The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts

Summary An immunoconjugate composed of natural interferon (nIFN) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFN/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFN/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFN on the injected tumors. Further enhancement was obtained when nIFN or nIFN together with Mc5 (at a dose 10 times larger than that present in nIFN/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of¹²⁵I-nIFN/Mc5 by the tumors was greater and its elimination slower than for¹²⁵I-nIFN alone or conjugated to irrelevant mouse IgG₁. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFN alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.     

Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: role of PI 3-kinase and ERK

Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: Role of PI 3-kinase and ERK. BACKGROUND: Lysophosphatidic acid (LPA) is a mitogenic lipid bound to albumin in the circulation and implicated in the induction of proximal tubular cell (PTC) injury in proteinuric states. In this study, we investigated the effect of LPA on proliferation of opossum kidney (OK) cells and the roles of the p85/p110 phosphatidylinositol 3-kinase (PI 3-kinase) and extracellular signal-regulated kinases (ERKs) ERK-1 and ERK-2 in LPA-induced proliferation. METHODS: [3H]-thymidine incorporation was used as an index of OK cell proliferation. PI 3-kinase and ERK activities were measured by in vitro kinase assays of immunoprecipitates from both wild-type OK cells and OK cells expressing a dominant negative p85 (Deltap85) subunit of PI 3-kinase in an inducible vector. RESULTS: LPA stimulated a marked increase in [3H]-thymidine uptake in wild-type and Deltap85 OK cells. OK cell PI 3-kinase activity was stimulated by LPA and was inhibited by expression of Deltap85. LPA-induced proliferation was inhibited by wortmannin and the induction of Deltap85 expression. These data suggest that LPA stimulates PI 3-kinase activity, which is essential for signaling the induction of proliferation. LPA also stimulated ERK activity (peak at 5 min, return to baseline by 60 min) maximally at a dose of 100 microM LPA. This increase was approximately 600% above basal and was similar to the effects of 10% fetal calf serum. The proliferative effect of LPA was decreased by the ERK-kinase (MEK) inhibitor PD98059 (5 microM), therefore suggesting that ERK as well as PI 3-kinase activation is important for proliferation. ERK activation by LPA was not affected by pretreatment with wortmannin or by the expression of Deltap85. PI 3-kinase activation by LPA was not affected by pretreatment with PD98059. CONCLUSIONS: We conclude that activation of PI 3-kinase is essential for the LPA-induced proliferation of OK cells and that ERK activation is also important. Therefore, they are both vital elements in separate signaling pathways leading to cell proliferation. LPA filtered into the proximal tubule in proteinuric states is likely to have profound effects on PTC growth.     

Informed consent, parental awareness, and reasons for participating in a randomised controlled study

BACKGROUND: The informed consent procedure plays a central role in randomised controlled trials but has only been explored in a few studies on children. AIM: To assess the quality of the informed consent process in a paediatric setting. METHODS: A questionnaire was sent to parents who volunteered their child (230 children) for a randomised, double blind, placebo controlled trial of ibuprofen syrup to prevent recurrent febrile seizures. RESULTS: 181 (79%) parents responded. On average, 73% of parents were aware of the major study characteristics. A few had difficulty understanding the information provided. Major factors in parents granting approval were the contribution to clinical science (51%) and benefit to the child (32%). Sociodemographic status did not influence initial participation but west European origin of the father was associated with willingness to participate in future trials. 89% of participants felt positive about the informed consent procedure; however, 25% stated that they felt obliged to participate. Although their reasons for granting approval and their evaluation of the informed consent procedure did not differ, relatively more were hesitant about participating in future. Parents appreciated the investigator being on call 24 hours a day (38%) and the extra medical care and information provided (37%) as advantages of participation. Disadvantages were mainly the time consuming aspects and the work involved (23%). CONCLUSIONS: Parents' understanding of trial characteristics might be improved by designing less difficult informed consent forms and by the investigator giving extra attention and information to non-west European parents. Adequate measures should be taken to avoid parents feeling obliged to participate, rather than giving true informed consent.     

Rearrangement of the T cell receptor gamma-chain gene in childhood acute lymphoblastic leukemia

We analyzed rearrangements of the T cell receptor gamma-chain (T gamma) gene as well as rearrangements of the T cell receptor beta-chain (T beta) gene and immunoglobulin heavy-chain (IgH) gene in 68 children with acute lymphoblastic leukemia (ALL). All 15 patients with T cell ALL showed rearrangements of both T beta and T gamma genes. Twenty-four of 53 non-T, non-B ALL patients (45%) showed T gamma gene rearrangements and only 14 of these also showed T beta gene rearrangements. Only a single patient rearranged the T beta gene in the absence of T gamma gene rearrangement. The rearrangement patterns of the T gamma gene in non-T, non-B ALL were quite different from those observed in T cell ALL, as 20 of 23 patients retained at least one germline band of the T gamma gene. In contrast, all T cell ALL patients showed no retention of germline bands. These data indicate that rearrangement of the T gamma gene is not specific for T cell ALL. Further, the results also suggest that T gamma gene rearrangement precedes T beta gene rearrangement. The combined analysis of rearrangement patterns of IgH, T beta, and T gamma genes provides new criteria for defining the cellular origin of leukemic cells and for further delineation of leukemia cell heterogeneity.     

Hyperphosphatemia,Phosphoprotein Phosphatases,and Microparticle Release in Vascular Endothelial Cells

Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.     

Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.     

C‐peptide and diabetic kidney disease

Kidney disease is a serious development in diabetes mellitus and poses an increasing clinical problem. Despite increasing incidence and prevalence of diabetic kidney disease, there have been no new therapies for this condition in the last 20 years. Mounting evidence supports a biological role for C‐peptide, and findings from multiple studies now suggest that C‐peptide may beneficially affect the disturbed metabolic and pathophysiological pathways leading to the development of diabetic nephropathy. Studies of C‐peptide in animal models and in humans with type 1 diabetes all suggest a renoprotective effect for this peptide. In diabetic rodents, C‐peptide reduces glomerular hyperfiltration and albuminuria. Cohort studies of diabetic patients with combined islet and kidney transplants suggest that maintained C‐peptide secretion is protective of renal graft function. Further, in short‐term studies of patients with type 1 diabetes, administration of C‐peptide is also associated with a lowered hyperfiltration rate and reduced microalbuminuria. Thus, the available information suggests that type 1 diabetes should be regarded as a dual hormone deficiency disease and that clinical trials of C‐peptide in diabetic nephropathy are both justified and urgently required.