Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography. A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.


DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.


Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis. The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.


Analysis of the structure of T4 bacteriophage-modified valyl-tRNA synthetase by limited proteolysis and isoelectric focusing. The new form of valyl-tRNA synthetase (EC 6.1.1.9) that appears immediately after infection of Escherichia coli with bacteriophage T4 was purified and subjected to mild proteolysis using five different proteases. The inactivation of aminoacylation activity was both more extensive and rapid than that obtained with valyl-tRNA synthetase purified from uninfected E. coli. The addition of bulk tRNA from E. coli B protected the phage-specific form of valyl-tRNA synthetase from proteolysis, but ATP and valine did not exhibit a similar protective effect. The characteristic property of phage-modified valyl-tRNA synthetase, resistance to denaturation by 4 M urea, remained unaffected during treatment with trypsin. This suggested that the phage-specific factor tau, known to be associated with the synthetase in phage-infected cells, was protected from proteolysis in the synthetase-tau complex. Comparison by isoelectric focusing of normal valyl-tRNA synthetase, the phage-specific form of this enzyme, and phage enzyme from which tau had been removed, revealed no differences in the isoelectric points of these three molecules. Based on these results a model was drawn for the structural changes occurring in valyl-tRNA synthetase after association with the phage factor tau.


Immunoadsorption of specific chicken oviduct polysomes. Isolation of ovalbumin, ovomucoid, and lysozyme messenger RNA. The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.


Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA. A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.


Bonding mechanism evidence in a ceramic--nonprecious alloy system. Bonding between porcelain and dental ceramic alloys is thought to be dependent upon the establishment of a continuous electron structure across the ceramic-metal interface (Pask, Proc. Porcelain Enamel Inst.,33,1,1971). Such a structure most likely results from the compatibility of metal ions at the metal surface saturated in an oxide form with the complex oxide structure of the ceramic matrix. Reaction zone compounds are expected to play a prominent role in the strength of the ceramic-metal bonds but thus far none have been detected or identified. The present study was centered on the determination of possible adherence zone compounds in four composite ceramic-metal couples examined by x-ray energy analysis. Elemental analysis of four couples revealed the presence of a predominant Al-Cr interaction resulting from the formation of an Al-Cr-O compound or mixed complex. The Cr ions were supplied by the Cr2O3 oxide layer at the metal surface and the Al was provided by the initial bonding-agent coating.


Posterior subtalar fusion. A preliminary report on a modified Gallie's procedure. A simple modification of Gallie's subtalar fusion is described as a salvage procedure in treating patients with pain from old fractures of the calcaneous involving the subtalar joint. Graft bone for the fusion is taken from the outer half of the calcaneus, thus avoiding disturbance of the tibia or iliac crest. Collapse of the donor site helps to narrow the widened heel present in these patients. The posterior approach allows the peroneal tendons to be freed from any adhesions, and at the same time release of the calcaneo-fibular ligament permits some correction of the valgus of the heel. The early results in six patients have been encouraging.


Triple arthrodesis by inlay grafting--a method suitable for the undeformed or valgus foot. A method of triple arthrodesis is described which involves inlay of the subtalar and midtarsal joints. It is applicable to the undeformed and valgus foot as is encountered in poliomyelitis, spasmodic flat foot, cerebral palsy and spina bifida. The operation was successful in controlling deformity and pain. The only significant complication was failure of fusion of the midtarsal joint which occurred in three of eighty-five feet (3-5%).


Total prosthetic replacement of a humerus for chronic osteomyelitis with a pathological fracture. Report of a case. A case is reported four years after successful total replacement of the left humerus for chronic osteomyelitis of six years' duration, with an axillary sinus that had persisted despite repeated operations and more distally a pathological fracture that had failed to united after bone grafting. The disability was such that the patient had requested amputation, but he returned to his previous work as a driver six months after the total replacement and there has been no recurrence of infection.


Arteriography in malignant neoplasms of the bowel. Eighty patients with gastrointestinal bleeding were investigated by angiography over a four-year period. Nine had malignant neoplasms of the bowel. Inconspicuous neovascularity was the most constant finding, best seen in leiomyosarcoma of the small bowel and adenocarcinoma of the colon. Neovascularity was not seen in a patient with reticulum cell sarcoma of colon detected by extravasation. When other studies including endoscopy and barium examinations have failed to explain acute or chronic bleeding, an appreciable number of intestinal neoplasms may be demonstrated by arteriography.


Phase-randomized laser illumination for microscopy. A simple apparatus is described that phase randomizes the output of a continuous argon ion laser, so that it may be used as a source of high intensity, monochromatic light for microscopy. The phase-randomizing device can be used with any laser, polarized or unpolarized, and of any desired power output and wavelength, including dye lasers for spectral studies. The randomizing system can be adapted to any light microscope and any optical system including bright-field, phase-contrast, Nomarski differential-interference, dark-field, and split-beam interference systems such as the Jamin-Lebedeff System. It can be used for a variety of photometric and photographic studies. The 514-nm wavelength appears to be relatively harmless to a variety of cells.


Chromatin behaviour during the mitotic cell cycle of Saccharomyces cerevisiae. Chromatin behaviour during the cell division cycle of the yeast Saccharomyces cerevisiae has been investigated in cells which have been depleted of 90% of their RNA by digestion with ribonuclease. Removal of large amounts of RNA from the yeast nucleus before treatment of the cells with heavy metal fixatives and stains permits chromatin to be visualized with extreme clarity in thin sections of cells processed for electron microscopy by conventional procedures. Spindle pole bodies were also visualized by this treatment, although the associated microtubules were not. Chromatin is dispersed during interphase and occupies the non-nucleolar region of the nucleus which is known to be Feulgen-positive from light microscopy. Because spindle microtubules are not visualized, direct attachment of microtubules to chromatin fibrils could not be verified. However, chromatin was not attached directly to the spindle pole bodies and kinetochore differentiations were not observed in the nucleoplasm. During nuclear division chromatin remains dispersed and does not condense into discrete chromatids. As the nucleus expands into the bud, chromosomal distribution to the daughter cells is thought to result from the separation of the poles of the spindle apparatus with attached chromatin fibrils. However, that such distribution is occurring as the nucleus elongates is not obvious until an advanced stage of nuclear division is reached and partition of the nucleus is nearly complete. Thus, no aggregation of chromatin into metaphase or anaphase plates occurs and the appearance of chromatin during mitosis is essentially the same as in interphase. These observations indicate that the marked changes in the topological structure of chromatin which characterize mitosis in the higher eukaryotes do not occur in S. cerevisiae.


Polyacrylamide gradient electrophoresis for protein purification on the milligram scale. A preparative-scale electrophoretic technique for protein fractionation and elution on a discontinuous gradient of acrylamide is described, which permits the separation and elution of a pure protein from a mixture containing 4-20 electrophoretically different proteins. The sharpness of the gradient electrophoretic resolution is demonstrated by the separation of proteins consisting of bovine serum albumin polymers and lactate dehydrogenase and enzymes such as acid phosphatase. The compositions of various discontinuous gradients of acrylamide and their application to enzyme purification are discussed. It was found that 60% of the enzyme activity loaded on the gel is recovered after gel fractionation and elution.


Use of omega-aminohexyl-sepharose in the fractionation of Escherichia coli B aminoacyl-tRNA synthetases. The usefulness of aminohexyl-Sepharose in purification of E. coli B aminoacyl-tRNA synthetases is presented. The purification factors for 14 synthetases lie in the range 3- to 94-fold and the recoveries of the enzymatic activity were 30-80%, depending on the enzyme.


Profiling of human body fluids in healthy and diseased states using gas chromatography and mass spectrometry, with special reference to organic acids. This review summarizes recent advances in the application of gas chromatography and mass spectrometry to the study of human diseases. Emphasis is placed upon the organic acid profiles of the various body fluids. Methods for sample work-up prior to separation and mass spectrometric analysis are reviewed, and artifacts and pitfalls are discussed. Organic acid profiles, obtained with packed or capillary columns attached to mass spectrometers with or without computer systems, have led to the discovery of new normal metabolites, new metabolic disorders, and to new knowledge about a number of other diseases. Stable isotopes and gas chromatography--mass spectrometry are suitable for quantitative analysis of many compounds in the body fluids, and well suited for investigation of metabolic pathways.


Esculin hydrolysis by Enterobacteriaceae. Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae.


Relationship of K1 antigen to biotype in clinical isolates of Escherichia coli. Two hundred and ninety-four isolates of Escherichia coli, including 105 from blood cultures, 94 from stools of hospital inpatients, and 96 from rectal cultures of healthy young adults, were biotyped by using the API-20E system and tested for the presence of K1 antigen. The overall frequency of K1 strains was 14.2% and was similar among the three sources. Forty-eight biotypes were observed, but two-thirds of all isolates, including two-thirds of the K1 strains, belonged to only five biotypes. Among the five commonest biotypes, the distribution of K1 strains was nonrandom, since 23 of the 27 K1 strains belonged to only two biotypes. Analysis of the O and H antigens of K1 strains indicated that this correlation of biotype with K1 antigen was due to a restricted number of serovars ("clones") that were repeatedly isolated from the population studied. These serovas included O18:K1:H7, O1:121:H6 and O16:K1:H6. Although a statistically significant correlation between biotype and K1 antigen was observed, the correlation was not sufficiently great to alow biotyping to be of significant predictive value as a marker for the K1 antigen.


Studies on neutralization of human serum bactericidal activity by sodium amylosulfate. The synthetic anticoagulant sodium amylosulfate (SAS) at concentrations of 125 to 2,000 microgram/ml failed to completely neutralize the bactericidal activity of 80 and 50% (by volume) fresh human serum. Furthermore, SAS failed to inhibit the alternative pathway of complement activation in 80% (by volume) fresh human serum that had been chelated with 0.01 M magnesium ions plus 0.01 M ethyleneglycol-bis(beta-aminoethylether)-N,N-tetraacetic acid. However, SAS at 250 to 1,000 microgram/ml effectively neutralized the bactericidal activity of 20% (by volume) fresh human serum. Therefore, SAS (at 250 to 1,000 microgram/ml) should be used only in blood samples that have been diluted at least fivefold (less than or equal to 20% [by volume]) in suitable broth media.


Pharyngeal carriage of group B streptococci: detection by three methods. The prevalence of pharyngeal carriage of group B streptococci was evaluated in patients with and without the complaint of a sore throat by three methods (blood agar plates, Columbia CNA agar plates, and a selective enrichment broth containing gentamicin and nalidixic acid). The overall carriage rate of group B streptococci was 12%, and there was no significant difference between the two groups of patients. The selective broth medium was more sensitive than the two solid agar plate methods in detecting carriage, and 37% of all group B streptococci were recovered solely from the broth. Use of the broth alone would have permitted a 94% detection of the group B streptococcal carriers.


Growth of cell wall-defective variants of Escherichia coli: comparison of aerobic and anaerobic induction frequencies. A method for quantitating the conversion of Escherichia coli to colony-forming, cell wall-defective (CWD) bacteria has been developed. The induction frequency, i.e., the percentage of the population recovered as CWD colonies was determined for 20 randomly selected clinical isolates of E. coli under aerobic and anaerobic incubation conditions. Penicillin (1,000 U/ML) was the inducing agent. The 20 strains segregated into three groups. Group I organisms produced CWD colonies with high frequency both aerobically and anaerobically. Grout II organisms showed a much higher induction frequency anaerobically than aerobically. Group III organisms were poor inducers. Thirty percent of the strains were group I, 50% were group II, and 20% were group III organisms. These data indicate that anaerobic conditions enhance the induction and growth of CWD E. coli in the research laboratory and suggest that anaerobic incubation may be important in recovery of medically significant CWD bacteria.


Improved dispenser for use in preparing prereduced, anaerobically sterilized medium. An improved, all glass, gravity-fed quantitative device for dispensing media during preparation of prereduced, anaerobically sterilized media with minimal loss due to oxidation of media is described.


Circulating immune complexes after renal transplantation. Correlation of increased 125I-Clq binding activity with acute rejection characterized by fibrin deposition in the kidney. To assess the role of circulating immune complexes in the pathogenesis of acute rejection, sera were measured for such complexes by the (125)I-C1(q) binding assay in 45 normal subjects, 24 allografted patients undergoing acute rejection, and in 11 allografted patients in a quiescent phase. Increased C1(q)-binding activity (C1(q)-BA) was detected in 14 patients with acute rejection, 9 of whom had renal biopsies showing fibrin deposition in the vasculature together with cellular infiltrates in the tubulo-interstitial structures; renal histology was not available in the other 5 patients. The other 10 patients with acute rejection, whose biopsies showed only cellular infiltrates, and the 11 patients in a quiescent phase posttransplantation did not have increased levels of serum C1(q)-BA. Of the group with increased serum C1(q)-BA, serial studies in eight patients showed a correlation between increased serum C1(q)-BA and the occurrence of rejection; with reversal by therapy, serum C1(q)-BA returned to within normal levels. Complexes from six patients were analyzed by sucrose density gradient ultracentrifugation to have sedimentation coefficients ranging from 15S to 18.4S. After acid dissociation and analysis by double-diffusion techniques, C1(q)-reactive complexes were shown to contain IgG. Immunofluorescent studies done in five renal biopsies from this group revealed granular deposits of immunoglobulin, and (or) less frequently, of complement in the glomeruli or the tubular basement membranes. The findings suggest that circulating immune complexes may mediate the type of acute rejection characterized by fibrin deposition in the kidney. The role of circulating immune complexes arising from the recipient's original kidney disease could be excluded in 10 patients with humoral rejection, inasmuch as the underlying renal pathology was of a "nonimmunologic" nature; this was corroborated by sequential studies in six patients in whom circulating immune complexes could not be demonstrated before rejection. The participation of administered antilymphocyte globulin (ALG) as an antigen also appears to be excluded in four patients, two who were not given ALG, and in two of whom episodes of rejection occurred unrelated temporally to ALG administration.


Phytohemagglutinin response in systemic lupus erythematosus. Reconstitution experiments using highly purified lymphocyte subpopulations and monocytes. THE PHYTOHEMAGGLUTININ (PHA) RESPONSE OF LYMPHOCYTES FROM UNTREATED PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) WAS STUDIED USING HIGHLY PURIFIED SUBPOPULATIONS OF CELLS INVOLVED IN THE TRANSFORMATION RESPONSE: T lymphocytes, B lymphocytes, and monocytes. Cell transformation was quantitated using both tritiated thymidine ([(3)H]-TdR) incorporation into DNA and cytofluorographic determination of cellular DNA content. Dose-response curves using six concentrations of PHA and five concentrations of cells over 0-5 days revealed a decrease in [(3)H]TdR by stimulated lymphocytes from some SLE patients. This decrease in [(3)H]TdR was paralleled by a decreased percentage of cells in S, G(2), and M phases of the cell cycle. However, abnormal response occurred entirely in those SLE patients who were hypocomplementemic. The etiology of the impaired response was further examined. Lymphocyte receptors for concanavalin A were studied using cytofluorography of lymphocytes stained with fluorescein-conjugated concanavalin A. The frequency distribution of concanavalin A receptors was similar in the normocomplementemic and hypocomplementemic lupus patients and in normals. The latex phagocytic activity of lupus macrophages was similar to normals when allogeneic normal plasma was used in the culture medium. Phagocytic activity became abnormal in the presence of SLE plasma. However, there was no difference in the [(3)H]TdR response or the percentage of cells in S, G(2), and M phases when T lymphocytes from the hypocomplementemic patients were stimulated on either autologous or normal allogeneic monocyte monolayers. Likewise, normal lymphocytes incorporated similar amounts of [(3)H]TdR and had similar percentages of cells in S, G(2), and M phases whether their T lymphocytes were stimulated on autologous or SLE monocyte monolayers. Highly purified subpopulations of B and T lymphocytes were obtained by density sedimentation or Fenwal Leuko-Pak passage of lymphocyte populations. The response to PHA by lymphocytes from the hypocomplementemic lupus patients could be seen to involve at least two abnormalities. One, in reference to normal lymphocytes, SLE T lymphocytes plus monocytes had an impaired response; two, SLE B lymphocytes plus SLE T lymphocytes plus SLE monocytes had an impaired response. Two patients in the hypocomplementemic group were treated with steroids. 5 days after steroid treatment was initiated, the percentage of cells in S, G(2), and M phases and the [(3)H]TdR response of PHA-stimulated lymphocytes returned to normal. The normalization of the [(3)H]TdR response was explained both by a return of purified T cells plus monocytes, purified B cells plus monocytes, and whole lymphocyte populations to normal responsiveness. These studies suggest that a steroid-correctable defect exists in T and B lymphocytes in SLE.


A role for prostaglandin E in defective insulin secretion and carbohydrate intolerance in diabetes mellitus. Prostaglandin E(2) (PGE(2)) infusion in normal humans inhibited acute insulin responses to a glucose (5 g i.v.) pulse (response before PGE(2) = 593 +/- 104%; during PGE(2) = 312+/-55%; mean+/-SE, mean change 3-5 min insulin,% basal, P < 0.005). This effect was associated with a decrease in glucose disappearance rates (K(G) before PGE(2) = 0.73+/-0.07; during PGE(2) = 0.49+/-0.06%/min, P < 0.025). Acute insulin responses to arginine (2 g i.v.) were not affected by PGE(2) (response before PGE(2) = 592+/-164%; during PGE(2) = 590+/-118%; P = NS). Infusion of sodium salicylate (SS), an inhibitor of endogenous prostaglandin synthesis, augmented acute insulin responses to glucose in normals (response before SS = 313+/-62%; during SS = 660+/-86%; P < 0.001). In adult-onset diabetes with fasting hyperglycemia, SS restored absent acute insulin responses to glucose (20 g i.v.) pulses (response before SS = 5+/-6%; during SS = 97+/-24%; P < 0.005). This was accompanied by a fourfold augmentation in second phase insulin secretion (second phase before SS = 1,696+/-430%; during SS = 5,176+/-682%; change 10-60 min insulin, muU/ml.min,% basal, P < 0.001) and by acceleration of glucose disappearance rates (K(G) before SS = 0.56+/-0.06; during SS = 1.02+/-0.17%/min, P < 0.005). These findings uniquely demonstrate that (a) PGE(2) inhibits glucose-induced acute insulin responses and decreases glucose disposal in nondiabetic humans and (b) SS restores acute insulin responses, augments second phase insulin secretion, and accelerates glucose disposal in hyperglycemic, adultonset diabetics. It is hypothesized that endogenous PGE synthesis may play a role in defective insulin secretion and glucose intolerance in diabetes mellitus.


Release of immunoreactive somatostatin from the pancreas in response to glucose, amino acids, pancreozymin-cholecystokinin, and tolbutamide. The effects of glucose, amino acids, pancreozymin-cholecystokinin, and tolbutamide upon the release of immunoreactive somatostatin (IRS) from the isolated perfused pancreas were studied. In seven experiments in which glucose was perfused either at a concentration of 100 or 350 mg/dl or at 25 mg/dl, IRS levels were significantly greater at the higher glucose concentrations. In three dose-response experiments in which the perfusing glucose concentration was increased at 30-min intervals from an initial concentration of 25 mg/dl to a final concentration of 300 mg/dl, progressive increases in IRS release were noted at glucose concentrations of 100 mg/dl and above. Perfusion of a 20 mM mixture of 10 amino acids also elicited a prompt and significant biphasic IRS rise in each of six experiments. In five experiments, 20 mM leucine evoked a similar response in mean IRS. Perfusion with 0.075 Ivy U/ml of pancreozymin-cholecystokinin, with or without the presence of a 1 mM 10-amino acid mixture, elicited a prompt rise in IRS with a pattern resembling that of insulin in a total of six experiments. Tolbutamide (0.75 mg/min) also stimulated IRS release in five of six challenges. The IRS responses to nutrients and to pancreozymin and their similarity to the insulin responses raise the possibility that, like insulin, pancreatic somatostatin may have an endocrine role related to nutrient homeostasis.


Interrelations of platelet aggregation and secretion. The mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of gamma-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of gamma-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are consistent with a simple model of platelet stimulus-response coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described.


The Kahn Test of Symbol Arrangement and criminality. Two groups of male Ss--33 incarcerated criminal recidivists and 33 trainees in a work placement bureau--were tested on two versions of the Kahn Test of Symbol Arrangement (KTSA). These were the original KTSA, which used the free responding technique, and a modified KTSA, which used the forced-choice responding technique. The groups were equated for average age and sociocultural status and did not differ in their intellignece scores. The results showed that the criminals scored significantly lower than their controls on the original KTSA with a symbol-pattern characterized by repetitive and concrete types of responses. The groups did not differ on the modified KTSA. The discriminative power of the free responding technique of the KTSA proved to be superior to that of the forced-choice in the area of identifying criminal recidivists.


Transcendental mediatation: a psychological interpretation. The authors suggest that Transcendental Meditation offers a great deal of promise for use in helping relationships. They also suggest that the technique might receive wider acceptance if it could be explained in other than a purely philosophical or mystical way. For that reason, in their article they offer a psychological interpretation of the TM process.


Psychological status after stroke as measured by the Hand Test. Forty-two patients with recent cerebral vascular accidents (cva) and 32 hospitalized patients without brain damage were administered the Hand Test. The CVA group consisted of 24 patients with right cerebral involvement, 10 patient with left cerebral involvement, and 8 patients with bilateral impairment. The mean age of the brain-damaged group was 48.3 years. The Hand Test was administered from .5 month to 3 months after the insult in the CVA group, and 1 month to 4 years after theinitial appearance of the chief complaint in the non-brain-damaged group. The median test was used to analyze the data. The brain-damaged group showed less aggressive, more withdrawn behavior and have greater difficulty in dealing with reality. The high tendency of brain-damaged patients to withdraw from reality can be viewed as a regressive process due to impaired cognitive functioning and emotional constriction.


Inkblot content and interpersonal distance. Four Ht content scales were related to a projective and a live measure of interpersonal distance. Based on previour research, negative correlations between human and barrier content and interpersonal distance were predicted, with positive correlations predicted between anxiety and hostility content and distance behavior. All four content scales were found to relate significantly to projective distance, but only anxiety and hostility correlated significantly with distance behavior. The content scales also were combined in regression equations to predict interpersonal distance. The projective measure of interpersonal distance correlated signifiantly with the live measure of distance. The results were interpreted as support for the construct validity of the inkblot scales.


Comparative clinical evaluation of freeze-dried skin allografts and autogenous gingival grafts in humans. Freeze-dried skin (FDS) allografts and fresh autogenous free gingival grafts (FGG) were placed in five paired sites in four patients. Both types of grafts healed uneventfully. Evaluation over a 1-year period demonstrated no statistically or clinically significant differences between the FDS and FGG when: a) increase in the width of keratinized gingiva, b) decrease in recession, c) decrease in sulcus depth, k) gain in attachment level, or e) percent shrinkage of the graft were compared. It was concluded that FDS performs essentially similar to FGG in the correction of mucogingival problems, and has the advantages of decreased patient morbidity (no donor site) and availability of abundant amounts of graft material when needed.


Gingival condition in areas of minimal and appreciable width of keratinized gingiva. Sixteen dental, dental hygiene, and dental assisting students and dental faculty members who had contralateral or unilateral areas of minimal (less than or equal to 1.0 mm) and appreciable (greater than or equal to 2.0 mm) widths of keratinized gingiva on mid-buccal plaque-free surfaces of mandibular bicuspids were examined. Gingival exudate amounts and clinical inflammation based on color change and/or swelling and bleeding on probing were evaluated. The results showed that gingiva with "appreciable" width as well as gingiva with "minimal" width of keratinized tissue exhibited only minute amounts of gingival exudate. Also, there were generally no clinical signs of inflammation for both types of tissue. From the groups of 16, six subjects were selected who had contralateral pairs of minimal and appreciable keratinized gingiva. They were instructed to cease oral hygiene in the lower bicuspid area for 25 days. At day 0, 4, 7, 11, 14, 18, 21, and 25, plaque, gingival exudate, and clinical gingival inflammation were evaluated. Results revealed increases in plaque, gingival exudate scores and clinical gingival inflammation over the 25-day period with no apparent difference between the areas with minimal and appreciable width of keratinized gingiva.


Comparative trial of single-dose versus twice-daily sisomicin in bacteriuric patients. Sisomicin was administered as a single daily intramuscular injection (160 mg) or as two daily injections (80 mg) to 50 patients with bacteriuria superimposed on chronic urologic diseases in a randomized controlled fashion. The administration of two daily doses was significantly more effective (P less than 0.01) in achieving cure than the injection of a single daily dose. The renal function, as expressed by creatinine clearance, became impaired significantly more often (P less than 0.05) in the patients receiving the single daily dose of sisomicin.


The clinical and histological evaluation of cresatin with calcium hydroxide on the human dental pulp. Metacresylacetate with calcium hydroxide in a methylcellulose base produces clinically and radiographically successful pulpotomies, when performed on sound primary teeth. Under sterile conditions, with proper surgical and restorative technique, pulp tissue will heal with metacresylacetate with calcium hydroxide-methylcellulose. The dental pulp will form on osteodentin bridge and the pulp adjoining the bridge will remain histologically viable and free of inflammation. Methylcellulose with sterile distilled water does not produce as favorable a histological reaction as metacresylacetate with calcium hydroxide-methylcellulose. No dentinal bridging was in evidence and there were present in the  radicular pulp varying degrees of inflammation.


Effect of pretreatment with ethanol containing fluoride on etched enamel surface as evaluated by scanning electron microscopy, optical microscopy, and tensile bond strength measurement. The effects of pretreatment with a fluoride-containing ethanol solution on etched enamel surfaces before application of sealant were examined using scanning electron microscopy, optical microscopy, and tensile bond strength determinations. The results suggested that when etched enamel surfaces are treated with ethanol containing no more than 250 ppm fluorine in potassium fluoride, there is no interference with the bonding of the sealant to the treated, etched anamel surface.


Electroless and chemical metal deposition on human tooth enamel. The feasibility of depositing a thin metal film onto tooth enamel has been demonstrated. The process employs chemical solutions containing ions of the metal to be deposited and suitable reducing agent solutions. The deposits are quite adherent and exhibit the normal properties of the bulk metal.


Letterer-Siwe disease: report of case.

Letterer-Siwe disease: report of case. Initial oral symptoms of Letterer-Siwe disease reveal the importance of a dentist in establishing this diagnosis. This case is important in that the patient was five months old, at which time an early diagnosis was made and treatment had begun. Because most cases occur in young children, a high level of suspicion is necessary, because the oral manifestations and radiographic findings in children may often resemble the periapical, periodontal or cystic involvements frequently seen first by the dentist. The oral lesions were necrotic, hemorrhagic, and degenerative gingival tissues in the maxillary molar area. Loss of alveolar bone was evident. There was precocious eruption of seven primary teeth, which were slightly mobile. Dentally, prophylaxis and fluoride applications were supportive measures.


The mechanism of tensile failure in a nickel-base casting alloy. Standard tensile specimens were cast from a nickel-base partial denture casting alloy and subsequently fractured in a tensile stress testing machine. Fracture sites were subjected to scanning electron microscopy in order to determine the exact mechanism by which failure had occurred. A metallographic examination of prepared specimens was used to demonstrate the distribution of phases within the alloy.


Surface chemical modification of hard tissues: I. Bone. Surfaces of bone were modified in a controlled manner by grafting or by adding interpenetrating polymeric side chains to the bone substrate. The properties of the hard tissue surface attained varied widely. The surface alteration may improve the ability of hard tissues such as bone or dentin to adhere to restorative materials.


The inception of photoelectric scintillation detection commemorated after three decades. The modern photoelectric scintillation detector emerged just 30 years ago out of the rubble of World War II. Kallmann in Germany had the original idea of combining an organic fluor (naphthalene), which is transparent to its fluorescent light, with an electronmultiplier phototube for the detection of single scintillation evants. Hofstadter at Princeton discovered that the inorganic thallium-activated sodium iodide scintillator has superior gamma detection efficiency and a high photoelectric yield for gamma spectrometry.


A filled pit and fissure sealant: 18-month results. One hundred and forty-three patients ranging in age from 5 to 8 years participated in a pit and fissure sealant study using a half-mouth design. At the end of 18 months, for all permanent first molars, there was a 75.9% effectiveness with a net gain of 82, which was statistically significant. The sealant was present on the surfaces to which it was applied in 74% of the teeth. Application time was 8 minutes per quadrant and 5.5 minutes per tooth. The effect of treatment was similar to that reported for an unfilled BIS-GMA sealant in patients with similar teeth and similar ages. Loss of sealant from the permanent maxillary first molars did not appear to contribute to the initiation of dental caries. However, in 6 of 38 mandibular tooth surfaces, sealant loss may have been a factor.


Direct bonding in the management of an ankylosed second deciduous molar. A simple and efficient bonding procedure has been described for the management of an ankylosed deciduous second molar. This has, in our experience, provided a satisfactory occlusal and periodontal environment in selected patients. Based on a limited number of cases, restorations have been maintained for about two years to date.


Orofacial problems associated with bilateral seventh cranial nerve palsy: report of case. Treatment of a patient with a bilateral facial nerve palsy consisted of extractions, construction of maxillary and mandibular complete dentures and, finally, the modification of the mandibular denture to assist in the support of the lower lip. Gratifying esthetic, physiologic, and psychological results were obtained.


Streptococcus group B: an old bug with new dangers. Infection of the newborn infant with Group B hemolytic streptococcus is increasing in importance as other pathogens decline. Such infection may appear within the first 48 hours of life and resemble respiratory distress syndrome, or around the tenth day, when signs of meningeal irritation may be present. Early onset disease may be due to any of the five serotypes of the Group B streptococcus and is fatal in a high percentage of cases. Late onset infection usually is due to serotype III. The epidemiologic aspects of early and late infection appear to differ, and a nosocomial source may be involved in the latter. Penicillin prophylaxis may be useful.


Insuring effective antimicrobial therapy: laboratory evaluation. Specialized tests for the susceptibility of pathogenic microorganisms are not indicated for most clinical infections. Primary attention should be focused on proper collection of representative culture material and subsequent accurate identification of the offending organism. Specific antimicrobial therapy can then be empirically instituted based upon previously known susceptibility patterns. An overview of the broth dilution, agar dilution, and agar (disk) diffusion techniques is presented. The absolute indications for these tests are as follows: (1) infections with gram-negative bacilli; (2) staphylococcal infections; and (3) enterococcal and other group D beta hemolytic streptococcal infections. The following situations are relative indications for susceptibility testing: (1) infections occurring in immunosuppressed hosts; (2) infections that by their location are more susceptible to certain types of antibiotics; and (3) infections that should be treated with a bactericidal rather than a bacteriostatic drug. The rationale and indications for the serum bactericidal test (SBT or Schlichter test) and antibiotic assay techniques are described. The cases reported illustrate appropriate application of the specialized tests for susceptibility.


Recurring digital fibrous tumor of childhood. A case of digital fibrous tumor of childhood is described. This tumor is multicentric, of possible viral origin, and is characterized histologically by intracytoplasmic inclusion bodies within proliferating fibroblasts. The tumor recurred despite wide local excision and skin grafting.


Epithelioid sarcoma of the hand and wrist: patterns of extension. The sites of local and remote extension of primary epithelioid sarcoma of the hand and wrist were studied in six patients. The tumor was found to spread to noncontiguous areas of skin, subcutaneous tissue, fascia, bone, and lymph nodes as well as by direct extension along fascial planes. Neither local nor remote extension of the tumor could be determined accurately or dependably by clinical examination, radiography, xerography, lymphangiography or radioisotope scanning. Wide en bloc excision of the tumor or amputation is recommended as the primary treatment of epithelioid sarcoma of the hand. Axillary node dissection is advised in all cases.


Allograft replacement of distal radius for giant cell tumor. Three cases of resection of the distal radius with allograft replacement for giant cell tumor of bone were reviewed. In one patient the tumor had penetrated the distal articular cortex; in another it had broken through the anterior cortex; in the third there had been recurrence of the tumor within a year of currettage and autogenous bone graft. In each case the allograft was glycerinized to help to preserve the viability of the articular cartilage and then it was frozen at -70 degrees C to decrease bone antigenicity. In all three patients rapid healing at the recipient-graft juncture took place, and none showed signs of rejection or of recurrence of the tumor. All three have a useful and relatively painless range of wrist motion. Distal radial resection and allograft replacement is recommended for giant cell tumor of bone if there has been spontaneous cortical or articular breakthrough, recurrence, or evidence of a rapidly enlarging lesion or a frankly malignant histologic appearance.


Scene segmentation techniques for the analysis of routine bone marrow smears from acute lymphoblastic leukemia patients. Automated analysis of lymphoblast cell morphology is being evaluated as a basis for predicting the response to therapy of patients with acute lymphoblastic leukemia. A new technique of scene segmentation particularly applicable to the "cluttered" images of cells in routine bone marrow smears is described. Morphologic characteristics of lymphoblasts found in bone marrow smears made at time of diagnosis were measured by an automated, interactive image-processing system using the new scene segmentation technique. These characteristics, on a patient by patient basis, are being compared to remission length and survival data to develop and test new prognostic methods.


An automated method of differential red blood cell classification with application to the diagnosis of anemia. A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.


A program system for interactive measurements on digitized cell images. Using a high precision image scanner and a PDP-8/F minicomputer, we have developed a program system for interactive measurements on microscopic images. By giving simple keyboard commands, the operator can run the image scanner and manipulate the digitized images. The interface between the operator and the microscope-computer system is a Tektronix 4010 graphic terminal. The system allows objects to be isolated and parameters to be calculated from each object, e.g., parameters characterizing shape of the object, irregularity in light transmission over the object, area, integrated light transmission, etc. Objects are isolated and parameters are calculated under complete operator control using interactive computer graphics technique. Calculated parameters may be stored in dedicated data records, which are stored in files for later statistical analysis. The system also includes a statistical evaluation part. Technically, the system consists of a command scanner, which translates commands into internal representation, a parser, which checks the syntax of the commands, and an interpreter, which executes the commands. The system is designed so that new commands can be added easily.


Translocations in the mosquito, Culex tritaeniorhynchus. Fifty-eight translocations were produced and studied using improved genetic and cytogenetic techniques now available for Culex tritaeniorhynchus. Data confirm previous chromosome-linkage correlation studies and indicated that the e locus is on the long arm (2R) of chromosome 2.


Automated classification of cells in electron microscopic images of lymphoreticular tissue. In classifying cells in tissue sections, one must consider the fact that only random projections of cells and of subcellular structures are available in the two-dimensional image. Therefore, measurement values that solely reflect the size of such projections cannot be taken on their own as a basis for cell classification. More complex morphologic features such as shape, texture and distribution pattern of cells and their components should be analyzed. Using cell nuclei as an example, the relationship between such features and geometric measurement values is evaluated. It can be shown that a well balanced combination of geometric parameters provides a suitable basis for reproducing the visual preclassification of lymphocytes in tissue sections. Moreover, using a cluster algorithm, which allows different levels of similarity to be defined, a hierarchical sequence of subclusters turns out, indicating the heterogeneity of the visually determined cell classes. Whether or not these subclusters can be correlated to functionally defined subpopulations of lymphocytes remains a matter for further investigation.


Bone marrow cell scene segmentation by computer-aided color cytophotometry. Computer scene segmentation of touching cell images in bone marrow, on the basis of color information, is achieved using digitized scans at three different wavelengths of light. With trivariate histograms and Euler's coordinate transformation, it is possible cytophotometrically to isolate, on the basis of chromatic differences, individual heterogeneous cells located in cell groups. The ability of the described computer methods to isolate correctly the touching cell images is determined by visual comparison of the cells as seen in the microscope and the computer-generated displays of the scanned and segmented scenes.


Mutagenicity of dieldrin in the Salmonella-microsome test. Dieldrin was assayed in vitro for its mutagenic activity in several strains of S. typhimurium with and without Aroclor 1254 treated mouse liver homogenate (S-9M). The mutagenicity of the insecticide was expressed maximally in all strains in the presence of liver S-9M and less so in three strains without liver microsomes.


A multi-spectral approach for scene analysis of cervical cytology smears. A multi-spectral approach for the scene analysis of cervical cytology smears, using multiple images of a scene digitized through suitably chosen color filters matched to the Papanicolaou stain, has been proposed here. This technique involves clustering of two-dimensional data for extracting cytoplasm of the epithelial cells. Its performance on an experimental data set of 233 scenes involving more than 10 types of normal and malignant epithelial cells has been compared with density and gradient thresholding techniques. This resulted in an approximate 83% rate of success compared to approximately 40% for the rest of the other techniques.


Segmentation of cervical cell images.

Segmentation of cervical cell images. A major problem in the automation of cervical cytology screening is the segmentation of cell images. This paper describes various standard segmentation methods plus one which determines a segmentation threshold based on the stability of the perimeter of the cell as the threshold is varied. As well as contour, certain structural information is used to decide upon the threshold which separates cytoplasm from the background. Once the cytoplasm threshold is found, cytoplasm and nucleus are separated by simple clustering into three groups, cytoplasm, folded cytoplasm and nucleus. These techniques have been tested on 1500 cervical cells that belong to one of eight normal classes and five abnormal classes. A minimum Mahalanobis distance classifier was used to compare results. Manually thresholded cells were classified correctly 66.0% of the time for the 13 class problem and 95.2% of the time on the two (normal-abnormal) class problem. The contour tracing technique was 52.9% and 90.0% correct, respectively.


High resolution analysis of cervical cells--a progress report. This paper presents preliminary results of research toward the development of a high resolution analysis stage for a dual resolution image processing-based prescreening device for cervical cytology. Experiments using both manual and automatic methods for cell segmentation are described. In both cases, 1500 cervical cells were analyzed and classified as normal or abnormal (dysplastic or malignant) using a minimum Mahalanobis distance classifier with eight subclasses of normal cells, and five subclasses of abnormal cells. With manual segmentation, false positive and false negative error rates of 2.98 and 7.73% were obtained. Similar experiments using automatic cell segmentation methods yielded false positive and false negative error rates of 3.90 and 11.56%, respectively. In both cases, independent training and testing data were used.


Estimating false positive and false negative error rates in cervical cell classification. The performance of a cell recognition system on unknown data is often estimated in terms of its error rates on a test set. This paper investigates methods for producing estimates of error rates in cervical cell classification. Classification performance curves calculated using these methods are given for several classification schemes used to classify 1500 cervical cells.


Quantitative extraction of morphologic cell parameters from the diffraction pattern. A new technique for the fast and quantitative extraction of cell parameters from diffraction patterns was applied to cervical gynecologic material. Based on radial scans through the diffraction pattern, this technique permits the simultaneous determination of the nuclear and cytoplasmic diameter by Fourier analysis of the radial scanning signals after compensation for the intensity falloff by an amplification which is proportional to the third power of the radial position in the diffraction plane. In addition, a correlation between the angle of the highest amplitude of the scanning signals and the coarseness of microstructures in the cell was observed. Illustrative examples of measurements on exfoliated cells of different type are presented.


The automated classification of mitotic phase for human chromosome spreads. A system has been developed that automatically recognizes the mitotic phase of human chromosome spreads for karyotyping. Suitable spreads are classified into one of five subphases of mitosis. Classification is performed on the basis of summed chromosome length and most probable chromosome width. Classification requires 100-500 msec. A television camera scans the spread through microscope optics; computer and special purpose electronics process the video signals to generate run length histograms. The histograms are used to determine mitotic phase. Unbanded spreads, 133, were classified with a 4.5% error rate. One hundred banded spreads were classified with a 15% error rate.


Computer-assisted karyotyping with human interaction. A system for machine assisted karyotyping and chromosome analysis has been developed. The system uses a drum- or TV-scanner as input device, runs provisionally in 32 K memory, and also allows human interaction on several stages. The accuracy with which banded chromosomes are karyotyped depends strongly on the type of classifier and varies from 40 up to 80%. The accuracy of the human assisted classifier (98%) comes close to that of a skilled technician (99.5%) using manual chromosomal analysis. Due to technical and memory limitations, the time necessary for the karyotyping of one cell is too long and depends on the interaction time; however karyotyping within 5 min, including human interaction, will be possible in the near future.


Uniform lateral orientation, caused by flow forces, of flat particles in flow-through systems. Recently, it was shown that the lateral orientation of sperm cells disturbs the deoxyribonucleic acid distribution measured by fluorescence in a laterally laser-illuminated flow system. The present results show how flat particles may be influenced to assume a uniform lateral orientation. This was achieved by choosing the geometrical dimensions of the hydrodynamic focusing flow path. High speed photographs of fixed chicken erythrocytes oriented in experimental chambers are presented.


Hydrodynamic orientation of cells.

Hydrodynamic orientation of cells. Chicken erythrocytes orient when caught in the confluence of two sheath-liquid streams. Preliminary results indicate that this phenomena might be used to align cells for morphologic analysis in flow.


A high efficiency flow cytometer.

A high efficiency flow cytometer. A flow chamber has been developed which collects about 60% of the total cell fluorescence for analysis compared to about 2.5% for conventional flow systems. The chamber, an ellipsoid of revolution, is gold-plated for increased reflectivity. Fluorochrome-stained cells enter the flow cell directly above the primary focus of the ellipsoid at the rate of 1000 cell/sec. A focused argon-ion laser beam enters the flow cell parallel to the semiminor axis and intersects the cell stream at the primary focus. Fluorescent light emanating from this point is reflected toward the secondary focus, where it exits the chamber for analysis. The high efficiency flow cytometer has been used to obtain nucleotide fluorescence distributions from samples of Micrococcus glutamicus bacteria stained with propidium iodide and of spermatozoa stained by the acriflavine-Feulgen procedure.


Identification of cell asymmetry and orientation by light scattering. Light scattering from chicken red blood cells has been used as a model system to identify the asymmetry of cells. The histogram for forward angle light scattering for these cells is bimodal, the signal size being dependent on the cell orientation. A dual orthogonal scatter system is used to conclusively demonstrate this orientational variation in signal. A third scattering system, using a single incident beam with two orthogonal detectors, is used to further characterize the orientational variation of the scatter signal. In this third system it is shown that the signal in a detector set 90 degrees from the incident beam collects light reflected from the cell surface. The optical selection of cells in specific orientations using these systems may circumvent the need to physically orient cell in flow systems.


Fluvo-metricell, a combined cell volume and cell fluorescence analyzer. A new flow through instrument that simultaneously measures cell volume (resistance pulse technique) and cell fluorescence in the same orifice will be described. The fluorescence pulses of the hydrodynamically focussed cells are picked up by the optics via the axial direction (principle of Dittrich and Goehde, Z Naturforsch 24b:360, 1969). There is no coordination problem between the fluorescence and the resistance pulses to be observed because a new type of transducer is used. The electronic system provides gating of one or two parameter histograms. Function tests are performed with the incorporated two-parameter test spectrum generator. Different examples of using the instrument in practice are shown. The volume that may be measured with an orifice of 70 micron diameter ranges between 4 and 1400 micron3 (1:350). Coefficients of variation of the fluorescence below 2% are measured.


Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system. A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.


Application of Fraunhofer diffraction theory to feature-specific detector design. Light scatter from epithelial cells in a slit-scan flow system is modeled using the Fraunhofer condition of scalar diffraction theory. Power spectra are calculated for successive positions of model cells in the line focus of a laser beam with a Fourier transform computer program. Using the calculated power spectra, detector configurations are designed to detect specific cell structures of interest. Detector configurations are tested in a static slit-scan scatter apparatus. Data indicating the ability to detect boundaries and cell orientation are discussed.


Imaging systems for correlation of false alarms in flow. False alarms, arising from a variety of sources, are the greatest remaining obstacle to development of an automated prescreening system for gynecologic cytology. This paper describes two correlation systems under development at the University of Rochester and discusses their utilization in the study of false alarms in slit-scan cytofluorometry. Both systems permit imaging of objects in flow and correlation between images and corresponding slit-scan contours. Correlation systems will permit a detailed study of false alarm causes and aid in the search for new features to assist in their recognition.


Experimental findings on gynecologic cell orientation and dynamics for three flow nozzle geometries. Gynecologic cell orientation and dynamics in sheath flow for three flow nozzle geometries are studied and compared. Cell orientations are classified into four categories based upon visual inspection of dark field laser stroboscopic photographs of cells in flow. Results are discussed in the context of slit-scan cytofluorometry.


Two-color immunofluorescence using a fluorescence-activated cell sorter. A technique for the analysis of fluorescein and rhodamine in a flow system using a single wavelength of excitation is described. Both optics and electronics are used to discriminate the fluorescein and rhodamine signals. This technique has been used to study the relationship between immunoglobulin M and immunoglobulin D on mouse splenic lymphocytes.


A modified technique to obtain uniform precipitation of lanthanum tracer in the extracellular space. A method for obtaining a uniform, dense precipitate of lanthanum nitrate to delineate extracellular space is described. Improvement of the previous technique is achieved by phosphate precipitation of lanthanum in the tissue carried out at low temperature. This method has been successfully applied to bone marrow.


[Morphology of the telencephalon of Salmo irideus (Teleostei). Golgi-impregnation study]. The telencephalon of Salmo irideus (Gibbons) was investigated by means of staining and silver impregnation techniques. It shows the fundamental plan characteristic for teleost species. The neurons in the various telencephalic nuclear areas are, in part, quite different with respect to their density of arrangement,migratory behavior, width of pericaryon and number of spines, inclusively. Neurons exhibiting a high degree of morphological differentiation (i.e. neurons of the stellate and pyramidal like type of differentiation) are located in the area dorsalis telencephali only. This telencephalic region is considered to be a functional homologon of limbic telencephalic structures of other vertebrates.


[Automatic pictorial analysis of normal and experimentally altered nervous tissue]. 1. Structural changes of the dorsal LGN of the albino rat following unilateral enucleation cause its decrease of volume. 2. Using the Quantimet 720 M and Nissl stained preparations we tried to explain the decreased volume by estimation of three parameters: The whole area of the cells, the number of neurons and the size distribution of cells. 3. The whole area and the number of neurons are constant. 4. The size distribution of cells changes significantly and indicates an alteration of the tissue surrounding the cells (development of the neuropil, loss of fibers). 5. The changes of the maxima of size distributions following enucleation suggest a diminution of the area of neuron somata, but are not significant. 6. Using more exact methods of measurements we want to get more detailed results.


[Ependymal structures in the telecephalon of Salmo irideus (Teleostei)]. The ependyma in the region of the telencephalon of Salmo irideus is composed of a great number of various elements: uni- and bipolar tancytes, ependymal astrogliacytes, ciliated ependymal cells, intraependymal neurons and neuron-like cells exhibiting ventricular contact. There are regional differentiations in the ventricular wall which can be attributed to certain structurally defined nuclear areas. When comparing the various regions of the ventricular wall in the telencephalon of Salmo irideus with appropriate regions of amphibians and reptiles, some, at least partially, far-going morphological similarities can be found. The ependymal differntiations could, thus, be regarded as an additional morphological criterium concerning the question of the homologization of the telecephalic nuclear areas with those of other verebrates. The abundnat occurrence of tanycytes of the bipolar type in the telecephalon appeared surprising; they are discussed to act in a preponderantly resorptive manner, as it is, above all, hitherto known for hypothalamic regions of other vertebrates. The morphological and functional characteristics of the neuronlike structures exhibiting ventricular contact are as yet not clear.


Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens. Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.


F(ab')2 reagents are not required if goat, rather than rabbit, antibodies are used to detect human surface immunoglobulin. The present report provides evidence that whole goat anti-human immunoglobulin, unlike similar reagents produced in the rabbit, binds both to the same number of and the same individual cells as the F(ab')2 fragments of rabbit or goat anti-human immunoglobulin. These results suggest that goat IgG has a lower affinity for the Fc receptors of human lymphocytes and monocytes than rabbit IgG. Because of this property, whole goat antibodies against human immunoglobulin can be used as simple, convenient relatively inexpensive reagents for the routine detection of immunoglobulin on cell surfaces by immunofluorescence microscopy. The preparation of F(ab')2 fragments of anti-immunoglobulin, which are necessary when rabbit antibodies are used, does not appear to -e required if goat antibodies can be empolyed. This observation has multiple practival applications in cellular immunology.


Immunosuppression by spleen cells from Moloney leukemia. III. Evidence for a suppressor cell that is not the leukemic, virus-producing cell. Spleens of mice bearing MuLV (Moloney)-induced leukemia contain cells that inhibit the antibody response of normal syngeneic lymphocytes to sheep RBC in Marbrook cultures. In order to determine whether these immunosuppressive cells are virus-infected tumor cells or normal cells we pretreated leukemic spleen cell suspensions with syngeneic mouse antiserum to Moloney leukemia antigen(s) (plus complement) and with rat anti-Moloney serum (plus complement). The cytotoxic treatment killed approximately 20% to 30% and 60% to 70% of the cells, respectively. The remaining viable cell population was tested for MuLV production (in an infectious center assay on S+L-fibroblasts), for lethal effect on newborn mice, and for immunosuppressive activity. After the treatment with anti-Moloney sera the number of MuLV-releasing cells decreased 10-fold and the leukemogenic potential in vivo decreased 100-fold as compared to leukemic spleen cells pretreated with nonimmune mouse and rat sera (plus complement). In contrast, the ability of the antisera-treated cells to inhibit anti-SRBC response remained undiminished. This indicates that, in part, the immunosuppressive cells in the leukemic spleen are normal, noninfected cells, involved, perhaps, in immune regulation.


Altered humoral immune response to sheep red blood cells by sheep erythrocyte-soluble hemolysate. Adult C3H/He mice were rendered unresponsive to a primary injection of sheep red blood cells (SRBC) by pretreatment with sheep hemolysate supernatant (SHS) or subfractions of SHS isolated by column chromatography. The following effects of SHS on the immune response were observed: SHS did not kill antigen-reactive cells, it did not prevent the release of antibody by cells actively synthesizing and secreting antibody, and SHS-induced tolerance was not inhibited or abrogated by methods which terminate or abolish tolerance. In addition, cell-mediated responses were not affected in animals whose humoral responses were suppressed; however, the secondary plaque-forming cell (PFC) response was enhanced by SHS treatment. SDS gel electrophoresis revealed SHS to contain several proteins ranging from 12,000 to approximately 500,000 daltons.


BCG-induced murine effector cells. II. Characterization of natural killer cells in peritoneal exudates. The cytotoxic activity in peritoneal exudates harvested from C57BL/6 mice 4 to 6 days after they had received viable Bacillus Calmette-Guérin (BCG) organisms i.p. was associatee with a nonadherent, nonphagocytic cell. The cytotoxic cell lacked demonstrable surface immunoglobulin and Thy 1 antigen and bore no readily detectable Fc receptors. Lytic activity was labile at 37 degrees C and was diminished after trypsinization of the effector cells. Preincubating effector cells with immune complexes was without effect on lytic expression. These features make it likely that the cytolytic activity was associated with "natural killer" (NK) cells, previously described in unimmunized mouse spleens and mesenteric lymph nodes. Whether BCG induced "activation" of resident NK cells, the de novo production of NK cells, or "homing" into the peritoneum of cells normally resident in other lymphoid tissue is not known.


Negative allogeneic effects in vitro. II. Mapping of histocompatibility differences leading to allosuppression. When splenic T cells from normal mice recognize alloantigens on primed spleen cells there is a dramatic suppression of the secondary antihapten PFC response of the latter cells. From studies using intra-MHC recombinants it appears that differences at K or D alone can elicit allosuppression. There is sometimes suppression to differences in the I region. Even small differences in H-2K, as seen in the H-2b mutants, are sufficient to lead to such negative allogeneic effects. Thus, as far as has been determined, the specificity of allosuppresive T cells is indistinguishable from that of cytotoxic effector cells. Negative allogeneic effects and positive allogeneic effects different in the degree to which they influenced the primary and secondary responses. In our experiments no evidence for positive allogeneic effects was seen in the secondary response to TNP-carrier even with cells differing at the I region only. In contrast the primary response to SRBC was much enhanced by allogeneic cells and little suppression of that response was seen. It is suggested that the allosuppression is distinct from cytotoxicity and may be a profitable model for the study of suppressor T cells.


Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response. Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis.


Expression of fetal antigens by normal human skin cells grown in tissue culture. Normal human sera are capable of causing complement-mediated lysis of normal human skin cells grown in tissue culture. This lytic reactivity can be completely removed by absorption with first trimester fetal tissue. Absorption with a variety of normal adult human tissues including lymphocytes, decidua, skin, and muscle are incapable of absorbing reactivity. Absorption of reactivity by fetal tissue is specific and not due to the introduction of anti-complementary or other nonspecific factors, as evidenced by the inability of simultaneous fetal absorption to remove reactivity from antisera with specificity for HLA antigens. Similarly, absorption of lytic sera with fetal calf serum proteins was incapable of removing reactivity against normal cells in tissue culture. It thus appears that normal human cells in tissue culture express antigens shared by the first trimester human fetus, but not present on a variety of adult human tissues. This "neoantigen" present on normal human cells when grown in tissue culture is a potential source of confusion and must be accounted for in searching for human tumor-specific antigens utilizing tissue culture cells.


Expression of protein HC on the plasma membrane of different human cell types. The surface expression of a recently described plasma glycoprotein called human complex-forming glycoprotein, hetergeneous in charge (protein HC) on a number of different human cell types was investigated. By means of direct and indirect immunofluorescence, protein HC was shown to be associated with the surface of virtually all cells of the investigated normal cell types including erythrocytes, peripheral blood B and T lymphocytes, and the human fibroblast lines HE 81, HE 31, and WI 38. When transformed and malignant cell populations were studied, it was found that some populations (e.g., the T cell line Molt-4) carried protein HC on the surfaces of very few cells, whereas other cell populations (e.g., chronic lymphocytic leukemia lymphocytes) carried the protein on most cells. Malignant cell populations with intermediary percentages of protein HC-positive cells were also found. Protein HC on the cell surface of normal peripheral blood lymphocytes could be redistributed by incubation of the cells with anti-protein HC-antiserum at 37 degrees C, and this reaction could be inhibited by sodium azide.


Destruction of tumor cells by BCG-activated alqolar macrophages. Alveolar macrophages obtained from Syrian golden hamsters were tested for their ability to destroy tumor cells. Only macrophages obtained from BCG immune animals rechallenged intratracheally with BCG five days before assay exhibited cytotoxic activity. Maximum destruction of tumor cells occurred after 5 days of incubation. Immunologic activation of macrophages was required to attain cytotoxic alveolar macrophages. Induction of inflammatory lung exudates by a variety of nonspecific irritants did not result in tumor cell destruction by macrophages. These observations may prove useful in designing an approach for immunotherapy of lung cancer.


Immunocycte stimulation in vitro by nontoxic bacterial lipopolysaccharide derivatives. Intact lipopolysaccharides (LPS), considered nonspecific enhancers of B cell responses, as well as nontoxic derivatives from Serratia marcescens LPS, were studied with regard to their ability to stimulate in vitro immune responses to a T-dependent antigen, sheep erythrocytes. Intact LPS, at a dose of 10 to 50 microgram, consistently enhanced the in vitro anti-SRBC immune response by normal splenocytes. The LPS also increased the background PFC response to SRBC in nonimmunized cultures. A chemically detoxified preparation derived from LPS (Mex B) had no stimulatory activity in vitro. A completely nontoxic, relatively small m.w., polysaccharide-rich preparation (PS), free of detectable lipid and protein, was stimulatory in vitro and at a dose of 10 microgram resulted in a 40 to 70% enhancement of the anti-SRBC response. The PS also stimulated an enhanced background response to SRBC as well as several other RBC species in nonimmunized cultures. PS had no mitogenic effect in vitro since addition of this bacterial derivative failed to stimulate thymidine incorporation into mouse splenocytes, as occurred with the intact LPS. The use of nontoxic preparations from gram-negative bacterial LPS for dissecting the stimulatory vs antigenic properties of bacterial products provides a model system for determining the role of a mitogenic stimulus in B cell activation.


Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures. Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.


Differential tumor immunogenicity of L1210 and its sublines. I. Effect of an increased antigen density on tumor cell surfaces on primary B cell responses in vitro. The differential immunogenicity of DBA/2 lymphoma L1210 and three L1210 sublines, each resistant to a different anti-leukemic agent (guanazole, methylglyoxal-bis-guanylhydrazone, and 4,4-diacetyldiphenylurea-bis-guanylhydrazone), was evaluated in vitro. Syngeneic spleen cells from nonimmunized DBA/2 mice were cultured in the presence of graded numbers of irradiated cells of L1210 or its sublines. The stimulation elicited a T-independent primary antibody response in vitro which was measured by determining the number of plaque-forming cells by using the immunizing lymphoma cells as target. Cells of all three sublines exhibited an increased immunogenicity, as compared to that of the parental L1210 cells, in eliciting the response directed to tumor-associated antigens which were common to all sublines. Dose-response experiments showed that high doses of the parental cells did stimulate responses which were detectable with subline cells as target. The results indicated that the differential immunogenicity of L1210 and its sublines, as demonstrated in the present assay system, is primarily quantitative, and was apparently due to increased amount or density of common tumor-associated antigens on the subline cells. The implications of these observations are discussed in relation to the possible mechanisms underlying the emergence of highly immunogenic drug-resistant sublines.


Characterization of suppressive immunoglobulin-binding factor (IBF). II. Purification and molecular weight determination of IBF produced by L-5178-Y theta-positive lymphoma. L-5178-Y thymoma cells were used to produce radioactive immunoglobulin-binding factor (IBF). For this purpose, the cells were internally labeled by incubation with radioactive amino acids and/or fucose. The supernatants contained radioactive material that bound to IgG-sensitized erythrocytes and suppressed the in vitro antibody response to sheep red blood cells. Upon filtration on Sephadex G-200 both the IgG-binding activity and the suppressive activity eluted at peaks of 140,000 and above 300,000 d. However, on SDS polyacrylamide gels, after precipitation with antigen-IgG-antibody complexes. IBF was found in a single peak of 80,000 d. This molecule could be dissociated in the presence of mercaptoethanol into a major unit of 40,000 d and a minor unit of 20,000 d. These data suggest that IBF is a molecule of 80,000 d, which contains chains of 40,000 d and probably 20,00 d linked by disulfide bridges. In cell supernatants, however, the factor exists in polymeric forms of 140,000 d and more than 300,000 d.


Radiographic measurement of topical corticosteroid-induced atrophy. Soft tissue x-ray techniques were used to measure skin thickness as influenced by the chronic usage of topical corticosteroids. In a double-blind study commercial preparations of 1% hydrocortisone (HC), 0.1% triamcinolone acetonide (TA), and a placebo cream were compared for their ability to produce atrophy in normal human forearm skin. After 8 weeks of topical application of the creams, only TA produced clinically apparent atrophy. The average percent decreases in skin thickness measured after 8 weeks of treatment with placebo, HC, or TA were 6.0%, 6.0%, and 17.1%, respectively. During the first week after cessation of treatment the clinical appearance of the skin began to improve and by 1 month all treated skin areas had essentially returned to pretreatment thickness.


Relative enterotoxigenicity of coliform bacteria. The enterotoxigenicity of 12 strains of coliform bacteria (Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli) isolated from the gastrointestinal (GI) tract of persons with either acute diarrhea or tropical sprue and of 13 strains of the same species isolated from urine (GU) cultures was determined. Fractions of heat-labile (LT) and heat-stable (ST) toxins of each strain were separated by ultrafiltration, and the effect of graded concentrations (range, 100 microgram-10 pg/ml) on water transport was assessed by in vivo perfusion in the rat jejunum. Enterotoxigenic activity was defined as inducement of net secretion of water. All 12 of the GI strains and six of 13 GU strains elaborated enterotoxins, but there was a millionfold quantitative difference in the potency of the toxins produced. All of the GI strains produced one or both forms of toxin, which had a minimal effective concentration of as low as 0.1-10 ng/ml, whereas the GU strains produced toxins of weak or, rarely, of intermediate potency.


Discrepancy between Clq deviation and Raji cell tests in detection of circulating immune complexes in patients with leprosy. Samples of serum from 45 patients with different clinical forms of leprosy and from 17 patients with systemic lupus erythematosus were studied in parallel for circulating immune complexes with use of two different in vitro tests adjusted to the same degree of sensitivity. The Clq deviation test relied upon the reaction of the complement component Clq with immune complexes. The Raji cell test detected complement-fixed immune complexes that bound to the complement receptors on cultured, bone marrow-derived lymphocyte-like Raji cells. Thirty (67%) of 45 patients with leprosy showed immune complexes according to the Clq deviation test; however, only two (7%) of the 30 samples of sera with positive Clq test results were positive by the Raji cell test. In contrast, 54% of 13 samples of sera from patients with systemic lupus erythematosus positive by the Clq test were positive according to the Raji cell test. Since Clq is known to react with DNA as well as with bacterial antigens, the Clq reaction may in fact be detecting antigenemia in many instances. Considerable caution is warranted in application of sensitive screening tests for assay of circulating immune complexes in various states of infectious diseases.


Gastroenteritis in children: a two-year review in Manitoba. I. Etiology. During two years, 1,217 children hospitalized with gastroenteritis at the Children's Centre in Winnipeg, Manitoba, Canada were studied. Bacterial pathogens were present in 25% of these children: enteropathogenic Escherichia coli in 120, Shigella in 139, Salmonella in 24, and multiple pathogens in 18. Rotavirus was detected in 54 (11%) of 472 patients examined. Rotavirus and enteropathogenic E. coli were the most common pathogens in infants, and Shigella was the most common in older children. Bacterial diarrhea occurred more commonly in summer, whereas rotavirus infection occurred more commonly in winter. Among 276 children screened, enterotoxigenic E. coli was found in three, and Aeromonas shigelloides that produced a similar toxin in two others. Enteroinvasive E. coli was not detected in 70 children. Organisms producing toxins "cytotoxic" to HeLa cells were isolated from three of 90 children. Screening for enterotoxigenic or enteroinvasive organisms was not productive of a significant number of pathogens, and, although screening for rotavirus did improve the number of etiologic diagnoses, the etiology of the majority of cases of diarrhea remained unknown.


Streptococcal infections that fail to cause recurrences of rheumatic fever. Prospective studies of recurrences of streptoccal infection and acute rheumatic fever were conducted among patients attending the acute rheumatic fever prophylaxis clinic (City of Memphis Hospitals, Memphis, Tennessee) between 1965 and 1972. The patient population consisted of 124 rheumatic children and adults, two-thirds of whom had evidence of rheumatic heart disease. A total of 104 immunologically documented streptococcal infections occurred during 235 patient-years of follow-up (44.3 infections per 100 patient-years) without a single recurrence of rheumatic fever. Immune responses tended to be modest, and 80% of the infections were subclinical. The majority of our group A streptococcal isolates were obtained from routine cultures of specimens from asymptomatic individuals. Many of these strains were "pyoderma" serotypes, whereas others exhibited a characteristic (production of opacity factor) recently reported to be associated with decreased immunogenicity. Several factors may have contributed to the low recurrence rate of acute rheumatic fever, including the age range of the population under study and immunologically significant infections with strains of Streptococcus that were not group A, but a major reason may be the possibility that the group A strains prevalent in this population have diminished rheumatogenic potential.


Virulence factors of enterotoxigenic Escherichia coli. Further evidence for the role of enterotoxigenic Escherichia coli as an etiologic agent of diarrhea is presented. A retrospective study of 71 cases of diarrhea in Mexican children demonstrated that greater than 40% of them harbored E. coli that produced heat-labile and/or heat-stable enterotoxin. The antigenic surface-associated colonization factor of E. coli strain H-10407 has been further characterized: this pilus-like antigen is produced under conditions of growth that repress the production of common pili of E. coli. The E. coli H-10407-type colonization factor pilus has been identified as one of the antigens possessed by a strain of E. coli that produced only heat-stable enterotoxin and that was responsible for an outbreak of pediatric diarrhea.


Antigens of Escherichia coli, human immune response, and the pathogenesis of urinary tract infections. Acute pyelonephritis (but not cystitis or "asymptomatic" bacteriuria) due to Escherichia coli induces serum antibodies to O-but rarely to K-antigens, especially not to the most common antigen, K1. Locally produced secretory IgA and IgG antibodies to O-and K-antigens appear in urine during most infections. The E. coli in urine of patients with asymptomatic bacteriuria are different from those in patients with acute pyelonephritis and cystitis and undergo continuous changes, presumably caused by the local antibody response. The E. coli become less virulent and are less able to attach to uroepithelial cells than E. coli causing acute symptomatic infections. Antibodies in urine prevent epithelial adherence. Parenteral and intravesicular injections of killed bacteria can protect against ascending pyelonephritis in rats. A few K-antigens dominate among E. coli that cause urinary tract infections. Vaccination of problem cases is a possibility because of the protective nature of K-antibodies. The mechanism of renal scarring that appears in some patients with urinary tract infections is unknown. Autoantibodies to the Tamm-Horsfall protein that increase after acute pyelonephritis or the cross-reactions noted between certain E. coli and antigens on the kidney may be involved.


Cross-reactive antigens: their potential for immunization-induced immunity to Gram-negative bacteria. The involvement of multiple species and serologic types in gram-negative bacteremia prompted evaluation of immunization with shared, cross-reactive antigens of gram negative bacilli. Active and passive immunization with Re chemotype mutants of Salmonella minnesota afforded significant protection against heterologous gram-negative bacilli and were considerably more effective than immunization with smooth S. minnesota or its Ra, Rb, Rc, Rd1 and Rd2 mutants. Since the lipopolysaccharide of the Re mutant is composed solely of 2-keto-3-deoxycotonate (KDO) and lipid A, the protective activity of antibody to the Re mutant and lipid A was evaluated. Immunization with Re mutant protected granulocytopenic rabbits against lethal bacteremia and protected mice against lethal challenge with heterologous endotoxins, whereas antibody to lipid A had no protective activity. In concomitant clinical studies, high titers of antibody to the Re mutant at the onset of bacteremia were associated with a significant diminution in the frequency of shock and death, which was independent of any effect of O-specific antibody.


Antibody to cell wall glycolipid of Gram-negative bacteria: induction of immunity to bacteremia and endotoxemia. Antiserum to the core glycolipid of gram-negative bacteria was prepared by immunization of rabbits with vaccine composed of killed cells of the uridine diphosphate galactose-deficient mutant (J5) of Escherichia coli O:111. Antiserum to J5 not only prevented death of animals from endotoxin but also prevented the local and generalized Shwartzman reactions. Antiserum to endotoxin also prevented renal cortical necrosis and disseminated intravascular coagulation during the evolution of the generalized Shwartzman reaction. Antiserum to be J5 mutant was successful in the treatment of overwhelming bacteremia produced by other gram-negative bacteria; in addition to bacteremia cause by coliform organism, antiserum to J5 was dramatically effective in treatment of bacteremia due to Pseudomonas aeruginosa. One injection of rabbit antiserum to J5 improved the survival rate from 15% in controls to 59% in treated animals (P less than 0.002). Active immunization with J5 vaccine was even more effective against pseudomonas bacteremia: such immunization improved the survival rate from 13% in controls to 92% in vaccinated rabbits. Since an antiserum effective against the J5 mutant of E. coli can be prepared safely in human subjects, such immunotherapy should be considered for patients with gram-negative bacteremia.


The infant rat as a model of bacterial meningitis. The pathogenesis of bacterial meningitis was studied in infant rats. Intranasal intoculation of greater than 10(3) Haemophilus influenzae type b resulted in an incidence of bacteremia that was directly related to the size of hte challenge inoculum. The temporal and quantitative relationship of bacteremia to meningitis indicated that bacteria spread to the meninges by the hematogenous route and that the magnitude of bacteremia was a primary determinant in the development of meningitis. In a sparate series of experiments, infant rats that were fed Escherichia coli strain C94 (O7:K1:H-) became colonized and developed bacteremia and meningitis, but invasive disease was rare when rats were fed E. Coli strain Easter (O75:K100:H5). A comparison of intranasal vs. oral challenge indicated that the nasopharynx was the most effective route for inducing H. influenzae bacteremia, whereas the gastrointestinal route was the more effective challenge route for the E. coli K1 serotype.


Chemically defined bacterial products with immunopotentiating activity. The adjuvant activity of two chemically well-defined bacterial products is reviewed: (1) lipopolysaccharides of gram-negative bacillis and their acylated detoxified derivatives, and (2) mycobacterial water-soluble fractions and synthetic analogues. Water-soluble adjuvant can substitute for mycobacteria in Freund's adjuvant, but if it is administered in saline, it has little activity. In contrast, lipopolysaccharide under the same conditions markedly increases the humoral antibody response. However, the use of lipopolysaccharide is limited by its toxicity. Water-soluble adjuvant treated with phthalic or succinic anhydride was shown to be an adjuvant when administered in saline. Furthermore, synthetic M-acetyl-muramyl-L-alanyl-D-isoglutamine also increased the humoral immune response when given in aqueous medium instead of in the usual water-in-oil emulsion. This compound, which has a small molecular weight, is not mitogenic, immunogenic, or toxic in mice, and was shown to have adjuvant activity even when given by the oral route.