Jane E. Ranchalis

Passive immune globulin therapy in the SIV/macaque model: early intervention can alter disease profile

One of the major questions in AIDS is the role that the host immune system and the virus play in the dynamics of infection and the development of AIDS in an infected individual. In order to test the role of antibody in controlling viral infection, high-dose SIV-immune globulin was passively transferred to infected macaques early in infection. Immune globulin purified from the plasma of an SIV-infected long-term non-progressor macaque (SIVIG) or a pool of normal immune globulin (normal Ig) was infused into SIVsmE660-infected macaques (170 mg/kg) at one and fourteen days post infection. Animals were monitored for SIV-specific antibodies, viremia, plasma antigenemia, and clinical course. All animals were infected by SIV. At 16 months post infection, five macaques in the combined control groups have been euthanized, one as a rapid progressor with debilitating disease at 20 weeks post infection. Four macaques from the comparison groups have signs of AIDS, accompanied by high and increasing levels of virus and p27 antigenemia. One of the ten control animals had a very low virus load in plasma and peripheral blood and lymph node mononuclear cells at all times tested and has remained disease-free. In the SIVIG treatment group, two macaques were euthanized at 18-20 weeks due to AIDS, rapid progressors to disease. Three macaques in the SIVIG group had an initial high level of virus in plasma, peripheral blood mononuclear cells (PBMC), and lymph node mononuclear cells (LNMC), which dropped to baseline at 6 weeks post infection and has remained very low or negative for 16 months, a disease profile which has not been observed in untreated animals in this model to date. These macaques have remained clinically healthy. The sixth treated animal is also healthy, with very low virus burden that is detectable only by nested set polymerase chain reaction (PCR). All SIVIG-treated macaques had no detectable p27 plasma antigenemia for the first 10 weeks of infection, demonstrating that the IgG effectively complexed with the virus. The immunological correlates in the treated animals include development of de novo virus-specific antibodies and/or cytotoxic T cell (CTL), both of which are hallmarks of long term non-progressors. The two SIVIG-treated macaques that progress to disease rapidly had no detectable de novo humoral immune responses, as is often seen in rapid HIV disease in humans. Envelope-specific and virus neutralizing antibodies alone were not sufficient to prevent disease progression, as the plasma of both non-progressors as well as progressors had high titers of envelope-specific and neutralizing antibodies against SIVsmE660. Poor clinical prognosis was associated with moderate to high and increasing virus loads in plasma, PBMC, and lymph nodes. Good clinical prognosis correlated with low or undetectable post acute viremia in the peripheral blood and lymph nodes. We hypothesize that SIVIG reduced the spread of virus by eliminating or reducing plasma virus through immune complexes during the first four to 8 weeks of infection and then maintaining this low level of viremia until the host immune response was capable of virus control. Reduction of virus burden early in infection by passive IgG can alter disease outcome in SIV infection of macaques. Modifications of this strategy may lead to effective early treatment of HIV-1 infection in humans.

Controlled release of TGF-β1 from a biodegradable matrix for bone regeneration

Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-beta 1 is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-beta 1 to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-beta 1 to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-beta 1 comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-beta 1 released was determined using several methods including 125I-labeled TGF-beta 1 as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-beta 1 released was directly proportional to both the TGF-beta 1 loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-beta 1 released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.

Human term placenta contains transforming growth factors

Abstract Growth factors of apparent molecular weights of 6,000, 10,000, 20,000 and one in excess of 30,000 daltons can be isolated from acid-ethanol extracts of human term placentas. Each size class of growth factor resembles transforming growth factor (TGF) in that it stimulates anchorage independent growth of normal rat kidney cells and competes with EGF for binding to EGF membrane receptors. The 6,000, 10,000 and 20,000 molecular weight polypeptides also resemble TGF in their acid and heat stability, and their requirement for intact disulfide bonds for growth promoting activity. By homologous radioimmunoassay, neither the 6,000 nor 10,000 dalton polypeptide is related to human epidermal growth factor (hEGF). The presence of these TGFs in ample concentrations (approximately 100 ng of EGF equivalents per term placenta for the 10,000 dalton polypeptide) indicates the usefulness of this tissue source for study of human TGFs.

γ‐Interferon‐Induced Activation of Latent Transforming Growth Factor‐β by Human Monocytes

Transforming growth factor-ps (TGFps), a family of at least four different homologous disulfide-linked homodimeric polypeptides, are potent modulators of cell growth and differentiation.’-3 Analysis of cDNA clones encoding mammalian TGFPs and appropriate cell culture supernatants indicates that the mature molecule is cleaved from the carboxy terminus of a larger glycosylated precurso+6 and is secreted in a latent, inactive form?-9 Transient acidification activates E F P ; however, the physiological mechanism(s) of TGFB secretion and activation, which initiate its potent autocrine and paracrine effects in vivo, are poorly understood. In this study, we asked whether cell association was involved in activation of ‘EFp. The ’’latentrecombinant complex used contains one dimeric TGFpl molecule associated with a disulfide-bonded dimeric remnant of the precursor. Fresh human monocytes stimulated by y-interferon (yIFN), activates the “latent” recombinant E F P l complex ( L m F P l ) in a dose-dependent manner. The activated X F p l released into the media is neutralized by a TGFPl monoclonal antibody and has a mass (24 kD) identical to native TGFB1. Thus, TGFSl activation by monocytes may require y IFN-mediated gene expression as well as a cell-associated processing event. We have recently reported the expression of simian ‘EFP type 1 cDNAIO in Chinese hamster ovary (CHO) cells using dehydrofolate reductase (dhfr) gene amplification.” One CHO cell transfectant, designated clone 17, secretes mg amounts of latent

Bone-derived and recombinant transforming growth factor β′S are potent inhibitors of tumor cell growth

Two naturally occurring chrondogenesis inducing peptides have been purified to homogeneity from demineralized bovine bone. Cartilage-inducing factors A and B are the bone-derived equivalents of transforming growth factor-beta types I and II. Both peptides exhibit identical biological activities in chondrogenesis assays and stimulate anchorage independent cell growth. In this study we show that both bone-derived factors are potent (ng/ml) inhibitors of both DNA synthesis and the anchorage independent growth of a variety of human and non-human tumor cells. Unique in this study is also a comparison of the activities of these polypeptide growth factors with recombinant transforming growth factor type I expressed in mammalian cells.

Novel delivery system for inducing quiescence in intestinal stem cells in rats by transforming growth factor β1

BACKGROUND/AIMS Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa. METHODS Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally. Control animals received phosphate-buffered saline only. The size of the villi was measured. Proliferating and mitotic indices were determined by quantifying immunohistochemical staining for proliferating cell nuclear antigen. RESULTS Alginate beads released no TGF-beta 1 in acid. However, in pH 7.4, TGF-beta 1 was released in an active form. Histomorphometrical analysis showed a marked reduction in villus height (50%-70%) in the intestinal mucosa of animals treated perorally with recombinant TGF-beta 1 in alginate beads. Also, the proliferating and mitotic indices were significantly reduced (P < 0.01) in these animals as compared with controls and other routes of administration. CONCLUSIONS This study shows that recombinant TGF-beta 1 administered using a novel oral delivery system induces stem cell quiescence in the intestinal mucosa of the rat.

Synthesis of an active hybrid growth factor (GF) in bacteria: transforming GF-α/vaccinia GF fusion protein

A hybrid gene encoding for a polypeptide consisting of the first 33 N-terminal amino acid (aa) residues of transforming growth factor-alpha (TGF-alpha) and a C terminus consisting of 20 aa residues of vaccinia growth factor (VGF) was chemically synthesized and expressed as a fusion protein in Escherichia coli. The primary structure of the hybrid gene product maintained the same positioning of the three disulfide bonds found in each parent molecule thus conserving the first two loop regions of TGF-alpha and the third loop region of VGF. After cleavage with CNBr its renatured biological activity was found to be comparable to TGF-alpha and VGF with respect to binding to the epidermal growth factor receptor, stimulation of DNA synthesis and induction of anchorage-independent growth of NRK cells in the presence of TGF-beta. Thus, we suggest that similar domains can be interchanged within the same family of molecules and equivalent functionality maintained.

Increase of Plasma Transforming Growth Factor Beta (TGFβ) During Immunotherapy with IL-2

Interleukin-2 (IL-2) is a lymphokine with pleiotropic activities on the immune system. When administered in vivo, besides inducing unrestricted tumor cytotoxicity, it is also responsible for the secondary release of other lymphokines, such as IL-1, TNF, and marrow growth factors, which may mediate some of the clinical toxicities (as well as therapeutic effects) seen during IL-2 immunotherapy. Among the clinical effects of IL-2, we previously reported thrombocytopenia and IL-2-induced in vitro inhibition of platelet aggregation accompanied by rapid secretion of alpha-granule components such as platelet factor 4 (PF4) and beta-thromboglobulin. Platelets constitute one of the largest storage forms of TGF beta. Preliminary evaluation of this factor in patients receiving IL-2 had indicated that plasma TGF beta activity increased in cancer patients following IL-2 therapy. We report a more detailed study of the quantitation of TGF beta activity in the plasma of 23 cancer patients treated with IL-2 immunotherapy. Of interest, we found that although elevation of the bioactive form of TGF beta occurred in most patients during IL-2 therapy, it was significantly higher in patients with clinical regression of tumor (p = .004). In the first 2 weeks of therapy increase of plasma TGF beta activity appeared to correlate with a decrease of platelet counts, suggesting that the factor may derive from the storage form of TGF beta contained therein.

Interleukin-2 (IL-2) Augments the Expression of Transforming Growth Factor Beta in Patients with Disseminated Cancer

Interleukin-2 (IL-2), originally discovered in the supernatant of lectin or antigen-stimulated T-cell cultures1-2, possesses a panoply of immunologic effects such as induction of paracrine growth of antigen-stimulated T-lymphocytes and of non-MHC restricted antitumor cytotoxic activity3-5. The mechanism involved in IL-2-induced signal transduction leading to these effects remains to be resolved6. The production of recombinant IL-2 has allowed the exploration of its biological effects as well as its therapeutic potential as an antitumor agent. Clinical Phase I and II studies of IL-2 in patients with disseminated cancer have shown that the lymphokine alone is able to induce often impressive regression of tumors typically insensitive to chemotherapy or radiation therapy7-10. Administration of IL-2 at tumoricidal doses, however, causes severe and limiting toxic side effects that often preclude its administration. Thrombocytopenia is universal toxicity of IL-2 therapy: it is not consequent to decreased marrow production but to peripheral sequestration of platelets11 and it is accompanied, in vitro12 and in vivo, by platelet degranulation (Paciucci, unpublished data) mediated by the release of thromboxan A-2 from IL-2-activated mononuclear cells in vitro12 and, perhaps, in vivo. Systemic platelet degranulation, in turn, is responsible for high circulating levels of other factors contained in alpha granules (such as PF-4 and beta thromboglobulin). Interestingly, PF-4 has recently been shown to have in vivo antitumoral effects through inhibition of tumor neovascularization13,14.