Various types of corticotectal neurons of cats as demonstrated by means of retrograde axonal transport of horseradish peroxidase. The retrograde labeling of cortical neurons with horseradish peroxidase (HRP) was used to investigate the morphological features of neurons in various cortical areas projecting to the superior colliculus in the cat. Corticotectal cells were found to be labeled in layer V of the entire cerebral cortex. The number of labeled cells and their locations varied according to the sites of injections of HRP in the colliculus. Most of the corticotectal cells identified in the present study were small (9--20 micrometer in diameter, 66%) and medium (20--40 micrometer, 30%) pyramidal neurons and only 4% of them were large (more than 40 micrometer). The labeled cells, 261 in total number, had somal diameters of 20.8 +/- 8.0 micrometer (mean and SD). The range of sizes of the labeled neurons was different in different cortical areas. For example, the labeled neurons in the Clare-Bishop area had a greater proportion of large diameter cells than in other areas. The present findings are largely in agreement with the previous data of anterograde degeneration methods with respect to the topographical correlation of the corticotectal projections. However, in some cortical areas, e.g., the sensorimotor and the first visual (area 17) cortex of the lateral surface of the hemisphere, relatively small numbers of corticotectal neurons appear to have been labeled by retrogradely transported HRP. The sparsity of the labeled neurons in certain cortical areas may reflect the existence of corticotectal neurons with axon collaterals supplying brain structures other than the superior colliculus.


Cortical neurons projecting to the pontine nuclei in the cat. An experimental study with the horseradish peroxidase technique. Horseradish peroxidase (HRP) injections in various portions of the cat pontine nuclei resulted in retrograde labeling of neurons in layer V of the ipsilateral cerebral cortex. Corticopontine neurons, pyramidal in type, have been found to be labeled in the entire cortex, confirming the previous findings of anterograde degeneration studies. Most (91%) of the labeled cells were 14--26 micrometer in diameter (mean 19.4 +/- 4.5 micrometer SD). Small (10--20 micrometer) and medium (20--40 micrometer) cells represent 51.5% and 47.7%, respectively, of the total number of the labeled neurons. The populations of the neurons of various sizes were almost identical in different cortical areas, and were different from the populations of corticoreticular and corticospinal cells. Corticopontine cells were well labeled in experimental cases of 3-days' survival time, confirming the topographical organization established previously by degeneration studies for this projection system. However, in cases of shorter survival time (20--27 h), the number of labeled neurons was very small. The relative paucity of labeled corticopontine neurons in the sigmoid and lateral gyri is discussed with reference to other cortical descending neurons (e.g., the corticotectal, corticoreticular and corticospinal) which have hitherto been identified morphologically as well as physiologically.


The anatomical substrate of callosal messages from SI and SII in the cat. Horseradish peroxidase (HRP) was injected into the first (SI) or second (SII) somatosensory areas of 21 adult cats. The radial and tangential (normal and parallel to the pial surface, respectively) distribution and morphology of the callosal neurons were studied. HRP injections were combined with single unit recording in the contralateral cortex in order to determine which part of the somatosensory periphery is represented within the regions containing callosal neurons, the callosal (efferent) zones, in SI and SII. The callosal zone of SI extends over the trunk and part of the forepaw representation. In the forepaw and hindlimb representations callosal neurons projecting only to the contralateral SII are found, while in the trunk representation callosal neurons projecting to contralateral SI or SII are found. The callosal zone in SII extends widely throughout the forepaw representation in this area and projects to the contralateral SII but not to SI. In both SI and SII the callosal neurons are mainly located in layer III. A few of them are also found in layer VI. They are very rare in other layers. Callosal neurons in layer III are mostly pyramidal but exceptionally stellate; in layer VI they are pyramidal, triangular, and occasionally stellate. These data indicate that transformations of the cortical somatosensory maps are achieved in the message sent through the corpus callosum. These transformations are i) determined by the extent and location of the callosal zones and perhaps by the distribution of callosal neurons within them, ii) different in different areas, iii) different in a same area, according to the cortical targets to which they are conveyed. The existence of callosal connections originated from areas of distal forepaw representation supplies a possible anatomical substrate for those types of intermanual transfer of tactile learning which depend upon the integrity of the corpus callosum.


Identification and distribution of the spinal and hypophyseal projection neurons in the paraventricular nucleus of the rat. A light and electron microscopic study with the horseradish peroxidase method. The distribution of labeled neurons in the paraventricular nucleus of the hypothalamus (PVN) was studied following injections of horseradish peroxidase (HRP) into the spinal cord (C8 to T1) or the hypophysis in the rat. Injections were also made in the spinal cord in another group of animals, which were subjected to water deprivation for a period of 3 days, and the PVN of these animals was examined with the electron microscope. Spinal projection neurons (paraventriculospinal tract, PVST, neurons) formed two groups; the dorsal and the ventral groups. They were located within the parvocellular part of the PVN and fused into one at the caudal level. The neurons of the dorsal group were well assembled whereas those of the ventral group were intermingled with paraventriculohypophyseal tract (PVHT) neurons, which were concentrated in the magnocellular part. Electron microscopic observations revealed that HRP-labeled neurons after spinal injections did not contain neurosecretory granules and that they were not affected by water deprivation. On the other hand, neurons containing a number of neurosecretory granules displayed a significant degree of dilatation of the endoplasmic reticulum as the result of water deprivation. These neurons contained no HRP granules. The present findings suggest that the PVST neurons are distinct from the PVHT neurons and that the neuronal groups of both systems form different cell columns within the nucleus.


An analysis of articulators.

An analysis of articulators. There are situations that do not demand absolute precision and duplication of all pathways. One example is complete denture construction. Denture base movement and tissue resiliency cannot registered accurately. Corrections for these conditions must be made directly. Instruments that use mechanical equivalents and/or accept positional registrations are suitable for this procedure. Instruments used for teaching purposes such as waxing exercises need not be capable of duplicating eccentric pathways. These instruments should be capable of demonstrating alterations of Bennett movement (immediate and progressive side shift) and protrusive inclinations. It would be most convenient and practical for dental schools to use the same instruments for complete dentures and occlusion courses. Static positional instruments are ideal for use in crown and bridge and operative departments, and in the treatment of patients in whom centric occlusion is to be preserved. Instruments that are fully adjustable for eccentric registrations are useful for demonstration purposes, postdoctoral teaching, and for patients requiring extensive reconstruction.


[Electron microscopic representation of the pili structure of Neisseria gonorrhoeae (author's transl)]. The technique of negative staining and ultra-thin section has been used for investigations of 30 Neisseria gonorrhoeae strains in order to represent the structure of pili (fimbriae) electron microscopically. The staining of the gonococci was effected by phosphotungstic acid (0,5%). The pili ascertained were 30 to 60 A thick. In course of in vitro passages up to 10. subculture morphological changes of the pili have been observed. The application of trisbuffer or solution of Hylase (hyaluronidase) showed not any improved results in comparison with buffered NaCl-solution as suspension medium. The investigation of ultra-thin sections showed that the structure of the pili could be exhibited not clearly. Therefore, these technique seems to be not suitable for qualitative representative of the pili.


Amniotic fluid levels of alpha-fetoprotein and other proteins in a case of Turner's syndrome of the fetus. A 34-year-old primigravida underwent genetic amniocentesis at 20 weeks gestation and the fetus was diagnosed as having Turner's syndrome. The concentration of alpha-fetoprotein in amniotic fluid was greatly elevated. Normal concentrations of amniotic fluid total protein, albumin and immunoglobulin indicated that the elevated level of alpha-fetoprotein was not the result of leakage through a hygroma, which has been previously suggested without supporting data in cases of Turner's syndrome of the fetus.


Alpha-fetoprotein: a marker for threatened abortion. The role of alpha-fetoprotein (AFP) as a marker for threatened abortion was investigated in 50 patients and compared with the roles of serum human chorionic somatomammotropin (HCS) or human placental lactogen (HPL) and human chorionic gonadotropin (HCG). Of the 50 patients admitted to the hospital with threatened abortion, 30 patients aborted and 20 gave birth to live-born babies. Twenty women aborted within seven days of admission. Eighty-two percent (82%) of the 20 had abnormal AFP values: one third were above the 97.5 percentile and two thirds were below the 2.5 percentile. The AFP values were normal in almost all cases in the group who aborted after seven days. The HCG level in serum was found to be the best indicator of threatened abortion. the AFP and HCS levels were equal indicators of threatened abortion, but were not as reliable as the HCG levels The AFP values are of more diagnostic use than the HCS values, however, because both abnormally low and high AFP levels can indicate an unfavorable course in pregnancy.


Ultrasonography in the detection of residual urine. Eleven patients with distended urinary bladders (10 diabetics with autonomic neuropathy and one patient with prostatic hypertrophy) were examined by ultrasonography in an attempt to define the volumes detectable by this technique. Incremental volumes of saline were instilled through indwelling Foley catheters with ultrasonographic examination at each volume. The ultrasonograms obtained were presented to two radiologists trained in ultrasonography, who were asked to identify the presence of residual urine. There was 97% certainty of recognizing the presence of 100-cc residual volumes and 100% certainty of recognizing 150-cc residual volumes. It is concluded that residual urine volumes greater than 100 cc are clinically detectable by ultrasonography, a noninvasive alternative for diagnosing this common diabetic condition.


Tissular immunoenzymatic detection of hepatic alphafetoprotein in human hepatomas. 5 human cases of hepatoma have been chosen with respect to their different seric alpha-fetoprotein (alpha-FP) level and histological characters. Cells producing alpha-FP have been studied with specific horseradish-peroxidase-labelled immunoglobulins. Ultrastructural examination shows that alpha-FP is present in the cytoplasm of some tumoral hepatocytes. alpha-FP is also present in the cytoplasm of some rare nontumoral hepatocytes of a nonsecreting hepatoma. Ultrastructural differences are described in tumoral hepatocytes according to the grade of differentiation of the tumoral cell population. alpha-FP production appears to be restricted to moderately differentiated tumoral hepatocytes. These observations led to the hypothesis that production of alpha-FP may transiently develop either during the differentiation of tumoral hepatocytes, or during the new differentiation of nontumoral hepatocytes involved in a proliferative process.


Profiles of serum complement in patients with hepatobiliary diseases. CH50 and the concentrations of C3, C4, C1 INH and factor B have been measured in sera from 34 control subjects and 178 patients with various hepatobiliary diseases, including primary biliary cirrhosis (PBC), chronic active hepatitis (CAH), cryptogenic cirrhosis (CC), alcoholic liver disease (ALD), Wilson's disease (WD), large duct biliary obstruction (LDBO) and viral hepatitis (VH). CH50 was decreased in CAH and CC. C3 was increased in PBC, LDBO and VH and decreased in CAH and CC. C4 was decreased in PBC, CAH, ALD and WD. C1 INH was increased in PBC, CAH, ALD, LDBO and VH. Factor B was increased in LDBO and VH and decreased in CC. In none of the patient groups was the mean C4 level increased or the mean C1 INH level decreased. All 5 indices of serum complement were lower in ascitic than nonascitic patients. Data on serum complement were similar in HBsAg positive and negative VH. Discriminant analysis facilitated separation of all the patient groups on the basis of complement data, except PBC and VA. Analysis of data using a within-group correlation matrix revealed a significant negative correlation between C4, the most discriminating variable of serum complement in CAH, and gamma-globulin concentration in CAH. The possible contribution of factors such as activation of complement, impaired hepatic synthesis of complement components, an acute phase response and cholestasis to altered serum complement profiles in different hepatobiliary diseases is discussed.


Studies on liver function under the influence of oral contraceptives. The effects of two sequential therapies using mestranol as an estrogen and chlormadinone acetate and norethisterone acetate, respectively, as a gestagen on the aminotransferase (GOT and GPT), alkaline phosphatase and alpha-amylase activities, the cholesterol, total bilirubin and total protein contents and the indocyanine green (Ujoviridin, VEB Chemiekombinat Bitterfeld) half-life were studied in two groups, each comprising 12 women of fertile age. The serum protein was separated by paper electrophoresis and both the thymol turbidity and zinc sulphate tests were performed during the first treatment cycle. An estrogen-induced rise in alanine aminotransferase (GPT) activity from 6.31 U/liter to 12.14 U/liter was observed during application of the mestranol/chlormadinone acetate therapy. The value dropped to 5.34 U/liter when chlormadinone acetate was administered as a gestagen in addition to the estrogen. No significant changes were noted in the other biochemical parameters. Sequential therapy involving application of mestranol and norethisterone acetate produced only statistically secured rises in the alpha 1-globulin content and the GPT activity. All other changes in the different parameters remained within the normal range and were statistically insignificant.


Biochemical and immunological heterogeneity of 100 A filament subunits from different chick cell types. The 100 A filament subunit proteins of chick fibroblasts and gizzard smooth muscle were compared. These proteins are major cellular components in these cell types, constituting up to 98% of the cell's total protein. Co-electrophoresis of cytoskeletal fractions of fibroblasts and smooth muscle revealed that the subunit proteins differed in their molecular weights: 58,000 daltons in fibroblasts and 55,000 daltons in smooth muscle. Cytoskeletal fractions from other cell types were also examined: chondroblasts contained the 58,000 dalton subunit, and cytoskeletons of skeletal muscle and cardiac muscle contained both 55,000 and 58,000 dalton proteins. Chick skin and rat kangaroo Pt K2 cells had more complex subunit patterns which resemble prekeratin. The peptide patterns resulting from proteolytic digestion of the 58,000 dalton protein of fibroblasts, the 55,000 dalton proteins of smooth muscle and PT K2 cells, and chick brain tubulin differed from one another. Two-dimensional electrophoresis of reconstituted gizzard smooth muscle 100 A filaments showed the 55,000 dalton subunit to be composed of two major components, differing in their isoelectric points. Antibodies prepared against electrophoretically purified 55,000 dalton subunit protein reacted in immunodiffusion against the original smooth muscle antigen and cytoskeletal fractions from skeletal and cardiac muscle, but not from fibroblasts, brain, liver, or skin cells. A specific antigenic determinant common to subunit proteins in smooth, skeletal, and cardiac muscle, is therefore indicated. A previously described antibody against fibroblast subunit protein reacted weakly against smooth muscle filament protein in immunodiffusion revealing the presence of a common antigenic determinant between the two subunit proteins. These data demonstrate striking antigenic and primary structural differences in 100 A filament subunits from even such closely related cell types as fibroblasts on the one hand and muscle cells on the other.


A comparative study of the efficiency of hydroxyprogesterone caproate and of chlormadinone acetate in the prevention of premature labor. The efficacy of caproate of hydroxyprogesterone and acetate of chlormadinone in preventing premature labore was compared in a controlled trial. The survey was based on 211 pregnant women with a high risk of premature delivery discovered during clinical examination. There are no significant differences between the two groups in either length of gestation, delay between the beginning of treatment and delivery or other parameters related to prematurity. The absence of evidence suggesting any significant difference between the two treatments can have three possible causes (which are discussed): the methodology, the inefficacy of the two products or the equivalent efficacy of the two products.


Immunofluorescent visualization of 100 A filaments in different cultured chick embryo cell types. Antibody prepared against the 55,000 dalton subunit of reconstituted chick gizzard 100 A filaments (anti-G55K) bound to the 100 A filaments of chick smooth muscle, cardiac muscle, and skeletal muscle cells, and to the 100 A filaments of Schwann cells and satellite glial cells of the peripheral nervous system. Anti-G55K did not bind to replicating presumptive myoblasts, fibroblasts, chondroblasts, pigment cells, neurons, or to central nervous system glial cells. This contrasted with the wider range of binding of antibody to the 58,000 dalton subunit of chick fibroblast 100 A filaments (anti-F58K) which bound to the 100 A filaments of all cell types examined except hepatocytes and skin epithelial cells. Anti-G55K) staining revealed a morphologically distinct distribution of 100 A filaments in the three types of muscle cells. Spindle shaped smooth muscle cells exhibited dense fluorescent staining near the poles of the cells, and also exhibited unique patches of fluorescent material after cytochalasin B and Colcemid treatment. In myotubes, the fluorescence was limited to longitudinal bundles of filaments between the striated myofibrils. Cardiac cells contained uniformly distributed fine filaments. Lastly, smooth muscle cells in various phases of mitosis bound the anti-G55K, whereas replicating presumptive skeletal myoblasts failed to bind the anti-G55K.


Selective expression of paternal human major histocompatibility antigens on the surface of hydatidiform mole cells. The typing of human major histocompatibility antigens (HLA) of two cases of invasive hydatidiform mole showed that the fibroblast-like cells as well as trophoblasts from the mole selectively expressed paternal HLA haplotype specificity but not maternal HLA on the surface. This result was completely in agreement with the notion of androgenetic origin of hydatidiform mole from the aspects of HLA specificity. Immunological implication of this finding and development of chorionic tumor were discussed in the light of fetomaternal relationship and host immune surveillance against the tumor.


Carcinoembryonic antigen and beta 2-microglobulin as serum tumor markers in women with genital cancer. A comparative study of carcinoembryonic antigen (CEA) and beta 2-microglobulin (beta 2-MG) in serum was made by radioimmunoassay in 77 women with genital cancer. With a positive level defined as 5 ng of CEA/ml and 3.0 microgram of beta 2-MG/ml, CEA was positive in 31% of the women with cancer of the corpus, 36% of those with cancer of the cervix and 36% of those with cancer of the ovary the corresponding figures for beta 2-MG were 6%, 27% and 56%, respectively. The additional use of beta 2-MG provided an increase in positive results, especially in cases of cancer of the ovary. A direct relationship between the extent of tumor and serum marker level was more evident for beta 2-MG than CEA. There was no correlation between serial levels of CEA and beta 2-MG in most patients. CEA levels appeared to predict subsequent tumor behavior more accurately in patients with good prognoses (ie, complete or partial tumor response), whereas beta 2-MG levels gave the same prediction in those with bad prognoses (ie, nonresponsive or progressive tumor).


[Prenatal diagnosis after abortus imminens--further progress of pregnancy and peculiarity of amniotic cell cultivation (author's transl)]. Among 1600 pregnancies, in which amniocentesis for prenatal diagnosis had been performed, were 73 cases with previous threatened abortion. In none of these abortion occurred after amniocentesis. 56 children have been born so far. There is evidence for a slight but not statistically significant increase in the number of multifactoral anomalies in this group. This study supports former findings of a higher prematurity incidence after vaginal bleeding in early pregnancy. If amniocentesis took place after imminent abortion, brownish discoloured amniotic fluid has been withdrawn in 53%. This phenomenon probably results from transfer of blood substances from hematomata between uterine wall and amniotic sac. Cultivation of amniotic fluid cells may be impaired by those degradation products, prolonging the cultivation period for an average of 2 days. Special methodological modifications to enhance the success of amniotic fluid cell cultivation in those heavily contaminated specimens are discussed. There seems to be no interference with AFP-estimation. Fetoscopy vision may be impaired.


Lactic dehydrogenase activity in degenerative neoplastic glial cells after actinomycin application in vitro. Glial tumors of glioblastoma type, cultured in vitro, have been exposed between the 7th and 14th day of growth to actinomycin C and K in a concentration of 5 x 10(-6) M. The developing degenerative changes in the neoplastic cells were observed after 6, 8, 12 and 24 hours after the addition of actinomycin to the culture of the tumor. It has been found, that the developing degenerative changes in the tumor cells are paralleled by a growing activity of the enzyme tested. The degenerative changes were described in the neoplastic cells, beginning from the accumulation of the enzyme activity in small granules of the cell processes up to very high activity of the enzyme in fragments of the breaking down cells. It is suggested that LDH activity is a good marker of cell form degenerative changes.


Influence of some aldehyde blocking agents on staining of depurinized DNA with cationic dyes. Rat liver, spleen and Walker carcinosarcoma imprints were subjected to depurinizing Feulgen hydrolysis and then treated with blocking agents of aldehyde groups. Such blockators as sodium bisulfite and hydroxylamine which multiplay additionally anionic groups in DNA and intensify the reactions with cationic dyes, ensuring anisotropic staining. Hydrazine lowers the binding of carionic dyes to DNA, instead phenylhydrazine, completely blocks both aldehyde and phosphate groups. When the imprints were treated with 2.4-dinitrophenylhydrazine, aldehyde and phosphate groups of apurinic acid were blocked, and DNA staining by cationic dyes occurred only on account of nitrogroups of the blocking agents which have been used. The staining reaction of cationic dyes after the use of anionogenic blocking agents of aldehyde groups is prospective not only for revealing DNA but also for several other compounds with natural or potential aldo- and ketogroups. However the reaction with phenylhydrazine can serve as a staining without removal of DNA prior to staining as an optional procedure.


[Percutaneous transhepatic bile duct drainage for obstructive jaundice (author's transl)]. Palliative percutaneous bile duct drainage was carried out in five patients with obstructive jaundice due to inoperable tumours. In four patients drainage was continued for a long period, in one patient for only one day. The bile duct was punctured by transhepatic cholangiography. It was then catheterised and a drainage catheter introduced into the duodenum in order to produce both internal and external drainage. This procedure, which carries few complications, produces a rapid reduction of jaundice and disappearance of pruritus. It does not increase the expectation of life, which depends on the underlying disease. Experience with this method may be of value in benign stenoses or in pre-operative drainage.


[Follow-up angiography after palliative renal artery occlusion with the GAW-(Gianturco-Anderson-Wallace)-spiral (author's transl)]. Follow-up angiograms were carried out in six patients after palliative renal artery embolisation for hypernephromas using a GAW-spiral. The following conclusions were reached: it is possible to obtain complete occlusion of the renal artery with cessation of haematuria, reduction in tumour size and in the extent of the pathological vessels. It is necessary, however, to place the spiral accurately in the vessel to be occluded and to embolise all major arteries supplying the tumour. Even when the renal artery has been completely occluded, tumour growth results in the development of varying extent. In order to reduce this collateral circulation, vessels to the tumour periphery should be embolised with a suitable substance before spiral occlusion is carried out.


[Prenatal diagnosis of congenital defects through the determination of alpha fetoprotein]. The prenatal diagnosis of neural tube defects is based on elevated alpha-fetoprotein (AFP) concentrations in the amniotic fluid as well as in the maternal serum. Amniocentesis has to be performed in those women who already gave birth to an affected child and have the high risk to bear another. On the other hand more than 90% of the newborns with anencephaly and spina bifida are born by mothers without any increased risk in anamnesis. Therefore maternal serum screening is indicated in all pregnant women to rule out a neutral tube defect. The pittfalls of a general serum screening are discussed an a survey of the datas from different international centers is presented.


[Alpha fetoprotein and HbF cells in the maternal blood and their modification through amniocentesis]. In 101 patients of the Frauenklinik der Medizinischen Hochschule Hannover we have measured the concentrations of fetal cells and alpha-fetoprotein before and after amniocentesis. There was a significant increase in both parameters corresponding to an arbitrary fetomaternal haemorrhage of 0,05--0,4 ml. AFP seemed to be more reliable than the fetal cell count. The site of placentation was of no influence on the fetomaternal haemorrhage. As less than 0,1 ml rhesus-positive fetal blood is enough to cause isoimmunization in mothers at risk we highly recommend to give immunoglobulin-anti-D to all unsensitized rhesus-negative mothers after amniocentesis.


PEP A9, a new, unstable variant in the peptidase A system. A rare peptidase A variant, tentatively designated PEP A9, was observed in six members of a German family, indicating autosomal codominant inheritance. The electrophoretic mobility is similar to that of PEP A 3-1, but it has very low in vivo stability. There is no apparent association with a disease state. A simple and sensitive staining reagent for PEP A was found in o-phthalaldehyde.


Detection of mycoplasma contamination in tissue cultures by fluorescence microscopy. The in situ staining method of Chen (1977) for the detection of mycoplasma contaminants in tissue cultures was tested in cultures of human skin fibroblasts after controlled contamination with Mycoplasma arginini. It is concluded that this method is reliable only at infection rates of 100% or higher, i.e., at one mycoplasma or more per tissue-culture cell.


Simplified cell microelectrophoretic method applied to the macrophage electrophoretic mobility test for cancer diagnosis. Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.


Split unresponsiveness to trinitrophenyl (TNP) determinant. Suppression of anti-TNP antibody responses by sensitization with picryl chloride. Contact sensitization by epicutaneous application of picryl chloride causes in mice a significant reduction of the antibody responses to immunogenic TNP-conjugates. This split unresponsiveness is along-lasting. It was found that hapten applied on the skin became attached to the serum proteins and the transfer of such a serum into normal recipients, while not influencing the ability of these animals to become contact-sensitized to PCl, rendered them unable to mount the anti-TNP antibody response. Possible mechanisms of split unresponsiveness to the TNP determinant induced by PCl treatment are suggested.


An investigation into the antigen-specificity of transfer factor in its stimulatory action on lymphocyte transformation. Dialysable transfer factor (TF) was prepared from the buffy-coat cells of donors with known cell-mediated reactivity to tuberculin (PPD), streptococcal protein (SKSD) and diphtheria toxoid (DT). The effect of such preparations on the transformation by these antigens of lymphocytes from tuberculin-negative donors was investigated. Transformation was determined as incorporation of tritiated thymidine. The concentrations of SKSD and DT were adjusted for different lymphocyte donors so as to give, in the absence of TF, a low index of transformation (less than 10-fold) comparable to that obtained with PPD. TF from tuberculin-positive donors stimulated antigen-induced transformation by on average approximately 2-fold whereas TF from tuberculin-negative donors generally had little effect. This was so not for PPD as antigen but also for SKSD and DT, and sensitivity of TF donor to SKSD of DT was not a determining factor. TF also frequently increased background transformation in the absence of antigen. Although a small effect, this ability tended to reflect the activity of TF in the presence of antigen. It is concluded that neither the whole nor any significant part of this enhancement of transformation can be ascribed to an antigen-specific factor. Tuberculin-positive donors apparently yield a higher level of non-specific factor and possible reasons for this are discussed. The factor active in transformation may be responsbile for the TF phenomenon in vivo.


Liver-specific and shared cell membrane antigens. Studies by light- and electron microscopy. Liver-specific and shared saline-insoluble cell surface antigens were localized by immunofluorescence as well as by light- and electron microscopic immunoenzyme techniques. Antisera against purified mouse liver cell membranes were surface membrane but not organ-specific. Variable quantities of shared antigens were present in endoderm- and mesoderm-derived organs but not in ectodermal nerve tissue. Species crossreactivity was observed for the rat. Repeated absorption produced liver-specific antisera that reacted with antigenic sites distributed along the entire hepatocyte and sinusoidal cell surfaces. For the precise localization as well as the detection of low concentrations of both liver-specific and nonspecific antigens, the ultrastructural visualization of reactive sites proved essential.


Binding properties of goat IgM anti-dinitrophenyl antibodies. Goats immunized over 2 months with low doses of 1 mg/kg of dinitrophenylated Salmonella typhimurium responded with low levels of anti-DNP antibodies restricted to the IgM class. The purified antibodies show low association constants (Ka between 10(4) of 10(5) l/M), a high degree of homogeneity (heterogeneity indices alpha between 0.7 and 0.9) and ten combining sites when tested against dinitrophenyl-lysine as ligand by equilibrium dialysis. These binding properties remained unchanged during the whole immune response. When after 9 months the animals received the same immunogen and DNP-BGG, the anti-DNP antibody response included antibodies in the IgG class.


Effect of rabbit anti-human B-cell antigens on the response of lymphocytes stimulated by blastogenic factor. It has been shown that specific antisera to B-cell determinants can block stimulation in the human mixed lymphocyte reaction. Therefore, it is of interest to study the effect of anti-human B-cell serum on blastogenic activities of the cell-free culture medium (CFM) derived from cultures of human blood lymphocytes. B-cell antigen was prepared from human B-cell line as a glycoprotein complex of mol. wt 27,000 and 33,000. Rabbit antisera to the B-cell antigen after absorption with human platelets or T-cell line (MOLT 4) was shown to react only against B cells but not T cells. The antisera suppressed human mixed lymphocyte reaction but did not affect the response of lymphocytes to PHA. The proliferative response of T-cell enriched population induced by blastogenic factor from lyphocyte cultures was markedly suppressed by the antisera. The inhibited reactivity irrespective of the source (autologous, allogeneic, mixed, T or B cells) of CFM. This is compatible with an effect caused by their interactions with the responding cell rather than blastogenic factor in the CFM. The results of the kinetic experiments suggest that addition of the antiserum at intervals after initiation of the culture only prevents the CFM stimulation of the responder cells that have not yet become committed to divide.


Immune responses in mice infected with lactic dehydrogenase virus. IV. Functional status of the macrophage during acute LDV infection. Macrophages from uninfected and lactic dehydrogenase virus (LDV)-infected mice were compared with respect to the affinity and number of their Fc receptors for IgG2a; no differences were found regarding these parameters. When the uptake of DNP-BGG by macrophages from uninfected and acutely LDV-infected mice was compared, again no differences were observed. However, when the per cent membrane-bound DNP-BGG was determined as a function of time after antigen uptake in these two groups, more DNP-BGG was found membrane-bound on the macrophages from the LDV-infected mice, than on uninfected macrophages. In view of the fact that humoral immunity is enhanced during acute LDV infection, these data provide a positive correlation between increased retention of membrane-bound antigen and enhanced humoral immune responses.


Regulatory mechanism of autoantibody production in mice to bromelin-treated isologous red blood cells. In the spleen of mice immunized with bromelin treated rat red blood cells (RBC), the number of PFC against bromelin treated isologous RBC increased, although immunization with non-treated rat RBC or bromelin treated isologous RBC gave no increase in number of these PFC. This immune response was found to be T-independent and the PFC developed are exclusively of direct or Ig-M type. In the secondary immune response, production of these PFC was depressed rather than increased. This can be thought of as one of the defence mechanisms against overproduction of autoantibodies in confrontation to foreign antigens cross-reactive with self antigenic determinants. This depressed secondary immune response can be adoptively transferred by primed spleen cells but no active suppressor effect was found. We concluded that clonal elimination by exhaustive differentiation may be operative in this depressed secondary immune response.


Hapten specific unresponsiveness in mice. I. Characteristics of unresponsiveness induced by 2,4,6-trinitrobenzene sulphonic acid. Injection of mice with the chemically reactive hapten 2,4,6-trinitrobenzene sulphonic acid (TNBSA) induces a specific unresponsiveness as judged by reduction or abolition of the anti-TNP response to TNP-KLH. The normal response to a nonrelated hapten, oxazolone, bound to the same carrier (OX-KLH) is unaffected. Reduction of the anti-TNP response was also observed after TNBSA treatment, in nude mice (nu/nu) and their littermates (nu/+) challenged with TNP-POL, an antigen to which the response is thymus independent. Injection of the chemically non-reactive hapten TNP-glycyl-glycine did not induce unresponsiveness. A similar failure was observed with TNP autologous red cells or serum proteins from mice previously injected with TNBSA. The specific unresponsiveness of spleen cells of TNBSA injected mice was maintained after their transfer into lethally irradiated syngeneic host mice. Finally tolerant cells do not inhibit specifically an adoptive anti-TNP secondary response.


Antigenic modulation of the cytophilic binding of guinea-pig IgG and IgM antibodies to homologous macrophages. The cytophilic binding of immune complexes by peritoneal exudate cells (PEC) from adjuvant-stimulated guinea-pigs was studied using 125I-labelled guinea-pig IgG1, IgG2 and IgM antibodies to the dinitrophenyl (DNP) group. The influence of hapten density upon cytophilic activity was studied by the addition of DNP-conjugated antigens to antibody in 2-200 molar ratios of DNP:antibody. Only IgG2 binding was enhanced by immune complex formation, and the increased binding of IgG2 anti-DNP was dependent on the number of DNP determinants per antigen molecule. Cytophilic activity with epsilon-DNP-L-lysine (DNP-LYS), alpha,epsilon-di-DNP-L-lysine (DNP-LYS-DNP), or DNP1-8-BSA was no greater than that seen in the absence of hapten. Increased cytophilic binding was noted only with DNP20-41-BSA. The binding of IgG2 and IgG2 anti-DNP:DNP-bovine serum albumin (BSA) complexes was inhibited by monomeric IgG2. The relative cytophilic capacities of guinea-pig immunoglobulins appeared as follows: IgG greater than IgG1 greater than IgM. IgG1 and IgM binding of DNP conjugates did not enhance their cytophilic activity; therefore, IgG1 and IgM cytophilic binding to PEC was considered biologically insignificant. This investigation provides further evidence that cytophilic binding of immune complexes to macrophages is due to the co-operative action of multiple Fc sites rather than a conformational change in the IgG2 antibodies, and serum proteins, notably complement components, can alter the binding and/or phagocytosis of IgG2 anti-DNP:DNP-BSA complexes.


Studies of human anti-IgM anti-IgG cryoglobulins. I. Patterns of reactivity with autologous and isologous human IgG and its subunits. Monoclonal human anti-IgG preparations purified from mixed IgM-IgG cryoglobulins were tested for their antigenic specificity by haemagglutination-inhibition assay. A panel of fourteen IgG preparations of the four gamma chain subclasses were prepared from myeloma sera and used as inhibitors of haemagglutination. Each of six IgM anti-globulins demonstrated different reactivity profiles with these IgG preparations. In addition, the fraction of the serum IgG which had bound to and cryoprecipitated with the IgM preparations, termed 'antigen-IgG', was purified and assayed for subclass content. The gamma chain subclasses found in the 'antigen-IgG' fractions showed that each IgM cryoprecipitated an IgG from serum which had different quantities of the subclasses present. These 'autologous' reactivity patterns were in instances different from the specificities expected from the results obtained with the myeloma proteins. When all antigen-IgG pools were tested with each IgM, some antiglobulins showed stronger reactivity with isologous than with their own, antigen-IgG pools. The IgM anti-IgG preparations were also compared in reactivity with IgG and its subunits in order to localize the antigenic determinant(s) with which these autoantibodies react. Heavy chains showed far greater reactivity than Fc fragment for 5/6 IgM preparations. Light chains, F(ab')2, pFc' and Fab were non-reactive. A relationship between the length of papain digestion and Fc reactivity was demonstrated. Based on the data, possible locations for the antigenic determinant(s) were considered.


alpha-Foetoprotein:immunoreactivity of the major oestrogen-binding component in mouse amniotic fluid. Competitive protein binding studies, using oestrone as the inhibitor, demonstrated that mouse amniotic fluid contains a high affinity oestradiol-binding component precipitable by monospecific rabbit anti-alpha-foetoprotein (AFP) in a double-antibody radio-immunoassay. On disc gel radio-electrophoresis the oestrogen-binding entity migrated as an alpha-1 protein which was immunoreactive to anti-AFP serum. Immunoprecipitation of protein-bound [3H]-oestradiol from solution was proportional to anti-AFP immunoglobulin G concentration. Rabbit antisera directed against other major proteins in amniotic fluid did not produce significant precipitation of radioactivity. Only when anti-AFP antiserum was the complexing protein and AFP, either crude or purified, was the binding protein did a substantial [3H]-oestradiol precipitate form. Our data suggest that oestradiol and anti-AFP do not bind at the same sites on the AFP molecule and that estradiol does not serve as a hapten in the production of anti-AFP serum in rabbits.


Studies of human IgM anti-IgG cryoglobulins: II. Isoelectric focusing characteristics of the reactive antigen and residual serum IgG pools. The co-isoelectric focusing characteristics of the antigen-IgG and residual IgG pools in the serum of patients with mixed IgM:IgG cryoglobulinemia have been determined. The antigen IgG purified from five IgM cryoglobulins and the residual IgG purified from serum depleted of cryoglobulin were radiolabelled and co-isoelectric focused in polyacrylamide gels in order to determine if the IgM anti-globulin reacted in order to determine if the IgM anti-globulin reacted with IgGs having unique physicochemical properties. Electrofocusing profiles of the IgG from four sera showed only minor differences when the antigen-IgG was compared to residual serum IgG. In one instance, the IEF profile of the antigen-IgG pool was markedly restricted and of a different charge dispersion than the residual serum IgG. This correlated with the limited antigenic specificity previously shown for the autologous IgM antiglobulin.


The specificity of heterologous antiserum against brain of nude mice. Serum prepared in rabbits against brain of nude mice was tested for its T-cell specificity by immunoperoxidase staining. The difference in activity of this anti-brain serum against early precursors and more differentiated precursors of the haemopoietic cell lines was investigated by CFU-s and CFU-c determination after serum incubation. The results showed that the activity of the anti-nude brain serum is completely comparable to serum prepared against brain of normal mice.


Protease inhibitors reduce mitogen induced lymphocyte stimulation. Proteolytic events might play a role during lymphocyte activation. Mouse spleen cells were therefore stimulated in serum-free cultures by PHA, ConA, LPS and dextran sulphate and the effect of various added protease inhibitors on [3H]-thymidine incorporation investigated. Both soybean inhibitor and Trasylol inhibited the response of the cells to all mitogens. The other inhibitors (antipain, leupeptin, ovomucoid, alpha-1 trypsin, alpha-2 macroglobulin) had little or no effect. The marked inhibitory effect of tosyl-lysine chloromethylketone could be neutralized by reduced glutathione, indicating an effect on intracellular glutathione rather than on proteases.


Immune responsiveness and oral immunization. The effects on the immune response of daily feeding of 30 mg of human serum albumin to rats have been studied. Feeding for periods of 17-20 days consistently resulted in a specific systemic hyporesponsiveness evident on subsequent parenteral immunogen challenge. Local secretory sites such as the major salivary glands were not made hyporesponsive as evidenced by the salivary antibody titres and the enumeration of glandular plaque-forming cells. The levels and classes of antibodies present in the secretions and sera were identified by passive haemagglutination, and a sensitive red-cell-linked antigen-antiglobulin reaction. By the use of radioimmunoassay and haemagglutination inhibition assays it was possible to quantitate the amount of antigen appearing in the circulation at varying times after feeding. It was shown in the use of oesophagectomised rats that absorption of small amounts of intact protein occurs from undefined sites in the oral cavity. Attempts were made to transfer the specific systemic hyporesponsiveness to syngeneic animals using spleen cell and serum transfers.


Immunogenetics of the McB1 macroglobulin allotype in cattle. The paper describes a cattle serum antigen (McB1) which is controlled by a dominant gene (McB1), independent from those controlling the two macroglobulin markers McA1 and McA2 as well as from that controlling the low-density lipoprotein marker Ld1A1. McB1 is located on a high-molecular-weight serum protein which, very likely, is an alpha2-macroglobulin.


Uninhibited growth and metastases of herpes simplex virus-transformed cells in virus-sensitized hosts. Four established tumour lines of hamster cells transformed by herpes simplex virus (HSV) but not shedding the virus were examined for continued expression of virus-associated antigens. Hamster or rabbits, appropriately immunized to the tumour cells, produced virus-neutralizing antibody. The serum titres were invariably low, suggesting that only small quantities of virion antigen were present in the cells. Hamster sensitized to HSV and resistant to virulent virus challenge did not reject low numbers of tumour cells, nor was the incidence of lung metastases significantly reduced. Virus-sensitized lymph-node cells, readily cytotoxic for HSV-infected hamster embryo fibroblasts, did not lyse any of three transformed lines tested in an 18-h 51chromium release test. Animals that had their tumour excised demonstrated no or only modest resistance to tumour rechallenge. Thus, virus-specific transplantation rejection antigen could not be detected in the HSV tumour lines although low levels of virus structural antigens were present.


Tumor-associated antigens of chemically-induced murine tumors; the emergence of MuLV and fetal antigens after serial passage in culture. Using radioiodinated Staphylococcus aureus protein A [125I]SPA to measure syngeneic, allogeneic and heterogeneic IgG bound to murine tumor cells, we performed a serological analysis of surface antigens of 8 solid tumors and 2 leukemias of BALB/c mice (3 chemically-induced colon carcinomas, 3 chemically-induced sarcomas, 1 murine leukemia virus (MuLV) induced leukemia, 1 irradiation induced leukemia, 1 spontaneous melanoma and 1 spontaneous sarcoma). We were able to detect and distinguish between at least five separate antigenic specificities on these tumors. Unique tumor-associated antigens were found on 3 of the tumors, MuLV related antigens on 8 tumors, fetal antigens on 7 tumors and two distinct common antigens on 7 tumors (common antigen 1 (CA-1) on 5 tumors and common antigen 2 (CA-2) on 2 tumors). Neither of the common antigens was found to be sarcoma, carcinoma or tissue-tupe specific. A number of tumors which did not originally express either MuLV or fetal antigens in primary cultures expressed these antigens after several serial passages in vitro.


A new virion precipitation test for oncovirus envelope antigens which detects common antigenic determinants in mammalian type-C viruses and Mason-Pfizer monkey virus. A method for the study of oncovirus envelope antigens was developed, bases on the precipitation of intact virions by a double antibody technique. The amount of precipitated virus was then measured as reverse transcriptase activity. The method was designated the virion precipitation test (VPT). It has been used for titration of antibodies to envelope antigens of oncoviruses. The study of envelop antigens of 11 different oncoviruses permitted their differentiation into the following groups: (1) murine type-C viruses: (2) feline type-C viruses; (3) simian type-C viruses; (4) the RD-114/BEV group; (5) Mason-Pfizer monkey virus (M-PMV); (6) bovine leukemia virus; (7) avian type-C viruses; (8) mouse mammary tumor virus. No common antigenic determinants were detected in the last three groups. Mammalian type-C viruses (RD-114, NIH-MuLV, G-MuLV) had common antigenic determinants in the envelope, as demonstrated with an anti-RD-114 serum. Mammalian type-C viruses also shared antigenic determinants with M-PMV. The relationship of type-C viruses to M-PMV decreased in the following order: RD-114--NIH-MuLV--G-MuLV. It was also shown that the endogenous xenotropic feline RD-114 virus was more closely related to xenotropic NIH-MuLV than to ecotropic G-MuLV. The nature of the common antigenic determinants, as demonstrated by VPT on the surface of mammalian type-C viruses and M-PMV, and their significance for the concept of oncovirus evolution are discussed.


Tissue CEA in premalignant epithelial lesions and epidermoid carcinoma of the uterine cervix: prognostic significance. Carcinoembryonic antigen (CEA) was studied by the indirect triple-bridge immunoperoxidase method in formalin-fixed paraffin-embedded tissue specimens from 191 patients with premalignant epithelial lesions or epidermoid carcinoma of the uterine cervix treated 12 years ago. The frequency of tissue CEA positivity was found to increase with advancing clinical disease in the following manner: mild dysplasia, 25%; severe dysplasia, 37%; carcinoma in situ, 60%; invasive carcinoma stage I, 60%; stage IIa, 65%; stage IIb, 80%; and stages III and IV, 69%. The prognostic significance of the tissue CEA positivity was studied in two groups of patients formed on the basis of clinical spread and treatment of the disease. The first group of 60 patients with stage I and IIa cancers had undergone radical surgery. The second group of 44 patients with more advanced carcinoma had been treated by radiotherapy alone. No significant difference in the survival rates was observed in either group between patients with CEA-positive and CEA-negative tumours. In the light of the absence of CEA from normal cervical epithelium, the increasing occurrence of CEA from premalignant lesions to advancing malignant growth suggests that CEA reflects an aggressive potential in premalignant lesions. However, the survival data on patients with CEA-positive and -negative invasive carcinomas suggest that CEA-positive cancers are not more malignant than CEA-negative cancers.


Ultrastructural and immunocytochemical localization of immunoglobulin synthesis in tumor cells in Hodgkin's disease. An immunocytochemical and ultrastructural study using the Fab fragment of an anti-human Ig antibody labelled with peroxidase was carried out on affected lymph nodes from five Hodgkin's disease patients. The tumor cells (Reed-Sternberg cells and Hodgkin cells) showed an exclusively hyaloplasmic granular staining. By comparing these grains with ribisome staining. By comparing these grains with ribosome staining of the endoplasmic reticulum of plasma cells it could be suggested that they are free risobomes. This ribosomal Ig synthesis is a major argument for the B lymphocyte nature of Reed-Sternberg and Hodgkin cells. The total absence of vacuole staining allows us to conclude that these cells are not histiocytic or macrophage derivatives.


Immunosurveillance in pre-malignant occupational bladder disease. A population of workers exposed to bladder carcinogens has been studied by comparing their immunoreactivity against bladder cancer cells and against a control cancer cell with that found in normal individuals and in patients with bladder cancer. Data are presented which indicate equivalent increases in specific reactivity against bladder cancer cells in clinically normal carcinogen-exposed workers and in patients with bladder cancer. Increases in reactivity are related to degree of exposure and also to early malignant changes in the urothelium. This indicates that immune recognition of tumour antigens does occur before development of overt malignancy.


Moloney lymphoma cells express a polyprotein containing the GAG gene-coded p15 and the Moloney leukemia virus-induced cell surface antigen (MCSA). The relationship between MCSA and the viral structural proteins in YAC Moloney lymphoma cells was further investigated by using membrane immunofluorescence and immunoadsorbent columns. As previously observed, MCSA showed only minimal capping and thereby differed in behaviour from gp70, p30 and p12 virion antigens. Whereas antibody-induced capping of gp70 did not change the membrane distribution of MCSA, co-capping was observed between MCSA and p30 and p12 gag protein antigens. This indicated that, whereas MCSA is distinct from gp70, it is linked to p30 and p12 on the membrane of living cells. It was then attempted to isolate MCSA on anti-p30 and anti-p15 immunoadsorbent columns from solubilized YAC cells. Contrary to what was expected, no MCSA was found to the anti-p30 column. On the other hand, a part (10-15%) of the MCSA was bound to the anti-p15 column, indicating that MCSA is linked to the gag p15.


Antiserum to murine leukemia virus recognizes novel cell surface molecules associated with growth control and transformation. Antiserum directed against murine leukemia virus also reacts with several external proteins present in rat cells transformed by a temperature-sensitive Rous sarcoma virus. Reaction of iodinated cell extracts with anti-MLV (murine leukemia virus) serum revealed the presence of a 200,000 dalton iodinated component detectable also by metabolic labelling with glucosamine only in serum-starved cultures restricted in the expression of transformation. A similar assay with iodinated cells that express the transformed phenotype revealed the preferential recognition of two components with an approximate molecular weight of 100,00 daltons as well as an additional 65,000-dalton external component. Growth of the transformed non-producer NT3-KR cells in the presence of inducers of C-type viruses leads to an increased synthesis of a 100,000-dalton glycoprotein (gp100) recognized by the anti-MLV serum which is also recognized by the antiserum in NRK-MSV-MLV transformed producer cells, in addition to a virus-like glycoprotein of 71,000 dalton (gp71). Absorption of the anti-MLV serum with monolayers of NT3-KR cells eliminated the ability of the serum to recognize the gp100 but not the gp71 from NRK-MSV-MLV-transformed producer cells. The mediation of post-translational changes in growth control is suggested by the transformation-dependent alteration in the molecular weight of the non-virion surface proteins recognized by anti-MLV serum in the rat cells used in this study.


Cell-mediated reactivity to antigens shared by Moloney-virus-induced lymphomas (LSTRA) and certain 3-methylcholanthrene-induced mouse sarcomas. Spleen cells (SC) both from BALB/c mice whose primary Moloney sarcoma virus (MSV)-induced sarcomas had spontaneously regressed and from normal, untreated BALB/c mice, were co-cultivated for 5 days with mitomycin-C-treated LSTRA cells; LSTRA is a BALB/c Moloney lymphoma which shares cell surface antigens with MSV-indiced sarcomas. These SC, referred to as CMR and CU cells, respectively, were shown to be cytotoxic to LSTRA cells in 3 h 51Cr-release assays; CMR cells showed, in most cases, the greatest lytic activity against LSTRA targets. The same SC were also reactive, in 20-h microcytotoxicity and 51Crassays, against target cells from a variety of transplanted sarcomas indiced by 3-methylcholanthrene (MCA) in Balb/c mice. The highest reactivity was seen when CMR or CU cells were tested against target cells from sarcoma lines that expressed an NB-ecotropic MuLV cross-reacting serologically with Moloney virus. Reactivity against isotope-labelled tumor cells expressing MuLV-associated cell surface antigens could be competititively inhibited by adding unlabelled tumor cells expressing such antigens. Finally, Winn assays were performed in which CMR cells strongly inhibited the outgrowth of cells from three sarcoma lines that express the NB-ecotropic MuLV. There was less but significant inhibition of cells from some other MCA sarcomas, either negative for the expression of MuLV-associated antigens or expressing the N-ecotropic endogenous BALB/c MuLV. CU cells enhanced tumor outgrowth in Winn assays at least as often as they inhibited it.


Interplexiform cell of the mouse retina: a Golgi demonstration. A neuron of the inner nuclear layer (INL) with some processes extending to the outer plexiform layer (OPL) and others to the inner plexiform layer (IPL) was discovered near the posterior pole of the mouse retina. The neuron's location and appearance are similar to the interplexiform cells of several other species. The relatively recent recognition of this cell type along with its characteristic of infrequent staining by the Golgi technique make the extent of its distribution among species uncertain. With each demonstration in a new species, the existence of the interplexiform cell as a sixth neuronal element of all vertebrate retinas becomes more assured.


[Nucleophagocytosis in monocytes--an immunocytologic phenomenon of anti-dna-antibodies]. In SLE-sera antinuclear antibodies pervent normal nuclear degeneration, i.e. lysis of free nuclei. In sera with high titers, small rigid LE-bodies develop rapidly, which are soon phagocytized in toto by neutrophil granulocytes and monocytes. Classic LE-cells as well as Tart-cells are formed. The foreign appearance of the phagosomes in the monocytes is a result of the different digestive processes occuring in these phagocytes. With decreasing serum activity, the LE-bodies expand somewhat more and become viscous. From these nuclei, relatively small portions are phagocytized fairly late. Sjögren-cells of the granulocytic as well as the monocytic type are generated. Nucleophagocytosis only occurs in the presence of antinuclear antibodies, regardless of whether it leads to LE-cell, Tart-cell or partial phagocytosis, i.e. Sjögren-cells of the granulocytic or monocytic type. A non-specific nucleophagocytosis does not exist.


Correlation between Alcian Blue stainig of glycosaminoglycans of cat nucleus pulposus and TEM x-ray probe microanalysis. The nucleus pulposus of cat intervertebral disc was examined after staining glycosaminoglycans with Alcian Blue and the results correlated with TEM X-ray probe microanalysis. In unstained sections a difference in copper levels between tissues and resin was detected. In tissue stained with Alcian blue before embedding, the copper levels were slightly increased and the morphological appearance of the intercellular material was amorphous. In sections restained after cutting, the relative levels of copper in the resin were considerably increased and tissue levels were significantly higher than in the resin. Moreover, the morphology of the intercellular material was altered from a rather amorphous material to a network. Sulphur levels behaved in similar manner to copper levels but any correlation between the elements was due to factors unrelated to glycosaminoglycan staining and probably resulted from contaminating sulphur.


Multiple intracellular peptidases in Neurospora crassa. Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.


Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs. Immunofluorescence, quantitative immunoprecipitation, and inhibition of bacterial agglutination and passive hemagglutination indicate that cross-reactive antigenic determinants are present on the surface of Rhizobium trifolii and clover roots. These determinants are immunochemically unique to this Rhizobium-legume cross-inoculation group. The multivalent lectin trifoliin and antibody to the clover root antigenic determinants bind competitively to two acidic heteropolysaccharides isolated from capsular material of R. Trifolii 0403. The major polysaccharide is an antigen which lacks heptose, 2-keto-3-deoxyoctulosonic acid, and endotoxic lipid A. The minor polysaccharide in the capsular material of R. Trifolii 0403 contains the same antigen in addition to heptose, 2-keto-3-deoxyoctonate, and lipid A. The acidic polysaccharides of two strains of R. trifolii share the clover r-ot cross-reactive antigenic determinant despite other differences in their carbohydrate composition. Studies with monovalent antigen-binding fragments of anti-clover root antibody and Azotobacter vinelandii hybrid transformants carrying the unique antigenic determinant suggest that these polysaccharides bind R. trifolii to the clover root hair tips which contain trifoliin.


Nuclear division in temperature-sensitive multicellular mutants of Wangiella dermatitidis. Temperature-sensitive morphological mutants of Wangiella dermatitidis were stained with mithramycin, a fluorescent nuclear stain. Multicellular forms of the mutants each contained two or more nuclei, documenting that nuclear division accompanied the yeast-to-multicellular form conversion.


Purification and characterization of a polyhook protein from Caulobacter crescentus. A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.


Lysogenic pneumococci and their bacteriophages. About half of pneumococci recovered from pediatric patients and one-third of isolates from adult patients yielded bacteriophages active against one or more of four noncapsulated indicator strains of pneumococcus. Strains of capsular types most frequently causing pediatric infections were associated with lysogeny. Classical restriction-modification phenomena have been demonstrated in vivo with some of the temperate phages, and correlation of restriction with the presence of one or the other of the two known pneumococcal restriction endonucleases has been established. The temperate phages differ serologically and in several other characteristics from virulent pneumococcal phages previously described. All pneumococcal phages so far studied can be classified into a minimum of three serological groups.


The isolation of avian viral RNA and polypeptides. From the same batch of virus, the four major avian viral structural proteins p27, p19, p15, and p12, the reverse transcriptase, the envelope glycoprotein gp85, and the high molecular weight 70 S RNA have been recovered. All proteins, except for gp85, have been purified by use of column chromatography procedures to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gels and isoelectric focusing. A new isolation procedure for p12 by affinity column chromatography takes advantage of its nucleic acid binding properties. The recovery of nondenatured viral structural proteins is demonstrated by the proteolytic activity revealed by p15. The purified proteins were used for the production of monospecific antibodies. The 70 S RNA served as source for the isolation of 35 S RNA subunits.


Reduction of GTP activation of adenylate cyclase system by its coupling to hormone receptor. We have examined the characteristics of the adenylate cyclase system from control and butyrate-treated cells. Butyrate treatment results in both an increased number of catecholamine receptors and an induction of a response to the hormone, as reported previously (Tallman, J.F., Smith, C.C., and Henneberry, R.C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 873-877); in addition, we found that the same treatment reduces the degree of activation of adenylate cyclase by GTP. We have demonstrated in two cell types that this decrease in GTP activation is inversely related to the degree of induction of the hormone response. Furthermore, in plasma membranes isolated from butyrate-treated cells, the hormone receptor is sensitive to GTP; i.e. GTP reduces the affinity of isoproterenol for the receptor. We propose that these changes reflect an interaction between the beta-adrenergic receptor and the nucleotide regulatory component and that this interaction represents, at least in part, the process of coupling. Several possible mechanisms which can account for the change in GTP activation are discussed in terms of our current understanding of the regulation of the adenylate cyclase system.


Reverse transcriptase-associated ribonuclease H does not require zinc for catalysis. omicron-Phenanthroline, a zinc chelating agent, is known to inhibit the DNA polymerase activity of cellular DNA-dependent and viral RNA-dependent DNA polymerases. We find that omicron-phenanthroline does not inhibit the reverse transcriptase-associated RNase H activity of retroviruses. Kinetic studies, using DNA template-primers as an inhibitor of RNase H, suggest that zinc does not play any role in template-primer binding by reverse transcriptase. These results also indicate a distinct binding site for the template and triphosphate substrates. Cellular RNase H from calf thymus and RNase H-II from Rauscher leukemia virus are likewise resistant to omicron-phenanthroline inhibition, implying non-involvement of zinc in the nucleic acid hydrolysis by these enzymes.


Electron microscopical and biochemical investigations on retra viruses in spleen tissue in malignancy. Simultaneous biochemical and electron microscopical investigations on surgically removed spleens yielded evidence for the presence of reverse transcriptase containing (Retra) virus in two patients with hematological malignancies with spleen involvement. In three other patients with hematological diseases and in one control patient, the spleens were negative in both assays. The results of these combined studies support the view, that retraviruses are present in human malignancies.


Interaction of digitalis and spironolactone with human sex steroid receptors. Spironolactone and digitoxin have previously been shown to interact with cytosol androgen and estrogen receptors, respectively, in the rat. The interaction of digitoxin with human uterine cytosol estrogen binding protein and spironolactone with human prostate and newborn prepuce cytosol dihydrotestosterone (DHT) binding protein has been analyzed in this study. Specific estradiol binding was found only in premenopausal uteri. The dissociation constant for estradiol binding was 0.6--2.3 X 10(-9) M (n - 12). Digitoxin in concentrations varying between 0.5--2.0 X 10(-6) M inhibited specific estradiol binding with a Ki of 2.0--7.3 X 10(-7) M (n = 9). The dissociation constants for DHT and the human androgen cytosol binding protein in prostate and newborn prepuce were 0.27--3.0 X 10(-8) M (n = 12) and 0.6--2.0 X 10(-8) M (n = 5), respectively. Spironolactone at concentrations of 0.3--2.0 X 10(-6) M competitively inhibited this binding with an affinity about one order of magnitude less than that of DHT. Digitoxin and spironolactone did not displace estradiol and DHT, respectively, from testosterone-estrogen binding globulin in male or female plasma. The interaction of digitoxin with the human uterus estrogen binding protein and spironolactone with the human prostate and prepuce androgen binding protein is similar to our previous observations in the rat, and may explain the weak estrogenic effects of digitoxin and spironolactone in man.


A new technique for Gram staining paraffin-embedded tissue. Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining.


The development of the hippocampus and dentate gyrus in normal and reeler mice. The histogenesis, the time of origin and the pattern of migration of the cells in the hippocampus and dentate gyrus, have been studied in normal and reeler mice. The earliest indication of a defect in the reeler hippocampus is seen on the fifteenth embryonic day (E15) which is at least 24 hours after the first indication of a defect in the neocortex. It is not until E18, that the dentate gyrus shows signs of its incipient abnormality. It appears then, that in both the hippocampus and the dentate gyrus the gene defect first manifests itself at the stage at which the definitive cellular layers are assembled. Experiments involving the injection of 3H-thymidine (3H-TdR) at different developmental stages have confirmed that the site and rate of cellular proliferation in the reeler hippocampus and dentate gyrus are normal, as is the initial pattern of cell migration. However, in the reeler dentate gyrus, most postnatal cell proliferation occurs ectopically and in the hippocampus the normal "inside-out" sequence of neurogenesis is reversed, the earliest pyramidal cells generated coming to lie superficially within the stratum pyramidale and the later formed cells being added at progressively deeper levels. There is no discernible gradient in the time of origin of the granule cells in the radial dimension of the reeler dentate gyrus, whereas there is an obvious "outside-in" gradient in the normal animal. The characteristic gradients in cell proliferation seen in the transverse and longitudinal dimensions of the normal dentate gyrus are, however, also evident in the reeler mouse. Taken together, these observations suggest that the reeler gene exerts its effect on neuronal position only in the radial dimension, and does so at a stage of development subsequent to the proliferation and initial migration of the relevant neurons. Timm's sulfide silver preparations indicate that the characteristic staining patterns seen in the dentate gyrus and hippocampus appear at the same time, and mature at the same rate in normal and reeler mice.


Comparative study of the linearity and sensitivity of electron capture and flame photometric detectors using a pesticide standard. A study was undertaken to compare and evaluate the different types of 63Ni and 3H electron capture (ECD) and flame photometric detectors (FPD) as part of the Canadian Check Sample Program  on Pesticide Residue Analysis. Twenty-seven laboratories were supplied with chlorpyrifos standard solutions for the determination of linear range and minimum detectable amounts (MDA). Each laboratory selected its own operating conditions except for the FPD study. Results for the FPD were compared with standardized flame conditions designed to optimize the exponential factor i, the S-mode. The MDA of the ECDs (pulsed and direct-current) ranged from 0.09 to 0.17 pg/sec for 63Ni and 3H sources respectively. The corresponding MDA for the linearized ECDs (pulse modulated or constant current) was 0.04 pg/sec and a linear range of 10(4) or greater with nitrogen as the carried gas. Use of argon/methane and a pulse width of 0.1 usec extended this range to 10(5) but the sensitivity was not as great as with nitrogen carrier gas. The practical limit of detector sensitivity was found to be instrument dependent depending primarily on the noise level. In the FPD study, operating in the P-mode gave an average MDA of 5.97 pg/sec and a linear range of 2 x 10(3). Similarly, the S-mode parameters were 81.1 pg/sec and 8 x 10(2) respectively with an average exponential factor of n 1.8. Attempts to apply optimized detector gas-flow conditions improved sensitivity and linearity in the S-mode only.


Carcinogenicity of hexachlorocyclohexane (BHC) in pure inbred swiss mice. Inbred Swiss mice were treated with technical BHC (1) orally with the diet or by intubation and (2) by skin painting. The total duration of the experiment was 80 weeks. There was no difference in body growth and mortality between the experimental and control groups. Symptoms of intoxication in the form of convulsions and tendency of circling in one direction with drooping ears were observed alongwith corneal opacity 40 weeks after the experiment. Oral BHC treatment resulted in a significant increase in the incidence of liver tumours and tumours of lymphoreticular tissue. Males and females were equally susceptible. Skin painting of mice did not significantly increase the incidence of tumour.


Treatment of Dupuytren's contracture by extensive fasciectomy through multiple Y-V--plasty incisions: short-term evaluation of 170 consecutive operations. The zigzag incision with Y-V closure has unique advantages for the treatment of Dupuytren's disease. Much skin can be mobilized to the longitudinal finger axis. There is rarely a need for skin grafting in spite of full correction of the deformity. This approach, combined with properly applied tamponade bulk dressing, allows for early use of the hand with minimal morbidity.


Mass action and acridine orange staining: static and flow cytofluorometry. We present results involving an approach to acridine orange staining of intact cells based on basic physicochemical considerations. We show by static microfluorometry of several in vitro and in vivo cell lines that the important parameters for such staining are the molar ratio (Formula: see text), and molar concentration of acridine orange. Differential nuclear DNA and cytoplasmic RNA staining are totally controlled by these two parameters. We show this by a physicochemical model of cell-dye interaction. Finally, we use the method to study the growth parameters of complex in vivo cell populations by automated multiparameter flow microfluorometry. We have explored also, both by static and flow systems, the effect on AO-cell staining of various cell pretreatments such as Triton X-100 and chelating agents.


Sedimentation velocity separation: a preparation method for cervical samples. A preparation procedure, aiming at monolayer deposition of cervical exfoliative material on glass slides for high resolution prescreening has been developed. The main features of this procedure are centrifugal deposition after suspension and sedimentation of samples over isopycnic medium of 1.026 density. Fractioning of the separation column after centrifugation at 50 X g yields two preparations with leukocytes, bacteria and cellular debris predominantly located on the first slide and epithelial cells on the second one. The degree of spatial cellular isolation as well as the amount of diagnostically relevant cells per slide seem to fit the requirements of automated high resolution analysis.


Cytomorphologic results of preparation experiments for monolayer deposition of cervical material. For automated prescreening methods by high resolution analysis serving as a detecting method in gynecologic mass screening programs a new monolayer deposition method of cervical material has been used. This method will be outlines briefly and the mode of evaluation as well as current cytomorphological findings will be presented. With regard to measurability of the slides prepared according to the new method a number of cytologic criteria were thought to be of particular importance. These criteria are delineated and compiled in a table. A form in which these criteria were listed was filled in by cytopathologists for each slide evaluated. When performing isolation and centrifugation procedures several new morphologic questions arose to the cytopathologist which can only partly be answered by now. If taking into account all criteria of evaluation it may be followed from the present experiences that slides of cervical material are much more suited for automated prescreening methods by high resolution analysis if prepared after isolation and centrifugation in macromolecular liquids than are conventional Papanicolaou smears or slides from suspensions with isolated cells that were not subjected to centrifugation procedures.


The preparation of cervical scrape material for automated cytology using gallocyanin chrome-alum stain. A method is described for preparing cervical scrape specimens for automated analysis on the Cerviscan prescreening system. In order to reduce the cellular clumping found in cervical scrape material, cells are collected in suspension, syringed to disaggregate the cell clumps, and then pipetted onto a glass to give a monolayer of cells. The cells are then stained with gallocyanin chrome-alum to give the required quantitation of nucleic acid content, using a rapid staining procedure. Experimental results are given which show that specimens prepared by this method are more suitable for automated analysis than the conventional Papanicolaou stained preparation.


A system for storage and retrieval of individual cells following flow cytometry. A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.


Flow cytometry as a new method for the measurement of electrophoretic mobility of erythrocytes using membrane charge staining by fluoresceinated polycations. The binding of FITC-labeled poly-L-ornithine and poly-L-lysine to fresh or neuraminidase treated human, rat or rabbit erythrocytes was investigated by simultaneous cell volume and cell membrane fluorescence measurements in a flow cytometer. The cell volume was converted into cell surface and the distribution curve of the fluorescence/micrometer2 cell surface was calculated from all histogram classes by a computer program. The mean fluorescence/micrometer2 cell surface as a measure of the density of the negative charges on the cell surface was directly proportional to the elctrophoretic mobility of the erythrocytes, showing that polycation binding can effectively be used for the measurement of the electrophoretic mobility of erythrocytes. The computer fitting of the experimental two parameter histograms by two dimensional Gaussian normal distributions was found to be a very efficient way of data reduction, and a good separation of overlapping cell clusters was possible even in the case of low total numbers of cells in the histogram.


Flow cytometry of mammalian sperm: progress in DNA and morphology measurement. Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.


Cytofluorometry of electromagnetically controlled cell dedifferentiation. Cellular morphology changes, which appear related to dedifferentiation (despecialization), have been produced in vitro in the nucleated red blood cell of the frog. This has been achieved by controlled alterations in the electrochemical environment of these living cells, both by a selective modification of the ionic concentrations of an isotonic amphibian Ringer solution, and by the electromagnetic induction of pulsating current having specific waveform parameters. Laser flow microfluorometry shows that the modified Ringer solution is able, per se, to partially trigger the process in the same time interval that certain induced current waveforms can significantly affect the number of cells in the so-called dedifferentiated state. It has also been found that, for a given waveform, the repetition rate appears to have a significant effect on the rate of cell change. Preliminary automated image analysis of cell smears suggests that dedifferentiated and normal cells have the same integrated optical density but different nuclear areas. In conclusion, it appears that, after the initial electrochemical trigger, the early stage of the process, when the cells move from a state of specialized function to one of less specific activity, is the unfolding of their chromatin supercoil, not involving DNA synthesis. Then cytofluorometry allowed us to identify, for the first time, fundamental modifications which occur in the cell nucleus under electromagnetic exposure.


Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry. Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.


Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye. The in vitro response of murine lymphocytes to allogeneic and mitogenic stimulation has been studied by using the nontoxic fluorescent DNA probe, Hoechst 33342, and a fluorescence flow cytometer-cell sorter. Under appropriate conditions, two peaks of fluorescent intensity, not related to cellular DNA content, can be seen. As early as 12 hr after culture set up, lymphocyte activation can be identified. It appears that Hoechst-labeled lymphocytes of higher fluorescence intensity represent those cells activated by allogeneic stimulation whereas cells obtained from the lower intensity peak are nonresponding lymphocytes.


Fluorescence polarization and pulse width analysis of chromosomes by a flow system. Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.e., differeing in DNA content. The emission anisotropy values for all chromosome classes was constant (emission anisotropy = 0.30), and the same value was obtained for purified DNA in solution. Pulse width was found to be a good parameter for resolving chromosomes as a function of total emission in the case of the smaller chromosomes and orientation (i.e., arm length) for large chromosomes. A simple theoretical model for predicting the pulse shapes generated by arbitrarily oriented, thin, rigid rods was developed and applied to the evaluation of the experimental data.


Quantitation of lymphocyte response to PHA by flow cytofluorometry. III. Heterogeneity of induction period. Early events in phytohaemagglutinin (PHA) stimulation of mouse splenocytes have been quantitated by using flow cytometry and supravital staining with acridine orange (AO). Increasing percentages of single cells with increased metachromatic (red) AO staining were demonstrated in cultures stimulated by PHA for up to 24 hr. These differences in staining could be eliminated by fixation with 1:1 ethanol/acetone before staining. Stimulated cells showed an increase in nonspecific esterase activity as measured by flow cytometry after supravital staining with fluorescein diacetate (FDA). The data reported show a heterogeneity in the per cell response of mouse splenocytes to PHA. The relationship between these data and the mechanism of mitogen stimulation is discussed.


Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry. The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.


Flow cytometric prescreening of cervical smears. One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.


Quantitative analysis of flow microfluorometric data for screening gynecologic cytology specimens. Gynecologic cytology specimens that included the entire spectrum of cervical cytology classification were stained with a combination of propidium iodide and fluorescein isothiocyanate, then analyzed using a flow microfluorometer to measure nucleic acid and protein content, respectively. Numerous descriptors of the resulting two parameter distribution (nucleic acid versus protein) were defined. These descriptors included assessment of the presence or absence of abnormal cells. They also included measures of the staining intensity and dispersion of the normal squamous cell population and the intensity of inflammatory response in the cell population. Relative percentages of inflammatory and epithelial cells were demonstrated to effect the screening performance of this system only in borderline lesions. Decision tree algorithms allowed optimization of the selected parameters for screening logic of normal-abnormal decisions on a specimen-by-specimen basis. In addition, quantitative definitions of specimen adequacy were determined. Appropriate controls for batch staining of specimens were evaluated. These results of applying pattern recognition techniques to flow microfluorometer multiparameter data demonstrate that considerably more information about cell populations and subpopulations can be extracted than heretofore possible.


Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters. Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.


Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum. Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.


Automatic cell identification and enrichment in lung cancer. III. Light scatter and two fluorescence parameters. Two fluorescence parameters and size are used in a flow through system to enrich sputum specimens for cancer cells. Human cells in sputum which are stained with acridine orange show a characteristic distribution of red and green fluorescence from which cancer cells can be localized. The peak enrichment is obtained by selectively sorting cells with the largest values of red and green fluorescence. Cancer cells located in other distribution regions having smaller fluorescence intensities show progressively diminished nuclear and cytoplasmic tinctorial features by Papanicolaou stain, consistent with the decreased intensity of red and green fluorescence.


The Heidelberg flow analyzer and sorter (HEIFAS) approach on the prescreening of uterine cancer. A dual laser flow system has been proved and used with a novel staining method for simultaneous quantitative DNA and protein analysis. A diamidinophenylindole compound (DAPI) for DNA has been employed in combination with sulforhodamine (SR 101) for protein. With this dye mixture various cell types of the cervical smear could be identified. Despite some overlapping, clusters of leukocytes, endocervicals, (para)basals, intermediates, superficials and dys/neoplastic cells together with artificial events could be discriminated. Up to now the problem of negative cells which create positive signals (false alarms) in the dys/neoplastic region could not be sufficiently solved.


Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract. Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.