Cell membrane-binding properties of group A streptococcal lipoteichoic acid.

Lipoteichoic acid (LTA) was extracted from group A streptococci, previously treated with hot HCl, by the phenol method. The extracted LTA was loaded on an isoelectric (IE) focusing column and two fractions were collected; one at pH 4.65 and the other at pH 2.95. Chemical analysis demonstrated that the unfractionated LTA contained alanine and glycerolphosphate at molar ratio of 1:10, and ester-linked lipids, but no detectable sugars or amino-sugars. The two IE fractions contained lipids but lacked alanine. The LTA and its IE fractions spontaneously adsorbed to human erythrocytes (sensitization) causing them to agglutinate in the presence of rabbit anti-LTA. The RBC-sensitizing and antigenic activities of IE fractions were equal to, or greater (for IE fraction at pH 4.65) than the unfractionated LTA, indicating that alanine is not involved in the sensitizing activity of LTA. Mild ammonia-hydrolysis abolished the RBC-sensitizing activity of LTA and its IE fractions. Chloroform-methanol-soluble material of the ammonia-hydrolysate lacked antigenic activity but blocked sensitization of erythrocytes by LTA. The water-soluble material of the hydrolyzed LTA retained antigenic activity, was not able to block sensitization by LTA, and its sensitizing activity was restored after esterification with fatty acids. These experiments indicate that ester-linked fatty acids (palmitic acid being the major one) are involved in the spontaneous adsorption of LTA to erythrocytes. The LTA, its lipid moiety, and anti-LTA blocked adherence of group A streptococci to human epithelial cells, suggesting that small amounts of LTA may reside on the streptococcal surface to mediate attachment and colonization of these organisms on mucosal surfaces in vivo.

The use of flow microbluorimetry in the analysis of the phenotype expression of mouse histocompatibility antigens.

Quantitation of the expression of cell surface antigens has hitherto been limited to analysis by either cytotoxicity tests or radioimmune assays (5, 15). We report here the use of a new methodology to analyze and quantitate the expression of mouse histocompabililty antigens (H-2 locus) in hybrid clones and parental cell types. The binding of fluorescein-tagged antibody is measured on a cell-to-cell basis in large viable cell populations using flow microfluorimetric techniques. These techniques have been used to measure hapten and immunoglobulin binding to lymphocyte populations (8, 9, 14). However, this is the first report in which these techniques have been used to examine the expression of the H-2 locus. The advantage of this approach is twofold: first, a large and statistically significant sample population may be analyzed one cell at a time, thus revealing the fine detail of heterogeneity in the expression of the cell surface markers within a population. Second, as has been demonstrated for analysis of specific components of the immune system, this method does permit fluorescence-activated sorting of cell types according to their different surface populations (8, 9, 14).

Magnetic microspheres prepared by redox polymerization used in a cell separation based on gangliosides.

A facile method is described for making magnetic microspheres that bind specifically to cell surfaces, in order to separate cells magnetophoretically. Control over the sizes of the spheres is effected by using their magnetic cores as part of a redox polymerization system. The use of the microspheres is demonstrated with a separation involving C-1300 neuroblastoma cells, 10% of which express the ganglioside GM1 in their membranes. The GM1-containing cells were separated with better than 99% purity, while the deficient cells were obtained at least 98% pure. The separation, which was carried out under sterile conditions, required only 6 minutes.

Nutrient retention and growth performance of chicks given low-phytate conventional or hull-less barleys.

1. The experimental barley samples included 4 hulled and one hull-less low-phytate barley cultivars and two commercial barley varieties as controls. 2. The diets were provided in meal form, with the experimental barley samples constituting the cereal source. Two additional treatments were added for each of the control varieties in which intermediate and recommended levels of phosphorus were provided. 3. A completely randomised design was used with 5 replicates of 5 chicks per treatment. The chicks were grown from 2 to 14 d of age with excreta collected over the subsequent 3 d. 4. Although total phosphorus levels were similar for all barley samples, there were large differences in their phytate content, which ranged from less than 0.5 to 13.8 g/kg. M2 955 hulled barley exhibited the lowest phytate and the highest phosphorus solubility. 5. There was a negative linear relationship between grain phytate and weight gain and with bone ash. The low-phytate hulled barleys M2 955 and the low-phytate hull-less barley (lpa1-1H) gave better feed conversion (8%) than controls. The hull-less low-phytate barley gave significantly higher total phosphorus (18%) and soluble phosphorus retention (23%) than the hull-less control. The low-phytate samples tended to give lower excreta phosphorus levels (total and soluble), but the effect was significant only for the hull-less samples. Amino acid retention was significantly higher for the low-phytate hull-less barley than the control (4%). 6. Overall, the results suggest that using low-phytate barley can result in similar growth while using less supplemental phosphorus, reducing waste phosphorus by more than 50%.

Influence of glutamine on the growth of human glioma and medulloblastoma in culture.

Cellular supply of glutamine, an essential substrate for growth, is derived from extracellular fluid and de novo synthesis. We investigated the relative importance of these sources to the growth of six human anaplastic glioma- and one human medulloblastoma-derived permanent cell lines. Exogenous glutamine was limiting for the proliferation of glioma-derived lines D-54 MG, U-118 MG, and U-251 MG. In contrast, medulloblastoma-derived line TE-671 and glioma-derived lines U-373 MG, D-245 MG, and D-259 MG grew in the absence of supplemental glutamine. Two cell lines with contrasting glutamine requirements, D-54 MG and TE-671, were used to explore the pharmacological interference with glutamine metabolism. DL-alpha-Aminoadipic acid, a reported glutamic acid analogue with gliotoxic properties, significantly inhibited the growth of both lines. These effects were reversed by increasing glutamine, suggesting that the major action of DL-alpha-aminoadipic acid is as a glutamine antagonist. In contrast, the glutamine synthetase inhibitor delta-hydroxylysine demonstrated activity only against TE-671. Acivicin and 6-diazo-5-oxo-L-norleucine, glutamine analogues available for clinical use, reduced the proliferation of both cell lines at pharmacological concentrations. Methionine sulfoximine, a glutamine synthetase inhibitor previously used clinically, produced marked growth inhibition only against TE-671. These findings indicate that the synthesis and utilization of glutamine are potentially exploitable targets for the chemotherapy of some human gliomas and medulloblastomas.

The emergence of West Nile virus in North America: ecology, epidemiology, and surveillance.

In late summer 1999, the first domestically acquired human cases of WN encephalitis were documented in the USA. Aggressive vector-control and public education efforts by state and local public health officials limited the extent of human involvement. The discovery of virus-infected, overwintering mosquitoes during the winter of 1999-2000, predicted renewed virus activity for the following spring, and prompted early season vector-control activities and disease surveillance efforts in NYC and the surrounding areas. These surveillance efforts were focused on identifying WN virus infections in birds and mosquitoes as predictors of the potential risk of transmission to humans. By the end of the 2000 mosquito-borne disease transmission season, WN virus activity had been documented as far north as the states of Vermont and New Hampshire, and as far south as the state of North Carolina. The ongoing impacts that WN virus will have on wildlife, domestic animal and human populations of the western hemisphere are not yet known. Plans are in place for public health officials and scientists to monitor the further expansion of WN virus with the establishment or enhancement of vector-borne disease surveillance and control programs throughout the eastern seaboard. The valuable lessons learned from the detection and response to the introduction of WN virus into NYC should prove useful if and when subsequent intrusions of new disease agents occur.

West Nile virus in the United States: guidelines for detection, prevention, and control.

The epidemic/epizootic of West Nile (WN) encephalitis in the northeastern United States in the summer and fall of 1999 was an unprecedented event, underscoring the ease with which emerging infectious pathogens can be introduced into new geographic areas in today's era of rapid transportation and increased movement of people, animals, and commodities. This epidemic/epizootic and the increased frequency of other exotic pathogens being imported into the United States raises the issue of whether local, state, and national public health agencies are prepared to deal with epidemics/epizootics of vector-borne infectious diseases. The overwintering of WN virus and the epizootic transmission in the summer of 2000 reinforces the need to rebuild the public health infrastructure to deal with vector-borne diseases in this country. This article summarizes guidelines for surveillance, prevention, and control of WN virus that were drafted in December 1999 to help prepare state and local health departments for monitoring WN virus activity in the spring and summer of 2000 and also summarizes the data collected from those surveillance systems through September 2000.

An evaluation of media for transport of tissues infected with Borrelia burgdorferi.

Currently, the best medium for culture of Borrelia burgdorferi, the etiologic agent of Lyme disease, is Barbour-Stoenner-Kelly (BSK), or its modifications. This medium is complex, expensive, and laborious to prepare. A recent report suggested that a less expensive and simpler medium, hypertonic Columbia broth, might be useful as a transport medium for human tissues infected with B burgdorferi. To test this observation, hypertonic Columbia broth, Amies broth, distilled water, physiologic saline, phosphate-buffered saline (PBS), and modified Stuart medium were compared with BSK II as transport media, using ear and tail tissue samples from B burgdorferi-infected laboratory mice and using holding times and temperatures simulating actual transport conditions. The results showed BSK II to be markedly superior to the other media tested, although B burgdorferi remained viable in a few tissue samples held at room temperature in hypertonic Columbia broth, physiologic saline, or PBS for up to 2 days. Barbour-Stoenner-Kelly II continues to be the best medium for transport of tissues infected with B burgdorferi.

Epidemic West Nile encephalitis in Romania: waiting for history to repeat itself.

Seroprevalence data suggest that West Nile virus activity in southern Romania dates to the 1960s or earlier. In the summer of 1996, southeastern Romania and especially Bucharest experienced an unprecedented epidemic of West Nile encephalitis/meningitis, with at least 393 hospitalized cases and 17 deaths. Contributing factors included a susceptible avian population and urban/suburban infrastructural conditions that favored the production of large numbers of Culex pipiens pipiens. The epidemic ended spontaneously in early autumn. Results of serosurveys conducted as the epidemic waned pointed to the recent, novel introduction of West Nile virus to Bucharest. During 1997-2000, 39 scattered human cases of clinical West Nile virus infection (mean, 10 per year; range, 5-14 per year)--including 5 (13%) fatal cases--were diagnosed serologically throughout the region, but epidemic disease did not recur. Results of limited ecologic surveillance efforts during 1997-2000 suggested the existence of numerous focal areas of enzootic West Nile virus activity within the region. The authors explore the possible factors that led to the 1996 epidemic, review the ecologic and human data gathered during the postepidemic period of 1997-2000, summarize the public health lessons offered by the epidemic and its aftermath, and speculate on the future of epidemic West Nile virus activity in southeastern Romania.

Distribution of neutralizing antibodies to California and Bunyamwera serogroup viruses in horses and rodents in California.

Neutralization tests were done on sera from 141 horses from high elevation regions of California. Antibody prevalences to Jamestown Canyon, snowshoe hare, and California encephalitis viruses in the California serogroup and Northway virus in the Bunyamwera serogroup were 55%, 43%, 18%, and 46%, respectively. In 51 horses from rural low elevation regions, seroprevalences were 31%, 35%, 35%, and 37%, respectively. Twenty-four horses from a suburban lowland area were seronegative, except for a single horse with a low titer to snowshoe hare virus. Seroprevalence to Jamestown Canyon and snowshoe hare viruses was associated with increasing age. Only 2 of 177 rodents from the Sierra Nevada had antibodies to Northway virus; none had antibodies to Jamestown Canyon or snowshoe hare viruses.

Isolation of Northway serotype and other Bunyamwera serogroup bunyaviruses from California and Oregon mosquitoes, 1969-1985.

Eight previously untyped Bunyamwera serogroup bunyaviruses that had been isolated from mosquitoes collected in California and Oregon between 1969 and 1985, were identified by cross-neutralization tests. Four viruses from Anopheles freeborni and a virus from Aedes sierrensis collected in Butte County in the Central Valley of California in 1970-71 were shown to belong to the Northway serotype. The existence of a Northway serotype virus in California had been inferred from previous serologic surveys of deer and horses, but these are the first Northway serotype viruses to be identified from the contiguous United States or from An. freeborni. This is also the first isolation of any virus from Ae. sierrensis. Two viruses from Culiseta inornata collected in Umatilla County, Oregon in 1969 may represent a new serotype in the Bunyamwera serocomplex of the Bunyamwera serogroup. The name "Stanfield" is proposed for this serotype, which is closely related to the Cache Valley and Northway bunyavirus serotypes. A virus from Ae. taeniorhynchus collected in San Diego County, California in 1985 was confirmed to belong to the Main Drain serotype. Previously, Main Drain serotype viruses in California have been associated principally with Culicoides variipennis.

Isolation of Jamestown Canyon virus from boreal Aedes mosquitoes from the Sierra Nevada of California.

More than 28,000 mosquitoes in four genera were collected from high elevation (greater than or equal to 1,000 m) areas of California during 1988-89 and tested for virus by plaque assay in Vero cells. Viruses were serogrouped by enzyme immunoassay and serotyped by cross-neutralization. Six strains of Jamestown Canyon virus in the California serogroup were isolated from three species of boreal Aedes in the Aedes communis group of the subgenus Ochlerotatus. All isolates were from mosquitoes collected in Alpine County at approximately 2,300 m elevation in the Sierra Nevada. These included one virus from a pool of male Aedes cataphylla collected in immature stages, which is evidence for vertical transmission; four viruses from adult female Ae. communis (sens. lat.); and one virus from adult female Aedes hexodontus. These are the first isolations of viruses from boreal Aedes mosquitoes in California and the first reported isolations of Jamestown Canyon virus from Ae. cataphylla or Ae. hexodontus.

Prevalence of neutralizing antibodies against California and Bunyamwera serogroup viruses in deer from mountainous areas of California.

Plaque reduction-serum dilution neutralization was used to evaluate the status of bunyavirus activity in deer in mountainous areas of California. Antibodies against 9 bunyaviruses were measured in 337 mule deer (Odocoileus hemionus hemionus, O. hemionus californicus, and O. hemionus inyoensis) and black-tailed deer (O. hemionus columbianus). More deer from high mountainous areas had neutralizing antibodies against Jamestown Canyon virus than did deer from low mountainous areas (23% vs. 9%; P less than 0.01). This finding is consistent with transmission by snow pool Aedes mosquitoes. Results for Jerry Slough virus were nearly identical to those for Jamestown Canyon virus, which is further evidence that these are strains of the same virus. Neutralizing antibodies against Northway virus were present in 26% of deer from high mountainous areas and 23% of deer from low mountainous areas, suggesting the involvement of a widespread vector, such as Culiseta inornata. Northway virus is not known to occur outside of Alaska and northwestern Canada. Low prevalences of antibodies were detected in deer to California encephalitis, La Crosse, and snowshoe hare viruses of the California serogroup; and Cache Valley, Lokern, and Main Drain viruses of the Bunyamwera serogroup.

An interstate outbreak of tick-borne relapsing fever among vacationers at a Rocky Mountain cabin.

In July 1995, an outbreak of acute febrile illness affected 11 (48%) of 23 family members from Nebraska and Kansas who had vacationed at a Colorado cabin in June. Similar symptoms were identified among five (17%) of 30 additional persons from Nebraska, Kansas, Florida, and Texas who had vacationed at the same cabin. Symptoms suggested tick-borne relapsing fever (TBRF). Although no spirochetes were detected in available blood smears from five case-patients, Borrelia hermsii was cultured from the blood of one case-patient and two chipmunks trapped near the cabin. Case-patients were more likely than non-ill cabin visitors to have slept on the floor (odds ratio [OR] = 28.0, 95% confidence interval [CI] = 3.0-258) or in the top bunk bed (OR = 5.2, 95% CI = 1.1-25.1). Tick-borne relapsing fever should considered in the differential diagnosis of fever in patients who have stayed overnight in mountain cabins in the western United States.

In vitro growth of glial cell-enriched and depleted populations from mouse cerebellum.

Glial cell-enriched and -depleted populations isolated from 10-day-old mouse cerebella have been grown in vitro. There are marked differences in the cellular morphology between these two populations. The glial cell-enriched populations are very heterogeneous with respect to cell size, morphology and processes, whereas the glial cell-depleted populations are very homogeneous, containing a cell type with a small cell body and predominantly bipolar processes. Further characterization of the cell types has been affected using antiserum prepared against GFA protein and tubulin. The immunocytochemical localization of these proteins clearly identifies astrocytes and neurons. The glial cell enriched populations contain several types of astrocytes and neurons in addition to cells of non-ectodermal origin, whereas the glial cell-depleted populations contain predominantly a single neuronal cell type, the granule cells.

Isolation of glial cell-enriched and -depleted populations from mouse cerebellum by density gradient centrifugation and electronic cell sorting.

Preparative amounts of populations enriched and depleted in glial cells have been isolated from 10-day-old mouse cerebella. Discontinuous density bovine serum albumin (BSA) gradients provide two distinct populations: the one derived from the 10-15% BSA interface is enriched in cyclic nucleotide phosphohydrolase (CNPase) activity, and a percentage of S-100, GFA protein, and NS-1 antigen-positive cells; the other, located as the 15-31% interface, contains a cell population depleted in these compounds. This report also describes the first use of flow microfluorimetry and electronic cell sorting techniques for the analysis and isolation of cell populations derived from the mammalian central nervous system. Glial cell-enriched and -depleted populations obtained by BSA density gradient centrifugation were analyzed for their forward angle light scattering properties and their capacity to bind anti-corpus anti-serum to the cell surface. The glial cell-enriched fraction shows a different frequency distribution of light scattering than the glial-depleted fraction. Anti-corpus callosum antiserum binds preferentially to the glial cell-rich fraction. Cells can be sorted into corpus callosum antigen-positive and -negative cell fractions and recovered with a viability of more than 95% as judged by trypan blue exclusion. Anti-corpus callosum-positive sorted cells are enriched in CNPase activity, S-100, and glial fibrillary acidic proteins.

Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools.

Pyruvate carboxylase is the predominant anaplerotic enzyme in CNS tissues, and thus provides for net utilization of glucose to generate citric acid cycle intermediates such as alpha-ketoglutarate and malate for replenishment of the neurotransmitter pools of glutamate, GABA and aspartate. Studies reported in this paper involving immunocytochemical and biochemical techniques demonstrate: (1) the enzyme is localized in astrocytes as visualized by immunofluorescence in sections of cerebellum and (2) the enzyme activity in astrocyte-enriched populations is 3 X higher than in granule cell-enriched populations isolated from the cerebellum; similarly activity in different synaptosomal preparations parallels that for glutamine synthetase. We conclude from these results that the enzyme pyruvate carboxylase is an astrocyte-specific marker. This localization substantiates some recent hypotheses for astrocyte functions, including CO2 fixation in the CNS and the replenishment of citric acid cycle intermediates by astrocytes as precursors for amino acid neurotransmitter pools.

Brain cell surface antigens detected by anti-corpus callosum antiserum.

When rabbits are injected with tissue homogenates of white matter from bovine corpus callosum, an antiserum is produced which reacts with the surface membrane of 20-30% of all cells obtained by trypsin-dissociation of cerebellum from 10-day-old mice. The antigen or set of antigens recognized by this antiserum is detectable on embryonic, early postnatal, and adult mouse brain, but not on liver, spleen, kidney, thymus and sperm. The antigen is expressed in different regions of the brain and also, in decreased amounts, on retina. In histological sections of cerebellum from 21-day-old mice the antigen is predominantly localized in white matter tracts. Whereas nervous tissue from chicken and rabbit does not carry detectable levels of the antigen, rat, bovine and human brains are antigen-positive.

Investigations on myelination in vitro: biochemical and morphological changes in cultures of dissociated brain cells from embryonic mice.

The incorporation of 35SO4(2-) and [3H]galactose into myelin-associated lipids, the activity of enzymes catalyzing the synthesis of these lipids, and the activity of 2',3'-cyclic nucleotide phosphohydrolase were determined in primary cultures of dissociated cells from brains of 15-day embryonic mice. These biochemical parameters of myelination were barely detectable before about 10 days in culture, but their activity increased in parallel after this time and reached a maximum at about 40 days in culture. The activities of the selected enzymes in homogenates of the cultured cells at their optimum age were of the same order of magnitude as the same enzymes derived from fresh brain. Scanning electron microscopic studies showed that the cells after adhering to the surface by the 4th day form aggregates and extensive membranes; the aggregates increase in size and coalesce to form nests of cells by the 15th day; the surface of the aggregates becomes smoother until by the 43rd day the entire surface is covered by and cells are buried in a membrane-like substance. These biochemical measurements and morphological data suggest that the cultures of dissociated cells from brain of 15 day embryonic mice provide a useful system for studying myelination and its regulation in vitro.

Occurrence and evolutionary significance of a California encephalitis-like virus in Aedes squamiger (Diptera: Culicidae).

More than 12,000 Aedes increpitus Dyar and 4,600 Aedes squamiger (Coquillett) were tested for the presence of arboviruses to test the hypothesis that there is a coevolutionary relationship between Aedes (Ochlerotatus) mosquitoes and California serogroup viruses. Five strains of a California encephalitis-like virus were isolated from adults reared from larvae of Ae. squamiger collected in January 1989 from a coastal salt marsh at Morro Bay, San Luis Obispo County, California. Viruses were isolated in Vero cell cultures and serotyped by cross-neutralization tests. These isolates represent the first arboviruses isolated from this species. On the basis of morphology, Aedes squamiger has been included in the Aedes stimulans group of the subgenus Ochlerotatus. Other species within the Ae. stimulans group are vectors of California (CAL) serogroup viruses elsewhere in North America. Analysis of isozyme variability supports the inclusion of Ae. squamiger in the Ae. stimulans group and suggests that coastal populations of Ae. increpitus are the closest California relatives of Ae. squamiger. Recovery of virus from Ae. squamiger reinforces the relationship between CAL serogroup viruses and Aedes (Ocherlotatus) mosquitoes. However, the failure to isolate virus from large samples of Ae. increpitus from coastal and low elevation inland habitats suggests a complex evolutionary history involving both vertical and horizontal transmission mechanisms.