Showing papers on "In vivo published in 1992"

Amyloid β-peptide is produced by cultured cells during normal metabolism

Abstract: Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.

Antisense c- myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo

Abstract: SYNTHETIC antisense oligonucleotides have been used to dissect gene function in vitro. Technical difficulties prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense c-myb oligonu-cleotide to suppress intimal accumulation of rat carotid arterial smooth muscle cells. Our results suggest that antisense oligonucleotides can be used to define the in vivo biological role of specific macromolecules in the blood vessel wall and could potentially serve as a new class of therapeutic agents for cardiovascular disorders.

Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule

Abstract: In vitro, when the B7 molecule on the surface of antigen-presenting cells binds to the T cell surface molecules CD28 and CTLA-4, a costimulatory signal for T cell activation is generated. CTLA4Ig is a soluble form of the extracellular domain of CTLA-4 and binds B7 with high avidity. CTLA4Ig treatment in vivo suppressed T cell-dependent antibody responses to sheep erythrocytes or keyhole limpet hemocyanin. Large doses of CTLA4Ig suppressed responses to a second immunization. Thus, costimulation by B7 is important for humoral immune responses in vivo, and interference with costimulation may be useful for treatment of antibody-mediated autoimmune disease.

Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1.

Abstract: Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo. When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed. This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy.

Adenovirus-mediated in vivo gene transfer and expression in normal rat liver.

Abstract: Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes. In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human α1–antitrypsin cDNA (Ad–α1AT) synthesized and secreted human α1 AT for 4 weeks. In rats, in vivo intraportal administration of a recombinant Ad containing the E. coli lacZ gene, was followed by expression of β–galactosidase in hepatocytes 3 days after infection. Intraportal infusion of Ad–α1AT produced detectable serum levels of human α1AT for 4 weeks. Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders.

IL-4 induces a Th2 response in Leishmania major-infected mice.

Abstract: The infection of mice with Leishmania major can cause either a fatal disseminated disease or a localized healing disease, depending on the genetic background of the mice. A strong correlation has been shown between disease outcome and the nature of the T cell response, with healer strains developing a Th1-like response and nonhealer strains a Th2-like response. The treatment of nonhealer BALB/c mice with a single dose of an anti-IL-4 antibody, given at the time of infection with L. major, allowed these mice to develop healing Th1-like responses, suggesting that IL-4 is required in BALB/c mice for the differentiation of Th cells into Th2 cells. Anti-IL-4 had to be present during the first 2 wk of infection to have this effect. Anti-IL-4 caused a marked shift from a Th2 to a Th1 pattern of cytokine expression within 4 days, in vivo, and injections of IL-4 had the opposite effect on the early response in healer C3H/HeN mice. These findings demonstrate that IL-4 can induce the development of Th2 response to L. major infection in vivo.

Dual role of tumor necrosis factor-alpha in angiogenesis.

Abstract: The role of tumor necrosis factor-alpha (TNF; cachectin) in angiogenesis has been controversial In vitro TNF inhibits proliferation of endothelial cells (EC) whereas in the cornea it appears to stimulate vessel growth The authors tested TNF in their recently developed disc angiogenesis system (DAS), designed to measure the proliferation of microvessels The DAS, implanted subcutaneously in mice, was either fitted with a central pellet containing mouse recombinant TNF (mrTNF), or exposed to mrTNF delivered subcutaneously by an osmotic minipump Low doses of mrTNF (001-1 ng) induced angiogenesis, which was maximum at 01 ng, whereas high doses (1, and 5 micrograms) inhibited it Subcutaneous mrTNF delivered at the (high) rate of 15-60 ng/hr for 14 days inhibited angiogenesis These results indicate bimodal, dose-dependent opposing effects and explain some of the in vitro versus in vivo paradoxical results TNF (native or exogenous) may have opposing effects on microvessels of neoplasms and inflammatory reactions, depending on its local tissue concentrations

In vivo cyclooxygenase expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and streptococcal cell wall arthritis.

Abstract: Cyclooxygenase (COX), or prostaglandin (PG) H synthase, plays a role in inflammatory diseases, but very limited data exist on the regulation of COX in vivo. We, therefore, studied the in vivo expression of COX in synovia from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), as well as joints of rats with streptococcal cell wall (SCW) and adjuvant arthritis. Extensive and intense intracellular COX immunostaining, which correlated with the extent and intensity of mononuclear cell infiltration, was observed in cells throughout RA synovia. Significantly less or equivocal staining was noted in OA and normal human synovia. Similarly, COX immunostaining was equivocal in the joints of normal and arthritis-resistant F344/N rats. In contrast, high level expression developed rapidly in euthymic female Lewis (LEW/N) rats throughout the hindlimb joints and overlying tissues including skin, preceding or paralleling clinically apparent experimental arthritis. COX was expressed in the joints of athymic LEW.rnu/rnu rats 2-4 d after injection of SCW or adjuvant but was not sustained. Physiological doses of antiinflammatory glucocorticoids, but not progesterone, suppressed both arthritis and COX expression in LEW/N rats. These observations suggest that, in vivo, (a) COX expression is upregulated in inflammatory joint diseases, (b) the level of expression is genetically controlled and is a biochemical correlate of disease severity, (c) sustained high level up-regulation is T cell dependent, and (d) expression is down-regulated by antiinflammatory glucocorticoids.

S-nitroso-glutathione inhibits platelet activation in vitro and in vivo.

Abstract: 1. The effect of S-nitroso-glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2. The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3. GSNO caused a concentration-dependent inhibition of collagen-induced platelet aggregation in vitro and decreased ADP-induced aggregation in the conscious rat. 4. Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5. The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6. The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.

Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis.

Abstract: Escherichia coli endotoxin (LPS) can induce the clinical syndrome of septic shock and renal cortical necrosis and can stimulate nitric oxide (NO) production from macrophages, vascular smooth muscle, and glomerular mesangial cells in vitro. NO is an endogenous vasodilator, which also inhibits platelet aggregation and adhesion. We therefore sought to determine whether LPS would stimulate NO production in vivo and, if so, whether this NO would modulate endotoxin-induced glomerular thrombosis. The stable NO end-products, NO2 and NO3, were measured in serum and urine collections from rats during baseline and after injection of LPS, with or without substances that modulate NO synthesis. The urinary excretion of NO2/NO3 was 1,964 +/- 311 nm/8 h during the baseline and increased to 6,833 +/- 776 nm/8 h after a single intraperitoneal injection of 0.1 mg/kg LPS (P < 0.05). The serum concentration of NO2/NO3 also significantly increased after LPS injection. Both the urine and serum stimulation was significantly prevented by the NO synthesis inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME). L-Arginine, given with LPS+L-NAME significantly restored the NO2/NO3 levels in the urine. Ex vivo incubation of tissues from rats treated with LPS demonstrated NO production by the aorta, whole kidney, and glomeruli, but not cortical tubules. Histological examination of kidneys from rats given either LPS or L-NAME alone revealed that 2 and 4.5% of the glomeruli contained capillary thrombosis, respectively. In contrast, rats given LPS+L-NAME developed thrombosis in 55% of glomeruli (P < 0.001), which was significantly prevented when L-arginine was given concomitantly. We conclude that LPS stimulates endogenous production of NO in vivo and that this NO is critical in preventing LPS-induced renal thrombosis.

Aerosol gene delivery in vivo.

Abstract: The ability to express transgenes selectively within the lung will greatly facilitate the development of gene therapy for a variety of human diseases. We have demonstrated that aerosol administration of a chloramphenicol acetyltransferase (CAT) expression plasmid complexed to cationic liposomes produces high-level, lung-specific CAT gene expression in mice in vivo. Significant levels of CAT activity are seen in the lungs for at least 21 days following aerosolization. In situ immunostaining for intracellular CAT protein reveals that the majority of airway epithelial and alveolar lining cells are transfected in vivo. Histological analyses show no apparent treatment-related damage. These results have important implications for the development of human gene therapy.

Gene transfer in vivo with DNA-liposome complexes: safety and acute toxicity in mice.

Abstract: DNA can be introduced into a variety of cell types after formation of liposomal complexes with cationic lipids. In this report, conditions have been established to optimize the production of DNA-liposome complexes that efficiently transfect cells. The safety and toxicity of this method of gene delivery have been assessed after in vivo administration, either by intravenous or direct intratumor injection. Nine to eleven days after intravenous injection, DNA was found primarily in heart and lung tissue by PCR analysis. No abnormalities were evident from histologic examination of tissue, examination of tissue-specific serum enzymes, routine biochemical parameters, or electrocardiographic monitoring. DNA-liposome complexes can therefore be used for the delivery of recombinant genes in vivo with minimal toxicity.

Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface antigens.

Abstract: Apoptosis, programmed cell death, was previously shown to be induced by the mAb anti-APO-1 (IgG3, kappa) by binding to the APO-1 cell surface Ag, a new member of the nerve growth factor/TNF receptor superfamily. To investigate the role of the Ig H chain Fc regions we compared induction of apoptosis by the original mAb IgG3 anti-APO-1 with anti-APO-1 F(ab')2 fragments and different anti-APO-1 isotypes (IgG1, IgG2b, IgG2a, and IgA) isolated by sequential sublining. We found that IgG3 was the most active isotype; IgG1, IgG2a, and IgA showed intermediate activity, and IgG2b and F(ab')2 were inactive. Cytotoxic activity of the inactive or less active antibody preparations was fully reconstituted by protein A, anti-mouse Ig, or anti-mouse Ig F(ab')2, respectively. Thus, APO-1-mediated induction of apoptosis was dependent on efficient cross-linking of APO-1 cell surface Ag, indirectly augmented by anti-APO-1 Fc-Fc self-aggregation. Because of their different in vitro activity we selected IgG3-, IgG2b-, and IgA anti-APO-1 to test their antitumor activity against solid human B lymphoblastoid tumors in SCID mice. The isotypes showed a different serum half-life (IgG3: 9.2-10.4 days, IgG2b: 1.9-2.6 days, and IgA: 14.1-29.2 h) and a different initial tumor localization 4 h after i.p. injection (IgG3 around the blood vessels, IgG2b homogeneously, and IgA heterogeneously distributed in the tumor). All antibody preparations induced tumor regression by induction of apoptosis, even IgG2b anti-APO-1 inactive in vitro without cross-linking. The activity of IgA anti-APO-1, which did not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity indicates that apoptosis may be used as the main if not the only mechanism of induction of tumor regression in vivo. As with in vitro, IgG3 anti-APO-1 was the most effective isotype also in vivo. This result suggests that cross-linking of APO-1 on the tumor cell surface may also be required for tumor regression by apoptosis in vivo. Taken together, our data show that selective targeting of apoptosis to tumors may be an efficient antitumor mechanism.

Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models.

Abstract: To provide an in vitro system for studying brain capillary functions, we have developed a process of co-culture that closely mimics the in vivo situation, by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier. To validate our in vitro blood-brain barrier model, we have compared the transport of ten different compounds across the in vitro model vs. transport across the blood-brain barrier in vivo. In vivo brain extraction (Eo) are measured according to the method of Oldendorf. The in vivo and in vitro Eo values show a strong correlation as indicated by the Spearman’s co-efficient (r = 0.88, p < 0.01). The in vitro blood-brain barrier permeability for glucose and leucine i.e. compounds that traverse the blood-brain barrier via carrier mediation is in the same range as the blood-brain barrier permeability in vivo indicating that in our model the glucose and leucine transporters are always present. The relative ease with which such co-cultures can be produced in large quantities would facilitate the investigations in delivery of nutriments across the blood-brain barrier, and the screening of new centrally acting drugs.

Platelet-Derived Growth Factor is Angiogenic In Vivo

Abstract: PDGF receptors have recently been found to be expressed in microvascular endothelium in vivo under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vivo. PDGF-BB was found to consistently induce an angiogenic response whereas PDGF-AA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endothelial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed.

Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo

Abstract: Phorbol esters stimulate and glucocorticoid hormones down-regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP-1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP-1 to its cognate DNA binding site in vivo. The DNA-protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP-1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos-Jun heterodimers or of purified AP-1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP-1 binding site in vivo. The hormone receptor acts without itself binding to DNA.

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

Abstract: The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1 overproduction in prostate cancer: effects on growth in vivo and in vitro.

Abstract: We found previously that transforming growth factor-beta 1 (TGF beta 1) mRNA levels are markedly elevated in rat prostate cancer (Dunning R3327 sublines) compared to levels in normal prostate. Our goal was to determine whether elevated expression of TGF beta 1 is biologically relevant to prostate cancer growth in vivo. We chose as our model the R3327-MATLyLu prostate cancer epithelial cell line, which produces metastatic anaplastic tumors when reinoculated in vivo. Our approach was to stably transfect MATLyLu cells with an expression vector that codes for latent TGF beta 1 and to isolate subclones of cells that over-expressed TGF beta 1 mRNA. We also isolated a subclone of MATLyLu cells transfected with a control vector lacking the TGF beta 1 cDNA insert. We then studied the growth of these cells in vivo and in vitro. Twenty days after sc inoculation of 10(6) cells in vivo, TGF beta 1-overproducing MATLyLu tumors were 50% larger, markedly less necrotic, and produced more extensive metastatic disease (lung metastases in 73% of all lobes and lymph node metastases in 88% of animals) compared to control MATLyLu tumors (lung metastases, 21%; lymph node metastases, 7%). Thus, TGF beta 1 produced in vivo is biologically active and can promote prostate cancer growth, viability, and aggressiveness, perhaps via effects on the host and/or on the tumor cells themselves. When followed in vitro, TGF beta 1-overproducing cells became growth inhibited, but this effect was transient as cells subsequently resumed proliferating. Growth inhibition was due to TGF beta, because it could be prevented by TGF beta-neutralizing antibody. Therefore, prostate cancer cells can activate and respond to secreted latent TGF beta 1, and although the cells are transiently inhibited in vitro, there is no net inhibition of growth. The ability of the cells to respond to endogenously produced TGF beta 1 suggests that TGF beta 1 overexpression enhances tumor growth in vivo at least in part via an effect of TGF beta 1 on the tumor cells themselves.

Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA).

Abstract: An automated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein was employed for chemotherapeutic-drug-sensitivity testing of tumor-cell suspensions from patients with leukemia. Fluorescence was linearly related to cell number, and reproducible measurements of drug sensitivity could be performed using fresh or cryopreserved leukemia cells. A marked heterogeneity with respect to chemotherapeutic drug sensitivity was observed for a panel of cytotoxic drugs tested in 43 samples from 35 patients with treated or untreated acute and chronic leukemia. For samples obtained from patients with chronic lymphocytic and acute myelocytic leukemia, sensitivity profiles for standard drugs corresponded to known clinical activity and the assay detected primary and acquired drug resistance. Individual in vitro/in vivo correlations indicated high specificity with respect to the identification of drug resistance. The results suggest that the FMCA may be a simple and rapid method for in vivo-representative determinations of chemotherapeutic drug resistance in tumor cells obtained from patients with leukemia.

In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides

Abstract: The c-myb protooncogene encodes proteins that are critical for hematopoietic cell proliferation and development. Disrupting c-myb function might, therefore, prove an effective therapeutic strategy for controlling leukemic cell growth. Antisense oligodeoxynucleotides have been utilized for this purpose in vitro, but their in vivo efficacy has not been reported. We therefore established human leukemia-scid mouse chimeras with K562 cells and treated diseased animals with phosphorothioate-modified antisense oligodeoxynucleotides. K562 cells express the c-myb protooncogene, which served as the target for the antisense DNA. They also express the tumor-specific bcr-abl oncogene that was utilized to track the human cells in the mouse host. Once circulating leukemic blast cells had been detected, the survival of untreated control mice was 6 +/- 3 days (mean +/- SD). The survival of animals treated for 7 or 14 days with either sense or scrambled-sequence c-myb oligodeoxynucleotides was not statistically different from the control animals. In distinct contrast, animals treated for similar lengths of time with antisense c-myb oligodeoxynucleotides survived at least 3.5 times longer than the various control animals. In addition, animals receiving antisense c-myb DNA had significantly less disease at the two sites most frequently manifesting leukemic cell infiltration, the central nervous system and the ovary. These results suggest that phosphorothioate-modified antisense DNA may be efficacious for the treatment of human leukemia in vivo, and by analogy, for the treatment of other human neoplasias.

Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses.

Abstract: Tricyclic antidepressants, such as amitriptyline (Elavil), and the nontricyclic agent, fluoxetine (Prozac), bind to growth-regulatory intracellular histamine receptors, associated with anti-estrogen binding sites in microsomes and nuclei. The prototype anti-estrogen binding site/intracellular histamine receptor ligand, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, inhibits normal cell proliferation in vitro but stimulates tumor growth in vivo. Because of their structural similarity to N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, we carried out studies to determine whether amitriptyline and fluoxetine stimulate tumor growth and/or development in rodents at concentrations relevant to the treatment of human depression (equivalent human dose range, approximately 100-150 mg/day for amitriptyline and approximately 20-80 mg/day for fluoxetine). All experiments were performed blinded. In studies of growth stimulation of transplantable syngeneic tumors, groups of mice were inoculated s.c. with C-3 fibrosarcoma cells or given i.v. or s.c. injections of B16f10 melanoma cells, followed 24 h later by daily i.p. injections of saline, amitriptyline, or fluoxetine. Tumor latency (fibrosarcoma), aggregate tumor weight (s.c. injected melanoma), or time to death from pulmonary metastasis (i.v. injected melanoma) was determined; drug-induced stimulation of DNA synthesis in C-3 fibrosarcoma cells in vitro was correlated with tumor growth acceleration in vivo. In a mammary carcinogenesis model, the effects of chronic saline, amitriptyline, or fluoxetine administration on the rate and frequency of development of mammary tumors in rats fed dimethylbenzanthracene (DMBA) were compared. Eight of 20 amitriptyline- or fluoxetine-treated mice developed fibrosarcoma tumors by day 5, as compared to none of 20 saline controls (P less than 0.002). Similarly, 20 of 21 DMBA-treated rats receiving the antidepressant drugs developed 33 mammary tumors by week 15 as compared to 5 tumors in 4 of 7 DMBA-treated rats receiving saline (P less than 0.001). For both models, tumor latency decreased 30-40% and, in the DMBA model, tumor frequency increased greater than 2-fold in the antidepressant-treated rats as compared to controls. Stimulation of fibrosarcoma growth in vivo correlated with a corresponding bell-shaped drug-induced increase in DNA synthesis in vitro. While the median time to death from pulmonary metastases did not differ among groups given i.v. injections of melanoma cells, a significant (P less than 0.01) stimulation of growth of s.c. injected melanoma was observed in mice receiving the antidepressants.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigen-pulsed dendritic cells can efficiently induce an antibody response in vivo.

Abstract: The aim of this study was to develop an immunization procedure avoiding external adjuvant. Data are presented showing that syngeneic dendritic cells (DC), which have been pulsed in vitro with antigen, induce a strong antibody response in mice. By contrast, antigen (Ag)-pulsed low-density B cells, although equally able to induce interleukin 2 secretion by an Ag-specific T cell hybridoma in vitro, only weakly prime the mice in vivo. Moreover, we show that the injection of Ag-pulsed DC induces the synthesis of isotypes similar to the immunoglobulin classes detected after immunization with the same Ag in complete Freund's adjuvant. Importantly, high amounts of IgG2a antibodies are produced, suggesting that T helper type 1 cells are activated. Collectively, these data indicate that DC can initiate a primary humoral response and that they may be used as physiological adjuvant in vivo.

The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia.

Abstract: Tumor necrosis factor (TNF) is a protein hormone implicated in the development of septic shock and other pathologic states. However, complexities inherent in detecting TNF synthesis by individual tissues have left the precise origins of this protein undefined. In addition, the possibility that localized TNF production may contribute to the pathogenesis of organ-specific diseases such as type I diabetes has not been explored in vivo. We have developed a transgenic mouse line bearing a reporter gene construct in which the TNF coding sequence and introns are replaced by a chloramphenicol acetyltransferase (CAT) coding sequence. In normal transgenic animals, CAT activity is expressed only in the thymus. When endotoxin is administered to the animals, CAT activity is also evident in kidney, heart, islets of Langerhans, spleen, lung, fallopian tubes, and uterus, but not in other organs. The biosynthesis of CAT in vivo correlated with tissue capacity to secrete TNF in vitro. Thus, TNF was secreted by all the tissues that expressed CAT, including lung, spleen, thymus, uterus/fallopian tubes, pancreatic islets, renal glomeruli, and cultured cardiac cells after exposure to endotoxin.

Heparin and modified heparin inhibit complement activation in vivo.

Abstract: Heparin regulates C activity in vitro, but has not been examined for this activity in vivo. The present study investigated the ability of commercial heparin and derivatized (N-desulfated, N-acetylated) heparin (Hep-NAc) with greatly diminished anticoagulant activity to inhibit C activation in guinea pigs. Catheters were placed in the right atrium of guinea pigs and kept patent with frequent saline flushes. The next day, heparin, Hep-NAc, or saline was given and 2.5 min later cobra venom factor or saline was given. Blood was drawn at intervals and assayed for total hemolytic C, C3 hemolytic activity, free hemoglobin, and activated partial thromboplastin time. Total hemolytic C and C3 activity decreased less rapidly in heparin- and Hep-NAc-pretreated animals than in non-pretreated animals, indicating that both heparins inhibited C activation. Heparin and Hep-NAc also inhibited cobra venom factor-induced hemolysis. This study demonstrates that commercial heparin and modified heparin inhibit C activation in vivo. This represents an important step in the development of an oligosaccharide drug to regulate C activation.

Hepatic Gene Therapy: Persistent Expression of Human α1-Antitrypsin in Mice after Direct Gene Delivery In Vivo

Abstract: The liver represents an excellent target organ for gene therapy. The current strategy for hepatic gene therapy involves the isolation of primary hepatocytes from a resected liver lobe, transduction of therapeutic genes in vitro followed by autologous hepatocellular transplantation. This ex vivo approach is a rather complex procedure in its entirety; thus, a simple method for direct gene delivery into hepatocytes in vivo has been developed. The procedure involves partial hepatectomy followed by the portal vein infusion of recombinant retroviral vectors. Histological analysis of hepatocytes after in vivo delivery of a recombinant retrovirus bearing the E. coli (β-galactosidase gene showed that 1–2% of the parenchymal cells were transduced. Direct hepatic transfer of human α1-antitrypsin cDNA under the transcriptional direction of the albumin promoter–enhancer led to constitutive expression of the human protein in the sera of recipients at concentrations of 30–1,400 ng/ml for at least 6 months. The ...

Monocyte chemotactic cytokine gene transfer modulates macrophage infiltration, growth, and susceptibility to IL-2 therapy of a murine melanoma.

Abstract: Tumor-derived chemotactic factors have been identified and suggested to play a role in the regulation of macrophage infiltration in neoplastic tissues. The present study was designed to assess the in vivo relevance of a tumor-derived chemotactic factor molecularly identified as monocyte chemotactic protein (MCP; alternative designations are JE and MCAF) by gene transfer in a murine melanoma. After gene transfer, MCP-producing melanoma clones showed a marked (twofold) increase in the percentage of tumor-associated macrophages compared with control clones and with the parent line: for instance, the percentage of tumor-associated macrophages was 20.9 +/- 1.5, 29.4 +/- 2.3, and 47.6 +/- 2.5 for the parent line, the control V14 clone, and the MCP-producing L12 clone, respectively. MCP-producing cells were tumorigenic but exhibited a slower growth rate in vivo (e.g., doubling time of 2.9 and 6.6 days for the control V14 and the MCP-producing L12 clone, respectively) with a prolongation of survival time. The in vitro growth rate of melanoma clones was unaffected by MCP gene transfer. The same difference between MCP-producing and control cells, in terms of macrophage infiltration and growth rate, was detected after implantation in athymic mice. Whereas the in vivo growth rate of MCP-expressing tumors was slower, after i.m. inoculation of small cell numbers (10(2) cells) MCP-producing cells were slightly, but significantly, more tumorigenic. Local administration of IL-2 had modest, but definite, antitumor activity in this model; MCP-producing cells were less susceptible to local IL-2 immunotherapy. These results demonstrate that a tumor-derived chemotactic cytokine can indeed play a role in the regulation of mononuclear phagocyte recruitment in neoplastic tissues and emphasize how tumor-associated macrophages can exert a dual influence in tumor-host interactions.

Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection.

Abstract: After infection of 1-day-old chickens, chicken anemia virus (CAV) causes a complete depletion of the thymic cortex by day 14. Since cell death can be caused either by necrosis or by apoptosis, we investigated which type of cell death occurs after in vivo and in vitro infections with CAV. Using electron microscopy and biochemical methods, we demonstrated that CAV induces apoptosis of cortical thymocytes after in vivo infection and of lymphoblastoid cell lines after in vitro infection. At day 13 after in vivo infection, virus-like particles were detected in apoptotic bodies that were absorbed by epithelial cells. These results show that apoptosis, a phenomenon that has been observed for a few other viruses, is also an important phenomenon during the pathogenesis of CAV.