A monoclonal antibody raised against an undecapeptide sequence from human transforming growth factor alpha recognizes a hexapeptide epitope in mitotic centrosomes.

A monoclonal antibody (mAbIIa138) raised against the second-loop undecapeptide (residues [21-31]) of human transforming growth factor alpha, known to be involved in binding to its cell receptor, was found to define a hexapeptide epitope (RFLVQE, residues [22-27]). In enzyme-linked immunosorbent assay testing mAbIIa138 did not react with either native or reduced human transforming growth factor alpha, indicating that conformational restraints in this protein interfered with the antigenicity of the cognate sequence. Despite its failure to react with human transforming growth factor alpha, this monoclonal antibody proved very interesting when used to immunostain mammalian cells. mAbIIa138 was found to bind with great specificity to the centrosomes of late-interphase and mitotic cells. The staining of the centrosomes was most intense when the centrosomes were active in organizing the mitotic spindle and faded during telophase as the spindle was disaggregated. The epitope that mAbIIa138 recognizes is thus in a centrosomal protein, denoted CSP alpha, involved in some aspect of mitotic spindle organization or function. Analysis of the antigenic stringency of the epitope, using an amino acid replacement set, showed that 25 individual single amino acid replacements were possible without significant loss of antigenicity. Data bank searches for proteins of possible relevance were unsuccessful, so CSP alpha is an as yet unsequenced protein, specific to the centrosome.     

Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells

BACKGROUND: The response of hepatocellular carcinoma (HCC) to therapy is often disappointing and new modalities of treatment are clearly needed. Active immunotherapy based on the injection of autologous dendritic cells (DC) co-cultured ex vivo with tumor antigens has been used in pilot studies in various malignancies such as melanoma and lymphoma with encouraging results. METHODS: In the present paper, the preparation and exposure of patient DC to autologous HCC antigens and re-injection in an attempt to elicit antitumor immune responses are described. RESULTS: Therapy was given to two patients, one with hepatitis C and one with hepatitis B, who had large, multiple HCC and for whom no other therapy was available. No significant side-effects were observed. The clinical course was unchanged in one patient, who died a few months later. The other patient, whose initial prognosis was considered poor, is still alive and well more than 3 years later with evidence of slowing of tumor growth based on organ imaging. CONCLUSIONS: It is concluded that HCC may be a malignancy worthy of DC trials and sufficient details in the present paper are given for the protocol to be copied or modified.     

Dendritic cell immunotherapy for stage IV melanoma

Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell 'vaccine' in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enrolled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings.     

Search for “Weapons of Mass Destruction” for Cancer - Immuno/ Gene Therapy Comes of Age

The complexity of a cancer, such as cell heterogeneity, and the existence of hypoxia, stromai cells and stem cells has so far prevented successful development and treatment of patients suffering from the later stages of cancers. At present, the use of conventional therapies, such as chemo/radio therapy is limited, and only therapies that are focused on utilizing the patient＇s immune response to combat against the disease appear to be the most reliable and promising. Two decades ago, cytokines were discovered to be able to activate the immune systems and mount an anti-tumour response. Then, dendritic cells were hailed as the most significant regulators of immunity and are employed in a variety of cancer management schemes. This review introduces current development in the field, focusing on combination of the components of ＇he rapidly growing fields of immunotherapy and gene transfer/therapy, providing useful and significant detailed information for readers of cellular and molecular immunology.     

Tumor metastasis biopsy as a surrogate marker of response to melanoma immunotherapy.

In patients undergoing immunotherapy for metastatic melanoma, the clinical response in immunotherapeutic trials may be partial or difficult to detect. Tumor metastasis biopsy allows direct characterisation of an anti-tumor immunological response. During a phase I/II trial of granulocyte macrophage colony stimulating factor (GM-CSF) transduced autologous melanoma immunotherapy, the cellular response was examined by immunohistochemical analysis in a limited number of tumor biopsies taken from patients who either responded or progressed. Clinical response was associated with tumor infiltration by CD4+ and CD8+ T-cells, macrophages and differentiated dendritic cells (DC), and expression of HLA-DR by the tumor cells. This tumor infiltration was associated with increased melanoma-specific peripheral blood precursor cytotoxic T-lymphocyte (pCTL) and the ability to obtain tumor-infiltrating lymphocytes in vitro. In contrast, progression or a lack of clinical response was associated with a lack of T-cell and DC infiltration into the tumor tissue in all such biopsies. Macrophages and eosinophils infiltrated these tumors, while T-cells and DC were present at some distance from the tumor. These preliminary data strongly suggest that the location and extent of T-cell and DC infiltration, as well as the expression of HLA-DR by tumor cells are associated with a clinical response in this form of melanoma immunotherapy.     

Treating prostate cancer: a rationale for targeting local oestrogens

Prostate cancer is the most commonly diagnosed cancer and the second most common cause of cancer-related death in men, and benign prostatic hyperplasia is the most common benign condition known to occur in ageing men. Oestrogen has been implicated in the development of prostate cancer, and offers a promising new avenue for treatment. Despite this, the role of oestrogens in the prostate is complex. This Perspective presents a rationale for a targeted approach for the treatment of prostate disease through the use of selective oestrogen-receptor modulators in conjunction with contemporary androgen-ablation therapy.     

The development and characterization of an HEK293-derived cell line for use in an intratumoral cytokine delivery system

As part of ongoing work to develop a method of cytokine delivery for use as an intratumoral depot, we noted that HEK293 cells, encapsulated in alginate, died within 24-48 h after in vivo, intratumoral implantation. We hypothesized that the highly hypoxic and acidic conditions found inside the tumor was the cause of the cells' premature demise. Therefore, we set out to develop a cell line, derived from HEK293, that would survive these hostile conditions. The HEK293 line was selected in 0.3-0.5% oxygen conditions over several weeks, followed by a further 6-week period of culture in alternating hypoxic and normoxic conditions. The most rapidly growing clones were selected and grown in normoxic conditions for several weeks to ensure their stability. The clones were then compared to the original line in terms of cell proliferation in normoxia and hypoxia, colony-forming efficiency, and morphological characteristics. The resulting line was able to proliferate in the harshest of conditions and continues to release its biological payload after alginate microencapsulation.     

Subfractionation of CsCl-purified H-1 parvovirus on metrizamide gradients.

The different density classes of H-1 parvovirus, collected within 30 hr of parection of parasynchronous cultures, following the standard CsCl purification step, have been shown to be heterogeneous. Rebanding of the denser form (HF, ? = 1.46 g/cm³) and the less dense form (LF, ? = 1.42 g/cm³) of infectious virus in the nonionic density generating solute, metrizamide, showed that both HF and LF virus bands were heterogeneous in density. The infectivity banded with isotopically labeled virus protein and DNA at 1.32 g/cm³ for both HF and LF virus. Amounts of protein and DNA which varied from preparation to preparation, but which were greater from the HF virus band, were distributed throughout the rest of the gradient, but predominated in a peak at a density of 1.2 g/cm³. The protein in this peak was without hemagglutinating activity but had the molecular weights and proportions of the H-1 virion proteins (VP1, VP2′, and VP2). The DNA was of the same size as H-1 DNA monomers and its proportion to the protein was similar to that of the infectious peak. The DNA was susceptible to micrococcal nuclease digestion. The nature of this noninfectious viral material thus seemed to be incompletely assembled virus. Radio-labeled H-1 virus collected after 72 hr of infection formed a discrete single peak in both CsCl (? = 1.42 g/cm³), and metrizamide gradients (? = 1.32 g/cm³). There was no significant amount of the 1.20 g/cm³ viral protein-DNA complex in these mature preparations.