A role for protein kinase Bbeta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes.

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.

Effects of two bisdioxopiperazines on mouse B-and T-cell function.

The immunosuppresive effects of the bisdioxopiperazines ICRF 154 and ICRF 159 on the function of bone marrow-derived lymphocytes (B cells) and thymusderived lymphocytes (T cells) were assessed and compared with those of cyclophosphamide (CPA). Mice given foreign erythrocytes with any of these drugs for 3 or 5 days showed suppressed antibody responses due to the inhibition of thymus-derived cooperating lymphocyte (T-helper cell) priming and inactivation of B cells in the spleen. In contrast, 3 days of drug treatment after the injection of allogeneic tumor cells only partially inhibited T cell-dependent cytotoxicity. Moreover, when irradiated tumor cells were given for 3 days with CPA or ICRF 154, enhancement rather than inhibition of cytotoxicity was the usual response. However, with both types of immunization, no thymus-derived cytotoxic lymphocytes (T killer cells) could be generated after prolonged treatment (6 daily injections) with any of the 3 drugs. Administration of drugs before antigen also resulted in selective drug action, i.e., a relative increase in the proportion of thy-1-positive cells in the spleen. After challenge in vivo or in vitro with erythrocyte antigens, the plaque-forming cell responses of these spleens were raised up to twofold, probably because they had higher T-helper cell activity than untreated controls. Similar pretreatment before immunization with allogeneic tumor cells also led to enhanced T-killer cell activity, but only in ICRF 154- and CPA-treated mice. These observations suggest that under certain conditions, the bisdioxopiperazines and CPA have selective effects on B-cell rather than T-cell function.

Recombinant human amyloid precursor-like protein 2 (APLP2) expressed in the yeast Pichia pastoris can stimulate neurite outgrowth.

The human amyloid precursor-like protein 2 (APLP2) is a member of the Alzheimer's disease amyloid precursor protein (APP) gene family. The human APLP2 ectodomain (sAPLP2) was expressed in the yeast Pichia pastoris and the recombinant sAPLP2 was purified from the culture medium in a single step by metal-chelating Sepharose chromatography. The neuritotrophic activity of APLP2 was compared to the APP isoforms sAPP695 and sAPP751 on chick sympathetic neurones. APLP2 had neurite outgrowth-promoting activity similar to that of the APP isoforms. This suggests that APP and APLP2 have a similar or related role and supports the idea of a redundancy in function between the APP-gene family proteins.

Adoptive T cell immunotherapy of MSV-induced tumours in nude mice. Part I. Biology of tumour regression and recurrence.

The induction of immunity to progressively growing murine sarcoma virus (MSV) tumours in nude (nu/nu) mice by reconstitution with immune T cells from syngeneic (+/+) donors has been studied. Whole spleen cell preparations served as the source of immune T cells. Transfer of immune, but not of normal, spleen cells resulted in partial or apparently complete regression of primary tumours and a related moderate to considerable extension of survival time. The dose, the time in days between immunization and transfer, as well as timing of the spleen cells in relation to tumour cell challenge, were all factors which influenced the effectiveness of the protective inocula. An unexpected consequence of even the very effective primary immunotherapy regimens, was secondary tumour development after varying tumour-free intervals. This was most frequently manifest as tumour recurrences at the original injection site either on their own or in combination with distant metastases. Such a relatively high frequency of tumour reappearance and metastatic spread contrasts markedly with the rare instances of secondary regrowth in normal immunocompetent mice. The present reconstitution system may therefore provide a new model for studying the inhibitory or stimulatory properties of T cells with respect to tumour regression and dissemination.

Adoptive T cell immunotherapy of MSV-induced tumours in nude mice. Part II. Sequential analysis of serum immune complexes and blocking activity in reconstituted mice in relation to tumour biology.

In seven separate experiments, nude (nu/nu) mice carrying established murine sarcoma virus (MSV) tumours were reconstituted with syngeneic (+/+) immune splenic T cells. These immune protected mice were randomly divided to provide smaller groups for serial exsanguination. At various time points mice were individually bled and CIC concentration and blocking activity of each individual serum was determined. Control sera were obtained from nu/nu and adult +/+ mice inoculated with tumour cells only, and from nu/nu mice protected with normal +/+ spleen cells. In all the mice studied, CIC and blocking appeared to be mutually independent parameters throughout the MSV tumour course. On the other hand, in immune protected mice considered alone or together with the control groups, CIC and time after tumour cell inoculation, but not tumour size, were significantly correlated. A significant relationship between blocking and tumour size was also established, although this only applied to immune protected mice. However, analysis of the combined data from sequentially bled immune protected mice in relation to different phases of tumour behaviour, did not support the notion that blocking, and more particularly the persistence of CIC, contribute to tumour regrowth and dissemination.

Placental alkaline phosphatase as a tumour marker in seminoma using the H17 E2 monoclonal antibody assay.

Serum samples from 62 patients with seminoma were assayed for placental alkaline phosphatase-like activity using the monoclonal antibody H17 E2, in order to evaluate its utility as a serum tumour marker. Fifteen of 16 patients (94%) with active seminoma had elevated serum PLAP levels. Sixteen of 46 (35%) of patients considered to be in remission had elevated PLAP levels (false positive rate 35%). Fifteen false positive results were considered attributable to concomitant smoking, and if these patients are excluded, only one false positive case was detected. In 7 out of 7 patients sequential PLAP assays reflected clinical response to treatment.

Fluorescent location of ornithine decarboxylase employing derivatives of the specific inhibitor alpha-difluoromethyl ornithine.

Two fluorescent derivatives have been made from alpha-difluoromethyl ornithine by linking the carboxyl group of the ornithine derivatives to fluorescent amines. alpha-difluoromethyl ornithine is a potent inhibitor of ornithine decarboxylase, an enzyme which plays an essential role in cell division. We have used these fluorescent derivatives as probes for ornithine decarboxylase in frozen sections of skin to locate the epithelial cells which are known to contain ornithine decarboxylase. The probes also located squamous cell carcinoma cells in human skin.

Internal carotid artery thrombosis associated with a fracture.

A case of occlusion of the internal carotid artery after craniocervical trauma with subsequent death of the patient is presented. The patient originally had a mandibular fracture; subsequently an altered sensorium and left-sided hemiparesis developed. The patient died. The typical "lucid interval" was noted. The changes in sensorium are similar to those associated with epidural and subdural hematomas.

The use of cancellous bone for closure of oroantral and oronasal defects.

Two patients who were successfully treated with cancellous bone grafts for closure of large oroantral or oronasal defects have been presented. In attempting closure of such large defects, it is important to follow certain principles. The oral, antral, and nasal mucosa must be free of infection and inflammation, continuity must exist between the oroantral and oronasal mucosa, and water-tight closure of the oroantral and oronasal flaps must be achieved. Preoperative and postoperative antibiotics and steroids are recommended.

Serum marker potential of placental alkaline phosphatase-like activity in testicular germ cell tumours evaluated by H17E2 monoclonal antibody assay.

A monoclonal antibody (H17E2) was used in a solid-phase localisation of enzyme activity (ILEA) assay to evaluate placental-like alkaline phosphatase (PLAP) as a serum marker of testicular germ cell tumours. Single or repeated assays were performed on 213 normal blood donor and a smaller number of term pregnancy and testicular cancer sera. The detection limit of PLAP by this system was 0.14 O.D. units equivalent to 0.04iul-1. Of 50 patients with established metastatic disease tested before treatment, 88% of 16 with seminoma, 54% of 13 with mixed seminoma and malignant teratoma and 33% of 21 with malignant teratoma had serum PLAP greater than 0.2 O.D. units. This compared to an incidence of 2% in non-smokers and of 29% in smokers who had been free of disease for more than 12 months. In 15 of 22 successfully treated patients, pre-treatment serum PLAP exceeded 0.2 O.D. units (mean 0.69 O.D.) and varying (53-97%) reductions in the initial levels occurred with treatment. These results with monoclonal antibody ILEA assay suggest that measurement of PLAP levels will be useful in the management of patients with germ cell tumours, particularly seminoma.

Circulating immune complexes as markers of response to chemotherapy in malignant teratomas and gestational trophoblastic tumours.

Concentrations of circulating immune complexes (CIC) were measured serially during chemotherapy of 22 patients with gestational trophoblastic tumours (GTT) and 11 patients with malignant teratoma (MT) by the polyethylene glycol precipitation and CIq solid-phase assays. Results were correlated with tumour response as measured by serum concentrations of human chorionic gonadotrophin (hCG) and alpha-foetoprotein (AFP). CIC concentrations correlated with disease status in the early stages of treatment in 4/22 patients with GTT and 5/11 with MT. CIC assays were less sensitive than hCG and AFP as a monitor of disease, and also less specific, in that 8 patients with GTT and 5 with MT developed raised CIC concentrations during chemotherapy in spite of sustained complete remission. Measurements of CIC concentrations by present methods are neither sufficiently sensitive nor specific to be of clinical value as a tumour marker in GTT and MT, and this casts doubt on their potential value in other malignancies. Attention should be directed to identification of the components of CIC, some of which may be more cancer-specific.

Independent expression in serum of three tumour-associated antigens: CA 125, placental alkaline phosphatase and HMFG2 in ovarian carcinoma.

Circulating levels of CA 125, the HMFG2 antigen and placental alkaline phosphatase were measured in patients with epithelial ovarian cancer. In 37 patients the antigens were assayed before operation and 161 follow-up samples from 41 patients were assayed at different times during treatment. These three human tumour-associated antigens were expressed independently of each other. Measurement of all three antigens, compared with measurement of CA 125 alone, resulted in a statistically significant improvement in the detection rate of patients with localized disease from 18% to 69%.

The human placental alkaline phosphatase gene and related sequences map to chromosome 2 band q37.

A human placental alkaline phosphatase (PLAP) cDNA was isolated from a lambda gt 10 library of the cell line HEp-2. Southern blots probed with a fragment of the cDNA clone showed that the human genome may contain more than one PLAP-related sequence. The PLAP probe showed person-to-person variation in banding pattern with a number of enzymes. Using a panel of human/rodent somatic cell hybrids the PLAP sequences were mapped to chromosome 2. In situ hybridization confirmed this assignment and localized the gene(s) to chromosome 2 band q37.

Monoclonal antibodies to placental alkaline phosphatase: preclinical evaluation in a human xenograft tumour model of F(ab')2 and Fab fragments.

Six new placental alkaline phosphatase monoclonal antibodies (MAbs) have been evaluated in order to select a potentially clinically useful antibody fragment for use in immunoscintigraphy or therapy. Initially, 3 antibodies were identified by trial pepsin digestion as likely to give satisfactory F(ab')2 yield. The corresponding intact antibodies were then compared for ability to localise human xenograft tumours in athymic mice. Of the best of these, designated 3F6, F(ab')2 and Fab fragments were then evaluated in similar xenograft experiments. In intact antibody biodistribution comparisons, 3F6 showed good tumour retention and satisfactory specific/non-specific ratios at 8 days. In similar fragment biodistribution experiments 3F6 F(ab')2 gave the highest tumour/blood ratio (10) and tumour/organ ratios (19) and the best specific/non-specific localisation. This fragment also showed higher absolute uptake in the tumour than intact antibody, 18.9% and 14.4% respectively of the injected dose. As expected, fragments showed much faster blood clearance rates than whole antibody. For Fab the in vivo instability by 6 hr was also demonstrated.

Placental-type alkaline phosphatase in cervical neoplasia.

Monoclonal antibodies reactive with placental-type alkaline phosphatase have formed the basis of methods for detection of this oncodevelopmental antigen in patients with pre-invasive and invasive cervical neoplasia, with or without evidence of papilloma virus infection. Disease-related elevations of placental-type alkaline phosphatase were not observed in patients' sera. Solubilised cervical smears or biopsy material, and cervical mucus swabs, often contained substantial amounts of this isoenzyme; however, there was no significant difference between any of the patient and control groups. Thus, serological and smear test assays for placental-type alkaline phosphatase were not useful in differential diagnosis of cervical lesions. However, its presence in most biopsy specimens, often at high levels, indicated possible application for in vivo radioimmunoimaging studies of invasive or metastatic cervical cancer.

Monoclonal antibody assay of serum placental alkaline phosphatase in the monitoring of testicular tumours.

A monoclonal antibody (H17E2) recognising both placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase was incorporated in a solid phase immunoassay. This was used to measure levels of PLAP in 257 sera from 148 patients with germ cell neoplasms of the testis. High levels of PLAP were found in all patients with active seminomas (mean 0.85 O.D.) compared to those in clinical remission (mean 0.20 O.D.) (P less than 0.0001). More importantly, changing levels of PLAP correlated with the course of disease in 79 samples from 33 patients with seminoma (P less than 0.0001). Elevated PLAP levels were also noted in patients in remission who were smokers (mean 0.32 O.D.) compared to non-smokers (mean 0.15 O.D.) (P less than 0.001). These data demonstrate that determination of PLAP levels using this sensitive immunoassay is an important new adjunct in the monitoring of the response to treatment in patients with seminoma.

Production of monoclonal antibodies to placental alkaline phosphatase: preliminary characterisation includes identification of one antibody reactive with routinely fixed histological preparations.

We describe the production and preliminary characterisation of a set of monoclonal antibodies (MAbs) raised against placental alkaline phosphatase (PLAP). Different forms of antigen presentation, PLAP or PLAP-like expressing whole cells, placental membranes or purified PLAP were used to immunise BALB/c mice. Initial screening was carried out against the immunising material by ELISA, against fresh frozen placental sections by immunostaining and against purified PLAP using an enzyme capture assay. The most successful fusions were those following whole cell immunisation, producing 27 antibodies that all reacted with both the placental and testicular form of enzyme. These all showed a broadly similar pattern of reactivity when tested against a range of human malignant cell lines. Further characterisation identified one antibody, 8B6, as strongly reactive with formalin-fixed paraffin-embedded placental sections. This antibody also performed well when tested against a range of normal and malignant routinely fixed tissue sections. Of 14 antibodies analysed for immunoglobulin isotype, 10 were of the IgGI subclass. In competitive binding studies with 7 antibodies to discriminate epitopes, at least 4 distinct binding sites were identified. By Scatchard analysis on 4 of these antibodies, binding constants of 3 were within the range 3.5-5.3 x 10(-9)M. Unusually the 4th antibody appeared to recognise 2 separate antigen sites with binding constants of 2.1 and 7.5 x 10(-9)M. In a preliminary study to compare patterns of reactivity of a selection of the new antibodies with a limited number of sera from smokers and seminoma patients, results indicate their potential for further typing within the placental group of enzymes.