The role of transbronchial lung biopsy in the treatment of lung transplant recipients. An analysis of 200 consecutive procedures. The purposes of this study were as follows: (1) to establish the positivity rate and complication rate of transbronchial lung biopsies in the treatment of lung transplant recipients; (2) to determine the sensitivity of transbronchial lung biopsy specimens for the diagnosis of clinically suspected acute rejection and cytomegalovirus pneumonia; and (3) to examine the results of surveillance transbronchial lung biopsies in clinically and physiologically stable recipients. Retrospective review and analysis of 203 consecutive procedures. Washington University Lung Transplantation Program, Washington University School of Medicine and Barnes Hospital, St. Louis, Mo. Fifty-five lung transplant recipients. Biopsies were done with 2-mm fenestrated forceps using fluoroscopic guidance. Two hundred three bronchoscopies with transbronchial lung biopsy were performed for clinical indications (n = 88), routine surveillance (n = 90), or follow-up of a previous biopsy (n = 25). Biopsy specimens showing acute allograft rejection were classified according to the scheme recommended by the Lung Rejection Study Group. The positivity rate and complication rate were determined for the procedures. In procedures performed for clinical indications, the sensitivity for the diagnosis of acute rejection and cytomegalovirus pneumonia was calculated by a decision-to-treat analysis. A specific histologic diagnosis was detected in 69 percent of the clinical procedures, 57 percent of the surveillance procedures, and 64 percent of the follow-up procedures. For clinical indications, the sensitivity of transbronchial lung biopsy was 72 percent for the diagnosis of acute rejection and 91 percent for the diagnosis of cytomegalovirus pneumonia. Surveillance biopsy specimens often showed clinically inapparent rejection or cytomegalovirus pneumonia. The overall complication rate was 8.9 percent; none of the complications were life threatening. Transbronchial lung biopsy is a useful and safe procedure in the treatment of lung transplant recipients. When performed for clinical indications, the procedure proved to be sensitive for the diagnosis of acute rejection and cytomegalovirus pneumonia. When performed for surveillance in clinically and physiologically stable recipients, the incidence of rejection and cytomegalovirus pneumonia was unexpectedly high; the potential clinical implications of these findings will require further study.


Pulmonary lymphangitic metastasis from breast cancer. Lymphocytic alveolitis is associated with favorable prognosis. More than 20 percent of patients with breast cancer have pulmonary lymphangitic spread at death. Previous reports have indicated that some patients may respond to aggressive chemotherapy that includes corticosteroids. Bronchoscopy with bronchoalveolar lavage (BAL) was used to assess pulmonary infiltrates in 14 patients with infiltrating ductal breast cancer. All patients had received prior cytotoxic or hormonal therapy. Following a diagnosis of pulmonary metastasis by BAL or biopsy specimen, all patients received prednisone and chemotherapy. BAL confirmed the diagnosis of metastatic tumor in 10 of 14 patients. The conditions of the remaining patients were diagnosed by one or more other bronchoscopy specimens. Patients were divided into two groups based on BAL lymphocyte percentage (10 percent is the upper limit of normal in our laboratory). Seven patients had > 10 percent lymphocytes (high lymphs: 26 +/- 11.6 percent [mean +/- SD]) compared with seven patients with normal lymphocyte percentage (low lymphs: 4 +/- 1.9 percent, p < 0.01). Six of seven patients with low lymphs died within three months of BAL compared with one in seven patients with high lymphs (p < 0.01). Within six months, all patients with low lymphs were dead whereas five of seven patients with high lymphs were alive, (p < 0.01). Comparing the two groups, there was no significant difference in percentage of BAL neutrophils, patient age, duration of disease prior to diagnosis, or arterial-alveolar oxygen gradient. In breast cancer patients with pulmonary lymphangitic metastasis, a lymphocytic alveolitis is associated with a better overall prognosis.


Soluble interleukin 2 receptors in patients with sarcoidosis. Possible origin. We determined levels of soluble interleukin 2 receptors (IL-2R) in patients with sarcoidosis and further examined their origin. Thirty-nine patients with sarcoidosis and 18 healthy control subjects were studied. Soluble IL-2R levels in serum were significantly higher (p < 0.01) in sarcoidosis than in control subjects. In sarcoidosis, levels of soluble IL-2R in serum were significantly higher (p < 0.05) in patients with active disease than those with inactive disease and were significantly (p < 0.01) correlated with serum angiotensin-converting enzyme (ACE) levels. IL-2R expression on monocytes and alveolar macrophages (AMs) was significantly (p < 0.01) increased in patients with sarcoidosis as compared with control subjects. Soluble IL-2R levels in the supernatants of cultured monocytes and AMs were higher in patients with sarcoidosis than in control subjects. Those of cultured T lymphocytes obtained from peripheral blood and bronchoalveolar lavage fluid were detected in some patients with sarcoidosis, while undetected in control subjects. Furthermore, soluble IL-2R in serum was significantly correlated with soluble IL-2R in the supernatants of cultured monocytes and AMs (p < 0.01 and p < 0.05, respectively). These results demonstrate that soluble IL-2R in serum is a useful index of the disease activity of sarcoidosis and is mainly derived from monocytes and AMs.


Pulmonary abnormalities in Klippel-Trenaunay syndrome. A histologic, ultrastructural, and immunocytochemical study. Klippel-Trenaunay (KT) syndrome is a rare, sporadic, congenital vascular disease of unknown etiology. We describe pulmonary findings in an 18-year-old male patient followed up since birth with the KT syndrome. The patient developed pleural and pericardial serous effusions that led to an open lung biopsy. Previous pulmonary findings have been limited to thromboembolic phenomena and pulmonary vein varicosities. On the other hand, reports of lymphatic hyperplasia, aplasia, and hypoplasia in KT have been limited to the extremities. For the first time, we describe lymphatic involvement of the lung in KT. The plexiform hyperplasia of the lymphatic channels with smooth muscle hyperplasia leading to lymphatic obstruction, pleural and pericardial effusions are new findings. The lymphatic nature of the plexiform channels was confirmed by immunohistochemistry. Von Willebrand factor and QD-END/10 monoclonal antibodies either did not react or reacted poorly with lymphatic endothelium, features used to distinguish lymphatic and venous endothelium. Ultrastructurally, the absence of basement membrane continuity further substantiated the lymphatic nature of the channels. From our findings, the lymphatic abnormality in the syndrome appears to be more generalized than previously thought. This entity should be distinguished from lymphangioleiomyomatosis to which it bears a superficial morphologic appearance.


Duck hepatitis B virus infection and duck hepatocellular carcinoma. To study the relationship between duck hepatitis B virus (DHBV) infection and duck hepatocellular carcinoma (DHCC), histological examination and DHBV DNA hybridization were performed in 875 ducks from three flocks in Qidong County. Among them, 34 suffered from hepatoma, including 23 hepatocellular carcinoma, 8 cholangiocarcinoma and 3 hepatocellular-cholangiocarcinoma. Of the 34 ducks with hepatoma 27 were positive for DHBV DNA in the liver and/or serum. DHBV DNA was demonstrated in neoplastic nodules of 22 ducks. Southern blot analysis showed that 13 cases were of the integrated pattern of DHBV DNA in neoplastic nodules. The paratumor tissues of 14 ducks with massive tumor were analysed at the same time. Five cases showed integrated pattern, 4 cases free pattern and the other 4 cases both integration and free pattern of DHBV DNA. The hybridization pattern of DHBV DNA in tumor nodule was different from that in paratumor regions in 11 cases and identical in 3 cases. DHBV antigen was positive in 13 tumor nodules and 21 paratumor tissues in the 34 ducks with hepatic tumor by both victoria blue and orcein stain methods. Advanced liver diseases were found in 30 out of the 34 ducks with hepatoma, including 12 cirrhosis and 18 chronic active hepatitis. In southern blot analysis of 122 DHBV DNA positive Qidong ducks without hepatoma, only free pattern of DHBV was seen, while 44 control ducks from Changchun were negative for DHBV DNA. Neither hepatic tumor nor liver diseases were seen in the control ducks. The results suggest that hepatocellular carcinoma in ducks is similar to that in human HCC. They have a high frequency of viral DNA integrated into the host genome and a liver disease background.


[Research on the relation between intrauterine infection and intrauterine growth retardation]. To investigate the relationship between intrauterine infection and IUGR, the following studies have been carried out: (1) measurement of specific antibodies of Toxoplasma (Toxo) and Cytomegalovirus (CMV) in the umbilical serum samples from 30 cases of IUGR with 26 normal newborn infants as controls; (2) follow-up exam on 14 positive cases in 17-25 months. The results showed that in 30 IUGR cases 14 were found to have Toxo and CMV infection, with an incidence significantly higher than that in the control group. One third of the infected patients were found to have retarded growth afterbirth and 63.63% of them appeared to have an increase in BAEP. This suggested that regular follow-up is indicated in these cases.


[Diagnosis and treatment of hemothorax in malignant trophoblastic tumors]. From 1949 to 1988, 32 cases of hemothorax were seen in our hospital. The incidence rate of hemothorax among the cases of choriocarcinoma and invasive mole in the whole series were 2.6% and 1.4% respectively. The most frequent symptoms were chest pain, cough, dyspnea, and hemoptysis. Before 1965, when 6-MP was the only agent used, 7 of the 16 patients with hemothorax died directly due to severe intrathoracic hemorrhage, from 1966 to 1988, when intravenous infusion of 5-FU and intrathoracic injection of 5-FU were used, only 4 of the 16 cases died, there was no death directly related to hemothorax. About 75% were followed up for more than 10 years, the longest duration of follow up being more than 28 years in 6 cases. Repeated examinations with serum hCG determination and chest film revealed no evidence of recurrence, nor pleural adhesion and thickening or pulmonocardiac diseases.


[Extensive and radical surgery for primary ovarian cancer]. From 1983 to 1990, 34 cases of primary ovarian cancer were treated with extensive and radical surgery. Seventeen cases belonged to stage I, 3 cases belonged to stage II and 14 cases were stage III. They consisted of 20 epithelial, 11 germ cell and 2 sex-cord tumors of ovary. The operative method was as follows: infracolic omentectomy, para-aortic and pelvic lymphadenectomy, dilateral salpingo-oophorectomy, total hysterectomy, removing of metastatic implants nd appendectomy. Post surgery, all patients received repeated combination chemotherapy and six cases received irradiation. Four patients underwent the second look operation. The incidence of metastasis of contralateral ovary, omentum, retroperitoneal nodes, uterus, fallopian tubes and appendix were 38.2%, 32.4%, 20.6%, 17.6%, 5.9% and 2.9% respectively. The 3 year survival rate of stage I and II was 92.9%, stage III was 40.0%. The 5 year survival rate of stage I and II was 90.9%, stage III was 50.0%. There was no serious complication both intra and post operation.


[The diagnostic value of headache in intracranial tumor]. 500 cases of intracranial tumor confirmed surgically and pathologically were studied. The aim of this study was to see the diagnostic value of headache in intracranial tumor. Four problems related to headache were studied in 350 cases along with other informations: Relationship between time of headache and tumor; Relationship between locality of headache and tumor; Relationship between degree of seriousness of headache and tumor; Relationship between headache and accompanying symptoms and signs. It was pointed out that headache is of certain diagnostic value in patients with intracranial tumor.


[Detection of hepatitis B virus DNA in leucocytes of primary hepatocarcinoma using polymerase chain reaction]. The detection of HBV DNA in sera and leucocytes of 30 primary hepatocarcinoma patients whom were negative for HBsAg by reverse passive hemagglutination using polymerase chain reaction (PCR) were reported. The detectable rates of HBV DNA in leucocytes and sera were 86.7% and 70% respectively and the serological indexes, 43.4%. These indicated that HBV DNA may existent in blood of the patients with negative serological indexes. It was common that HBV DNA presented in peripheral leucocytes of primary hepatocarcinoma, indicated that extrahepatic sites of the HBV genome, and perhaps replication of HBV virus, existed. It was of great importance for the role of HBV DNA in lemology and epidemiology, etiology of hepatocarcinoma, diagnosis and treatment of infection as well as standard of cure.


[An experimental study on the mechanism of impairment of cell membrane during hemorrhagic shock in dogs]. Dogs were bled and maintained at an arterial pressure of 6.7 kPa for 3 hours. During hemorrhagic shock, red blood cell and serum were assayed for Na+, K+ and Ca++ ion level. RBC and hepatic tissue were together assayed for adenosine triphosphatase (ATPase) activities, free radicals. Results showed that: (1) at 15 minutes after hemorrhagic shock. ATPase activities in RBC and hepatic tissue was significantly elevated. While ratio of sodium and potassium (Na+/K+) in RBC was showed no significant difference; (2) ATPase activities in RBC and liver were decreased, Na+/K+ in RBC was elevated at first after hemorrhagic shock; (3) the signals of free radicals were increased during hemorrhagic shock. The signals of free radicals and ATPase activities in cell membrane was negative correlated. While signals of free radicals and Na+/K+ in RBC was positive correlated. Based on these findings, it is proposed that: free radicals after hemorrhagic shock is an important factor responsible for decrease of ATPase activities and dysfunction of ions active transport in cell membrane.


Growth factor-stimulated phosphorylation cascades: activation of growth factor-stimulated MAP kinase. Protein phosphorylation is an important mechanism in the response of cells to growth factors by which signals can be conveyed from cell surface receptors to intracellular targets. In addition to stimulation of protein tyrosine phosphorylation, activation of growth factor receptors having protein tyrosine kinase activity leads to dramatic alterations in the levels of protein serine/threonine phosphorylation. Several growth factor-stimulated serine/threonine-specific kinases have been identified as potential mediators of such signalling. MAP (microtubule-associated protein) kinase has emerged as a very interesting member of this group, because it activates a separate kinase, pp90rsk, which is also growth factor-stimulated. MAP kinase itself appears to be regulated by protein phosphorylation, because it can be inactivated by protein phosphatases. We have identified two 60 kDa proteins that promote the phosphorylation and full activation of MAP kinase in a manner paralleling its activation by growth factors in intact cells. These 'MAP kinase activators' are themselves stimulated by growth factors, suggesting that they function as intermediates between the MAP kinase and cell surface receptors in a growth factor-stimulated kinase cascade. Identification of the components of this protein kinase cascade reveals a mechanism by which at least some of the effects of receptor tyrosine kinases can be mediated through serine/threonine phosphorylation.


The synaptic activation of NMDA receptors and Ca2+ signalling in neurons. Long-term potentiation (LTP) in the hippocampus is a model system for understanding the synaptic basis of learning and memory. We have studied the mechanism of induction of LTP using voltage-clamp techniques and confocal imaging of Ca2+ in rat hippocampal slices. In the Schaffer collateral-commissural pathway the neurotransmitter L-glutamate activates two classes of ionotropic receptor, named after the selective ligands AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and NMDA (N-methyl-D-aspartate). During low frequency transmission the excitatory postsynaptic potential (EPSP) is mediated predominantly by AMPA receptors. NMDA receptors play a minor role because their ion channels are substantially blocked by Mg2+, and this block is intensified by GABA-mediated synaptic inhibition. During high frequency transmission the GABA-mediated inhibition is depressed, by mechanisms initiated by GABAB autoreceptors. This allows a greater contribution from the NMDA receptors, through which Ca2+ enters the dendrites of the postsynaptic neurons to initiate a cascade of biochemical processes which ultimately result in enhanced synaptic efficiency.


The inositol 1,4,5-trisphosphate receptor. Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from its intracellular stores. The InsP3 receptor has been purified and its cDNA has been cloned. We have found that the InsP3 receptor is identical to P400 protein, first identified as a protein enriched in cerebellar Purkinje cells. We have generated an L-fibroblast cell transfectant that produces cDNA-derived InsP3 receptors. The protein displays high affinity and specificity for InsP3. InsP3 induces greater Ca2+ release from membrane vesicles from transfected cells than from those from control L-fibroblasts. After incorporation of the purified InsP3 receptor into lipid bilayers InsP3-induced Ca2+ currents were demonstrated. These results suggest that the InsP3 receptor is involved in physiological Ca2+ release. Immunogold labelling using monoclonal antibodies against the receptor showed that it is highly concentrated on the smooth-surfaced endoplasmic reticulum and slightly on the outer nuclear membrane and rough endoplasmic reticulum; no labelling of Golgi apparatus, mitochondria and plasmalemma was seen. Cross-linking experiments showed that the receptor forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region contains the critical sequences for InsP3 binding. We have investigated the heterogeneity of the InsP3 receptor using the polymerase chain reaction and have found novel subtypes of the mouse InsP3 receptor that are expressed in a tissue-specific and developmentally specific manner.


Persistent signalling and changes in presynaptic function in long-term potentiation. Long-term potentiation (LTP) is an example of a persistent change in synaptic function in the mammalian brain, thought to be essential for learning and memory. At the synapse between hippocampal CA3 and CA1 neurons LTP is induced by a Ca2+ influx through glutamate receptors of the NMDA (N-methyl-D-aspartate) type (see Collingridge et al 1992, this volume). How does a rise in [Ca2+]i lead to enhancement of synaptic function? We have tested the popular hypothesis that Ca2+ acts via a Ca(2+)-dependent protein kinase. We found that long-lasting synaptic enhancement was prevented by prior intracellular injection of potent and selective inhibitory peptide blockers of either protein kinase C (PKC) or Ca2+/calmodulin-dependent protein kinase II (CaMKII), such as PKC(19-31) or CaMKII(273-302), but not by control peptides. Evidently, activity of both PKC and CaMKII is somehow necessary for the postsynaptic induction of LTP. To determine if these kinases are also involved in the expression of LTP, we impaled cells with microelectrodes containing protein kinase inhibitors after LTP had already been induced. Strikingly, established LTP was not suppressed by a combination of PKC and CaMKII blocking peptides, or by intracellular postsynaptic H-7. However, established LTP remained sensitive to bath application of H-7. Thus, the persistent signal may be a persistent kinase, but if so, the kinase cannot be accessed within the postsynaptic cell. Evidence for a presynaptic locus of expression comes from our studies of quantal synaptic transmission under whole-cell voltage clamp. We find changes in synaptic variability expected to result from enhanced presynaptic transmitter release, but little or no increase in quantal size. Furthermore, miniature synaptic currents in hippocampal cultures are increased in frequency but not amplitude as a result of a glutamate-driven postsynaptic induction. The combination of postsynaptic induction and presynaptic expression necessitates a retrograde signal from the postsynaptic cell to the presynaptic terminal.


Inositol lipids and phosphates in the proliferation and differentiation of lymphocytes and myeloid cells. It is established that receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate is an essential signalling reaction in the responses of many haemopoietic cells to stimuli: examples include platelet activation, antigen-driven initiation of cell proliferation in mature B and T lymphocytes and histamine release by mast cells, and chemotaxis and oxygen radical generation by neutrophils. However, the roles of inositol lipids and phosphates in the development of haemopoietic and immune cells are less well understood. This paper discusses three such situations: the sequential employment of phosphatidylinositol 4,5-bisphosphate hydrolysis and cyclic AMP accumulation as two signals essential to the action of the B lymphocyte-stimulatory cytokine interleukin 4; the involvement of antigen receptor-triggered inositol lipid hydrolysis in apoptotic elimination of immature anti-self T lymphocytes in the fetal mouse thymus; and the possible role of changes in the levels of abundant inositol polyphosphates in the differentiation of HL-60 promyelocytic cells and of normal human myeloid blast cells.


Regulation of phosphoinositide and phosphatidylcholine phospholipases by G proteins. Two G proteins that regulate phosphoinositide phospholipase C in liver plasma membranes have been purified to homogeneity in both the heterotrimeric and dissociated forms. The heterotrimers contain a 42 kDa or 43 kDa alpha subunit and a 35 kDa beta subunit. The alpha subunits are not ADP-ribosylated by pertussis toxin and are closely related immunologically to members of the recently identified Gq class of G proteins. The specific phosphoinositide phospholipase C isozyme that responds to the G proteins has been determined to the beta 1 isozyme. GTP analogues stimulate phosphatidylcholine hydrolysis in rat liver plasma membranes. The nucleotide specificity and Mg2+ dependency of the response indicate that it is mediated by a G protein. Phosphatidic acid, diacylglycerol, choline and phosphorylcholine are the products, indicating that both phospholipase D and C activities are involved. Activation of phospholipase D is also indicated by the enhanced production of phosphatidyl-ethanol in the presence of ethanol.


[An etiology study on the 1986 epidemic of acute hemorrhagic conjunctivitis in China]. An epidemic of Acute Hemorrhagic Conjunctivitis (AHC) broke out in Shanghai, Henan and Fujian provinces of China in 1986. Twenty four strains of etiologically suspected virus were then isolated but not correctly identified. In 1990 9 out of 24 strains from these three places were re-examined by us in our laboratory, and found feasible to be neutralized by CA 24V antiserum, but not by antiserum to EV 70 as once reported by Henan. It was also found that these viruses (one strain from each of these three places) produced pathogenic changes in suckling mice, showed definite immuno-fluorescence with Mcabs, and were neutralized by McAbs against CA 24V which was isolated from Beijing by our laboratory in 1988. It is evident that all the isolates isolated from Shanghai, Henan and Fujian provinces in 1986 were Coxsackie-virus A 24 variant (CA 24V).


Risk of gastric cancer among Korean familial adenomatous polyposis patients. Report of three cases. Gastric cancer has been recognized as an extracolonic manifestation in patients with familial adenomatous polyposis (FAP). In Korea, gastric cancer is the most common malignant neoplasm. In a recent survey, we collected data from 72 Korean patients with FAP. Among them, three (4.2 percent) were found to have associated gastric cancer. This incidence of gastric cancer in our series is much higher than the previous reports from Japan and other countries. The expected cumulative incidence of gastric cancer among these 72 patients was 0.44, which gives the standardized incidence ratio of 6.9 (95 percent CI, 1.4-20.1). This difference in incidence of gastric cancer was statistically significant (P less than 0.05), which implies that patients with FAP are at significantly higher risk of developing gastric cancer compared with the general population in Korea. These findings confirm an increased risk of gastric cancer in FAP patients, even in a region where gastric cancer is highly prevalent.


HCV RNA and antibody to HCV core protein in Japanese patients with chronic liver disease. We evaluated the prevalence of hepatitis C virus (HCV) RNA and antibody (anti-HCVcore) to the putative HCV core protein in Japanese patients with chronic liver disease. Sera were screened by solid-phase enzyme immunoassay with a recombinant HCV core protein and by the reverse transcription-polymerase chain reaction (RT-PCR) test which directly detects the HCV genome. Anti-HCV core was detected with high titers in 95% (69/73) of chronic non-A, non-B hepatitis, and 94% (65/69) of anti-HCVcore-positive patients had the genome. Anti-HCVcore was also found with lower titers in 24% (10/41) of chronic hepatitis B virus carriers, and three of them had the genome. Only one (3%) of the 35 patients negative for anti-HCVcore tested positive to RT-PCR. These findings indicate the overwhelming prevalence of HCV infection in Japanese patients with chronic non-A, non-B hepatitis and a close relationship between the presence of anti-HCVcore and chronic hepatitis C in this population.


Arteriovenous malformation of the pancreas associated with hepatocellular carcinoma. A case report and review of the literature. A case of pancreatic arteriovenous malformation (AVM) with hepatocellular carcinoma is reported. The patient, a 56-year-old Japanese man, was asymptomatic. The pancreatic lesion was found incidentally during an evaluation for hepatocellular carcinoma. Celiac arteriogram demonstrated tortuous feeding arteries, a racemose intrapancreatic stain, which disappeared before the venous phase, and early portal filling.


[The relationship between carcinoembryonic antigen (CEA) expression and histogenesis of gastric cancer (application of immunohistochemical and mucin histochemical techniques)]. The distribution of CEA and mucin in gastric specimens of 134 cases was examined by immunohistochemical and mucin histochemical techniques. The results showed that in 85.58% of the gastric cancers CEA was positive, including all mucinous adenocarcinomas, signet-ring cell carcinomas and papillary adenocarcinomas. In tubular adenocarcinoma, there was a tendency of increased expression of CEA with the degree of cell differentiation. The positive rate of CEA in intestinal type of gastric cancer was higher than that in gastric type and stem cell type. Intestinal metaplasia with colonic type sulphomucin had a higher positive rate than that without sulphomucin. The positive rate of CEA in cancers secreting sulfomucin was higher than that in cancers without sulfomucin. It suggested that gastric cancers expressing CEA was histogenetically related to colonic type intestinal metaplasia and cancers without CEA expression might be evolved from gastric proper epithelium.


[Observation of the in vivo tumor cell lysis mediated by NK cells]. Natural killer cells/large granular lymphocytes (NK/LGL) separated on discontinuous Percoll gradient from rat spleen cells were injected iv to rats (7 x 10(7) cells/rat) 3 days following iv inoculation of 2 x 10(6) Walker-256 cells. Two and 4 hr after NK/LCL injection, animals were sacrificed and the lungs examined by light and immunoelectron microscopy. The latter was done using colloidal gold-labelled polyclonal antibody against purified rat LGL cytoplasmic granules. At 2 hr following iv NK/LGL, in addition to the scattered individual tumor cells and minute tumor foci, many lymphocytes were seen accumulating in the small pulmonary vessels and capillaries. This was not observed in tumor-inoculated control rats. At 4 hr, many extravasated lymphocytes reached the lung parenchyma, some of which had attached to the tumor cells. Immunoelectromicroscopically, lymphocytes were found in intimate contact with the tumor cells with the cytoplasmic gold particles clustering at the cell contact site. Gold particles could also be seen closely adherent to the plasma membrane of degenerating tumor cells. This is the first in vivo demonstration of the role of cytotoxic granules of NK cells in tumor cell lysis.


[Immunohistochemical investigation of serum proteins in human brain gliomas]. Paraffin sections of forty cases of human brain gliomas were immunohistochemically stained for five serum proteins by PAP techniques. In all gliomas P-Alb and Alb were present within the tumor interstitium. Cer was observed around tumor vessels and in highly anaplastic tumors consisting of polymorphic glioma cells. LDL and FN were almost restricted to blood vessels and necrotic areas. These results indicate that the occurrence and distribution of serum proteins in gliomas depend on the molecular weight of serum proteins, serum concentration and the differentiation of tumors. Serum proteins accumulated in peritumoral brain tissue, in reactive astrocytes and, occasionally, in neurons. Therefore, it can be assumed that in human brain gliomas serum proteins extravasate also mainly across tumorous blood vessels and reach the peritumoral tissue.


[Inhibitory effect of refined Amorphophallus konjac on MNNG-induced lung cancers in mice]. 550 seven-wk-old LACA mice were used in 3 batches for studying the inhibitory effect of refined Amorphophallus konjac (Konjaku powder) on MNNG-induced lung cancers. The mice (within each batch) were randomly allocated into four groups, namely, positive control (MNNG), Amorphophallus konjac (A. K.), complex (MNNG+A. K.), and blank control (C) groups. In MNNG group, MNNG (250 micrograms) was injected intravenously once every five days for seven times in each mouse, the total dosage of MNNG being 1.75 mg. In A. K. group, according to w/w, 8% A. K. was well mixed into 92% common diet for long-term breeding. In the complex group, MNNG was given as that in MNNG group and the mice were kept as those in A. K. group. The mice in MNNG group and in C group were all maintained on common diet. The results showed different degrees of inhibitory and preventive effect of refined A. K. on MNNG-induced lung cancers. Refined A. K. not only exerted effect on the number of induced cancer and precancerous lesions, causing a drop in cancer rate from 70.87% to 19.38% and the mean number of cancer and precancerous lesions in each animal, but also altered the constituent ratio of the kinds of tumors, showing a decrease in malignancy (adenoma with malignant change), absence of adenocarcinoma, and relative increase in benign adenoma. The results of experiments in 3 batches also exhibited good reproducibility as well as absence of adverse reaction to Konjaku powder.


[Combined moving strip whole liver irradiation and traditional Chinese medicine for large liver cancer]. This paper reports the result of large liver cancer treated by moving strip whole liver irradiation from 1980 to 1988. The 5-year survival rate was 30.83% +/- 7.77% and the median survival time was 25.8 months. Analysis of factors affecting prognosis showed: 1. The higher the midplane tissue irradiation dose, the longer the survival (P less than 0.001) and 2. Patients with greater than or equal to 8 less than 13 cm tumor diameter and/or greater than or equal to 50% less than 75% tumor/liver volume ratio had longer survival than those with greater than or equal to 13 cm diameter and/or greater than or equal to 75% tumor/liver volume ratio (P less than 0.001). Traditional Chinese medicine was indispensable as a supplement to this treatment. Both clinical and experimental study suggested that this technique could improve the patient's final outcome.


[Improved cryosurgery for primary liver cancer]. Cryosurgery with liquid nitrogen was employed in treating 70 patients with primary liver cancer (PLC). There were 25 cases with small PLC (less than or equal to 5 cm). The postoperative course was uneventful in all. The 1-5 year survival rates were 59.1% (39/66), 37.5% (24/64), 27.0% (17/63), 17.5% (10/57), 12.5% (7/56), respectively in the whole series. The 1-5 year survival rates were 48.4% (15/31), 16.1% (5/31), 6.5% (2/31), 6.5% (2/31), 3.2% (1/31) for 31 cases treated 1973-1977, and 68.6% (24/35), 57.6% (19/33), 46.9% (15/32), 30.8% (8/26), 24.0% (6/25) for 39 cases treated 1978-1989. It is suggested that hepatic cryosurgery is a promising and safe treatment for non-resectable PLC. The remarkable improvement in survival in the latter time period might be attributed to the increased incidence of small PLC, the combined use of hepatic artery ligation and/or regional chemotherapy by hepatic artery cannulation and multimodality treatment.


[Effect of combined use of chemotherapeutic agents and hematopor--phyrin derivative (HPD) on human gastric cancer cell line in vitro]. The interaction between (HPD) photodynamic therapy and 4 cytotoxic drugs on human gastric poorly differentiated mucinous adenocarcinoma cell line MGc 80-3 was studied. As the cells were blocked by 4 cytotoxic drugs (vincristine, mitomycin, bleomycin A5 and 5-Fluorouracil) in different phases, HPD was located in different sites in these cells. The photodynamic effect was potentiated when the cytotoxic drugs were administered before but not after the application of HPD plus light. The synchronous cells would absorb HPD more than the asynchronous cells.


[Significance and application of anti-malignant hepatoma MAb HAb18 in radioimmunal diagnosis of human hepatocellular carcinoma]. In this study, 125I or 131I labelled intact IgG and its F(ab')2 fragments of an antihuman hepatoma monoclonal antibody, HAb18, was used for in vivo radioimmunoimaging of malignant hepatomas. Clear imaging of the tumor was obtained in 168 hours for intact IgG or in 72 hours for F(ab')2 fragments after iv injection or via selective catheterization of the arteriae hepatica communis. In addition, 99mTc-PHY (500 microCi) was simultaneously injected for static scanning of the liver, with which the tumor appeared as defects. All the 20 patients in this study were operated and the lesions were pathologically examined to verify the imaging results. The tumor: liver isotopic ratios were 2.15 +/- 0.15 and 2.63 +/- 0.21 for intact IgG and F(ab')2 fragments, respectively. In other vital organs, such as heart, brain, spleen, lungs and kidneys, as well as non-hepatoma neoplasms, including 3 cases of liver cavernous hemangioma and 2 cases of gastric cancer metastasized to the liver, no radioisotopic concentration was observed. Both the labelled IgG and F(ab')2 fragments had the same targeting potential, but a better contrast was obtained with F(ab')2 fragments. Furthermore, its clearance rate was faster than intact IgG. The smallest tumor diagnosed with this antibody was 0.5 cm in diameter and the positive rate for imaging primary hepatoma was 86.7% (13/15). The results obtained in this study promisingly indicate that HAb18 antibody may become the first choice for the early radioimmunodetection of human hepatoma.


[Role of colposcopy in the diagnosis of human papillomavirus infection of the uterine cervix]. Among 6706 women screened by cytology, only 9 (0.13%) showed evidence of human papillomavirus infection (HPVI). In 133 women examined by colposcopy for abnormal cytology or/and suspected lesions on the cervix, 41 (30.8%) showed subclinical papillomavirus infection (SPI) while 17.4% and 5.3% showed HPVI by histopathology and cytology, respectively. The conformation rate between colposcopy and pathology was 69.6%. Sixty-nine specimens out of 133 colposcopy guided biopsies were assayed by HPV-DNA dot hybridization with 6B/11, 16, 18 probes to detect the presence of HPV-DNA in the cervical specimens. Thirty-nine (56.5%) gave a positive result. The colposcopic predictive value of positive result for HPVI was 76.7%. The difference between colposcopy (59%) and pathology (20.5%) is statistically significant (P less than 0.01). These results suggest that colposcopy is superior to cytology and histopathology for the detection of SPI in the cervix. In colposcopy HPV-DNA positive women, aceto-while-epithelium was most common (28.2%). As it is difficult to differentiate SPI from cervical intraepithelial neoplasia especially the Grade I lesion by colposcopy, discrimination criteria are proposed together with the chief colposcopic features of SPI.


[Radiotherapy of ectopic ACTH syndrome]. From 1984 to 1990, 6 patients with histologically and hormone assay proved ectopic ACTH syndrome were treated. All these lesions occurred in the chest: 4 thymic carcinoids and the other 2 bronchial carcinoids. The patients all had Cushing's syndrome. Treatment consisted of operation plus radiotherapy. The results showed four completely relieved, one improved and is alive with tumor for 4 years and the other relieved for 2 years and 8 months, then died of local recurrence. The indications, dose and method of radiotherapy are discussed.


[Primary small cell carcinoma of the esophagus--report on 8 patients]. Eight patients with primary small cell carcinoma of the esophagus, a rare tumor, were treated by radiotherapy from June 1986 to July 1989. Five of 6 patients who received a radical dose of 4200-5500 cGy/5-6 wk were living without any recurrence for 40, 38, 27, 25 and 16 months. One died of hepatic metastasis 31 months after diagnosis. Two patients who did not complete radiation therapy also died of hepatic metastasis 3 and 5 months after diagnosis. Incidence, pathologic classification, clinical manifestations and treatment are discussed with a review of literature.


[Combination of surgery, radiotherapy and chemotherapy for rectal cancer--a 423 cases report]. From June 1975 to June 1985, 423 patients with rectal carcinoma were randomized into 4 groups: 1. Surgery alone (S), 2. Pre-operative radiotherapy plus surgery (RS), 3. Surgery plus postoperative chemotherapy (SC), and 4. Preoperative radiotherapy plus surgery followed by postoperative chemotherapy (RSC). The 5-year survival rates of the combination groups: RS-34.4% (55/160); SC-47.5% (19/40); RSC-52.4%-(33/63) were all superior to that of S alone-28.8% (40/160). The difference between RSC and S alone was particularly significant (P less than 0.01). Further studies are warranted.


[Monoclonal antibody A12 against gastric cancer produced in immuno-reconstituted nude mice]. A monoclonal antibody (mAb) A12 against gastric cancer was prepared in immuno-reconstituted nude mice from human ductal adenocarcinoma of stomach, Sy86B. mAb A12 could react with the majority of gastric cancer tissues (24/27-88.9%) but only cross react with a few normal tissues tested. The corresponding antigen of mAb A12 (antigen A12) was expressed at higher levels and usually in more than 50% of the cancer cells. mAb A12 may be a valid preparation in targeting therapy of gastric cancer. Preliminary analysis of antigen A12 showed that it is a oncofetal antigen probably of glycolipid or glycoprotein in nature.


[Morphometric analysis of gastric dysplasia and malignancy]. One hundred and eleven specimens of dysplasia and carcinoma of gastric mucosa were studied morphometrically with automatic IBAS image analysis system. A total of 12 parameters of histologic and cellular changes were measured. The results showed that index of structural atypism, gland area, gland perimeter, gland maximum diameter, nucleus-gland ratio index and nuclear area increased in the order of intestinal metaplasia, mild dysplasia, moderate dysplasia, severe dysplasia and carcinoma. These data indicate that computer-assisted morphometry can offer objective criteria in the differential diagnosis of gastric dysplasia and carcinoma.


[Mucin core peptide expression in malignant and non-malignant colorectal tissues]. Three human mucin cDNAs (Muc-1, Muc-2, Muc-3) have recently been cloned and sequenced. The major portion of each mucin consists of sequences repeated in tandem along the protein. Three mucins are distinct due to differences in tandem repeat length, lack of sequence homology and different chromosomal locations of their genes. Since altered mucin glycosylation occurs in cancer resulting in exposure of core carbohydrate, we postulated that increased exposure or other alteration of core peptide structure may occur in cancerous tissues. Antibodies against Muc-1, Muc-2, Muc-3 tandem repeats were used for immunohistochemical analysis of normal, non-malignant and cancer tissues. The results indicate that in normal tissues, only Muc-2 expressed, while in cancerous tissues all three mucin core peptides were significantly accumulated, All of the three mucin core peptides increasingly expressed in adenoma, dysplasia epithelium and active ulcerative colitis (pre-malignant lesions), but not in the hyperplastic polyps, ischemic colitis and quiescent ulcerative colitis (non-malignant diseases).


[Relationship between serum CA125 level and second-look findings in ovarian cancers]. Serum CA125 levels were monitored in 30 patients with epithelial ovarian cancer prior to second-look laparotomy. All patients had undergone cytoreductive surgery and multiple courses of chemotherapy and with no clinical and radiologic evidence of tumor. All ten patients who had had negative second-look finding gave normal serum CA125 levels (less than or equal to 35 u/ml). Of the 20 patients who were positive in second-look, 11 had normal CA125 levels, with a false negative rate of 55% including 67% (4/6) patients with microscopic evidence of tumor and 50% (7/14) patients with gross residual tumor. All 9 patients with elevated serum CA125 levels were positive in their second-look. 73% (8/11) patients with residual tumor having greatest diameter less than or equal to 2 cm had normal CA125 with a median value of 13 u/ml. 33% (3/9) patients with tumors having the greatest diameter greater than 2 cm had normal CA125 and the CA125 median value was 114 u/ml. These results show that the size of the residual tumor found in second-look was related to the serum CA125 level. As CA125 level within normal limits gave more false negatives, the necessity of second-look can not be judged by serum CA125 assay though elevated CA125 levels do predict the presence of tumor.


[Relation between female genital human papilloma virus (HPV) infection and cancer of uterine cervix in Xiang Yuan County, a high incidence area of cervical carcinoma]. Six thousand seven hundred and ten women were screened in a high incidence area, Xiang Yuan County, Shanxi province to study the relation between cervical cancer and female genital HPV infection (HPVI). Invasive cervical cancer was diagnosed by histopathology in 15 subjects, CIS in 44 and HPVI in 110. Morphologic features of HPVI were found in 23.9% (80/334) of the biopsies from abnormal cervix and lower genital tract. A total of 20.3% lesions had morphologic feature of HPVI associated with CIN, CIS and ICC. One hundred sixty eight biopsies were assayed for the presence of HPV 6B/11, 16, 18 DNA by dot blot hybridization. Altogether, 40.5% biopsies were found to have HPV DNA. The positivities of HPV DNA in cancerous and non-cancerous groups were 62.8% and 32.8%, respectively. The positivity of HPV DNA and the grade of cervical lesions showed a statistically significant correlation (P less than 0.01). Of those with positive HPV DNA HPV 16 was the most frequent type (66.2%). This study further demonstrated that Xiang Yuan is one of the highest risk areas of cervical cancer in China. It is suggested that there is a close association between cervical cancer and HPV infection, especially of type HPV 16, of the female genital tract.


[Possible relationship between acute viral myocarditis and dilated cardiomyopathy]. To explore the possible relationship between acute viral myocarditis and dilated cardiomyopathy (DCM), 35 acute diffuse viral myocarditis (ADVM) patients with cardiac enlargement were studied for 6 years on average. The results showed that: (1) In 22 ADVM patients, the dilated hearts had returned to normal on X-ray films. The other 13 cases still had cardiac enlargement complicated with various degrees of cardiac insufficiency (NYHA II/III) and ECG abnormalities. The manifestation of these 13 patients resembled those seen in the early stage of DCM. (2) Serum neutralizing antibody titres of Coxsackie B virus (CBV) in 35 ADVM patients after 6 years observation on average were significantly higher than those cases with cardiac enlargement induced by other causes (P less than 0.01). High neutralizing antibody titres (greater than or equal to 320) to CBV were more common among the patients with ADVM 65.7% vs 25.7% (P less than 0.05). The results indicate that some cases of ADVM might develop into DCM.


Application of a synthetic analogue of leu-enkephalin (Dalargin) in alcohol withdrawal. The effects of 1 mg of a synthetic analogue of leu-enkephalin (Dalargin) injected intramuscular twice daily for 3 consecutive days were studied in 20 patients with alcohol withdrawal syndrome. The results suggest that the therapeutic effects of Dalargin in alcohol withdrawal syndrome are mainly connected with the changes in the dopamine-, serotonin-, GABA- and opioid-ergic neurotransmitters systems.


Evaluation of outpatient prescribing for substance misusers in Kuwait: the need for a rational approach. The outpatient follow-up care of 140 substance misusers in Kuwait was evaluated. Treatment has been traditionally based on substitution pharmacotherapy with no set objectives or criteria for outcome evaluation. Patients' structural characteristics, mode of referral, clinical diagnoses, medication prescribed and screening urine samples for drugs were examined for meaningful associations between these variables. Benzodiazepines were by far the most prescribed (94%) medication. Only 40% of the urine test results were positive for drugs containing different combinations of medications and street drugs. Unlike patients who received benzodiazepine, those who received tricyclic antidepressant medication were significantly more likely to associate with predictors of improvement. There is a need for better training of doctors and careful selection of patients in order to implement treatment strategies based on good doctor-patient relationships and well defined objectives for treatment and clear criteria of outcome.


Changes in benzodiazepine-receptor activity modify morphine withdrawal syndrome in mice. The effects of different benzodiazepine-receptor ligands on morphine withdrawal were studied: a benzodiazepine agonist, flunitrazepam; a benzodiazepine antagonist, flumazenil; a partial inverse benzodiazepine agonist, Ro 15-4513; and a partial benzodiazepine agonist, Ro 16-6028. Benzodiazepine-ligands were administered i.p. 30 min before naloxone-induced morphine withdrawal syndrome. Jumping behavior was significantly increased by Ro 15-4513 at 10 and 20 mg/kg. Flunitrazepam decreased jumps at all the doses used. Wet dog shakes were decreased by flumazenil and Ro 15-4513 and increased by Ro 16-6028 (only at the highest dose) and flunitrazepam. Our results show that the activation of the benzodiazepine receptor by agonists or high doses of partial agonists decreases jumping and increases wet dog shake behaviour, while the antagonists or the partial inverse agonists enhance jumping and decrease wet dog shakes. These modifications could be interpreted as an attenuation in the severity of the morphine withdrawal syndrome by benzodiazepine agonists.


A pharmacological comparison of parathyroid hormone receptors in human bone and kidney. While abundant information is available characterizing PTH receptor properties in other species, data on human PTH receptors is very limited. We have been interested in the possibility that tissue-specific differences among human PTH receptors (i.e. bone vs. kidney) might exist. We have, therefore, compared pharmacological profiles for a wide array of PTH and PTH-related peptide (PTHrP) analogs in human osteoblast-like cells (SaOS-2) and human renal cortical membranes (RCM) using radioiodinated (Tyr36)hPTHrP(1-36)NH2 as a probe for PTH receptor function. The rank order of receptor affinity for 10 PTH/PTHrP receptor agonists tested was very similar in the bone and kidney assay systems. Binding affinity for these peptides was greater in human (h)RCMs and SaOS-2 membranes than in SaOS-2 intact cells. The relative binding affinities for (Tyr36)hPTHrP(1-36)amide, hPTH(1-34), bovine (b)PTH(1-34), and rat PTH(1-34) were similar in human RCMs, SaOS-2 membranes, and SaOS-2 cells. bPTH(1-84) and hPTHrP(1-74) both manifested lower receptor affinity than the amino-terminal analogs. Seven PTH/PTHrP receptor antagonists were also studied in this homologous human assay system. The binding affinity for hPTHrP(7-34)NH2 was 2- to 3-fold greater than that for (Tyr34)bPTH(7-34)NH2 in RCM and SaOS-2 membranes. However, in SaOS-2 cells, a striking reversal in the relative binding affinities was observed; the PTHrP(7-34) analog was nearly 3-fold less potent than (Tyr34)bPTH(7-34)NH2, underscoring a difference between intact and broken cell preparations. Two hybrid PTH-PTHrP receptor antagonists demonstrated similar relative affinity to each other in the human bone and kidney assay systems. Affinity cross-linking of receptors in human renal and skeletal tissues demonstrated an indistinguishable dominant 85-kilodalton receptor protein. We conclude that the binding and bioactivity profiles of a broad array of PTH and PTHrP peptides are very similar or identical in human renal and skeletal tissues. Differences relating to intact vs. broken cell preparations accounted for some variation in potency. These studies emphasize the importance of employing homologous assay systems to study PTH receptor function and the existence of interspecies differences among PTH receptors. The results support the possibility that PTH receptors in human bone and human kidney are very similar if not identical.


The effect of blockade of kappa-opioid receptors in the medial basal hypothalamus and medial preoptic area on luteinizing hormone release during midpregnancy in the rat. The objective of this study was to determine if kappa-opioid receptors present in the medial preoptic area (MPOA) and medial basal hypothalamus (MBH) are involved in opioid peptide suppression of LH secretion during midpregnancy (day 13-16) in the rat. Nor-binaltorphimine (nor-BNI), a selective antagonist of brain kappa-opioid receptors, was applied directly to the MPOA or MBH for 3.5 h by means of push-pull perfusion. Nor-BNI perfusion in the MBH produced a dose-dependent increase in LH pulse frequency as well as increases in blood LH level. The effect on amplitude could not be determined, since too few pulses occurred in cerebrospinal fluid-treated control rats. Nor-BNI perfusion in the MPOA also increased LH pulse frequency. Moreover, in the majority (62%) of rats perfused with nor-BNI in the MPOA, the final 1.5 h of perfusion were unexpectedly characterized by an increase in LH that was of greater magnitude and more prolonged than an LH pulse and produced an elevation in blood LH levels. This delayed LH response did not occur in any rat perfused in the MBH. Perfusion with nor-BNI in the diagonal band of Broca had no significant effect on LH secretion. The LH responses observed during nor-BNI perfusion in the MPOA or MBH were not due to spread to the third ventricle and subsequent diffusion via the cerebrospinal fluid to another brain site, since perfusion with nor-BNI in an area of the ventral thalamus close to the third ventricle had no effect on LH release. These results provide support for the involvement of kappa-opioid receptor-mediated mechanisms in both the MPOA and MBH in the suppression of LH secretion during midgestation in the rat.


The placenta, PGE2 and parturition.

The placenta, PGE2 and parturition. It is proposed that prostaglandin E2 (PGE2), secreted by the fetal placenta of the sheep, acts as a circulating regulator of the physiological function of many fetal organs (tissue) in a way analogous to catecholamines in the adult. The specificity of PGE2 action in different tissues is determined by three different receptor subtypes which regulate intracellular calcium concentrations via the IP3 pathway, or cyclic AMP concentrations via the adenylcyclase system. The placenta, by secreting PGE2 (and possibly other factors such as adenosine), modifies the function of key organ systems allowing the fetus to survive and develop in the aqueous environment of the uterus. During fetal development, fetal organs and metabolic pathways can mature while their function is suppressed by placental PGE2. At birth, by ligating the cord and removing the placenta as the source of these inhibitory substances, the newborn is able to adapt readily to its new environment with fully-functional, mature organ systems. This paper discusses how placental PGE2 may regulate fetal breathing movements, whether the removal of placental PGE2 is involved in the initiation of continuous breathing at birth, and whether it suppresses the activity of the peripheral chemoreceptors during fetal life. The ability of PGE2 to maintain a widely patent ductus arteriosus, to suppress non-shivering thermogenesis, to stimulate fetal insulin secretion and to suppress the hepatic gluconeogenic pathway in the fetus is also discussed. Finally, the ability of PGE2 to activate the fetal hypothalamo-pituitary-adrenal axis is discussed, raising the possibility that the placenta also plays a key role in the initiation of birth in this species.


Posttranscriptional up-regulation of thyrotropin-releasing hormone (TRH) receptor messenger ribonucleic acid by TRH in COS-1 cells transfected with mouse pituitary TRH receptor complementary deoxyribonucleic acid. We found previously that the level of endogenous TRH receptor (TRH-R) mRNA in pituitary (GH3) cells and the level of mouse TRH-R mRNA in GH3 cells stably transfected with mouse pituitary TRH-R cDNA are down-regulated by TRH. This down-regulation is caused by TRH stimulation of TRH-R mRNA degradation via a mechanism that appears to involve protein kinase-C. In this report we study regulation of TRH-R mRNA in monkey kidney (COS-1) cells transiently transfected with mouse pituitary TRH-R cDNA. In transfected COS-1 cells, TRH and phorbol 12-myristate 13-acetate (PMA) caused increases in the level of TRH-R mRNA. In contrast, TRH caused only a small transient increase in the level of the mRNA for the neomycin resistance gene, which was cotransfected with TRH-R, and did not affect the level of the mRNA for glyceraldehyde phosphate dehydrogenase, an endogenous gene. The increases in TRH-R mRNA caused by TRH and PMA were inhibited to similar extents by H-7 (1-[5-isoquinolinesulfonyl]2-methyl piperazine dihydrochloride), an inhibitor of protein kinases. The effect of TRH was observed in cells transfected with expression vectors in which TRH-R cDNA was controlled by cytomegalovirus or Rous sarcoma virus promoters. There was no effect of TRH or PMA on the rate of transcription of the transfected TRH-R cDNA. In contrast, TRH caused the rate of degradation of TRH-R mRNA to decrease from 8.0% to 5.1%/h. Hence, TRH, most likely via a protein kinase-C-mediated mechanism, up-regulates TRH-R mRNA levels in transfected COS-1 cells by decreasing the rate of TRH-R mRNA degradation. Since TRH and PMA down-regulate TRH-R mRNA in GH3 cells, posttranscriptional regulation of TRH-R mRNA is a cell-type specific process.


Forskolin-induced homologous desensitization via an adenosine 3',5'-monophosphate-dependent mechanism(s) in human osteoblast-like SaOS-2 cells. We have reported that pretreatment of human SaOS-2 osteoblast-like cells with forskolin (Fsk; 10(-5) M) for 4 h strikingly inhibited subsequent cAMP responsiveness to a second challenge with Fsk (Fsk-induced homologous desensitization) without altering the responses to PTH or vasoactive intestinal peptide (VIP). Pretreatment with PTH acutely augmented Fsk responsiveness, despite desensitizing the cells to rechallenge with PTH. The present studies were performed to investigate the mechanism of this differential desensitization. Fsk-induced desensitization was not mimicked by 1,9-dideoxyforskolin, a Fsk analog that does not activate adenylate cyclase (AC) but does reproduce certain cAMP-independent effects of Fsk. Fsk-induced homologous desensitization was also completely blocked in a cAMP-resistant mutant SaOS-2 cell line (Ca 4A), in which protein kinase-A (PKA) is not activated by endogenous cAMP. However, pretreatment with PTH (or VIP), which induced a large increase in cAMP, did not attenuate, but, rather, increased, the subsequent cAMP response to Fsk. Potentiation by PTH was also observed in Ca 4A cells. Pretreatment of SaOS-2 cells with pertussis toxin (100 ng/ml) for 12 h strikingly inhibited the initial cAMP response to Fsk, although Fsk-induced homologous desensitization was still clearly observed. Pretreatment with cholera toxin (1 microgram/ml) completely prevented Fsk-induced homologous desensitization. Combinations of maximal concentrations of Fsk plus hormones such as human PTH, human PTH-related peptide, or VIP elicited cAMP responses that were much more than additive, an effect not observed with combinations of hormones alone. We conclude that 1) Fsk-induced homologous desensitization of the AC response of SaOS-2 cells to a second challenge with Fsk is dependent upon activation of PKA; 2) one or more pertussis toxin-sensitive G-proteins contribute to full AC activation by Fsk, but are not involved in homologous desensitization; 3) augmentation by PTH (or VIP) pretreatment of Fsk-dependent AC activation involves an effector(s) other than PKA. These results provide further evidence that the regulation of AC responsiveness in SaOS-2 cells by PTH or VIP is complex and cannot be explained by activation of PKA alone.


Secretion of melanin-concentrating hormone and neuropeptide-EI from cultured rat hypothalamic cells. Rat melanin-concentrating hormone (MCH) is a homolog of the peptide first isolated from salmon pituitary glands which produces melanosome aggregation within melanophores of teleost fish as well as interacting with the hypothalamic-pituitary axis. We have previously characterized the rat and human MCH counterparts as identical 19-amino acid peptides showing approximately 70% peptide sequence identity to salmon MCH. Immunoreactivity for MCH has been found in high concentrations within cell bodies of the dorsolateral hypothalamus, with projections to the hippocampus, brainstem, posterior pituitary, and cerebral cortex. We have adapted a cultured cell model for studying MCH secretory responses of hypothalamic cells obtained from 7-day-old rats. MCH and the MCH precursor-derived peptide neuropeptide glutamic acid isoleucine (NEI) were secreted from these cells after 2 days of culture and for up to 22-24 days of culture. The secreted peptides were confirmed by HPLC analysis and RIA to be identical to MCH isolated from rat hypothalamic tissue and to the sequences of MCH and NEI predicted from the MCH precursor. MCH and NEI secretion was stimulated 3- to 5-fold by 8-bromo-cAMP and 8-bromo-cGMP. Dexamethasone produced a dose-dependent increase in cell content of both MCH and NEI and an increase in MCH secretion. The present study is the first to demonstrate the existence of the predicted peptide NEI in a biological system and indicates that cultured neonatal rat hypothalamic cells are a useful model for the study of MCH/NEI release in vitro.


In vivo effects of adrenocorticotropin on hamster adrenal steroidogenic enzymes. The hamster, a rodent possessing adrenal 17 alpha-hydroxylase activity, was used to study the effect of ACTH on the regulation of cortisol formation in vivo. The characterization of the mRNA and protein of hamster adrenal steroidogenic enzymes revealed close similarities between this animal and other mammalian species. The hamster adrenal RNA hybridized in a single band to cDNA probes for bovine adrenal P450scc, P450(17 alpha), P450c21, to mouse adrenal P450(11 beta), and to pig testis 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the areas of 2.2, 2.0, 2.3, 2.0, and 2.1 kilobases, respectively. Immunoblotting analyses revealed the presence of single protein bands reacting with antibodies to bovine P450scc, P450c21, porcine P450(17 alpha), or human placental 3 beta HSD in the areas of 52, 55, 51, and 41 kilodaltons, respectively, whereas two protein bands were detected at 48 and 52 kilodaltons with the antibody to bovine P450(11 beta). After stimulation with ACTH injected at 5-h intervals over 20 h, plasma cortisol levels, which were already increased 2.5 h after the first injection, remained elevated for the duration of treatment and returned to control values 15 h after the last injection. The ratios of plasma cortisol to corticosterone were 1.5, 3.9, and 7 at 0, 2.5, and 5 h after the first injection and continued to rise to a value of 11 at 15 h after multiple injections. This ratio returned to control values 15 h after cessation of either the short term (one injection) or long term (five injections) treatment, indicating a control mechanism favoring cortisol formation upon ACTH stimulation. Of the adrenal enzyme systems examined, only three were directly affected by ACTH treatment. The mRNA level of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase, the key precholesterol regulatory step, increased after ACTH administration within 2.5 h and remained elevated during the entire study period. ACTH provoked a rapid and sustained increase in P450scc mRNA levels, which decreased very slowly after cessation of treatment without reaching control values 30 h after the last injection. Meanwhile, ACTH treatment caused no changes in the amount of adrenal cytochrome P450scc protein during treatment and 30 h after its cessation. Therefore, we postulate that factors other than newly synthesized P450scc protein participate in the control of this rate-limiting step. The high P450scc mRNA levels observed suggest stabilization of mRNA and posttranscriptional events affecting its catabolism.(ABSTRACT TRUNCATED AT 400 WORDS)


Inhibitory action of ethanol on L-type Ca2+ channels and Ca(2+)-dependent guanosine 3',5'-monophosphate accumulation in rat pinealocytes. It has previously been shown that the K+ potentiation of vasoactive intestinal peptide-stimulated cAMP and cGMP responses was inhibited by ethanol in rat pinealocytes, suggesting an inhibitory action of ethanol on the voltage-dependent Ca2+ channels (VDCC). In this study, using the whole cell version of the patch clamp technique, we found that ethanol reduced the amplitude, but did not change the voltage dependence or the time course of activation or inactivation of the L-type VDCC. The inhibitory effect of ethanol on this current was concentration dependent, and ethanol (100 mM) resulted in a 40% inhibition of this current. However, in fura-2-loaded cells, total increases in intracellular Ca2+ ([Ca2+]i) caused by ethanol and BayK 8644 did not differ from the [Ca2+]i signal caused by BayK 8644 alone, suggesting that the inhibitory action of ethanol on VDCC may not be related to a reduction in [Ca2+]i. Although there was no change in the total [Ca2+]i signal, ethanol (25-200 mM) dose-dependently inhibited the potentiation effects of depolarizing concentrations of K+ and BayK 8644 on the isoproterenol-stimulated cGMP, but not the cAMP, response. Therefore, the cGMP response appears to be more sensitive to the inhibitory action of ethanol, and a site distal to elevation of [Ca2+]i of importance to the potentiation mechanism may be inhibited by ethanol. This was confirmed by the finding that ethanol was effective in inhibiting the A23187 potentiation of isoproterenol-stimulated cGMP response. These results suggest that 1) the L-type VDCC was inhibited by ethanol; 2) the Ca(2+)-mediated potentiation of the isoproterenol-stimulated cGMP response was sensitive to the inhibitory action of ethanol; and 3) although ethanol inhibits the VDCC, it alone cannot explain the inhibitory effect of ethanol on BayK 8644- and K(+)-mediated potentiation of the isoproterenol-stimulated cGMP response.


Secretion and regulation of a neuropeptide-processing enzyme by AtT-20 cells. The mouse anterior pituitary-derived cell line AtT-20 has been widely used to study the biosynthesis and secretion of peptide hormones, such as ACTH, and peptide-processing enzymes, such as carboxypeptidase-E (CPE). Although AtT-20 cells do not express dynorphin (Dyn), previous studies using gene transfer have revealed that these cells are capable of processing pro-Dyn into peptides such as Dyn-B-13. A Dyn-converting enzyme (DCE) has been identified in AtT-20 cells; this enzyme processes Dyn-B-29 to Dyn-B-13. By several criteria, the enzyme activity secreted from AtT-20 cells is similar to the previously characterized enzyme activity in rat brain and bovine pituitary. In AtT-20 cells, the DCE activity and CPE activity are localized to a similar secretory compartment. Upon stimulation with a beta-adrenergic agonist, a phorbol ester, a calcium ionophore, or forskolin, the secretion of DCE activity was increased; this rise was parallel to the secretion of CPE activity and ACTH. These data suggest that DCE activity is in the regulated pathway of secretion. In AtT-20 cells treated with glucocorticoid for up to 7 days, cellular levels of beta-endorphin decreased to half the control levels. In contrast, the levels of DCE activity did not decline in response to this treatment, suggesting that the enzyme activity was not coregulated with the endogenous hormone. Taken together, the data presented here support a role for DCE in posttranslational processing of regulatory peptides.


Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium, and lanthanum, but not to nickel: evidence provided by fluorescence changes in fura-2-loaded cells. Cobalt (Co), nickel (Ni), manganese (Mn), cadmium (Cd), and lanthanum (La) are commonly used as calcium (Ca) channel blockers, but some of them, besides reducing Ca entry, also traverse Ca channels and can exert effects intracellularly that confound interpretation of functional responses. Because of this and our need to use Ca channel blockers in an ongoing analysis of Ca channel activity in the regulation of the cytosolic free Ca concentration ([Ca2+]i) and secretion in melanotrophs, we assessed whether the cations mentioned enter these cells. This was done by incorporating the fluorescence for changes that would signal the presence of the cations in the cytosol. In cell-free solution, where the probe and cations can interact freely, Mn, Co, and Ni all quench fluorescence, whereas Cd and La act in a Ca-like manner. When tested on fura-2-loaded melanotrophs in basal (unstimulated) conditions, Mn, Co, and Cd each yielded corresponding signals, thereby showing that they had penetrated the cells. By contrast, Ni caused no quenching of fluorescence even in melanotrophs exposed to 100 mM K+ to recruit additional Ca channels. Ni, therefore, did not penetrate the cells. However, as expected, Ni quenched fluorescence when given artificial access to the cytoplasm by ionomycin. Ni blocked spontaneous entry of Mn, Co, and Cd. It also lowered [Ca2+]i in unstimulated melanotrophs, consistent with blockade of spontaneous Ca entry. Like Ni, La lowered basal [Ca2+]i in unstimulated melanotrophs without penetrating the cells; however, unlike Ni, it penetrated when the melanotrophs were exposed to high potassium. We conclude that Ni is the most specific of the Ca channel blockers tested and that results obtained with Mn, Co, Cd, and La must be interpreted with reserve.


Identification and functional studies of a specific peptide YY-preferring receptor in dog adipocytes. Specific binding sites for peptide YY (PYY) and neuropeptide Y (NPY) as well as functional responses were identified in dog adipocytes. Studies were carried out using the radioligand [125I-Tyr1]monoiodo-PYY on crude adipocyte membranes. [125I]PYY bound to dog adipocyte membranes with a high affinity (156 +/- 24 pM) and binding capacity of 314 +/- 48 fmol/mg protein. Competition studies revealed a higher affinity of the binding sites for PYY than NPY (inhibition constants were 118 +/- 17 pM and 300 +/- 53 pM, respectively, P < or = 0.001). NPY analogs displaced [125I]PYY specific binding with the following order of potency: NPY-(13-36) > NPY-(18-36) > NPY-(22-36) >> [Leu31-Pro34]NPY. Neither adrenergic nor adenosine agents (activating or inhibiting other antilipolytic systems) interacted with [125I]PYY binding sites. So [125I]PYY binding was specific, saturable, and reversible. Lipolysis experiments performed with PYY, NPY, and NPY analogs confirm the relative order of potency found in competition experiments. The data agree with the definition of PYY-preferring receptor which resembles a Y2 receptor subtype since NPY-(13-36), a specific Y2 receptor agonist, inhibited binding and lipolysis in a similar way to PYY, whereas [Leu31-Pro34]NPY did not. No difference was observed in the antilipolytic response between IC50 values measured on omental, perirenal, and subcutaneous fat deposits. Moreover, PYY and NPY (10(-6) M) significantly attenuated forskolin-stimulated cAMP levels, involving inhibition of adenylyl cyclase as a transmembrane signaling mechanism. Cross-linking of bound [125I]PYY to membranes indicated that the mol wt of the receptor was 62K. The relative importance of such a receptor on fat cells alongside another powerful antilipolytic receptor--the alpha 2-adrenoceptor--is discussed.


The effect of streptozotocin diabetes on the vasoactive intestinal peptide receptor/effector system in membranes from rat ventral prostate. All of the components of the neuropeptide vasoactive intestinal peptide (VIP) signal transduction system were underexpressed in rat prostatic membranes 6 weeks after streptozotocin-induced diabetes. Binding studies with [125I]VIP showed decreases of 86% and 62% in the binding capacity of the high and low affinity classes of VIP receptors in diabetes. Affinity labeling experiments indicated that the main form of VIP receptor was 51 kilodaltons in control rats and 45 kilodaltons in diabetic animals. The efficacy of VIP and forskolin in stimulation of adenylyl cyclase activity as well as the potentiating effect of GTP on VIP action were also reduced in diabetes, as was the expression of the alpha-subunit of the guanine nucleotide-binding regulatory proteins Gs and Gi (studied by ADP ribosylation with cholera and pertussis toxins). Gi function was lost in diabetes, as assessed with experiments on guanyl-5'-yl-imidodiphosphate potentiation of forskolin activity. These disturbances together with previous findings argue for VIP playing a role in the diabetic neuropathy of the genitourinary tract.


Gradual progress of ACTH deficiency in a child with panhypopituitarism associated with pituitary stalk transection. We present here a 13-year-old male with hypopituitarism which accompanied an insidious and gradual progress of ACTH deficiency. ACTH deficiency finally led to an overt crisis of adrenal insufficiency at the age of 12 years and 7 months. This patient is unique because the insidious and gradual progress has been proved by not only the laboratory results but also the clinical course for over 13 years. The cause of panhypopituitarism including ACTH deficiency is thought to have existed before or at the delivery because of the stalk transection seen on the magnetic resonance image (MRI). At the crisis, his laboratory results suggested that he had secondary adrenal insufficiency, whereas he showed normal adrenal function proved by the insulin tolerance test (ITT) at the age of 4 years. Abrupt crisis of secondary adrenal insufficiency developed at the age of 12 years, although he had been well until the crisis.


Intracranial and intraspinal dissemination of an ACTH-secreting pituitary tumor. A case of a 29-year-old man with an ACTH-producing pituitary tumor disseminated into the subarachnoid space is described. After total adrenalectomy for Cushing's disease at the age of 15, Nelson's syndrome developed. Transsphenoidal adenomectomy at 17 and 21 years of age, pituitary irradiation and medical therapies with sodium valproate, baclofen and bromocriptine failed to lower his plasma ACTH level. Multiple intracranial and intraspinal tumors associated with the symptoms of left hemiparesis developed. The removal of a tumor grown at the level of C1-3 was performed with successful palliation of his symptoms. Histologically, the tumor cells showed sinusoidal, papillary and diffuse patterns with a preponderance of the former over the latter two, although the papillary pattern predominated in the primary pituitary tumor. Immunohistochemical analysis demonstrated most cells to be positive for ACTH in the metastatic tumor as well as the primary adenoma. The clinical significance of his course is discussed with a review of 11 reported cases with metastatic ACTH-producing pituitary tumors.


Vasoactive intestinal peptide enhances dopamine accumulation in primary cell culture of rat hypothalamus. The effect of vasoactive intestinal peptide (VIP) and PHI-27 on dopamine accumulation in cultured rat hypothalamic cells was investigated. VIP enhanced [3H]dopamine accumulation dose dependently. This effect was significant at 10(-8)-10(-5) M VIP with a concomitant increase in intracellular cyclic AMP (cAMP), and reached its plateau level at 10(-6) M VIP. VIP increased [3H]dopamine accumulation significantly within 15 min. PHI-27 and dibutyryl cAMP ((Bu)2-cAMP) also enhanced [3H]dopamine accumulation. These results suggest that VIP enhances dopamine accumulation in hypothalamic cells by increasing intracellular cAMP.


The early phase of sodium channel gating current in the squid giant axon. Characteristics of a fast component of displacement charge movement. A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligo forbesii. This component is characterized by relaxation time constants typically shorter than 25 microseconds. The charge displaced accounts for about 10% (or 2 nC/cm2) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above +20 mV and below -100 mV. This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of 1 e- and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.


Phagolysosomal pH in alveolar macrophages. We studied phagolysosomal pH in alveolar macrophages (AM) using fluorescein-labeled yeast (FYP) and silica particles (FSP) as probes. Fluorescence intensities from the ingested test particles were measured on populations of AM using fluorescence spectrometry and on individual phagolysosomes using fluorescence microscopy. Measurements were performed on rabbit AM, which had been incubated with FYP or FSP (in vitro procedure). We also instilled FYP or FSP via the trachea into rabbit lungs and after 1 day, 1 week, 1 month, and 3 months lavaged the lungs and measured the pH in AM (in vivo procedure). Phagolysosomal pH was independent of the number and size of the fluorescent particles. Measurements of populations of AM with fluorescence spectrometry and of individual phagolysosomes with fluorescence microscopy gave similar average pH. For the FYP, pH decreased during the first day after lavage both in the in vitro and the in vivo procedures. For the FSP, pH was unchanged during the same period. After 1 day pH was similar for both particles. Electron microscopy showed a larger number of lysosomes in contact with phagosomes and a higher percentage of vacuolated phagosomes for FYP than for FSP. In the in vivo procedure, pH was unchanged at least up to 1 month, and this pH was lower than that in the in vitro procedure. The difference was probably due to conditions at the time of phagocytosis. Particles retained in the lung parenchyma were within AM, and their location within the AM appeared unchanged from 1 week up to 3 months.


Effects of instilled fibrogenic particles on the clonal growth of murine pulmonary alveolar macrophages. Murine pulmonary alveolar macrophages (PAM) form macrophage colonies in vitro with colony-stimulating factors, which stimulate the clonal growth of radioresistant alveolar colony-forming cells (AL-CFC). The toxic effects of fibrogenic mineral dust particles on AL-CFC were investigated after intratracheal instillation into mice. Exposure to either crocidolite asbestos or silica (Min-u-sil) induced a significant depletion of AL-CFC as well as a decrease in PAM recovery compared to either untreated or titanium dioxide-exposed animals. Such effects were also noted with different doses (50-200 micrograms/animal) of instilled particles. The plating efficiency of AL-CFC was depleted in PAM exposed to fibrogenic particles in vitro, but not when exposed to nonfibrogenic titanium dioxide particles. These results indicate the toxic effects of fibrogenic dust particles on the clonal growth of PAM, cells which play a role in the clearance of inhaled particles from the lung and in subsequent pathologic processes.


Silica-exposed lung fluids have a proliferative activity for type II epithelial cells: a study on human and sheep alveolar fluids. The type II pneumocyte changes in silicosis are characterized by hyperplasic and hypertrophic epithelial cells, and increased surfactant phospholipids in the bronchoalveolar lavage fluids (BALF). To assess the proliferative activity of alveolar lining fluids, BALF were applied on type II cell cultures. The growth-promoting activity was studied by tritiated thymidine incorporation for 24 h, and the cell number was measured by an electronic counting after a 48-h exposure time. Human BALF from 3 subsets of workers exposed to silica, staged according to ILO classification (silica exposed-workers without disease: hSWD n = 6; workers with simple silicosis: hSS n = 7; workers with confluent silicosis: hCS n = 5), were compared to healthy volunteers (hC n = 6). Sheep BALF from our model of silicosis and control animals (sS and sC) were studied at months 0, 6, and 24 of exposure. A clear enhancement was found in type II cell DNA synthesis under the effect of either normal and silicotic human or sheep BALF, in comparison to the negative control (p less than .05). In addition hSWD and hSS BALF as sS BALF at 20% dilution (peak activity) were significantly more stimulating than the normal alveolar fluids from the same species (p less than .05). The highest sheep BALF stimulatory activity was found at month 6 (170% of increase vs control, p less than .05) and clearly correlated with the high cellularity of BALF. The thymidine incorporation was supported by changes in cell counts. Sheep silicotic BALF run through G50 columns identified at least 3 molecular weight (MW) areas of mitogenic activity between 30 and 5 kDa. Biochemical characteristics of growth factors in the above MW range (PDGF, FGF, TGF alpha, EGF) were tested. Increased mitogenic activity of type II cells eluted from heparin sepharose columns loaded with silicotic sheep BALF, at 0.5 and 1 M NaCl, corresponded to the removal areas of PDGF- and acidic FGF-like heparin-binding molecules. The high proliferative activity on type II cells of the latter two molecules, alone or in combination with other growth factors, was demonstrated in vitro (greater than 9 x control). In conclusion, a stimulatory activity for type II cell growth was found in the normal human and sheep alveolar lining fluid. This activity was clearly enhanced in the early stages of human and sheep silicosis. The BALF type II cell growth factors had biochemical characteristics consistent with the PDGF- and FGF-like molecules.


Effects of inhaled alpha-emitting actinides on mouse alveolar macrophages. The effects of inhaled alpha-emitting actinides on the alveolar macrophage (AM) population of the rodent lung are reviewed and, in particular, of the effects of 239PuO2 on murine AM. The effects discussed include changes the AM pool size, macrophage diameter, mobility, phagocytic competence, and enzyme content. Finally, similarities in the dose-response relationships for the induction of nuclear aberrations by alpha emitters and in the induction of lung tumors by the same materials are noted.


Intrathoracic distribution and transport of aerosolized silica in the rat. Short-term exposure of rats to aerosols of the silicon dioxide, cristobalite, leads to pulmonary inflammation persisting several months. Clearance of particles occurs during the first two weeks after cessation of exposure, after which there is little additional clearance in the whole lung. In the present studies, quantitation of silica in lung compartments at selected times following exposure indicated movement of particles between the alveolar space and the lung tissue per se, with increased alveolar silica content associated with decreased silica content in the tissue compartment. Further, changes in the silica content in the alveolar compartment were generally associated with fluctuations in the alveolar macrophage population. Silica accumulated linearly in the mediastinal lymph nodes and thymus for several months after cessation of exposure, while negligible amounts were found in kidney, spleen, liver, and blood. A compartmental model was used to describe the distribution and translocation kinetics of the inhaled silica in the lung and extrapulmonary tissues.


Influence of surfactant components and exposure geometry on the effects of quartz and asbestos on alveolar macrophages. Bovine (BAM) and rat (RAM) alveolar macrophages were incubated in vitro with DQ12 quartz or UICC chrysotile asbestos either alone or in the presence of dipalmitoyl lecithin (DPL). The reaction of the cells of both species to the untreated dust particles was similar qualitatively and quantitatively, with a loss of viability and release of lactate dehydrogenase and N-acetyl-beta-glucosaminidase after 20 hr of incubation. In the presence of DPL, the toxicity of quartz to BAM disappeared completely, whereas the protective influence of the phospholipid was distinctly diminished in the case of RAM. The presence of lavage fluid was less effective than that of pure DPL. There was no protective influence of DPL with asbestos either for BAM or for RAM. The effects of phagocytizable, suspended quartz particles were compared with the effects of the same type of particles fixed on a glass surface to exclude the possibility of phagocytosis. The effect of the suspended particles on the viability and release of enzymes was more pronounced than that of the fixed particles. On the other hand, superoxide anion production was stimulated to a much higher degree by the fixed quartz particles. This could be explained by the continuing contact of the outer cell membrane with the silica surfaces, whereas free particles were rapidly phagocytized. The release of lysosomal enzymes induced by fixed quartz particles was a secondary phenomenon following cell death.


Radiation enhances silica translocation to the pulmonary interstitium and increases fibrosis in mice. The effects of whole body irradiation (WBR) on particle clearance and the development of pulmonary fibrosis have been investigated. Using carbon, clearance is accomplished by polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM), and only a few particles reach the interstitum. However, in preirradiated mice, the usual eflux of inflammatory cells is much delayed so that more free carbon remains in the alveoli, and by 1 week, many particles cross the epithelium to be phagocytized by interstitial macrophages. Carbon is found in the peribronchiolar interstitium 6 months later with no evidence of fibrosis. In the present study, mice received 1 mg silica intratracheally 2 days after 6.5 Gy WBR when the white blood cell count was low. A much-reduced AM and PMN response was found in the following 2 weeks compared to the reaction to silica alone, and many silica particles reached interstitial macrophages. In this case, macrophage activation by silica was associated with fibroblast proliferation, and by 16 weeks, much more pulmonary fibrosis was produced than after silica or irradiation only. This was measured biochemically and correlated with a large increase in retained silica in the irradiation-silica group. The results indicate that radiation inhibits the inflammatory response to particle instillation, resulting in greater translocation of free particles to the pulmonary interstitium. In the case of silica, the greater, prolonged interaction with interstitial macrophages leads to a much exaggerated fibrotic reaction.


Transport of particles of colloidal gold within and from rat lung after local deposition by alveolar microinjection. Because inhalation and intratracheal instillation deposit particles throughout the respiratory tract, these methods of administration give little information on the movement of particles within the lung and no direct information on the clearance kinetics from locally defined sites within alveolar tissue. Approximately 0.05 microL of 195Au-labeled gold colloid was administered to 32 rats by microinjection into a small volume of subpleural alveoli. Its fate was studied by whole-body counting and serial sacrifice over 15 months. The kinetics of clearance from the subpleural deposition site showed that there was no rapid removal of particles, and the main clearance process was defined by an exponential term with a half-time averaging 583 days. There was a wide variation between individual animals. The distribution of 195Au at sacrifice showed that the gold colloid was nearly all retained within the respiratory tract. The particles were not appreciably redistributed throughout the lung volume, so most of the material not cleared from the lung remained close to the deposition site. At the later times after microinjection, much of the gold colloid was associated with thickened pleura and adjoining septae.


Overview of pulmonary alveolar macrophage renewal in normal rats and during different pathological processes. We report experimental results on pulmonary alveolar macrophage (PAM) renewal in healthy rats and in rats treated with particles introduced in the lungs. Morphometric studies showed that the lungs of normal rats of the strain used in our study contain 20 x 10(6) PAM, 50 x 10(6) monocytes in alveolar capillaries, and about 3 x 10(5) interstitial macrophages. Pulse labeling with a tritiated thymidine (3HT) gave a labeling index of 0.4% for the monocytes, of which a few could be observed in mitosis within alveolar capillaries. These monocytes are likely to be the PAM precursors. The daily input (greater than 4%) by PAM proliferation exceeds PAM loss by migration to the upper respiratory tract (2.5%). The life span of PAM was measured by sequential counting of lavaged cells after labeling with [125I]iododeoxyuridine instilled intratracheally. The pulmonary lavage procedure used allowed us to recover at least 80% of the whole PAM population. A daily loss of PAM of 8-9% was measured, of which loss by death in the endoalveolar compartment was estimated at 5-6%. During the pathological processes studied, several parameters of PAM renewal were shown to be modified. PAM migration to the upper respiratory tract was frequently inhibited, PAM cytotoxicity was observed, and PAM proliferation increased in some cases and decreased in others. Under most of the pathological conditions investigated, the renewal of endoalveolar macrophages appeared quite different from that in normal rats, and direct blood monocyte migration to the endoalveolar compartment became a major component of PAM renewal.


Modulation of eicosanoid production by human alveolar macrophages exposed to silica in vitro. Repeated inhalation of silica dust can lead to inflammation and fibrosis in human lung and in experimental animal models. The alveolar macrophage is believed to play a pivotal role in this process. Numerous macrophage-derived growth factors, cytokines, and arachidonic acid metabolites have been shown to contribute to inflammation and fibrosis. The objective of this study was to determine the eicosanoid production by human alveolar macrophages in response to silica exposure in vitro and to assess the contribution of alveolar macrophages to silica-induced fibrosis and inflammation. Macrophages were obtained from healthy volunteers and were incubated for 3 or 24 hr in the presence of silica (100, 60, and 0 micrograms/mL). Supernatants were removed for eicosanoid analysis. Eicosanoids were analyzed by both high performance liquid chromatography and radioimmunoassay. The data suggest that silica causes an increased release of leukotriene B4, leukotrienes C4/D4/E4, and 5-hydroxyeicosatetraenoic acid (5-HETE) after 3 hr and decreases in prostaglandin E2 and thromboxane B2 production after 24 hr of exposure to 100 micrograms/mL silica. In addition, 12-HETE and 15-HETE production remained unchanged at either time point. These opposing effects seen with the metabolites of lipoxygenase and cyclooxygenase pathways could contribute to silica-induced fibrosis. The pattern of eicosanoid production after exposure to silica was different from that obtained when macrophages were stimulated with lipopolysaccharide for 3 or 24 hr, indicating that the response to the particles was not just due to general cellular activation.


The relation of the accumulation of cadmium in human placenta to the intake of high-fibre grains and maternal iron status. Exposure to cadmium via the diet is known to depend to a large extent on the intake of cereal grains, particularly the high-fibre fractions of wheat. Subjects with low iron status absorb more cadmium than those with better iron status. The purpose of the present study was to determine to what extent cadmium accumulation in human placenta is affected by the intake of grain fibre and maternal iron status during pregnancy. Thirty-nine pregnant women participated in the study. In each trimester the women were requested to complete a dietary history and to allow blood samples to be taken for haemoglobin, serum ferritin and serum thiocyanate determinations, the latter as a marker for smoking. At delivery the whole placenta was taken for the determination of the cadmium concentration. The 32 women who had serum thiocyanate levels less than 70 mumol/l, who had completed at least one dietary history and from whom a blood sample was obtained in the third trimester, were included in the final statistical analyses. In the group of women who consumed less than the median intake of grain fibre and had more than 15 micrograms ferritin/l serum in the third trimester, the placenta cadmium concentration was nearly half that in the placentae of women who had consumed more grain fibre or had lower iron status in late pregnancy.


Do beans and oat bran add to the effectiveness of a low-fat diet? The effects of consuming an increased amount of soluble fibre as oat bran or beans were examined in 40 free-living hypercholesterolaemic men and women. The subjects were initially established on a low-fat background diet (29% of energy from fat) and then 55 g low-fibre oat bran, 55 g high-fibre oat bran or 80 g mixed cooked beans were added to their diet in random order for 6 week periods. Body weight and overall composition of the diet did not change. Plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) were unchanged. High-density lipoprotein cholesterol (HDL-C) was significantly higher on all three intervention diets than on the lower fibre run-in diet. Supplementation of a moderately low-fat diet with palatable quantities of oat bran or beans without changing the overall fat intake does not appear to significantly lower cholesterol but may have a benefit by increasing HDL-C and reducing the ratio of LDL-C to HDL-C.


Increase in plasma progestagen concentrations in the mare after foetal injection with CRH, ACTH or betamethasone in late gestation. Maternal plasma progestagen concentrations increase about 20 days before parturition. The major contributors to the increase are reduced metabolites (ie 5 alpha-pregnanes). Precocious increases (ie less than 310 days of gestation) in these metabolites may occur in abnormal pregnancies. The effects of CRH, ACTH or betamethasone administered to the foetus at gestational ages ranging from about 250 to 320 days were examined. Sixteen healthy pony mares were used for foetal injection employing aseptic techniques. Water or normal saline were used as controls. Maternal plasma progestagen concentrations were measured using a commercial radioimmunoassay (RIA) progesterone kit and results were confirmed using gas chromatography-mass spectrometry (GC-MS). Results demonstrated clearly that an increase in maternal plasma progestagen concentrations occurred after injection of ACTH, CRH or betamethasone to the foetus, irrespective of gestational age. A comparable increase was not observed in the control animals. Of the 16 mares in which the foetus was injected, 13 produced viable foals at gestational ages ranging from 307 to 339 days whereas 3 mares delivered non-viable foals at 284 to 306 days gestation. The results support the hypothesis that the pre-parturient rise in progestagens occurring in the mare is the result of foetal adrenocortical activity.


Contribution of Na+,K(+)-ATPase to focal epilepsy: a brief review. The authors review some of their experimental data on the contribution of Na(+)- and K(+)-dependent adenosine triphosphatase (Na+,K(+)-ATPase) to focal epilepsy. It has been previously demonstrated that high extracellular K+ concentration increases glial Na+,K(+)-ATPase specific activities in normal conditions while this was not observed in neuronal preparations. At this time, it was hypothesized that this molecular mechanism could play a role in removing K+ released in the extracellular space during neuronal firing. These results have therefore been investigated in acute and chronic epileptogenic lesions of cats with freeze lesion. It was demonstrated that within the primary (F) and the secondary or 'mirror' (M) focus the K+ activation of the glial Na+,K(+)-ATPase dramatically decreased compared to both control animals (C) and the perifocal (PF) non epileptogenic area. Similar results were observed in man when using specimens of anterolateral temporal neocortex obtained during temporal lobectomies in patients with intractable temporal lobe epilepsy, compared with postmortem human specimens or control brain tissues. The modifications of the level of phosphorylation of partially purified Na+,K(+)-ATPase was also investigated in the epileptic cortex in these two experimental conditions. The catalytic subunits were resolved by sodium dodecylsulfate (SDS) gel electrophoresis and their phosphorylation levels were measured in the presence of various concentrations of K+ ions which dephosphorylate the catalytic subunit. K(+)-induced dephosphorylation was decreased in primary and secondary foci of acutely lesioned cats. Those alterations, due to a decreased affinity for K+, were limited to the alpha (-) subunit. In cats with chronic lesions, the dephosphorylating step of the Na+,K+-ATPase catalytic subunit recovered to normal affinity for K+.(ABSTRACT TRUNCATED AT 250 WORDS)