) fibroblasts transfected to secrete equivalent amounts of GM-CSF were admixed with either D5 melanoma or MCA 207 sarcoma and inoculated s.c. into the flanks of C57BL/6 mice. Vaccine-primed lymph node (LN) cells were examined for in vivo antitumor reactivity in an adoptive transfer model. At fibroblast: tumor cell ratios of < or=1, allogeneic and syngeneic granulocyte macrophage colony-stimulating factor-secreting fibroblasts enhanced T-cell reactivity to tumor cells. However, at ratios of 2.4, the adjuvant effect induced by granulocyte macrophage colony-stimulating factor was not evident. Instead, we observed increased alloreactivity of primed LN cells against 3T3 targets as assessed by cytotoxicity and cytokine release assays, which was not observed with syngeneic fibroblasts. Moreover, with increasing numbers of allogeneic fibroblasts, there was a skewing of the T-cell Vbeta repertoire. These latter cells responded to tumor stimulation with the release of greater amounts of interleukin 10, which may account for the diminished antitumor reactivity observed in vivo. Allogeneic fibroblasts transduced to secrete interleukin 2 or IFN-gamma also induced diminished tumor reactivity of primed LN cells. Syngeneic fibroblasts are superior to allogeneic fibroblasts as vehicles to deliver cytokines in tumor vaccines.