Does ND-YAG laser extend the indications for resection of pulmonary metastases? Surgery forms part of a combined oncological concept in the management of pulmonary metastases. The following questions are relevant for its role: Does survival depend on the type of primary tumor? Are there any prognostic factors? What are the limits on radical resection? We analyzed retrospectively 657 patients who had undergone 759 resections of pulmonary metastases between 1973 and 1990. After conducting in vitro and in vivo experiments with the non-contact neodymium aluminum garnet (Nd-YAG) laser with a generating and delivery power of 10-120 W at the site of operation for 0.1-9.9 s, we have treated 65 patients by laser resection and/or vaporization since January 1990. Our preferred surgical approach was median or transverse thoracotomy. The 5-year survival of all resected patients was 30%, ranging from 21% (soft tissue sarcoma) to 60% (testicular carcinoma). Statistically significant differences in prognosis were seen related to the type of primary tumor, the disease-free interval, the caval or portal type of metastatic spread, the number of metastases and the potential degree of radical resection. The potential degree of conventional radical resections (wedge, anatomical sub-/segmental) was negatively influenced by the number of metastases (n > 9: 79% "radical" surgery = 38% 5-year survival). Laser treatment allowed parenchyma-preserving resection in cases of metastases of more than 0.5 cm in diameter, and vaporization in smaller ones. Resection with the intention of achieving complete remission was possible in up to 72 unilateral metastases. The complication rate was comparable to conventional resections.


Successful revascularization of large isolated tracheal segments. The omentum has been shown to be of use in clinical and experimental revascularization of tracheobronchial anastomoses. We have evaluated the possibility of revascularizing large and completely isolated tracheal segments while preserving the main tracheal characteristics. Ten experiments were performed in dogs, introducing 10-14 cartilage ring tracheal segments enveloped in omentum into the abdomen. Revascularization resulted in all cases with preservation of tracheal consistency. In only two cases were small mucosal necrotic zones observed. The experimental model thus appears to be of use in the revascularization of large tracheal segments, with excellent preservation of both cartilage and mucosa.


Effects of prostaglandin E2 and cyclic AMP on uptake of immunoglobulin G complexes by cultured macrophages. The uptake of immune complexes by macrophages (MP) may be important in disease states in which circulating immune complexes are increased. We undertook the present study to determine the effects of prostaglandin E2 (PGE2) and adenosine 3',5'-cyclic monophosphate (cAMP) on the uptake of immunoglobulin G complexes (IgG complexes) by MP. The uptake of 125I-IgG-gold particles (IgG-gold) was measured in the established MP cell line J774.16 following pretreatment with PGE2, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), PGE2 plus IBMX, the cyclooxygenase inhibitor indomethacin (IM), and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP). Preincubation with PGE2 at concentrations from (10(-12)) to (10(-5) M) revealed significantly diminished uptake of IgG-gold at (10(-6)) and (10(-5) M) (in counts per min per well, PGE2 [10(-6) M], 5,401 +/- 140; control, 17,150 +/- 493, p less than 0.001); (PGE2 [10(-5) M], 3,835 +/- 172; control, 17,150 +/- 493, p less than 0.001). IBMX (10(-3) M) alone did not significantly alter IgG-gold uptake (IBMX, 14,450 +/- 1938; control, 14,840 +/- 995, p less than 1.0). PGE2 (10(-6) M) plus IBMX (10(-3) M) significantly suppressed IgG-gold uptake (in counts per min per well, PGE2 plus IBMX, 3,659 +/- 129; control 18,296 +/- 486, p less than 0.001). PGE2 (10(-6) M) alone also suppressed IgG-gold uptake versus control (PGE2, 4,578 +/- 105; control, 18,296 +/- 486, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


The prostacyclin analogues, cicaprost and iloprost, increase cytosolic Ca2+ concentration in the human erythroleukemia cell line, HEL, via pertussis toxin-insensitive G-proteins. In the human erythroleukemia cell line, HEL, prostaglandin E2 (PGE2) and the stable prostacyclin analogue, iloprost, increase cytosolic Ca2+ concentration ([Ca2+]i) via pertussis toxin-sensitive and -insensitive pathways. Unlike iloprost, the stable prostacyclin analogue cicaprost (ZK 96480), is devoid of agonistic properties at prostaglandin E2 receptors. We compared the effects of cicaprost, iloprost and PGE2 on [Ca2+]i in HEL cells. Cicaprost, iloprost and PGE2 were similarly potent to increase [Ca2+]i in HEL cells. However, unlike the effects of PGE2, those of the prostacyclin analogues were not inhibited by pertussis toxin. The prostaglandins studied increased [Ca2+]i through both mobilization from internal stores and Ca2+ influx from the extracellular space. Prostacyclin analogue- and PGE2-induced rises in [Ca2+]i were desensitized in a homologous manner. Additionally, there was cross-desensitization between cicaprost and iloprost, but not between the prostacyclin analogues and PGE2. Our data suggest that in HEL cells (i) cicaprost and iloprost act through prostacyclin receptors and (ii) that these receptors couple to pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (iii) resulting in an increase in [Ca2+]i by Ca2+ mobilization from internal stores and sustained influx.


Long-term outcome and sequelae in aggressive lymphoma patients treated with the LNH-80 regimen. One hundred patients with aggressive malignant lymphoma treated with the LNH-80 regimen were evaluated for long-term survival, late relapse and long-term sequelae. The LNH-80 regimen consisted of three intensive courses of adriamycin, cyclophosphamide, vindesine and bleomycin, followed by consolidation and final intensification, as previously described. Of the eighty-four patients who achieved CR after induction, fifty-two are alive in continuous complete remission with a median follow-up of 9.2 years. Twenty-nine CR patients (35%) relapsed. Sixty-six percent of the relapses occurred during the first 18 months following the end of treatment but late relapses were observed up to 7 years off-therapy. The fifty-two long-term responders were evaluated for their ability to resume work, sexual function, fertility and presence of long-term sequelae. Of the 41 patients who worked before diagnosis of their disease, 66% had resumed their normal jobs and 24% had retired. Sexual activity was considered to be satisfactory by 66% of the patients. Eleven of the 15 patients (73%) who wanted children had between one and three children. Seven patients (14%) were considered to have mild long-term sequelae but all long-term survivors reported having an acceptable quality of life. Five of the patients who reached CR (6%) had second neoplasias. The LNH-80 regimen allowed 52% of the patients to benefit from long-term disease-free survival with minor long-term toxicity.


Vehicle pollutants: effects on the lung. As concern about pollution of the environment and the consequent effects on health grows nationally and internationally, the role of the nurse as health promoter is increasing in importance. The Royal College of Nursing responded to this concern by establishing an Environmental Working Party, of which the author is a member. This article considers one type of pollution, that caused by vehicles, and its effects on the functioning of the lung. The author argues that nurses need to be familiar with environmental determinants of health if they are to work towards upholding a 'health' service.


Chemotherapy with MACOP-B and VACOP-B for intermediate- and high-grade non-Hodgkin's lymphoma: clinical results and analysis of prognostic factors. Eighty-three previously untreated patients with aggressive non-Hodgkin's lymphomas were treated with either MACOP-B (23 patients) or VACOP-B (60 patients) as originally described by Klimo and Conners [1, 2]. Their median age was 46 years. Thirty-seven patients had stage I or II and 46 stage III or IV disease. The tumor histopathology was reviewed in all cases. Sixty-five patients had intermediate grade and 18 high-grade non-Hodgkin's lymphomas according to the International Working Formulation. The rate of complete response was 74% for MACOP-B and 76% for VACOP-B. At the time of analysis the duration of follow-up was 50 months for the MACOP-B and 22 months for the VACOP-B group. The actuarial three-year progression-free survival was 35 +/- 10% for the MACOP-B group, 48 +/- 11% for the VACOP-B group, and 46 +/- 7% for all patients. Treatment mortality was 6%. A univariate and a multivariate analysis of selected pretreatment parameters and of regimen demonstrated that stage III or IV, high-grade lymphoma, and elevated serum LDH, but not the type of regimen, are significantly associated with poor progression-free survival in our patient population.


The Second International Symposium on Hodgkin's Disease. Workshop III: Recommendations for future clinical trials. In the opinion of the participants of the workshop, the following problems should be addressed with high priority: 1st, a clear definition of the 10%-15% of patients who should receive radiotherapy only; 2nd, reduction of toxicity of combined modality approaches for patients in localized stages with risk factors; and 3rd, definition of a poor-prognosis subgroup of patients in stages IIIB/IV for whom more efficient treatment programs must be developed. Dose intensification in the form of ABMT as the prominent example of high dose intensity should be tested in these patients. If this approach proves its efficacy, the concept of dose intensification could be extended to other patients with advanced disease. Results of recent trials in Hodgkin's disease suggest that the cure rates which increased considerably between 1960 and 1980 have reached a plateau. Further improvements that can be realistically expected will be small and studies to prove such progress need large numbers of patients ('megatrials') to avoid 'false negative' results. Such megatrials could be performed as large international or even intercontinental trials. To some extent, overviews could substitute for megatrials, if sufficient high-quality randomized studies addressing a specific question are available for a meta-analysis. The installation of an international steering committee was recommended, which should stimulate and coordinate world-wide efforts for the solution of the remaining problems in the treatment of Hodgkin's disease.


Long-term gonadal dysfunction and its impact on bone mineralization in patients following COPP/ABVD chemotherapy for Hodgkin's disease. Only limited data is currently available on long-term gonadal toxicity and its impact on bone mineralization in men and women treated for Hodgkin's disease. The present study was therefore conducted to evaluate gonadal toxicity and bone loss in 49 patients with Hodgkin's disease 2-10 (median 5.37) years after chemotherapy. Most patients were treated with the COPP/ABVD regimen +/- irradiation according to the protocols of the German Hodgkin Study Group. Blood samples were tested for gonadotropins (FSH, LH), gonadal steroids, parathyroid hormone, osteocalcin, and calcitonin. Bone mineral density was measured using single- and dual-energy quantitative computed tomography as well as single-photon absorptiometry. FSH serum levels were significantly increased in 21/27 (80%) men demonstrating germ-cell aplasia. 13/15 (86%) men showed azoospermia after the COPP/ABVD regimen. In contrast, testosterone levels were within normal limits in all men tested, suggesting normal Leydig-cell function. 17/22 (77%) women exhibited increased FSH and LH levels, indicating premature ovarian failure. Women with therapy-induced ovarian failure had a significantly lower trabecular (98 +/- 34) and cortical (292 +/- 48 mg/cm3) spinal bone density than those with normal ovarian function. Men showed no evidence of bone loss after therapy. These data suggest severe gonadal toxicity in both men and women treated with the COPP/ABVD regimen. In female patients, drug-induced ovarian failure has a significant impact on bone mineralization.


Role of additional radiotherapy in advanced stages of Hodgkin's disease. Although radiotherapy is widely used as additional treatment following chemotherapy, its precise role has never been clearly proven. Relapses tend to occur in previously involved bulky sites. Non-randomized studies may suggest a positive effect of the addition of radiotherapy. This effect however, might also be caused by selection. Randomized studies have not resulted in a survival advantage for the patients treated with additional radiotherapy compared to no further treatment or additional chemotherapy. The SWOG study 7808 suggest a 20% benefit in remission duration for the nodular sclerosis histology subgroup. Definitive conclusions have to wait for more mature results of randomized studies including the ongoing EORTC study and the possibility to perform an overview of all studies.


Polymorphonuclear leukocyte adhesion to endothelial cells is inhibited by resting platelets. The effect of human platelets on the adhesion of polymorphonuclear leukocytes (PMNs) to cultured endothelial cells was investigated. Resting platelets inhibited the adhesion of PMNs stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4), and tumor necrosis factor-alpha (TNF-alpha). Platelets similarly inhibited PMN adhesion induced by endothelial cell activation with TNF-alpha. The inhibitory effect depended on platelet number, was not associated with detectable platelet activation, and was also exerted by paraformaldehyde-fixed platelets. Moreover, supernatants of U46619- or thrombin-stimulated platelets were ineffective, thus excluding a role for constituents released as a result of the platelet-release reaction. Strong inhibition of PMN adhesion was exerted by platelet lysates. The inhibitory activity associated with lysates was sedimentable, heat sensitive, and not dialyzable through a membrane with a molecular-weight cutoff of 8,000; it was directed toward PMNs and was not due to cytotoxic effects or a general inhibition of PMN responsiveness to stimulation, since enzymatic release from activated PMNs was unaffected by platelet lysates. Finally, the activity was not prevented by specific adenosine inhibitors and anti-P-selectin monoclonal antibody. These data suggest that resting platelets can exert an inhibitory effect on PMN adhesion to the vessel wall during inflammatory and thrombotic conditions.


Injury of mouse lymphocytes caused by exogenous methyl linoleate hydroperoxides in vitro. The incubation with methyl linoleate hydroperoxides (MLHPO), a model of lipid peroxides, depressed DNA, RNA and protein syntheses of mouse thymic lymphocytes and increased the amount of thiobarbituric acid-reactive substances in lymphocytes. These phenomena were also found in the splenic lymphoblasts in the DNA synthetic phase (S-phase) obtained by mitogen. Prior culturing with all-rac-alpha-tocopherol increased DNA synthesis in splenic lymphoblasts. Electron microscopically, cytoplasmic micro-organelles of splenic lymphoblasts in the S- and G2-phases were markedly destroyed as compared with nuclei. No discernible changes were observed in not-blastotransformed lymphocytes under these experimental conditions. These findings indicate that thymic lymphocytes and splenic lymphoblasts are affected by exogenous lipid peroxides, and cytoplasmic micro-organelles of splenic lymphoblasts might be markedly damaged by exogenous lipid peroxides as compared to their nuclei.


Pharmacological modulation of the endotoxin-induced increase in plasminogen activator inhibitor activity in rats. Pharmacological modulation of the in vivo induction of plasminogen activator inhibitor type-1 (PAI-1) synthesis was studied in rats using the induction of PAI-1 by endotoxin as a model system. Both the cyclooxygenase inhibitors acetylsalicylic acid and indomethacin enhanced PAI-1 induction. The combined cyclooxygenase-lipoxygenase inhibitor, BW755C, dose-dependently inhibited induction. Since five other lipoxygenase inhibitors, a phospholipase inhibitor, an inhibitor of leukotriene formation and dexamethasone had no effect on the endotoxin-induced increase in PAI-1 synthesis, the effect of BW755C could not be ascribed to its known pharmacological properties. In addition, induction of PAI was enhanced by isobutyl-methylxanthine, a phosphodiesterase inhibitor, but not, however, by other phosphodiesterase inhibitors, or by forskolin or NG-nitro-L-arginine, suggesting an effect of isobutyl-methylxanthine other than through cyclic nucleotides. Heparin and hirudin had no effect either. Overall, the data showed that the induction of PAI-1 synthesis by endotoxin in vivo can be up- and down-regulated pharmacologically, but the mechanisms involved remain elusive.


Contact factor proteases and the complexes formed with alpha 2-macroglobulin can interfere in protein C assays by cleaving amidolytic substrates. Plasma from women taking combined oral contraceptives and cold-activated plasma contain proteases which cleave chromogenic substrates in protein C assays in the absence of protein C activators such as Protac. This spontaneous activity makes a background substraction necessary and makes protein C (PC) assays less accurate. We investigated two commonly used substrates < Glu-Pro-Arg-pNA (S-2366) and 2AcOH.H-D-Lys(Cbo)-Pro-Arg-pNA (PC substrate) and found that cold-activated normal and protein C-deficient plasmas gave absorbance values up to 300 times higher than buffer blanks. FXIa cleaves these substrates but activity was not blocked by corn or lima bean trypsin inhibitors, soy bean trypsin inhibitor (SBTI), hirudin or epsilon-amino-n-caproic acid (EACA). Kaolin activation of normal, FXI, FIX, FVIII, FVII and protein C-deficient, but not of FXII or prekallikrein (PKK)-deficient plasmas led to cleavage of chromogenic substrate for protein C. The protein C substrates were cleaved by purified kallikrein and alpha- and beta-FXIIa. Immunoabsorption with alpha 2-macroglobulin (alpha 2M) antibodies removed 60% of the alpha 2M and 70% of the activity on PC Substrate. Gel filtration of normal plasma on Sephadex G-150 gave a single peak of protein C activity and antigen in the included volume. After cold activation of the fractions, a second protein C-like peak appeared in the void volume, but with no detectable protein C antigen. This peak coincided with alpha 2M (chromogenic and ELISA) and plasma kallikrein (S-2302), but FXII (measured with a substrate insensitive to kallikrein) eluted separately.(ABSTRACT TRUNCATED AT 250 WORDS)


Antenatal care for the normal patient.

Antenatal care for the normal patient. Screening protocols for low-risk obstetric patients are used to identify those at risk for adverse perinatal outcome. Gestational diabetes screening is best performed by administering 50 g of glucose in the nonfasting state, with plasma glucose values above 140 mg/dL at 1 hour considered abnormal. Assay of maternal serum alpha-fetoprotein is primarily used to identify pregnancies at risk for neural tube defects and other structural anomalies. However, an elevated value may also identify patients at increased risk for a number of abnormal pregnancy outcomes, even when the fetus is free from defects. Patients who have previously had a cesarean section are now strongly encouraged to undergo a trial of labor and to attempt a vaginal birth. Such a course is not without risk, and catastrophic complications can develop. Careful patient selection, appropriate counseling, and close management are necessary in this situation.


Lectin characterization of cystogenesis in the SBM transgenic model of polycystic kidney disease. The generation of a novel transgenic mouse model of polycystic kidney disease with a construct (SBM) that links the coding region of the c-myc proto-oncogene to the simian virus 40 enhancer and beta-globin promoter was previously reported (see reference 1). In order to determine the site of origin and histogenesis of renal cysts in this model, lectin/immunohistochemical and electron microscopic studies on mice of varying ages (from birth to adulthood) are described here. Cysts are detectable at birth and increase in number and diameter with age. Cysts predominantly involve the collecting tubules of young transgenic mice but progressively affect the proximal tubules with advancing age. A minority of cysts are of distal tubular origin in all age groups studied. Tubular hyperplasias are primarily reactive with proximal tubular markers and appear to precede the development of proximal tubular cysts in adult mice. This particular phenotypic evolution of polycystic kidney disease with advancing age suggests that the cystogenic potential of the transgene is modulated by yet unidentified tubular segment-specific responses.


Evidence for sense RNA-mediated protection to PVYN in tobacco plants transformed with the viral coat protein cistron. The coat protein (CP) cistron of the tobacco veinal necrosis strain of potato virus Y (PVYN), supplemented with translational start signals, was cloned into an Agrobacterium tumefaciens Ti transformation vector. Transformation of tobacco leaf discs resulted in 99 transgenic lines which were subsequently analysed for the presence and expression, at both the transcriptional and translational level, of the CP-gene. Although CP-specific RNA transcripts were produced in all plants no CP could be detected by several sensitive immunological techniques. Upon mechanical inoculation of progeny lines of self-pollinated original transformants (S1) with PVYN, protection levels of 20 and 95%, respectively, could be observed in two out of ten lines tested. This level of protection increased to 100% in the S2 progeny obtained from self-pollination of virus-protected S1 plants. Transformation of tobacco leaf discs with a PVYN CP construct from which the ATG start codon had been removed by site-directed mutagenesis resulted in 57 transgenic lines that all produced CP-specific transcripts. Mechanical inoculation with PVYN of S1 progeny plants of several of these lines resulted in resistance to a similar level and extent as in the S1 progeny of plants transformed with the intact CP cistron. The results obtained strongly suggest that the resistance observed in the transgenic plants is principally based on the presence of PVYN CP RNA sequences rather than on the accumulation of viral coat protein.


Clinical application of radioimmunoimaging in the detection of tumors--current status in China. In this article, the development of radioimmunoimaging (RII) in China is reviewed. Since 1984 conventional heterologous specific antibodies (Abs), such as anti-carcinoembryonic antigen (CEA) and anti-alphafetoprotein (AFP) Abs, have been used to detect cancers of the colon, ovary, lung, liver, etc. Later on, Abs against specific tumor cell-associated antigens have been produced in many institutions and applied clinically, with quite satisfactory results (detection rate ranging from 80-90%). Recently, human monoclonal antibodies (McAbs) are also being prepared. Finally, the existing problems to be overcome are discussed.


Pilot production of high-titer interferon and virus with a microcarrier suspension culture system. We have successfully cultured four cell lines--L929, BHK-13, BHK-21 and CHO-K1 using an MC-1 type microcarrier made in our academy. With the microcarrier in a concentration of 5 mg/ml, the cell density was about 30 x 10(4) cells/ml; after 3 days in suspension culture, the cells could proliferate to 1 x 10(6) cells/ml. At this time, when L929 cells were primed with 25 IU/ml MuIFN for 14-24 h and then superinduced with NDV, cycloheximide (20 micrograms/ml) and actinomycin D (2 micrograms/ml), the titer of IFN reached approximately 10(5) IU/ml (10(5) IU/mg of protein, expressed in specific activity). When VSV was inoculated into the other three cell line cultures, the viral titer reached 6 Log TCID50/ml or much higher. The yield with the CHO-K1 cell line was the highest, reaching titers of 7-8 Log TCID50/ml. These titers were similar to those seen in stationary culture. With trypsin-citrate solution and a more rapid stirring speed, the cells could be satisfactorily released from the microcarriers and reattached on fresh ones. These experiments show that the microcarrier suspension culture system is suitable for producing large scale, high-titer, low-cost vaccines and IFNs, both natural and recombinant, using genetically-engineered CHO cell lines.


The ligand for c-kit, stem cell factor, stimulates the circulation of cells that engraft lethally irradiated baboons. Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) > or = 500/microL, WBC > or = 1,000/microL, and an absolute neutrophil count (ANC) > or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.


In vivo effects of interleukin-6 on thrombopoiesis in healthy and irradiated primates. We have studied the in vivo effects of recombinant human interleukin-6 (rhIL-6) on hematopoiesis in eight healthy and nine irradiated cynomolgus monkeys. Of the healthy animals, three received rhIL-6 alone (10 micrograms/kg/d, subcutaneously [SC]), one received rhIL-6 in combination with rhIL-3 (10 micrograms/kg/d, SC), one received rhIL-6 in combination with recombinant cynomolgus granulocyte-macrophage colony-stimulating factor (rcGM-CSF; 10 micrograms/kg/d, SC), two received rhIL-6 in combination with recombinant human granulocyte-CSF (rhG-CSF; 10 micrograms/kg/d, SC), and one received rhIL-6 in combination with recombinant human leukemia inhibitory factor (rhLIF; 10 micrograms/kg/d, SC). All animals were treated for at least 2 weeks with rhIL-6 or the above mentioned combinations. rhIL-6 alone significantly increased the peripheral blood platelet counts (2- to 3.5-fold). The platelets reached a plateau between days 10 and 15 of treatment. No synergistic effects on platelet numbers were observed when rhIL-6 was combined with rhIL-3, rcGM-CSF, rhG-CSF, or rhLIF. In addition to rhIL-6, only rhLIF increased the platelet numbers when administered alone. To test whether rhIL-6 might also protect the animal from thrombocytopenia or shorten the time of thrombocytopenia after irradiation, we treated nine animals with total body irradiation (3.8 Gy). Six of the animals were additional treated with rhIL-6 (4 with 10 micrograms/kg/d; and 2 with 100 micrograms/kg/d) from day -1 or +1 to day 28 post irradiation. In these animals, rhIL-6 at the same dose effective in healthy animals (10 micrograms/kg/d) was not capable of protecting the animals from platelet nadir. However, when pegylated rhIL-6 was used at a dosage of 100 micrograms/kg/d post irradiation, the mean of the nadirs was 71,000/microL as compared with 39,000/microL in control animals and the time of thrombocytopenia was shorter (3 v 5 days). In all animals (healthy and irradiated), rhIL-6 did not increase the number of bone marrow megakaryocytes but induced a right shift of DNA ploidy in megakaryocytes. These data suggest that IL-6 acts as "thrombopoietin"-like activity, but not as "megakaryocyte-CSF"-like activity.


Human arterial smooth muscle cells synthesize granulocyte colony-stimulating factor in response to interleukin-1 alpha and tumor necrosis factor-alpha. Vascular smooth muscle cells (SMC) are a major cell type comprising the walls of blood vessels. We report the synthesis of granulocyte colony-stimulating factor (G-CSF) by cultured human SMC obtained from the internal mammary artery and thoracic aorta. Interleukin-1 alpha (IL-1 alpha) greatly increased in a dose-dependent manner the amount of this cytokine produced by the SMC, with tumor necrosis factor-alpha (TNF-alpha) being less effective. Newly formed G-CSF could be detected in culture supernatants within 6 hours after IL-1 alpha or TNF-alpha treatment. Northern blot analysis of SMC stimulated with IL-1 alpha and TNF-alpha showed an increase in the amount of mRNA for G-CSF as compared with control cells. Enhanced G-CSF mRNA levels were observed when SMC were treated with cycloheximide in the absence or presence of added cytokine. In vasculitis, the walls of blood vessels become inflamed as evidenced by a leucocytic infiltrate usually dominated by polymorphonuclear neutrophil leukocytes (PMNs). G-CSF is known to stimulate PMNs, and our findings raise the possibility that G-CSF made by SMC contributes to the development of vasculitis lesions.


G19.4(alpha CD3) x B43(alpha CD19) monoclonal antibody heteroconjugate triggers CD19 antigen-specific lysis of t(4;11) acute lymphoblastic leukemia cells by activated CD3 antigen-positive cytotoxic T cells. A highly purified, 300-Kd bispecific monoclonal antibody (MoAb) heteroconjugate was prepared by covalently linking the anti-CD3 MoAb, G19.4, to the anti-CD19 MoAb, B43. Dual-color staining techniques and multiparameter flow cytometry confirmed that this alpha CD3 x alpha CD19 heteroconjugate was able to bind to both CD3+ T cells and CD19+ t(4;11) acute lymphoblastic leukemia (ALL) cells. T-cell-mediated lysis of freshly isolated primary bone marrow blasts from nine newly diagnosed ALL patients with a t(4;11)(q21;q23) chromosomal translocation were studied with 51Cr-release assays. Picomolar concentrations of alpha CD3 x alpha CD19 MoAb heteroconjugate effectively triggered lysis of CD19+ t(4;11) ALL cells by interleukin-2-activated CD3+ peripheral blood T-cell (PBTC) effectors but did not augment the cytolytic activity of the same effectors against CD19- T-ALL cells. In contrast to the alpha CD3 x alpha CD19 heteroconjugate, neither the alpha CD3 x alpha CD3 homoconjugate control nor the alpha CD19 x alpha CD72 heteroconjugate control facilitated the cytolysis of t(4;11) ALL blasts. Occupation of the target CD19 binding sites on t(4;11) ALL blasts by preincubation with excess unconjugated alpha CD19 MoAb abrogated the potentiating effects of the alpha CD3 x alpha CD19 heteroconjugate on PBTC-mediated cytolysis. Thus, the cell type-specific cytolysis of t(4;11) ALL blasts by PBTC effectors is dependent on both the alpha CD19 and alpha CD3 moieties of the alpha CD3 x alpha CD19 heteroconjugate. To our knowledge, this is the first description of an effective bispecific antibody that facilitates the T-cell-mediated lysis of t(4;11) ALL blasts.


Human peritoneal mesothelial cells produce many cytokines (granulocyte colony-stimulating factor [CSF], granulocyte-monocyte-CSF, macrophage-CSF, interleukin-1 [IL-1], and IL-6) and are activated and stimulated to grow by IL-1. To investigate the role of peritoneal mesothelial cells in regulating hematopoiesis, as well as inflammation, healing, and tissue regeneration processes, long-term cultures of peritoneal mesothelial cells from human endocavitarian fluids were established. The purity of the cell population was assessed by morphologic and immunocytochemical criteria. Five peritoneal mesothelial cell cultures were analyzed for cytokine expression. Macrophage colony-stimulating factor (M-CSF), granulocyte-CSF (G-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6 transcripts were constantly but variably detected throughout the culture period, while granulocyte-monocyte-CSF (GM-CSF) expression started as the cell culture aged. No IL-2, IL-3, IL-4, IL-5, or IL-7 transcripts were detected in the same samples. Corresponding cytokine activities were detected in the supernatants of the cultures. Peritoneal mesothelial cells proliferated after the addition of exogenous IL-1 beta or IL-1 alpha, whereas the addition of recombinant GM-CSF, G-CSF, M-CSF, or IL-6 failed to trigger proliferation. IL-1 receptor type I transcripts were detected in peritoneal mesothelial cells. Moreover, IL-1 was able to upregulate the expression of the genes that code for G-CSF, GM-CSF, IL-1 alpha, and IL-1 beta in these cells. These data indicate that peritoneal mesothelial cells produce many cytokines and suggest that IL-1 is a regulatory molecule for peritoneal mesothelial cells.


Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death. In the absence of appropriate stimuli, polymorphonuclear neutrophils (PMN) undergo programmed cell death (PCD), also termed apoptosis. We show that granulocyte-macrophage colony-stimulating factor (GM-CSF), but not the chemotactic factors formyl-methionyl-leucyl-phenylalanine (FMLP), recombinant human (rh) C5a, transforming growth factor (TGF)-beta, and interleukin-8 (IL-8), or other cytokines including IL-3, IL-4, IL-6, and G-CSF, maintains viability of PMN in culture by preventing these cells from undergoing PCD. Prevention from PCD by GM-CSF was associated with induction of RNA and protein synthesis in PMN. Inhibition of RNA and protein synthesis by actinomycin-D and cycloheximide impeded the protection of apoptosis by GM-CSF. Similarly, neutralization of GM-CSF biologic activity by a specific antiserum abrogated GM-CSF-mediated inhibition of PCD.


A simple method for hemoglobin measurement in cell culture system. A simple method of hemoglobin analysis in a cell culture system is described. Hemoglobins synthesized in cell cultures are labeled with radioactive amino acids. The cell extract containing radiolabeled hemoglobin is mixed with A, F, S, C, hemoglobin markers and separated by cellulose acetate electrophoresis. Individual bands of hemoglobin are cut from the gel and analyzed for radioactivity. This method is especially useful for determination of newly synthesized minute amount of hemoglobin in cell extracts that are difficult to visualize by staining procedure.


Role of tachykinins in the control of pancreatic secretion and circulation. Tachykinins (TK) are family of peptides including substance P (SP), substance K (SK) and neuromedin K (NK) that have been found in the nerves of the gastrointestinal tract and proposed to act as neurotransmitters to affect the motor, secretory and circulatory functions of the gut, but little is known about their action on the pancreas. In this study three series of tests were carried out to determine the action of SP, SK and NK on pancreatic secretion in conscious dogs and amylase release from the dispersed rat pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects of TK in anesthetized dogs. SP, SK and NK infused i.v. in graded doses (0.12-1.0 microgram/kg per h) in conscious dogs stimulated pancreatic protein outputs reaching, respectively, 38% and 23% of the maximal response to CCK (40 pmol/kg per h). HCO3- outputs were also significantly increased but the highest response did not exceed about 5% of secretin (328 pmol/kg per h) maximum. Cholinergic blockade by atropine abolished the pancreatic responses to tachykinins. When added at various concentrations (10(-11)-10(-7) M) to the incubation medium of rat dispersed pancreatic acini, SK, SP and NK increased in concentration-dependent manner the release of amylase from the resting pancreatic acini and augmented the enzyme release induced by CCK-8 and by urecholine. In anesthetized dogs infused with a background dose of secretin (82 pmol/kg per h), addition of SP, SK and NK caused an immediate and dose-dependent increase in the pancreatic blood flow, oxygen consumption and pancreatic secretion accompanied by a dose-dependent decrease in arterial blood pressure. This study shows that TK are potent pancreatic circulatory stimulants and moderate secretagogues both in vivo and in vitro, acting, at least in part, via cholinergic pathway.


Occurrence of ovulation after intracerebroventricular infusion of substance P in 6-OHDA pretreated female rats. Substance P (SP) infused into the third cerebral ventricle blocks spontaneous ovulation in female rats, probably through catecholaminergic neurons. The studies presented in this paper were undertaken to investigate whether SP exerts its suppressing effect on ovulation in 6-hydroxydopamine (6-OHDA) pretreated female rats. After 8-12 days following 6-OHDA pretreatment female rats were infused, on the day of proestrus, with vehicle and all animals were found to ovulate or with a solution containing 5 nmol of SP and 89% of female rats were then found to ovulate. In the group pretreated with vehicle and subsequently infused with SP, ovulation was found to occur only in 25% of animals. The obtained results indicate that spontaneous ovulation in 6-OHDA-pretreated female rats cannot be blocked by i.c.v. administration of SP, and it may be concluded that SP exerts its suppressing effect through the monoaminergic neurons.


Influence of suramin on some DNA-directed enzymes and primary cultures of chicken embryo and rat cells. The influence of suramin on O6-alkylguanine-DNA alkyltransferase (AT), DNase I and poly(adenosine diphosphate ribose)polymerase (PADPR) as well as on primary cultures of rat and chick embryo cells was examined by using some short-term tests. AT and DNase I were inhibited by suramin in a dose-dependent manner (DE50 = 65 and 100 micrograms/ml, respectively). PADPR activity was increased over a concentration range of 40-320 micrograms/ml. At concentrations above 40 micrograms/ml suramin decreased scheduled and unscheduled DNA synthesis. At doses of below 20 micrograms/ml the substance slightly stimulated unscheduled DNA synthesis in embryonic cells. Suramin enhanced nucleoid sedimentation and diminished the viscosity of alkaline cell lysates. From the present results it is concluded that suramin, at clinically relevant concentrations, is able to interact with enzyme systems which are critical to important nuclear functions and to interfere--in a cell specific manner--with histones and/or matrix proteins, resulting in greater chromatin compactness.


The level of serum granulocyte colony-stimulating factor in cancer patients with leukocytosis. The level of serum granulocyte colony-stimulating factor (G-CSF) obtained from patients with leukocytosis (greater than 10,000/microliters) between May 1989 and April 1991 was measured by enzyme immunoassay. Studied were 18 patients with malignant neoplasms (median age, 64 years) and 14 patients with hematologic disease (median age, 59 years). Increased serum G-CSF values ranging from 70 to 374 pg/ml were noted in 7 of 15 lung cancer cases, a case of malignant thymoma and a blastic crisis of chronic myelogenous leukemia. The rest of the cases showed a normal value (less than 60 pg/ml). There was no correlation between the neutrophil count and G-CSF level. In lung cancer cases with high G-CSF values, neither a characteristic histologic type nor common elevation of tumor markers could be seen. The neutrophil alkaline phosphatase score was significantly increased and hypercalcemia was presented in high G-CSF cases. G-CSF may contribute at least in part to unknown leukocytosis observed in malignant neoplasms, especially in lung cancer.


Hypersensitivity pneumonitis induced by hexamethylene diisocyanate. A case of hypersensitivity pneumonitis (HP) induced by hexamethylene diisocyanate (HDI) is described. Serial determinations of the lymphocyte surface phenotypes by two-color assay revealed the following: 1) increased activated cytotoxic T lymphocytes in the bronchoalveolar lavage fluid (BALF), and 2) increased percentage and absolute number of non-major histocompatibility complex (MHC)-restricted natural killer (NK) cells in the peripheral blood (PB) during the recovery phase of the disease. These findings were considered to be related to the activity of the disease.


New polymer alloy dialysis membranes with varying permeabilities and sievings. The superstructure of polymer alloys that is responsible for their function is best controlled by varying the polymers, polymer blend ratio, solvents, temperature, or all four. Dialysis membranes made of polymer alloys can have varying pure water and solute permeabilities, pore sizes, pore size distributions, and mechanical strengths when wet. Resistance to heat and mass transfer can also vary. The optimal superstructure design of a polymer alloy dialysis membrane enables it to remove efficiently newly identified uremic toxins with higher molecular weights from patients on hemodialysis. The authors prepared new hollow fiber dialysis membranes using a polyethersulfone/polyacrylate (PEPA) polymer alloy, and evaluated pure water and solute permeabilities and reflection coefficients that are dependent upon the superstructure of the PEPA membranes. The new PEPA membranes are asymmetric, with skin layers on either side. The values for pure water permeability and overall mass transfer coefficients for urea, creatinine, and vitamin B12 are quite adequate for hemodialysis. The reflection coefficients for substances with molecular weights greater than 3,000 are strongly dependent upon the conditions under which the membranes were prepared. The values for cytochrome C permeability ranged from 0.42 to 0.71 microns/s.


Assessing and counteracting the prooxidant effects of anticancer drugs. The relationship between peroxide generation and respective cellular damage, triggering various biochemical consequences is first discussed. Then we review the prooxidant effects of various anticancer drugs including anthracyclines and bleomycin, platinum derivatives and the N- and S-mustards. We present and discuss some experimental results on peroxidase inhibition by drugs such as zinc salts, almitrine, deferoxamine, which had previously been tested as efficient in vivo treatment on chlormethine intoxication. In an overview we propose that not only ionizing radiations and anticancer drugs, but also promoters and initiators of cancer might all generate free radicals, in turn triggering oxidative processes generating endogenous peroxides, then probably amplifying the deleterious biological response. The possible limitations of drug therapies decreasing peroxide generation are presented.


Peptides reactive with a transmission-blocking monoclonal antibody against Plasmodium falciparum Pfs25: 2000-fold affinity increase by PEPSCAN-based amino acid substitutions. Based on PEPSCAN analyses, a peptide derived from the 25-kDa surface protein of P. falciparum sexual stages (LDTSNPVKT, amino acids 122-130) was recently shown to react with transmission-blocking monoclonal antibodies. Subsequently, a pinbound construct (FDDTDPIKK; six amino acids substituted) was designed that reacted 1000 times better with the transmission-blocking monoclonal antibody 32F81 than with the parent LDTSNPVKT peptide. While this construct was obtained through single amino acid replacement studies, it now is shown that the combined contribution of the various substitutions is necessary to give the total effect. Free peptides comprising LDTSNPVKT or FDDTDPIKK were shown to inhibit the interaction of the transmission-blocking monoclonal antibody with plate-bound peptides, when prepared by conventional synthesis procedures. Compared with peptides that contained LDTSNPVKT, similar levels of inhibition could be achieved with an average 500-fold lower molar amount of peptides containing the FDDTDPIKK sequence. Affinity constants of the peptides for the transmission-blocking monoclonal antibody ranged from 1.3 x 10(5), for peptides that contained LDTSNPVKT, to 2.6 x 10(8), for peptides that contained FDDTDPIKK. The structural relationship between the epitope of the 25-kDa surface protein and the peptides was demonstrated by competition experiments with the transmission-blocking monoclonal antibody 32F81. Again, peptides comprising the newly designed sequence FDDTDPIKK competed about 200 times better than peptides with the parent LDTSNPVKT sequence.


Evaluation of soluble CD 14 and neopterin as serum parameters of the inflammatory activity of pulmonary sarcoidosis. CD14 represents the most specific marker for monocytes/macrophages. It has been demonstrated in vitro that monocytes/macrophages lose this antigen upon activation. Results of studies investigating the expression of membrane-bound CD14 on the surface of monocytes/macrophages in sarcoidosis patients are controversial. To investigate whether the soluble form of CD14 reflects monocyte/macrophage activation in sarcoidosis, serum levels of soluble CD14 were determined concurrently with other serum markers of monocyte/macrophage activation (neopterin, angiotensin-converting enzyme) in 50 consecutive patients with bioptically confirmed sarcoidosis. The patients were allocated to three groups according to disease activity and therapy. The soluble interleukin-2 receptor in serum and the CD4/CD8 ratio in lavage fluid were used to monitor T-lymphocyte activation. No significant differences in serum or bronchoalveolar lavage levels of soluble CD14 were observed in patients with active or inactive sarcoidosis. Despite the presence of normal soluble CD14 serum concentrations a correlation with serum neopterin and angiotensin-converting enzyme was found in active sarcoidosis (soluble CD14 versus neopterin, rs = 0.61 and 0.65, P < 0.05 and 0.01, respectively; soluble CD14 versus angiotensin-converting enzyme, rs = 0.6 and 0.72, P < 0.02 and 0.003, respectively). A correlation between soluble CD14 and parameters of T-cell activity was not demonstrated. We therefore conclude that soluble CD14 in serum is not a useful clinical parameter in establishing disease activity in sarcoidosis. Neopterin and angiotensin-converting enzyme serum concentrations are parameters with higher sensitivity, although specificity remains very low. The exact role of CD14 antigen in sarcoidosis requires further investigation.


Multiple peptide synthesis on polypropylene membranes for rapid screening of bioactive peptides. A new multiple peptide synthesis technique that allows several hundred peptides to be synthesized on polypropylene membranes in one working day has been developed. Peptides are assembled as spots on a membrane sheet sandwiched between two 96-reaction well templates. Activity measurements can be made either with the membrane-bound peptides, for example, using an enzyme linked immunosorbent assay (ELISA), or after cleavage from the membrane. The method is simple and economical in the use of reagents, and peptides are of high quality, making possible the synthesis of rather long (> 20 residues) peptides. This method has been used to identify antigenic determinants recognized by antisera to bovine myelin proteolipid protein.


Granulocyte colony-stimulating factor treatment in AIDS patients. Frequent complications of human immunodeficiency virus infection are hematopoietic failure and poor tolerance of myelosuppressive drugs. Reasons for neutropenia resulting from hematopoietic failure are infection of the bone marrow and hematotoxicity of treatment with zidovudine, ganciclovir, sulfonamides, and interferons. Moreover, tumor necrosis factor-alpha, transforming growth factor-beta and interferon-gamma have been shown to suppress proliferation of bone marrow cells. Both granulocyte (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) increase neutrophil counts and ameliorate phagocytic and bactericidic function of neutrophils. We report eight cases of AIDS patients with serious infections and neutropenia (< 750 cells/microliters), who were treated concomitantly with recombinant human G-CSF (3-4 micrograms subcutaneously per kilogram body weight daily). G-CSF treatment was well tolerated in all patients and showed no side effects or disturbances of other lineages than neutrophils. Life-threatening bacterial infections were treated successfully by stimulating the neutrophil immune system. This therapy shortened the duration of subsequent treatment with antibiotics. Since human immunodeficiency virus infects CD4-positive monocytes and macrophages, which are stimulated by GM-CSF, G-CSF seems to be the cytokine of choice, if stimulation of the neutrophil lineage is warranted.


Expression of GD2-epitopes in human intracranial tumors and normal brain. Two monoclonal antibodies (mabs) were raised against ganglioside GD2 (Gtri2) and tested on human intracranial tumors and normal brain by immunohistochemical methods both in frozen and paraffin embedded sections. In normal brain structures, astrocytes were visualized with both mabs (BW 625 and BW 704) almost exclusively in the subventricular and subpial layer. A minor amount of myelin sheaths in well defined localisation was only recognized in frozen sections. Consequently in astrocytic tumors of different grades of malignancy (WHO I-IV), astrocytes were depicted in their characteristic shape and arrangement around vessels. In addition, staining was observed in meningiomas and schwannomas, but not in pituitary adenomas or metastatic carcinomas. In meningioma und schwannoma the staining was restricted to the cellular periphery and was again present in frozen section material and absent in paraffin embedded tissue. In astrocytes, reactive and neoplastic, obviously fibrous processes and cytoplasm were distinctly stained both in frozen and paraffin embedded sections. It is concluded that some neuroectodermal derived cells as well as myelin of defined localisation express GD2 on the membrane surfaces and in the cytoplasm. The latter may primarily be the case in fibrous astrocytes, which were stained in reactive and pathologic conditions. The reaction can be used as diagnostic tool in astrocytic tumors; its positive therapeutic significance is hampered by the fact that (1) not all cells in heterogeneous tumor populations express the epitope and (2) there are normal structures which do so.


Rat lymphoid cell--derived histamine and 5-hydroxytryptamine releasing factor. The in vitro production of histamine releasing factor (HRF) by lymphoid cells of rats, both normal and infected with Nippostrongylus brasiliensis, has been studied. Spleen cells and thymocytes were cultured either alone or in the presence of mitogen (PHA, 10 and 50 micrograms/ml) and the dialysed cell-free supernatants were tested for histamine releasing activity on rat peritoneal and pleural mast cell in vitro. We found that spleen cells and thymocytes of normal rats stimulated with PHA in 24 h cultures generated a factor which released histamine and 5-hydroxytryptamine from mast cells, and this ability was potentiated following N. brasiliensis infection of rats - lymphoid cells donors. Pleural mast cells were more sensitive to the action of HRF than peritoneal cells. Rat HRF had an apparent m.w. of 50,000 to 70,000 daltons as determined by gel chromatography and was a heat stable protein inducing histamine release from homologous mast cells in a very rapid (complete in 1-2 min at 37 degrees C), dose and temperature dependent secretory process.


PreS antigen expression and anti-preS response in hepatitis B virus infections: relationship to serum HBV-DNA, intrahepatic HBcAg, liver damage and specific T-cell response. The diagnostic value of preS antigens and anti-preS antibodies during Hepatitis B virus (HBV) infections have not yet been clearly elucidated. Therefore, the objectives of this study were: 1) to better understand the clinical significance of the expression of both preS1 and preS2 antigens (preS1Ag and preS2Ag) in the serum of chronic HBsAg carriers, and 2) to define the respective role of antibody responses to HBs-, preS2- and preS1-specific determinants in the course of acute hepatitis B (AH-B) infections with different outcomes. Our data showed that the serum preS1Ag/HBsAg ratio correlated well with the level of viral replication (serum HBV-DNA as monitored by PCR assay and liver HBcAg), especially in anti-HBe positive chronic carriers. The complete eradication of virions required a persistent antibody response to conformation-dependent HBs-epitopes, temporally associated with a vigorous T cell response to nucleocapsid antigens. Recovery from hepatitis B can be achieved when there is no early antibody response to preS2- and preS1-proteins, which was found to be transient, concomitant with a flare-up of the liver disease, and preceding anti-HBs production. Information on the patterns of preS antigens and their antibodies remained clouded because of the varying specificities and sensitivities of research methods used in studies to date. We have, therefore, developed original Polyclonal-Monoclonal RadioImmunoAssays (PAb-MAb RIAs) by using monoclonal antibodies (MAbs) having previously well-defined specificities. We could thus detect and quantify simultaneously the three distinct antigenicities of the HBV envelope, HBsAg, preS2Ag and preS1Ag, with the same sensitivity. In this way, the preS1Ag/HBsAg and preS2Ag/HBsAg ratios can be calculated to estimate the serum expression of both preS1Ag and preS2Ag in relation to total HBsAg activity during different stages of chronic HBV infection. For optimal management of the state of HBV replication in chronic viral infection, the detection of HBV-DNA in serum was monitored by the Polymerase Chain Reaction (PCR) assay. We extended our work by investigating the clinical significance of antibody response to preS-specific determinants in patients with AH-B showing different outcomes in both natural course or response to alpha-interferon therapy. In a first attempt, we chose to use the Western Immuno-Blotting Assay (WIBA) to obtain a qualitative assessment of the nature of preS antibody responses. Finally, the cell-mediated immune response to HBV antigens was also studied in several patients with self-limited AH-B leading to a relevant finding which may help to clarify the mechanisms responsible for complete clearance of HBV.


HBc and HBe specificity of monoclonal antibodies against complete and truncated HBc proteins from E. coli. We have prepared and used monoclonal antibodies against various populations of full-length and truncated hepatitis B core proteins in order to distinguish between epitopes of HBcAg and HBeAg. Our results show that various epitopes are specific for the different proteins. Certain epitopes, however, are ubiquitous to HBc/e proteins and these are probably exposed on the surface of HBc particles.


Clinical evaluation of indocyanine green video-angiography in the diagnosis of choroidal neovascular membrane associated with age-related macular degeneration. The Authors compared ICG video-angiographic findings in 85 eyes of 78 cases of presumed choroidal neovascular membrane (CNM) associated with age-related macular degeneration with those diagnosed by fluorescein angiography. The detection rate of CNM was 68% with indocyanine green (ICG) video-angiography and 59% with fluorescein angiography. ICG video-angiography can be especially useful for identifying hemorrhagic or exudative detachments of the retinal pigment epithelium. In these cases, detection of CNM by ICG video-angiography was generally far superior to that by fluorescein angiography. However, some CNM in which leakage was identified with fluorescein angiography could not be visualized by ICG video-angiography.


Properties of a recombinant yeast-derived hepatitis B surface antigen containing S, preS2 and preS1 antigenic domains. Yeast cells have been engineered to express mixed HBsAg particles containing the S and a modified large (L*) protein. Their construction resulted in reduced protease sensitivity, reduced glycosylation and complete inactivation of the polymerized human albumin binding site. The particles exposed the S, preS1 and preS2 antigenic determinants and induced an immune response against the three domains. Highly purified preparations have been obtained and are presently being tested in human volunteers.


Antigenicity of synthetic peptides derived from C100 protein of hepatitis C virus. Synthetic octapeptides spanning the 119-147 region of the Hepatitis C Virus (HCV) C100 protein were tested on HCV positive sera. The 138-145 region proved to be antigenic and possibly able to avoid undesired cross-reactions.


Localization of hepatitis C virus antigen(s) by immunohistochemistry on fixed-embedded liver tissue. Using two sources of primary antibodies, we immunohistochemically stained hepatitis C virus-related antigen(s) on fixed-embedded liver specimens. These antigens were localized in the cytoplasm of hepatocytes. The results obtained serologically correlated well with immunohistochemistry.


Identification of HCV-associated antigen(s) in hepatocytes. HCV-associated antigens (HCAg) were localized morphologically using immunofluorescence methods. Fluorescence was found in the cytoplasm of individual liver cells or in groups of cells. Nuclear fluorescence was not observed. Blocking and absorption studies suggest that HCAg is related to nucleocapsid or envelope proteins.


Fine specificity of the human T cell response to hepatitis B virus core antigen. The fine specificity of the human T cell response to the hepatitis B virus core antigen (HBcAg) was investigated in 23 patients with acute hepatitis B virus (HBV) infection using a panel of short synthetic peptides covering the entire core region. An immunodominant T cell epitope which was recognized by all except one patient, was identified within the core sequence 50-69. Two further important T cell recognition sites were represented by the amino acid sequences 1-20 and 117-131, which were stimulatory for the T cells of 69% and 73% of the patients, respectively. T cell recognition of the synthetic peptides was HLA class II restricted because the peptide-induced T cell proliferation was inhibited by anti-HLA class II but not by anti-HLA class I monoclonal antibodies. These findings may be relevant to the development of future preventive and therapeutic strategies against HBV infection.


HCV and blood transfusion.

HCV and blood transfusion. Posttransfusion hepatitis remains a threat to transfusion therapy. Testing for increased ALT levels has been used in an attempt to reduce this risk. Presence of the infectious agent, hepatitis C virus (HCV), appears to be a much more sensitive criterion. Stored serum samples from transfusion blood as well as recipients of transfusion were tested by ELISA, RIBA and PCR for the presence of HCV. The results show that RIBA and PCR are about equally sensitive and are able to detect HCV positivity in many sera that might have been otherwise transfused. Routine screening for the presence of virus will dramatically reduce the danger of hepatitis infection to transfusion patients.


Hepatitis in children with thalassemia major. Since thalassemia major patients are transfusion dependent, they are at a particularly high risk of contracting post-transfusion hepatitis. In this study, 36 transfusion-dependent children were followed up for evidence of viral hepatitis. Of 23 with increased ALT levels, 17 were anti-CMV and 12 were anti-HCV positive, 9 were positive for both CMV and HCV. Of 13 children with normal transaminase levels, 5 were CMV positive and 3 were HCV positive. These results show that CMV may be a very common cause of non-A, non-B hepatitis in transfusion dependent thalassemic children.


Prevalence of antibodies to hepatitis C virus in patients with hepatocellular carcinoma in Austria. In 12 of 54 (22%) patients with histologically verified hepatocellular carcinoma, antibodies to hepatitis C virus were found. In patients with hepatocellular carcinoma the frequency of anti-hepatitis C virus positivity was similar whether cirrhosis (6 of 22 patients (27%)) was present or not (2 of 15 (13%)). Out of 54, 23 patients (43%) were negative both for hepatitis B or C markers. Out of 53, 22 (42%) had positive hepatitis B markers, 8 of 22 were HBsAg positive. Patients with hepatocellular carcinoma and cirrhosis had a higher percentage of hepatitis B virus markers than patients with cirrhosis without hepatocellular carcinoma. Our of 70 patients with cirrhosis but without hepatocellular carcinoma, 24 (34%) had antibodies to hepatitis C virus. Our data of similar frequencies of antibodies to hepatitis C virus in patients with hepatocellular carcinoma or with liver cirrhosis but without hepatocellular carcinoma indicate that at least in Austrian patients, hepatitis C virus infections are not an important factor for development of hepatocellular carcinoma.


Serotype A and B strains of bean common mosaic virus are two distinct potyviruses. The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.


Potyviruses, monoclonal antibodies, and antigenic sites. Virus-specific and cross-reactive monoclonal antibodies have been produced to at least 19 different aphid-transmitted potyviruses. This report summarizes the development of these monoclonal antibodies as well as presents information on the delineation of the virus-specific and group-common epitopes defined by these monoclonal antibodies. Virus-specific and group-common antigenic determinants were mapped by a variety of techniques, including analysis of antigen: antibody reactivity patterns, determination of N-terminal vs. trypsin-resistant core peptide-specificity, immunoanalysis of overlapping synthetic peptides, and immunoanalysis of bacterially expressed coat-protein gene products. Of those monoclonal antibodies that have been examined, monoclonal antibody-defined virus-specific epitopes are virion surface-located within the 30+ amino acid amino terminus, whereas the group-common epitopes are found in the trypsin-resistant core protein not usually located on the virion surface, as has been shown previously with certain polyclonal antibodies. New information is presented on the analysis of bean yellow mosaic virus amino terminal epitopes as well as on the identification of amino terminal antigenic determinants shared between strains of bean yellow mosaic virus and pepper mottle virus. A recommendation on the evaluation and use of a panel of potyvirus broad-spectrum reacting monoclonals as reference monoclonal antibodies for the detection and classification of aphid-transmitted potyviruses is also presented.


The immune response to and expression of cross-reactive retroviral gag sequences in autoimmune disease. To examine the immune response to retroviral gag sequences in autoimmune disease, we measured antibody levels to synthetic peptides representing the major epitopes on HTLV-1 p19 gag and a homologous sequence on the endogenous retrovirus, HRES-1, in sera from 121 patients with autoimmune disease and 52 healthy controls. In the absence of HTLV-1 antibodies, using a conventional diagnostic assay, significantly elevated levels of antibodies to the HTLV-1 peptide were found in 23% of multiple sclerosis and 20% of anti-Sm antibody positive systemic lupus erythematosus patients. Elevated antibody levels to HRES-1 were found in 32% of Sjögren's syndrome and 19% of multiple sclerosis patients. Evidence of reactivity with both HTLV-1 and HRES-1 was found in human sera and cross-reactivity demonstrated with affinity purified rabbit anti-peptide antibodies. Expression of HRES-1, detected by antibodies and Northern blots, was found in lymphoblastoid cells, salivary gland biopsy sections and salivary gland epithelial cells in culture. This study confirms previous reports of low levels of anti-retroviral gag antibodies in autoimmune disease. The cross-reactions support the concept that reports of elevated HTLV-1 antibodies may be due to an endogenous agent such as HRES-1. The expression of HRES-1 salivary gland may explain its antigenicity in a small proportion of Sjögren's syndrome patients as well as suggesting mechanisms whereby it may contribute to the chronic inflammation of autoimmune disease.


Effect of substance P on detrusor muscle in rats with diabetic cystopathy. Diabetic cystopathy (DCP) is a well known complication in diabetes mellitus (DM). In the present experimental study, DM was induced in rats by streptozotocin and DCP was confirmed on cystometry. In vitro studies on detrusor strips from diabetic rats showed an increased contractile response to substance P (SP) compared with controls, indicating denervation supersensitivity. A decreased response to capsaicin in diabetic detrusor strips indicated a decreased neuronal content of SP or a diminished number of SP-containing sensory nerves. This suggests that DM induces alterations in nerves containing SP which may be at least partly responsible for sensory loss and the development of DCP.


Cholinergic, monoaminergic and peptidergic innervation of the primary visual centers in the brain of the lizards Gekko gecko and Gallotia galloti. In order to study the relationship between retinal projections and immunohistochemically identified neurotransmitter systems in the primary visual centers of the brain in lizards, intraocular injections of horseradish peroxidase were combined with immunohistochemistry. Antibodies raised against six substances were applied: choline acetyltransferase (ChAT), serotonin (5-HT), tyrosine hydroxylase (TH), dopamine (DA), substance P (SP), and leu-enkephalin (LENK). In the primary visual centers of the lizards Gekko gecko and Gallotia galloti, notable overlap was observed between retinofugal fibers with: 1) ChAT-immunoreactive fibers in almost all primary visual centers; 2) 5-HT-immunoreactive fibers in the ventral lateral geniculate body and the basal optic nucleus; 3) TH-immunoreactive fibers in the nucleus ovalis and the dorsal lateral geniculate body; 4) SP- and LENK-immunoreactive fibers in the perirotundal belt; and 5) TH- and SP-immunoreactive fibers in the pretectal posterodorsal nucleus. The latter nucleus also contains dopaminergic cell bodies that lie outside the retinal target area but have dendrites extending into it. Several differences were noted in the distribution of 5-HT, TH-, DA-, and LENK-immunoreactive fibers in the tectum of the midbrain in the two species studied. Distinct laminae of 5-HT-immunoreactive fibers (layer 9) and TH- and DA-immunoreactive fibers (layers 9 and 11) are present in G. gecko but absent or, at least, less distinct in G. galloti. On the contrary, the optic layers in the tectum of G. galloti show a rather dense plexus of LENK immunoreactive fibers, whereas the corresponding layers in G. gecko are devoid of LENK-immunoreactivity. Since only a very few ChAT immunoreactive fibers were observed in the optic nerve of G. galloti, most of the observed immunoreactive fibers in the primary visual centers are considered to have an extraretinal origin. Putative sources of the cholinergic, the monoaminergic, and the peptidergic innervation of the primary visual centers in reptiles include the isthmic nucleus, the raphe nuclei, the substantia nigra and the nucleus of the posterior commissure, as reported in other amniotes.


The formation of a 'pseudo-nerve' in silicone chambers in the absence of regenerating axons. The formation of a regenerate between sciatic nerve segments or stumps inserted into Y-tunnelled silicone chambers was studied under conditions where regenerating axons were prevented from entering the chamber. This was accomplished by using an isolated segment of the nerve as a proximal insert. After one week, a cellular regenerate spanned the proximal and distal inserts. The size of the regenerate increased if circulation was preserved in the distal inserts. At four weeks, a perineurium-like sheath surrounded the regenerate and longitudinally oriented Schwann cell columns could be observed throughout the regenerate. A similar 'pseudo-nerve' formed towards a piece of distally inserted tendon. Thus, the information required for the formation of a nerve-like structure is inherent to the non-neuronal cells entering the chamber. Schwann cells, in contrast to regenerating axons, do not exhibit preferential growth towards nervous tissue.


The C-terminal fragment of substance P enhances dopamine release in nucleus accumbens but not in neostriatum in freely moving rats. The in vivo microdialysis technique was used to study the effects of carboxyl or amino terminal sequences of substance P on the extracellular concentrations of dopamine, its metabolites dihydroxyphenylacetic acid and homovanillic acid, as well as on 5-hydroxyindoleacetic acid, in neostriatum and nucleus accumbens of freely moving rats. The i.p. administration of 37 nmol/kg of the substance P C-terminal heptapeptide analog [pGlu5, MePhe8, Sar9]SP5-11 (DiMe-C7) caused an increase in extracellular dopamine concentrations in nucleus accumbens but not in neostriatum. The administration of the equimolar dose of the heptapeptide N-terminal fragment substance P 1-7 (SP1-7) did not have an effect in either structure. No changes were observed in the extracellular concentrations of the metabolites after the administration of either substance. These results are discussed with respect to the reinforcing effects of substance P and its C-terminal sequence, which may be mediated via dopamine release in the nucleus accumbens.


Serial reconstruction of beta-protein amyloid plaques: relationship to microvessels and size distribution. The suggestion that the amyloid plaques in Alzheimer disease are formed by abnormal leakage from microvessels is mainly based on the finding that many plaques are topographically associated with microvessels. However, because the microvessel network is dense and amyloid plaques are numerous, the frequently observed association may result from chance contact, especially for larger plaques. Therefore, we determined the frequency of this association as a variable of plaque size. If all the amyloid plaques are associated with microvessels, a constant and high rate of association would be expected for all plaque sizes. On the other hand, if the association is a chance contact, larger plaques would show more frequent contact than smaller ones. Sections were double-immunostained for amyloid plaques and microvessels with antibodies raised against beta-protein and collagen type IV, respectively. Amyloid plaques were reconstructed using 12 serial sections (7 microns thick) from the entorhinal cortex of two Alzheimer patients. With reconstruction we determined the size distribution of amyloid plaques as well as the influence of size on vascular association. All the amyloid plaques larger than 42 microns were associated with microvessels, however, the smaller the amyloid plaques, the less frequently they were associated with microvessels. Interestingly, although diffuse amyloid plaques occur in all size classes, core-containing amyloid plaques have a more discrete size. We conclude that the topographical relationship between amyloid deposition and capillaries does not support the leakage theory for amyloid plaque formation.


Three types of sodium channels in adult rat dorsal root ganglion neurons. Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.


Release of neuropeptide FF (FLFQPQRF-NH2) from rat spinal cord. Neuropeptide FF (FLFQPQRF-NH2), originally isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. Neuropeptide FF (NPFF) is highly localized in the dorsal spinal cords where there are also specific NPFF binding sites. Furthermore, there have been studies indicating that NPFF may participate in the regulation of pain threshold in the spinal cord. However, whether NPFF can be released from the spinal cord is not known. The present experiments, using an in vitro superfusion of an isolated whole rat spinal cord, demonstrated that high concentrations of KCl or substance P caused a release of NPFF immunoreactive material (IR) from the spinal cord into the perfusion medium in a calcium-dependent manner. Substance P (1-11) also produced a detectable release of NPFF-IR in vivo although the response was quite variable. The released NPFF-IR was analyzed by an HPLC study and found to consist of NPFF and other minor immunoreactive peptides. Further studies with substance P-related peptides showed that the in vitro release of NPFF-IR could also be induced by substance P (1-7) but not by [pGlu5,Me-Phe8,Sar9]-substance P (5-11) or substance K. These results suggest that the specific substance P receptor (SP-N), which is recognized by both substance P (1-11) and substance P (1-7) rather than the tachykinin receptor, is involved in NPFF secretion from the spinal cord. In view of the role of substance P (1-11) and substance P (1-7) in sensory transmission, the results of this study further support the role of NPFF in the modulation of antinociception in the spinal cord.


Kindling causes changes in the composition of the astrocytic cytoskeleton. Changes in the astrocytic cytoskeleton were examined in amygdala kindled rats using immunocytochemical techniques. One week following kindling, there was a dramatic increase in immunoreactivity to glial fibrillary acidic protein and vimentin in astrocytes throughout amygdala, pyriform cortex and hippocampus bilaterally. Since these changes occurred in anatomical sites involved in the propagation of kindled seizures, the observed cytoskeletal reorganization in astrocytes may signify important functional alterations in the kindled brain.


Expression of insulin-like growth factor I by astrocytes in response to injury. Astrocytes are known to express several growth factors in response to injury and neurological disease. Insulin-like growth factor I (IGF-I) induces astrocytes to divide in vitro and is expressed by developing, but not adult astrocytes both in vivo and in vitro. We tested whether IGF-I is re-expressed by reactive astrocytes in response to injury. We found that astrocytes surrounding the lesioned parenchyma after introduction of a cannula through the cerebral cortex, hippocampus and midbrain contain high levels of immunoreactive IGF-I, as determined by immunocytochemistry using a highly sensitive and specific anti-IGF-I monoclonal antibody. Interestingly, the contralateral hippocampus also contained IGF-I positive astrocytes although in substantial lower numbers. Intact animals showed no detectable IGF-I immunoreactivity in astrocytes. IGF-I was detected at the first time point tested after the lesion was made, 1 week, and for at least 1 month thereafter. Reactive astrocytes expressing high levels of glial fibrillary acidic protein were found in a much wider distribution all along the lesioned area and beyond. We conclude that mechanical injury of the brain induces a specific pattern of expression of IGF-I by a subpopulation of astrocytes. These findings suggest that IGF-I is participating in the response of astrocytes to injury.


Localization of amyloid precursor protein in selective postsynaptic densities of rat cortical neurons. One of the hallmarks of Alzheimer pathology is extracellular deposition of beta-amyloid protein (BAP) which is derived from a larger glycoprotein called amyloid precursor protein (APP). Although APP has often been described as a surface membrane protein, such a localization has not previously been demonstrated at the light or electron microscopic level. We now report the results of immunoelectron microscopy using three specific antibodies against different synthetic fragments of APP. All three antibodies demonstrated a major localization to organelles such as the Golgi apparatus, endoplasmic reticulum and vesicular-like structures. A minor proportion of staining with all three was on selective postsynaptic membranes of asymmetrical synapses, whereas staining of presynaptic membranes was not observed. The morphological evidence suggests that one role of APP may be in association with the function of selective synapses.


Lindane-induced convulsions in NMRI and OF1 mice: antagonism with (+)MK-801 and voltage-dependent calcium channel blockers. The convulsant profile of lindane was investigated in OF1 and NMRI mice lines in relation to other convulsants acting at the GABAA and NMDA receptor complexes. Thus, a specific GABA-gated chloride channel blocker, PTX, a GABAA receptor antagonist, PTZ, and an excitatory amino acid receptor agonist, NMDA, were used. Antagonism of the convulsant effects of each of these drugs was investigated with (+)MK-801, a blocker of the NMDA-operated cation channel, and with nifedipine, a voltage-dependent calcium channel antagonist. While no differences in potency for PTX or PTZ to induce seizures were observed between OF1 and NMRI mice, lindane was approximately 80 and 90% more potent in its ability to induce seizures and lethality, respectively, in OF1 than in NMRI mice. Brain lindane concentrations at the moment of convulsion, measured after ED100 doses of lindane (400 and 200 mg/kg for NMRI and OF1 mice, respectively), did not differ between OF1 and NMRI mice, suggesting that the different potency of lindane between these mouse lines is a consequence of pharmacokinetic factors. Furthermore, (+)MK-801 antagonized seizures induced by either lindane, PTX or PTZ with similar potencies in both mouse lines. These results, coupled with the different pharmacokinetics of lindane in OF1 and NMRI mice, suggest that the distinct effects of lindane in these mice are not mediated by different activities at either NMDA or GABAA receptor complexes. Nonetheless, nifedipine antagonized lindane-induced seizures with a three-fold higher potency in NMRI than in OF1 mice. In contrast, nifedipine failed to antagonize PTX and PTZ convulsions in both OF1 and NMRI mice. These results suggest that besides the GABAA receptor complex other mechanisms related to calcium mobilization may be involved in the convulsant action of lindane.


Senile plaques in cerebral amyloid angiopathy show accumulation of amyloid precursor protein without cytoskeletal abnormalities. The abnormal neurites that surround beta-amyloid in senile plaques (SP) in Alzheimer disease contain beta-amyloid precursor protein (beta APP) or abnormal filaments which react with antibodies to tau. Occasionally, beta APP and abnormal filaments are present in the same neurite. Whether both types of abnormal neurites are reactive to the presence of beta-amyloid or they are instead independent from each other is unknown. To begin to clarify this issue, we comparatively studied beta APP and tau-epitopes in SP from cases of classical Alzheimer disease and cases of cerebral amyloid angiopathy, with SP but without neurofibrillary pathology. In subjects with cerebral amyloid angiopathy, about one-third of SP, the same percentage as in Alzheimer disease, were beta APP reactive in the absence of tau-reactivity. beta APP epitopes were ultrastructurally localized in dense bodies of probable lysosomal origin, adjacent to the core of SP. These results demonstrate that beta APP and tau-reactive cytoskeletal alterations occur independently in the neurites of SP. The presence of beta APP in dystrophic neurites of SP and the localization of beta APP in lysosomes suggest that beta APP containing dystrophic neurites may play a role in the extracellular deposition of amyloid.


Preferential activation of [3H]phorbol-12,13-dibutyrate binding by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) in neonatal striatal cell cultures. Activation of excitatory amino acid receptors increased [3H]phorbol-12,13-dibutyrate ([3H]PdBu) binding in four week cultures of striatal cells from postnatal day 7 rat pups (PN7), and in PN7 cells co-cultured the fourth week with striatal cells from postnatal day 1 rat pups. Kainate (KA), trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), and N-methyl-D-aspartate (NMDA) increased [3H]PdBu binding equally in both types of cultures, but alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) increased binding by 3-fold in the co-cultures. Thus, [3H]PdBu binding in these two types of striatal cultures offers a simple model system for studying the regulation of AMPA/KA receptor responses.


A selective and extremely potent antagonist of the neurokinin-1 receptor. Sendide [Tyr6,D-Phe7,D-His9]-substance P(6-11) has been examined by measurements of ligand binding to crude membrane fractions and by functional tests on the spinally mediated behavioral response. Sendide potently displaced [3H]-labeled substance P (SP) binding to mouse spinal cord membranes in a competitive manner. In vivo, sendide, intrathecally co-injected with SP, competitively antagonized SP-induced scratching, biting and licking. The behaviors elicited by physalaemin, septide and [Sar9, Met(O2)11]-SP were also reduced by co-administration of sendide. Large doses of sendide were needed to reduce the action of neurokinin A, D-septide, neurokinin B and eledoisin. The in vitro and in vivo pharmacological profile of sendide demonstrated that it is a selective and extremely potent antagonist of the neurokinin-1 receptor.


Ultrastructural examination of enkephalin and substance P input to cholinergic neurons within the rat neostriatum. Enkephalin and substance P-containing inputs to cholinergic perikarya were examined in the rat neostriatum using an ultrastructural immunocytochemical double-labeling protocol. Sections of rat neostriatum were double-labeled for either choline acetyltransferase (ChAT) and substance P or ChAT and enkephalin using silver intensified colloidal gold and peroxidase as labels. Regions containing both ChAT-positive neurons and peroxidase reaction product were identified in the light microscope prior to sectioning for electron microscopy. Substance P-containing terminals which contained round synaptic vesicles and made symmetrical synaptic contacts were commonly observed in the neostriatum. Substance P synapses onto ChAT-positive perikarya and dendrites were frequently observed: up to 5 synaptic contacts were observed onto a ChAT-positive dendrite. Enkephalin labeling was also seen in a population of axon terminals containing round synaptic vesicles and exhibiting symmetrical synaptic specializations. In contrast to substance P-containing terminals, relatively few synaptic contacts were observed onto ChAT-positive labeled perikarya and dendrites although enkephalin-labeled terminals were seen in frequent contact with perikarya and dendrites of unlabeled spiny neurons. Since enkephalin and substance P are contained within different populations of striatal spiny neurons, the results of the present study suggest that these two types of neurons differ in their intrinsic striatal connections.


A new co-culture method utilizing a dual compartment cell-allocation apparatus for observation and identification of synapse formation in vitro. A new co-culture method for observation and identification of synapse formation utilizing dissociated retinal and tectal neurons from 7-day-old chick embryos was developed using a glass apparatus which we named 'Dual Compartment Cell-Allocation Apparatus.' This apparatus enabled us to culture these two types of cells by forming two parallel lanes, each with a dimension of 200 microns by 15 mm and with an interval of 200 microns separating the two lanes, on a collagen-coated cover slip. Infiltration and/or migration of retinal cells into the tectal cell lane was minimal with only 0.15% identified and confirmed with the fluorescence-dye labeling method. Growth of neurites connecting the two lanes was observed within 48 h of culture in a serum-free medium. Double-immunostaining of the culture preparations with anti-neurofilament and anti-synaptophysin antibodies revealed the presence of synaptophysin-hot spots on neurofilament-positive processes connecting the retinal neurons with the tectal neurons, thus confirming the formation of synapses between the neurons of the retinal lane and the neurons of the tectal lane. The dual compartment cell-allocation apparatus has proven itself to be advantageous and useful for studying the formation of synapses between two types of neurons from different origin.


Dextromethorphan antagonizes the acute depletion of brain serotonin by p-chloroamphetamine and H75/12 in rats. A role for calcium in p-chloroamphetamine-induced neurotoxicity has been inferred previously from protective effects of dextromethorphan. We found that dextromethorphan reduces rat brain concentrations of 5-hydroxyindoleacetic acid and blocks the acute, non-neurotoxic depletion of brain serotonin by p-chloroamphetamine and by H75/12. Inhibition of the membrane transporter on brain serotonin neurons by dextromethorphan in vivo might explain its protective effect against p-chloroamphetamine neurotoxicity.


Organization of layers II-III connections in human visual cortex revealed by in vitro injections of biocytin. In the search for cortical mechanisms subserving psychological phenomena, a better understanding of human cortical circuitry is crucial. In this report we describe aspects of intrinsic connectivity of supragranular layers in human visual cortex, revealed by extracellular injections of the anterograde tracer biocytin in vitro. Human cortical slices were obtained from visual association cortex in the posterior-medial portion of the dorsal bank of the occipital lobe, removed during neurosurgical tumor ablations. Small iontophoretic injections of biocytin into layers II-III revealed intense Golgi-like staining of axonal projections emanating from the injection sites. Vertically descending axons are grouped in bundles 20 microns in diameter which are spaced 15 microns apart. Some of these axons enter the white matter and send long oblique and horizontal collaterals. The main horizontal spread of the axons could be observed in layers II-III and V. The bulk of projections extends to a distance of 1.5 mm in layers II-III and 1.1 mm in layer V. Few individual axons could be observed at greater distances. In contrast, layer IV is almost devoid of horizontal connections, forming a clear gap between supra- and infragranular layers. Axon collaterals in the infragranular layers project mostly in a descending oblique direction with long horizontal collaterals in lower layer VI.


Failure of anticytokeratin 18 antibody to improve flow cytometric detection of bladder cancer. Bladder washing specimens containing inflammatory or squamous cells have been difficult to accurately analyze with single-parameter DNA flow cytometric (FCM) methods. The anticytokeratin 18 antibody, CK5, was used in a multiparameter assay of 275 bladder washing and voided urine specimens to immunoselect only the bladder transitional cells for DNA analysis. Flow cytometric detection of transitional cell carcinoma was increased by immunoselection of CK5-positive cells in specimens from patients with disease. Unfortunately, a similar increase in hyperdiploid cells in pathologically benign specimens was observed, which resulted in a false-positive rate of 45%. In some instances, multiparameter FCM assays with CK5 could detect aneuploid cell populations not clearly evident by single-parameter analysis. However, the results from this study of the hyperdiploid cell fraction showed that the increased sensitivity resulting from the use of CK5 was not clinically useful because of the decrease in specificity.


Changes in ventricular 1,2-diacylglycerol content in rats following monocrotaline treatment. 1,2-Diacylglycerol may initiate cardiac hypertrophy, probably by activating protein kinase C. To test this hypothesis we determined the 1,2-diacylglycerol content of hypertrophied tissue. Rats were treated with monocrotaline and developed severe right ventricular hypertrophy followed by congestive heart failure. 1,2-Diacylglycerol content and fatty acid composition, DNA concentrations, and RNA concentrations in the right ventricle from monocrotaline treated rats were compared with values obtained from the left side or from control rats. During the first week, the right ventricle showed no significant change in 1,2-diacylglycerol content and a small increase in RNA concentration. However, the 1,2-diacylglycerol content was significantly increased by 55% at two weeks after monocrotaline injection, when DNA and RNA synthesis was also enhanced to its highest level when compared with control rats (37% and 18%, respectively). At four weeks after monocrotaline injection, conversely, the 1,2-diacylglycerol content was decreased by 25% in the right ventricle from monocrotaline treated rats, most of which had pleural and peritoneal effusions indicating congestive heart failure, although RNA synthesis was sustained at a high level. The fatty acid composition of 1,2-diacylglycerol did not differ significantly between the right and left ventricles or control rat ventricles. These results suggest that 1,2-diacylglycerol accumulation is associated with development of hypertrophy in monocrotaline treated rats. In contrast, at a stage of congestive heart failure 1,2-diacylglycerol production decreased, suggesting that intracellular transduction mechanisms may be attenuated.


Prevention of experimental autoimmune encephalomyelitis in Lewis rats by treatment with an anti-rat CD5 antibody (OX19). Treatment of Lewis rats with a single dose of OX19 antibody, specific for rat CD5, uniformly prevented the development of experimental autoimmune encephalomyelitis (EAE). This protective effect had several notable characteristics: (1) it persisted for at least 10 days; (2) it could be achieved with either high doses of the antibody (> 200 micrograms) or lower doses (100-200 micrograms), which did not deplete T cell populations; and (3) the treated animals were able to mount comparable T cell responses to both myelin basic protein and myelin-unrelated antigens. In addition, antibody treatment consistently prevented the development of adoptively transferred EAE, suggesting that enhanced suppressor cell activity may have contributed to the protection. Antibodies such as OX19 appear capable of blocking the development of EAE, and perhaps other autoimmune diseases as well.


Effect of interferon-gamma on myelin basic protein-specific T cell line proliferation in response to antigen-pulsed accessory cells. We have shown previously that treatment of SJL/J mice with anti-interferon-gamma monoclonal antibody (mAb) exacerbated experimental allergic encephalomyelitis (EAE) only if administered at the time of encephalitogenic challenge. Here we investigate the role of interferon-gamma (IFN-gamma) and anti-IFN-gamma mAb in the early events of T cell activation in vitro. Pretreatment of murine peritoneal exudate cells (PEC) with IFN-gamma led to a significant increase in their ability to activate myelin basic protein (MBP)-specific, short-term T cell lines. When exogenous IFN-gamma was added to cocultures of T cells and MBP-pulsed PEC, the antigen-specific T cell proliferation was considerably reduced. Anti-IFN-gamma mAb added to these cultures neutralized the inhibitory effect of the exogenous IFN-gamma on T cell proliferation but had no visible effect on class II MHC expression by the antigen-pulsed PEC present in the same cultures. A reduction in T cell proliferation was also observed when the T cells were treated with IFN-gamma prior to coculture with the MBP-pulsed PEC. These results demonstrate that, on one hand, IFN-gamma enhances the ability of PEC to induce antigen-specific T cell proliferation but, on the other hand, acts on the T cells themselves by inhibiting their proliferation in response to the antigen-pulsed PEC. This may explain why treatment with anti-IFN-gamma antibody in vivo induces EAE exacerbation.


Anaesthetic implications of 32% Dextran-70 (Hyskon) during hysteroscopy: hysteroscopy syndrome. This review describes the properties and side effects of Hyskon and the implications for the patient and anaesthetist during hysteroscopy. The amount of Hyskon absorbed is dependent on the injection pressure, the extent of tissue trauma, the seal of the hysteroscope around the cervix, and the duration of infusion. The mechanism of pulmonary oedema after absorbtion of Hyskon is fluid overload, and not injury to pulmonary capillary endothelium. The haematological effects are primarily due to haemodilution. However, case reports suggest that Dextran 70 may cause a syndrome resembling disseminated intravascular coagulation. The allergic response to Hyskon consists of both an anaphylactic and an anaphylactoid component. It is recommended that hysteroscopy with Hyskon be limited to 45 min, and that all possible measures be taken to minimize tissue trauma and bleeding. The volume of Hyskon should be limited to less than 500 ml, since pulmonary oedema and coagulopathy have been described with even lesser amounts. The cumulative volume of Hyskon should be monitored frequently and the patient should be closely monitored for signs of impending pulmonary oedema.