Immunogene therapy with interleukin-2-secreting fibroblasts for intracerebrally metastasizing breast cancer in mice.

OBJECT: Breast cancer is the second leading cause of cancer-related death in American women. Brain metastases occur in 15 to 30% of patients with breast cancer, and this usually results in death. Despite the availability of surgery, radiotherapy, and chemotherapy, the prognosis for patients with breast cancer that has metastasized to the intracerebral region remains poor. This study was designed to determine if an intracerebrally metastasizing breast tumor could be treated successfully with a cellular vaccine consisting of allogeneic fibroblasts (H-2K) modified to secrete interleukin (IL)-2. METHODS: The authors used EO771 breast cancer cells, derived from a spontaneously arising breast-cancer tumor in C57BL/6 mice. The authors first determined the length of survival of C57BL/6 mice that had been injected with varying numbers of EO771 cells into the right frontal lobes and found that 100% of those animals that received a dose of 10(4) cells died within 41 days, whereas 100% of the group that received 10(3) cells died within 23 days. Based on these results, 5 x 10(4) EO771 cells were injected into the right frontal lobe of C57BL/6 mice and the animals were treated intracerebrally with a single intratumoral injection of 10(6) allogeneic fibroblasts genetically engineered to secrete IL-2. The allogeneic fibroblasts transfected with the IL-2 gene formed large quantities of IL-2 as measured by an enzyme-linked immunosorbent assay. Control groups of animals were treated with either allogeneic fibroblasts transfected with the retroviral vector, but not the IL-2 gene, or with media. The effects of this treatment on the animal's survival and the tumor's histopathological characteristics were investigated. The results indicate a significant prolongation (p < 0.005) of survival in animals with intracerebrally metastasizing breast cancer treated only with IL-2-secreting allogeneic fibroblasts. Tumor did not develop in four of 10 animals in the IL-2-treated group, and these animals were rechallenged at 90 days by intracerebral injection of tumor, but no treatment cells, and compared with four naive animals receiving intracerebral tumor. Again, animals that previously had been treated with IL-2-secreting fibroblasts had a markedly prolonged survival (p < 0.05) compared with control animals following a second challenge with tumor cells. Histopathological examination revealed smaller tumors associated with lymphocytic infiltrations in the treated animals. CONCLUSIONS: This work represents a new treatment for breast cancer that has metastasized to the brain in which a cellular vaccine consisting of allogeneic fibroblasts genetically engineered to secrete cytokines is used as a novel means for delivery of immunogene therapy and demonstrates the induction of long-term immunity against tumor.

Cytokine immunogene therapy.

The prognosis for patients with either a primary or metastatic brain tumor is poor. Clearly new forms of therapy to improve the long-term survival of patients with malignant brain tumors are urgently needed. The authors are in the process of developing a new and novel form of treatment for primary and metastatic brain tumors in which they use genes involved in growth repression. In particular most tumors fail to induce an antitumor immune response strong enough to kill the tumor. Under appropriate circumstances, however, immunity can be produced in unique structures on the tumor cells known as antigens. To prepare the vaccine, genes are transferred into a fibroblast cell line that causes the cell to produce cytokines, the potent proteins known to stimulate the immune system. These cells are subsequently injected into the tumor bed, resulting in the development of an antitumor immune response. In experiments described in this manuscript, the authors have investigated a number of ways of augmenting the immune response by administering this type of cellular vaccine. They found that mice with a primary intracerebral glioma, melanoma, or breast cancer treated with this allogeneic cytokine-secreting vaccine survived significantly longer than untreated mice. Additionally the vaccine was found to stimulate a systemic antitumor immune response, as shown by immunocytotoxic studies, histopathological examination, and delayed immune memory responses. In summary, these results indicate that immunogene therapy is a promising new targeted therapy for the treatment of intracerebral malignant tumors.

Prolongation of survival of mice with glioma treated with semiallogeneic fibroblasts secreting interleukin-2.

OBJECT: The current prognosis for patients with malignant brain tumors remains poor, and new therapeutic options are urgently needed. We previously have shown that prolongation of survival can be achieved in C57BL/6 mice (H-2b) with a syngeneic intracerebral or subcutaneous glioma when treated with allogeneic mouse fibroblasts (H-2k) genetically engineered to secrete interleukin-2 (IL-2). Like other antigens, tumor-associated antigens are recognized by cytotoxic T lymphocytes in the context of determinants specified by the major histocompatibility complex class I locus. Because the rejection of allogeneic major histocompatibility complex determinants has the property of an immune adjuvant, immunotherapy of glioma with a cellular immunogen that combines the expression of both syngeneic class I determinants and allogeneic antigens could have advantages over an immunogen that expresses syngeneic or allogeneic determinants alone. METHODS: To investigate this question in a mouse glioma model, we further modified allogeneic mouse fibroblasts (H-2k), not only for IL-2 secretion, but also for the expression of H-2Kb class I determinants. We tested the immunotherapeutic properties of these semiallogeneic cells (LM-IL-2/H-2Kb) in C57BL/6 mice with Gl261 glioma in both subcutaneous and intracerebral glioma models. RESULTS: C57BL/6 mice with either a subcutaneous or intracerebral glioma treated solely by injection of these IL-2-secreting semiallogeneic cells had significantly prolonged survival rates compared with untreated mice or mice treated with cells secreting only IL-2 or cells lacking the H-2Kb determinants. In some instances, the mice treated with the semiallogeneic cells survived indefinitely, suggesting total eradication of the glioma. When a 51Cr release assay was used, the specific immunocytotoxicity measured by release of isotopes from labeled Gl261 glioma cells coincubated with spleen cells from mice immunized with the semiallogeneic IL-2-secreting cells was significantly higher than that of spleen cells from nonimmunized mice or mice immunized with allogeneic cells lacking syngeneic major histocompatibility complex determinants. In addition, antibody depletion studies using monoclonal antibodies against CD8+ and natural killer/lymphokine-activated killer cells demonstrated a specific CD8+ immunocytotoxic response in animals immunized with the semiallogeneic IL-2-secreting cells compared with only a natural killer/lymphokine-activated killer response in mice immunized with the allogeneic IL-2-secreting cells. CONCLUSION: The augmented immune response against glioma in mice treated with the semiallogeneic IL-2-secreting cells points toward a new form of immunotherapy, "immuno-gene therapy," for patients with malignant intracerebral glioma.

Development of a new mouse brain tumor model using implantable micro-cannulas.

The objective of this study was to develop a brain tumor model in a mouse where gene therapy could be delivered either directly into a pre-established tumor bed or prophylacticly prior to tumor delivery (protective treatment). Micro-cannulas were constructed from metal tubing, implanted into the right frontal lobe of mice, and then secured in place in the skull with cement. Experiments evaluating the usefulness, reproducibility and morbidity of the system were performed. It was found that tumor cells could reproducibly be delivered into the right frontal lobe of the mice. Two tumors could be precisely delivered into the same area following injections at different times. Repeat injections were performed without a stereotaxic frame and without the need for repeat intracerebral needle tracts. There were no noticeable side effects of maintaining the cannulas in place for long periods of time. In summary, this system is useful for studying the effects of various treatment strategies on established brain tumors in a mouse model which more closely simulates the clinical situation. It obviates the need for time consuming stereotaxic procedures or repeat invasive intracerebral injections.

Loss of heterozygosity on chromosome 11q13 in two families with acromegaly/gigantism is independent of mutations of the multiple endocrine neoplasia type I gene.

Familial acromegaly/gigantism occurring in the absence of multiple endocrine neoplasia type I (MEN-1) or the Carney complex has been reported in 18 families since the biochemical diagnosis of GH excess became available, and the genetic defect is unknown. In the present study we examined 2 unrelated families with isolated acromegaly/gigantism. In family A, 3 of 4 siblings were affected, with ages at diagnosis of 19, 21, and 23 yr. In family B, 5 of 13 siblings exhibited the phenotype and were diagnosed at 13, 15, 17, 17, and 24 yr of age. All 8 affected patients had elevated basal GH levels associated with high insulin-like growth factor I levels and/or nonsuppressible serum GH levels during an oral glucose tolerance test. GHRH levels were normal in affected members of family A. An invasive macroadenoma was found in 6 subjects, and a microadenoma was found in 1 subject from family B. The sequence of the GHRH receptor complementary DNA in 1 tumor from family A was normal. There was no history of consanguinity in either family, and the past medical history and laboratory results excluded MEN-1 and the Carney complex in all affected and unaffected screened subjects. Five of 8 subjects have undergone pituitary surgery to date, and paraffin-embedded pituitary blocks were available for analysis. Loss of heterozygosity on chromosome 11q13 was studied by comparing microsatellite polymorphisms of leukocyte and tumor DNA using PYGM (centromeric) and D11S527 (telomeric), markers closely linked to the MEN-1 tumor suppressor gene. All tumors exhibited a loss of heterozygosity at both markers. Sequencing of the MEN-1 gene revealed no germline mutations in either family, nor was a somatic mutation found in tumor DNA from one subject in family A. The integrity of the MEN-1 gene in this subject was further supported by demonstration of the presence of MEN-1 messenger ribonucleic acid, as assessed by RT-PCR. These data indicate that loss of heterozygosity in these affected family members appears independent of MEN-1 gene changes and suggest that a novel (tissue-specific?) tumor suppressor gene(s) linked to the PYGM marker and expressed in the pituitary is essential for regulation of somatotrope proliferation.

Survival and toxicity of an allogeneic cytokine-secreting fibroblast vaccine in the central nervous system.

OBJECTIVE: In previous studies, we determined that C57BL/6 mice (H-2b) with intracerebral (i.c.) malignant glioma or melanoma treated with allogeneic fibroblasts genetically engineered to secrete interleukin (IL)-2 results in prolonged survival and a cellular antitumor response when the treatment is administered intratumorally (via i.c. injection). For this study, we evaluated the survival and toxicity of the cytokine-secreting cellular vaccine administered directly into the central nervous system in both syngeneic (C3H H-2k) and allogeneic (C57BL/6 H-2b) mice using bioassay and polymerase chain reaction techniques. METHODS: First, brain sections were prepared at varying time intervals (2-60 d) after i.c. injection of the IL-2-secreting fibroblasts (H-2k) into allogeneic and syngeneic mice, and the presence of the cells was detected by polymerase chain reaction for the neomycin gene, which was used as a selection marker for the gene transfer. As a second means of assessing survival of the IL-2-secreting cells, an in vitro bioassay technique was used. Brain sections were prepared at varying time intervals (2-60 d) after i.c. injection of the IL-2-secreting cells into allogeneic and syngeneic mice. Cells were disassociated and grown in media containing neomycin. RESULTS: A positive signal by polymerase chain reaction was no longer detectable 14 days after injection into allogeneic C57BL/6 (H-2b) mice. In contrast, under similar circumstances, the IL-2-secreting cellular vaccine could be detected for more than 55 days in histocompatible (C3H) syngeneic mice (H-2k). Allogeneic cells could be recovered from tissue culture for only 2 to 5 days after i.c. injection of the modified cells. In contrast, syngeneic cells were recovered for up to 28 days after i.c. injection. At no time did the mice exhibit signs of neurological deficit or adverse affects on their survival (>60 d). CONCLUSION: An allogeneic cytokine-secreting cellular vaccine has a limited lifespan in the central nervous system, surviving only 14 days or less, and has no apparent adverse effects. These results indicate the advantage of using a modified allogeneic cell line as the delivery vehicle for gene therapy in the treatment of an i.c. neoplasm.

Intracerebral versus subcutaneous immunization with allogeneic fibroblasts genetically engineered to secrete interleukin-2 in the treatment of central nervous system glioma and melanoma.

OBJECTIVE: The purpose of this study was to determine the optimal route of delivery of gene therapy for an intracerebral (IC) tumor. In previous studies, treatment of an IC tumor with the IC administration of a cellular vaccine consisting of allogeneic fibroblasts genetically engineered to secrete cytokines prolonged survival. Systemic delivery of gene therapy is of significant clinical interest. METHODS: In this study, allogeneic fibroblasts engineered to secrete interleukin (IL)-2 (LM-IL-2 cells) were administered either subcutaneously or intracerebrally to C57BL/6 mice with IC glioma. In addition, fibroblasts genetically engineered to express (antibody-defined) melanoma-associated antigens and to secrete IL-2 (RLBA-IL-2) were injected either intracerebrally or subcutaneously into mice bearing IC melanoma. RESULTS: The results indicate a significant prolongation of survival in mice with IC glioma treated intracerebrally with LM-IL-2 cells, relative to the survival of mice with IC glioma treated subcutaneously with LM-IL-2 cells or untreated mice with glioma. The specific release of isotope from 51Cr-labeled glioma cells coincubated with spleen cells from animals treated either subcutaneously or intracerebrally with LM-IL-2 cells was significantly greater than the release of isotope from glioma cells coincubated with spleen cells from nonimmunized mice. In a similar fashion, the survival of mice with IC B16 melanoma immunized intracerebrally with RLBA-IL-2 cells was significantly longer than nonimmunized mice injected with B16 cells alone. In contrast, the survival of mice with IC melanoma treated by subcutaneous injection with RLBA-IL-2 cells was not significantly different than that of untreated mice. Using a 51Cr-release assay, the specific release of isotope from labeled B16 cells coincubated with spleen cells from mice immunized either intracerebrally or subcutaneously with RLBA-IL-2 cells was significantly higher than that of B16 cells coincubated with cells from nonimmunized mice. CONCLUSIONS: Direct IC administration of fibroblasts genetically engineered to secrete IL-2 was more effective in prolonging survival than peripheral subcutaneous administration in the treatment of mice with IC glioma or melanoma.

Insulin-like growth factors in central nervous system tumors.

Insulin-like growth factors (IGFs) appear to play a role in the development of tumors in general and brain tumors in particular. Specific receptors for IGFs have been identified in normal human and rat brain, and evidence suggests that components of the IGF signal transduction system may play a role in the transformation process. Secretion of IGFs by a variety of human brain tumors has been confirmed, and these growth factors appear to have an autocrine stimulatory effect on these tumors. IGFs circulate in the blood stream bound to at least six distinct binding proteins which may modulate the effects of these growth factors on target tissues. Sex steroids may also regulate the behavior of certain brain tumors such as meningiomas at least in part through their effects on the expression of IGFs and their binding proteins. Recently, antisense gene technology against certain IGFs or their receptors have resulted in potent antitumor effects in the case of several gliomas, although the mechanism for this remains unclear.

Prolonged survival of mice with glioma injected intracerebrally with double cytokine-secreting cells.

A novel approach toward the treatment of glioma was developed in a murine model. The genes for both interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were first transfected into a mouse fibroblast cell line that expresses defined major histocompatibility complex (MHC) determinants (H-2k). The double cytokine-secreting cells were then cotransplanted intracerebrally with the Gl261 murine glioma cell line into syngeneic C57BL/6 mice (H-2b) whose cells differed at the MHC from the cellular immunogen. The results indicate that the survival of mice with glioma injected with the cytokine-secreting allogeneic cells was significantly prolonged, relative to the survival of mice receiving equivalent numbers of glioma cells alone. Using a standard 51Cr-release assay, the specific release of isotope from labeled Gl261 cells coincubated with spleen cells from mice injected intracerebrally with the glioma cells and the cytokine-secreting fibroblasts was significantly higher than the release of isotope from glioma cells coincubated with spleen cells from nonimmunized mice. The cellular antiglioma response was mediated by natural killer/lymphokine-activated killer and Lyt-2.2+ (CD8+) cells. The increased survival of mice with glioma and the specific immunocytotoxic responses after immunization with fibroblasts modified to secrete both IL-2 and IFN-gamma indicate the potential of an immunotherapeutic approach to gliomas with cytokine-secreting cells.

Fibroblasts genetically engineered to secrete cytokines suppress tumor growth and induce antitumor immunity to a murine glioma in vivo.

The genes for interleukin (IL)-2, interferon (IFN)-gamma, or both IL-2 and IFN-gamma were introduced into a mouse fibroblast cell line (LM) expressing defined major histocompatibility complex determinants (H-2k). The cytokine-secreting cells were then co-transplanted with the Gl261 murine glioma cell line (H-2b) into syngeneic C57BL/6 mice that differed at the major histocompatibility complex from the cytokine-secreting cells. The period of survival of mice with glioma treated with IL-2- or IL-2/IFN-gamma-secreting allogeneic cells was significantly prolonged (P < 0.025) relative to the survival of mice receiving equivalent numbers of tumor cells alone or mice with glioma treated with nonsecreting fibroblast (LM) cells. Gliomas in the treated mice had an extensive lymphocytic cell infiltrate. Using a 51Cr release assay, the specific release of isotope from labeled Gl261 cells co-incubated with spleen from mice injected with the glioma cells and IL-2-secreting fibroblasts was higher (P < 0.001) than the release from glioma cells co-incubated with spleen cells from nonimmunized mice. Significantly higher levels of release (P < 0.005) were found in the group immunized with fibroblasts secreting both IL-2 and IFN-gamma. Based upon the effect of monoclonal antibodies for T-cell subsets on the antiglioma response, the immunity was mediated predominantly by natural killer/lymphokine-activated killer cells.

Radioimmunoassay of insulin-like growth factors I and II in the cerebrospinal fluid of patients with pituitary and other central nervous system tumors.

Tumor cells are characterized by abnormalities in growth and metabolism, including the autocrine secretion of certain growth factors. On the basis of our previous demonstration of the production of insulin-like growth factors (IGFs) and their binding proteins by central nervous system (CNS) tumors, we asked whether the levels of IGFs in the CSF may be altered in patients with pituitary and other CNS tumors and may reflect autocrine secretion. We used specific radioimmuoassays for IGF-I and -II and measured these growth factors in the cerebrospinal fluid (CSF) from 26 patients with tumors located adjacent to the ventricular system. The tumors included were eight pituitary tumors (five nonsecreting, three growth hormone secreting), five gliomas, two meningiomas, five medulloblastomas, three metastases, and three other tumors. CSF from patients without tumors served as controls. For radioimmunoassay, CSF was treated with acetic acid overnight and IGF-binding proteins were separated from IGFs by C-2 solid phase cartridge extraction. The pituitary tumors were characterized by significantly elevated levels of IGFs in the CSF. In nonseceting pituitary tumors, the levels of IGF-I in the CSF were similar to normal levels, whereas IGF-II levels were significantly elevated. In acromegalic patients, levels of both IGF-I and -II in the CSF were significantly elevated compared with normal levels and compared with levels in patients with nonsecreting tumors. In contrast, the levels of IGFs in the CSF from most of the primary and metastatic CNS CNS tumors did not significantly differ from normal values. In summary, although IGFs may contribute to the regulation of cell growth in primary CNS tumors, CSF levels are not elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of the growth hormone and insulin-like growth factor I axis to medical castration in women with polycystic ovarian syndrome.

OBJECTIVE: To assess the impact of ovarian hormone deprivation on serum concentrations of GH and insulin-like growth factor I (IGF-I) in women with polycystic ovarian syndrome (PCOS). DESIGN: Growth hormone and IGF-I levels were measured before, during, and at the end of a 6-month period of treatment with leuprolide acetate (LA) depot. Normal cycling and ovariectomized women served as controls. SETTING: The Clinical Research Center of Northwestern University Medical School. PATIENTS: Six women with PCOS, seven normal cycling women, and six ovariectomized women. INTERVENTIONS: The PCOS and normal cycling subjects were treated with six consecutive monthly injections of LA depot 3.75 mg IM. MAIN OUTCOME MEASURES: Circulating concentrations of GH and IGF-I. RESULTS: After 3 months of treatment, GH levels decreased modestly in the PCOS group; however, these changes were not significant. No further decline was observed at 6 months. Growth hormone levels were unaffected in the normal cycling group. Levels of IGF-I remained unchanged in both groups. Mean baseline levels of both GH and IGF-I in the ovariectomized group were lower than those of the normal cycling and PCOS groups at all time points, but not significantly so. CONCLUSIONS: Medical castration fails to significantly alter serum GH and IGF-I levels in either normal cycling or women with PCOS. These data indicate that GH secretion is not modulated by hormones of ovarian origin in either group, at least over the period of time studied.

Proliferative growth of neonatal cerebellar cells in culture: regulation by male and by maternal serum in late gestation.

Neonatal cerebellar cells were utilized as a model system to examine the effect of 20 day pregnant rat serum on proliferative growth in the CNS. Cells were prepared by mechanical dissociation and cultured as mixed cells or populations enriched in astrocytes or oligodendrocytes. Cell proliferation was estimated by measurement of DNA, protein, and/or mitochondrial reductase activity (MTT). When mixed cells were incubated with 10% male rat serum, both total DNA and protein content increased after 6 days of culture. By contrast, neither of these parameters were altered in cultures incubated with 10% pregnant serum. When cells were incubated with either male or pregnant sera, changes in MTT activity paralleled changes in protein content. Graded concentrations of pregnant serum (5-20%) added to mixed cell cultures produced consistently lower MTT values when compared with identical concentrations of male serum. In addition, MTT activity was diminished in both astrocytes and oligodendrocytes incubated with graded concentrations of pregnant sera when compared with similar concentrations of non-pregnant sera. When potential effects of these different sera on the cell cycle were examined, an increase in the number of cells in the S and G2/M phase was similar, and DNA doubling began to increase at 96 hrs in the presence of either male or 20 day pregnant sera. Thus the inhibition of cell growth by pregnant serum was not likely a result of either cytotoxicity or a delay of entry of cells into the cell cycle. To examine whether this inhibition of cell growth may reflect the effect of pregnant serum on endogenous growth factor production, we tested the production of IGF-II by cerebellar cells. production of an endogenous source of IGF-II was apparent using an RNAse protection assay and was noted using Slot Blot analysis of mRNA extracted at sequential times during cell incubation. Mixed cell cultures also secreted immunologically defined IGF-II. These observations are consistent with the previous demonstration that the fraction of pregnant serum which bound IGF-II also inhibited cell growth. The inhibitory effect of pregnant serum was diminished by preincubating aliquots of sera with graded concentrations of IGF-I prior to adding sera to tissue culture medium. Pregnant serum inhibition was also diminished by prolonging incubation times beyond 6 days. The blunting of pregnant serum inhibition may have been consequent to either a continuing production of endogenous growth factors or to the potential emergence of resistant cells due to prolonged tissue culture incubation.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of insulin-like growth factor (IGF) and glucose transporter-1 and -3 mRNA in CNS tumors.

Glucose metabolism is increased in CNS tumors and correlates with malignant grade. We have previously investigated the role of IGFs in regulating CNS tumor growth and metabolism. In the present study we examined total cellular RNA from human CNS tumors for the presence for glucose transporter (Glut) and IGF mRNA. Human meningiomas and gliomas were frozen in liquid nitrogen at the time of surgery and then stored at -80 degrees C. Total cellular RNA was prepared by acid-guanidinium phenol-chloroform extraction and 20 micrograms of RNA was loaded for agarose-formaldehyde gel electrophoresis and transfer. RNA integrity in 5 meningiomas and 2 gliomas was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNA and hybridization with a cDNA probe for beta-actin. For analysis, membranes were hybridized to radioactively labeled human Glut-1, Glut-3, IGF-I, and IGF-II cDNA probes, and mRNA transcripts were identified by autoradiography. All 7 tumors expressed Glut-1 and Glut-3 mRNA and Glut-3 appeared to be more abundant in meningiomas. IGF-II mRNA was detected in 2 of 6 meningiomas and in both gliomas. IGFs may play an important role in the regulation of glucose metabolism in CNS tumors. IGFs and specific glucose transporters may prove useful as markers of malignancy and potential targets for future therapy.

Insulin-like growth factors and binding proteins in the fetal rat: alterations during maternal starvation and effects in fetal brain cell culture.

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction. Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [125I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating levels of insulin, insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins in the small for gestational age fetal rat.

Insulin-like growth factors (IGFs) are important regulators of somatic growth in childhood and circulate in association with specific binding proteins (IGFBPs). Insulin is important for the regulation of IGFs and IGFBPs in postnatal life and is required for normal growth in utero. We asked whether alterations in the availability of IGFs and/or their BPs may contribute to impaired fetal growth 24 h after bilateral uterine artery ligation when circulating levels of insulin are low and fetuses are small for gestational age (SGA). Bilateral uterine arterial ligation or sham surgery was performed on maternal rats on day 19 of gestation (term = 21.5 days). One day after ligation, fetuses were SGA compared to shams (2.9 +/- 0.1 vs. 3.5 +/- 0.1 g/fetus, respectively; P < 0.001), and liver weight was reduced (242 +/- 9 vs. 300 +/- 6 mg/liver; P < 0.001). Serum levels of both insulin and IGF-I were reduced approximately 50% in SGA litters compared to those in shams (P < 0.001) and correlated with each other (P < 0.02). IGF-I levels also correlated with fetal body and liver weights (P < 0.005 for each) even after controlling for the effects of insulin. Insulin levels correlated with body and liver weights (P < 0.02 and P < 0.03, respectively), but these relationships were not significant after controlling for the effects of IGF-I. Serum levels of IGF-II were not significantly reduced in SGA and did not correlate with fetal weight or insulin or IGF-I levels. [125I]IGF-I binding assay demonstrated that the availability of IGFBPs was increased in SGA serum, and Western ligand blotting indicated that circulating levels of 32K IGFBPs were increased in SGA fetuses compared to shams. Densitometric analysis indicated that levels of 32K IGFBPs were 4-fold greater in SGA litters (P < 0.001 vs. shams), and the level of 32K IGFBPs correlated with fetal body and liver weights (P < 0.05 for each) and with levels of both insulin and IGF-I (P < 0.001 for each), but not with levels of IGF-II. Immunoblotting with newly developed antiserum against rat IGFBP-1 confirmed that levels of immunoreactive 32K IGFBP-1 are increased in SGA serum, and immunoprecipitation studies confirmed that IGFBP-1 accounted for the rise in 32K IGFBPs in SGA serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin and insulin-like growth factors in central nervous system tumors. Part V: Production of insulin-like growth factors I and II in vitro.

The authors have previously reported the presence of insulin-like growth factor (IGF) receptors in central nervous system (CNS) tumors and the production of IGF's and their binding proteins by CNS tumors in situ. This study was designed to investigate whether CNS tumor cells are capable of autocrine secretion of IGF-I and IGF-II in vitro. Production of IGF's was studied by specific radioimmunoassay of tumor-cell-conditioned serum-free media from 34 CNS tumors: 12 gliomas, 12 meningiomas, and 10 miscellaneous tumors. Normal human serum and cerebrospinal fluid served as controls. Insulin-like growth factor I was detected in five of 12 meningiomas but in none of the gliomas studied. In contrast, IGF-II was detected in four of 12 gliomas and in six of 11 meningiomas studied. Four miscellaneous tumors produced IGF-I and/or IGF-II. These results suggest that CNS tumors differentially produce IGF-I and IGF-II in vitro. Preferential production of IGF's may be an important marker of the tumor-cell differentiation or malignancy and may be useful as a clinical diagnostic tool. These results add further support to the concept that IGF's may play a role in the regulation of the behavior of CNS tumors.

Diagnostic accuracy of AIDS-related CNS lesions.

Because of the high incidence of neurological complications seen in patients with acquired immunodeficiency syndrome (AIDS), an increasing number of these cases are being referred to neurosurgeons for consideration of intracranial biopsy. To better determine the need for biopsy in these patients we evaluated the accuracy of non-tissue-based neurological diagnoses in AIDS patients who subsequently had a final diagnosis on the basis of biopsy or postmortem brain examinations. The records of 56 AIDS patients who had undergone either autopsy or brain biopsy were retrospectively reviewed. Of the ten patients who underwent biopsy, three were found to have a lesion that was different from the suspected diagnosis and that resulted in a change in treatment. Thirty patients with neurological symptoms had postmortem brain examinations. In the case of the 12 patients who had carried specific diagnoses and received treatments based on those diagnoses, only six diagnoses (50%) were proven correct at autopsy. Of the 18 cases that did not record a specific antemortem diagnosis, in only 5 were normal brains reported, while the others reported a variety of nonspecific or infectious findings. Twelve patients without neurological symptoms had postmortem brain examinations and only six of these (50%) had normal or slightly atrophic brains. Case reports of the others noted nonspecific findings most of which were suggestive of subacute HIV encephalitis. The poor rate of diagnostic accuracy in this series suggests that biopsy should be considered for atypical lesions or those that do not respond to empiric therapy. The use and relative sensitivities of various diagnostic studies are also discussed.