Edward Lewin

INTRALAMINAR DISTRIBUTION OF Na+‐K+ ADENOSINE TRIPHOSPHATASE IN RAT CORTEX

PREVIOUS publications from this laboratory have described the intracortical distribution of Mg2+-activated ATP phosphohydrolase (Mg-ATPase) activity in rat somatosensory cortex (HESS and POPE, 1959) and in human frontal cortex (HESS and POPE 1961). In those experiments, enzyme activity was determined in a medium containing Mg2+, Na+, and K+ in concentrations of 7, 40, and 7 rnM/l., respectively, and no attempt was made to measure separately the stimulation of Mg2+-activated ATPase activity produced by Na+ plus K+ and its distribution within the cortex. It was appreciated, however, that the concentrations of Na+ and K+ had a marked effect upon the activity of Mg2+-activated ATPase in brain tissues, and a systematic study was made of this phenomenon (HESS and POPE, 1957; HESS, 1962). The work of many investigators now supports the idea that a Na-K ATPase is involved in the active linked transport of Na+ and K+ in certain cell membranes. SKOU (1957; 1960) first separated a particulate enzyme of this sort from crab nerve, and similar enzymes have been prepared from human erythrocytes (POST, MERRITT, KINSOLVING and ALBRIGHT, 1960), mammalian brain (J~RNEFELT, 1961 ; DEUL and MCILWAIN, 1961), kidney cortex (WHITTAM and WHEELER, 1961), and the electric organ of Electrophorus electricus (GLYNN, 1962). Because of the potential fundamental significance of such an enzyme for cortical function, we decided to determine in rat cortex the intralaminar distribution of the Na-K ATPase, as distinct from ATPases fully activated by Mg2+ alone. Accordingly, the enzymic hydrolysis of ATP in the presence of (a) Mg2+ alone, and (b) optimal concentrations of Mg2+, Na+, and K+ was studied in adjacent 20 ,u thick frozen sections sampled at 80 ,u intervals throughout the cortex. The Na-K ATPase activity was derived by subtracting (a) from (b). The different intracortical distributions obtained are consistent with distinct differences in the cytological localizations and physiological functions of the two types of ATPases.

INTRALAMINAR DISTRIBUTION OF CEREBROSIDES IN HUMAN FRONTAL CORTEX.

THE biochemical architecture of human cerebral cortex has been the subject of a number of investigations in this laboratory. The intracortical distributions of a number of enzymes were determined in cortical biopsy specimens obtained during prefrontal lobotomy operations (POPE, CAVENESS and LIVINGSTON, 1952; POPE, 1959; HESS and POPE, 1960, 1961). More recently, experiments designed to provide similar information in regard to certain biochemical structural components were carried out upon human autopsy material. The components include cerebrosides, proteolipid proteins, residue proteins, RNA, DNA and gangliosides. The rationale for the choice of these substances for study in cortex and other regions of brain was discussed (HESS and THALHEIMER, 1965), and a scheme for their simultaneous assay outlined. Cerebroside, one of the groups of brain lipids first isolated by THUDICHUM (1901), was selected because of its suggested relationship to stainable myelin. When cerebrosides have been assayed in grey matter by methods that exclude gangliosides, variably low concentrations have been found relative to their abundance in white matter (ROSSITER, 1955; TINGEY, 1956; SVENNERHOLM, 1957). If cerebroside is a constituent of myelin, however, significant amounts should be present in cortex, as stainable myelinated axons are clearly evident. Furthermore, the pattern of the intracortical concentration of cerebroside should follow the myeloarchitecture of the region of cortex studied. This article describes a quantitative microchemical study of human eulaminate frontal cortex that demonstrates the degree of parallelism between cerebroside concentration and myeloarchitecture. A preliminary report has been made (LEWIN and HESS, 1963).

INTRALAMINAR DISTRIBUTIONS OF PROTEOLIPID PROTEIN AND RESIDUE PROTEIN IN HUMAN FRONTAL CORTEX

INVESTIGATIONS on the biochemical architecture of mammalian cerebral cortex have been in progress for a number of years in this laboratory. The principles and techniques of quantitative histochemistry (LINDERSTRBM-LANG, 1939), as applied to the study of cortex (POPE, 1952; POPE, CAVENESS and LIVINGSTON, 1952), are being used. The aim has been to outline the biochemical composition of the cortex in relation to its anatomical constitution in health and disease. Recent studies have been designed to determine the intralaminar distributions of biochemical structural components in human frontal cortex obtained at autopsy. The distribution of cerebrosides within frontal cortex was reported previously to show a high degree of correlation with the cortical myeloarchitecture ( L E V ~ N and HESS, 1965). Proteolipid proteins (PLP) were chosen for comparison with cerebrosides as an index of myelinated fibres in nervous tissue. The exceptional abundance of these chloroform-methanol-soluble lipoproteins in white matter of adult animals first suggested that they might be the lipoprotein complex of myelin (FOLCH and LEES, 1951). Consonant with this idea, investigations on developing mouse brain showed that PLP became detectable in significant amounts at the time stainable myelin appeared (FOLCH-PI, 1955; FOLCH, CASALS, POPE, MEATH, LEBARON and LEES, 1959). Further support for this localization came from studies of bovine brain myelin fractions prepared by sucrose density gradient centrifugation (AUTILIO, NORTON and TERRY, 1964). However, the distribution of PLP in grey and white matter, nerve roots, and peripheral nerve does not entirely parallel that of the lipid constituents of myelin (AMADUCCI, 1962; AMADUCCI, PAZZACLI and PESSINA, 1962). Small amounts of PLP were isolated from non-neural tissues (FOLCH and LEES, 1951), and perhaps in nervous tissues they are not confined to myelin sheaths. It seemed, therefore, of considerable interest to observe the extent to which the distribution of PLP followed that of cerebrosides in the layers of cerebral cortex. The term residue protein is used to refer to that protein remaining in the tissue after removal of PLP by chloroform-methanol (2: 1, v/v). Particularly in white

A Paradigm for the Control of Influenza

in the intervention community. Furthermore, LAIV was 1.7 times more effective in the prevention of proven influenza virus infection in the target group than among those who received TIV and was 6 times more effective than among those who did not receive vaccine.

771 COMPARATIVE IMMUNOGENICITY OF GROUP C NEISSERIA MENINGITIDIS VARIANTS AND ESCHERICHIA COLI K92 CAPSULAR POLYSACCHARIDES IN ADULT VOLUNTEERS

We studied three structurally and antigenically similar capsular polysaccharides: Group C Neisseria meningitidis O-acetyl positive (OAc+) and negative (OAc−) variants, and the cross-reacting E. coli K92 for their ability to induce Group C meningococcal antibodies in adults. All three polysaccharides elicited specific serum antibodies. The OAc− variant was the most immunogenic. Geometric mean pre-immunization anticapsular antibody levels were 1.4 μgm/ml, 0.8 μgm/ml, and 1.2 μgm/ml for groups receiving OAc−, OAc+ and E. coli K92 respectively. Geometric mean antibody titers 3 weeks and 2 months post immunization were 41.7 μgm/ml for OAc−, 22.8 μgm/ml for OAc+, and 7.0 μgm/ml for E. coli K92 (p = 0.001 for OAc− and OAc+ versus K92). No Group C meningococci or cross-reacting organisms were isolated from repeated NP cultures, but one individual demonstrated persistant rectal carriage of E. coli K92. Antibodies elicited by either Group C polysaccharide were bactericidal for OAc+ and OAc− organisms. Absorption of OAc+ antisera with OAc− polysaccharide did not remove all bactericidal antibody. The superior immunogenicity and distinct biochemical characteristics of the OAc− variant support further study in children and infants.

PHARMACOKINETICS OF AMIKACIN IN CHILDREN

Pharmacokinetics of aminoglycoside antibiotics vary both within and between different age groups. To determine proper dosing level and interval for children, sequential pharmacokinetics were studied in 10 patients (age = 8.7 yrs). All children had normal renal function. Mean weight and surface area were 29.5 kg and 0.99 m2 respectively.A dose of 7.5 mg/kg was administered IM q12h. Utilizing an acetyl transferase radioenzymatic assay, sequential serum levels were determined and were as follows (μg/ml ± SD) : Oh=0; ½h=13.0 ± 4.0; 1h=12.2 ± 3.3; 2h=8.9 ± 3.3; 4h=3.1 ± 1.8; 6h=1.5 ± 1.3; 8h=< 1.0. Drug was undetectable in serum after 8 hrs. No accumulation was observed. Mean peak serum: mean urine (0-12h) ratio was 1:20. There was no evidence of toxiclty.Utilizing this dosage regimen, the t½ in our patients (1.7h) was 23% shorter than that in adult subjects (2.2h). The mean peak serum level (13μg/ml) was 35% lower than in adults (20μg/ml).These data suggest that children require larger doses of amikacin administered at more frequent intervals than those utilized in this study.

HISTOPATHOLOGY Of EXPERIMENTAL BACTERIAL MENINGITIS IN RABBITS

Despite extensive use of the rabbit to study the pathophysiology of bacterial meningitis (BM) and pharmacokinetics of new antimicrobials, the histopathology of the disease in this model has not been delineated. In order to define these abnormalities, 20 NZ wh rabbits were inoculated intracisternally by a percutaneous technique with normal saline (Controls, N=6), S. pnemnoniae type III (Spn) (N=5 before {NV} and N=2 after (V) passage in mice) and H. inf. type b (HII8). Inocula wore 3.810 CFU, 4.310CFU and 8.410 CFU, respectively. The brain was removed from the skull within 10 min after death, fixed immediately in 10% formalin for 2 uks and sectioned coronally at 10 uniform levels from the frontal lobes to the brain stem. Sections were stained by H&E and graded blindly for severity of leptomeningitis (0=nl ← 5=severe) and the presence of parenchymal involvement (cerebritis, abscess formation, ventriculitis, vasculitis and choroid plexitis).3/5 rabbits with HIIB and 5/7 with Spn demonstrated at least 1 parenchymal lesion. These were of mild degree with the exception of 1 rabbit (HIIB) who died at 24th. There was no consistent relationship between the degree of CSF pleocytosis, ↑ lac or (I and the severity of histopathologic changes observed.

1136 COLONIZATION AND INFECTION RELATED TO CONTAMINATED ENTERAL FEEDINGS

Although tube feeding formulae are known to frequently be contaminated with bacteria, few cases of sepsis related to tube feedings have been reported. A premature infant admitted to the Special Care Nursery appeared to develop sepsis in this manner. She had received hyperalimentation for one month followed by enteral tube feedings of PregestimilR formula. One month later, she became septic; E. cloacae and K. pneumoniae were isolated from the blood. E. cloacae and P. aeruginosa were isolated from the stool. Samples of the formula, which had been distributed to the patient unit, as well as a portion which had remained in the pharmacy after mixing, were cultured and found to contain the following: E. cloacae (105 cfu/ml), P. aeruginosa (3-5×104 cfu/ml), K. pneumoniae (2-8×104 cfu/ml), and P. calcoaceticus, var. antratus (.5-8×104 cfu/ml). Since the formula was found to be contaminated while still in the pharmacy where it had been mixed, investigations focused on the preparation of the formula. Formula had been prepared from a powder using utensils and a technique considered “clean” but not sterile. Changes were introduced to insure a 3 minute hand scrub prior to mixing, the use of sterile and/or disposable utensils, disinfection of the work areas and the mixing of formula under a laminar flow hood. Subsequent cultures of formula remained negative and no further cases were seen over the ensuing 12 months.

TRANSMISSIBILITY OF TYPE B HEPATITIS IN A SPECIAL CARE NURSERY

The opportunity to investigate nurse-infant hepatitis B virus (HBV) transmissibility arose when a nurse in the special care nursery (SCN) developed acute Type B hepatitis. This nurse had close contact with all 31 neonates in the SCN during the month prior to the onset of her clinical hepatitis. Six months later sera were obtained from 24 of the 31 Infants and from 22 of their mothers. Sera were analyzed for HBeAg, anti-HBc and anti-HBs. The nurses serum and saliva were strongly positive for HBsAg; her serum antl-HBc = 1:32. Neither HBeAg nor anti-HBe was detectable in her serum. No infant or mother demonstrated evidence of HBV infection. These data show that a nurse caring for infants in a SCN did not transmit HBV to any of the neonates with whom she had direct contact despite the fact that she had acute icteric hepatitis, HBeAg in her serum and saliva, and ongoing viral replication. The fact that the nurse was HBeAg negative may have influenced the results.