Prediction and conformation by synthesis of two antigenic sites in human haemoglobin by extrapolation from the known antigenic structure of sperm-whale myoglobin. The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.


Interaction of aspartate aminotransferase with mercurochrome. Relationship of an exposed thiol group of the enzyme to the active centre. Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 x 10(4) M-1 - min-1 and 2.15 x 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 x 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule.


Detection of carcinoembryonic-like antigen on melanoma cells by leucocyte-dependent-antibody assays. CEA-like antigen has been detected on the surface of some melanoma cells using a rabbit antiserum raised against CEA antigen in 51Cr-release leucocyte-dependent cytotoxic-antibody (LDA) assays. The CEA antigen used for production of the antiserum was shown to be immunologically pure by immunoelectrophoresis procedures and reacted with a reference serum to CEA. The specificity of the CEA antiserum for CEA on the melanoma cells was further shown by removal of reactivity to melanoma cells after absorption on CEA affinity columns. LDA activity to CEA was also shown in a serum taken during pregnancy. No CEA LDA activity was found in several melanoma sera. These findings suggest that CEA may have biological significance in tumour rejection, and that CEA assays may be of value in monitoring disease activity in melanoma patients.


Epidermal abnormalities in Refsum's disease. A female patient with severe Refsum's disease is described. She had striking ichthyosis and detailed investigations into the structure of the epidermis were performed. Light microscopy and transmission electron microscopy revealed lipid-containing vacuoles in the epidermis. Scanning electron microscopy of the stratum corneum showed disruption and the presence of microvilli on individual horn cells consistent with a high output state of epidermal cell production.


Binding of zinc to alpha-2-macroglobulin and its role in enzyme binding activity. Physicochemical studies performed on alpha-2-macroglobulin were correlated with the biological activities of this protein. Equilibrium dialysis of the binding of 65Zn by alpha-2-macroglobulin at pH 7.9 showed heterogeneous binding which could be attributed to two classes of binding sites. The site of greatest affinity for zinc had an apparent stoichiometry (n1 in gatoms/mol of alpha-2-macroglobulin monomer) of 12 and an apparent association constant (K1) of 3.06.10(7). The second binding site had an n2 of 60 and K2 of 1.32.10(5). The trypsin binding activity of alpha-2-macroglobulin did not depend on the presence of zinc in this protein since all but traces of this metal could be removed by EDTA without loss of trypsin binding activity. Saturation of site 1 with zinc did not affect the trypsin binding activity of alpha-2-macroglobulin, but binding of the metal by site 2 progressively decreased the trypsin binding activity by causing an irreversable association of the alpha-2-macroglobulin molecules. Removal of excess zinc from alpha-2-macroglobulin did not restore its trypsin binding activity. Our results also indicate that the high zinc content of alpha-2-macroglobulin (320--770 microgram/g protein) reported in the literature is an artifact and that native alpha-2-macroglobulin contains approximately 150--180 microgram Zn/g protein.


Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean. Five protease inhibitors, I--V, in the molecular weight range 7000--8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I--V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that  inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.


Heparin cofactor activity during and after extracorporeal circulation with bubble and membrane oxygenators. Heparin cofactor activity as measured by an amidolytic method has been investigated in 48 patients undergoing cardiac surgery with extra-corporeal circulation (E.C.C.). Heparin cofactor activity is reduced soon after the start of E.C.C., due to hemodilution but it returns towards initial levels at the completion of the procedure and 3 hours later. Thus heparin cofactor does not seem to play a role in the heparin rebound phenomenon. In our patients bubble oxygenators did not modify heparin cofactor activity more than did membrane oxygenators.


Effects of porcine plasmin on the coagulation and fibrinolytic systems in humans. Pig plasmin (Lysofibrin) was given to 11 patients with phlebographically verified venous thrombosis, 2 of whom were treated two and three times, respectively. The effect on coagulation and fibrinolytic parameters was studied. The platelet count, Owren's P&P (prothrombin plus factors VII and X), plasminogen, factor XIII, and antithrombin III did not change during the treatment. All patients developed a proteolytic activity demonstrable on both unheated and heated fibrin plates. The fibrinogen decreased successively to very low levels, and parallel to this an increase in fibrin/fibrinogen degradation products was found. The factor VIII and factor V activities decreased immediately after each Lysofibrin infusion but normalized rapidly again. The factor VIII molecule, however, retained its reactivity to rabbit antiserum against factor VIII. Immediately after the plasmin infusion a decrease of both alpha2-macroglobulin (alpha2-M) and the rapidly reacting alpha2-antiplasmin was observed. alpha2-M decreased successively and in several of the patients values were unmeasurable for a period of some days. A complex formation between pig plasmin and the alpha2-antiplasmin was demonstrated in crossed immunoelectrophoresis. The complexes were rapidly cleared from the circulation. No interaction between the pig plasmin and the inhibitor of the plasminogen activation, alpha1-antitrypsin or inter-alpha-inhibitor, was found.


[Spectral characteristics of human white blood cells fluorochromed with acridine orange]. It has been shown that the ratio between the intensity of luminescence band in the red spectrum region (640 nm) and that in the green one (530 nm) of acridine orange fluorochromed cells fixed under certain conditions alpha=I640/I530 is a specific character. The latter can be used for automatic classification of bone marrow cells and perypheric blood and for diagnostics of some pathological states of the cell. It has been found that the type of the changes of the ratio of alpha=I640/I530 at photochemical bleaching of fluorochromed cells under irradiation (436 nm) depends on the level of cell differentiation. Completely differentiated mature cells are characterized by a simultaneous decrease of luminescence intensity, both in the red (640 nm) and green (530 nm) spectrum regions. In undifferentiated cells (especially at the blast stage) a decrease of luminescence intensity in the red region (640 nm) is accompanied by an increase of the luminescence intensity in the green region (530 nm) which may serve as an additional specific character. The descovere effect of photobleaching of undifferentiated cells is suggested to be due to the photodestruction of dimers of acridine orange bound with monohelical regions of DNA.


[Mechanism determining the variability of the immunologic specificity spectrum of antibodies and other immunoglobulins (theory)]. An analysis of the theories of immunity-the germ line theory, the translocations and recombinations theory, the somatic mutation theory, and the reverse translation theory advanced earlier shows that their postulates afford no answer to any of the central questions of immunology, such as: 1) The origin of genetic information which codes the entire multimillion totality of immunologically different antibodies and antigen-recognizing receptors of the immunological system B and T cells. 2) The causes of sharp differences in both, the resolving power and mechanisms of recognition of antigenic determinant by antibodies and B cell receptors, on the one hand, and of macromolecular antigens as such by antigen-recognizing receptors of T cells, on the other 3) The essence of the mechanisms by means of which the T cell receptors recognize and distinguish the macro-molecular antigens as such. A new theory is advanced which in terms of the principle of cross stereocomplementarycity determining the regularities of mutual specific recognition by polynucleotides and polypeptides coded by them and also on the basis of some biophysical, virological phenomena explains the physico-chemical and genetic basis of immunological phenomena mentioned above.


Acute phase reactant proteins in prostatic cancer. A profile of acute phase reactant proteins has been studied in patients with cancer of the prostate as an aid to diagnostic staging and therapy control. A linear discriminant function analysis incorporating serum acid phosphatase, prealbumin, alpha1 antitrypsin, alpha1 acid glycoprotein and haptoglobin allows the correct identification of metastatic disease in 88.6% of patients. The profile may also serve to augment other parameters in the assessment of the physiological effect of oestrogen treatment and show whether the prescribed medication is being taken.


Metastatic orchioblastoma: alpha1-fetoprotein in diagnosis and combination chemotherapy in treatment: a case report. A case in which orchioblastoma metastasised to a lymph node at the left renal hilum is described. The metastasis was not detected by bipedal lymphography, but was associated with a rise in the serum level of the alpha1-fetoprotein. The metastasis responded to combination chemotherapy, including adriamycin, and residual tumour was excised at laparotomy. Tumour regression was associated with a fall in serum alpha1-fetoprotein which may be of value as a tumour marker substance in the diagnosis and monitoring of therapy in this tumour.


Serum dihydrotestosterone in patients with benign prostatic hypertrophy. Serum dihydrotestosterone (DHT) and testosterone were measured by radioimmunoassay in 24 normal men aged 60 to 80 years and in 25 patients with benign prostatic hypertrophy in the same age range. There was a significant increase in the level of dihydrotestosterone in patients with prostatic hypertrophy but no significant difference was found between the testosterone levels of the 2 groups. The scatter of the individual values for DHT indicated that this measurement has little practical clinical application in the diagnosis of prostatic hypertrophy. It is suggested that the increase in DHT may be due, at least in part, to a prostatic contribution to the circulating level and the relevance of this observation in relation to the aetiology of benign prostatic hypertrophy is discussed.


Neuronal transport in salamander nerves and its blockade by colchicine. Neuronal transport and the effects of colchicine on it has been studied in salamander spinal nerves. Cholinesterase (ChE) accumulation above the cut region of a nerve at 12.5 degrees C was shown to depend upon two processes. One caused a transient increase which declined to zero by 24 h; the other was explained by axoplasmic transport. At 22 degrees C the transient change was not observed, but the rate of accumulation attributable to transport increased. The Q10 for this transport over the range 12.5 degrees C--22 degrees C is approximately three. The ChE accumulation in the sensory component of the mixed nerve was about equal to that in the motor. The rate of fast axoplasmic transport of labeled leucine was 56 mm/day at 22 degrees C; if ChE moves at the same rate, then only 7% of the total enzyme is carried by fast axoplasmic transport. The transport of ChE was reduced by at least 50% when nerves were bathed in a 75 mM solution of colchicine for 30 min; this treatment is known not to cause subsequent degeneration of these nerves. The rate of slow flow of labeled material after bathing the nerve trunk in tritiated colchicine was found to be approximately 0.5 mm/day.


The visual cortex of the opossum: the retrograde transport of horseradish peroxidase to the lateral geniculate and lateral posterior nuclei. The visual cortex of opossum was studied by injecting horseradish peroxidase into the cortex and identifying labeled neurons in the thalamus. The results show that the lateral geniculate nucleus projects to area 17 in a topographical manner: the rostral lateral geniculate is represented in caudal striate cortex, and the dorsal extremity of the lateral geniculate, which probably corresponds to the zero vertical meridian, is represented along the border of area 18. Small injections in area 17 produced restricted bands of labeled neurons across the medial-lateral extent of the lateral geniculate, suggesting a greater precision in the topography than previously shown by retrograde degeneration studies. Following injections into area 17, labeled cells were also found in the lateral posterior nucleus. Injections of peristriate cortex produced labeled cells in the lateral posterior nucleus, as well as the lateral intermediate, posterior and intralaminar nuclei. Since the lateral posterior nucleus receives visual projections from the superior colliculus, the results show two visual pathways: the geniculo-striate path projecting just to core area or area 17, and a more diffuse parallel path that projects to both the core and belt. Whether or not this overlap is characteristics of the mammalian prototype it seems to be present in widely separated species.


[Isolation of alpha-1-macroglobulin (alpha 1 M) and alpha-2-macroblogulin (alpha 2 M) from rabbit blood]. A purification process of rabbit alpha 1 M and alpha 2 M from plasma was described: first the platelets, the fibrinogen, the plasminogen and the low-density lipoproteins were eliminated; then alpha 1 M and alpha 2 M were purified by gel filtration on Sephadex G 200 and by chromatography on DEAE-cellulose. These purified materials were then isotopically labeled allowing the study of the proteins metabolism.


Tumour antigens associated with primary mediastinal choriocarcinoma. Various biochemical markers of cancer were investigated in two men aged 26 and 45 years with primary mediastinal choriocarcinoma. The daily excretion of urinary human chorionic gonadotropin (HCG) and the serum concentration of the beta-subunit of HCG were elevated in both patients, but carcinoembryonic antigen, Regan isoenzyme, alpha1-fetoprotein, serum pregnancy-associated globulin and human chorionic somatomammotropin were not detectable. Comparison of the results of the investigation of biochemical markers of this rare neoplasm in these two men with those published previously illustrates the discordance in the expression of biochemical markers of primary mediastinal choriocarcinoma.


Clinical pharmacology of bleomycin following intravenous infusion as determined by radioimmunoassay. The clinical pharmacology of bleomycin administered by continuous intravenous infusion over a 4 to 5 day period was examined by nine patients. Patients receiving 30 units per day attained an average steady state plasma level of 145.8 (+/- 43.1) ng/ml bleomycin. Elimination of bleomycin was initially described by first order rate kinetics (t 1/2 = 1.32 +/- 0.39 hour). However, at times greater than 12 hours following termination of infusion, a second elimination phase was observed (t 1/2 = 8.9 +/- 2.7 hour). There was also a high correlation between renal bleomycin clearance and creatinine clearance. The importance of renal clearance was indicated in a patient with renal impairment. This patient attained a steady state bleomycin concentration of 1046 ng/ml and exhibited a terminal elimination half-life of 33 hours. Overall plasma clearance of bleomycin (Qbeta) was generally greater than renal clearance, indicating that a nonrenal clearance mechanism was also important in bleomycin elimination. This nonrenal mechanism became especially apparent during renal failure.


Combination chemotherapy with bleomycin, cyclophosphamide and dactinomycin for the treatment of osteogenic sarcoma. Thirteen patients with osteogenic sarcoma were treated with multiple drug chemotherapy consisting of bleomycin, cyclophosphamide and dactinomycin. The dosage schedule used was: bleomycin 12 mg/m2/day, cyclophosphamide 600 mg/m2/day, and dactinomycin 450 microgram/m2/day. All drugs were given intravenously for two consecutive days. Treatment was repeated every 2 weeks. Toxicity included severe nausea and vomiting (managed with antiemetics and intravenous hydration) and manifestations of bone marrow depression. Of 13 patients, eight were previously treated with high dose methotrexate with citrovorum factor rescue, cyclophosphamide and Adriamycin. Of these eight, three patients had objective evidence of tumor regression (37.5%). Five of five previously untreated patients had objective evidence of tumor regression. The overall response rate in osteogenic sarcoma patients to BCD was 61.5%. The combination of BCD appears to be more active against osteogenic sarcoma than cyclophosphamide alone or Adriamycin alone. The relative safety with which BCD can be administered makes this combination a valuable adjunct to high dose methotrexate with citrovorum factor rescue and Adriamycin in the treatment of osteogenic sarcoma.


Bleomycin-sensitivity test: application for human squamous cell carcinoma. Bleomycin (BLM)-inactivating enzyme activity, which is probably a parameter for the efficacy of this antibiotic in cancer therapy, was determined in biopsies from human carcinomas in the head and neck region. Twenty-three cases were studied. It was found that highly differentiated as well as moderately differentiated squamous cell carcinomas had low extractable activities of this enzyme, comparably to those found in normal skin tissue. Slightly differentiated as well as undifferentiated carcinomas (only one case) had increased enzyme activity. Parallel experiments estimating the total extractable thiol content in the biopsies gave no obvious correlation. The results are discussed in the light of the reported BLM efficacy in the treatment of differently differentiated squamous cell carcinomas.


Multiple agent chemotherapy including bleomycin of non-Hodgkin's lymphoma during pregnancy. The association of non-Hodgkin's lymphoma and pregnancy is a rarely reported occurrence. We describe the case of a female patient in which a wide-spread lymphoma of mixed lymphohistiocytic diffuse type was diagnosed in the second trimester of the pregnancy. Combination chemotherapy (cyclophosphamide, vincristine, prednisone and bleomycin) was started at approximately the twenty-first week of fetal life. A prompt remission was obtained and a normal full term infant was born. Chemotherapy of progressive lymphoma in women late in pregnancy appears to be rational with little risk of fetal malformation or morbidity.


Comparison of oncophilic radiopharmaceuticals, *I-fibrinogen, 67Ga-citrate, 111In-bleomycin, and *I-bleomycin in tumor-bearing mice. The pharmacokinetics of 67Ga-citrate, 111In-bleomycin, *I-bleomycin, and *I-fibrinogen were compared in a murine KHJJ tumor model in order to assess their relative potential as agents for in vivo detection of cancer. Although all four agents have been reported to be clinically efficacious, in this tumor model, *I-fibrinogen and 67Ga-citrate had the greatest tumor accumulation with maximum concentrations of 11.7% and 10.5% respectively. However, both these radiopharmaceuticals cleared slowly from the blood and animal. The maximum tumor concentrations of 111In-bleomycin and *I-bleomycin were 2.9% and 2.6% respectively, but *I-bleomycin had the advantage of rapid clearance from the blood and animal. 67Ga-citrate did not achieve its maximum tumor concentration until 24 hours after administration, whereas the other radiopharmaceuticals achieved maximum tumor concentration within several hours of administration. From these observations 123I-bleomycin seems to deserve clinical trials in patients. 123I-fibrinogen appears to have significant oncophilic potential if its clearance from the animal can be accelerated without altering its accumulation in the tumor.


Physicochemical approach to the purification of human alpha1-fetoprotein from the ascites fluid of a hepatoma-bearing patient. A method for the purification of human alpha1-fetoprotein from the ascites fluid of a hepatoma-bearing patient is described that is capable of yielding large quantities of pure alpha1-fetoprotein within a relatively short period of time. The technique is based entirely on the physicochemical properties of the alpha1-fetoprotein molecule and uses sequential purification steps: ion-exchange chromatography on DEAE-Sephadex A-50, molecular-sieve chromatography on Sephadex G-200, negative-affinity chromatography on Sepharose-Blue Dextran, positivepaffinity chromatography on concanavalin A-Sepharose and, finally, molecular-sieve chromatography on Sephadex G-100. The efficiency of the entire procedure in its present form is 15% of the alpha1-fetoprotein activity of the starting preparation from ascites fluid. The purity of the final product was shown by polyacrylamide gel electrophoresis, radioimmunoelectrophoresis, and determinations of the NH2-terminal and COOH-terminal amino acid residues of the alphs1-fetoprotein isolated. Amino acid analysis of the final product revealed a composition very similar to those reported for alpha-fetoprotein preparations that have been previously isolated by the use of immunochemical technology.


[Carcinoma of the thyroid gland in childhood]. In a surgical series of 54 carcinomas of the thyroid in children under 15 years of age (22 males, 32 females), differentiated carcinoma was disclosed in 50 cases. Of these, the pattern was purely papillary in 5, purely follicular in 3 and mixed, i.e. papillary and follicular, in 42 cases. There was an admixture of a solid component in 25 of the mixed cases. In one case the tumour was solid medullary carcinoma with amyloid and 3 cases appeared to be anaplastic carcinomas. In 21 cases the material available for microscopical examination was both that of the primary focus and of a metastasis. In only 2 of such cases the pattern was identical in the primary carcinoma showed a purely follicular pattern in the metastases. In the remaining 18 cases the primary carcinomas appeared as structurally uniform but showed an admixture of the other differentiated component or of a solid one in the metastases. The reverse also occurred in some cases. Therefore it appears to be impossible to assess the pattern of the primary growth from its metastasis. No essential difference was disclosed in the tendency of histologically different neoplasms to metastasize. During orientational histological examination of the removed glands intrathyroidal metastases were revealed in 66%. The discrimination between the two extreme and consequently even the mixed variety of differentiated carcinomas is of no practical significance in childhood because there is no obvious correlation between the histological pattern and formation of metastases, as well as between histology and induction of hormonal function. Age and sex seems to play a more significant role in the biological behaviour in children. The incidence of carcinoma is higher in females, whereas the biological behaviour seems to be worse in males.


Invasion of erythrocytes and antigenic variation. Consideration of information published on the immunology of malarial parasites has highlighted an unusual problem associated with the penetration of erythrocytes by merozoites, namely, how to reconcile strain and intrastrain antigenic variation in the parasite surface with recognition of specific receptors on red cells. This problem should now be susceptible to experimental investigation.


Proper use of skin tests with food extracts in diagnosis of hypersensitivity to food in children. This study was undertaken to determine the proper use of skin tests with food extracts in diagnosis of hypersensitivity to food in children. Cutaneous reactions evoked by graded amounts of food extracts were compared with results of double-blind food challenge and in vitro release of histamine from leucocytes. A 3 mm or greater weal reaction in skin tests by puncture technique using food extracts of 1:20 w/v concentration was found to indicate the degree of hypersensitivity likely to be associated with clinically significant hypersensitivity reactions to food. Proper use of this simple technique will facilitate accurate diagnosis of food hypersensitivity in children by identifying the group among whom all positive reactions to food challenges will be found. Nevertheless, double-blind food challenge is essential to establish a diagnosis of symptomatic hypersensitivity to food.


Determination of some L-3,4-dihydroxyphenylalanine and dopamine metabolites in urine by means of mass fragmentography. We describe a mass-fragmentographic method for determination in urine of the following metabolites of L-3,4-dihydroxyphenylalanine and dopamine: vanillactic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol. Deuterated analogs were used as internal standards. The method is fast, reproducible, sensitive, and selective, and does not require the use of time-consuming clean-up procedures. Normal excretion values in terms of creatinine, expressed as a function of age, as well as values obtained for patients with neurogenic tumors, a patient during therapy with L-3,4-dihydroxyphenylalanine, and a patient receiving dopamine are presented and discussed.


Serum alphafetoprotein in cystic fibrosis of the pancreas. Serum alphafetoprotein concentrations were measured by three different types of radioimmunoassay in 30 patients with cystic fibrosis of the pancreas and in 55 controls. The highest value obtained in cystic patient was 10.2 ng/ml and in a control 10.8 ng/ml. These are within published normal limits. Previously reported large increases in serum AFP in patients with cystic fibrosis and in heterozygote carriers have not been confirmed.


Antigenic analysis of the IgA component of LDH-IgA immunoglobulin complexes. LDH-IgA complexes present in some human sera were purified by using affinity chromatography on 5'-AMP-Sepharose 4-B. The IgA component of the purified complexes was analysed in haemagglutination-inhibition tests. Antigenic determinants specific for alpha1 and alpha2 heavy chains and for kappa and lambda light chains were found. Earlier studies suggested that the IgA component is of kappa light chain type only. These different results are discussed. It is suggested that the LDH association site is located in the Fab fragment of the IgA component.


Intra-vital staining as an aid to parotid gland surgery. Successful parotid surgery depends on the careful identification and dissection of the facial nerve, and to prevent recurrence all tumours should be excised with a wide margin of normal tissue. These two requirements are facilitated by the technique of intra-vital staining of the parotid gland with 2.5% methylene blue solution.


Malaria antigen-specific T-cell responsiveness during infection with Plasmodium falciparum. Protective immunity against Plasmodium falciparum develops only after several years of repeated exposure to the malarial parasite. We therefore investigated the possibility that acute malaria was associated with malarial antigen-specific immunosuppression. Peripheral lymphocytes of West Africans with and without P. falciparum infections were tested for their in vitro proliferative responses to a preparation of P. falciparum antigen. There was no significant difference between the magnitude of the proliferative response of lymphocytes from infected as compared to normal Africans, although the responses from both African groups were significantly higher than responses from a group of European controls. Furthermore, no soluble inhibitor of antigen-specific proliferation was present in plasma of infected patients. These observations strongly suggest that if the sluggish development of protective immunity in malaria is based upon infection-related immunosuppression, this occurs without affecting the proliferative responsiveness of specific sensitized, circulating T cells. Preliminary observations also indicate that Europeans residing in Africa and taking malaria prophylaxis may acquire sensitized T cells without experiencing clinically apparent infections.


Blocking of histamine release from human basophils in vitro by the ATPase inhibitor, ethacrynic acid. Ethacrynic acid, a known inhibitor of both Na+--K+ and Mg2+-activated ATPases, effectively inhibits histamine release from antigen-challenged human basophils in vitro. Ouabain, an inhibitor specific for Na+--K+-activated ATPases, shows no effect upon the quantity of histamine released from the antigen-challenged basophils. Ethacrynic acid also effectively inhibits Ca2+--ionophore A23187-induced release, implying it inhibits the Ca2+-dependent secretory stage of the histamine-release process. Inhibition of ATPases and histamine release by ethacrynic acid both require the presence of the olefinic bond in the ethacrynic-acid molecule. Possible utilization of analogues of ethacrynic acid as anti-allergic drugs and as a device to investigate the ATPase system of histamine-releasing cells is suggested.


Cell-mediated hypersensitivity in multiple sclerosis and other neurological diseases. Using a direct macrophage migration inhibition test the hypersensitivity against encephalitogenic protein and phytohaemagglubinin in normal persons, multiple sclerosis patients and patients with other diseases of the central nervous system were examined. It proved that the vast majority of patients were sensitised to brain antigen. The percentage of positive tests and the percentage of migration inhibition was related to the activity of the disease. No differences were found between lymphocytes of multiple sclerosis patients and of patients with the other neurological diseases patients. Foetal calf serum was proven to depress the hypersensitivity to phytohaemagglutinin as did multiple sclerosis serum on normal lymphocytes. The results did not support the hypothesis that multiple sclerosis is caused by a cell-mediated auto-immune process.


EEG and haemodialysis. A structural survey of EEG spectral analysis, Hjorth's EEG descriptors, blood variables and psychological data. Computerized EEG was performed in 20 patients with renal failure before and after haemodialysis (HD), applying spectral analysis and Hjorth's EEG descriptors in EEG quantification, correlation and factor analysis as statistical procedures to analyse the connections of EEG, blood variables and psychological performance. The main results were: (1) Moderate uraemic encephalopathy -- according to Kiley's (1971) standards -- was present in most of our patients, before and after HD. (2) Before HD, EEG slowing was most strongly connected with the creatinine level and EEG acceleration with hyperkalaemia, which in most cases accompanied a high urea level. (3) Significant EEG changes after HD were: decrease of percentage delta activity, increase of Hjorth's 'mobility', decrease of Hjorth's 'complexity'. (4) The theta/alpha ratio (Matousek 1968) was significantly correlated with the patient's general clinical state after HD. (5) Visual discrimination, memory and maximal tapping speed improved significantly after HD. Only Hjorth's EEG parameters were correlated with test performance in that patients with low voltage and fast EEGs did worse in visual discrimination.


EEG spectra in XYY and XXY men.

EEG spectra in XYY and XXY men. Ten XYY, 13 XXY, and 45 control XY men were located in a birth cohort of tall men (non-institutionalized), and their EEGs were subjected to computer analyses. The XYY men showed a significantly slower alpha activity and more power in the 3.3--9.6 c/sec band than their XY controls. There were no consistent EEG differences between the XXY men and their controls.


Correlations between psychophysical magnitude estimates and simultaneously obtained auditory nerve, brain stem and cortical responses to click stimuli in man. Responses from the auditory nerve, brain stem auditory nuclei and cortex, as well as subjective responses to click stimuli at 10 intensities, were recorded simultaneously in the same human subjects. For various measures of the responses, the power-law exponents of their intensity functions were calculated, along with their statistical significances. The electrophysiological and psycho-physical functions were compared for similarity. On average, the exponents of the intensity functions of amplitudes of the auditory nerve and earlier brain stem responses were highly significant, showing similarity across subjects and similarity with the exponents of the subjective estimates. However, a closer examination proved this similarity to be superficial, since magnitude estimates showed an appreciable intersubject and intersession variability while the auditory nerve and brain stem responses were approximately constant. All other electric response measures either had exponents which were not significant or showed even poorer correlation with the subjective response. It is proposed that the type of electrical activity recorded in this study may not be the proper set of neural parameters which give rise to the loudness estimate.


The influence of natural sleep on focal spiking in experimental temporal lobe epilepsy in the monkey. In 12 rhesus monkeys, made epileptic by injection of alumina cream into the temporal lobe structures, the changes of spike frequency were studied in wakefulness and natural sleep on 78 occasions. Five monkeys had a focus in the lateral surface of the temporal lobe and seven had a focus in the mediotemporal structures. Basic cortical and subcortical EEG patterns, recorded through chronic indwelling electrodes, showed fairly consistent changes in the course of natural sleep, allowing a classification of the sleep into stages. During slow wave sleep, the focal spikes increased in all 12 cases. The increase was more prominent during the light stage of slow wave sleep in the neocortical foci and during deep stage in the mediotemporal foci. During REM sleep, the mediotemporal foci showed a marked decrease of spiking, whereas changes in the neocortical foci were inconsistent.


An objective indicant of binocular vision in humans: size-specific interocular suppression of visual evoked potentials. Evoked cortical potentials (VEPs) to grid patterns flashed to one eye were suppressed in amplitude when grid patterns were continuously presented to the other eye. The degree of interocular suppression of VEPs was influenced by the stereoacuity of the subjects. VEPs were obtained to each of two grid sizes flashed to one eye (individual squares subtending 15 and 60 min of arc) and changes in amplitude of these VEPs were considered as a function of four stimuli continuously presented to the other eye (diffuse light, 15, 30, and 60 min of arc squares in grids). Interocular suppression of VEPs was greater (a) when the continuously presented grid was of high (38.00 mL) as compared to low (00.38 mL) intensity, (b) when the continuous and flashed grids were of the same as compared to different sizes, and (c) in six subjects who had good as compared to six subjects who had poor binocularity. Eleven of the twleve subjects could be classified correctly as having good or poor binocularity on the basis of statistically significant interocular suppression of VEPs. The results were interpreted in terms of centrally located binocular neurons responsive to specific grid sizes or spatial frequencies and the decreased functioning of such neurons in subjects with poor binocularity.


Changes in visual evoked potentials of schizophrenics after addition test. Visual evoked potentials were obtained for schizophrenics, neurotics and patients with affective disorders, and comparison between those before and after an addition test for 15 min was made. Reduction in amplitude and prolongation of latency of the potential were observed in schizophrenics after the test, while there was no change after that in neurotics and patients with affective psychosis. In schizophrenics, 4 types of changes in amplitude of the potential could be seen after the test. Namely, marked reduction occurred in A1 (A1 type), in A3 with fast recovery (A3f type), in A3 with slow recovery (A3s type), and in A5 (A5 type). (A1, A3, A5: Averaged potentials evoked by 1c/sec flashes which started 1 min, 3 min and 5 min after the end of the test.) Each type closely corresponded to paranoid schizophrenia, atypical hebephrenia, typical hebephrenia and simple schizophrenia, respectively.


Short latency potentials recorded from the neck and scalp following median nerve stimulation in man. Short latency evoked potentials were recorded from sites overlying the cervical and thoracic vertebrae, the clavicles, mastoid processes and cerebral cortex, following percutaneous stimulation of median nerve fibres at the elbow, wrist and fingers in 23 normal subjects. At least four major early components each with simultaneous positive and negative constituents, plus the first component (N20) of the cortical response, were all found to be mediated by sensory afferent fibres with conduction velocity 65--75 m/sec in the forearm of one subject. Study of the distribution of these potentials, using reference electrodes located at Fz or over the lower part of the spine, has led to the proposal of generator sites in the brachial plexus (N9), spinal roots or dorsal columns (N11), spinal grey matter or brain stem (N13), and brain stem or thalamus (N14). Comparison with intrathecal recordings in man lends support to the view that N11 and N13 are generated in or adjacent to the spinal cord. It is hoped the findings may extend the clinical applications of a non-invasive technique for investigating the afferent sensory pathways in man.


Electroencephalographic studies with i.v. methaqualone in man. Methaqualone injected i.v. has been reported to be a safe short-acting anaesthetic agent and a muscle relaxant in man. It has been employed in a variety of operative procedures and to modify convulsions in the management of tetanus and in electroconvulsive therapy. Safety, usefulness and the marked central effects of the drug led us to study its central effects by EEG in man. Discrepancies between clinical and EEG signs were thus seen. EEG patterns resembled those after barbiturates and the effect was dose dependent. Differences between methaqualone and barbiturates are discussed. The EEG patterns were potentiated by thioridazine and antagonised by imipramine.


Automatic noctural sleep sampling: a useful method in clinical electroencephalography. The clinical usefulness of obtaining a sleep record in epileptic patients is well established. However, in many clinical EEG laboratories, it is difficult to obtain records at night during the patient's natural sleep. Thus, most sleep EEGs are recorded in the EEG laboratory during daytime and are induced by drugs. A 16 channel cable-telemetry system is described which automatically samples throughout the night the EEG of patients while sleeping in their room or ward without the aid of any medication. The electrodes and the cable-telemetry unit are applied to the patient in the afternoon and the system is checked. The patient is then instructed to plug the system into a wall box upon retiring and to disconnect it when he wakes up in the morning. The EEG samples are automatically written out during the night on one of the EEG machines located in the EEG laboratory to which the system is connected via spectral intra-hospital wiring. The duration of the samples and of the intervals separating them can be varied according to the circumstances. We have found this method to be very helpful in the investigation of epileptic patients, especially those considered for surgical treatment.


A quantitative measurement of tremor.

A quantitative measurement of tremor. A quantitative measurement of tremor was achieved by means of separate analysis of frequency and amplitude, using an EEG spectrum analyser and an amplitude histogram plotter (giving distribution curve), with magnetic tape storage. Tremors were registered bilaterally from most proximal finger joints and elbow joints.


Inhibition of antibody production in plasmacytoma cells by antigen. Tissue culture-adapted MOPC 315 mouse plasmacytoma cells which secrete monoclonal anti-DNP IgA antibody, were cultured with a variety of DNP-carrier conjugates and the production of IgA measured by pulse labeling with [3H]leucine and by plaque-forming cell assays. DNP-coupled bovine, human and rabbit gamma-globulins (containing 9 to 12 DNP groups per 50 000 mol wt of carrier) inhibited the synthesis and secretion of IgA by 40 to 80%. This inhibition was specific, since unconjugated globulins were ineffective, non-IgA proteins and DNA synthesis remained unaffected, and DNP-globulins had no effect on X5563 cells which do not bind DNP. The degree of suppression of antibody production dependen on the concentration and epitone density of the antigen and on the duration of exposure of cells to it, and was no attributable to absorption of secreted antibody by cell-bound antigen. Comparably substituted DNP conjugates of F(ab')2 and intact globulins were equally inhibitory. The inhibition of antibody synthesis by DNP-BGG was reversible following removal of the antigen. This phenomenon is similar in many respects to the antigen-induced blockade of normal antibody-secreting cells, and provides a valuable model system for analyzing the mechanisms of antigen-mediated cellular inactivation.


Analysis of the alloimmune properties of a recombinant genotype in the major histocompatibility complex of the chicken. In a population of (CB X CC)F1 X WB hybrids, a chicken was found with a presumably recombinant haplotype, BR1, whose antigenic products detectable by hemagglutination contained determinants derived from both parental haplotypes, i.e. B1 (from CB) and B2 (from CC). This recombinant bird and its progeny from different crosses were tested by skin grafting, graft-vs.-host (GVH) and mixed lymphocyte reactions (MLR). The results define two regions of the B system, the B-F and the B-G. The B-F region determined serologically defined antigens, histocompatibility antigens, and controlled the GVH and MLR reactions, while the B-G region was responsible only for synthesis of serologically detectable antigens.


A myeloma hybrid producing antibody specific for an allotypic determinant on "IgD-like" molecules of the mouse. A hybrid cell line was produced by fusing a mouse myeloma line with spleen cells from BALB/c mice immunized with B10 cells. The hybrid line grew in tissue culture and in syngeneic mice and produced IgM antibody specific for "IgD-like" molecules of mice with the Igb haplotype. The concentration of monoclonal antibody in the serum of tumor-bearing animals reached about 2 mg/ml and gave cytotoxic titers of up to 1:800 000. The derivation of the line, some properties of the antibody secreted and the nature of its antigenic target are described.


Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyosine50. IV. In vitro activity and immunochemical properties. The responsiveness of BALB/c spleen cells to the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT), the copolymer of L-glutamic acid50-L-tyrosine50 (GT) and the corresponding complexes with methylated bovine serum albumin (MBSA) has been studied in vitro in a modified Mishell-Dutton culture system. Cultures of BALB/c spleen cells respond to GAT, GAT-MBSA and GT-MBSA over a wide dose range. Contrary to in vivo findings BALB/c spleen cell cultures respond to GT in vitro although in a much narrower dose range. Lymphoid extracts from GT-primed BALB/c or B10.BR mice (GT-TSF) specifically suppress the GT-MBSA response in vitro. This culture system has allowed us to further investigate immunochemical properties of both BALB/c and B10.BR GT-TSF-GT-TSF from both strains display affinity for GT-Sepharose and bear determinants encoded by the I region of the H-2 complex. Moreover, we demonstrate that the suppressive activity from B10.BR mice is a molecule or a molecular complex which displays affinity for GT and bears determinants of the I-J subregion of the H-2k haplotype.


Ly and Ia phenotype of suppressor T cells induced by graft-vs.-host reaction. Suppressor T cells were generated in (CBA X C57BL)F1 mice undergoing graft-vs.-host (GvH) reaction and treated with anti-Ly and anti-Ia antisera to determine their membrane antigen phenotype. Pretreatment of CBA-induced GvH suppressor T cells with anti-Ly-1.1, anti-Ly-2.1 or anti-Iak antisera plus complement abrogated their suppressive properties when tested against primed F1 spleen cells in vitro. In contrast, concanavalin A (Con A)-induced suppressor T cells were insensitive to anti-Ly-1.1 serum. It is concluded that GvH-induced suppressor T cells are Ly-1+,2+,3+,Ia+, and thus distinct from Con A or antigen-induced suppressor T cells.


Identification of distinctive stromal elements in erythroid and neutrophil granuloid spleen colonies: light and electron microscopic study. Erythroid and granuloid spleen colonies from 1100 R irradiated mice transfused with 5 X 10(4) syngeneic bone marrow cells, fixed in paraformaldehyde-glutaraldehyde followed by osmium tetroxide, were processed for light and electron microscopy. Light microscopic examination of day 7 erythroid colonies revealed distinctive reticular cells indigenous to the erythroid colony; they were not observed outside the confines of the erythroid colony or in granuloid colonies or within the spleens of control irradiated mice that had not been transfused with bone marrow cells. Examinations of sections stained with a newly developed differential stain for thick sections (1 mu) revealed unique staining properties for these reticular cells. Ultrastructural features and the intimate contact of the proerythroblasts and the reticular cells are described. Microscopic examination of day 10 neutrophil granuloid spleen colonies harvested from polycythemic mice showed distinctive stromal cells, different from those of erythroid colonies. These distinctive cell to cell relationships constitute a possible morphological basis for the function of the erythroid and granuloid hemopoietic inductive microenvironments.


Myeloid membrane markers. I. Expression of marker antigens specific for murine granulocytes and their precursor cells. Bone marrow cells were investigated by immunoelectromicroscopy and by quantitative photometric immunoradioautography with a rabbit antiserum against murine bone marrow cells. The serum was absorbed with murine spleen, liver and thymus cells until it no longer reacted with thymocytes and lymph node cells in a quantitative complement fixation test. The antiserum stained granulopoietic but not erythropoietic or lymphopoietic cells. The density of the myeloid antigen on single cells increased with the differentiation from immature to mature granulopoietic cells. While the increase of label was statistically not significant at the level of differentiation from promyelocyte to myelocyte and metamyelocyte to band neutrophil, there was a remarkable gain of label from myeloblast to promyelocyte and from band neutrophil to segmented neutrophil. This was evident under the electron microscopy using peroxidase-labeled antibodies and could be measured quantitatively with photometric immunoradioautography using 125I-labeled antibodies.


The staining of starch gels with Coomassie brilliant blue G250 perchloric acid solution. Coomassie Brilliant Blue G250 dissolved in dilute perchloric acid solution was used to stain protein bands in starge gels. The bands were visible within min, no destaining was required and the gels could be stored indefinitely.


Feulgen positive nucleoli in Epon semithin sections: fact or artifact? Epon semithin sections from glutaraldehyde fixed tissues show stained nucleoli after treatment with the Schiff's reagent. The possibility that aldehyde groups already present could account for this positive staining reaction is briefly discussed.


Function of macrophages in genetic control of immune responsiveness. Macrophages serve an essential but poorly understood role in the cellular and molecular events that underlie immune competence. Antigenic proteins are now known to bind initially to macrophages prior to their recognition by T lymphocytes. Antigen uptake by macrophages is a metabolism-dependent event that results in an association of the antigen or a fragment thereof with a product of genes linked to the major histocompatibility complex of the species. For recognition of this associative form of antigen and self to result in cell proliferation, a direct physical interaction of antigen-bearing macrophage and lumphocyte must occur. Soluble forms of the altered antigen complexed to self may, however, function in nonproliferative T cell activation phenomenon. Using antigens of defined structure, it is possible to derive data which indicate that genetic control of immune responsiveness resides at the level of the antigen-presenting cell, thus indicating that these latter cells have profound discriminatory influences on host immune competence.


Cell-mediated immunity in vasectomized rhesus monkeys. Cell-mediated immunity in rhesus monkeys that had been vasectomized for 2, 4, 7, or 11 years was measured by lymphocyte blastogenesis following stimulation with concanavalin A, phytohemagglutinin (PHA), and pokeweed mitogens. Several of the 7- and 11-year vasectomized animals had significantly reduced PHA reactivity when compared with control animals, and the percentage of animals with reduced PHA reactivity increased with time after vasectomy.


Plasma beta-thromboglobulin in diabetes mellitus. The plasma beta-thromboglobulin (betaTG) content was measured in 56 diabetic patients with known complications of this disease, including neuropathy, retinopathy, and ischemic skin lesions. Although two patients were found to have elevated levels beyond the normal range, there was no significant difference between the diabetic group as a whole and the group of 35 controls. The significance of these findings with regard to the proposed contribution of small-vessel platelet sequestration in the pathogenesis of late complications of diabetes mellitus is discussed.


Replication of primate oncornavirus SSV-1 on MRC-5 human diploid cells. MRC-5 human diploid cells infected with Simian Sarcoma Virus from woolly monkey (SSV-1) were not transformed but an efficient replication of non transforming SiLV was demonstrated. Increase of virus reverse transcriptase activity paralleled cell replication during successive passages. Preliminary results concerning the influence of viral infection on the life span and the karyotype of MRC-5 diploid cells will be reported and several implications of these findings discussed.


Inhibition of leucocyte migration by potassium chloride extracts of human tumor cells in ascites and from tissues. Human gastric cancer-associated antigens were solubilized from fresh ascites and surgical specimens by 3M KCl extraction. The antigenicity of these extracts was measured by leucocyte migration inhibition assay with an improved modification devised in this laboratory. Antigens extracted mutually reacted with autologous and allogeneic leucocytes taken from cancer patients. The out of 11 (27%) patients and 7 out of 28 (25%) patients with various cancers showed positive migration index values against 13 ascites tumor cell antigens in the autologous and allogeneic reaction systems, respectively. Reactions were positive in 4 out of 10 patients with gastric cancer against autologous antigens, and in 10 out of 26 (38%) of the patients against allogeneic tumor antigens. On the other hand, one patient with gastric cancer reacted well with various sources of antigens extracted from allogeneic cells of gastric cancer, while the other patient with gastric cancer did not react with any of five gastric cancer antigens tested so far. These findings suggested that the cell-mediated immune response of patients with gastric cancer against autologous and allogeneic tumor antigens extracted was detectable in the test system. It is considered at present that the eM KCl antigen may be relate to human leucocyte antigen, and some common antigenic reactants capable of reacting with several gastric cancer may be involved in this reaction.


Reverse transcriptase and thymidine kinase as markers for tumorigenicity and viral contamination of cells. Continuous monitoring of enzymes, particularly those involved in nucleic acid synthesis could be a useful means of detecting infections and abnormalities in cells in culture. Model systems using mouse (3T3), human (MRC-5) and chick embryo cells infected with RNA tumour viruses were studied. Reverse transcriptase activities were determined by the incorporation of (3H) nucleotides into synthetic primer-templates or into complementary DNA of endogenous RNA and characterised by their specificity for primer-templates dT12-18.rAn, dG12-18.rCn, dT12-18.DAn and dG10.rCmn, their requirements for metal ions and inhibition by antisera. Measurement of reverse transcriptase is a more sensitive method than the COFAL test for the detection of RAV infection of chick cells. Iododeoxyuridine, bromodeoxyuridine and dexamethasone, which can induce latent C viruses, have no effect on MRC-5 cells; no increases in reverse transcriptase were detected and no C particles were seen by electron microscopy. Solid tumours developed in immunosuppressed mice injected s/c with 3T3 and MRC-5 cells chronically infected with MLV but none formed after injection of cells or virus suspension alone. Thymidine kinase activities of WI-38 and MRC-5 cells are greatly increased by infection with CMV or transformation with SV40. Mammalian tumours and tumour cell lines also show a high specific activity of cytoplasmic thymidine kinase.


Tests for RNA tumor viruses in cell substrates used in virus vaccine production. Test to detect RNA tumor viruses in cell substrates were reviewed with respect to advantages and disadvantages. Viral induction, electron microscopy, reverse transcriptase assays, density gradient centrifugation, and radioimmunoassays were performed on several cell substrates now in use or being considered for use in vaccine production. RNA tumor viruses were not detected by any of the methods except in the positive control cultures.


Inhibitory effect of tumor-bearing state on the generation of in vivo protective immune T cells in a syngeneic murine tumor system. Tumor-specific immunity mediated by thymus-derived lymphocytes (T cells) was established in C3H/He mice against syngeneic X5563 plasmacytoma. Splenic T cells from mice, which exhibited resistance to tumor challenge, revealed a potent in vivo tumor-neutralizing activity as well as in vitro cytotoxicity. In contrast, spleen cells from mice 7 days after tumor cell inoculation (7-day tumor-bearing mice) exhibited no protective activity when assayed by in vivo tumor-neutralization test, whereas these cells exhibited almost comparable in vitro cytotoxic activity to that from the tumor-resistant mice. Any suppressor cell activity was not detected in 7-day tumor-bearing animals. While a slight in vivo protective activity was observed in the spleen cells from 14-day tumor-bearing mice, this activity was still significantly weaker than that of spleen cells from mice similarly inoculated with tumor 14 days before but whose tumor had been removed 7 days before the assay. The development of in vivo protective immunity in tumor-resected mice was suppressed by intravenous inoculation with 7000 R X-irradiated tumor cells. These results indicate that in vitro reactivity of immune T lymphocyte population is not always correlated with in vivo protective immunity, but there is a substantial decrease in in vivo immune capability of T cells from tumor-bearing animals, and that this suppression may be ascribed to the presence of a large amount of tumor-associated transplantation antigens rather than to suppressor cell activity.


Immunohistochemistry of carcinoembryonic antigen: characterisation of cross-reactions with other glycoproteins. In the course of demonstrating carcinoembryonic antigen (CEA) in normal human small intestine cross-reactivity of specific antiserum against red blood cells, vascular endothelium, and Paneth cell granules was noted. Pretreatment of sections with periodic acid eliminated these cross-reactions without affecting the staining of CEA, indicating that the antigenic determinants shared between CEA and other glycoproteins are in the carbohydrate portion of the molecules. These findings emphasise the caution with which immunohistochemical results should be regarded even when they are apparently well controlled.


Myopathy in Whipple's disease.

Myopathy in Whipple's disease. We report a patient with Whipple's disease who developed a myopathy that improved during antibiotic therapy. The muscle biopsy showed mild type 2 fibre atrophy, type 1 fibre preponderance, variability in fibre size, and changes in the myofibrillar pattern of affected fibres. Interfascicular macrophages contained PAS-positive material. With the electron microscope these macrophages contained membranous inclusions and bacillary bodies, similar to those seen in the jejunal biopsy.


Effect of histamine receptor antagonists on bile damage to the gastric mucosa of canine Heidenhain pouches. The net ionic fluxes of H+ and Na+ were examined in Heidenhain pouches during damage with sodium taurocholate solution. The increase in ionic flux which occurred with taurocholate could be prevented in animals given H1 and H2 histamine antagonists in combination. The results suggest that histamine mediates changes in the transmucosal ionic flux produced by bile acid.


Inhibitory effect of various cytostatics and cycloheximide on acute experimental pancreatitis in rats. Cycloheximide, 5-azacytidine, and 4-methoxybenzoyl-beta-bromoarylate (Cytembena) block the development of experimental acute pancreatitis in rats when mediated by the administration of 5% bile solution into the pancreas in vivo. Six hours after drug treatment the pathological changes, evaluated macroscopically or using histological sections of the pancreas, were significantly decreased. The drugs affected the amount of abdominal fluid and lowered its lipase and amylase activity. The known inhibitory mechanism of the active drugs and the possible advantage of cycloheximide for clinical use are briefly mentioned.


A receptor for 'self' on lymphocytes.

A receptor for 'self' on lymphocytes. A large population of lymphocytes is able to form rosettes with syngeneic, allogeneic or closely related xenogeneic erythrocytes. Similar results were found with spleen cells from mice, rats and rabbits. The highest numbers were found in mice where up to 30% of lymphocytes bound autologous erythrocytes. Rosette formation is probably due to stereospecific cell surface receptors since erythrocytes of distant xenogeneic origin were not recognized. Rosette forming cells do not seem to be restricted to the B-cell or T-cell compartment since mouse thymus cells as well as spleen cells from congenitally athymic (nude) mice bound erythrocytes to a similar degree.


Marsupial immunoglobulins: the distribution and evolution of macropod IgG2, IgG1, IgM and light chain antigenic markers within the sub-class Metatheria. The distribution within Australian and American marsupials of the heavy and light chain antigenic markers identified by antisera to purified quokka (Setonix brachyurus) immunoglobulins is described. Markers for IgM and IgG2 constant region determinants as well as for light chains were widely distributed in Australian species and were also detected in Didelphis, the American opossum, thus indicating a long-term structural conservatism of some immunoglobulins within the marsupials. More detailed analysis of the distribution of quokka IgG2 determinants by quantitative precipitation and sequential absorption procedures suggested that there had been a gradual and cumulative acquisition of these markers with time. The presence of IgG2 markers in species separated for 130 million years (quokka and opossum) suggested that IgG2 was the ancestral IgG present before the divergence of these separate lines. The origin of IgG1 remains obscure as it appears to be limited to a small group of closely related diprotodont marsupials suggesting a recent origin.


Antigens in penicillin allergy. I. A radioimmunoassay for detection of penicilloylated protein contaminants in penicillin preparations. This communication presents a sensitive and discriminative method for detection of protein impurities in penicillin preparations. Antibodies against various penicilloyl derivatives of high avidities and specificities raised in rabbits were coupled to microcrystalline cellulose. The amount of penicilloyl antigen present in a sample was calculated from the relative uptake of a radioiodinated penicilloylated albumin competing with the sample for binding to the antipenicilloyl immunosorbent. As little as 0.048 pmol/ml of penicilloylated human serum albumin could be detected. The accuracy of the determination was within +/- 23% (p less than 0.05). The pronounced specificities against the penicillin side chains demonstrated by the various immunosorbents were not displayed by the antibodies in passive cutaneous anaphylaxis experiments in guinea pigs. Furthermore, the immunosorbents showed the same pattern of specificity against monomeric penicillins as for penicilloylated proteins, but the former were considerably less efficiently recorded. The relatively small quantities of protein impurities in penicillin preparations, however, necessitated a separation from penicillin prior to analyses with the RIA. This was accomplished by fractionation on Sephadex G-50 fine, ginving a recovery of 80-90% of 0.1-2.5 ppm of penicilloylated protein.


Rosette-forming mast cells in rat anaphylaxis. Immunological characteristics of mast cell rosettes. Peritoneal mast cells from immunized rats can form rosettes with antigen-coated sheep red blood cells. The receptor responsible for this active rosette formation is shown to be IgE cytophilic antibody: rosettes are inhibited by previous contact of mast cells with antigen, or with anti-IgE antiserum; the kinetics of mast cell rosettes following a primary immunization with ovalbumin and Bordetella pertussis vaccine is similar to the kinetics of reaginic antibody response. Furthermore, a reaginic serum can induce passive rosette formation. There is no correlation between cell-bound and circulating IgE.


III. Mediators of allergic reactions. Endogenous and exogenous stimulating or suppressor substances. Mediators of experimental hypersensitivity pneumonitis. Using a physiologic model of hypersensitivity pneumonitis where depressions of arterial oxygen tension in unimmunized rabbits are monitored following aerosol challenge with Aspergillus terreus spores, attempts were made to assess the nature of the cellular and pharmacologic mediators of the impairment. Unlike normal animals no PaO2 depressions were obtained following aerosol challenge in either rabbits deficient in C6 or in rabbits made thrombocytopenic with antiplatelet serum. Such aerosols were also shown to produce platelet count depressions of up to 45% in normal rabbits. Finally, in vitro evidence of histamine release was obtained following incubation of Aspergillus extract, platelets and autologous serum from unimmunized rabbits. It was concluded that Aspergillus-induced pulmonary disease may be initiated by platelet release of mediators such as histamine stimulated by nonspecific complement activation.


Enhancement by a serum factor of immunoglobulin-mediated histamine release from human leukocytes. When serum was fractionated on Sephadex G-200, the material eluted in the second and thir major peaks had a very pronounced capacity to enhance IgE- as well as IgG-mediated histamine release from the leukocytes of both normal and allergic donors. Unseparated serum on the other hand had a low capacity to stimulate anti-IgE-induced histamine release. Besides resulting in a higher histamine release, pretreatment with serum fractions also increased the rate of histamine release. Further purification revealed that the stimulating activity of the material in the second peak was mediated by a trypsin-sensitive component, probably active in low concentration.


Stimulation of the in vitro immune response by factors present in the human serum. Two distinct factors which stimulate the recovery of plaque-forming cells in the vitro immune response have been found in human serum. One acts in the presence of fetuin and the other augments the recovery of plaque-forming cells in its absence. The molecular weight of the former is higher than 1 X 10(5). The two factors could be separated from each other by ammonium sulphate fractionation. Large differences were found in the amount of these factors in sera obtained from different human donors.


Sodium fluoride--a stimulus for a calcium-triggered secretory process. Calcium triggers the secretion of histamine from mast cells after previous exposure of the cells to sodium fluoride. The secretory process can be divided into a fluoride-activating step and a calcium-induced secretory step dependent on cellular metabolic energy. The secretory response induced by compound 48/80 in the absence of extracellular calcium was found to decrease after fluoride pretreatment of the cells. The response was, however, unaffected, provided calcium was introduced to the cells simultaneously with compound 48/80.


Non-responder basophils in highly ragweed-sensitive subjects. 12 of 93 patients with untreated seasonal ragweed rhinitis, strong skin sensitivity and 2+ to 4+ RAST gave insignificant (less than or equal to 14%) histamine release from their leukocytes on challenge with ragweed allergen.


Antigens in penicillin allergy. II. The influence of the number of penicilloyl residues on the antigenicity of macromolecules as determined by radioimmunoassay (RIA), passive cutaneous anaphylaxis (PCA) and antibody induction. The present communication reveals a relationship between the epitope density of penicilloylated protein antigens and their antigenic activities in a radioimmunoassay (RIA), in passive cutaneous anaphylaxis (PCA) and in inducing antibody formation in mice. In the RIA and PCA a critical number of 2-4 penicilloyl residues per protein molecule was noted. At this level small changes in the number of substituents considerably influenced the antigenic activities. The molecular weight and the nature of the carrier proteins, myoglobin, bovine serum albumin (BSA) and dimeric BSA also affected the threshold concentration for efficient antigenic activity. The results with the RIA and PCA were significantly correlated to each other. Using penicilloylated BSA as immunizing antigen in mice it was found that an epitope number higher than 11 penicilloyl residues per protein molecule induced significant antibody formation after a single injection. Antigens with a lower degree of penicilloyl substitution were less immunogenic. An antigen carrying 0.6 penicilloyl residues per BSA molecule did not induce penicilloyl-specific antibodies even after three injections. The capacity of heavily penicilloylated proteins to induce and elicit penicillin allergy as revealed by the present results stresses the importance of limiting their presence in penicillin preparations.


Serum-derived immunosuppressive substances. III. Regulation of the immune response by human serum alpha-globulin fractions: the dose-response relationship. Suppression of the plaque-forming cell response in mice following injection of substances fractionated from pooled normal serum alpha-globulin was investigated. The dose-response relationship for fractions obtained by ion-exchange chromatography show that a single preparation has both enhancing and suppressive activities which are revealed at different doses. Whether this observation reflects the sum of activities of a number of molecular species remains to be determined. The immune responses to both thymus-dependent (heterologous erythrocytes) and thymus-independent (DNP-ficoll) antigens are suppressed while the response to the thymus-independent antigen lipopolysaccharide is enhanced. Thus, the cellular locus for immunosuppression cannot be exclusively on the T cell, and the magnitude of the action of the two populations (T and B cells) remains unclear.


The effect of phosphatidyl serine on the activation stage of histamine release induced by neutrophil cationic protein. Histamine release from rat mast cells induced by cationic protein (band 2) from rabbit neutrophil lysosomes occurs in Ca++-deficient medium. At higher concentrations of Ca++ the release is inhibited. Strontium not only supports, but also enhances the release of histamine in the absence of Ca++. Progressive enhancement of release occurs between 1.8 and 14.4 mM Sr++. The release of histamine from mast cells, activated at low temperature (0-4 degrees C) in the presence of 14.4 mM Ca++ and then washed prior to incubation at 37 degrees C, is inhibited. However, if phosphatidyl serine (PS) (10 microgram) is present with 14.4 mM Ca++, the inhibition is reversed. There is also inhibition of release when cells, activated in the presence of 1.8 mM Ca++, are incubated in the second stage with 14.4 mM Ca++, but this inhibition is less pronounced than when the 14.4 mM Ca++ is in the activation stage. PS enhances the release in the presence of both Ca++ and Sr++. The presence of PS in the activation stage enhances the release, but there is no significant enhancement when cells activated in the absence of PS are washed and incubated in the presence of PS. This suggests that PS enhancement of histamine release occurs at the activation stage, probably through the efficient delivery of calcium to the membrane sites, thereby increasing the efficacy of the membrane perturbation by band a protein.


Pharmacologic profile of a new antiallergic compound PRD-92-Ea. PRD-92-Ea [5,5-Dimethyl-11-oxo-5H, 11H-(2) benzopyrano (4,3-g) (1) benzopyran-9-carboxylic acid ethanolamine], was an active antiallergic compound in rat and monkey experimental models of immediate hypersensitivity. It inhibited, in a dose-dependent manner, the rat PCA reaction after both intravenous and oral administration. It also inhibited the degranulation of rat peritoneal mast cells after antigenic challenge. PRD-92-Ea was also active in preventing bronchoconstriction in Ascaris-sensitive Rhesus monkeys after intravenous, topical and oral administration. Using chopped monkey tissues, it was found that PRD-92-Ea prevented histamine release from the respiratory mast cells, but not from the cutaneous mast cells. No reason for this dichotomy of effect is known. PRD-92-Ea showed antagonistic activity against the allergic mediators released from mast cells. In order of decreasing potency it was active against SRS-A (monkey lung), PGF2alpha, PGE2, serotonin, bradykinin and histamine. Apart from its antiallergic effects PRD-92-Ea had no other significant pharmacological activity.


Studies on the mechanism of tachyphylaxis to disodium cromoglycate. The tachyphylaxis to disodium cromoglycate's (DSCG) inhibition of antigen-induced histamine release is readily demonstrable utilizing passively sensitized rat lung fragments. This tachyphylaxis to DSCG is evident whether or not calcium is present during drug preincubation. An attempt to relate the mechanism of tachyphylaxis to the DSCG-induced release of an endogenous cellular inhibitory material was unsuccessful insofar as could be demonstrated by an effect on mediator release.


Further studies on the tachyphylaxis to DSCG. The effects of concentration and temperature. The tachyphylaxis to DSCG's inhibition of anaphylactic histamine release has been demonstrated in passively sensitized rat lung fragments. The induction of tachyphylaxis appears to depend on the concentration of the drug and the length of pretreatment. Tachyphylaxis is relatively independent of the concentration of the second exposure to DSCG. The development of tachyphylaxis is highly temperature dependent; it can be prevented by cooling the tissues to 2-4 degrees C after a brief (30 sec) preincubation with DSCG. It is suggested that DSCG inhibits histamine release by binding to 'receptor' site(s). Once the site is occupied by DSCG, it is modified by a temperature-dependent process, thus losing the ability as a 'receptor' for inhibition of histamine release. The tachyphylaxis is the result of such a modification.


Differences in antigenicity of Escherichia coli strains isolated from patients with various forms of urinary tract infections. The antibody amounts to crude O antigens were analyzed using the enzyme-linked immunosorbent assay (ELISA) and the indirect hemagglutination (IHA) technique in 42 sera from 6 rabbits. The rabbits were hyperimmunized with one of the WHO Escherichia coli standard strains O1, O2 or  O75. In the antibody assays the O antigens were obtained from E. coli strains isolated from 15 patients with asymptomatic bacteriuria (ABU), from 3 patients with acute pyelonephritis and from the WHO E. coli strains used for the immunization. The strains from the patients belonged to serogroup O1, O2 or O75. Significantly higher IgG and IgM antibody levels were detected with the ELISA when using the O1 and O2 but not O75 antigens from the pyelonephritis and WHO standard strains than when using the antigens from the ABU strains. Using the IHA technique the O1, O2 and O75, IgG and IgM antibody titers against the antigens from the pyelonephritis and WHO standard strains were significantly higher than those against the antigens from the ABU strains. In inhibition experiments the antigens from the strains causing pyelonephritis inhibited the reaction between antigen on solid phase and antibodies in solution much more efficiently than did antigens from strains causing ABU.


Antigenic correlations between chicken brain organelles, thymocytes and bursacytes. Rabbit antisera to chicken whole brain and brain microsomes, synaptic membranes, and synaptic vesicles were used in cytotoxicity and immunofluorescence assays against chicken bursacytes and thymocytes. Brain organelles, bursacytes and thymocytes have shown to possess common antigenic determinants. The common antigen is present in small amounts on thymocytes and can be demonstrated only by immunofluorescence.


Demonstration of epidermis-specific heteroantigens in thymic epithelial cells. Heterologous anti-epidermal cell serum was obtained by immunizing rabbits with enzymatically dispersed, viable epidermal cells of guinea pigs, followed by absorption of the antiserum with red blood cells (sheep and guinea pig), lymphoid cells and liver powder (guinea pig). Immunofluorescence demonstrated that the antiserum reacted specifically with stratified squamous epithelial cells and thymus epithelial cells including Hassall's bodies of the guinea pig, monkey and man. It is suggested that the epithelial cells of the skin and thymus have common heteroantigens.


IgE-type antibodies to Ascaris antigens in man. IgE antibodies to Ascaris antigens were detected by radioallergosorbent test (RAST) in the serum obtained from a person experimentally sensitized with Ascaris suum antigens prepared from the body fluid of worms. This serum passively sensitized human skin in vitro and histamine release from the sensitized skin was observed by challenge with antigens. The allergenicity of A. suum antigens was present over a very broad spectrum of molecular size. The present results suggest that specific IgE antibodies to Ascaris antigens might be in Ascaris-infected patients and allergic symptoms which belong to type 1 reaction might be mediated by IgE in these patients.


Pharmacologic regulation of antigen-induced mediator release from canine lung. The ascaris antigen-induced release of histamine and a slow-reacting substance of anaphylaxis (SRS-A) from passively sensitized fragmented canine lung is further characterized. Histamine and SRS-A were released within 30 sec of antigen challenge, reached a maximum at 7 and 10 min, respectively, and thereafter appeared to remain constant to 30 min. Contractions of guinea pig ileum produced by canine SRS-A were competitively antagonized by the SRS-A antagonist FPL-55712. Indomethacin and deuterium oxide enhanced antigen-induced SRS-A release from canine lung but had little effect on histamine release. The ability of several chemically novel 'antiallergic agents' to inhibit mediator release was evaluated. Inhibition of histamine release, and to a lesser extent SRS-A release, by one of these compounds was shown to vary with time and temperature. It is concluded that fragmented canine lung, while disclosing some qualitative pharmacological differences from other species, is a useful in vitro model of immediate hypersensitivity reactions.


Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique. In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.


Intravesical pressure in benign prostatic hypertrophy. Synchronous pressure and flow studies were carried out on 51 men over 50 years of age. The subjects were divided into groups based on degree of bladder outlet obstruction. Eight patients were also examined six to ten weeks after prostatectomy. Following multiple pressure and flow recordings, the mean premicturition resting intravesical pressure, the mean intravesical pressure at the commencement of voiding (the commencing intravesical pressure), the mean maximum intravesical pressure and the mean intravesical pressure at the end of voiding (mean cessation intravesical pressure) were determined for each subject. The maximum intravesical pressure varies in the same individual. A mean maximum intravesical pressure of 75 mm Hg and above indicates moderate or severe bladder outlet obstruction. However, this pressure is not of value in the determination of clinically doubtful or mild obstruction. None of the other intra-vesical pressures is of value in the diagnosis of bladder outlet obstruction. Six to ten weeks following successful prostatectomy the mean maximum intravesical pressure fell to normal limits.


Diagnostic and prognostic value of the cystograms of patients with prostatic adenoma: distance between symphysis pubis and urinary bladder. Presurgical cystograms of prostatectomized patients were compared as regards their diagnostic and prognostic usefulness. Because of the lack of fully objective prostatic adenoma symptoms, the authors concluded the distance between the urinary bladder and symphysis pubis to be an important radiological symptom in prostatic adenoma, being the measure of inflammation of the prostate and tissues of this region.


Studies on human hyperplastic prostates maintained in organ culture. Prostates from patients with prostatic hyperplasia were maintained in organ culture for periods up to 8 days. Explants were disaggregated and studied after 2, 4, and 8 days of culture, as well as before culture. Although the data varied from patient to patient, short periods of culture (2 to 4 days) resulted in increased numbers of cells with histochemically demonstrable acid phosphatase; however, by 8 days of culture, cells with histochemically demonstrable acid phosphatase decreased in number. The ratio of acid phosphatase to beta-glucuronidase expressed per cell or per milligram of protein decrease; with time in culture.


Effectiveness of small-group self-instruction in radiographic anatomy. A small-group self-instructional program with 11 slide-tape units was designed to teach medical students radiographic anatomy concurrently with their course in gross anatomy. The effectiveness of the program was examined by administering an objective multiple choice examination at three medical schools. One school offered the slide-tape program alone; one, the slide-tape program plus lecture-demonstrations; and one acted as a control. The results indicate that students will, on their own, learn some radiographic anatomy. With exposure to an objectives-based, small-group slide-tape program, the students can learn significantly more. Such a slide-tape program plus lecture-demonstrations can further enhance learning. Student attitudinal results were uniformly positive toward this slide-tape program.


Intracellular reduction of the cupric ion of bleomycin copper complex and transfer of the cuprous ion to a cellular protein. The cupric ion of the bleomycin copper complex has been shown to be reduced and transferred to a cellular protein by the following mechanism: Bleomycin approximately Cu2+ leads to Bleomycin plus Cu+ X Cu+ plus M leads to M approximately Cu+. The intracellular reducing agents (X) are suggested to be sulfhydryl compounds, because their action is inhibited by N-ethylmaleimide. The active group of the cellular protein (M) that binds with the cuprous ion is suggested to be a sulfhydryl group. The action of the bleomycin copper complex in causing DNA fragmentation in cells can be explained by the mechanism presented in this paper. This mechanism in cells is also supported by the temperature dependency of the action of the bleomycin copper complex on cells.


Amplifications of phleomycin and bleomycin-induced antibiotic activity in Escherichia coli by aromatic cationic compounds. A wide range of aromatic compounds has been shown to amplify phleomycin-induced cell killing in Escherichia coli. They include acridines, acridinium chlorides, dihydroanthracenes, anthracenes, dianthracenes, phenanthridinium salts, phenazinium chlorides, phenoxazones, triphenyl methane dyes, benzoquinolizinium chloride, diphenylmethane derivatives, stilbene and diphenyl derivatives. Low concentrations of these amplifiers also amplified the DNA breakage and degradation effects of phleomycin. The minimum structural specification for activity as an amplifying agent is suggested. A representative sample of compounds effective as amplifiers of phleomycin also amplified the antibiotic effects of bleomycins B4 and B6. The amplifiers described are known to vary in their ability to penetrate and accumulate in different organisms or tissues. This suggests the possibility of developing a series of antibiotic regimes using these amplifiers (or the large number of derivative compounds also likely to be active) where the therapeutic index is determined by the properties of the amplifier chosen rather than of the phleomycin or the bleomycin.


The revised structure of capreomycin.

The revised structure of capreomycin. The total structures of capreomycins IA and IB were determined mainly from the results of the comparative study on the NMR spectra of capreomycins and tuberactinomycins, resulting in a revision of the formerly proposed structure. The beta-lysine residue as a branched part was revealed to be linked with the beta-amino group of the alpha, beta-diaminopropionic acid residue in the cyclic pentapeptide moiety in a different manner than in tuberactinomycins.


Electrogenic behavior of synaptic vesicles from Torpedo californica. Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles.


A total internal-reflection technique for the examination of protein adsorption. A total internal-reflection fluorescence (TIRF) technique for examining protein adsorption at solid-liquid interfaces is described. For the representative case of adsorption of bovine gamma-globulin onto silicone rubber, the applicability of the technique to serveral important aspects of protein adsorption phenomena is demonstrated. Specifically, in addition to the tightly adsorbed protein layer observed herein and previously by maany others, the presence of a loosely adsorbed protein layer was also noted under conditions of either static or flowing protein solutions. Taking advantage of the continuous real-time measurements possible with TIRF, a rate constant for the protein adsorption step during laminar flow was estimated at both 25 and 37 degrees C. These results serve to demonstrate that the TIRF technique represents a significant and versatile new tool for the study of protein adsorption phenomena.


Adsorption of plasma proteins on hydrophobic surfaces. I. Albumin and gamma-globulin. The adsorption of albumin and gamma-globulin from solution at the liquid/air and liquid/liquid interfaces has been studied by the pendant drop technique as a model for the solid-liquid system. It is concluded that each protein adsorbs in a constant manner at the air, isooctane, and methylene iodide interfaces at equilibrium (1 hr), and the surface tension components of the protein layer presented to the hydrophobic phase have been found. The range of hydrophobic surfaces for which such constant behavior might be expected has been deduced, and it is shown that most common polymers lie within this range. A new model for protein adsorption is proposed, and the implications for biomaterials are discussed.


Stimulation of heme accumulation and erythroid colony formation in cultures of chick bone marrow cells by chicken plasma. A fibrin clot culture system with high plating efficiency is described for the growth of erythroid cells from chick bone marrow. Erythroid colonies grown in the absence of adult chicken plasma (spontaneous colonies) were either benzidine-negative or weakly benzidine-positive. Colonies grown in the presence of chicken plasma were 90% strongly benzidine-positive and 40% more abundant than spontaneous colonies. Plasma from anemic chickens was more effective than control  plasma in inducing heme accumulation (heme-stimulating activity) and in increasing the number of erythroid colonies (colony-stimulating activity). Spontaneous colonies from 48-h cultures were transformed into benzidine-positive colonies by exposing them for 6-10 h to chicken plasma.


Identification of a chromosome that controls malignancy in Chinese hamster cells. A chromosome that controls malignancy in Chinese hamster cells has been identified by analysis of the Giemsa banding pattern of a malignant cell line transformed by simian virus 40 (SV40), non-malignant revertants from this line, segregants from the revertants that were again malignant and a cell line transformed by methylcholanthrene. The malignant cell line transformed by SV40 was near diploid and had gained additional material of chromosome 3. Revertants with a suppression of malignancy and malignant revertants from which they were derived. Malignancy of these cells was associated with the ability to form colonies in agar. Cells of a line transformed by methylcholanthrene were malignant, formed almost no colonies in agar and the only chromosome change from the normal diploid chromosome banding complement was the addition of a long arm of chromosome 3. The results indicate that chromosome 3 carriers gene(s) that control malignancy in Chinese hamster cells in cell lines transformed by a viral or a chemical carcinogen and that malignancy was induced in both cell types by an increase of these genes.


Clinical evaluation of commercial conjugates for direct immunofluorescence of herpes simplex virus. Commercially prepared antibody conjugates that are now available allow extensive application of direct immunofluorescence for diagnosing herpetic infections. Five of these conjugates--Microbiological Associates bivalent herpes antiserum (MBA-bivalent), MBA anti-herpes types 1 (MBA-1) and 2 (MBA-2), and Flow Laboratories anti-herpes types 1 (Flow-1) and 2 (Flow-2)--were compared for antibody titer, diagnostic accuracy, and nonspecific background staining on known infected tissue. Four of these were tested simultaneously on 75 clinical specimens. No attempt was made to differentiate between herpes simplex virus type 1 or type 2 infections. All antisera were comparable in titer and exhibited fluorescence with both herpes simplex virus types 1 and 2 antigens. The most significant difference was excessive nonspecific staining consistently seen with the MBA-2 conjugate. Overall, Flow-2 and MBA-bivalent antisera were preferable.


Characterization of the binding of a potent synthetic androgen, methyltrienolone, to human tissues. The potent synthetic androgen methytrienolone (R 1881), which does not bind to serum proteins, was utilized to characterize binding to receptors in human androgen responsive tissues. Cytosol extracts prepared from hypertrophic prostates (BPH) were utilized as the source of receptor for the initial studies. High affinity binding was detected in the cytosol of 29 of 30 samples of BPH (average number of binding sites, 45.8+/-4.7 fmol/mg of protein; dissociation constant, 0.9+/-0.2 nM). This binding had the characteristics of a receptor: heat lability, precipitability by 0-33% ammonium sulfate and by protamine sulfate, and 8S sedimentation coefficient. High affinity binding was also detected in cytosol prepared from seminal vesicle, epididymis, and genital skin but not in non-genital skin or muscle. However, similar binding was demonstrated in the cytosol of human uterus. The steroid specificities of binding to the cytosol of male tissues of accessory reproduction and of uterus were similar in that progestational agents were more effective competitors than natural androgens. Binding specificities in cytosol prepared from genital skin were distinctly different and were similar to those of ventral prostate from the castrated rat in that dihydrotestosterone was much more potent than progestins in competition. Thus binding of R 1881 to the cytosol of prostate, epididymis, and seminal vesicle has some characteristics of binding to a progesterone receptor. When the nuclear extract from BPH was analyzed, high affinity binding was demonstrated that conformed to the specificities of binding to an androgen receptor. Here dihydrotestosterone was a more potent competitor than progestational agents. Similar patterns of binding were detected in the crude nuclear extracts from seminal vesicle, epididymis, and genital skin but not in uterus, muscle, or non-genital skin. We conclude that the androgen receptor is not demonstrable in the cytosol of prostate, epididymis, or seminal vesicle of non-castrated men but can be measured in the cytosol of genital skin and the nuclear extracts of androgen responsive tissues. Because steroid hormones exert their major influence within the nucleus of target tissues, the measurement of nuclear receptor may provide valuable insight into the regulation of growth of target tissues.


Immunologic and nonimmunologic generation of superoxide from mast cells and basophils. Mediator release from rat peritoneal and human lung mast cells as well as human leukemic basophils was examined to determine whether super-oxide (O(-) (2)) was concomitantly generated. Immunologic or nonimmunologic stimulation of each preparation induced parallel release of histamine and O(-) (2) within 2 min. O(-) (2) production was quantitated by superoxide dismutase (SOD)-inhibitable chemiluminescence and cytochrome c reduction. SOD was detected in basophil and mast cell lysates and was also released by rat mast cells stimulated by anti-IgE. Secretory granules isolated from purified rat mast cells released histamine, O(-) (2), and SOD upon exposure to cations. Thus, both superoxide radicals and SOD may play a role in host defenses involved in immediate hypersensitivity reactions.


Bleomycin-induced diffuse interstitial pulmonary fibrosis in baboons. Pulmonary fibrosis was induced in eight baboons with bleomycin; five untreated animals were controls. After 45-65 U/kg of bleomycin, lung volumes and diffusing capacity were reduced, and static lung pressure-volume curves were shifted to the right. Right middle lobes were resected at this time in five bleomycin-treated and two control animals. Compared to controls, right middle lobes from bleomycintreated animals had increased weight and contained increased amounts of total protein, collagen, elastin, and DNA; synthesis of collagen and noncollagen protein were also elevated. Occasional alveolar septae were edematous and infiltrated by mononuclear inflammatory cells; a slight increase in collagen was demonstrable histologically. Four of six treated animals died with extensive diffuse interstitial fibrosis after 95 U/kg of bleomycin. Biochemical analyses revealed significantly elevated lobar contents of dry weight, protein, elastin, and collagen. Two animals survived 95 U/kg of bleomycin and were terminated 6 mo after treatment. In these animals, physiologic studies were indicative of restrictive lung disease, but lung histology was nearly normal. Lung weight, total protein, and DNA had returned to control values, but collagen and elastin were increased in amount and concentration. Bleomycin induces an intense inflammatory response in the lung. During this inflammation, connective tissue proliferation occurs in concert with proliferation of other tissue components. Cessation of bleomycin treatment is followed by resolution of inflammation manifested by decreases in tissue mass, cellular content, and nonconnective tissue protein. Collagen and elastin deposited during inflammation are less successfully removed during resolution, leading to a stage characterized by increased concentrations of these proteins. A similar sequence of tissue alterations may occur in idiopathic diffuse interstitial fibrosis of man in response to various lung injuries.


Human papovavirus in Papanicolaou smears of urinary sediment detected by transmission electron microscopy. A technique is described whereby inclusion-bearing cells identified by light microscopy in stained smears of urinary sediment were reprocessed for examination in the electron microscope. The nuclei of the abnormal cells were found to contain numerous virus particles, 35 nm in diameter, which morphologically resembled papovaviruses. The technique was applied in this case to identify further the virus producing the cytopathic changes in the Papanicolaou smear. It could be particularly valuable for retrospective studies of mounted cytological or histological material when suitable specimens are no longer available for virological investigation.