Microwave tumour coagulation plus in situ treatment with cytokine-microparticles: induction of potent anti-residual tumour immunity.

After local microwave coagulation and subsequent intra-tumoural injection of microparticles encapsulating interleukin-2 and granulocyte-macrophage colony-stimulating factor, the anti-tumour efficacy against subcutaneous Lewis lung carcinoma in syngeneic mice was evaluated. This treatment elicited a potent systemic anti-tumour immunity that protected treated mice from re-challenge with the same tumour cells and caused the distal tumours in a bilateral tumour model to be rejected. Cytotoxicity assay indicated that both T- and natural killer cells acted as the effector cells in the anti-tumour immunity. These data highlight the feasibility of microwave-pre-treated in situ cancer vaccination for clinical use.

A tumour vaccine of fixed tumour fragments in a controlled-release vehicle with cytokines for therapy of hepatoma in mice.

BACKGROUND: Cytokines can be strong potentiators for a tumour vaccine, but they have very short life in vivo when administered as a solution. AIMS: To evaluate the slow release of interleukin 2 from a cytokine-vehicle in vitro and in vivo and to evaluate the anti-tumour activity of a new tumour vaccine in vivo. METHODS: The tumour vaccine was composed of formalin-fixed Hepa 1-6 hepatoma tissue fragments, tuberculin and a lipid based vehicle containing granulocyte-macrophage colony-stimulating factor and interleukin 2. The quantity of interleukin 2 release from the cytokine-vehicle in vitro and in vivo was determined by a proliferation assay with CTLL-2 cell line. Hepa 1-6 hepatoma model system with C57BL/6J mice was used to examine protective and therapeutic anti-tumour effect of the vaccine. RESULTS: Release of interleukin 2 from the cytokine-vehicle lasted 5 days in vitro and 3 days in vivo. The vaccine protected 67% of mice from a Hepa 1-6 cell challenge and had a therapeutic effect by prolonging the life span of mice bearing established Hepa 1-6 tumours of 5 mm in diameter. Of the treated mice, 20% became completely tumour-free. CONCLUSIONS: Formalin-fixed tumour fragments and cytokines in controlled-release vehicle are useful in the rational design of tumour vaccines.

In-situ visualization and quantification of mineralization of cultured osteogenetic cells.

An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, beta-glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.

Totally synthetic polymer with lectin-like function: induction of killer cells by the copolymer of 3-acrylamidophenylboronic acid with N,N-dimethylacrylamide.

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation.

Transient infiltration of neutrophils into the thymus in association with apoptosis induced by whole-body X-irradiation.

Generally, the process of apoptosis does not cause leakage of noxious cytosolic contents and is therefore non-inflammatory. However, as previously shown, macrophages ingesting apoptotic CTLL-2 cells produced pro-inflammatory cytokines, particularly interleukin-8 (IL-8) and macrophage inflammatory protein-2 (MIP-2), a murine IL-8 homolog. This predicted that rapid and massive apoptosis may induce neutrophil accumulation in vivo. In this study, we tested this prediction by inducing apoptosis by whole-body X-irradiation in mice. After exposure to 4 Gy X-ray irradiation, mice exhibited considerable apoptosis of thymic cells, which was associated with transient infiltration of neutrophils as well as MIP-2 mRNA expression. In contrast, in p53-deficient mice in which irradiation-induced apoptosis was suppressed, as has been reported, infiltration of neutrophils into the thymus was less than that found in p53+/+ mice. Taken together, these results suggest that massive and rapid apoptosis can result in infiltration of neutrophils.

Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatory cytokines.

It is generally believed that apoptosis is not associated with inflammation. To explore the possibility that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation, we examined the cytokine production by thioglycollate-induced peritoneal exudate cells (PEC) upon interaction with a murine T cell clone (CTLL-2) cultured in the absence of interleukin-2 (IL-2). Coculturing of PEC with apoptotic CTLL-2 cells led to the production of not only anti-inflammatory cytokines but also pro-inflammatory ones, notably MIP-2, at the mRNA level, and neutrophils were accumulated in vivo in response to the culture supernatant. Our findings suggest the possibility that apoptosis may be associated with leukocyte infiltration in vivo in some situations.

Modulation by lipopolysaccharide of inflammatory cytokine production by two T cell lines.

In this study, the modulation of inflammatory cytokine production by lipopolysaccharide (LPS) in two T cell lines, RL-male-1 and L5178Y-ML was examined. Both cell lines produced interleukin 1 (IL-1) activity in response to LPS, which was largely independent of the presence of serum. The IL-1 activity induced was neutralized by an anti-IL-1beta antibody, but not by an anti-IL-1alpha antibody. Induction of IL-1alpha and beta was also confirmed by polymerase chain reaction of reverse-transcribed mRNA (RT-PCR), although in RL-male-1 cells, IL-1alpha mRNA was constitutively expressed and somewhat enhanced by LPS. RT-PCR analysis revealed that these cell lines also upregulated tumour necrosis factor alpha (TNF-alpha) and IL-1 receptor antagonist mRNAs in response to LPS, although RL-male-1 cells expressed TNF-alpha mRNA constitutively. Flow cytometric analysis showed that, although these cells expressed Thy-1 antigen, they hardly expressed CD14 and gammadelta T cell receptor. In conclusion, LPS modulated inflammatory cytokine production in these T cell lines.

Novel biological response modifiers: phthalimides with tumor necrosis factor-alpha production-regulating activity.

Novel N-substituted phthalimides (2-substituted 1H-isoindole-1,3-diones) were prepared, and their effects on tumor necrosis factor-alpha (TNF-alpha) production by human leukemia cell line HL-60 stimulated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) were examined. A structure-activity relationship study of the N-phenylphthalimides and N-benzylphthalimides revealed that their enhancing effect on TPA-induced TNF-alpha production by HL-60 cells and their inhibiting effect on OA-induced TNF-alpha production by HL-60 cells are only partially correlated.