Interleukin 12 augments the liver-associated immunity and reduces liver metastases

BACKGROUND/AIMS: It has been reported recently that in vivo administration of interleukin-12 (IL-12) augments the cytotoxic activity of natural killer (NK)/T cells and shows a powerful anti-tumor activity. In this study, we evaluated that the IL-12 effect on liver-associated immunity and in vivo efficacy on the hepatic metastases in a rat model. MATERIALS AND METHODS: Varying amounts of mouse recombinant IL-12 were injected intraperitoneally for 5 days to adult male Fischer rats and hepatic sinusoidal lymphocytes (HSL) were collected. Purified HSL are spontaneously cytolytic to both conventional NK-sensitive target (YAC-1) and NK-resistant target (RCN-H4) tumor cells. RESULTS: IL-12 was found to increase the number of HSL and the cytolytic activity against these target cells in a dose-dependent fashion. Flow cytometric analysis showed that IL-12 caused an increase of CD8+ subpopulation in HSL and a double staining study revealed that the increased subpopulation was not CD3+8+ (cytotoxic T cell) fraction, but actually CD3-8+ (NK cell) fraction. Experimental liver metastases was markedly reduced in rats treated intraperitoneally with IL-12. CONCLUSION: These results demonstrate that IL-12 augments the cytolytic activity of HSL and suggests this cytokine as an attractive choice for liver metastases therapy.     

Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy

OBJECTIVE: We examined the prevalence, correlates, and predictive value of an abbreviated somatization index, based on specific symptom thresholds, in primary care patients using services at a university-affiliated clinic. METHOD: We interviewed 1456 patients with a survey instrument that included the Composite International Diagnostic Interview (CIDI) to elicit symptoms and diagnoses of several psychiatric disorders as well as demographic information and a measure of disability. Statistical analyses examined the relationship of abridged somatization with physical functioning and various demographic and diagnostic factors. RESULTS: About one fifth of this primary care sample met the abridged somatization criteria. "Somatizers," defined according to these criteria, had significantly higher levels of psychiatric comorbidity and disability than "nonsomatizers". Analyses taking into account the number and type of organ/body systems represented by the unexplained symptoms showed that this dimension adds specificity to the prediction of outcomes. Thus, regardless of the total number of medically unexplained symptoms, abridged somatization with unexplained symptoms attributable to four or more organ/body systems showed the strongest association with disability and psychopathology. CONCLUSIONS: Abridged Somatization is a frequent syndrome in primary care that is strongly associated with psychopathology and physical disability. Our research also yielded a new series of abridged somatization subtypes (eg, "discrete" vs. "comorbid" and "simple" vs. "polymorphous") that may effectively separate among various psychopathologies, and may become useful tools for future research with somatizing patients.     

Interleukin 10 production by human melanoma

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.     

Laminin-alpha1-chain sequence Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg (LQVQLSIR) enhances murine melanoma cell metastases

We earlier screened overlapping synthetic peptides from the globular domain of the laminin alpha1 chain to identify active sites for cell attachment. We report here that one of the active cell-adhesion peptides, AG-73 (Arg-Lys-Arg-Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg-Thr; RKRLQVQLSIRT) causes B16F10 murine melanoma cells to metastasize to the liver, a site not normally colonized by these cells. Increases in liver metastases and in lung colonization are observed in immune-deficient beige/nude/xid and in C57Bl/6 mice with this peptide. This metastatic activity was observed with i.v. and with i.p. peptide injections, regardless of tumor cell or of peptide-injection times. In vitro, the AG-73 peptide enhances tumor cell adhesion, migration, invasion, and gelatinase production, and blocks laminin-1-mediated cell migration. AG-73 was found to significantly inhibit cell adhesion to a proteolytic laminin-1 fragment, E3, containing the AG-73 sequence. Cell attachment to AG-73, the E3 fragment, and laminin-1 involved cation-dependent receptors. We report that a laminin peptide has the novel and unexpected activity of causing B16F10 melanoma cells, a lung selected cell line, to metastasize to the liver. The minimal active sequence of AG-73, LQVQLSIR, could be one of the most important biologically active sites of laminin-1, especially in promotion of the malignant phenotype. Activation of the malignant phenotype by this peptide provides a significant new model for understanding metastatic mechanisms.     

Abrogation of B16 melanoma metastases by long-term low-dose interleukin-6 therapy

We investigated the antitumor effects of human recombinant interleukin-6 (hrIL-6) on the highly metastatic B16 melanoma clone F10.9. These tumor cells were found to have very low levels of IL-6 receptors and in vitro IL-6 had no effect on cell proliferation or on the expression of MHC class I antigens. However, in vivo IL-6 was active against the metastatic growth of this tumor in mice, presumably through indirect immune effects. Low-dose IL-6 (1-10 micrograms/day), in three daily injections, 4 days a week, for 3 weeks, strongly inhibited the formation of experimental lung metastases following intravenous tumor cell inoculation. IL-6 therapy could be started even 10 days after tumor injection, when metastases are already established. Moreover, IL-6 treatment of mice bearing F10.9 tumors in the footpads resulted in complete protection against pulmonary spontaneous metastasis and in long-term survival. Histology confirmed the absence of micrometastases in most of the IL-6-treated animals. Analysis of the cytolytic activity of splenocytes at different times during therapy of tumor-bearing mice revealed significant lysis (up to 42%) of the melanoma F10.9 cells in the mice receiving IL-6 but not in the control mice.     

Treatment of lung metastases from malignant melanoma with IL-2 inhalation therapy

Seven patients with pulmonary metastases of malignant melanoma were treated with inhalation therapy with 36 million IU interleukin-2 for six months. Inhalation therapy was combined with four bolus infusions of DTIC at a dose of 850 mg/m2 once every four weeks. Response rates were 71.4% with 2 patients achieving a complete remission (CR), 2 partial remissions (PR), 1 stable disease (SD), and 2 progressing disease (PD). Therapy was well tolerated with low toxicity. Six of the patients developed cough; one patient was slightly feverish. We conclude that inhalation therapy of lung metastases is a promising addition to the therapeutic arsenal against malignant melanoma.     

Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer

Metastases of breast cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of suicide gene therapy in metastatic breast cancer, we used the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) administration to treat breast cancer, generated by an adenocarcinoma cell line MOD in syngeneic mice. The bystander effect of HSV-tk + GCV on tumor cell killing was illustrated by demonstrating complete regression of subcutaneous tumors consisting of 90% parental tumor cells and 10% HSV-tk transformed tumor cells. To establish a model of breast cancer metastases in the liver, tumors were generated by intra-hepatic implantation of MOD cells in syngeneic animals. Two weeks after tumor cell implantation, replication defective adenoviral vectors expressing HSV-tk (ADV.tk), or beta-galactosidease (ADV. beta-Gal) were injected intratumorally, followed by buffer or GCV administration. Treatment with ADV.tk + GCV resulted in significant regression of tumor (P < .001), as assessed by computerized morphometric analysis of residual tumor. This was reflected as a significant prolongation of survival in treated animals (P < .001). These results demonstrate that ADV-mediated suicide gene therapy in vivo can be incorporated in a comprehensive treatment strategy for liver metastases of breast cancer.     

Immunotherapy, including gene therapy, for metastatic melanoma

Current standard therapy for distant metastatic melanoma is ineffective and often compromises the quality of a patient's life. Immunotherapy is briefly reviewed in relation to its many forms: from local non-specific to the more recent specific vaccines, including those using specific melanoma peptides (e.g. from the proteins encoded by melanoma-associated gene (MAGE)) and those involving genetically transduced autologous melanoma cells using retroviral vectors in vitro. The mode of action of genetically transduced melanoma cells incorporating the granulocyte macrophage colony stimulating factor (GM-CSF) gene (GVAX) is presented as a paradigm for cytokine-mediated strategies. Trials of GVAX and other cytokine gene strategies are under way in Brisbane, Boston and Amsterdam, and some interim perspectives on the clinical outcomes and immunological mechanisms involved are sketched. Some of the compounding problems in immunotherapeutic strategies for cancer are identified, and possible adjunct manoeuvres for overcoming them are discussed.     

Detection of melanoma metastases with thallium-201 scintigraphy

The design and development of a simulator for endovascular repair of abdominal aortic aneurysm (AAA) is described. The simulator consists of an interchangeable model of a human AAA based on computed tomography data and is produced by means of computer-aided design and manufacture (CAD/CAM) techniques. The model has renal, iliac, and femoral arteries, and is perfused with a temperature controlled blood-analog fluid under simulated physiological flow conditions. "Fluoroscopic imaging" is simulated by a computerized imaging system that uses visible light. A movable video camera relays images in the antero-posterior and lateral planes of the AAA to a monitor. The imaging system allows "arteriography" and "road-mapping" to be performed so as to facilitate accurate deployment of endovascular stent-grafts. The system has been used for teaching and demonstrating endovascular techniques to clinicians, as well as the evaluation of new stent-graft devices. Its successful incorporation into endovascular workshops has demonstrated its role in the training of clinicians in endovascular repair of AAA.     

Gastric metastases of melanoma: two case reports

Primary hydatid cyst of the brain in adults is rare and can pose various diagnostic problems. Multiplicity of these cysts is even rarer. The most important diagnostic tool is computed tomography scanning of the brain. Hydatid cyst should be included in the differential diagnosis when a cystic brain lesion is found in patients from an endemic echinococcosis area.     

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy

In addition to its role as a survival factor, nerve growth factor (NGF) has been implicated in initiating apoptosis in restricted cell types both during development and after terminal cell differentiation. NGF binds to the TrkA tyrosine kinase and the p75 neurotrophin receptor, a member of the tumor necrosis factor cytokine family. To understand the mechanisms underlying survival versus death decisions, the TrkA receptor was introduced into oligodendrocyte cell cultures that undergo apoptosis in a p75-dependent manner. Here we report that activation of the TrkA NGF receptor in oligodendrocytes negates cell death by the p75 receptor. TrkA-mediated rescue from apoptosis correlated with mitogen-activated protein kinase activation. Concurrently, activation of TrkA in oligodendrocytes resulted in suppression of c-jun kinase activity initiated by p75, whereas induction of NFkappaB activity by p75 was unaffected. These results indicate that TrkA-mediated rescue involves not only activation of survival signals but also simultaneous suppression of a death signal by p75. The selective interplay between tyrosine kinase and cytokine receptors provides a novel mechanism that achieves alternative cellular responses by merging signals from different ligand-receptor systems.     

Optimal scheduling of interleukin 12 and chemotherapy in the murine MB-49 bladder carcinoma and B16 melanoma

The antitumor activity of interleukin (IL)-12, a naturally occurring cytokine, has been demonstrated in several murine solid tumors. Animals bearing established B16 melanoma or MB-49 bladder carcinoma were used to study the most effective scheduling of recombinant murine IL-12 (rmIL-12), along with systemic chemotherapy. rmIL-12 (0. 45, 4.5, or 45 microgram/kg) was more effective as a single agent when administered to mice bearing the MB-49 bladder carcinoma at the highest dose for 11 doses rather than for 5 doses. In combination with chemotherapy (Adriamycin, cyclophosphamide, or 5-fluorouracil), rmIL-12 administration did not increase the toxicity of the chemotherapy, and there was increased antitumor activity with each rmIL-12-drug combination. Administering rmIL-12 (45 microgram/kg) on days 4-14, along with Adriamycin, cyclophosphamide, or 5-fluorouracil on days 7-11, resulted in 2.2-2.7-fold increases in tumor growth delay, compared with the chemotherapy alone against the primary tumor, and a marked decrease in the number of lung metastases on day 20. Because the B16 melanoma grows more slowly than the MB-49 bladder carcinoma, allowing multiple courses of chemotherapy, cyclophosphamide could be administered. The rmIL-12 (45 microgram/kg)-cyclophosphamide combination regimen that was most effective overlapped 2 days with the terminal portion of the chemotherapy treatment. There was a parallel increase in the response of the primary tumor and metastatic disease to the lungs. Administration of rmIL-12 to animals bearing the MB-49 bladder carcinoma or the B16 melanoma was compatible with coadministration of chemotherapy at full dose without additional toxicity.     

IL-12 gene therapy protects mice in lethal Klebsiella pneumonia

IL-12 is a proinflammatory cytokine that has recently been shown to have beneficial effects in the setting of acquired host immunity. To determine the role of IL-12 in innate immunity against Gram-negative bacterial organisms, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally (i.t.), resulting in the time-dependent expression of IL-12 mRNA (p35 and p40) and protein within the lung. Passive immunization of animals with anti-IL-12 serum i.p. at the time of K. pneumoniae inoculation resulted in a 12-fold increase in K. pneumoniae CFU in lung homogenates at 48 h, as compared with animals receiving control serum. In addition, treatment of Klebsiella-infected mice with anti-IL-12 Abs significantly decreased both short and long term survival. To assess the effect of compartmentalized IL-12 overexpression on outcome in Klebsiella pneumonia, animals were treated i.t. with 5 x 10(8) PFU of a nonreplicating adenoviral vector containing a human cytomegalovirus promoter and cDNAs coding for the p35 and p40 subunits of IL-12 inserted into the E1 and E3 domains (Ad5mIL-12), respectively. In vivo transfection with Ad5mIL-12 resulted in 45% long term survival in Klebsiella pneumonia, whereas no animals with Klebsiella pneumonia receiving control adenovirus survived. Moreover, treatment with anti-IFN-gamma Abs or soluble TNF receptor:Ig construct partially and completely attenuated survival benefits observed in animals receiving Ad5mIL-12, respectively. In conclusion, endogenous IL-12 is a critical component of antibacterial host defense, and the compartmentalized overexpression of IL-12 using recombinant adenoviral gene therapy represents a safe and effective approach to deliver IL-12 to the lung in the setting of murine Klebsiella pneumonia.