Expression of a Forssman antigenic specificity in the preimplantation mouse embryo. A monoclonal antibody recognizing a Forssman antigenic specificity has been shown to react with cells of the preimplantation mouse embryo. The antigen is believed to be carried on glycolipid molecules on teratocarcinoma stem cells. This antigen is first detected on the trophectoderm of the early blastocyst. The topography of the expression on the trophectoderm is striking and novel. The antigen is no longer found on these cells after the blastocyst has hatched from the zona pellucida in utero. Inner cell masses are antigen-positive at all times. This is the first study of the distribution of a single antigenic determinant in early mouse embryogenesis.


Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization. We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.


A gene (Bevi) on human chromosome 6 is an integration site for baboon type C DNA provirus in human cells. Human VA-2 cells infected with baboon type C virus were cloned and fused to Syrian hamster cells, and 33 primary hybrid colonies were obtained. These cells segregated human chromosomes and retained the complete hamster genome. Assays for type C viral p30 antigen and reverse transcriptase were performed in conjunction with analyses of 30 gene-enzyme systems representing 22 different human chromosomes. The results comfirmed that a gene, Bevi, previously assigned to human chromosome 6, dominantly controls baboon type C virus expression in hybrid cells. Representative hybrid colones were studied by nucleic acid hybridization techniques for the presence of integrated proviral DNA using complementary 3H-DNA transcripts of the baboon viral RNA genome. For each of 12 clones examined, there was a concordance between the presence of human chromosome 6, the presence of baboon type C proviral DNA sequences and virus expression. Clones which segregated chromosome 6 as judged by isozyme and karyological analyses lost detectable proviral DNA sequences and failed to produce virus. No syntenic association between the replication of baboon virus and the presence of 21 other human chromosomes was deteced. We conclude that Bevi is a preferred integration site for the baboon type C provirus in the human genome.


[Experience with a technic using basic fuchsin for the demonstration of early myocardial ischemia]. A series of 156 patients dying suddenly and unexpectedly was analysed with respect to the occurrence of fibres staining with basic fuchsin with the method of Lie et al. (6). The findings were correlated with the cause of death, coronary sclerosis and heart weight. Positive staining was revealed in the form of contraction bands or perinuclear segments. No statistically significant correlation was demonstrated between the occurrence of positive fibres on the one hand and regressive myocardial changes due to coronary and extracoronary insufficiency or other causes on the other hand.


Epidemiology of endemic goitre in western Colombia. This paper reports on recent epidemiological observations in western Colombia, which further demonstrate the presence of naturally-occurring goitrogens contaminating water supplies in areas where goitre persists despite prolonged and continuous iodine supplementation. 'Prospective' and 'cross-sectional' studies in 41 localities where the populations have been on a uniform and adequate iodine supplementation for the last 10-20 years indicate that, in the endemia of western Colombia, environmental factors other than nutritional iodine deficiency are responsible for differences in goitre prevalence. Further epidemiological studies to determine the causal factors for the persistence of the endemia established a correlation between the sources of drinking water and goitre prevalence rates. Organic compounds containing sulfur with marked thionamide-like antithyroid activity were isolated from water supplying endemic goitre districts, and results are presented supporting the hypothesis that sedimentary rocks rich in organic matter are the main source of water-borne goitrogens. Bacteriological investigations showed that the presence of Klebsiella pneumoniae in drinking water and bacterial concentration were related significantly with goitre prevalence only in the presence of other variables, particularly the presence of sedimentary rocks. In the light of these epidemiological observations and experimental studies it may be concluded that, at present, endemic goitre in western Colombia is not due to nutritional iodine deficiency, but that water supplies are contaminated with sulfur-bearing organic compounds with thionamide-like antithyroid activity most probably deriving from sedimentary rocks rich in organic matter and that these compounds are the main factor underlying the endemia.


Nomenclature for factors of the HLA system, 1977. This article gives the decisions of the WHO nomenclature committee on leukocyte antigens, in particular concerning (a) the upgrading of certain HLA-A and HLA-B specificities to full HLA status, (b) the designation of new provisional specificities of the HLA-B, HLA-C, and HLA-D loci, and (c) the establishment of a nomenclature for the new specificities identified by serological techniques on B lymphocytes.


[Degradation of ribosomal RNA from Escherichia coli by ribonuclease U2 and in vitro transcription of "RNA-fragments" into complementary DNA]. Under well defined conditions ribosomal RNAs purified from Escherichia coli can be degraded by ribonuclease U2 giving rise to RNA fragments of 60--70 nucleotides. In vitro, these fragments are efficiently transcribed into a complementary DNA by DNA polymerase RNA dependent, partially purified from extracts of E. coli. In vivo, "RNA-fragments-U2" inhibit the development of plant tumors.


Mechanisms of ventricular parasystole.

Mechanisms of ventricular parasystole. Eight cases of ventricular parasystole are reported. In all these cases, regardless of whether parasystole seems intermittent or "continuous," the presence of second degree entrance block of the Mobitz type I was suggested. Parasystole alternated with concealed extrasystolic bigeminy showing occasional reentrant extrasystoles. Such intermittent parasystole appears to originate in the reentrant path of extrasystoles. Reentrant extrasystolic bigeminy was seen in a comparatively rapid sinus rhythm, whereas parasystolic bigeminy was seen in a comparatively slow sinus rhythm. The difference between the interectopic intervals during parasystolic bigeminy and during (manifest or concealed) extrasystolic bigeminy was comparatively small so that occasionally the difference was not distinct; on such an occasion the case showed a seemingly "continuous" parasystole. These observations strongly suggest the possibility that most cases of parasystole, whether intermittent or "continuous," may be governed by incomplete entrance block of the second degree.


Double irregular ventricular parasystole: rate-dependent entrance block and "supernormal" exit conduction. An analysis of electrocardiograms from a patient with spontaneous double irregular ventricular parasystole is presented. Irregularity in one of the two parasystoles was produced by intermittence based on rate-dependent (phase 3) entrance block, and in the other parasystole it was attributed to "supernormal" exist conduction. Critical analysis of electrocardiograms revealed that first degree block, rate-dependent block, and "supernormal" conduction in the exit pathway may account for the alterations in the arrangement and manifestation of the parasystolic beats. An electrocardiographic approach to these properties of the parasystolic structure and demonstration of double ventricular parasystole with irregularity in both parasystoles has not previously been found in the literature.


46,XY pure gonadal dysgenesis with non-fluorescent Y chromosome. A 46,XY karyotype with a non-fluorescent Y chromosome was found in an infantile girl aged 16 with primary amenorrhea. Identification of the Y chromosome was made by different staining techniques and a photometric scanning method. The histology of the streak gonad also indicated the Y character of the chromosome. The authors' interpretation is a 46,XY pure gonadal dysgenesis with a non-fluorescent Y chromosome.


Ontogenic development and tissue distribution of renal proximal tubule brush border membrane antigens in the dog. Using an antibody raised against highly purified dog renal proximal tubule brush border membranes, we have investigated the tissue distribution and ontogenic development of antigens sharing specificity with those in the renal brush border membrane. The antigens have been found in the small intestine, gall bladder, epididymis, pancreas, salivary gland, lacrimal gland, liver and spleen. Examination of embryos at various stages of gestation has revealed that the antigens make their appearance early in organogenesis, and are present in the cytoplasm as well as on the membrane during foetal development. Absorption studies have shown that at least two populations of antigens are present in the kidney: one appears to be kidney-specific and the other is shared by many epithelia. Maltase has been identified as one of the shared antigens.


Class-specific antibody titres (ELISA) against the primary immunogen Helix pomatia haemocyanin (HPH) in man. Using the indirect ELISA technique, IgM, IgG and IgA antibody titres against the primary test immunogen Helix pomatia haemocyanin (HPH) could be measured. All twenty-seven normal volunteers (age range 26-74 years) developed maximal or high IgM, IgG and IgA antibody titres 2 weeks after primary immunization with 1.0 mg HPH subcutaneously. Lower dose immunization resulted in submaximal responses. Titre kinetics in the three Ig classes correlated in height and time per person. There was no significant difference in antibody response between two adult age groups. Secondary immunization after 6 weeks, again with 1.0 mg HPH, when high titres were still present, resulted in a small titre increase in all three Ig classes. Comparison with the tanned red cell agglutination technique used previously, in combination with the 2-mercaptoethanol treatment of sera, showed the unreliability of 7S titres measured thus early in the primary immune response. Investigation of four patients with humoral immunodeficiency confirmed the class specificity of the test.


Gamma heavy chain disease in man: synthesis of a deleted gamma3 immunoglobulin by lymphoid cells in short and long term tissue culture. Bone marrow cells were obtained from a patient with gamma heavy chain disease (HCD) whose serum contained a deleted immunoglobulin heavy chain. Incubation of the marrow cells with radioactive amino acids in short term tissue culture resulted in the synthesis of the labelled HCD protein. A permanent cell line was established from the peripheral blood of the patient. Similar labelling studies with the cell line and its cloned progeny demonstrated the synthesis of a protein identical in size and antigenicity to that synthesized by the marrow cells and found in the patient's serum. These experiments clearly demonstrated that, in this case of heavy chain disease, the deleted protein was the synthetic product of a clone of malignant lymphoid cells.


Dynamic ventriculography in patients with ischaemic heart disease. II. Influence of postextrasystolic potentiation on left ventricular function. The effect of postextrasystolic potentiation [PESP] on left ventricular [LV] function was assessed by means of left ventricular cineangiography in 26 patients with ischaemic heart disease. Ventricular extrasystoles were induced by a catheter inserted into the LV during injection of the contrast agent. The values of parameters of segmental and global function of LV, found in potentiated and nonpotentiated sinus contractions, were compared. Segmental disturbances present at rest in 13 patients improved or disappeared after extrasystole in 8 patients. Improvements in contraction also took place in segments with normal resting contraction. Global LV function improved significantly; an improvement was observed even without an increase in the LV end-diastolic volume in the postextrasystolic beat. The advantages and limitations of the method for preoperative estimation of the effect of intended myocardial revascularization on the LV function are discussed.


[The effect of early appearing extrasystoles on systemic and coronary circulation]. In 10 anaesthesized dwarf pigs with an open thorax the effects of extrasystoles on systemic and coronary circulation were investigated. The actual effect of an extrasystole depends on the time of its onset. Early extrasystoles hamper the cardiac blood perfusion, with decreases in the stroke volume, mean arterial pressure, coronary blood flow, and elevation of coronary resistance. Extrasystoles setting on later are of minor haemodynamic and coronarodynamic importance. The effects of bigeminy and 2:1 extrasystolia are more adverse than the consequences of extrasystoles with later onset. With cumulation of several consecutive extrasystoles also adverse the haemodynamic coronarodynamic sequelae increase, in dependence on the magnitude of the quotient coupling time /extrasystolic excitation period [ greater than 1.1], or of the quotient: duration of pre- and postextrasystolic intervals/ normal interval of heart action [ greater than 1.5]. The authors point out the importance of haemodynamic findings during early extrasystoles in ischaemic heart disease.


Immunological characterization of Na+ and K+ mediated structural states of rat kidney Na+-k+-ATPase. As different structural states of the Na+-K+-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of different ligands on the reaction between highly purified membrane-bound Na+-K+-ATPase and specific antibodies was investigated. The antigen antibody reaction was registered by measuring the antibody inhibition of Na+-K+-ATPase activity. It was found that Na+ decreased and K+ increased the antibody inhibition of the Na+-K+-ATPase activity of the membrane-bound enzyme if both Mg++ and ATP were present during the antigen antibody reaction. These effects were not observed if ATP was replaced by ADP or by the ATP analog adenylyl (beta-gamma-methylene) diphosphonate. If a solubilized enzyme preparation with the same specific activity was used the effects of Na+ or K+ which were demonstrated in the membrane-bound enzyme could not be detected. The study suggests that the Na+-K+-ATPase structure is altered by Na+ and K+, provided Mg++ and specifically the nucleotide ATP are also present. These structural changes are likely to occur during Na+-K+-transport and do not seem to be necessarily linked to the Na+, K+ and Mg++ stimulated ATP splitting of the enzyme.


Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU. The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques.--Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7--10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.


Localization of the synaptonemal complex under the light microscope. The use of osmium tetroxide fixation followed by postreatment with p-phenylenediamine gives an opportunity of locating the synaptonemal complex (SC) under the light microscope in mouse testes and Allium cepa anthers. When semi-thin sections from these materials were observed under phase contrast optics or dark field microscopy, fine threads in the pachytene nuclei were clearly visible. Post-staining of semi-thin sections with ammoniacal silver increased the contrast of the SC and allowed for observations using a bright field illumination. Ultrathin sections of osmium tetroxide/p-phenylenediamine treated material showed that, under the electron microscope, this technique stains preferentially elements of the synaptonemal complex, while the surrounding chromatin remains unstained.


Interaction of anthracyclines with DNA and chromosomes. Daunomycin and adriamycin were previously found to produce Q-like banding patterns on chromosomes. The interaction of several anthracyclines with both natural and synthetic DNAs and chromosomes has been investigated in more detail. Daunomycin fluorescence is almost completely quenched by natural DNAs with varying base composition from 31 to 72% G-C and by the alternating polymer poly-d(G-C).poly-d(G-C). In contrast, daunomycin fluorescence is quenched by only 50% when the dye interacts with synthetic A-T polymers. Thus, differential quenching of daunomycin fluorescence can account for the production of bright bands at contiguous A-T sequences along the chromosome. Slight differences in fluorescence quenching between the repeating and homopolymeric A-T duplex DNAs were observed which can be attributed to differences in affinity of daunomycin for these DNAs. The aminosugar moiety of daunomycin, daunosamine, increases the binding of daunomycin to DNA and also enhances chromosome banding.--Nogalamycin, which displays no differential quenching with the different DNAs in solution, also fails to produce bands on chromosomes.--These findings suggest that non-random nucleotide sequence arrangements along the chromosome are a basic determinant for dye interaction to produce the observed banding patterns. Specific banding procedures may determine the accessibility of these sites within the chromosomal DNA.


Biochemical and cytological studies of bleomycin actions on chromatin and chromosomes. Interaction of bleomycin with nuclei isolated from a variety of mammalian cells resulted in the release of nucleosomes. When isolated mononucleosomes (core plus linker) were re-treated with bleomycin, no further degradation of DNA occurred. The results suggest that the bleomycin cleavage sites in chromatin are present only in the linker region and that there are probably only one or two cleavage sites per linker. The repeat lengths of nucleosomal DNA released by bleomycin from nuclei of different species are different; this variability is considered to reflect the length of the linker. Incorporation of BrdU into DNA did not alter the bleomycin action on nucleosomes. When mitotic cells were held at metaphase for a prolonged period, bleomycin caused a gradual disintegration of chromosomes, although the bleomycin cleavage sites in metaphase chromosomes were found to be the same as those in interphase nuclei.


Chromatographic and electrophoretic heterogeneity of the cytochromes P-450 solubilized from untreated rat liver. When the hepatic microsomes prepared from one or two untreated male rats were incubated for 3 hr at room temperature in buffered Emulgen 911, cholate, glycerol, and EDTA, almost complete recovery of the cytochromes P-450 in solubilized form was obtained. The intact cytochromes P-450 in this preparation were retained by Sephadex G-200. They could be resolved by anion-exchange chromatography into four fractions with 70--80% recovery of cytochrome P-450 and little or no conversion to P-420. In the process, the p-450 hemoproteins were separated from cytochrome b5, NADPH-cytochrome c reductase, and hemoglobulin. Each of the four fractions of cytochrome P-450 could be further resolved by electrofocusing in polyacrylamide into numerous protein bands, more than eight of which stained for heme. Extracts from focused gels retained P-450 spectral character. Mixtures of the various fractions electrofocused additively. Added hematin focused only in the acidic region of the gels. Although these results indicate extensive heterogeneity in the cytochromes P-450, the complexities of electrofocusing in the presence of detergent warrants cautious interpretation. These methods, however, should provide a basis for subsequent chemical, physical, catalytic, and immunological investigations of the heterogeneity, and its significance.


A comparison between naturally occurring macroamylasaemia and macroamylasaemia induced by hydroxyethyl-starch. Macroamylasaemia was produced in vitro by incubation of hydroxyethylstarch with serum, and in vivo by intravenous infusion of hydroxyethylstarch. Gel filtration on Sephadex G-100 revealed distinct differences in molecular size distribution between such hydroxyethylstarch-induced macroamylase and the usual form of naturally occurring macroamylase which was observed in a few patients from our hospital. Further studies demonstrated that the gel filtration elution pattern of amylase activity in serum containing hydroxyethylstarch-induced macroamylase is significantly altered with time in vitro and in vivo, probably because of an enzymatic degradation of the hydroxyethylstarch components of the macromolecular complexes. In a healthy volunteer the serum amylase activity was elevated to a maximum of 797 u/l and the renal clearance rate of amylase was diminished to a minimum of 0.3 ml/min after infusion of 500 ml of a 6% solution of hydroxyethylstarch, as compared to 300 u/l, and 0.95 ml/min, respectively, during the  pre-infusion period.


In vitro induction of experimental autoimmune orchitis: characterization of a primary T lymhocyte response against testicular self antigens. Lymphocytes from normal, young adult rats were autosensitized in vitro against trypsin-dissociated autologous testis cells. The autoimmune response had been assayed both by determination of the lymphocyte proliferation, using a (3H)thymidine incorporation test, as well as by determination of their specific cytostatic activity against monolayer-forming testis cells which was measured in a terminal 51Cr uptake assay. The actively responding cells in this system are T lymphocytes. Non-T cells can exert additional, non-specific cytostasis. The immune specificity of the in vitro experimental autoimmune orchitis (EAO) has been demonstrated on two levels. First, the responding T lymphocytes were found to specifically adhere around cells from one particular testis subpopulation which morphologically resemble Sertoli cells. The rosetting lymphocytes respond only to testicular self antigen, but not to allogeneic lymphocytes. Second, specificity to the effector T lymphocytes was revealed in transfer experiments. Autosensitized EAO effector T cells cytostatically affected only major histocompatibility gene complex (MHC)-compatible testis cells. Their reactivity against MHC-different testis cells or MHC-compatible nontesticular fibroblasts was significantly decreased which suggests both tissue as well as MHC specificity of the EAO response.


Quantification of antigen-reactive cells among human T lymphocytes. The number of antigen-reactive cells among human peripheral blood T lymphocytes was estimated by a limiting dilution analysis. Antigen-induced lymphocyte activation was measured by means of incorporation of tritiated thymidine [3H]dThd. We have studied the frequency of memory T cells for the bacterial antigens tuberculin PPD and tetanus toxoid in immune donors, as well as the frequency of alloantigen-reactive T cells. In 11 different donors, the observed frequencies of the antigen-reactive T cell ranged between 1:300 and 1:16 000 for PPD; for tetanus toxoid values, between 1:750 and 1:11 500 were obtained in 5 different donors. The frequency of alloantigen-reactive T cells was found to be higher: between 1:200 and 1:600 (n = 10). For 3 donors, the estimated frequencies proved to be reproducible over a period of several months. Finally, a correlation could be demonstrated between the frequency of PPD-reactive T cells and the [3H]dThd incorporation of 4 X 10(4) PPD-stimulated lymphocytes.


Evidence for the endogenous production of T cell receptors bearing idiotypic determinants. Unprimed thymus-derived (T) cells from spleens of either conventional TEPC 15+ (T15+) BALB/c mice or neonatally suppressed T15- BALB/c mice were used to reconstitute neonatally suppressed T15- BALB/c nude mice and conventional T15+ BALB/c nude mice, respectively. In so doing, the idiotypes produced by phosphorycholine (PC)-specific bone marrow-derived (B) cells were dissociated from idiotypes putatively produced by PC-specific T cells. Subsequent to priming of the reconstituted BALB/c nude mice with either anti-T15 antisera or PC-MOPC 315, their splenic T cells were assessed for PC-specific helper activity and expression of the T15 idiotype. Our results indicate that expression of the T15 idiotype by T cells as determined by (a) the ability to prime PC-specific helper cells with anti-T15 antibodies, and (b) the ability to inhibit PC-specific helper function with anti-T15 antibodies, depended on the idiotype of the unprimed donor T cell source and not that of the B cells in the reconstituted recipients. We conclude that the idiotype-bearing T cell receptor specific for PC is not a passively adsorbed B cell product.


[Radioimmunoassay for plasma betamethasone and its application for clinical study (author's transl)]. A sensitive and specific radioimmunoassay for plasma betamethasone has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone-3-oxime-human serum albumin (BM-3-oxime-HSA) conjugate. The sensitivity was 20 pg and a standard curve was established with a useful range from 20 pg to 4 ng. The cross reactivity of all tested endogenous steroids was less than 0.2%. Cortisol with a cross reactivity of 0.14% caused slight interference at very high concentrations, but this factor is negligible when plasma cortisol level is less than 30 microgram/dl. The calculated interference by cortisol was less than 10%, and plasma not receiving BM consistently gave blanks which were less than 20 pg/tube. Reliability criteria were satisfactory. By this method plasma BM could be measured directly in dichloromethane extract of plasma. The plasma concentrations of BM were measured in normal subjects and patients with liver diseases following oral administration of BM. The peaks of the plasma concentrations for 1.0 mg and 1.5 mg of BM were 345 +/- 40 (n = 3) and 710 +/- 200 ng/dl (n = 5) respectively within 2 hours after administration in normal subjects. After the peak level, plasma BM rapidly fell and disappeared 24 hours after administration in all examined normal subjects. In patients with chronic active hepatitis, the peak levels for 1.0 mg and 1.5 mg of BM were 428 +/- 48 (n = 4) and 837 +/- 83 ng/dl (n = 7) respectively within 2 hours after administration. However, the peak levels of plasma BM were higher than those of normal subjects, and the disappearance of BM from the blood was markedly delayed, reaching a level of 318 +/- 88 nad 622 +/- 148 ng/dl respectively for 1.0 mg and 1.5 mg of BM at 5 hours. The relatively high plasma concentration of BM, ranging from 135 to 170 ng/dl was maintained even 24 hours after administration in all patients with chronic active hepatitis. The disappearance of cortisol from the blood also rapidly fell in normal subjects, but was markedly delayed in patients with chronic active hepatitis. There was good correlation between the severity of the liver disease as measured by the ICG retention at fifteen minutes and removals of BM and cortisol from the blood.


Ontogeny of macrophage function. I. Phagocytic activity and A-cell activity of newborn and adult mouse peritoneal macrophages. Phagocytic activity and immune-participating A-cell activity of newborn and adult mouse macrophages were investigated under in vitro conditions. Thioglycollate medium-stimulated newborn (SNB) or adult (SA), and nonstimulated adult (NA) peritoneal macrophages were used. Immune complexes of sheep erythrocytes (SRBC), aldehyde-fixed SRBC, and latex beads were employed in phagocytosis tests. A-cell activity was estimated as the capacity to support primary or secondary responses of macrophage-deprived adult spleen cells to SRBC. Results obtained were: 1) phagocytic activity of NA macrophages was highest and that of SNB macrophages was higher than SA macrophages, 2) no difference was observed in A-cell activity for secondary response among SNB, SA, and NA macrophages, and 3) SNB macrophages were lacking in A-cell activity for primary response. These results suggest that the substantial A-cell function should be evaluated in primary responses, and that the A-cell and pahgocytic functions do not necessarily accompany each other.


Nature of the phosphotungstic acid-chromic acid (PACP) stain for plasma membranes of plants and mammalian sperm. The selective staining of plasma membranes of plants and porcine spermatozoa given by a mixture consisting of 1% phosphotungstic acid in 10% chromic acid (PACP) applied following periodic acid destaining of glutaraldehyde-osmium tetroxide-fixed electron microscope sections is reduced or eliminated by prior extraction of the tissues with lipid solvents, including ethanol. The ethanol-soluble fraction of sperm contains constituents which restore the PACP-staining reaction when added to ethanol-extracted and lyophilized sperm. Analysis of the ethanol extracts by thin layer chromatography revealed two major components which reacted with both phosphotungstic acid (PTA) and alpha-naphthol detection reagents. These PTA-positive constituents were concentrated in plasma membranes of sperm; components with similar mobilities were found in fractions of plasma membranes from plants. Addition of the PTA-positive constituents from either sperm or plants to extracted and lyophilized sperm restored the PACP staining. The findings are interpreted to mean that one or more low molecular weight constituents (saccharides or glycolipids), rather than glycoproteins, concentrated in plaslma membranes are responsible for the unique PACP staining in both plants and porcine sperm.


[Electron microscopical demonstration of polysaccharides in rotifers (Aschelminthes) with the use of OsO4-ferricyanide (author's transl)]. Three species of rotifers were fixed with glutaraldehyde and postfixed in a OsO4-ferricyanide solution. This procedure results in staining of the sarcoplasmic reticulum (SR) and the surface coat of muscle cells as well as in staining of the surface coat of glands and nerve cells. Section-staining with leadcitrate increased the electron opacity of the precipitates, which are interpreted as polysaccharides. The partial decrease of staining of polysaccharides, which sometimes is observed, may be related to processes of function. The precipitates within the SR are considered as glycogen-beta-particles.


Biological horizons in molecular microscopy. The purpose envisaged in this report is not to provide a comprehensive monography but rather to give a survey, especially for biologists, of the state of the art and of current research trends in molecular microscopy. Following a brief discussion of the obvious discrepancy between instrumental capabilities and the limits of biologically significant information, a definition of the diversifying field is attempted. Four main topics are discussed. First, recent progress in the field of "low noise" specimen supports is reviewed. It is emphasized that a minimum background structure is an important but not the sole criterion for a satisfactory support. It is the ability to adsorb molecules in a predeterminable and orderly fashion which will attract wider attention in the future; positional and orientational order figure as crucial points in the strategem of low dose microscopy. Second, the problem of achieving adequate contrast without the expense of an unfaithful representation of molecular structures is discussed. Contrast is a problem of optimum imaging modes as well as of preparatory techniques. The third topic of discussion is specimen dehydration. Several avenues to circumvent or at least to alleviate dehydration artifacts are outlined. The last chapter focusses on the most fundamental problem in molecular microscopy:radiation damage. A brief synopsis of the physical and physico-chemical processes involved in damaging interactions is given and an attempt is made to tesselate the true picture of radiation damage to lipids and proteins. This might serve as a guidance in assessing the degree of structural fidelity to be expected for a given electron dose. Possibilities to overcome the radiation damage problem are adumbrated.


Alpha-fetoprotein concentration in maternal serum and abortion via VA and PGF2alpha. Most women undergoing abortion by vacuum aspiration (VA), extraamniotic prostaglandin instillation or vaginal prostaglandin suppositories showed a significant rise in their serum alpha-fetoprotein (AFP) concentrations during the procedure. We suggest this rise is due to a fetomaternal hemorrhage during the abortion procedure. Few women undergoing VA before 9 weeks' gestation showed this rise in serum AFP. To avoid fetomaternal hemorrhage during pregnancy termination, we recommend abortion by VA in early pregnancy.


Immunosuppressive factors in serum of patients with gastric carcinoma. Sera from patients with gastric carcinoma markedly suppressed the normal lymphocyte response to phytohemagglutinin (PHA) when compared with normal sera. The effect of this inhibition was well correlated with the degree of advance of the disease. Fractionation of normal and cancerous sera was carried out by DEAE-cellulose column chromatography using a continuous concave gradient. The elution patterns of the two kinds of sera were quite similar and separated into nine fractions. Protein analysis by immunoelectrophoresis and immunodiffusion showed no difference in subfractions between normal and cancerous sera. However, inhibitory activities of cancerous sera on PHA response of normal lymphocytes were found in alpha-globulin, beta-globulin, and alpha2-macroglobulin fractions. On the other hand, in the fractions from normal serum, alpha- and beta-globulin fractions showed a similar degree of inhibition to PHA response of normal lymphocytes, but alpha2-macroglobulin fraction had no inhibitory activity. Such inhibitory fractions showed an immunosuppressive activity not only to PHA response of normal lymphocytes in vitro, but also due to the induction of splenic plaque-forming cells in vivo. The electrofocusing patterns of alpha2-macroglobulin fractions obtained from normal and cancerous sera showed a remarkable difference in protein composition. These results indicated that the function of lymphocytes may be regulated by a variety of humoral factors and, especially in patients with gastric carcinoma, the cell-mediated immunity may be modified by some factors not present in normal serum.


Beta2-microglobulin levels of serum and ascites in malignant diseases. Serum beta2-microglobulin levels were measured by radioimmunoassay in patients with various malignant neoplasms, ascitic patients, and also patients with definite or suspected hepatoma showing variable levels of serum alpha-fetoprotein. Elevated serum beta2-microglobulin levels greater than 2.5 mg/liter were found in various malignant neoplasms, especially in multiple myeloma (66.6%) and hepatoma (60.4%) The ascites/serum ratio of beta2-microglobulin levels in the patients with malignant ascites is significantly higher than in those with non-malignant ascites. However, ascites/serum ratios of total protein, IgG, albumin, creatinine levels were not significantly different between the two groups. Levels of serum beta2-microglobulin were correlated well with those of alpha-fetoprotein in the patients with definite or suspected hepatoma (r=0.72, P less than 0.001). From these results it was concluded that (1) high levels of serum beta2-microglobulin in these patients could be attributed to its hyperproduction by tumor cells or by the cells which had been infiltrated and activated, (2) it is useful to estimate the ascites/serum ratio of beta2-microglobulin levels in differentiating malignant from non-malignant ascites, and (3) it might suggest that a function of beta2-microglobulin is in some way related to that of alpha-fetoprotein, and the alpha-fetoprotein-synthesizing cells secrete a great deal of beta2-microglobulin, although its function remains unclear.


Effect of x-irradiation of recipients on in vivo neutralization of syngeneic tumors by spleen cells nonspecificially immunized with allogeneic tumors. Spleen cells from WKA rats immunized with allogeneic Donryu tumor (AH66), when examined by the Winn test, inhibited lethal growth of admixed antigenically unrelated syngeneic tumor (KMT-17) only when a mixture of spleen cells and tumor cells was injected into pre-irradiated recipients. On the other hand, spleen cells specificially sensitized to KMT-17 inhibited growth of admixed KMT-17 cells either in irradiated or non-irradiated rats.


The influence of serum content in the medium on the survival of human bone marrow in suspension in vitro. Samples of human bone marrow cells from patients with various diseases were cultivated in vitro by means of a simple stationary suspension method. Medium Eagle MEM with the addition of allogeneic serum and borosilicate glass were used. The cells survived significantly longer in samples with 50 per cent of serum than in samples with 30 per cent of serum only. Monocytes showed the longest survival (max. 95 days) in cultures with 50 per cent of serum. Myelocytes and segmented neutrophils as well as normoblasts survived till 45th day and plasmocytes till 66th day. Mitoses in monocytes were found till 50th day. Moreover so called "satellitosis" of plasmacytes around a macrophage was observed in cases with reactive plasmacytosis.


The effect of endotoxin on the colony stimulating activity (CSA) in sera of mice. We found one peak of a new molecular form of CSA after chromatography on Sephadex G-200 in the serum of mice treated with endotoxin. Total activity and two peaks of CSA were much higher than controls.


Inhibition of absorption capacity of HLA antigens on in vitro hormone-treated lymphocytes. Hydrocortisone, Agostilben, Agovirin, Neolutin and Superanabolon partially to completely inhibit the absorption capacity of HLA antigens on the lymphocytes in vitro. The effect is directly proportional to the duration of lymphocyte treatment and the type of hormone, yet unrelated to the type of antigen. The importance of this phenomenon for HLA antigen typing in hormones-treated patients is discussed.


Angio-immunoblastic lymphadenopathy. Report of a case with pleural effusion. A 78-year old man is presented with a characteristic case history and physical and laboratory findings typical of angio-immunoblastic lymphadenopathy with dysproteinemia (AILD). The disease had an acute onset with constitutional symptoms, generalized lymphadenopathy and hepato-splenomegaly. The presence of a large pleural effusion was of particular interest. Histologically the distinctive feature was a pronounced proliferation of small blood vessels and immunoblasts in the lymph nodes. Management of patient with AILD is problematic. Since AILD is a non-neoplastic process, symptomatic treatment with small doses of steroids, if necessary, would seem to be the best therapeutic approach. However, in our patient, as well as in other cases reported in the literature, a rapid and long standing (18 months so far in our case) complete remission was obtained with a short cycle of chemiotherapy.


[Non-secreting plasmacytomas].

[Non-secreting plasmacytomas]. In non-secernent plasmocytomas there are no characteristic changes of serum protein. Diagnostic difficulties may be overcome by cytomorphological examinations, immunofluorescence and electron microscopy. By referring to 4 own observations the value of the electron microscopic characteristics of plasma cells is demonstrated. Subtile investigations in protein diagnostics are required for avoiding incomplete monoclonal immunoglobulins which may greatly enter the kidneys to be overlooked. Generally the prognosis of non-secernent plasmocytomas is not worse than that of other forms.


[Immunoglobulins and glycoproteins in lymphogranulomatosis]. In the serum of 27 patients with malignant lymphogranulomatosis the authors determined the serum level of glycoproteid-carbohydrate components (hexose, hexosamine, sialin acid, and seromucoid) and the concentration of 11 different glycoproteids. In the early stage of the disease the immunoglobulin level is moderately increased in the serum, whereas a diminution can be observed in stage IV. The concentrations of ceruloplasmin, alpha-2-macroglobulin and orosomucoid were already increased significantly in stage III. The increase did not continue in stage IV. In the final stage of the disease the concentrations of alpha-1-antitrypsin and haemopexin turned out to be increased considerably. A significant decrease in the transferrin level could be registered in stage III with this diminution also continuing in the further course. Changes of beta-C-globulin and haptoglobin concentrations could not be evaluated statistically. The content of carbohydrate components in the glycoproteids will already increase in the early stage of the disease with this increase continuing in the further course. Among histological types there was a more significant increase of immunoglobulins in those forms rich of lymphocytes.


[Clinical treatment of chronic lymphatic leukemia by means of extracorporeal blood irradiation]. A chronic lymphatic leukaemia (CLL) with hepato-splenomegaly was treated in a 76 years old female patient by means of extracorporeal irradiation of blood (ECIB), after which a complete involution of the tumour in the spleen had taken place. Leukocytes initially increased to 35,500/mm3, amounting to 10,700/mm3 later on. The patient died of pneumonia in general cachexia. No signs of CLL could be found macroscopically after autopsy. An involution of the lymphatic infiltration could be identified microscopically in the bone-marrow.


[Contribution to central nervous system tumor diagnosis by means of cytocentrifuged cerebrospinal fluid]. The cytocentrifugation of the liquor cerebrospinalis provides a useful diagnostic tool to differentiate benign from malign diseases of the central nervous system. The method is illustrated with liquor analyses in the field of pediatric oncology: medulloblastoma, leptomeninx sarcoma, plexuspapilloma, astrocytoma, Abt-Letterer-Siwe, ALL, reticulum cell sarcoma, Ewing sarcoma, retinoblastoma. The speed and ease of the processing, the highest possible cellular yield and the good preservation of the cellular characteristics are great advantages of this new method.


[The appearance of immature cell leukemias after radiologic and cytostatic intensive therapy of Hodgkin's lymphoma]. Among 106 patients with Hodgkin's lymphoma in stage IIIB and IVB treated from September 1969 to June 1977 by a working team for hematology and oncology at the clinic and outpatients clinic of the Medical School of Erfurt, there were two cases (=1.9%) showing a final transition to leukaemia with immature cells. A third neoplasia which had developed in a former field of irradiation was additionally observed in one patient. The various possibilities arising from an oncologic effect of ionizing rays and those substances having a cytostatic effect are discussed. After frequent reports on the occurrence of secondary tumours following intensive radiological and cytostatic therapy of advanced Hodgkin's lymphoma the enhanced risk of a secondary tumour being induced by this combined therapy cannot be excluded. Various conclusions are drawn from that by the authors.


[Maintenance of anti-streptococcal activity of haptoglobins after addition of hemoglobin]. The antibody-like activity of haptoglobin types hp 2-2 and hp 2-1 for streptococci having the T4 antigen is not reduced by saturation with hemoglobin.


[Method for the stabilization of E-rosettes with glutaraldehyde]. A technically simple method of fixing E-rosettes is represented. Under the influence of glutaraldehyde of 0.5% the E-rosettes being themselves very fragile assume a solid form and may be firmly spread to smears. Comparative examinations made on fresh and panoptically stained preparations showed a good accordance of results. The advantages of this method consist in its easy handling, possible simultaneous or alternative application of two counting procedures and the possibility of having the test results registered in archives.


[A new method for the determination of thrombocyte function in patients and in preserved blood]. In a circulating thrombocytic plasma the pressure in front of a defined mesh filter depends on the combined capacity of adhesion and aggregation of thrombocytes. A new clinical in vitro test is based on this principle which comes nearest to the conditions to be found in hemostasis dependent on thrombocytes. Judging from the preliminary results of investigations performed on stored blood, blood donors, patients with thrombocytopenia, disturbances of blood flow and patients under anticoagulant therapy, the time until the pressure rise after administering ADP and the maximal pressure amplitude proved to be the most significant parameters of the new procedure.


[Results of a new platelet function test in various clinical diseases and in preserved blood]. In healthy persons, patients with thrombocytopenia and patients with arterial disturbances of blood flow with and without administration of Falithrom the pressure recording was carried out after the combined aggregation and adhesion of thrombocytes. The reaction time particularly observed after administration of ADP revealed marked differences in the groups examined, likewise the maximal pressure amplitude in front of the pumped through filter. When Falithrom was administered the thrombocyte functions were clearly impaired. The reduction of the capacity of platelets to obstruct filter pores can impressively be proved even in those thrombocytes stored in ACD-AG-plasma.


No change of whole blood of plasma viscosity in cardiac patients after subcutaneous calcium heparin. Whole blood viscosity, plasma viscosity and haematocrit were studied in a group of cardiac patients before and during subcutaneous heparin treatment. No significant change was noted in any of the parameters investigated. Relative viscosity (whole blood viscosity/plasma viscosity ratio) was also unaffected. These data indicate that heparin has no effect on the rheology of blood in vitro.


[Measurements of storage-induced morphologic changes in preserved blood by means of light scattering]. This study is concerned with changes in diffuse backscattering of white light caused by laminarly streaming or resting banked blood stored for periods up to 7 weeks. The most pronounced changes, i.e. a steep rise of scattering intensity, were found at the beginning of storage. Under streaming conditions such as resulting in a maximum of backscattering and effective disaggregation of rouleaux the temporal changes of scattering intensity obviously were not simply related to morphological properties of the blood. In addition, there was considerable individual variation in the amount of backscattering from various preserves. This invalidates an assessment of banked blood by means of light scattering by a single-data type measurement.


[Evaluation of internationally used blood stabilizers for effective erythrocyte substitution]. Haemotherapy developed in the direction of transfusing pure blood cell preparations, if possible and enlarging the therapy with plasma fraction considerably. Quality losses of haemotherapeutics will already occur when they are prepared. This is mainly due to the conditions of blood collecting, blood stabiliser, duration and storage temperature from the blood collecting to further processing as well as biological variability of the composition of the donor's blood. The amount of substrate available to erythrocytes differs in various blood stabilisers. Deplasmatized erythrocyte concentrates can be used after several weeks of storage, if a high glucose concentration is present in the blood stabiliser. In CPD media the function of erythrocytes, the oxygen supply of the tissue, will remain intact a week longer than in ACD media. This effect will be increased by xylitol and pyruvate as well as by adding bicarbonate simultaneously. In future a primary importance will have to be attached to an improved storage of erythrocytes in the form of resuspended buffy coat-free erythrocyte concentrates.


[Effect of dihydroxyacetone on metabolic parameters and survival of resuspended erythrocytes after storage at 25 degrees and 4 degrees]. Erythrocyte concentrates from CPD blood were resuspended after separating the leukocyte-thrombocyte layer and stored at 4 degrees C and 25 degrees C. The solutions for resuspension differed in the content of substrate, pH, condition of sterilization and the resuspension volume used, 1 or 4 volumes of cell concentrate to 1 volume of resuspension solution. At 4 degrees C, there was no effect of DHA on the the 2.3 bisphosphoglycerate content (P2G content). The decreased activity of triokinase at low temperature, phosphate deficiency and a partial disintegration of DHA during the autoclave as well as the difference of the commercial DHA preparations are discussed as underlying causes. At 25 degrees C DHA delayed the P2G decrease during storage. The enhanced triokinase activity above 15 degrees C is principally considered to be the cause for it. High rates of haemolysis, rapid ATP decrease, survival rates of 56% after a storage of three weeks for erythrocyte resuspensions with DHA in used compositions render a clinical application impossible at present. It could be well suitable as a substrate for resynthesizing P2G. An addition of ascorbate + nicotinamid + adenin at a storage temperature of 4 degrees C had no influence on the P2G content. A resuspension solution on the basis of xylitol with additions of sorbitol, bicarbonate, inorganic phosphate, pyruvate, adenine and guanosine showed the best properties in the content of P2G and ATP, lactate formation and survival rate during a storage at 4 degrees C.


The anticlastogenic effect of various combinations of cysteamine, AET, HCT, and amino acids on chromosome damage by trenimon and bleomycin in human lymphocytes in vitro. The protective activity of the combined application of anticlastogens against the chromosome-damaging action of Trenimon and bleomycin was studied by analyzing more than 32000 metaphases from cultures of human peripheral lymphocytes. Screening tests with the combinations cysteine/cysteamine/AET, cysteine/methionine/asparagine, cysteine/serine/HCT, and AET/HCT, with Trenimon as clastogen, in no case revealed a hyperadditive (synergistic) effect. From the results of detailed analyses of the action of the combination AET/HCT it was concluded that the (nonsynergistic) anticlastogenic effects observed were induced due to intracellular biologic mechanism, and not due to a reaction in the culture fluid between clastogens and anticlastogens. Although the observations gained with Trenimon or bleomycin differed in some respects, the anticlastogens apparently act via a mechanism at least common to AET and HCT, i.e., they manifest their effect in lymphocyte cultures by a limited interaction with the process of aberration formation, rather than by influencing repair processes, which are blocked by caffeine.


Concerning the linkage relationships of the Gc and MNSs loci. Lod scores for the linkage relationships of the Gc and MNSs loci are presented for data from a number of published pedigrees and 103 new families. Linkage may be excluded at a recombination frequency of less than 25% in males and 30% in females.


Histochemical demonstration of collagen fibers in ascorbic-acid-fed cell cultures. Nine cultures of fibroblast cell types and 13 epithelial-like cell types were maintained for 1 week in media supplemented with L-asborbic acid (50 microgram per ml). All fibroblast-like cultures produced extracellular fibers that stained positively by a silver-impregnation reticulin stain. Nine of the 13 epithelial-like cultures produced fibers that stained positively for reticulin. Nearly all cultures not supplemented with ascorbic acid showed no fiber staining. Those few lines that stained positively for reticulin in the absence of ascorbic-acid supplementation demonstrated only slight reticulin formation. Reticulin from one fibroblast culture and one epithelial culture was examined by electron microscopy, and the silver-impregnated fibrils were morphologically identical to collagen. The reticulin was digestible with collagenase, providing further evidence that the silver-impregnation reticulin stain identifies collagen in culture. The demonstartion of collagen can be performed easily in histology laboratories using Formalin-fixed cells, and provides a means of assaying a functional property of cells in culture which is characteristic of connective tissue fibroblasts in general as well as certain specialized epithelia.


Cholera toxin effects on fluid secretion, adenylate cyclase, and cyclic AMP in porcine small intestine. The effects of cholera toxin on mucosal cyclic nucleotide concentrations and on net fluid secretion in the porcine small intestine are reported. Cholera toxin causes net secretion of fluid into the small intestine of weanling pigs, and secretory rates are dependent on the dose of the toxin placed in intestinal loops. Intestinal secretion due to cholera toxin exposure was not consistently accompanied by elevated concentrations of mucosal cyclic AMP or cyclic GMP. Net fluid fluxes in individual loops did not correlate with mucosal cyclic AMP concentration in the same loop. Jejunal adenylate cyclase was activated to a lesser extent in pigs, compared with rabbits, after in vivo treatment with cholera toxin. In vitro activation in cell-free homogenates was similar for both species. Papaverine was similar to cholera toxin in causing fluid secretion without cyclic AMP accumulations, but 3-isobutyl-1-methyl xanthine significantly increased cyclic AMP concentration and induced fluid secretion in pigs. Weanling pigs appeared to differ from rabbits in having a secretory response to cholera toxin which was independent of elevations in total mucosal cyclic AMP concentration.


Possible role of Fc receptors on cells infected and transformed by herpesvirus: escape from immune cytolysis. Receptors for the Fc portion of nonimmune immunoglobulin G were demonstrated on B103 rat brain neuroma cells infected with herpes simplex virus type 1 (HSV-1) KOS by a radioimmunoassay using 125I-labeled heat-aggregated Fc fragments. Immune F(ab')2 fragments specific for HSV antigens competed efficiently for Fc binding sites, suggesting that the binding of Fc fragments to infected cells is specific for viral cell-surface antigens. It has been suggested that the binding of immune complexes to Fc receptors on the surfaces of tumor cells in vivo plays a role in protecting these cells from immune destruction. In vitro evidence is presented for the ability of aggregated immunoglobulin G molecules bound to cell-surface Fc receptors to protect both HSV-infected and HSV-transformed cells against complement-dependent and cell-mediated immune lysis.


Hepatitis B surface antigen-specific cell-mediated immune responses in human chronic hepatitis B surface antigen carriers. The presence of hepatitis B surface antigen (HBsAg) and antibody (anti-HBs), hepatitis B e antigen (HBeAg) and antibody (anti-HBe), the nature of T-cell function, and specific cell-mediated immunity to HBsAg were determined and evaluated serially in groups of subjects with chronic HBsAg carrier states and in seronegative controls. The techniques of in vitro lymphocyte transformation, spontaneous rosette formation, radioimmunoassay, reverse passive hemagglutination, passive hemagglutination, rheophoresis, and liver function tests were employed for these studies. For the lymphocyte transformation assay, multiple concentrations of phytohemagglutinin and purified HBsAg were used as stimulants. Cell-mediated immunity to HBsAg was detectable in 50% of the chronic HBsAg carriers (responders) at one or more concentrations of HBsAg. The remaining carriers (nonresponders) and controls failed to manifest HBsAg-specific lymphocyte transformation activity. The profile of the responders was characterized by elevated serum glutamic pyruvic transaminase levels, the presence of anti-HBe, high HBsAg titers, and the conspicuous absence of HBeAg in the serum. The nonresponders were characterized by normal serum glutamic pyruvic transaminase levels, the presence of HBeAg and anti-HBe, and lower HBsAg titers. These observations demonstrate the presence of specific cell-mediated immunity to HBsAg in chronic HBsAg carriers who manifest biochemical evidence of liver disease.


Stimulation of cyclic AMP secretion in Vero cells by enterotoxins of Escherichia coli and Vibrio cholerae. The morphological response of Vero cells to Escherichia coli heat-labile enterotoxin was similar to that of cholera toxin and was accompanied by increases in the intracellular level of cyclic AMP. The effects of both enterotoxins were enhanced by the presence of phosphodiesterase inhibitor and inhibited by heat or specific antisera. Accumulation of cyclic AMP preceded the morphological response.


Significance of noncapsular antigens in protection against experimental Haemophilus influenzae type b disease: cross-reactivity. A bacterial strain, tentatively identified as an Actinobacillus species, was found to asymptomatically colonize the pharynx of some rats and to bear cell wall antigens which cross-react with noncapsular antigens of Haemophilus influenzae type b (HIb). The cross-reacting antigens appeared to be a heterogeneous mixture with varying molecular size and charge. The antigenic moieties are probably carbohydrate in nature. Antisera raised with this strain had both immunochemical and biological (bactericidal, opsonizing, and protective against experimental infection) activity against HIb. These findings lend further evidence to the idea that noncapsular antigens are important in the induction of resistance to HIb disease. The findings also raise the possibility of using bacteria which cross-react with noncapsular antigens for immunization against HIb disease in humans through nasopharyngeal or enteric colonization.


New surface-associated heat-labile colonization factor antigen (CFA/II) produced by enterotoxigenic Escherichia coli of serogroups O6 and O8. Enterotoxigenic Escherichia coli (ETEC) belonging to serogroups O6 and O8 do not possess the H-10407-type colonization factor antigen (CFA/I). However, these frequently isolated ETEC were found to possess a second and distinct heat-labile surface-associated colonization factor antigen, termed CFA/II. Whereas CFA/I mediates mannose-resistant hemagglutination of human group A erythrocytes, CFA/II does not. CFA/II mediates mannose-resistant hemagglutination of bovine erythrocytes, and mannose-resistant hemagglutination is rapid only at reduced temperature (4 degrees C). Because CFA/II, like CFA/I, is spontaneously lost by many ETEC isolates in the laboratory, it was possible to produce specific anti-CFA/II serum by preparing antiserum against living cells of a prototype strain (PB-176) and adsorbing this serum with living and heat-treated cells of its CFA/II-negative derivative strain PB-176-P. This serum, which neutralized the colonization factor activity of CFA/II-positive strains in infant rabbits, was employed to confirm the presence of CFA/II on ETEC which exhibited mannose-resistant hemagglutination of bovine but not human erythrocytes. CFA/II, like CFA/I, mediates adherence of the bacteria to the mucosal surface of the small intestine, as demonstrated by indirect immunofluorescence. CFA/II appears to be an important virulence factor for humans since CFA/II-positive ETEC are frequently isolated from diarrhea cases, particularly travelers' diarrhea, in Mexico; these ETEC were not uncommon in a collection of isolates from Bangladesh. The O6:H16 strain of ETEC responsible for an outbreak of diarrhea in the United States was also shown to be CFA/II positive. CFA/I and CFA/II were never found on the same serotypes of ETEC, but 98% of the heat-stable and heat-labile enterotoxin-producing ETEC belonging to the frequently isolated serogroups O6, O8, O15, O25, O63, and O78 were positive for either CFA/I or CFA/II.


Progressive vaccinia: immunological aspects and transfer factor therapy. Progressive vaccinia is a rare and serious complication of smallpox vaccination. Depressed immune function can generally be found as an underlying disorder; thus adequate immuno-correction may be expected to be therapeutically effective. Humoral and cell-mediated immunity was repeatedly examined in one case throughout the course of the disease. Results indicated partial deficiency of cell-mediated immunity. No therapeutic effect was achieved by using human antivaccinia immunoglobulin and N-methylisatin beta-thiosemicarbazone. Transfer factor therapy was also attempted. Treatment with a non-specific transfer factor preparation was followed by a transitory clinical improvement. A specific transfer factor preparation given during the last month of life, however, had no therapeutic effect. The patient died on the 145th day after vaccination. Autopsy findings pointed to combined immune deficiency.


Tumours of the urinary bladder accidentally diagnosed during prostatectomy. By analysing 1472 prostatectomies, the authors have stated a coexistence of tumour of the bladder in 31 cases (2.11%). In 11 patients (0.75%) the tumours were accidentally diagnosed, as late as during the operation itself. Eight patients were operated transvesically. The tumour was diagnosed immediately after opening the bladder. The tumour was removed first and then the adenoma. Three patients were operated on according to Millin's method, their tumours having been detected only after enucleation of adenoma of the prostate. In five cases cancer of the bladder was diagnosed and in six papillary tumour of the bladder was found. The bladder cancers were treated by partial excision of the bladder or by deep electrocoagulation. The authors maintain that the small percentage of accidentally diagnosed tumours is no indication for a routine performance of cystoscopy in every case of adenoma of the prostate before intended prostatectomy.


Biochemical methods for the assessment of the selectivity of proteinuria. The authors have elaborated a biochemical technique for the assessment of the selectivity of proteinuria by means of the clearances of ceruloplasmin, haptoglobin and albumin. The results obtained in 69 patients were compared to those established by the method of Cameron and Blanford, as well as by the clearance of alpha2-macroglobulin. No significant differences were found in the indices of proteinuria, as determined by the proposed biochemical technique and by the method of Cameron and Blanford. Thus the diagnostic value of both methods to assess the selectivity of proteinuria could be considered as equal. The additional measurement of the selectivity of proteinuria by means of proteins with considerably high molecular weight (haptoglobin, alpha2-macroglobulin) gave the possibility of recording cases with severely impaired glomerular permeability. In this respect, the use of the clearance of haptoglobulin, particularly the phenotype Hp 2--2, provided valuable diagnostic information in cases in which the routine methods gave borderline values for the index of proteinuria.


[Florid oral papillomatosis. A contribution to cytostatic therapy]. Based on the recent clinical experience in treating florid oral papillomatosis (FOP), the only successful therapy appears to be the use of cytostatic agents. The case report of a patient with clinically and histologically confirmed FOP is described to emphasize the advantages of administering Bleomycin intra-arterially (external carotid artery) by means of a Holter catheter. This catheter is used to release pressure in the treatment of internal hydrocephalus and is designed to remain in place for a number of years. The successful treatment of FOP points out the possibility of employing a Holter catheter for administering cytostatic agents in the treatment of malignant neoplasms in the facial area. Cytophotometric examinations of the prickle cells within the FOP mass showed a diploid pattern of DNA distribution similar to that of benigh tumors. However, it is not possible to definitely establish the malignant or benign nature of FOP in this manner.


The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC. Treatment of tissue sections with enzymes wich degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. Both Streptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of both Streptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, and Streptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.


The aldehyde fuchsin and colloidal iron staining reactions in the canine thyroid C cell. The cytochemically reactive groups which are responsible for Aldehyde Fuchsin (AF) and colloidal iron (CI) staining of C cells were investigated in the canine thyroid gland. To this end, stains for proteoglycans and peptide groups were utilized in conjuction with hydrolysis of glycosidic and amide bonds. In addition, the following procedures were used: acetylation, benzoylation, nitrozation, aldehyde blockade, sulphydryl blockade, methylation and mild acid hydrolysis. No acidic proteoglycan, sialic acid, polyphosphate or polysaccharide ester sulphate were detected in C cells; the results suggest that AF staining, after an oxidation step, and CI staining are due to polypeptides. Sulphydryl and carboxyl groups together are necessary for mediating the attachment of AF in C cells and it is adduced that this attachment is due to the combined charges of sulphonic and carboxylic acids. Methylation and acetylation inhibit CI staining and those staining reactions that depend upon carboxylic acid (TB) and hydroxyl groups (PAS) for their dye attachment in C cells. Acid hydrolysis, which increases the demonstration of carboxylic acid in C cells, decreases the attachment of hydroxyferric ions. I speculate that this inhibition is due to extraction of iron sites in the C cell and conclude that it is not solely carboxylic acids in C cells that are responsible for CI staining.


Concanavalin A-peroxidase-diaminobenzidine-periodic acid-m-aminophenol-fast black salt K: a method for the dual staining of neutral complex carbohydrates. A method has been developed for the dual staining of neutral complex carbohydrates in light microscopy. It combines a concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB) method with a period acid-m-aminophenol-Fast Black salt K (PA-AP-FBK) sequence. With the combined method it is possible to stain alpha-D-glycosyl and alpha-D-mannosyl residues brown and 1,2-glycol groups of neutral complex carbohydrates blackish purple. The validity of the method has been confirmed with appropriate histochemical controls and enzyme digestions on test tissues.


The histochemistry of thiols and disulphides. II. Methodology of differential staining. The reduction of disulphide bonds by various mercaptans and tri-n-butyl-phosphine (TBP) has been examined in paraffin sections of rat tissues. A "re-reduction' procedure demonstrating any residual disulphides shows that nearly equivalent endpoints are reached by all of the reagents at pH 8.5 and room temperature, though at greatly differing rates. TBP is the reductant of choice in that it acts rapidly, cannot cause the thiolation which is more or less pronounced with certain mercaptans and least reverses the prior alkylation of native thiol groups by iodoacetate or N-substituted malemides. Supporting studies establish that, except in highly compact structures, native as well as generated thiol groups can be visualized with satisfactory completeness and specificity by N-(4-aminophenyl)maleimide followed by a diazotization and coupling sequence. These findings provide the basis for the selective staining of disulphides, either alone or differentiated from native thiols in the same section.


The histochemistry of thiols and disulphides. III. Staining patterns in rat tissues. With the aid of new staining methods, thiol groups produced by the reduction of disulphide bonds were positively distinguished from pre-existing groups in paraffin sections of several organs of the rat. Good preservation of structures in which the natural thiol-disulphide balance had been maintained was sought by fixing the tissues in neutral formalin containing an organomercurial. After dissociation of the resulting mercaptide bonds that protected the native thiols, these were shown in one colour and then disulphide sites in another within the same sections. Intracellular granules and extracellular membranes rich in disulphides thereby stood out in red against the predominantly blue labelling of the cellular ground plasm. Intimate mixtures of the two forms in some places and the presumed transformation of thiols to disulphides in others, notably the keratinizing epithelium of the tongue, were readily seen. Supplemented by separate visualization of thiols and disulphides along with suitable controls for specificity of staining, the results obtained diverged in some major respects from those of previous investigations.


Conjugation of lectins with fluorochromes: an approach to histochemical double labeling of carbohydrate components. Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyan ate (TRITC) are reported. 20-microgram of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.


On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid. A reappraisal. The specificity of the alcoholic acidic silver nitrate staining method for the histochemical localization of ascorbic acid was reappraised. It was found that the method is by and large better suited for the localization of ascorbic acid in both animal and plant tissues due to its greater specificity, which is ensured by employing reagent made in carbon dioxide saturated glass distilled water as well as by carrying out the reaction at a low temperature (0-4 degrees C) and at a pH of 2-2.5.


Tallysomycin, a new antitumor antibiotic complex related to bleomycin. III. Antitumor activity of tallysomycins A and B. The antitumor activity of tallysomycins A and B was determined in five experimental tumor systems in mice. Tallysomycins A and B were highly active against B16 melanoma, sarcoma 180 ascites tumor and Lewis lung carcinoma, and moderately active against P388 leukemia but were without effect on lymphoid leukemia L1210. The antitumor activity of tallysomycin A was 2 to 3 times that of tallysomycin B and 3 to 17 times that of bleomycin. Tallysomycin A was about 1.5 and 4 times more toxic for mice than tallysomycin B and bleomycin, respectively, in terms of subacute LD50 values.


Bibliographic control of nonprint educational material. AVLINE is an on-line data base maintained by the National Library of Medicine containing references to over 5,000 nationally available nonprint materials. Each title is screened for technical quality, processed according to NLM's categorization, and undergoes descriptive and subject cataloging. In addition to the bibliographic control, an assessment is made of educational potential and content validity by the peer appraisal process of the Association of American Medical Colleges (AAMC). AVLINE is available through the computer network that supports the MEDLINE system. The data base offers the use of a wide range of sophisticated retrieval possibilities.


The in vivo transfer of antigen-induced airway reactions by bronchial lumen cells. Rhesus monkeys with airway responses to aerosol challenge with Ascaris antigen constitute a primate model of inhalant asthma. Previous studies have shown that bronchial lavage cells from airway-reactive animals will release histamine or a slow-reactive substance of anaphylaxis after challenge with antigen. Because these bronchial lumen cells are the first cells in contact with inhaled antigen, they may play a role in induction of antigen-induced airway responses. To evaluate this possibility, bronchial lumen lavage cells from animals with airway reactivity were transferred to the bronchial lumens of animals with negative airway responses to antigen challenge. The transfer of the bronchial lumen cells resulted in transient airway reactivity of the recipients to aerosol antigen challenge. It is suggested that the mast cells which constitute a component of the bronchial lumen cells may be the active cell alone, or in combination with other cells, which results in this primate immunoglobulin E-mediated airway response and its transfer to nonreactivie recipients.


Histamine release reaction of basophils in chronic granulocytic leukaemia. Induction by heterologous anti-immunoglobulin E, concanavaline A, and phytohaemagglutinin; effect of heavy water. Basophils possess membrane bound IgE molecules, and immunological activation leads to a secretory process with cell degranulation and histamine release. Heterologous anti IgE, concanavaline A, and phytohaemagglutinin are potent non-cytotoxic releasing agents. They operate by a mechanism similar to that of immunological activation. Heavy water is not a histamine releasing inducer but it increases histamine release of the cells. We studied the histamine release reaction of leukaemic basophils in 10 patients and found a physiological response such as that previously reported with normal human basophils.


Histological demonstration of copper and copper-associated protein in chronic liver diseases. Liver copper concentrations in percutaneous biopsy specimens were measured by neutron activation analysis and compared with histological staining for copper by rubeanic acid and rhodanine, and with copper-associated protein stained by orcein. Liver copper concentrations were elevated in 31 of 35 biopsies from patients with primary biliary cirrhosis (PBC), and discrimination between normal and elevated liver copper was correct in 32 of the 35 biopsies by staining with rubeanic acid, and 31 of the 35 by staining with rhodanine. Orcein staining of copper-associated protein was positive in 33 of the 35 biopsies. All 17 biopsy specimens from patients with Wilson's disease had high liver copper concentrations, but only nine had positive staining for copper, and six were orcein positive. Similarly, histological stains gave little indication of the liver copper concentrations in tissue from 16 patients with chronic active hepatitis. Staining of liver sections can be useful in detecting elevation of liver copper in PBC, but not in Wilson's disease, where the absolute concentration must be measured. Excess copper appears to accumulate in the liver in different chemical forms in PBC and Wilson's disease.


The islands of Calleja: organization and connections. The islands of Calleja (IC) in the rate are composed of seven small groups of granule cells in the polymorph layer of the olfactory tubercle and one large group, the insula magna, which lies along the border between septum, nucleus accumbens and nucleus of the diagonal band. The cytoarchitecture and neuronal morphology of the IC and surrounding cells, studied using Nissl-stained and Golgi-Kopsch material, are described. In addition, the afferent and efferent connections of the IC were analyzed using fluorescence histochemistry, the autoradiographic tracing method, and the anterograde and retrograde horseradish peroxidase methods. Topographically organized projections to the IC from the dopamine-containing cells of the substantia nigra-ventral tegmental area are demonstrated by the glyoxylic acid fluorescence histochemical method and the autoradiographic tracing technique. Anterograde and retrograde horseradish peroxidase studies provide evidence for reciprocal, topographically organized interconnections between the IC and the septum, nucleus accumbens, amygdala and piriform cortex. These observations indicate that the IC constitute a unique population of granule cells, located in the olfactory tubercle, innervated by dopamine neurons of the mesencephalon and interconnected with olfactory and non-olfactory components of the basal forebrain.


Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials. In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.


Immunocytochemical localization of oncodevelopmental proteins in human germ cell and hepatic tumors. In a combined tissue and serum study alpha-1-antitrypsin (AAT) and alpha-fetoprotein are demonstrated in parallel within tumor tissue inclusions in both endodermal sinus (yolk sac) tumors and malignant hepatomas, and AAT is demonstrated as a marker in both neoplastic and preneoplastic liver lesions occurring in oral contraceptive users, all in association with normal serum AAT phenotype. The tumor inclusions in the first two instances differ immunocytochemically from AAT liver cell globules found in inherited AAT deficiency, which are unreactive for alpha-fetoprotein. It is concluded that unlike the molecular basis of storage associated with AAT phenotypic variation, the tumor inclusions reflect a separate, nongenetic mechanism of AAT storage, which may be epigenetic in nature. AAT and alpha-fetoprotein both are synthesized normally in yolk sac and fetal liver, a parallelism which disappears soon after birth. The reexpression of both proteins in two distinct tumor types arising from endodermal origins (yolk sac and liver), suggests that these markers may represent reemerging fetal gene products, a phenomenon previously proposed only for alpha-fetoprotein, a prototypic "oncofetal antigen."


Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas. An indirect immunoperoxidase method was first used to localize mouse mammary tumor virus (MMTV) antigens in paraffin sections of mammary tumors of Paris RIII and CD8F1 mice. By using the same method, an antigen with cross-reactivity to a group-specific antigen (gp52, a 52,000 dalton glycoprotein) of MMTV was detected in paraffin sections of human breast carcinomas. The specificity of this reaction with antibody against MMTV was examined by absorption of the IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and prufied gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 73 of 191 (38%) breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. A positive reaction of uncertain specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.


Palladium chloride as a stain for elastin at the ultrastructural level. Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.


An evaluation of Coomassie Brilliant Blue as a stain for quantitative microdensitometry of protein in section. The specificity and stoichiometry of the binding of Coomassie Brilliant Blue (CBB) to protein in section has been examined using both frozen protein matrices and plant material. The maximum adsorbance of the stain, bound and in solution, was found to be 620 nm although variation in the results at this wavelength necessitated measurements to be made at 600 nm. After enzyme treatments of sectioned plant material embedded in resin, all CBB-binding biological material was shown to be sensitive to non-specific protease. The relationship between optical density at 600 nm and section thickness was tested statistically against the Lambert-Beer law, using microdensitometry of cryostat-sectioned, frozen genatine solution. The analyses showed conclusively that, under these conditions, CBB adheres strongly to the Lambert-Beer relationship. CBB may thus be considered as a very specific protein stain, eminently suited both to cytological observation and quantitative microdensitometry.


Antigen-specific regulation of myeloma cell differentiation in vivo by carrier-specific T cell factors and macrophages. Previous studies demonstrated that: i) the TNP-binding myeloma MOPC-315 differentiated during in vivo growth in diffusion chambers (DC) implanted i.p. into normal BALB/c mice, and ii) the myeloma cell differentiation was regulatable by carrier-specific presentation of TNP to MOPC-315 cells in carrier-primed mice. In those studies, promotion and suppression of MOPC-315 differentiation occurred in the presence of carrier-specific helper and suppressor activities, respectively. In the present studies, we demonstrate that carrier-specific regulation of MOPC-315 differentiation can be adoptively transferred to normal mice with carrier-primed T lymphocytes. In addition, the induced regulation of MOPC-315 differentiation is abrogated when macrophages are not present with MOPC-315 cells in the DC. These studies establish the immunologic basis of myeloma cell regulation and suggest that soluble, carrier-specific helper and suppressor factors of T cell origin regulate MOPC-315 differentiation directly or in collaboration with macrophages.


Rabbit latent group a allotypes: characterization and relationship to nominal group a allotypic specificities. Latent group a allotypes were detected with a sensitive radioimmune inhibition assay. Sera, IgG preparations, and antibody fractions containing these allotypes inhibited the binding of insolubilized allotypic antisera to various radiolabeled antigens including IgG pools, homogeneous antibodies, and, in the case of a3, a VH fragment from a3/b4 IgG. Several different group a antiallotypic sera were used in the assays and all gave similar results. Comparison of inhibition curves for nominal and latent allotypes indicated that the full spectrum of allotypic subspecificities may be expressed in latent allotypes. Hemagglutination studies carried out with five sera containing high levels of latent allotypes confirmed the results obtained with the radioimmunoassay and indicated that inhibition values did not, at least in four of the five samples studied, reflect the presence of antiallotype antibodies.


Nature of the antigenic complex recognized by T lymphocytes. VI. The effect of anti-TNP antibody on T cell responses to TNP-conjugated macrophages. In this paper we examined the effect of anti-TNP antibody on guinea pig T cell proliferation in response to TNP-modified macrophages in vitro. The addition of anti-TNP to TNP-modified macrophages immediately after conjugation inhibited their ability to stimulate TNP-specific T cell proliferation. This inhibition appeared to be specific for the TNP response since anti-TNP had no effect on the ability of TNP-modified macrophages pulsed with either PPD or TNP-Ova to stimulate efficient PPD or Ova T cell responses. On the other hand, anti-TNP had no effect on the TNP-specific response to TNP-modified macrophages that had been cultured overnight before addition to primed T cells or to macrophages which had been pulsed with TNP-Ova. We also demonstrated that the same TNP-specific T cell subpopulation responds to both freshly TNP-modified macrophages and overnight cultured TNP-modified macrophages. These results suggest that the relevant TNP-determinants recognized by T cells are not exposed on the macrophage surface and raise the possibility that macrophages must process membrane-conjugated TNP to create the immunogen recognized by T cells.


Nature of the antigenic complex recognized by T lymphocytes. VII. Evidence for an association between TNP-conjugated macrophage membrane components and Ia antigens. In the present study we examined the effects of anti-sera directed against guinea pig Ia antigens on the ability of TNP-conjugated macrophages to stimulate TNP-specific T lymphocyte proliferation. Treatment of macrophages with anti-Ia sera for 1 hr before, 1 hr immediately after, or as late as 24 hr after TNP-modification resulted in a reduced ability to stimulate the TNP-specific T cell. The inhibition produced by anti-Ia sera was specific and did not result from interference with the ability of macrophages to process TNP-conjugated membrane antigens in a nonspecific manner. Brief treatment with anti-Ia serum did not result in inhibition of Ia-antigen synthesis nor could evidence of carry-over of anti-Ia antibody into the lymphocyte cultures be obtained. These results demonstrate that anti-Ia sera interfere with the development of a TNP-specific immunogen on the macrophage surface and strongly suggest that an association exists between TNP-modified membrane proteins and Ia antigens on the macrophage surface.


Inosiplex, a stimulating agent for normal human T cells and human leukocytes. The influence of inosiplex upon various in vitro leucocyte assays was studied in normal individuals. It was found that the drug increases the response of bidirectional and unidirectional mixed lymphocyte cultures at concentrations of 200, 300, and 500 microgram/ml. It also significantly increases the percentage of active T rosettes (concentration range: 50 to 500 microgram/ml) and the percentage of autologous red cell T rosettes (concentration: 100 microgram/ml). In contrast, inosiplex did not modify the percentage of total T rosettes and EAC rosettes. Inosiplex increases the number of nonadherent leucocytes in the leucocyte adherence inhibition test at a concentration range between 100 and 300 microgram/ml. Finally, inosiplex also increases the percentage of monocytes phagocytizing yeast at a concentration between 50 and 500 microgram/ml. These data indicate that inosiplex enhances the function of normal human T cells, monocytes, and possibly neutrophils. Therefore inosiplex appears to have immunostimulant properties.


Independent analysis of T helper and T killer cell function in young and old NZB mice. With age, NZB mice lose their ability to develop a cytotoxic response after alloimmunization in vitro. This decline is shown to coincide with a diminution of T-helper cell activity as assessed by proliferation in mixed lymphocyte culture or in response to PHA. When cytotoxic T cell precursors are activated with the polyclonal activator Con A, there is no reduction in the number of cytotoxic effector T cells that develop. No autoreactive cytotoxic cells are seen in Con A-activated cultures. These findings are related to previous work on cell-mediated immunity in NZB and B/W mice.


Cell-mediated cytotoxicity and serum-mediated blocking: evidence that their associated determinants on human tumor cells are different. The preceding paper showed that patients with gliomas may have lymphocyte-mediated cytotoxic activity (LMC) directed against at least two determinants on the glioma cell surface. The present study showed that serum from patients with gliomas could block this LMC. The blocking activity, however, was specific for different determinants on the glioma cell than those to which the LMC was directed. Blocking activity was specific for tumor cells homotypic to those of the serum donor. It was effective, however, in blocking the cytotoxic activity against these cells of lymphocytes from patients with tumors either homotypic or heterotypic to that of the serum donor. Likewise, although patients with glioblastomas or melanomas had LMC against fetal glial cells, sera from such patients were unable to block the LMC against these fetal glial targets. The specificity of the blocking activity was confirmed by absorption of the sera with various normal and neoplastic cells. These studies have thus shown an immunologic functional dichotomy among different determinants on the glioma cell surface.


Radioimmunoassay for the detection of hepatitis e antigen (HBeAG) and antibody (anti-HBe). A solid phase micro-immunoradiometric assay (micro-SPIRA) for the detection of hepatitis e antigen (HBeAg) and antibody has been developed. Chimpanzee anti-HBe/2 was developed by repeated immunizations with purified antigen containing HBeAg/1 and HBeAg/2. An anti-HBe/2 titer of 1:4 was determined by immunodiffusion (ID) analysis. Anti-HBe/1 was not detected. The anti-HBe IgG used in the assay was purified from plasma by a combination of DEAE-cellulose and affinity chromatography. The sensitivity of the micro-SPIRA for antigen and antibody was 193 ng/ml and 65 ng/ml, respectively. By comparing relative endpoint titers obtained by ID to micro-SPIRA, it was determined that micro-SPIRA for antigen and antibody is 320 and greater than 1300 times more sensitive, respectively, than ID. The specificity of the assay was ascertained by the examination of various non-B specimens. The application of the assay to a panel of 50 hepatitis B surface antigen (HBsAg)-positive specimens resulted in an increase in positivity of 18% for antigen and 22% for antibody.


Lymphocyte-mediated activation of fibroblast proliferation and collagen production. Among the many activities of antigen-triggered lymphocytes may be the control of fibroblast function. Thymus-dependent lymphocytes challenged with the specific antigen, dinitrophenylated ovalbumin or tetanus toxoid produced a nondialyzable factor(s) capable of causing dermal fibroblasts to undergo DNA synthesis. These fibroblasts, which exhibit basal proliferative levels in the absence of serum, responded to the lymphocyte factor with maximal thymidine incorporation at 48 to 72 hr. In addition, these activated fibroblasts significantly increased their production of protein of both the collagenous and noncollagenous types. This increase in protein synthesis preceded maximal proliferation. Thus, the fibroplasia consisting of increased numbers of fibroblasts and of increased collagen deposition associated with chronic inflammatory diseases may be the direct consequence of a specific antigenic challenge.


Apparent lack of H-2 restriction of allograft rejection. BALB/c ByJ (H-2d) mice immunized with tail skin grafts of either B10.D2 (H-2d) or B10 (H-2b) demonstrated similar second set rejection of B10.D2 tail skin. This apparent lack of H-2 restriction was not due to the induction of a new population of cytotoxic T lymphocytes (Tc) since 450R given 24 hr before the challenge graft did not abrogate the second set reactivity. Host macrophage processing or anti-Qa-2 reactivity was also not the explanation for the lack of H-2 restriction since immunization of BALB/c Li mice with either B10.D2 or B10 tail skin grafts resulted in second set rejection of B10 tail skin. Shared public H-2 specificities of H-2d and H-2b may result in cross-reactive Tc, thus causing the apparent lack of H-2 restriction. However, no H-2 restriction of allograft rejection is observed when only one public H-2 specificity is shared between the recipient and the allogeneic challenge graft (H-2f and H-2k combination). These results suggest that H-2 restriction of T cell cytotoxicity has little relevance in allograft rejection because 1) one public H-2 specificity is sufficient to cause cross-reactivity or 2) Tc are not the major effectors of allograft rejection.


Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. I. Lyt-2.1 and Lyt-3.1 antigenic determinants reside on separable molecular species. Anti-Lyt-2.1 and anti-Lyt-3.1 sera were employed for sequential precipitation of NP-40 extracts of 125I-labeled C57BL/6-Lyt-2a, Lyt-3a thymocytes (Lyt-2.1, Lyt-3.1) to determine whether these alloantigenic determinants are present on the same or different molecular species. Treatment of extracts with anti-Lyt-3.1 serum and SaCI completely precipitated both Lyt-3.1 and Lyt-2.1-specific components, whereas treatment with anti-Lyt-2.1 serum reduced by approximately 37% the quantity of labeled species subsequently precipitable by anti-Lyt-3.1 serum. When 125I-labeled thymocytes were subjected to mild trypsinization before NP-40 extraction, the quantity of radioactive components precipitated by anti-Lyt-2.1 serum was essentially unchanged, but that of anti-Lyt-3.1-precipitable components was greatly reduced. Moreover, sequential precipitation of extracts of trypsinized thymocytes with anti-Lyt-2.1 and anti-Lyt-3.1 sera demonstrated that these molecular species were precipitated independently. Thus 1) Lyt-2.1 and Lyt-3.1 antigenic determinants appear to reside on different molecular species; 2) some Lyt-2.1- and Lyt-3.1-positive molecules appear to be complexed with each other in the NP-40 extract; and 3) this association of Lyt-2.1- and Lyt-3.1-positive species was dependent upon components that were labile to trypsinization of intact thymocytes.


The correlation of bacteriophage types of Mycobacterium tuberculosis with guinea-pig virulence and in vitro-indicators of virulence. Among 58 isoniazid-sensitive strains of Mycobacterium tuberculosis from India, Burma and East Africa, 23 were of phage type A, 31 of type I (intermediate), 4 of type B and none of type C. Type I strains differed from type A strains in being attenuated in the guinea-pig, susceptible to H2O2, sensitive to thiophen-2-carboxylic acid hydrazide and resistant to thiacetazone and p-aminosalicylic acid; the content of strongly acidic lipids and of sulphatide lipids was low and the attenuation indicator lipid was present. The pattern of results with the type B strains did not correspond to the patterns for types A or I. Strains of type I appear to be a distinct group within the species M. tuberculosis.